1. Immunotherapeutics Master’s Programme: Engineering antibody molecules - 1 University of Nottingham 11th 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)
CD52 IlexOncology/Genzyme (Campath® humanisation)
CD4 TolerRx/Genentech (for induction of tolerance)
CD4 BTG (improved method of humanisation)
CD3 BTG /TolerRx (immunosuppression and tolerance)
VAP-1 BioTie / University collaboration
RhD NBS / University collaboration
HPA-1a NBS / 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
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 + +/ - + +/ -

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. a a a a a a-GPI Human Fcg Receptors and their Activities
IgG1=IgG3
>>IgG4
>>>IgG2
IgG1
IgG2=IgG3
>>IgG4
IgG1
IgG3
IgG4>>IgG2
IgG1=IgG3
>>>>>>>
IgG4,IgG2
FcgRI FcgRIIa FcgRIIb FcgRIIc FcgRIIIa FcgRIIIb
(CD64) (CD32) (CD32) (CD32) (CD16) (CD16) a a a a a
a-GPI
ITAM-
ITIM-
ITAM-
ITAM-
ITAM- g2 g2
Inhibitory
Affinity High Low-Med Low-Med Low-Med Low Low-Med
(108 M-1) (2x106 M-1) (5x105 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%
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
Db mutation
Dc mutation
Da mutation

23. Residues at key positions in mutated constant regions

24. 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

30. 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

31. 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

32. Complement-mediated lysis of mononuclear cells45
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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. Binding to the cell line 3T6 + FcgRIIa 131H, measured by flow cytometry10 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

40. Binding to the cell line 3T6 + FcgRIIb1*, measured by flow cytometry10 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

41. 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*

42. 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

43. 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

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 (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.

46. 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.

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. 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.

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
Mouse VH
Human 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, (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.

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

62. 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%

63. 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

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,
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,
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
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

70. 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

71. 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!