A melanoma-specific V-H antibody cloned from a fusion phage library of a vaccinated melanoma patient

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (V-H) and light chain variable domain (V-L) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells, In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections, The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus, An unexpected finding is that V86 contains a complete V-H domain hut only a short segment of a V-L domain, which terminates before tile CDR1 region, This V-L deletion resulted from the occurrence in the V-L cDNA of a restriction site, which was cleaved during construction of the scFv library, Thus V86 is essentially a V-H antibody, The effect of adding a V-L domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to V-lambda or V-kappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones, None of tile V86-V-lambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-V-kappa clones, it was generally weaker than the binding of V86. These results indicate that most of the V-L domains in the original scFv library reduce or eliminate tile affinity of V86 for melanoma cells, Accordingly, V-H libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the Incompatibility of most randomly paired V-H and V-L domains.