Generation of human IgG,IgA,and IgM anti-melanoma monoclonal antibodies utilizing lymphocytes of an actively immunized melanoma patient

Abstract

Active specific immunotherapy with irradiated allogeneic melanoma cells has been shown to enhance the humoral immune response in melanoma patients. An increased titer of melanoma-binding antibodies was demonstrated in sera of immunized patients. Lymph node cells and splenocytes isolated from an actively immunized melanoma patient were fused with the human-murine heteromyeloma cell lines SHMD-33, SPM4-0, and SBC-H20. A group of human anti-melanoma monoclonal antibodies (MABs) were generated from the SHMD-33 fusion. Isolated MABs (one IgG₂ one IgA, and two IgM) have been stable in cultures for more than 12 months and have produced human immunoglobulins at 0.2–0.9 Ug/ml/day. As shown by solid phase radioimmunoassays, the MABs react with autologous tumor cells and allogeneic melanoma tumors, including the cell line that was used for immunotherapy. In immunocytochemical assays, all four MABs react with a number of melanoma tumor cell lines. The IgG₂ and IgA MABs stained preferentially melanoma tumor cells. In contrast, the IgM MABs cross-reacted with a broad panel of tumor cells from colon, prostate, pancreas, lung, and other human tumors. The MABs appear to be directed to intracellular rather than membrane-associated antigens as shown by immunofluorescence assays on live and permeabilized cells. The IgG₂ antibody recognizes a 70 kDa antigen in melanoma cell lysates by Western immunoblotting. The target antigens for the other MABs have not yet been defined. Stability in culture and strong binding to melanoma tumor cells provide the basis for evaluating the potential of these human MABs. The IgG₂ MAB, in particular, may prove useful for diagnostic and therapeutic applications in humans. This study emphasizes the efficacy of primed B lymphocytes isolated from immunized patients for the generation of human MABs of different isotypes.