Engineering Antibodies (1) MSc Programme University of Nottingham 14 th February 2005

1. Engineering Antibodies (1)MSc 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. Antibody based immunotherapeutics

3. IgG is the preferred class

4. Schematic view of IgG domains

5. Antibody fragments can also be used

6. Antibodies can be derived from immunised animals

7. The antibody immune response in-vivo can be T-cell dependent or independent

8. Antibody fragments can also be selected from in-vitro systems such as phage expression

9. Cycles of selection and mutation can give an artificial in-vitro immune response based simply on binding affinity

11. The Selection of IgG Fc Regions for appropriate effector functions:The role of isotypes and polymorphisms

12. Effector functions of human IgG IgG1 IgG2 IgG3 IgG4 Complement activation Classical pathway +++ + +++ - Alternative pathway + - - - Fc receptor recognition +++ +++ ++ Fc RI g - ++ ++ Fc RIIa, 131R/R g - - + + ++ Fc RIIa, 131H/H g - ++ ++ + Fc RIIb g - + + Fc RIII +/ +/ g - -

14. Unlike mouse the human IgG subclasses are very similar in sequence but they still have different properties

18. The IgG receptor FcRn Interaction with FcRn and with Protein A through similar region FcRn is important for IgG half-life and transport

19. Transferring motifs between human subclasses Db mutation Dc mutation Da mutation

20. Residues at key positions in mutatedconstant regions

21. Summary of terminology Mutant residues a D Residues 327, 330, 331 of IgG4 b D Lower hinge of IgG2; omitting Gly236 c D Lower hinge of IgG2; including Gly236 : 2613 29 Armour et al. Eur J Imm 1999;

27. Test systems: antibodies with CD52 and a-RhD specificities • Short, GPI-anchored glycoprotein • Found on T cells and some B cells, granulocytes and eosinophils • About 45 x 104 molecules/cell • Good target for CDC and ADCC • Humanised variable domains of CAMPATH-1 antibody used • Range of antibodies with same variable domains already existed CD52

28. Test systems: antibodies with CD52 and a-RhD specificities • Protein complex on erythrocyte membrane • 1 - 3 x 104 molecules/cell • Provides opportunities for use of agglutination and rosetting assays • Target for ADCC • Used variable domains of Fog-1, a human IgG isolated from hyperimmunised, RhD- blood donor a-RhD

29. Complement-mediated lysis of mononuclear cells 45 CAMPATH-1 antibodies 40 G1 35 G1D a 30 G1D b G1D c 25 G1D ab % specific Cr release 20 G1D ac 15 G2 G2D a 10 G4 5 G4D b G4D c 0 -5 1 10 100 0.1 m antibody, g/ml

30. Complement-mediated lysis withCAMPATH-1 G1(6.3 mg/ml),inhibited by CAMPATH-1 G2Da 25 20 15 10 % specific Cr release 5 0 -5 1 10 100 1000 G2Da concentration, g/ml m

31. Binding to the FcgRI-bearing cell line, B2KA, measured by fluorescence staining G2 G1Da CAMPATH-1H antibodies at 100 mg/ml G4 G1Db G1Dc G1

32. Binding to the FcgRIa-bearing cell line, B2KA, measured by fluorescent staining CAMPATH-1 160 antibodies 140 G1 120 G1D a G1D b 100 G1D c G1D ab mean fluorescence 80 G1D ac 60 G2 G2D a 40 G4 20 G4D b G4D c 0 0.001 0.01 0.1 1 10 100 antibody, g/ml m

33. 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

34. Inhibition of chemiluminescent response to clinical sera by Fog-1 G2Da 280 240 200 G1 anti-D serum A anti-D serum B 160 anti-D serum C % chemiluminescence anti-D serum D 120 anti-D serum E anti-C+D serum anti-K serum 80 40 0 0 10 100 1000 G2Da concentration, mg/ml

35. Binding to the cell line 3T6 + FcgRIIa 131R, measured by flow cytometry 100 Fog-1 antibodies 90 G1 G1Da 80 G1Db 70 G1Dc G1Dab 60 mean fluorescence G1Dac G2 50 G2Da 40 G4 G4Db 30 G4Dc G1Dg 20 IgA1,k 10 0.1 1 10 100 antibody concentration, mg/ml

36. Binding to the cell line 3T6 + FcgRIIa 131H, measured by flow cytometry 90 Fog-1 antibodies 80 G1 G1Da 70 G1Db G1Dc 60 G1Dab mean f luorescence 50 G1Dac G2 40 G2Da G4 30 G4Db 20 G4Dc G1Dg 10 IgA1,k 0.1 1 10 100 antibody concentration, mg/ml

37. Binding to the cell line 3T6 + FcgRIIb1*, measured by flow cytometry 260 Fog-1 antibodies G1 210 G1Da G1Db G1Dc 160 G1Dab mean fluorescence G1Dac G2 110 G2Da G4 60 G4Db G4Dc G1Dg 10 IgA1,k 0.1 1 10 100 antibody concentration, mg/ml

38. Binding to different forms of FcgRII 120 FcgRIIa 131R 100 FcgRIIa 131H FcgRIIb1* 80 percentage of G1 binding 60 40 20 0 G1 G4 G2 G4Db G1Dc G4Dc G1Dg G1Db G1Da G2Da G1Dab G1Dac antibody constant region 0% = binding of IgA1,k

39. G1 G1D ab G2 G2D a G4 G4D b Activity of Fog-1 antibodies in ADCC 120 100 80 60 % RBC lysis 40 20 0 -20 0.1 1 10 100 1000 10000 antibody concentration, ng/ml

40. Inhibition by Fog-1 antibodies of ADCC due to Fog-1 IgG1 (at 2ng/ml) 45 40 G2 35 D a G2 30 25 % RBC lysis 20 G1D a 15 10 • G1 D c G1Db, G1Dab, G1Dac, G4, G4Db, G4Dc { 5 0 0.0001 0.001 0.01 0.1 1 10 100 1000 10000 inhibitor antibody concentration, ng/ml

41. Summary of antibody activities

42. Effect of mutations cannot always be predicted from wildtype antibody activities • Complement lysis: • IgG2 activity is only ~3-fold lower than that of IgG1 • but placing IgG2 residues in IgG1 (Db, Dc) eliminates lysis. • FcgRIIa 131H binding: • IgG1 and IgG2 show equal binding • but G1Db and G1Dc activities are 30-fold lower. • IgG1 binding may depend heavily on the mutated regions. Other subclasses may have additional sites of interaction with the effector molecules.

43. Db and Dc mutants The 3 pairs of Db and Dc mutants show reduced activity in all functions assayed but the residual levels of activity differ: Db slightly more active in FcgRIIa 131H and 131R binding Dc more active in FcgRI binding, monocyte activation FcgRIIIb NA1and NA2 binding and ADCC These mutants differ only by -/+ G236. This must affect the ability of the FcR to accommodate the IgG2 lower hinge.

44. What of the immunogenicity of therapeutic antibodies?

45. Bad News • Universal tolerance to all self-antigens does not exist. • Auto and allo-immunity are common observations • Human proteins can be immunogenic in humans. (e.g. recombinant insulin, EPO and Factor VIII) • Human antibodies can be immunogenic in humans (anti-idiotype and anti-allotype) and this applies to chimeric, humanised and fully human antibodies.

46. Good News • Auto and allo-immunity are common observations but these immune reponses can be modified and regulated. • Human antibodies can be immunogenic in humans but this immunogenicity varies from antibody to antibody for complex reasons, and is probably more dependent on the mode of action, and not just the way they were made (i.e. chimeric, humanised or fully human).

47. Antigenicity and Immunogenicity • Antigenicity is simply an ability of a molecule to be recognised by a pre-existing T-cell receptor (TCR) or a B-cell receptor (antibody) • But once an antigen is recognised by a receptor it can either be immunogenic or tolerogenic. • The same antigen can sometimes induce tolerance and sometimes provoke an immune response depending upon factors such as mode of administration and uptake by and co-stimulation of antigen presenting cells (APCs).

49. Immunogenicity • Immunogenicity of T-cell dependent antigens relies on presentation by professional APCs (e.g. Dendritic cells). • Dendritic cells (and other APCs) acquire antigen through use of innate receptors including complement receptors and Fc receptors, thus allowing recognition and uptake of immune complexes.

50. Induction of tolerance to therapeutic antibodies • Benjamin,R.J., Cobbold,S.P., Clark,M.R., & Waldmann,H. (1986) J. Exp. Med. 163, 1539-1552. Tolerance to rat monoclonal antibodies: implications for serotherapy • Observation • Relatively easy to tolerise mice, with de-aggregated human immunoglobulin or with rat immunoglobulin, despite large differences in the constant region sequences between mouse, human and rat. • However in mice which are tolerant of soluble rat IgG2b, administration of antibodies which bind to mouse cell surface antigens provokes a strong anti-idiotype response. • Explanation • Is this a function of the inherent immunogenicity of immune complexes? • Aggregated antibody is more likely to activate complement and to bind to low affinity Fc receptors.