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by Mike Clark, PhD Department of Pathology Division of Immunology

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1 Engineering Antibodies (1) MSc Programme University of Nottingham 14th February 2005
by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK

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

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

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

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

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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 mutated constant regions

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

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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 25 G1D c G1D ab % specific Cr release 20 G1D ac 15 G2 G2D a 10 G4 5 G4D b G4D c -5 0.1 1 10 100 antibody, m g/ml

30 Complement-mediated lysis with CAMPATH-1 G1(6
Complement-mediated lysis with CAMPATH-1 G1(6.3 mg/ml), inhibited by CAMPATH-1 G2Da 25 20 15 % specific Cr release 10 5 -5 1 10 100 1000 G2Da concentration, m g/ml

31 CAMPATH-1H antibodies at 100 mg/ml
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 mean fluorescence 80 G1D ab G1D ac 60 G2 40 G2D a G4 20 G4D b G4D c 0.001 0.01 0.1 1 10 100 antibody, m g/ml

33 Chemiluminescent response of human monocytes to sensitised RBC
Fog-1 140 antibodies 120 G1 100 G1D a G1D b 80 G1D c % chemiluminescence 60 G1D ab 40 G1D ac G2 20 G2D a G4 -20 G4D b 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 80 anti-K serum 40 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 80 G1Da G1Db 70 G1Dc G1Dab 60 mean fluorescence G1Dac 50 G2 G2Da 40 G4 G4Db 30 G4Dc 20 G1Dg 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
10 20 30 40 50 60 70 80 90 0.1 1 100 antibody concentration, mg/ml mean f luorescence G1 G1Da G1Db G1Dc G1Dab G1Dac G2 G2Da G4 G4Db G4Dc G1Dg IgA1,k Fog-1 antibodies

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

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

39 Activity of Fog-1 antibodies in ADCC
120 100 80 G1 G1D ab G2 G2D a G4 G4D b 60 % RBC lysis 40 20 -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 G2 D a 30 25 % RBC lysis 20 G1D a 15 10 G1 D c G1Db, G1Dab, G1Dac, G4, G4Db, G4Dc { 5 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).

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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, 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.

51 Antibody selection and design
The choice of antibody constant region is largely dictated by functional requirements of the antibody. But what about the V-regions ?

52 The V-region Mythology
Chimaeric 65% Human ? Humanised 95% Human ? This commercial marketing mythology is based on an assumption that mouse and human antibody sequences are unique. However a study of the Kabat database shows that there is high sequence homology for antibodies from different species.

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54 Kabat database variability of VH sequences
Mouse VH Human VH

55 Are chimaeric, humanised and fully human antibodies so very different in sequence?

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57 Possible to select alternative V genes for humanisation
Gorman,S.D., Clark,M.R., Routledge,E.G., Cobbold,S.P. & Waldmann,H. P.N.A.S. 88, (1991) Reshaping a therapeutic CD4 antibody. Routledge,E., Gorman,S., & Clark,M. in Protein engineering of antibody molecules for prophylactic and therapeutic applications in man. (Ed. Clark,M. ) Pub. Academic Titles, UK (1993) pp Reshaping of antibodies for therapy. Gorman et al recognised that homology also extended through the CDR regions not just the framework regions Homology to Kol was increased from 69% to 89% by the humanisation process.

58 The same strategy can be applied to almost any V-region

59 Sequence homologies of some rodent, humanised and human sequences
Antibody comparisons FR CDR Whole V murine versus human germline Campath-1G (68/87) 78% (14/34) 41% (82/121) 68% Anti-Tac (67/87) 77% (14/29) 48% (81/116) 70% OKT3 (12/32) 38% (81/119) 68% humanised versus human germline Campath-1H versus germline (78/87) 90% (8/34) 24% (86/121) 71% Anti-Tac versus germline (77/87) 89% (91/116) 78% OKT3 versus germline (76/87) 87% (6/32) 19% (82/119) 69% human versus human germline Fog-1 RhD versus germline (23/37) 62% (100/124) 81%

60 Homologies for antibody heavy chain V regions
compared with human germline sequences Sorted by homology Antibody Specificity V-region Homologous VH JH Length Matches Homology FOG-1 RhD Human V4-34 JH6A 124 100 0.807 anti-Tac CD25 Humanised HV1F10T JH6a 116 91 0.784 Mouse JH4D 84 0.724 anti-TNFa TNF-alpha VI-4-IB JH3B 117 0.718 Campath-1H CD52 DP-71_3D197D- 121 86 0.710 Campath-1G Rat DP-34_DA-10 85 0.702 OKT3 CD3 b25 119 82 0.689 DP-7_ 81 0.681 HD37 CD19 6M27 83 0.669 anti-CD20 CD20 DP-7_21-2- JH2

61 What of the Emperor’s new clothes?
Appropriate selection of sequences of antibody Constant and Variable regions is likely to be only one factor controlling the immunogenicity of therapeutic antibodies. However it is the final sequence of the antibodies which matters and not the route by which they were made. For example it is possible to come up with alternative humanised sequences for the same antibody. Similar sequences can often be found for mouse, rat and human variable regions within the databases. Even fully human antibodies may contain unusual motifs or structures as a result of the somatic recombination and junctional diversity combined with somatic hypermutation.

62 What determines immunogenicity?
Classical Self vs non-Self (Peter Medawar) Aquired neonatal tolerance to antigens. Danger Hypothesis (Polly Matzinger) Cell killing (inappropriate, non-apoptotic) Inflammation (cytokine release) Pattern recognition (Charles Janeway) Innate receptors for infectious pathogens Complement activation and fixation of C3 (Fearon) ( Fc receptors for immune complexes)

63 The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al 1995 Transplantation 60, Normal Mouse (no antigen) CD3 Transgenic (cell surface) Human IgG1 - +++ Aglycosyl IgG1 +

64 The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al 1995 Transplantation 60, The human IgG1in the CD3 transgenic mice was able to kill target cells, to activate complement, to bind to FcR and to cause cytokine release. Whereas the aglycosylated antibody was poor in these functions and produced only a weak immune response. Is this a special case or can it be generalised to other antibodies? Is it consistent with the Matzinger “Danger Hypothesis” as applied to therapeutic administration of recombinant antibodies?

65 Elimination of the immunogenicity of therapeutic antibodies
Elimination of the immunogenicity of therapeutic antibodies. Gilliland et al 1999 J.Immunol 162, Took CAMPATH antibody and mutated a key residue in the CDR region so as to prevent cell binding to CD52. This variant could be used to tolerise CD52 transgenic mice so that they no longer mounted an immune response to the wild type CAMPATH

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67 Factors likely to influence immunogenicity of therapeutic antibodies
Murine constant regions V-region sequences Human Ig allotypes Unusual glycosylation Method of administration Frequency of administration Dosage of antibody Patients' disease status Patients' immune status Patients' MHC haplotype Specificity of antibody Cell surface or soluble antigen? Formation of immune complexes with antigen Complement activation by antibody Fc receptor binding by antibody Inflammation and cytokine release

68 Will the idiotype always be immunogenic? Take home message to remember
The idiotype will obviously always be unique and thus antigenic. However it may be possible through mode of use to influence whether this antigenic idiotype is immunogenic or tolerogenic! Take home message to remember In immunological terms antigenicity is certainly not the same as immunogenicity!


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