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