Development of passive immunity for Ebola virus infections through convergence of science amp
technology mice primates and plants
Gene Garrard Olinger Jr PhD MBA
Principal Advisor Science MRIGlobal Inc
Nov 3 2014
Acknowledgements Mapp
Natasha Bohorova
Ognian Bohorov
Andrew Hiatt
Do Kim
Michael Pauly
Jesus Velasco
Kevin Whaley
USAMRIID
James Pettitt
Julia Biggins
Lisa Hensley
Gene Olinger
PHAC
Gary Kobinger
Defyrus
Jeff Turner
Jane Ennis
Icon Genetics
Yuri Gleba
CBR International
Jeanne Novak
Miles Brennan
Kevin Hennegan
Jessica Egner
Kentucky BioProcessing (KBP)
Barry Bratcher
Hugh Haydon
Steve Hume
Ashley Johnson
Josh Morton
Katrina Whelan
Cara Working
Funding
NIAID AI61270 AI72915
UAMRMC 17-02-2-0015
DTRA 410007-08-RD-B
2
Interpreting Technology Hype When new technologies make bold promises how do you discern the hype from whatrsquos commercially viable And when will such claims pay off if at all
Filovirus Viral Hemorrhagic Fevers bull Ebola and Marburg
bull First case 1976 Zaire bull Negative sense enveloped ssRNA virus bull Filamentous morphology bull Epidemiology
bull Natural host is unknown bull Transmission associated with close contact (blood or body fluids)
bull Clinical Features bull Incubation period 4-21 days bull Abrupt onset of nonspecific symptoms bull Liver function impaired bull Bleeding amp dysregulated coagulation (clotting) bull Deathshock 6-9 days after onset bull Case fatality rates high (40-90) bull No apparent immune response in fatal cases
Subtypes Zaire Sudan Ivory Coast Gabon Reston Uganda
Filovirus Structure
RNA
L
NP
VP35
VP40 VP30
Envelope
VP24
GP12
Glycoprotein (GP) spikes exist as
trimers Structure is incompletely
described crystallography is
available only for a portion of EBOV
base GP2
Virus-associated host cell proteins
(specifically or nonspecifically
incorporated into virions) have neither
been described nor excluded
diameter 80 nm
avg length 665 nm
viral envelope only GP
ectodomain (GP1 and most
of GP2) is known to be
exposed on exterior surface
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Acknowledgements Mapp
Natasha Bohorova
Ognian Bohorov
Andrew Hiatt
Do Kim
Michael Pauly
Jesus Velasco
Kevin Whaley
USAMRIID
James Pettitt
Julia Biggins
Lisa Hensley
Gene Olinger
PHAC
Gary Kobinger
Defyrus
Jeff Turner
Jane Ennis
Icon Genetics
Yuri Gleba
CBR International
Jeanne Novak
Miles Brennan
Kevin Hennegan
Jessica Egner
Kentucky BioProcessing (KBP)
Barry Bratcher
Hugh Haydon
Steve Hume
Ashley Johnson
Josh Morton
Katrina Whelan
Cara Working
Funding
NIAID AI61270 AI72915
UAMRMC 17-02-2-0015
DTRA 410007-08-RD-B
2
Interpreting Technology Hype When new technologies make bold promises how do you discern the hype from whatrsquos commercially viable And when will such claims pay off if at all
Filovirus Viral Hemorrhagic Fevers bull Ebola and Marburg
bull First case 1976 Zaire bull Negative sense enveloped ssRNA virus bull Filamentous morphology bull Epidemiology
bull Natural host is unknown bull Transmission associated with close contact (blood or body fluids)
bull Clinical Features bull Incubation period 4-21 days bull Abrupt onset of nonspecific symptoms bull Liver function impaired bull Bleeding amp dysregulated coagulation (clotting) bull Deathshock 6-9 days after onset bull Case fatality rates high (40-90) bull No apparent immune response in fatal cases
Subtypes Zaire Sudan Ivory Coast Gabon Reston Uganda
Filovirus Structure
RNA
L
NP
VP35
VP40 VP30
Envelope
VP24
GP12
Glycoprotein (GP) spikes exist as
trimers Structure is incompletely
described crystallography is
available only for a portion of EBOV
base GP2
Virus-associated host cell proteins
(specifically or nonspecifically
incorporated into virions) have neither
been described nor excluded
diameter 80 nm
avg length 665 nm
viral envelope only GP
ectodomain (GP1 and most
of GP2) is known to be
exposed on exterior surface
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Interpreting Technology Hype When new technologies make bold promises how do you discern the hype from whatrsquos commercially viable And when will such claims pay off if at all
Filovirus Viral Hemorrhagic Fevers bull Ebola and Marburg
bull First case 1976 Zaire bull Negative sense enveloped ssRNA virus bull Filamentous morphology bull Epidemiology
bull Natural host is unknown bull Transmission associated with close contact (blood or body fluids)
bull Clinical Features bull Incubation period 4-21 days bull Abrupt onset of nonspecific symptoms bull Liver function impaired bull Bleeding amp dysregulated coagulation (clotting) bull Deathshock 6-9 days after onset bull Case fatality rates high (40-90) bull No apparent immune response in fatal cases
Subtypes Zaire Sudan Ivory Coast Gabon Reston Uganda
Filovirus Structure
RNA
L
NP
VP35
VP40 VP30
Envelope
VP24
GP12
Glycoprotein (GP) spikes exist as
trimers Structure is incompletely
described crystallography is
available only for a portion of EBOV
base GP2
Virus-associated host cell proteins
(specifically or nonspecifically
incorporated into virions) have neither
been described nor excluded
diameter 80 nm
avg length 665 nm
viral envelope only GP
ectodomain (GP1 and most
of GP2) is known to be
exposed on exterior surface
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Filovirus Viral Hemorrhagic Fevers bull Ebola and Marburg
bull First case 1976 Zaire bull Negative sense enveloped ssRNA virus bull Filamentous morphology bull Epidemiology
bull Natural host is unknown bull Transmission associated with close contact (blood or body fluids)
bull Clinical Features bull Incubation period 4-21 days bull Abrupt onset of nonspecific symptoms bull Liver function impaired bull Bleeding amp dysregulated coagulation (clotting) bull Deathshock 6-9 days after onset bull Case fatality rates high (40-90) bull No apparent immune response in fatal cases
Subtypes Zaire Sudan Ivory Coast Gabon Reston Uganda
Filovirus Structure
RNA
L
NP
VP35
VP40 VP30
Envelope
VP24
GP12
Glycoprotein (GP) spikes exist as
trimers Structure is incompletely
described crystallography is
available only for a portion of EBOV
base GP2
Virus-associated host cell proteins
(specifically or nonspecifically
incorporated into virions) have neither
been described nor excluded
diameter 80 nm
avg length 665 nm
viral envelope only GP
ectodomain (GP1 and most
of GP2) is known to be
exposed on exterior surface
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Filovirus Structure
RNA
L
NP
VP35
VP40 VP30
Envelope
VP24
GP12
Glycoprotein (GP) spikes exist as
trimers Structure is incompletely
described crystallography is
available only for a portion of EBOV
base GP2
Virus-associated host cell proteins
(specifically or nonspecifically
incorporated into virions) have neither
been described nor excluded
diameter 80 nm
avg length 665 nm
viral envelope only GP
ectodomain (GP1 and most
of GP2) is known to be
exposed on exterior surface
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Olinger Laboratory
bull Vaccines bull Candidate Filovirus vaccines
bull VEE Replicon (VRP) amp Synthetic Vaccines bull Novel vaccine platforms (antigenformulation)
bull Immune Correlates bull Antibody Responses bull Cellular responses bull Biomarkers (early predictors of vaccine or disease outcome)
bull Filovirus
bull Lassa virus
bull Therapeutics bull Small Molecules (drugs)ndash Repurposing of approved drugs bull Passive Immunotherapy ndash monoclonal antibody(s)
Converging Science Disciplines and Innovation
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Animal Models for VHFV- Animal Rule
bull Mouse Guinea Pig Nonhuman Primate (NHP)
Critical link that may be difficult to
achieve with animal data
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Functional overlaps and distinctions among antibodies elicited by infection andor vaccination
(Alan L Schmaljohn Current HIV Research 2013 Vol 11 No 5)
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Venezuelan Equine Encephalitis Replicon (VRP) Expressing Filovirus Glycoprotein Genes
Phase I Clinical Experience with Platform bull HIVgag VRP (AVX101)
ndash HIV uninfected healthy adults 108 vaccine recipients ndash NIH HVTN
bull Flu HA (H3) VRP (AVX502) ndash Adults 18-40 yo 192 vaccine recipients ndash Adults gt65 yo 20
bull CMV VRP (AVX601) ndash CMV seronegative adults 18-40 yo 32 vaccine recipients
bull CEA VRP (AVX701) ndash Patients with CEA+ malignancies 20 vaccine recipients
bull PSMA VRP ndash At MSK in patients with prostate cancer lt18 vaccine recipients
bull Total ~390 subjects have received multiple doses of VRP vaccines to date
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Filovirus Vaccine Induced Immunity
Obtaining Correlates of Protection for Human Disease
What are the Protective Innate amp
Adaptive Immune Responses
B Cell
Dendritic Cells CD4 CD8
Macrophage
NK Cell
Antibody
CTL
Vaccinate
animal
Lethal
Filovirus
challenge
Cr51
+ CD8
CTL
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Correlate of Immunity-Mechanism of Action (MOA)
Vaccinate Mice
Lethal Challenge
Protected
Cells Serum (Antibody)
Adoptive transfer into unvaccinated mouse
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Survival of Mice Vaccinated to Ebola and Identification of Protective Immune Responses
SurvivorsTotal () Protection
Replicon BALBc C57Bl6 Sera Transfer
GP 910 (90) 1010 (100) 3240 NP 1010 (100) 1516 (93) 140 VP24 1515 (100) 040 (0) 020
VP30 2525 (100) 2525 (100) 020 VP35 3840 (95) 4040 (100) 020 VP40 1515 (100) 1620 (80) 020
Lassa N 030 (0) 030 (0) 040
Optimized Vaccine platform
No protection observed in C57Bl6 mice vaccinated with VP24 expressing VRP
Protection data improved based on conservative replicon titer estimation
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
14
Generation of Ebola Zaire Monoclonal Antibodies
bull BALBc mice were injected subcutaneously at the base of the neck with 2 x 106 focus-forming units of Venezuelan equine encephalitis (VEE) virus replicons encoding the glycoprotein (EboGP-VRP) from the Mayinga isolate of the Zaire strain of Ebola virus Fusionhybridization of P3X63Ag8653 myeloma cells with spleen cells from immunized mice as previously described (Stiles et al Toxicon 29 1195-1204 1991)
bull Boosted SC and then IV
bull Screen using IFA whole antigen ELISA
bull Cloning assess in mouse model for protection
bull Non-neutralizing protective mabs
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
15
Generation of Ebola Zaire Specific Monoclonal Antibodies
gt1700 Supernatants (ELISA IFA)
600 positives
40 cloned 27 immunoprecipitated GP
18 tested for protection in mice
90-100 60 0-30
N= 9 2 7
IgG2a IgG1 IgG1 IgG2a (3) IgG2b IgG3 IgA
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Properties of Murine derived Ebola GP Monoclonal Antibodies
MAb Specificity Survival Days given Isotype
1 13F6 Zaire GP1 (401-417) 90-100 -1+1 G2a ATQVEQHHRRTDNDSTA
2 6D8 Zaire GP1 (389-405) 90-100 -1+1 G2a
KLDISEATQVE 50-60 +2
3 12B5 Zaire GP1 (477-493) 60-80 -1+1 G1 GKLGLITNTIAGVAGLI
4 13C6 Zaire IC Sudan GP1 sGP conformational 90-100 -1+1+2 G2a
5 6D3 Zaire IC GP1 sGP conformational 80-100 -1+1+2 G2a
Reference Wilson J Hevey M Bakken R Guest S Bray M Schmaljohn A and Hart MK 2000 Science 287 1664-1666
Linear Epitope Confirmational Epitope
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
17
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Summary of MB-003 mAbs bull Discovered at USAMRIID1
ndash 3 mAbs against non-overlapping epitopes on GP
ndash 1 is cross-reactive with Sudan and Ivory Coast
ndash Protective in mice pre- and post-exposure (up to +2 days)
bull Humanizationchimerization of mAbs (DARPA)
bull RAMP production initial in vivo testing (NIAID U01)
bull CHO production to compare with RAMP-derived mAb (NIAID SBIR)
bull Process development and RAMP manufacture of MB-003 for NHP studies (Phase 2 SBIR)
bull NHP efficacy studies (DTRA) 1 Wilson JA et al Science 2000 Mar 3287(5458)1664-6
18
mAb ELISA reactivity to Ebola strains
Immuno-precipitation
Western blot reactivity
Epitope group
In vitro neutralization
Z S IC R
h13F6 + - - - GP1 GP1 1 No
c6D8 + - - - GP1 GP1 2 Yes
c13C6 + + + + GP1 sGP negative 4 Yes
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Mechanism of action in vivo (mouse studies)
19
Study Result Conclusion
Mice treated with cobra venom factor were protected from challenge
Complement is not required for protection
C3 knockout mice were protected from challenge
Complement is not required for protection
Aglycosylated mAb provides significant but reduced protection from challenge
Steric hinderance (classic neutralization) and glycan dependent Fc functions both play a role in protection
SCID and SCID beige mice offer a similar delay day to death (to each other) from challenge while mAb is present in serum
Lack of B T and NK cells donrsquot antibody delay to death
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Antibodies against ebolaviruses cluster at 3 sites
1H3
13C6
glycan cap mucin
13F6
6D8
14G7
KZ52
16F6
4G7
2G4
base But none yet against
the receptor-binding site
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
So how do these protective non-neutralizing antibodies function in vivo bull ADCC
bull ADCrsquo ndash less likely
bull Bind and inhibit sGP and free GP ndash alter pathogenesis
bull Others
Novel Functioning Antibodies
bull ldquoCell Targeting Antibodiesrdquo
bull Multiple functions- ldquoMulti-functional Effector Antibodiesrdquo
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Three Main Issues
bull Unknown MOA for antibody protection
bull Production plant production and glycosylation
bull Prior studies showing mAbs ineffective
Need sufficient quantities for NHP efficacy studies 100 mgkg x 4 kg NHP x 3 NHP = 1200 mg
Ultimate goal Scalable manufacturability regulatory approved method production amp purification processes
wwwmedicinevirginiaedu
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
bull Production
bull Cell line characterization
bull Automated Purification
bull QA-Control
Increasing Laboratory Scale of mAb production
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Historic concerns with plant production
bull GMO and field crop production systems
bull Potential immunogenicity of plant sugars
bull Regulatory risk
24
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Clinicalregulatory experience of plant-derived biopharmaceuticals
Company Plant system Product Clinical stage
ProtalixPfizer Carrot Enzyme replacement Licensed
Medicago Nicotiana Influenza vaccine Phase 2
Meristem Corn Enzyme Phase 2
Planet Nicotiana Anti-caries mAb Phase 2
IconBayer Nicotiana mAb cancer vaccine Phase 1
Large Scale Biology Nicotiana scFv cancer vaccine Phase 1
iBio Nicotiana Influenza vaccine Phase 1
Monsanto Corn Cancer therapy Phase 1
25
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Monoclonal Antibodies to Ebola Virus Glycoprotein
Molecular Rescue ldquoHumanizationrdquo amp isotype optimization
mAb variable regions were de-immunised and joined with human constant regions to make IgG1
Antibody-based Therapeutic for Ebola virus
Y Y
Y
Formulation and Efficacy Testing
mAbs production in Nicotiana benthamiana-based rapid antibody manufacturing platform (RAMP)
Mechanism of Action Studies
Repeat process for other Biothreat agents
Optional Cell Production of mAbs
Murine mAbs (13C6 6D8 and 13F6) protect mice from Ebola challenge (Wilson JA et al Science 2000)
mAbs were expressed in mammalian tissue culture (NS0 CHO) amp and in N benthamiana using the Magnifection system
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
MappKBPICON partnership
Mapp bull Discovery bull Characterization bull Construct
development bull Preclinical testing bull Clinical bull Regulatory
KBP bull Process
development bull Expression
optimization bull In process analytics bull cGMP production bull CMC preparation bull Regulatory
ICON bull Expression technology bull Glycan technoology bull cGMP experience
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Small scale production CHO and RAMP (Rapid Antibody Manufacturing Platform)
RAMP magnICON deconstructed viral vectors (Icon Genetics) results in self-replicating viral-like spread of mRNA containing the mAb genes
CHO cells in a Wave bioreactor
Clarification Protein A affinity chromatography
Q Sepharose 28
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Transient production in Nicotiana benthamiana using RAMP
- magnICON deconstructed viral vectors (Icon Genetics)
- results in self-replicating viral- like spread of mRNA containing the mAb genes
Giritch et al PNAS 2006
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Lab scale RAMP production
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Rapid antibody manufacturing platform (RAMP) tobacco plant system overview
31
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
MB-2003 mAbs process flow chart
Harvest Extraction Clarification Filter press
Chromatography Protein A Sartobind Q CHT
Aseptically filled in a Class 100 cleanroom
UltrafiltrationDiafiltration
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Large scale manufacturing in Nicotiana
- More than 1 acre of indoor controlled growth space - cGMP production at KBP in 2011 - gt 100 grams per lot - Multiple kilogram annual capacity in place
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Schematic presentation of reactions catalyzed by β14 galactosyltransferase (GalT) N-acetylglucosaminyltransferase III (GnTIII) and core α16 fucosyltransferase (FUT8)GlcNAc N-acetylglucosamine UDP Uridine diphosphate GDP guanosine diphosphate
Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody Alexandra Castilho1 Natasha Bohorova2 Josephine Grass3 Ognian Bohorov2 Larry Zeitlin2 Kevin Whaley2 Friedrich Altmann3 and Herta Steinkellner1PLoS One 2011 6(10) e26040 Epub 2011 Oct 24
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
GlcNAc
IgG Asn297
Mannose core glycans ndash plants and mammals
plant glycans Xylose Fucose (α13)
Fucose (α16) mammalian glycans
IgG glycosylation
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
2014 Current State of the Art in Plant Pharmahellip Host plant engineering for ldquohumanized glycosylationrdquo
Fuc
Sialic Acid
Plant Human
Gal
Xyl
Remove Xyl amp Fuc
Add Gal Multi-antennary
Add Sialic Acid
bull Multiple groups have genetically engineered tobacco to alter post-translational protein modifications
bull The work of Steinkellner and colleagues pioneered removal of Xylose and Fucose transferases and addition of mammalian enzymes to give tobacco the entire human sialylation pathway
bull Engineered host plants is mitigating regulatory concerns
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Pilot studies
Comparing CHO and RAMP
37
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectant Larry Zeitlina1 James Pettittb Corinne Scullyb Natasha Bohorovaa Do Kima Michael Paulya Andrew Hiatta Long Ngoc Herta Steinkellnerd Kevin J Whaleya and Gene G Olingerb aMapp Biopharmaceutical San Diego CA 92121 bDepartment of Virology US Army Medical Research Institute of Infectious Diseases Frederick MD 21702 cDepartment of Medicine Beth Israel Deaconess Medical Center Boston MA 02215 and dDepartment of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences 1190 Vienna Austria
20690ndash20694 | PNAS | December 20 2011 | vol 108 | no 51
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS
Zeitlin L et al PNAS 201110820690-20694
copy2011 by National Academy of Sciences
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
RAMP-derived mAbs improved potency
0
10
20
30
40
50
60
70
80
90
100
1 10 100
s
urv
ival
micrograms of mAb
h-13F6 CHO
h-13F6 RAMP
h-13F6 agly
murine 13F6CHO ED50 = 055 mgkg RAMP ED50 = 015 mgkg
Enhanced Potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant Zeitlin L Pettitt J Bohorova N Kim
D Pauly M Hiatt A Ngo L Steinkellner H Whaley KJ Olinger GG Proc Natl Acad Science USA 2011 Dec 20 108 (51) 20690-4
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques Gene Garrard Olinger Jra12 James Pettitta1 Do Kimb Cara Workingc Ognian Bohorovb Barry Bratcherc Ernie Hiattc Steven D Humec Ashley K Johnsonc Josh Mortonc Michael Paulyb Kevin J Whaleyb Calli M Leara Julia E Bigginsa Corinne Scullya Lisa Hensleya3 and Larry Zeitlinb2 aDivision of Virology United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702 bMapp Biopharmaceutical Inc San Diego CA 92121 and cKentucky BioProcessing LLC Owensboro KY 42301
20690ndash20694 | PNAS | October 15 2012 |
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Pilot NHP testing
1000 pfu IM Zaire (Kikwit)
Dosing +1 h 4 d 8 d
42
No virus detected by RT-PCR or plaque assay in treated survivors
Olinger G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Initiation of treatment of rhesus macaques with MB-003 at 24 or 48 h postinfection provides
protection against EBOV Delayed treatment with MB-003RAMP (1667 mgsdotkgminus1sdotmAbminus1) was
given 24 h (purple arrows) and 48 h (green arrows) after being challenged im w
Olinger G G et al PNAS 201210918030-18035
copy2012 by National Academy of Sciences
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Research Article
Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail James PettittLarry Zeitlin Do H Kim Cara Working Joshua C Johnson Ognian Bohorov Barry Bratcher Ernie Hiatt Steven D Hume Ashley K Johnson Josh Morton Michael H Pauly Kevin J Whaley Michael F Ingram Ashley Zovanyi Megan Heinrich Ashley Piper Justine Zelko and Gene G Olinger
Science Translational Medicine 21 August 2013
Vol 5 no 199 pp 199
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Symptom Initiated Treatment of Ebola Zaire
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
27 33 39 45 51 57 63 69 75 81 87 93 99 105 111 117 123
1 2 3 4 5 7 8 9 10
Positive PCR Result DSD EUA Temperature increase
Hours post infection Treatment delayed due to EUA PCR errorrepeat
MB-003 treatment initiation Control NHP N
HP
survivor
non-survivor
Symptom Initiated Treatment of Ebola Zaire
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
NHP efficacy experiments for MB003
47
Time treatment initiated post-
challenge
Challenge stock
(1000 pfu) Dosing
Survival (n)
P-value vs controls
(inc historic)
Survivors with minimal to no clinical
morbidity
1 h Kikwit P3 50 mgkg x 3 100 (3) lt 002 100
24 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
48 h Kikwit P4 50 mgkg x 4 67 (3) lt 005 100
103-119 h Kikwit P4 50 mgkg x 3 43 (7) lt 003 67
Olinger GG Jr et al Proc Natl Acad Sci U S A 2012 Oct 30109(44)18030-5 Pettitt J et al Sci Trans Med 2013 21 August 2013 Vol 5 Issue 199
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Pharmacokinetics of MB-003 in NHP (non-GLP)
48
c13C6 c6D8 h13F6
AUC (μgmlday) 1239 + 73 1849 + 211 2539 + 171
Cmax (μgml) 272 + 13 369 + 55 249 + 27
Beta half-life (day) 74 + 09 71 + 11 83 + 07
- Single IV dose of 50 mgkg of MB-003 infused over 30 min - Samples tested by antigen specific ELISA - PK parameters estimated with WinNonlin 63
c13C6
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Pharmacokinetics in efficacy experiments (therapeutic study)
49
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Research Article
Successful Treatment of Ebola VirusndashInfected Cynomolgus Macaques with Monoclonal Antibodies Xiangguo Qiu Gary Wong Lisa Fernando Jonathan Audet Alexander Bello Jim Strong Judie B Alimonti and Gary P Kobinger
Science Translational Medicine 13 June 2012
Vol 4 no 138 pp 138
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Defyrus MappBio Joint Venture
Plants (KBP)
US Army
PHAC
IP IP
CHO Cells (NRC)
Plants (KBP)
CHO Cells (CMO)
ClientStockpile (Lab accident biodefense)
Current Production
Manufacturing Options
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Optimal Combinations MB-003 ZMAb
6D8 2G4 13F6 13C6
Eboviximab - ZMAPP
1H3 4G7
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp Xiangguo Qiu Gary Wong Jonathan Audet Alexander Bello Lisa Fernando Judie B Alimonti Hugues Fausther-Bovendo Haiyan Wei Jenna Aviles Ernie Hiatt Ashley Johnson Josh Morton Kelsi Swope Ognian Bohorov Natasha Bohorova Charles Goodman Do Kim Michael H Pauly Jesus Velasco James Pettitt Gene G Olinger Kevin Whaley Bianli Xu James E Strong Larry Zeitlin amp Gary P Kobinger
Nature 514 47ndash53 (02 October 2014) doi101038nature13777
Received 05 August 2014 Accepted | 21 August 2014 | Published online 29 August 2014
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
PHAC (Kobinger) testing of plant-derived mAbs in Guinea Pigs
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
1 Comparison of two cocktails (n=6 per group) 13C6 + 2G4 + 4G7 (cocktail 1) 13C6 + 1H3 + 4G7 (cocktail 2) PBS control (n=1) + mAb control (n=1) 2 mAb dosing begins 3 days post-infection (50 mgkg) with additional doses days 6 and 9 3 Animals are generally RT-PCR positive on day 3 The assay used as PHAC is identical to the one used by the CDCDoctors without Borders on the ground in Africa
PHAC (Kobinger) testing of MB-003ZMAb cocktails in NHPs
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
0 5 10 15 20 25
Post-infection (days)
Challenge 1000xLD50 EBOV strain Kikwit Backtitre 4000 TCID50 per NHP
mAb cocktail 50mgkg of 13C6 2G4 and 4G7 (Group A n=6) 50mgkg of 13C6 1H3 and 2G4 (Group B n=6) PBS or mAb isotype control (Group C n=1 each)
Termination of experiment at 27 days post-infection
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
mAb dosing
SURVIVAL Dosing initiated 3 days post-challenge
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
ZMapp
bull Combination is optimal of the six monoclonal antibodies
bull 13C6 is a multi-functional antibody that can target infected cells virus and sGPssGP
bull Antibodies recognize current outbreak isolate of EBOV
bull Useful post-exposure and as a therapeutic
bull Safety and efficacy demonstrated in macaques
bull Further development is on-going
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Gene Garrard Olinger Jr PhD MBA Principal Science Advisor MRIGlobal Inc High Containment Coordinator (Contractor) Office 1A-120 National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility Division of Clinical Research 8200 Research Plaza Fort Detrick MD 21702 Office Phone +1-301-631-7208 Cell Phone +1-240-344-0847 Fax +1-301-631-7389 Email geneolingernihgov golingermriglobalcom
Disclaimer Opinions interpretations conclusions and recommendations are those of the author and are not necessarily endorsed by the US Army US Government MRIGlobal Mapp Biopharmaceutical or by Boston University Disclosure Gene Olinger serves as a scientific consultant to MAPP and is a founder of Phoenix Research Group (PRG)
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
Director of High Containment Training Adjunct Associate Professor of Medicine Department of Infectious Diseases Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus golingerbuedu 617-414-8918
Collaborative Core Boston University School of Medicine National Emerging Infectious Diseases Laboratories Medical Campus
Global Cambio Simple Sustainable Solutions
