Since its introduction in 1975, the well-known Kohler and Milstein technique (Nature 256:495, 1975) for the production of mouse hybridoma cells has made it possible to produce large quantities of mouse antigen-specific monoclonal antibodies that are useful in a number of investigative, diagnostic and therapeutic applications. The mouse hybridoma cells, which are initially produced by the fusion of antibody-producing cells (B-lymphocyte cells, hereinafter referred to as B-cells) with malignant, transformed B-cells (in vivo transformed, myeloma cells from mice afflicted with myeloma or plasmacytoma) are capable of producing large quantities of monoclonal antibodies with predetermined specificities.
Using the Kohler and Milstein technique, a B-cell and a plasmacytoma cell are fused using, for instance, polyethylene glycol, lysolecithin or Sendai virus as the cell-fusing agents. A selectable marker must be present in the fused cells to enable them to be selected from parent cells and other non-hybridoma cells. As an example, the plasmacytoma fusion partner is generally deficient in an enzyme, (for instance, hypoxanthine-guanosyl phosphoribotransferase (HGPRT)) that is necessary for growth of the fused cell in certain media (hypoxanthine-, aminoprotein-, and thymidine- containing medium or HAT medium). This enzyme deficiency enables the resultant hybrids to be selected for their ability to grow in such media. This insures that only B-cell:plasmacytoma cell hybrids are recovered since neither parental cells (nor hybrids comprising B-cell: B-cell and plasmacytoma: plasmacytoma cell) can survive in selective media.
Murine antibodies produced with the Kohler and Milstein technique are generally unsuitable for administration to human subjects as in-vivo therapeutic agents, e.g., to provide passive immunity to an infectious agent. The extension of the Kohler and Milstein hybridoma technology to the production of human monoclonal antibodies has been limited, largely due to: (1) the lack of good human plasmacytoma cells for use as fusion partners; (2) the low frequency of cell fusion events ("fusion efficiency"); and (3) the relative scarcity of B-cells circulating in human blood and producing specific antibodies against antigens of interest (and the inherent difficulties in isolating such cells). These factors make it difficult to obtain hybridoma cell lines secreting human monoclonal antibodies of a predetermined specificity.
Casali et al. (Science 234:476-479, 1986) disclosed a method which represents some progress toward making human monoclonal antibody-producing cells. Normal B-cells obtained from peripheral human blood were pre-selected for their specificity to a given antigen using Fluorescence-Activated Cell Sorting (FACS). Positively selected clones were then established as lymphoblastoid cells in vitro by infecting such cells with Epstein-Barr virus (EBV). The EBV infected cells produced antigen-specific human monoclonal antibodies. However, the method of Casali et al. has several significant drawbacks which impair its usefulness: (1) the amount of monoclonal antibodies produced by the Casali et al. cells is relatively low, and (2) the antibody producing cells are relatively unstable and some clones stop antibody production prematurely. In addition maintenance of the antigen-specific antibody production requires repeated cloning of the cells, a time-consuming and inefficient procedure given the low clonogenic (i.e. growth) properties of the resultant lymphoblastoid or lymphoblastoid cell lines (LCL); (3) large-scale production and purification of the monoclonal antibodies is inefficient in view of the long doubling time and high serum requirements of the LCL; and (4) the LCL produced by this process cannot be grown as tumors in animals. Such tumor cell growth permits the amplification and purification of antibodies from ascitic fluids, an efficient method for large scale antibody production that is widely used in making murine monoclonal antibodies. Finally, the Casali et al. method does not dispense with the requirement for identifying a human B-cell specific to a certain antigen.
Copending U.S. patent application Ser. No. 041,803 (allowed) filed Apr. 23, 1987 of Riccardo Dalla-Favera discloses a method for the production of human monoclonal antibody-producing cells. Specific B lymphocytes are selected using the method of Casali et al. (supra), infected with Epstein Barr virus (EBV) and transfected with activated c-myc DNA sequences. The resultant cells are tumorigenic (i.e. can grow in semisolid medium and animals such as rats or mice) and clonogenic and produce monoclonal antibodies of a predetermined specificity. However, it was found that these cells still produce relatively low amounts of antibody because the transfected lymphoblastoid cells had not undergone differentiation.
Currently there is no convenient and reliable system available for the production of human monoclonal antibodies wherein the monoclonal antibody-producing cells are stable, highly malignant and which can be readily manipulated to produce high antibody titers.
It, is therefore an object of the present invention to provide a method for the production of tumorigenic human cells that are capable of producing human monoclonal antibodies.
A further object of the present invention is to provide a transformed lymphoblastoid cell that is useful as a fusion partner in the production of human monoclonal antibodies.
Another object of the present invention is to provide a transformed lymphoblastoid cell that demonstrates high level proliferative, differentiation and antibody production properties.
Another object of the present invention is to produce a new human cell line comprising human B-cells infected with Epstein-Barr virus and which have at least one exogenous activated K-, N- or H-ras oncogene DNA sequences.