Engineering Antibodies (2) Immunotherapeutic Examples MSc Programme University of Nottingham 14 th February 2005 by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK

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) CD52IlexOncology/Genzyme (Campath® humanisation) CD4TolerRx/Genentech (for induction of tolerance) CD4BTG (improved method of humanisation) CD3BTG /TolerRx (immunosuppression and tolerance) CD18Millennium Pharmaceuticals VAP-1BioTie / University collaboration RhDNBS / University collaboration HPA-1aNBS / 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 Fc  R-mediated cell destruction unable to activate complement

RhD HPA-1a

Chemiluminescent response of human monocytes to sensitised RBC 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

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

Inhibition by Fog-1 antibodies of ADCC due to clinically relevant polyclonal anti-RhD (at 3ng/ml) inhibitor antibody concentration, ng/ml % RBC lysis G1  ab G2 G2  a G4 G4  b

VAP-1 HuVAP antibody

Selectins IgSF 4. Migration Free flow Infection Chemokine signal Integrin 1.Capture and rolling 2. Activation 3. Stationary adhesion Endothelium Multistep paradigm of neutrophil adhesion

Role of VAP-1 Selectin Anti VAP-1 Modified Fc region VAP-1 Amines Toxic aldehydes & H sVAP-1

Capillary flow system

Neutrophil adhesion assay VAP-1 3. Ultra-rapid stationary adhesion  2 Integrin Fc 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 ClarkDept of Pathology Kathryn Armour Chris Kirton Cheryl Smith Lorna WilliamsonNational Blood Service & Transfusion Medicine