title: Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma.
abstract: The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted cross-reactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens.
authors.affiliation: Active Biotech Research AB, Lund, Sweden. Jesper.Tordsson@activebiotech.com
authors.forename: J M
authors.initials: JM
authors.lastname: Tordsson
authors.forename: L G
authors.initials: LG
authors.lastname: Ohlsson
authors.forename: L B
authors.initials: LB
authors.lastname: Abrahmsén
authors.forename: P J
authors.initials: PJ
authors.lastname: Karlström
authors.forename: P A
authors.initials: PA
authors.lastname: Lando
authors.forename: T N
authors.initials: TN
authors.lastname: Brodin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000951:Antigens, Neoplasm / Q000276:immunology*; D003429:Cross Reactions; D004768:Enterotoxins / Q000627:therapeutic use*; D000939:Epitopes / Q000276:immunology; D005260:Female; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000627:therapeutic use*; D007167:Immunotherapy; D000922:Immunotoxins / Q000627:therapeutic use*; D008252:Macaca fascicularis / Q000276:immunology*; D008545:Melanoma / Q000276:immunology / Q000628:therapy*; D051379:Mice; D016513:Mice, SCID; D009130:Muscle, Smooth / Q000276:immunology; D009368:Neoplasm Transplantation; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000627:therapeutic use; D013045:Species Specificity; D013047:Specific Pathogen-Free Organisms; D018089:Superantigens / Q000627:therapeutic use*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D004768:Enterotoxins; D000939:Epitopes; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D018089:Superantigens; D000099216:Chondroitin Sulfate Proteoglycan 4; C055966:immunoglobulin Fv; C026066:enterotoxin A, Staphylococcal