Immortalization of Human Lymphocytes from a Tumor-involved Lymph Node1

Lymphocytes isolated from a regional draining lymph node of a patient with a carcinoma of the vulva were fused with human UC 729-6 cells. The generated human-human hybridomas were tetraploid with the 21p+ chromosomal marker of UC 729-6, expressed HLA derived from the patient's cells, and have been stable in culture for 2 yr. Supernatant* from six immunoglobulin G (IgG)-secreting hybridomas were found to have broad reactivity profiles with human carcinoma cells and no reactiv ity with hematopoietic cell lines (Band I -cells, myelomas, leukemias, lymphomas), red blood cells, peripheral blood lymphocytes, and normal fibroblasts. All six human IgG monoclonal antibodies reacted with the A431 cell line, an epidermoid carcinoma of the vulva, similar to the patient's cancer. The supernatant of one hybridoma, which phenotyped as having T-cell parentage, enhanced the cloning efficiency of human hybrids, suggesting the presence of a growth factoids). In serum-supple mented cultures these hybrids secreted 200 ng to 3 MRof IgG/ml/10' cells/24 h and in serum-free medium, an enhanced production of 1 to 9 MRof IgG/ml/106 cells/24 h. One IgG monoclonal antibody, VLN3G2, precipitated a single chain protein with an apparent molecular weight of 48,000 from Nonidet P-40 extracts of A431 cells. Altogether, the data suggest that regional draining lymph nodes of cancer patients are highly immunoreactive and contain B-cells whose immunoglobulin recognizes putative tumor-associated antigens.