Pokeweed mitogen-stimulated human lymphocytes fused to LICR-2 (HMY2) generate human-human hybridomas producing monoclonal IgG antibodies reactive to human breast carcinoma and malignant melanoma
Affiliations: Wright State University School of Medicine and Fort Hamilton-Hughes Hospital, Hamilton, Ohio, USA
Note: [] Address reprint requests to: Dr. Richard F. Martin, Director of Pathology, Fort Hamilton-Hughes Hospital, 630 Eaton Avenue, Hamilton, OH 45013, USA. A version of this paper was presented at the First International Conference on Human Antibodies and Hybridomas, Orlando, FL, USA, 18–20 April 1990.
Abstract: Human-human hybridomas were generated using pokeweed mitogen–stimulated lymphocytes from the regional lymph nodes of cancer patients by fusion to the LICR-2 human myeloma cell line . A total of 35 fusions, using the regional lymph node lymphocytes of cancer patients, resulted in hybrid growth in 23% of wells plated with 21 IgG ELISA positive clones, 6 of which have maintained stable human monoclonal antibody production. Mononuclear cells were separated on Ficoll-Paque and grown for 3–4 days in 1% pokeweed mitogen and fused to the LICR-2 human myeloma cell line. Human-human hybridoma producing membrane reactive IgG antibodies have been isolated and react to the following cancers: breast; melanoma. Twenty-seven fusions from 8 breast carcinoma patients resulted in 13 ELISA positive IgGs, 3 of which were stable after cloning. A total of 5,071 wells were plated after polyethylene glycol fusion with resultant hybrid growth in 1210 wells (24% hybrid growth) after hypoxanthine-aminopterin-thymidine selection. In 8 fusions using regional lymph node lymphocytes of other types of cancer, including 6 fusions using lymphocytes from malignant melanoma patients, there were 1,580 wells plated with positive growth in 20% of the wells (311 wells). Of these, 8 clones were ELISA positive and 3 stable clones all producing IgG anti-melanoma antibody were isolated. The overall hybrid frequency was 43×10−7 fused lymphocytes (39×10−7 non-breast and 45×10−7 breast). A total of 21 IgG-producing clones were identified to crude membranes of allogeneic tumor cell lines and stable antibody production was achieved for 6 (29% stable clones). Pokeweed mitogen stimulation of lymph node lymphocytes with subsequent fusion to the LICR-2 is an effective methodfor rescuing human hybridomas producing membrane reactive human monoclonal antibodies.
Keywords: human monoclonal antibodies, hybridomas, breast melanoma, pokeweed mitogen
