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Immunotherapeutics Master’s Programme: Engineering antibody molecules - 1 University of Nottingham 11 th February 2009 by Mike Clark, PhD Department of.

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Presentation on theme: "Immunotherapeutics Master’s Programme: Engineering antibody molecules - 1 University of Nottingham 11 th February 2009 by Mike Clark, PhD Department of."— Presentation transcript:

1 Immunotherapeutics Master’s Programme: Engineering antibody molecules - 1 University of Nottingham 11 th February 2009 by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK

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

3 Declaration of interests (rights as an inventor) CD52IlexOncology/Genzyme (Campath® humanisation) CD4TolerRx/Genentech (for induction of tolerance) CD4BTG (improved method of humanisation) CD3BTG /TolerRx (immunosuppression and tolerance) VAP-1BioTie / University collaboration RhDNBS / University collaboration HPA-1aNBS / University collaboration

4 Antibody based immunotherapeutics

5 IgG is the preferred class

6 Schematic view of IgG domains

7 Antibody fragments can also be used

8 Antibodies can be derived from immunised animals

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

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

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


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

14 Effector functions of human IgG IgG1IgG2IgG3IgG4 Complement activation Classical pathway++++  Alternative pathway  +  Fc receptor recognition Fc  RI +++  ++ Fc  RIIa, 131R/R ++   Fc  RIIa, 131H/H ++++  Fc  RIIb ++  + Fc  RIII + +/  + 


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

17 Human IgG Fc Receptors CD64 CD32 CD16

18 Fc  RI Fc  RIIa Fc  RIIb Fc  RIIc Fc  RIIIa Fc  RIIIb (CD64) (CD32) (CD32) (CD32) (CD16) (CD16)   -GPI ITAM - ITIM - ITAM -  Inhibitory Affinity High Low-Med Low-Med Low-Med Low Low-Med (10 8 M -1 ) (2x10 6 M -1 ) (5x10 5 M -1 ) Alleles -- IIA-131H -- -- IIIA-158V NA1 IIA-131R IIIA-158F NA2 HH 25% HR 50% RR 25% VV 20% VF 40% FF 40% IgG1=IgG3 >>IgG4 >>>IgG2 IgG1 IgG2=IgG3 >>IgG4 IgG1 IgG3 IgG4>>IgG2 IgG1=IgG3 >>>>>>> IgG4,IgG2 Human Fc  Receptors and their Activities Slide courtesy of Bill Strohl, Centocor, September 2008



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

22 Transferring motifs between human subclasses  b mutation  c mutation  a mutation

23 Residues at key positions in mutated constant regions

24 Summary of terminology Mutant residues  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






30 Test systems: antibodies with CD52 and  -RhD specificities Short, GPI-anchored glycoprotein Found on T cells and some B cells, granulocytes and eosinophils About 45 x 10 4 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

31 Test systems: antibodies with CD52 and  -RhD specificities Protein complex on erythrocyte membrane 1 - 3 x 10 4 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  -RhD

32 -5 0 5 10 15 20 25 30 35 40 45 0.1 110100 antibody,  g/ml % specific Cr release G1 G1  a G1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  b G4  c CAMPATH-1 antibodies Complement-mediated lysis of mononuclear cells

33 -5 0 5 10 15 20 25 1101001000 G2  a concentration,  g/ml % specific Cr release Complement-mediated lysis with CAMPATH-1 G1(6.3  g/ml), inhibited by CAMPATH-1 G2  a

34 Binding to the Fc  RI-bearing cell line, B2KA, measured by fluorescence staining G2 G1  b G1  c G4 G1  a G1 CAMPATH-1H antibodies at 100  g/ml

35 0 20 40 60 80 100 120 140 160 0.0010.010.1110100 antibody,  g/ml mean fluorescence G1 G1  a G1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  b G4  c CAMPATH-1 antibodies Binding to the Fc  RIa-bearing cell line, B2KA, measured by fluorescent staining

36 Chemiluminescent response of human monocytes to sensitised RBC -20 0 20 40 60 80 100 120 140 050001000015000200002500030000 antibody molecules/cell % chemiluminescence G1 G1  a G1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  b G4  c Fog-1 antibodies

37 Inhibition of chemiluminescent response to clinical sera by Fog-1 G2  a 280 0 40 80 120 160 200 240 0101001000 G2  a concentration,  g/ml % chemiluminescence G1 anti-D serum A anti-D serum B anti-D serum C anti-D serum D anti-D serum E anti-C+D serum anti-K serum

38 Binding to the cell line 3T6 + Fc  RIIa 131R, measured by flow cytometry 10 20 30 40 50 60 70 80 90 100 0.1110100 antibody concentration,  g/ml mean fluorescence G1 G1  a G1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  b G4  c G1  g IgA1,  Fog-1 antibodies



41 Binding to different forms of Fc  RII antibody constant region percentage of G1 binding 0% = binding of IgA1,  0 20 40 60 80 100 120 G1 G1  aG1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  bG4  c G1  g Fc  RIIa 131R Fc  RIIa 131H Fc  RIIb1 *

42 Activity of Fog-1 antibodies in ADCC -20 0 20 40 60 80 100 120 0.1110100100010000 antibody concentration, ng/ml % RBC lysis G1 G1  ab G2 G2  a G4 G4  b

43 Inhibition by Fog-1 antibodies of ADCC due to Fog-1 IgG1 (at 2ng/ml) inhibitor antibody concentration, ng/ml % RBC lysis 0 5 10 15 20 25 30 35 40 45 0.00010.0010.010.1110100100010000 G1  a G1  c G2  a G1  b, G1  ab, G1  ac, G4, G4  b, G4  c {

44 Summary of antibody activities

45 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 (  b,  c) eliminates lysis. Fc  RIIa 131H binding: IgG1 and IgG2 show equal binding but G1  b and G1  c 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.

46  b and  c mutants The 3 pairs of  b and  c mutants show reduced activity in all functions assayed but the residual levels of activity differ:  b slightly more active in Fc  RIIa 131H and 131R binding  c more active in Fc  RI binding, monocyte activation Fc  RIIIb 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.

47 What of the immunogenicity of therapeutic antibodies?

48 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.

49 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).

50 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).


52 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.

53 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. Induction of tolerance to therapeutic antibodies

54 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 ?

55 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.


57 Kabat database variability of VH sequences Human VH Mouse VH

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


60 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, 4181-4185 (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. 13-44 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.

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

62 Antibody comparisonsFRCDRWhole 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(67/87) 77%(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%(14/29) 48%(91/116) 78% OKT3 versus germline(76/87) 87%(6/32) 19%(82/119) 69% human versus human germline Fog-1 RhD versus germline(77/87) 89%(23/37) 62%(100/124) 81% Sequence homologies of some rodent, humanised and human sequences

63 AntibodySpecificityV-region Homologous VH JHLengthMatchesHomology FOG-1RhDHumanV4-34JH6A1241000.807 anti-TacCD25HumanisedHV1F10TJH6a116910.784 anti-TacCD25MouseHV1F10TJH4D116840.724 anti-TNFaTNF-alphaMouseVI-4-IBJH3B117840.718 Campath-1HCD52HumanisedDP-71_3D197D-JH4D121860.710 Campath-1GCD52RatDP-34_DA-10JH4D121850.702 OKT3CD3Humanisedb25JH6a119820.689 OKT3CD3MouseDP-7_21-2-..JH6a119810.681 HD37CD19Mouse6M27JH4D124830.669 anti-CD20CD20MouseDP-7_21-2-JH2121810.669 Homologies for antibody heavy chain V regions compared with human germline sequences Sorted by homology

64 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.

65 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)

66 The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al 1995 Transplantation 60, 847-853 Normal Mouse (no antigen) CD3 Transgenic (cell surface) Human IgG1 -+++ Aglycosyl IgG1 -+

67 The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al 1995 Transplantation 60, 847-853 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?

68 Elimination of the immunogenicity of therapeutic antibodies. Gilliland et al 1999 J.Immunol 162, 3663-3671 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


70 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 Factors likely to influence immunogenicity of therapeutic antibodies

71 Will the idiotype always be immunogenic? 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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