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Theo Rispens

ADA to ada Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies. Theo Rispens. Humanized. Human. Mouse. Chimeric. 30% mouse. 3-5% mouse. HAMA. HACA. HAHA. HAHA. :. Immunogenici ty therapeutic antibodies. Infliximab. Adalimumab.

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Theo Rispens

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

  2. Humanized Human Mouse Chimeric 30% mouse 3-5% mouse HAMA HACA HAHA HAHA : Immunogenicity therapeutic antibodies Infliximab Adalimumab 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 www.biologicals.sanquin.nl Bartelds et al 2007

  4. ADA issues to address using mAbs • clonality of the response • B cell epitopes • Immune complex formation • Neutralization • Standardization

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

  6. All monoclonal antibodies are derived from different precursor B-cells

  7. All mAbs bind to an overlapping epitope

  8. mAbs neutralize adalimumab

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

  10. Antibody structure VH VL Fab 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) (105-117) (118-128) • 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) (105-117) (118-128) • ——————————————> ——————————> ———————> ————————> ————————> —————————> ———————————> ———————> ——————————> • 1 10 15 16 23 26 27 38 3941 46 47 55 56 65 66 74 75 80 84 85 89 96 97 104 105 111112 117 118 128 • |........|....| |......|..| |..........| |.|....| |.......| |........| |.......| |....|...| |...|......| |......| |.....|1|....| |.........| • adaVH • EVQLVESGG.GLVQP GRSLRLSCAAS GFTF....DDYA MHWVRQAP GKGLEWVSA ITWN..SGHI DYADSVE.G RFTISRDNAK NSLYLQMNSLRA EDTAVYYCAKVSYLSTASSLDY WGQGTLVTVSS • IGHV3-9*01 • EVQLVESGG.GLVQP GRSLRLSCAAS GFTF....DDYA MHWVRQAP GKGLEWVSG ISWN..SGSI GYADSVK.G RFTISRDNAK NSLYLQMNSLRA EDTALYYCAKD YFDY WGQGTLVTVSS • (Homo sapiens) • A T H D EV 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) (105-117) (118-128) • 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) (105-117) (118-128) • ——————————————> ——————————> ———————> ————————> ————————> —————————> ———————————> ———————> ——————————> • 1 10 15 16 23 26 27 38 3941 46 47 55 56 65 66 74 75 80 84 85 89 96 97 104 105 11112 117 118 128 • |........|....| |......|..| |..........| |.|....| |.......| |........| |.......| |....|...| |...|......| |......| |.....||....| |.........| • adaVL • DIQMTQSPSSLSASV GDRVTITCRAS QGI......RNY LAWYQQKP GKAPKLLIY AA.......S TLQSGVP.S RFSGSG..SG TDFTLTISSLQP EDVATYYCQRYNR....APYT FGQGTKVEIK. • IGKV1-27*01 • DIQMTQSPSSLSASV GDRVTITCRAS QGI......SNY LAWYQQKP GKVPKLLIY AA.......S TLQSGVP.S RFSGSG..SG TDFTLTISSLQP EDVATYYCQKYNSAP YT FGQGTKLEIK. • (Homo sapiens) • R A R R V • |—————————————————————————————————————————————————————V-REGION——————————————————————————————————————————————————————————————||——J-REGION—|

  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

  15. Results gradients Prot A • Most sera contain complexes • Complexes are small • These complexes are not rapidly cleared

  16. prot. A ELISA and ABT vs affinity

  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

  18. Acknowledgements Reade 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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