Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma.

Abstract

This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2-derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human antibody that detected nuclei, nucleoli, cytoskeletal elements, Golgi complex, or other cytoplasmic components were also isolated in this study. One of these antibodies detected an intracellular antigen that is restricted to cells of neuroectodermal derivation, and a second antibody reacted primarily with cells of epithelial origin. Using these methods to isolate and analyze human monoclonal antibody, it should now be possible to define the repertoire of the humoral immune response to melanoma.

The Use of Insulin-Enriched Culture Medium and Human-Human Hybridoma Formation: A Preliminary Study

“…Since the first human-human hybridoma fusion re ported by Olsson and Kaplan [1980], several other in vestigators have confirmed the capability of similarly generating human monoclonal antibodies of various specifications [Croce et al, 1981;Houghton et al, 1983].…”

Human autologous tumor-specific T cells in malignant melanoma

“…Human antibodies recognizing tumor-associated determinants expressed on melanoma and other tumor cells, but not on normal cells, have been reported [1][2][3][4] and several of them have been immortalized either by fusing the antibody-producing cells with human or mouse myeloma cell lines [1] or by transforming these cells with Epstein-Barr virus [3]. Certain of the human anti-melanoma antibodies have been used for in vivo therapy of patients with malignant melanoma [3,4].…”

Mining human antibody repertoires

“…22 Given the genetic instability of heterohybridomas, the use of alternative fusion partners, particularly human lymphoblastoid cell lines and human myeloma cells, has been explored extensively. [23][24][25][26][27][28] While in principle successful, the yield of viable hybridomas is typically too poor for practical applications. In addition, such hybridomas often secrete permutated Igs derived from both fusion partners.…”