Engineering Antibodies (2)
Immunotherapeutic Examples
MSc Programme University of Nottingham
14th February 2005
by
Mike Clark, PhD
Department of Pathology
Division of Immunology
Cambridge University
UK
www.path.cam.ac.uk/~mrc7/
University Research Programmes
•
Immunosuppression

•
Tumour Therapy

•
CD52, CD3, CD4, synergistic CD45 pair
Allo and auto-immunity

•
CD52 (Campath), bispecific CD3
Organ Transplantation

•
CD4, CD3, monovalent CD3, CD52 (Campath)
RhD, HPA-1a
Chronic Inflammation

CD18, VAP-1
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)
• CD18
Millennium Pharmaceuticals
• VAP-1
BioTie / University collaboration
• RhD
NBS / University collaboration
• HPA-1a
NBS / University collaboration
The antibody isotype is important
Chimeric and humanised
Rat IgG2b is effective in therapy
Human IgG1 also effective in therapy
Antibodies (eg CD52 Campath) can be
effective in killing cancer cells (BCLL)
Fetomaternal alloimmune
thrombocytopenia
• Maternal IgG raised against fetal platelet alloantigens can
cross the placenta and cause fetal platelet destruction
• If the fetal platelet count falls dangerously low, cerebral
hemorrage or death may result
• Current therapies are intrauterine platelet transfusion and
maternal therapy with high dose IVIG
Can a protective antibody be
developed?
• 90% severe cases FMAIT are due to antibodies
against the alloantigen HPA-1a on GPIIIa
• Single B cell epitope (Leu-33) could be blocked
to prevent the binding of harmful antibodies
• Outcome depends on antibody titre


Williamson et al. Blood 1998; 92: 2280
Jaegtvik et al. Br J Obs Gynae 2000; 107: 691
Ideal properties of an antibody for
FMAIT therapy
• HPA-1a specificity (B2 variable regions)
• able to cross the placenta
• inactive in FcgR-mediated cell destruction
• unable to activate complement
RhD
HPA-1a
Chemiluminescent response of human
monocytes to sensitised RBC
Fog-1
antibodies
% chemiluminescence
140
120
G1
100
G1D a
G1D b
80
G1D c
60
G1D ab
40
G1D ac
20
G2
G2D a
0
G4
-20
G4D b
0
5000
10000
15000
20000
25000
30000
G4D c
antibody molecules/cell
Inhibition of chemiluminescent response due to
2 mg/ml Fog-1 G1 by other Fog-1 antibodies
100
90
G1D b
% chemiluminescence
80
G1D c
70
G1D ab
60
G1D ac
50
G2
40
G2D a
30
G4D b
20
G4D c
10
0
0.1
1
10
inhibitor concentration, mg/ml
100
1000
Inhibition by Fog-1 antibodies of ADCC due to
clinically relevant polyclonal anti-RhD (at 3ng/ml)
120
100
% RBC lysis
80
G1D ab
G2
G2D a
G4
G4D b
60
40
20
0
0.1
1
10
100
1000
inhibitor antibody concentration, ng/ml
10000
HuVAP antibody
VAP-1
Multistep paradigm of neutrophil adhesion
1. Capture
and rolling
Free flow
2. Activation
3. Stationary
adhesion
4. Migration
Selectins
Chemokine
signal
Integrin
IgSF
Endothelium
Infection
Role of VAP-1
sVAP-1
Modified
Fc region
Anti VAP-1
Selectin
VAP-1
Amines
Toxic aldehydes
& H202
Capillary flow system
Neutrophil adhesion assay
1. Capture and FcR
ligation
2. Activation and integrin
expression
Fc receptor
3. Ultra-rapid stationary
adhesion
2 Integrin
Anti VAP-1 IgG
VAP-1
IgSF like
motif
Microslide
Flow
Human IgG1 wildtype anti-VAP-1 antibody
HuVAP mutated anti-VAP-1 antibody
Brief Acknowledgements
Mike Clark
Kathryn Armour
Chris Kirton
Cheryl Smith
Dept of Pathology
Lorna Williamson
National Blood Service
& Transfusion Medicine