A general procedure is described for the production of human monoclonal antibodies from peripheral blood lymphocytes immunized in vitro against T-cell-dependent antigens. These lymphocytes immunized in culture were used to produce human-human or human-mouse hybridomas secreting monoclonal antibodies specific for digoxin, hemocyanin, a recombinant fragment of the gpl20 envelope glycoprotein of human immunodeficiency virus (PB1), or a melanomaassociated antigen (p97). Depletion of a lysosome-rich cell population, containing large granular lymphocytes, monocytes, cytotoxic T cells, and a subset of CD8-positive T cells, was shown to be crucial before the cells could be immunized in vitro. This depletion was accomplished by treating the peripheral blood lymphocytes with the lysosomotropic agent L-leucine methyl ester. In addition, the in vitro immunization had to be supported by interleukin 2, y-interferon, and B-cell growth and differentiation factors, derived from irradiated, pokeweed-mitogen-stimulated human T cells. The production of human monoclonal antibodies from primary, antigenspecifically activated peripheral lymphocytes might obviate the need to immunize volunteers or patients.

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Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3 x KS1/4 and OKT3 x KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h 51Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10-10,000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.