Alexander GabibovMechanisms of Antigen degradation

Workshop "Biomedical technologies at Skokovo. Opportunities and challenges"

Antigen degradationmachinery

AAntintiBBodiesodies

Catalytic Antibodies

ENENzymeszymes

ABzymesABzymes

Catalytic Antibodies. Historical Background

Five ways to obtain catalytic antibodies

Five ways to obtain catalytic antibodies

Immunization by transition

state analog of reaction

Production of antiidiotypicantibody

Reactive immunization

Induction of autoimmunedisease

Screening of phage-display libraries

Belogurov et al, BioEssays 2009

Organophosphorous poisons

The major target of organophosphorous toxins are cholinesterase-like enzymes;

Extremely low LD50 value

OPC-associated mortality is 200,000 people per year;

There are real threats of acts of terrorism, for example sarin attack in Tokyo underground at 20 March of 1995

9A8 may covalently accept and destroy anti-acetylcholine esterase poisons from blood stream 9A8 may covalently accept and destroy anti-acetylcholine esterase poisons from blood stream

Kolesnikov et al, PNAS 2000

3D Structure of the 9A8 Antiidiotypic Antibody Active Site

• Superposition of the active sites of esterolytic abzymes 9A8 (green) and 17E8 (blue).

• Ser99 - His35 diades are indicated.

• Hydrogene bonds are indicated by dashed lines.

Soman

r9A8 interact with soman-MCA

RFU

Time, h

Combinatorial approachRational design

High resolution 3D Effective screening

Directed evolution

-streptavidin molecule

-BSA molecule

-Biotin group

-Phosphonate group

Control wellExperimental well

Phage pull after reaction with Bt-X

phosphonate

Nonspecific sorbsion

Nonspecific and

specific sorbsion

Trypsin elution

Phage amplificatio

n

Next rounds of selection

Bt-X

Reactive (covalent) selection

A.17 Catalytic Antibody

Biotin-X-phosphonate

Griffin.1 scFv library

+

Screening for biotin-X binding

Conversion into the full-size human antibody

crystal

Reshetnyak et al, JACS 2007

A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base

A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base

Comparison of active site cavities of natural and created de novo biocatalysts. Chemically selected reactibody A.17 possess deepest substrate binding niche. Cross-section views of the active center of esterolytic antibodies 49G7, TEPC15, aldolase antibody 33F12, choline esterases AChE and BChE and antibody A.17 complexed with their ligands.

Tyr59

Tyr53

Tyr33

Tyr34

Trp109 Tyr37

Trp92

Trp48

Phe100

OP compound

P

O

O

O

NO2

HN

Tyr37

OH

P(R)

P

O

OO

HN

Tyr37

P(S)

SN2

Tyr59Tyr53

Tyr33

Tyr34

Trp109

Asn105

Ala107

Phe100

Trp92

Trp48

Cl-

2.5 Å 3.3 Å

3.2 Å

Ser51

2.8 Å

Tyr37

The pre-existing primitive active site of the A.17 antibody stereo-selectively interacts with P(R)-isomer of the phosphonate molecule

TyrTyr

+Tyr

Tyr

TyrTyr

++

A.17 antibody hydrolyzes organophosphorus pesticide – paraoxon by multi-step covalent catalysis.

Covalent intermediate

Amount of released p-nitrophenol in case of the reaction with paraoxon is evidently higher than

concentration of active sites

The reaction rate increased linearly with hydroxylamine concentration. It allows to define that

dephosphorylation is rate limiting step

k2 = 1.1 ± 0.1 x10-1 min-1

k3 = 1.6 ± 0.2 x10-2 min-1

k4 = 1.26 ± 0.09 x10-3 min-1

1576.3

1578.530+

1579.430+

1580.330+

1581.331+

1584.21585.9

1590.930+

1591.830+

1592.7 1593.81603.2

19+

1575 1580 1585 1590 1595 1600 1605 m/z0.0

0.5

1.0

1.5

2.0

8x10Intens.

FAB_A17_000001.d: +MS

1578.430+

1580.915+

1583.030+

1584.230+

1585.9 1587.5 1589.0

1590.930+

1595.730+

1603.230+

1575 1580 1585 1590 1595 1600 1605 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

8x10Intens.

FAB_A17_Paraoxon_000001.d: +MS

1578.5m/z=30

1578.5m/z=30

1583.0m/z=30

Δm=135 ( )

(unmodified Fab A.17)

(phosphonylated Fab A.17)

(unmodified Fab A.17)

The reaction mechanism of paraoxon hydrolysis by A.17 antibody proceeds through the phosphotyrosine covalent

intermediate.

Perspectives: generation of artificial biocatalysts in vivo

MESSAGE• Basically ALL Autoantigens may serve as substrates for autoantibodies. Kolesnikov et al. PNAS, 2000

Ponomarenko et al. Meth. Immunol., 2002Kozyr et al. Imm.Lett., 2002Ponomarenko et al. PNAS, 2006Ponomarenko et al. Biochemistry 2006Ponomarenko et al. Biochemistry, 2007Belogurov et al. J.Immunology, 2008Durova et al. Molecular Immunology, 2009Belogurov et al. Autoimmunity, 2009

Belogurov et al, BioEssays 2009

CDRH3

DNA

Possible catalytic residues

Schuster et al, Science, 1992, Gololobov et al, PNAS, 1995; Gololobov et al. Mol Immunol. 1997.

Structural Similarity Between BV04-01 and MRL-4 anti-DNA Autoantibodies: DNA-binding and DNA-cleaving Activities are

Germline-Encoded

Do Myelin-Directed Antibodies Predict Multiple Sclerosis?N EJM, 2003

The B-Cell – Old Player, New Position on the TeamNEJM, 2008

Multiple sclerosis

B-cells as one of the key players in the MS

Environmental hypothesis of MS induction. EBV virus involvement.

Environmental hypothesis of MS induction.EBV virus involvement.

Antibodies selected from MS Phage-display library are crossreactive towards both, Myelin Basic Protein and EBV latent membrane protein 1.

Abzymatic Site-Abzymatic Site-specific MBP specific MBP hydrolysishydrolysis

The cleavage sites are localized inside the encephalitogenic epitopes The cleavage sites are localized inside the encephalitogenic epitopes

MBPMBPMusse et al, PNAS 2006

Ponomarenko et al, PNAS 2006

Toxin

Autoreacti

veB-Cell

B-Cell Receptor (BCR), fragments of myelin basic protein (MBP)

BCR

MBPCD4CD25High TOLERANCE

Specific B-Cells depletion in MS

C D A B

Autoantibody binding pattern

Specific B-Cells depletion in MS

Fc-based immunotoxins are shown to be the best in the presented set due to the excellent specific/unspecific cytotoxicity ratio

MDS score versus days post disease induction (surrounding pictures). Peptides were applied at days 7-11 (120 μg/rat per day) after disease induction by nasal route. Maximum clinical score in each group of rats, median and 95% confidential interval (in the middle). NS - not significant

Belogurov et. al Autoimmunity

Administration of MBP peptides to DA rats with EAE

Clinical trials of MBP46-62 formulation in DA rats

• SUV liposomes 50-90 nm mimicking virus particles

• Interaction with APC cells • Dose-dependent effect

PP IPIP

The level of IP in the brain is dramatically elevated during EAEdevelopment in SJL/J mice.

Immunoproteasome localization in mice brain. LMP2 and LMP7 are carried by

different cells.

MBP hydrolysis by proteasome and enzymes. LC-ESI approach.

Enhanced release of encephalitogenic peptide by immunoproteasome. LC-ESI approach with isotope labeled

peptide.

• Immunoproteasome marks oligodendrocytes for CTL (immune system)

• Some kind of target designation

• How can we prevent this process? The answer is Inhibitors or siRNA

Expression of LMP7 immunosubunit is significantly decreased by siRNA

administration

Low-weight proteasome inhibitors

Low-weight inhibitors efficiently inhibit immunoproteasome in vitro and ameliorate EAE

in vivo

Immunoproteasome as a target for MS treatment

Der Mensch als Industriepalast (Man as Industrial Palace) Stuttgart, 1926. Chromolithograph. National Library of Medicine. Fritz Kahn (1888-1968) Kahn’s modernist visualization of the digestive and respiratory system as "industrial palace," really a chemical plant

M.M. Shemyakin & Yu.A. OvchinnikovInstitute of Bioorganic Chemistry,

Russian Academy of Sciences

Authors:

Alexandre G. Gabibov, Alexey A. Belogurov Jr., Ivan V. Smirnov, Inna N. Kurkova, Ekaterina Kuzina, Alexey Kononikhin, Alexey Stepanov, Natalie A. Ponomarenko, Andrey V. Reshetnyak

COLLABORATIONRUSSIA

Prof. Eugene NikolaevInstitute of Biochemical Physics RAS

Prof. Alexey BoykoMoscow MS Center

Prof. Dmitry Knorre,Prof. Olga Fedorova, Dr. Nikita Kuznetsov

Dr. Dmitry GenkinDr. Dobroslav Melamed

USAProf. Al Tramontano, UC Davis, Medical school

National Institute of Allergy and Infectious Diseases National Institutes of Health Dr. Herbert C. Morse III;Ciphergen Biosystems, Inc

FRANCEProf. Daniel Thomas, Alain Friboulet, Drs. Dominidue Pillet, Marjorie

Paon, Berangere Avalle.

The University of Technology, CompiegneProf. Patrick Masson, Drs Eugénie Carletti, Florian Nachon

Département de Toxicologie – CRSSA Institut de Recherche Biomédicale des Armées