The specific aims of this project are twofold. A hypoxanthine-aminopterin thymidine (HAT)-sensitive, nonimmunoglobulin secreting human myeloma cell line will be developed and characterized for specific application to human hybridoma production, and a tissue culture medium will be developed (not requiring conditioned medium or fetal bovine serum) to support the growth of this cell line. Both developments will make possible the use of human cell-derived monoclonal antibodies for cancer diagnosis and therapy. Monoclonal antibodies produced by the hybridoma technique of Kohler and Milstein have already been shown to have both diagnostic and therapeutic application. In tumor models, monoclonal antibodies conjugated to radionuclides, cytotoxic drugs or toxins, or even unconjugated monoclonal antibodies have demonstrated inhibition of tumor growth and in some cases regression of established tumors. However, due to the nonavailability of a nonsecretory, HAT-sensitive in vitro adapted human myeloma line for human-human fusions, mouse antibodies have been used for the most part. The multiple injection of foreign proteins such as mouse antibodies may result in an anaphylaxis, immune complex vasculitis, and/or the production of blocking antibodies in the patient. These problems would theoretically be reduced or eliminated in a hurnan-human system.HAT-sensitive mutants are being developed from a cell line, K737 (Lozzio), which was derived from the pleural effusion of a patient with multiple myeloma. The K737 cells will undergo 8-Azaguanine treatment in semisolid agar to develop the HAT-sensitive hypozanthine-phosphoribusyl-transferase deficient (HPRT-) mutants. During Phase 11, the HAT-sensitive HPRT- mutants selected from Phase I will be further characterized as to growth properties, morphology, and fusion efficiency. The cell line will undergo further mutation in order to select for a HAT-sensitive HPRT- mutant that does not secrete immunoglobulin. Immunoglobulin levels will be tested by the principal investigator while karyology studies will be done by Dr. Lozzio. Also, during Phase I and 11, a tissue culture medium based on a low-serum medium (HSI-LoSM) will be developed to grow the cells without conditioned Fnedium and fetal bovine serum. The medium will be further tested against various other cell lines with particular emphasis on supporting the growth of established human and mouse hybridomas. This medium would allow for the production of monoclonal antibodies in culture without interference of fetal bovine serum proteins.National Cancer Institute
