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22-09-08 ADA to ada Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies Theo Rispens.

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Presentation on theme: "22-09-08 ADA to ada Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies Theo Rispens."— Presentation transcript:

1 ADA to ada Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies Theo Rispens

2 Immunogenicity therapeutic antibodies
Chimeric 30% mouse Infliximab Adalimumab Human Mouse Humanized 3-5% mouse monoclonal antibodies: mouse mAb (OKT3) proved quite immunogenic. According to dogma self – non-self: make antibodies more human like. However, antibodies to adalimumab can still be formed : HAMA HACA HAHA Immunogenicity:

3 Formation of antibodies to adalimumab
Treatment of rheumatoid arthritis with adalimumab: effective in 60-70% of patients Free anti-drug antibodies (ADA) in 17% of patients after 6 months of treatment ADA formation leads to impaired treatment efficacy Bartelds et al 2007

4 ADA issues to address using mAbs
clonality of the response B cell epitopes Immune complex formation Neutralization Standardization What happens once ADA are formed?

5 Making human monoclonal antibodies
Schouwenburg et al., ARD 2012

6 All monoclonal antibodies are derived from different precursor B-cells
R – Replacement S – silent Multiple clones SHM; IgG1/4; Th2 High affinities Polyclonal, broad response

7 All mAbs bind to an overlapping epitope
However, also restricted....

8 mAbs neutralize adalimumab

9 TNF- completely inhibits binding of ADA from patient sera
Schouwenburg et al., ARD 2012

10 Antibody structure Fab Fc VH VL
- restricted response: one single epitope on adalimumab? Fc

11 Adalimumab: possible antigenic determinants
FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT FR4-IMGT (1-26) (27-38) (39-55) (56-65) (66-104) ( ) ( ) A B BC C C' C'C" C" D E F FG G (1-15) (16-26) (27-38) (39-46) (47-55) (56-65) (66-74) (75-84) (85-96) (97-104) ( ) ( ) ——————————————> ——————————> ———————> ————————> ————————> —————————> ———————————> ———————> ——————————> | |....| |......|..| | | |.|....| | | | | | | |....|...| |...|......| |......| |.....|1|....| | | adaVH EVQLVESGG.GLVQP GRSLRLSCAAS GFTF....DDYA MHWVRQAP GKGLEWVSA ITWN..SGHI DYADSVE.G RFTISRDNAK NSLYLQMNSLRA EDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS IGHV3-9*01 EVQLVESGG.GLVQP GRSLRLSCAAS GFTF....DDYA MHWVRQAP GKGLEWVSG ISWN..SGSI GYADSVK.G RFTISRDNAK NSLYLQMNSLRA EDTALYYC AKD YFDY WGQGTLVTVSS (Homo sapiens) A T H D E V V SL |—————————————————————————————————————————————————V-REGION—————————————————————————————————————————————————————————||———N————||———J-REGION——| FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT FR4-IMGT (1-26) (27-38) (39-55) (56-65) (66-104) ( ) ( ) A B BC C C' C'C" C" D E F FG G (1-15) (16-26) (27-38) (39-46) (47-55) (56-65) (66-74) (75-84) (85-96) (97-104) ( ) ( ) ——————————————> ——————————> ———————> ————————> ————————> —————————> ———————————> ———————> ——————————> | |....| |......|..| | | |.|....| | | | | | | |....|...| |...|......| |......| |.....||....| | | adaVL DIQMTQSPSSLSASV GDRVTITCRAS QGI......RNY LAWYQQKP GKAPKLLIY AA S TLQSGVP.S RFSGSG..SG TDFTLTISSLQP EDVATYYC QRYNR....APYT FGQGTKVEIK. IGKV1-27*01 DIQMTQSPSSLSASV GDRVTITCRAS QGI......SNY LAWYQQKP GKVPKLLIY AA S TLQSGVP.S RFSGSG..SG TDFTLTISSLQP EDVATYYC QKYNSAP YT FGQGTKLEIK. R A R R V |—————————————————————————————————————————————————————V-REGION——————————————————————————————————————————————————————————————||——J-REGION—| close to germ-line

12 inhibition of anti-ada to wild-type adalimumab by ada Fab variants
adalimumab mutant Fab single-point mutants of adalimumab selected for enhanced TNF binding adalimumab ADA adalimumab

13 inhibition of anti-ada to wild-type adalimumab by ada Fab variants.
adalimumab mutant Fab values represent binding relative to uninhibited: 100% = no inhibition = no binding by ada Fab variant = mutation affects binding adalimumab ADA adalimumab

14 mAbs: immune complexes with adalimumab
fine specificity differs larger immune complexes can be formed (but still only the size of tetramers)

15 Results gradients Prot A
Mainly small IC (very large IC will not be detected in this assay). Most sera contain complexes Complexes are small These complexes are not rapidly cleared

16 ELISA and ABT vs affinity
prot. A

17 Conclusions ADA to ada is a diverse response
broad range of unrelated clones Extensive SHM / affinity maturation Multiple epitopes on adalimumab response is also very restricted All antibodies bind competitively to overlapping epitopes >98% of antibody response neutralizes TNF-α formation of small and larger immune complexes (humanization restricts/focusses B cell response, does not necessarily eliminates the response) multiple B cell epitopes: no easy fix. But: mutants that still bind TNF can be made that do not bind the existing response. Are these less immunogenic?

18 Acknowledgements Reade Sanquin Research Gertjan Wolbink
Margret de Koning Charlotte Krieckaert Margret Bartelds Mike Nurmohamed Genmab Rob de Jong Esther van Buren Tom Vink Bayer Christian Votsmeier Sanquin Research Pauline van Schouwenburg Margreet Hart Simone Kruithof Els de Groot Marieke van Ham Gertjan Wolbink Diana Wouters Lucien Aarden Sanquin Diagnostics Services Desiree van der Kleij Henk de Vrieze Astrid van Leeuwen


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