29
Page 1 A New Site Specific Antibody Conjugation Using Bacterial Transglutaminase ADC Summit, San Francisco On 15 th October, 2013
Page 1
A New Site Specific Antibody Conjugation
Using Bacterial TransglutaminaseADC Summit, San Francisco
On 15th October, 2013
October 2013 Page 2
From Chemotherapy to Homogeneous ADCsImproving the Therapeutic Index
Second Generation:engineered Cysteine conjugation
• Increased tumor delivery• Decreased normal tissue exposure• Heterogeneous PK
• Homogeneous PK• Unstability questiomark
• unpaired cystein• thiol-maleimide linkage
First Generation:Lysine or Cysteine conjugation
0 1 2 3 4 5 6 7 8 9
DAR
0 1 2 3 4 5 6 7 8 9
DAR
0 1 2 3 4 5 6 7 8 9
DAR
October 2013 Page 3
• Aglycosylated mAb
• Conjugation on endogenous Q295 and possibly on N297Q
• Lower uptake by FcR+ cells might improve tumor-specific targeting and limitoff-target toxicity
• LLQG Tag to createconjugation site
• POC (Strop et al., Chem. Biol., 2013)
• Effector function preserved
Next Generation ADCsHomogeneous and Stable
Transglutaminase (TG)
N297S
QQQ Q
N297Q
Unnatural Amino Acid
• Expanded genetic code to incorporate orthogonal side chains
• POC (Axup et al., PNAS, 2012)
• Specific production system
Oxime ligation
O O
TAG
TAG
TAG
TAG
TAGTAG
NOR
NOR
LLQG Tag Single point mutation
October 2013 Page 4
Bacterial Transglutaminase (BTG)Site-specific and Stoichiometric Enzymatic Conjugation
• BTG catalyses reactions betweenglutamine and lysine
• BTG recognizes exclusivelyendogenous Q295 located in Fcregion of aglycosylated IgG
• N297Q mutation provides 2 additionalsites for conjugation
Jeger et al., Angew. Chem. Int. Ed., 2010
N297QQ295
N297QQ295
N297SQ295
N297SQ295
PNGase
Single Point Mutation
NH2
BTG
NH2
BTG
NH2
BTG
N297Q295
N297Q295
N297QQ295
N297QQ295
Q295N297SQ295
N297S
Q295N297
Q295N297
October 2013 Page 5
Q 295 is barely exposed and partially hidden by the carbohydrate
N 297Q 295
Carbohydrate
CH2
CH3
BTG Ligation Site in Fc Structure Before and After Carbohydrate Removal
Degree of freedom is improved when carbohydrates are absent
October 2013 Page 6
BTG Coupling Reaction
BTG is calcium independant Acylenzyme intermediate formation Release of ammonia
K-substrate’s attack of thioester bond
Isopeptide bond formation
BTG
BTG
Page 7Page 7
One-step Approach
October 2013 Page 8
One-step Approach
Aglycosylated IgGSingle mutation N297S or N297Q
x 2 for N297Sx 4 for N297Q
BTG
NH2-step1-
= toxin
October 2013 Page 9
N297S coupling with NH2-step1a-vc-PAB-MMAELC/MS (ESI-qTOF)
145267
DAR 0
146610
147975
DAR 2
DAR 1
0
2
4
6
4x10
0
2
4
6
4x10
145000 145500 146000 146500 147000 147500 148000 148500 149000 m/z
DAR=1.8
• 10 equivalents of toxin/site• 24 hours
• M/Z shift = 2708• Theoretical 2 x M Toxin = 2706
October 2013 Page 10
N297Q coupling with NH2-step1a-vc-PAB-MMAELC/MS (ESI-qTOF)
145349
DAR 0
148042 149407
150763
DAR 4
DAR 2DAR 3
0
2
4
6
84x10
0
1
2
3
4x10
145000 146000 147000 148000 149000 150000 151000 152000 m/z
• m/z shift = 5415• Theoretical 4x M Toxin = 5412 DAR=3.7
• 20 equivalents of toxin/site• 24 hours
Page 11Page 11
Two-step Approach
October 2013 Page 12
Two-step Approach
x 2 for N297Sx 4 for N297Q
= toxin
Aglycosylated IgGSingle mutation
N297S or N297Q
BTG
NH2-step1-R
Z-step2-
October 2013 Page 13
145281
145680
149056
0
200
400
600
Intens.[%]
0
200
400
600
[%]
0
100
200
300
400
[%]
144000 145000 146000 147000 148000 149000 150000 151000 152000 m/z
Coupling with Azide Linker and DBCO ToxinN297S-step1a-click-step2-vcMMAE
DAR 2
• m/z shift = 399• Theoretical2x M Linker = 402 Da
• m/z shift = 3775• Theoretical 2x M Linker-toxin = 3774 Da
* Ion fragmentation from MS
• 10 eq. of linker NH2-step1-N3 per site• 24 hours
• 1.5 eq. of DBCO-step2-vcMMAE per site• 4 hours
DAR=2.0
October 2013 Page 14
Coupling with Azide Linker and DBCO Toxin N297Q-step1a-click-step2-vcMMAE
145358
146159
152907
0
250
500
750
Intens.[%]
0
200
400
600
[%]
0
200
400
[%]
144000 146000 148000 150000 152000 154000 m/z
DAR 4
DAR=4.0
• m/z shift = 801• Theoretical 2x M
Linker = 804 Da
• m/z shift = 7550• Theoretical 2x M Linker-toxin = 7548 Da
• 10 eq. of linker NH2-step1-N3 per site• 24 hours
• 1.5 eq. of DBCO-step2-vcMMAE per site• 4 hours
Page 15Page 15
Preclinical POC
October 2013 Page 16
Tools for POC
• Naked antibodyo SGN30 (cAC10) targeting CD30
o SGN30S or SGN30Q with 2 or 4 coupling sites
• Intermediates: various linkerso Structure of spacer (size, hydrophobicity):
step1a, b or c
o Reactive groups for click chemistry: -R, -R’, -R’’
• BTG-ADCso -vc-PAB-MMAE for all conjugates
o One-step: NH2-step1-vcMMAE
o Two-step: DBCO-step2-vcMMAE
• Comparatoro ADCETRIS®, Brentuximab vedotin
QQ QQQ Q
SGN30S SGN30Q
Page 17Page 17
Stability in Buffer and in Plasma
October 2013 Page 18
0,02,04,06,08,010,012,014,016,018,0
0 20 40 60 80
HMWP, SEC
area %
DaysSGN30S‐sp1b‐click‐sp2‐vcMMAE SGN30Q‐sp1b‐click‐sp2‐vcMMAESGN30S‐sp1a‐click‐sp2‐vcMMAE SGN30Q‐sp1a‐click‐sp2‐vcMMAESGN30S‐sp1a‐vcMMAE SGN30Q‐sp1‐vcMMAEADCETRIS®
0,02,04,06,08,010,012,014,016,018,020,0
0 5 10 15 20
HMWP, SEC
area %
DaysN297S‐sp1a‐R N297Q‐sp1a‐R N297S‐sp1b‐R'
N297Q‐sp1b‐R' N297S‐sp1a‐R'' N297Q‐sp1a‐R''
N297S‐sp1a‐R'
BTG-ADCs Stability in BufferHMWP by SEC at +40°C
• Linker has significant impact on intermediates stability
• R’ reactive group abandoned
• All BTG-ADCs (except step1b) showed less soluble aggregates than ADCETRIS®
NOGO
GO
Intermediates BTG-ADCs
Good Quality Attribute
October 2013 Page 19
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 10 20 30 40 50 60 70 80
DAR
Days
SGN30S‐sp1b‐click‐sp2‐vcMMAE SGN30Q‐sp1b‐click‐sp2‐vcMMAE
SGN30S‐sp1a‐click‐sp2‐vcMMAE SGN30Q‐sp1a‐click‐sp2‐vcMMAE
SGN30S‐sp1a‐vcMMAE SGN30Q‐sp1‐vcMMAE
BTG-ADCs Stability in BufferDAR at +40°C
October 2013 Page 20
Ex Vivo Plasma Stability
• ADCs spiked in plasma
• Plasma types: rat (Wistar), cynomolgus and human
Paramagnetic beads coated with streptavidin
Anti-HumanCk(nanobody coupled to biotin)
Magnet
CD30 coupled to biotin
Rodents Human and cynomolgus
LC/MS(ESI-qTOF)
DAR
Affinity capture
October 2013 Page 21
BTG-ADCs ex vivo Stability in Wistar Rat PlasmaDAR over one week
No DAR variation observed over one week at 37°C
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 20 40 60 80 100 120 140 160 180
Average DA
R
HoursSGN35S‐sp1a‐vcMMAE SGN35Q‐sp1a‐vcMMAE
SGN35S‐sp1a‐click‐sp2‐vcMMAE SGN35Q‐sp1a‐click‐sp2‐vcMMAE
October 2013 Page 22
BTG-ADCs ex vivo Stability in NHP and Human PlasmaDAR over one week
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 50 100 150 200
DAR
HoursSGN30S‐sp1a‐click‐sp2‐vcMMAE SGN30Q‐sp1a‐click‐sp2‐vcMMAESGN30S‐sp1a‐vcMMAE SGN30Q‐sp1a‐vcMMAE
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 50 100 150 200
DAR
Hours
Cynomolgus Human
Page 23Page 23
PK Study
October 2013 Page 24
DaysTo
tal a
ntib
ody
(µg/
ml)
0 10 20 30 400.1
1
10
100
1000
SGN30SSGN30S-sp1a-click-sp2-vcMMAE SGN30Q-sp1a-click-sp2-vcMMAE
ADCETRIS
BTG-ADCs PK in Wistar Rat
n=4
0,00,51,01,52,02,53,03,54,0
0 5 10 15
Average DA
R
Days
n=4
Conjugated antibody Total antibody
Unit SGN30Q-sp1a-click-sp2-vcMMAE
SGN30S-sp1a-click-sp2-vcMMAE SGN30S ADCETRIS®
DAR N/A 4.0 2.0 N/A ~4Half-Life days 8.5 12.0 9.6 8.5
Cl ml/h 0.099 0.071 0.088 0.168
Page 25Page 25
In Vitro and In Vivo Efficacy
October 2013 Page 26
BTG-ADCs in vitro Efficacy
Conc (µg/ml)
Lum
ines
cenc
e
0.001 0.01 0.1 1 10 1000
100000
200000
300000
400000
0Conc (µg/ml)
Lum
ines
cenc
e
0.001 0.01 0.1 1 10 1000
100000
200000
300000
400000
0
ADCETRIS IgG1S-sp1a-click-sp2-vcMMAESGN30S-sp1a-click-sp2-vcMMAE SGN30Q-sp1a-click-sp2-vcMMAESGN30S-sp1a-vcMMAE SGN30Q-sp1a-vcMMAE
RAJI-CD30+
SGN30S-sp1a-click-sp2-vcMMAE
SGN30Q-sp1a-click-sp2-vcMMAE
SGN30S-sp1a-vcMMAE
SGN30Q-sp1a-vcMMAE ADCETRIS®
DAR 2.0 4.0 2.0 4.0 ~4
EC50 RAJI-CD30+ (ng/ml) 5.1 2.0 5.4 2.3 1.4
EC50 KARPAS 299 (ng/ml) 11.2 3.1 14.6 4.7 2.4
KARPAS 299
October 2013 Page 27
BTG-ADCs in vivo Efficacy
Days
Tum
or v
olum
e (m
m3)
0 10 20 30 40 500
500
1000
1500
SGN30QIgG1S-sp1a-click-sp2-vcMMAE
ADCETRISSGN30Q-sp1a-vcMMAE
• Karpas 299 (S.C.) in SCID mice• Dose 0.6mg/kg, I.V., q4d X4, • Treatment started when tumor ~100mm3
• 9 mice per group
October 2013 Page 28
Summary
• ADCs with DAR of exactly 2.0 or 4.0 from minimally modified antibody scaffold, i.e. with a single point mutation
• Rapid and versatile process appropriate for testing various linkers and toxins in HTS
• BTG two-step process yields to quantitative coupling using only 1 to 2 molar excess of toxin per site, making it a cost-efficient and scalable process
• BTG-ADCs are stable ex vivo in human and cynomolgus plasma and in vivo in rat, without DAR variation. In addition, BTG-ADCs clearance is lower compared to Adcetris®
• BTG-ADCs with DAR=4.0 show equivalent in vitro and in vivo efficacy compared to Adcetris®
October 2013 Page 29
Acknowledgment
• Innate Pharmao Delphine Bregeon
o Christian Belmant
o Angélique Boedec
o Hélène Rispaud
o Sandra Savard-Chambard
o Naouel Lovera
o Agnès Represa
o Mélody Sapet
o Céline Delcambre
o Sophie Ingoure
o Sylvia Trichard
o Stéphane Delahaye
o Cécile Bonnafous
o Nicolas Viaud
o Mathieu Bléry
o Stéphanie Zerbib
o Benjamin Rossi
• ETH/PSI (Zurich)o Patrick Dennler
o Aris Chiotellis
o Eliane Fisher
o Roger Schibli
• PIT2 (Marseilles)o Sega N’Diaye
o Claude Villard
o Daniel Lafitte
o Laurent Gauthier
o Lukas Vollmy
o Carine Paturel
o François Romagné
