Immunotherapeutics Examples Master’s Programme: Engineering antibody molecules - 2 Nottingham University 9 th February

1. ImmunotherapeuticsExamplesMaster’s Programme: Engineering antibody molecules - 2 Nottingham University 9th February 2009 by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK www.path.cam.ac.uk/~mrc7/

2. Breakdown of Protein Therapeutics Estimated Market based on the sales of the top-selling biologics drugs.* Slide courtesy of Bill Strohl, Centocor, September 2008

3. Examples of Therapeutic Antibody-relatedProducts on the Market Slide courtesy of Bill Strohl, Centocor, September 2008

4. Sales of Existing Commercial Monoclonal Antibodies through 2006 Bioloogic Year OKT3 1986 --- /////// ReoPro® 1994 --- 1995 --- 1996 Rituxan® 1997 Zenapax® 1997 Remicade® 1998 Enbrel® 1998 Herceptin® 1998 Simulect® 1998 --- 1999 Mylotarg® 2000 Campath® 2001 Zevalin® 2002 Xolair® 2003 Raptiva® 2003 Amevive® 2003 Bexxar® 2003 Humira® 2003 Erbitux® 2003 Avastin® 2004 Tysabri® 2004 Actemra® 2005 Orencia® 2005 Lucentis® 2006 Vectibix® 2006 * $271K $3788K * $3768K $4442K $3020K * $115K * * $533K $90K * * $2020K $1069K $2372K * * $107K $380K * * >$80M in 2006 $0.5B $1.0B $1.5B $2.0B $2.5B $3.0B $3.5B $4.0B 2006 Sales Slide courtesy of Bill Strohl, Centocor, September 2008

5. Fc fusion protein Human MAb Humanized MAb Chimeric MAb Murine MAb Current and Projected Number of Marketed Monoclonal Antibodies and Fusion Proteins If all (unlikely) current Phase III candidates were to be approved in next 3-4 years (100% POS) 60 60 75% POS (industry average) would yield approx ca. 55 marketed Mabs and Fc fusion proteins 50 50 If ~50% of current Phase III candidates are approved in next 3-4 years (50% POS) 40 40 If all current BLAs are approved and result in marketed biologics Cumulative number of MAbs and Fc fusion proteins approved 30 30 Current status as of Sept 2008; N=26 20 20 10 10 1995 2000 2005 2008 2010 Year (Current status) Slide courtesy of Bill Strohl, Centocor, September 2008

6. Form and Source of Existing Commercial and Phase III Monoclonal Antibodies Monoclonal Antibody Format Murine Chimeric Humanized Human Monoclonal Antibody Source Murine hybridoma Humanized mouse hybridoma Phage displayed human antibody library Total Marketed Mabs = 22 Total Phase III Mabs = 30 3 3 6 2 11 11 2 14 20 16 1 8 1 6 2 4 6 8 10 12 14 16 18 20 Number of Monoclonal Antibodies Slide courtesy of Bill Strohl, Centocor, September 2008

7. Time Required for Maturity of Technologies 11 years Murine hybridoma Kohler and Milstein, 1975 Muronomab-OKT3®, 1986 10 years Chimeric antibodies Morrison et al., 1984 ReoPro®, 1994 11 years HumanizedCDR- grafted antibodies Jones et al., 1986 Zenapax®, 1997 9 years Fc fusion protein Capon et al., 1989; First Fc fusion Enbrel®, 1998 12 years Human antibodies from phage display libraries McCafferty et al., 1990 Humira®, 2002 10 years Human antibodies from transgenic humanized mice Lonberg et al., 1994; Green et al., 1994 Vectibix®, 2004 Antibodies with modified, muted Fc function 13 years Alegre et al., 1994; (first OKT3 ala-ala) Soliris®, 2007 1980 1985 1990 1995 2000 2005 2010 1975 Slide courtesy of Bill Strohl, Centocor, September 2008 Year

8. Spectrum of IgG Activities Substantially Moderate High muted activity ADCC • FcgRI,II,III- • very active • Complement activity • FcgRI,II,III- • silent • No comple- ment activity • FcgRI,II,III- • silent • Complement • activity • FcgRI,III-silent • FcgRIIa-active • Little Comple- • ment activity • FcgRI,II,III- • active • Complement activity IgG2m4 IgG2-4 IgG4ala-ala Aglycosylated IgG1 Engineered IgG1 Standard IgG2 Standard IgG1 Non-Oncology, Cytokines, Oncology Non-Infectious other soluble receptor Diseases cell targets targets; ID surface target Slide courtesy of Bill Strohl, Centocor, September 2008

9. Summary: Examples of “fit-for-purpose” Mabs and Fc Fusions with Isotypes other than IgG1 Slide courtesy of Bill Strohl, Centocor, September 2008

10. Otelixizumab • Started out as a depleting monovalent rat antibody for use in immunosuppression of graft rejection. • Clark,M., Bindon,C., Dyer,M., Friend,P., Hale,G., Cobbold,S., Calne,R., & Waldmann,H. Eur. J. Immunol. 19, 381-388 (1989) The improved lytic function and in-vivo efficacy of monovalent monoclonal CD3 antibodies. • Abbs,I.C., Clark,M., Waldmann,H., Chatenoud,L., Koffman,C.G. & Sacks,S.H. Therapeutic Immunology 1, 325-331 (1994) Sparing of the first dose effect of a monovalent anti-CD3 antibody used in allograft rejection is ssociated with diminished release of pro-inflammatory cytokines. • Humanised as a monovalent depleting antibody. • Routledge, E.G., Lloyd, I., Gorman, S., Clark, M. & Waldmann, H. Eur. J. Immunol. 21, 2717-2725 (1991) A humanized monovalent CD3 antibody which can activate homologous complement. • Converted to a non-depleting form by modification of the Fc region. • Bolt,S., Routledge,E., Lloyd,I., Chatenoud,L., Pope,H., Gorman,S.D., Clark,M. & Waldmann,H. Eur. J. Immunol. 23, 403-411 (1993) The generation of a humanised, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties

14. The antibody isotype is important

20. Chimeric and humanised

22. Rat IgG2b is effective in therapy

23. Human IgG1 also effective in therapy

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

27. NOVEL ANTIBODIES TO TREATFETO-MATERNAL ALLOIMMUNE THROMBOCYTOPENIA? Lorna M Williamson, Kathryn Armour, Mike Clark Departments of Haematology & Pathology, University of Cambridge/National Blood Service

28. 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

29. Alloantigen positive fetal platelets (ab) Alloantigen negative Mother (bb) Fetal platelet material Feto-maternal Alloimmune Thrombocytopenia

30. Feto-maternal Alloimmune Thrombocytopenia Platelet destruction by maternal HPA alloantibodies Maternal platelet antibodies Anti-a

31. FMAIT causes neonatal purpura

32. FMAIT causes intracranial haemorrhage in utero

33. 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

34. 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

35. Proof of concept applicationAlloimmune diseases as a target • We have been developing blocking antibodies for use in alloimmune disorders such as Feto-maternal Alloimmune Thrombocytopenia and Haemolytic Disease of the Newborn. • The desire is to have antibodies that block killing and are also not cytotoxic in their own right. • The properties demanded for such antibodies are applicable to other blocking or inhibitory therapies making use of antibodies or antibody Fc regions.

36. Summary of antibody activities Mutants able to block function of active antibody Used in HuVAP & by Rinat/Pfizer Tested in volunteers

37. RhD HPA-1a

38. 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

39. 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

40. 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

41. No triggering of cell destruction in vivo

42. Red cell survival study in normal volunteers Compare intravascular survival of RBC coated with Fog-1 G1 and Fog-1 G1Dnab in human volunteers. 99mTc G1 Label and coat autologous RBC 51Cr G1Dnab Design allows • simultaneous comparison in same donor • assessment of survival over several days (51Cr) • gamma camera imaging of sites of red cell accumulation (99mTc) • Armour et al, Blood 2006;107:2619-2626

43. RBC incubated with antibodies at 50 mg/ml,giving 75% saturation of RhD sites.

44. 7 7 G1 G1Dnab 6 6 5 5 4 4 plasma count, % injected dose 3 3 2 2 1 1 0 0 -1 -1 1 10 100 1000 10000 1 10 100 1000 10000 time after injection, min Plasma counts subject 2 subject 4 subject 1 subject 3 subject 5

45. G1-coated cells Complete, irreversible clearance by 200 min Appearance of plasma radiolabel Accumulation in spleen and, at high coating levels, in liver Total cell clearance and destruction G1Dnab-coated cells Clearance incomplete and transient No appearance of plasma radiolabel Accumulation in spleen but not in liver even at high coating levels No destruction of red cells but sequestration in the spleen

46. Next Step? Completed in-vitro safety testing of anti-platelet antibody with similar Fc region to anti-RhD Completed testing in an in-vivo mouse model of platelent survival About to start a new volunteer study using anti-platelet antibody

47. Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia J. Clin. Invest. 118(8): 2929-2938 (2008) Cedric Ghevaert et al • B2G1Δnab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. • The response of monocytes to B2G1Δnab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. • In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. • In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia • F(ab′)2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies

48. Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopeniaJ. Clin. Invest. Cedric Ghevaert, et al. 118:2929 doi:10.1172/JCI34708

49. Summary • Fc regions with reduced functions • Based on existing human subclass sequences • Tested in humans • Suitable for use in inhibiting killing, cellular adhesion or blocking cytokine receptors. • Fc regions with enhanced activity • Single amino acid change over wild-type sequence (H268E) • Potential for applications where enhancement in cytotoxicity and cellular activation is desirable

50. Acknowledgements Kathryn Armour Chris Kirton Cheryl Smith University of Cambridge Richard Farndale Mike Peters David Parry-Jones NBS Cambridge/Division of Transfusion Medicine Lorna Williamson Cedric Ghaevert Lotta Joutsi-Kornhonen Simon Bowden Sandy Preston Steve Garner Nick Watkins Peter Smethurst Willem Ouwehand NBS, Bristol Andrew Hadley Belinda Kumpel Marion Scott Craig Turner Keith Williams NBS FOR FUNDING