Engineering Antibodies (2)Immunotherapeutic Examples

MSc Programme University of Nottingham

14th February 2005 by

Mike Clark, PhDDepartment of Pathology

Division of Immunology

Cambridge University

UK

www.path.cam.ac.uk/~mrc7/

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

• 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 FcR-mediated cell destruction

• unable to activate complement

RhDHPA-1a

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

Inhibition of chemiluminescent response due to 2 g/ml Fog-1 G1 by other Fog-1 antibodies

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

inhibitor concentration, g/ml

% c

hem

ilum

ines

cenc

e

G1 b

G1 c

G1ab

G1ac

G2

G2a

G4b

G4c

Inhibition by Fog-1 antibodies of ADCC due to clinically relevant polyclonal anti-RhD (at 3ng/ml)

0

20

40

60

80

100

120

0.1 1 10 100 1000 10000

inhibitor antibody concentration, ng/ml

% R

BC

lysi

s

G1 ab

G2

G2a

G4

G4 b

VAP-1

HuVAP antibody

Selectins IgSF

4. Migration

Free flow

Infection

Chemokinesignal

Integrin

1. Captureand rolling

2. Activation 3. Stationaryadhesion

Endothelium

Multistep paradigm of neutrophil adhesion

Role of VAP-1

Selectin

Anti VAP-1

Modified Fc region

VAP-1

Amines

Toxic aldehydes & H202

sVAP-1

Capillary flow system

Neutrophil adhesion assay

VAP-1

3. Ultra-rapid stationaryadhesion

2 IntegrinFc receptor

IgSF like motif

1. Capture and FcR ligation

2. Activation and integrin expression

Anti VAP-1 IgG

Microslide

Flow

Human IgG1 wildtype anti-VAP-1 antibody

HuVAP mutated anti-VAP-1 antibody

Brief Acknowledgements

Mike Clark Dept of Pathology Kathryn Armour Chris Kirton Cheryl Smith

Lorna Williamson National Blood Service& Transfusion Medicine