Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines.

Abstract

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-ll; IgG secreting) was mutagentreated with low-level V-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 JIM ouabain. Surviving cells, which arose at an optimal frequency of i0-5, were subcloned by limiting dilution and screened for anti-TT production. Out of395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, K chain) at a maximum concentration of 3-6 #g/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal Blymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.The production of human monoclonal antibodies expressing a desired specificity has proved successful with methods that use either the lymphotropic Epstein-Barr virus (EBV) to immortalize antigen-specific B cells (1)(2)(3)(4)(5) or the classical hybridoma technique, whereby human blood lymphocytes are fused with murine (6) or human myeloma cell lines (7,8). To overcome difficulties inherent in each technique (9), we examined the possibility of combining them. We have described a technique whereby lymphocytes from normal donors immunized with tetanus toxoid (TT) were preselected for antigen-binding cells, subsequently transformed with EBV, and cloned (10). In the present study, an EBV-transformed clone B6 was fused with a human lymphoblastoid cell line ofthe B-cell type (B-LCL), KR-4, to rescue high amounts of anti-TT antibody production. The resulting hybridomas were found to be more stable, to have a higher cloning efficiency, and to secrete =8-fold more specific antibody compared with the parental B6 clone. In addition KR-4 had a 25-fold higher frequency of hybridization with EBVtransformed B cells compared with nontransformed B cells. MATERIALS AND METHODSCell Lines. B6 is an anti-TT antibody-producing, EBV-transformed, cloned cell line that was previously established in our laboratory (10). GM 1500 6TG A-li is a 6-thioguanine-resistant (SGuaR) [hypoxanthine/aminopterin/thymidine (HAT)-sensitive] human B-cell line, which was generously provided by H. Koprowski at The Wistar Insti...

Production of a polyspecific human monoclonal antibody reacting with an epidermal antigen

“…In examining the FMC-Hl clone we were surprised to discover that it lacked characteristics typical of human hybridomas which are usually near tetraploid and often secrete both of the parental immunoglobulin types (19,24). The FMC-Hl line was near diploid and secreted only IgM.…”

“…Fusion ofthe established FMC-HI line (described m this report) to the HAT-sensitive, ouabain-resistant KR-4 cell line (19) was performed using a similar protocol.…”

Highly efficient procedure for production of human monoclonal antibodies: Establishment of hybrids between Epstein-Barr virus-transformed B lymphocytes and heteromyeloma cells by use of GIT culture medium.

“…With human-mouse hybrids, the stable production of human monoclonal antibodies is limited to the lambda type, because preferential loss of human chromosome No. 2 (assigned to the kappa chain) usually occurs in human-mouse hybrids during cultivation (Kozbor et al 1982). In our experiment, on the contrary, (mouse x human) heteromyeloma SHM-D33 was chosen as fusion partner, and this may be why our six monoclonal antibodies were of the kappa type.…”

B cell responses to HIV and the development of human monoclonal antibodies

“…General strategies Rodent MoAbs are generally produced by the fusion of immune spleen lymphocytes and a suitable non-secreting myeloma partner to form hybrid cell lines [152], This strategy can also be applied to hMoAbs but there are numerous problems to be overcome [reviewed in 153,154], In particular, there are limitations in the availability of immune lymphocytes, while the human equivalent of the rodent myeloma cell has yet to be found, although substitutes have been tried, often themselves hybrids of human and murine cells [153,154], An alternative strategy is to use Epstein-Barr virus (EBV), a B lymphotropic herpes virus that induces polyclonal stimulation of B cells, followed in a small percentage of cells by their transformation to immortalized cell lines [155], Many investigators now use a combination of viral transformation to increase the proportion of specific antibody-secreting cells followed by fusion with a murine or hybrid partner cell [111,[156][157][158], An alternative strategy is to employ molecular cloning methods to identify and isolate the sequences for specific antibody and to re-express these in a suitable cell [159,160], Starting material may be transiently secreting EBV lines, immune B cells selected for their specificity or even the germ line sequences which are then screened for suitable reactants [161], Table 5 summarizes the hMoAbs presently reported with specificities for HIV antigens. They include antibodies reactive with the major structural proteins of gag and env but none reacting with the regulatory proteins.…”