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22-09-08
ADA to ada
Functional analysis of the anti-adalimumab response using patient-derived monoclonal
antibodies
Theo Rispens
Immunogenicity therapeutic antibodies
Mouse
: HAMA HACA HAHA HAHA
Immunogenicity:
Humanized
3-5% mouse
Chimeric
30% mouse
Human
Infliximab Adalimumab
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 2007www.biologicals.sanquin.nl
ADA issues to address using mAbs
- clonality of the response
- B cell epitopes
- Immune complex formation
- Neutralization
- Standardization
Making human monoclonal antibodies
Schouwenburg et al., ARD 2012
All monoclonal antibodies are derived from different precursor B-cells
All mAbs bind to an overlapping epitope
mAbs neutralize adalimumab
TNF- completely inhibits binding of ADA from patient sera
Schouwenburg et al., ARD 2012
Antibody structure
VL
VH
Fab
Fc
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 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) (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 EDVATYYC QRYNR....APYT FGQGTKVEIK.
IGKV1-27*01
DIQMTQSPSSLSASV GDRVTITCRAS QGI......SNY LAWYQQKP GKVPKLLIY AA.......S TLQSGVP.S RFSGSG..SG TDFTLTISSLQP EDVATYYC QKYNSAP YT FGQGTKLEIK.
(Homo sapiens)
R A R R V
|—————————————————————————————————————————————————————V-REGION——————————————————————————————————————————————————————————————||——J-REGION—|
inhibition of anti-ada to wild-type adalimumab by ada Fab variants
- single-point mutants of adalimumab
- selected for enhanced TNF binding
adalimumab
adalimumab
ADA
adalimumab mutant Fab
inhibition of anti-ada to wild-type adalimumab by ada Fab variants.
values represent binding relative to uninhibited:
100% = no inhibition = no binding by ada Fab variant = mutation affects binding
adalimumab
adalimumab
ADA
adalimumab mutant Fab
mAbs: immune complexes with adalimumab
Results gradients Prot A
0 5 10 15 200
20
40
% B
ind
ing
fraction
Most sera contain complexes
Complexes are small
These complexes are not rapidly cleared
ELISA and ABT vs affinity
prot. A
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
Acknowledgements
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
Reade
Gertjan Wolbink
Margret de Koning
Charlotte Krieckaert
Margret Bartelds
Mike Nurmohamed
Genmab
Rob de Jong
Esther van Buren
Tom Vink
Bayer
Christian Votsmeier