immunotherapeutics master’s programme: engineering antibody molecules - 1 university of nottingham...
TRANSCRIPT
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Immunotherapeutics
Master’s Programme: Engineering antibody molecules - 1
University of Nottingham 11th February 2009
by
Mike Clark, PhDDepartment of Pathology
Division of Immunology
Cambridge University
UK
www.path.cam.ac.uk/~mrc7/
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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
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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
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Antibody based immunotherapeutics
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IgG is the preferred class
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Schematic view of IgG domains
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Antibody fragments can also be used
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Antibodies can be derived from immunised animals
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The antibody immune response in-vivo can be T-cell dependent or independent
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Antibody fragments can also be selected from in-vitro systems such as phage expression
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Cycles of selection and mutation can give an artificial in-vitro immune response based
simply on binding affinity
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The Selection of IgG Fc Regions for appropriate effector functions: The role of isotypes and polymorphisms
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Effector functions of human IgG
IgG1 IgG2 IgG3 IgG4Complement activation
Classical pathway +++ + +++ Alternative pathway +
Fc receptor recognitionFcRI +++ +++ ++
FcRIIa, 131R/R ++ ++ FcRIIa, 131H/H + + ++ FcRIIb ++ ++ +
FcRIII + +/ + +/
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Unlike mouse the human IgG subclasses are very similar in sequence but they still
have different properties
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Human IgG Fc Receptors
CD64 CD32 CD16
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FcRI FcRIIa FcRIIb FcRIIc FcRIIIa FcRIIIb(CD64) (CD32) (CD32) (CD32) (CD16) (CD16)
-GPIITAM-
ITAM- ITIM- ITAM-ITAM-
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%
IgG1=IgG3>>IgG4
>>>IgG2
IgG1IgG2=IgG3
>>IgG4
IgG1IgG3
IgG4>>IgG2
IgG1=IgG3>>>>>>>
IgG4,IgG2
Human Fc Receptors and their Activities
Slide courtesy of Bill Strohl, Centocor, September 2008
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The IgG receptor FcRn
Interaction with FcRn and with Protein A through similar region
FcRn is important for IgG half-life and transport
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For FcRI binding:
231 238IgG1, IgG3 A P E L L G G PIgG2 . . P V A - . .IgG4 . . . F . . . .sequence introduced into IgG1, IgG4
. . P V A - . .or . . P V A G . .
For C1q binding:
318 327 331IgG1, IgG3 E Y K C K V S N K A L P A PIgG2 . . . . . . . . . G . . . .IgG4 . . . . . . . . . G . . S Ssequence introduced into IgG1, IgG2
. . . . . . . . . G . . S S
Transferring motifs between human subclasses
b mutationc mutation
a mutation
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Residues at key positions in mutatedconstant regions
Antibody 233 234 235 236 327 330 331
G1 E L L G A A PG1a E L L G G S SG1b P V A - A A PG1c P V A G A A PG1ab P V A - G S SG1ac P V A G G S SG2 P V A - G A PG2a P V A - G S SG4 E F L G G S SG4b P V A - G S SG4c P V A G G S S
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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
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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 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
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Test systems: antibodies with CD52 and -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
-RhD
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-5
0
5
10
15
20
25
30
35
40
45
0.1 1 10 100antibody, g/ml
% s
peci
fic
Cr
rele
ase
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
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-5
0
5
10
15
20
25
1 10 100 1000
G2a concentration,g/ml
% s
peci
fic
Cr
rele
ase
Complement-mediated lysis withCAMPATH-1 G1(6.3 g/ml),
inhibited by CAMPATH-1 G2a
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Binding to the FcRI-bearing cell line, B2KA, measured by fluorescence staining
G2
G1bG1c
G4
G1a
G1
CAMPATH-1H antibodies at 100 g/ml
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0
20
40
60
80
100
120
140
160
0.001 0.01 0.1 1 10 100
antibody, g/ml
mea
n fl
uore
scen
ce
G1
G1a
G1 b
G1 c
G1 ab
G1 ac
G2
G2 a
G4
G4 b
G4 c
CAMPATH-1 antibodies
Binding to the FcRIa-bearing cell line, B2KA, measured by fluorescent staining
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Chemiluminescent response of human monocytes to sensitised RBC
-20
0
20
40
60
80
100
120
140
0 5000 10000 15000 20000 25000 30000
antibody molecules/cell
% c
hem
ilum
ines
cenc
e
G1
G1a
G1b
G1c
G1ab
G1ac
G2
G2a
G4
G4b
G4c
Fog-1 antibodies
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Inhibition of chemiluminescent response to clinical sera by Fog-1 G2a
280
0
40
80
120
160
200
240
0 10 100 1000
G2a concentration, g/ml
% c
hem
ilum
ines
cenc
e G1anti-D serum Aanti-D serum Banti-D serum Canti-D serum Danti-D serum Eanti-C+D serumanti-K serum
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Binding to the cell line 3T6 + FcRIIa 131R, measured by flow cytometry
10
20
30
40
50
60
70
80
90
100
0.1 1 10 100
antibody concentration, g/ml
mea
n fl
uore
scen
ce
G1
G1a
G1b
G1c
G1ab
G1ac
G2
G2a
G4
G4b
G4c
G1g
IgA1,
Fog-1 antibodies
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Binding to the cell line 3T6 + FcRIIa 131H, measured by flow cytometry
10
20
30
40
50
60
70
80
90
0.1 1 10 100
antibody concentration, g/ml
mea
n f
luor
esce
nce
G1
G1a
G1b
G1c
G1ab
G1ac
G2
G2a
G4
G4b
G4c
G1g
IgA1,
Fog-1 antibodies
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Binding to the cell line 3T6 + FcRIIb1*, measured by flow cytometry
10
60
110
160
210
260
0.1 1 10 100
antibody concentration, g/ml
mea
n fl
uore
scen
ce
G1
G1a
G1b
G1c
G1ab
G1ac
G2
G2a
G4
G4b
G4c
G1g
IgA1,
Fog-1 antibodies
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Binding to different forms of FcRII
antibody constant region
perc
enta
ge o
f G
1 bi
ndin
g
0% = binding of IgA1,
0
20
40
60
80
100
120G
1
G1
a
G1
b
G1
c
G1
ab
G1
ac G2
G2
a
G4
G4
b
G4
c
G1
g
FcRIIa 131R
FcRIIa 131H
FcRIIb1*
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Activity of Fog-1 antibodies in ADCC
-20
0
20
40
60
80
100
120
0.1 1 10 100 1000 10000
antibody concentration, ng/ml
% R
BC
lysi
s
G1G1abG2G2aG4G4b
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Inhibition by Fog-1 antibodies of ADCC due to Fog-1 IgG1 (at 2ng/ml)
inhibitor antibody concentration, ng/ml
% R
BC
lysi
s
0
5
10
15
20
25
30
35
40
45
0.0001 0.001 0.01 0.1 1 10 100 1000 10000
G1 a
G1 c
G2 a
G2
G1b, G1ab, G1ac, G4, G4b, G4c
{
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Summary of antibody activities
Mutants of : G1 G2 G4wt a b c ab ac wt a wt b c
Binding to:FcRIFcRIIa R/RFcRIIa H/HFcRIIb1*FcRIIIb NA1FcRIIIb NA2
Monocyte actnComplement lysisADCC
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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.
FcRIIa 131H binding: IgG1 and IgG2 show equal binding
but G1b and G1c 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.
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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 FcRIIa 131H and 131R binding
c more active in FcRI binding, monocyte activation FcRIIIb 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.
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What of the immunogenicity of therapeutic antibodies?
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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.
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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).
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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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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.
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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
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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 ?
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The V-region Mythology
Chimaeric65% Human ?
Humanised95% 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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Kabat database variability of VH sequences
Human VH Mouse VH
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Are chimaeric, humanised and fully human antibodies so very different in sequence?
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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.
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The same strategy can be applied to almost any V-region
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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 (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
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Antibody Specificity V-regionHomologous
VHJH Length Matches Homology
FOG-1 RhD Human V4-34 JH6A 124 100 0.807
anti-Tac CD25 Humanised HV1F10T JH6a 116 91 0.784
anti-Tac CD25 Mouse HV1F10T JH4D 116 84 0.724
anti-TNFa TNF-alpha Mouse VI-4-IB JH3B 117 84 0.718
Campath-1H CD52 Humanised DP-71_3D197D- JH4D 121 86 0.710
Campath-1G CD52 Rat DP-34_DA-10 JH4D 121 85 0.702
OKT3 CD3 Humanised b25 JH6a 119 82 0.689
OKT3 CD3 Mouse DP-7_21-2-.. JH6a 119 81 0.681
HD37 CD19 Mouse 6M27 JH4D 124 83 0.669
anti-CD20 CD20 Mouse DP-7_21-2- JH2 121 81 0.669
Homologies for antibody heavy chain V regionscompared with human germline sequences
Sorted by homology
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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.
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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)
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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 - +
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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?
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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
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Murine constant regionsV-region sequencesHuman Ig allotypes
Unusual glycosylation
Method of administrationFrequency of administration
Dosage of antibody
Patients' disease statusPatients' immune status
Patients' MHC haplotype
Specificity of antibodyCell surface or soluble antigen?
Formation of immune complexes with antigen
Complement activation by antibodyFc receptor binding by antibody
Inflammation and cytokine release
Factors likely to influence immunogenicity of therapeutic antibodies
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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!