Combinatorial Antibodies Against Human Malignant Melanoma

Abstract

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for >7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10⁸ phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a >10⁶-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.