This invention relates to a process for making human antibody-producing B lymphocytes in tissue culture. More specifically, this invention relates to the antigen specific induction and regulation of antibody synthesis in cultures of human peripheral blood mononuclear cells. Such antibody producing cells can be used to make hybridomas using cell fusion techniques for producing monoclonal antibodies for therapeutic and diagnostic purposes.
The knowledge of mechanisms that govern the humoral immune response has been greatly advanced by the culture technique, initially developed by Mishell and Dutton for murine spleen cells, which permitted the study of antibody production in virto, Mishell et al., J. Exp. Med. 126: 423, 1967. Application of this technique developed for murine cells to the study of human lymphocytes has been less successful. Human B lymphocytes are obtained from peripheral blood, tonsils, spleens, and lymph nodes. While tonsil and spleen cells have been shown to produce antibodies against certain antigens [sheep red blood cells (SRBC)] in vitro under conditions which are similar to the conditions commonly used in experiments with murine spleen cells, peripheral blood lymphocytes (PBL) did not produce antibodies when cultured and tested in the same way, Hoffman, et al., Nature 243: 408, 1973. As peripheral blood lymphocytes (PBL) represent the only source of lymphocytes which is readily accessible, progress in the study of the humoral immune response in man has been hampered by the lack of technique which permits in vitro sensitization of peripheral blood lymphocytes.
If one disregards the well-established assay that measured the polyclonal production of antibodies which does not depend on antigen, Fauci et al., J. Exp. Med. 144: 674, 1926, success has been reported only by two groups. In the first case, infection with the Epstein-Barr virus (EB virus) was required to enable PBL to produce antibodies in specific response to SRBC or horse red blood cells (HRBC), Luzatti et al., Nature 269: 419, 1977. In the second case, Dosch et al., J. Immunol. 118: 302, 1977, no additional stimulus was used, but the hemolytic plaques in the standard assay has been questioned.
With respect to the use of EB virus, although it stimulates response in Human B lymphocytes, it apparently operates through an extraordinary pathway which cannot be reproduced with more benign mitogens. Furthermore, the usefulness of antibody producing cell lines generated using EB virus, for treatment of human patients is questionable as one would always be in danger of the effect of such viruses. EB virus is known to be tumor producing and also to cause mononucleosis. For these same reasons, laboratory personnel do not want to handle them either.
It has been found that one can use natural pathways of antibody production to generate antibody producing cells from human B lymphocytes. This avoids the need to deal with such extraordinary pathway mitogens as the tumor producing EB virus, with their attendant dangers to personnel working with the virus and patients receiving a possibly contaminated or otherwise dangerous end product. The production of antibody producing cells from Human B lymphocytes (or B-cells) is based on the realization that it is helper cells (macrophages and helper T cells) that support the B cell responses and it is suppressor lymphocytes, present in the B lymphocytes source which suppress or interfere with Human B lymphocytes activation. The effect of the suppressor lymphocytes can be overcome either by their destruction or removal from the Human B lymphocytes or by use of a B cell mitogen to overcome their effect.