Engineering Antibodies (2) Immunotherapeutic Examples MSc Programme University of Nottingham 14 th February 2005

1. Engineering Antibodies (2)Immunotherapeutic ExamplesMSc Programme University of Nottingham14th February 2005 by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK www.path.cam.ac.uk/~mrc7/

2. University Research Programmes • Immunosuppression • CD4, CD3, monovalent CD3, CD52 (Campath) • Tumour Therapy • CD52 (Campath), bispecific CD3 • Organ Transplantation • CD52, CD3, CD4, synergistic CD45 pair • Allo and auto-immunity • RhD, HPA-1a • Chronic Inflammation • CD18, VAP-1

3. Declaration of interests (rights as an inventor) • CD52 IlexOncology/Genzyme (Campath® humanisation) • CD4 TolerRx/Genentech (for induction of tolerance) • CD4 BTG (improved method of humanisation) • CD3 BTG /TolerRx (immunosuppression and tolerance) • CD18 Millennium Pharmaceuticals • VAP-1 BioTie / University collaboration • RhD NBS / University collaboration • HPA-1a NBS / University collaboration

8. The antibody isotype is important

14. Chimeric and humanised

16. Rat IgG2b is effective in therapy

17. Human IgG1 also effective in therapy

18. Antibodies (eg CD52 Campath) can be effective in killing cancer cells (BCLL)

22. Fetomaternal alloimmune thrombocytopenia • Maternal IgG raised against fetal platelet alloantigens can cross the placenta and cause fetal platelet destruction • If the fetal platelet count falls dangerously low, cerebral hemorrage or death may result • Current therapies are intrauterine platelet transfusion and maternal therapy with high dose IVIG

23. Can a protective antibody be developed? • 90% severe cases FMAIT are due to antibodies against the alloantigen HPA-1a on GPIIIa • Single B cell epitope (Leu-33) could be blocked to prevent the binding of harmful antibodies • Outcome depends on antibody titre • Williamson et al. Blood 1998; 92: 2280 • Jaegtvik et al. Br J Obs Gynae 2000; 107: 691

24. Ideal properties of an antibody for FMAIT therapy • HPA-1a specificity (B2 variable regions) • able to cross the placenta • inactive in FcgR-mediated cell destruction • unable to activate complement

25. RhD HPA-1a

26. Chemiluminescent response of human monocytes to sensitised RBC Fog-1 140 antibodies 120 G1 G1D a 100 G1D b 80 G1D c % chemiluminescence 60 G1D ab 40 G1D ac G2 20 G2D a 0 G4 -20 G4D b 0 5000 10000 15000 20000 25000 30000 G4D c antibody molecules/cell

27. 100 90 G1D b 80 G1D c 70 G1D ab 60 G1D ac 50 G2 % chemiluminescence 40 G2D a 30 G4D b G4D c 20 10 0 0.1 1 10 100 1000 inhibitorconcentration, m g/ml Inhibition of chemiluminescent response due to 2 mg/ml Fog-1 G1 by other Fog-1 antibodies

28. Inhibition by Fog-1 antibodies of ADCC due to clinically relevant polyclonal anti-RhD (at 3ng/ml) 120 100 80 G1D ab G2 G2D a 60 % RBC lysis G4 G4D b 40 20 0 0.1 1 10 100 1000 10000 inhibitor antibody concentration, ng/ml

29. HuVAP antibody VAP-1

30. Capture • and rolling 2. Activation 3. Stationary adhesion Free flow 4. Migration Integrin Chemokine signal Selectins IgSF Infection Multistep paradigm of neutrophil adhesion Endothelium

31. Amines Toxic aldehydes & H202 VAP-1 Role of VAP-1 sVAP-1 Modified Fc region Anti VAP-1 Selectin

32. Capillary flow system

33. 1. Capture and FcR ligation 2. Activation and integrin expression 3. Ultra-rapid stationary adhesion Fc receptor Anti VAP-1 IgG 2 Integrin IgSF like motif Microslide Flow Neutrophil adhesion assay VAP-1

34. Human IgG1 wildtype anti-VAP-1 antibody

35. HuVAP mutated anti-VAP-1 antibody

36. Brief Acknowledgements Mike Clark Dept of Pathology Kathryn Armour Chris Kirton Cheryl Smith Lorna Williamson National Blood Service & Transfusion Medicine