Antibody producing human-human hybridomas. I. Technical aspects.

Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested.

Sensitivity of cloned high- and low-metastatic murine Lewis lung tumor cells to lysis by cytotoxic autoreactive cells.

Cloned malignant cell lines from primary tumor sites (two lines) and from a lung metastatic focus (one line) were established 14 and 28 days after s.c. inoculation of Lewis lung tumor cells into C57BL/6 mice. Cloning was done in semisolid agarose culture medium and individual clones expanded in liquid medium. In vivo malignancy of the three lines was assured by graft experiments to healthy C57BL/6 recipients. Morphology, cloning efficiency in agarose, and growth rate were the same for all three lines. However, the cloned line from a metastatic focus had a higher metastatic rate (number of lung metastases per 10(5) injected tumor cells) compared to the two other cell lines. T-lymphocyte-depleted mononuclear spleen cells from mice grafted with tumor cells 2 to 4 weeks previously were found to be cytotoxic to cells from all three lines, whereas unfractionated mononuclear spleen cells from the same animals had weak or no cytotoxic activity. The cytotoxicity of T-lymphocyte-depleted spleen cells was found to include other malignant and nonmalignant target cells of C57BL/6 origin, but not allogeneic cells. In mixing experiments, splenic T-lymphocytes inhibited the cytotoxic activity of non-T-lymphocytes. The high- and low-metastatic tumor cell lines were found to be equally sensitive to lysis by T-cell-depleted spleen cells, suggesting that the effector cells (cytotoxic autoreactive cells) may have a significant antineoplastic potential.