UC 729-6, a human lymphoblastoid B-cell line useful for generating antibody-secreting human-human hybridomas.

Abstract

UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B-cell line, was fused with normal and malignant human lymphocytes. Parent UC 729-6 cells were diploid with a 21p+ marker chromosome, expressed surface and cytoplasmic IgM Kc, and doubled every 17 hr. The resulting human-human hybridomas were pseudotetraploid containing the 21p+ marker, doubled every 20-30 hr, and secreted 3-9 jig of human Ig per ml per 106 hybrid cells for >9 months in continuous culture. Human Ig-secreting hybridomas were generated in 88% (14/16) of the fusions carried out and were cloned by limiting dilution (one cell per three wells) without the use of feeder layers. The mean fusion frequencies (number of wells, plated at 105 cells per well, showing hybrid growth per 106 lymphocytes fused) of UC 729-6 with normal lymphocytes ranged from 0.45 to 2.9 and with malignant B lymphocytes, from 3 to 10. Analysis of the human-human hybridomas derived from lymphocytes isolated from regional draining lymph nodes of cancer patients revealed several that secreted human monoclonal antibody that reacted by an enzyme immunoassay with some carcinoma cell lines but not with normal fibroblast cell lines. These data suggest that (i) UC 729-6 can be fused with human lymphocytes to generate stable human-human hybridomas, some of which secrete antibody reactive to human cell surface antigens, and (ii) UC 729-6 can be used to rescue Ig from nonsecretory malignant B cells and thereby allow for the production of anti-idiotype antibodies.The use of somatic cell hybridization to produce monoclonal antibodies (mAbs) of predefined specificity, as pioneered by Kohler and Milstein (1), has revolutionized many areas of human biology. However, since most of these mAbs are of rodent origin their application to in vivo use in humans may be limited. Therefore, the production of human mAbs could potentially be of considerable value and in many instances may offer advantages over mAbs of other species.Currently available human mAbs have been obtained by mouse-human hybridizations (2-4), by Epstein-Barr virus (EBV) transformation of human immune lymphocytes (5-7), and by human-human hybridization (8-10) and has recently been reviewed (11). However, each of these approaches suffers from individual difficulties and each has met with limited success. Mouse-human hybrids preferentially lose human chromosomes and, therefore, are genetically unstable (12, 13). EBV transformation yields cell lines that produce small quantities of Ig (5-7). The reproducibility of generating human-human hybridomas is limited by the lack of a suitable fusion partner in that currently available cell lines for fusion (8)(9)(10) 6327The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

“…We therefore transfected cDNA for either mouse or human CD8 into COS 7 cells and determined whether these cells could bind the HLA class I-expressing human B lymphoblastoid cell line, UC, (29). Detectable binding of UC cells to human CD8 expressing COS 7 cells could be observed using phase contrast microscopy ( Fig.…”

“…Transfected COS 7 cells were washed once with PBS and 10"SS-cysteine-labeled B cells, UC (29), were added to each 35-mm dish in 1 .0 ml PBS supplemented with 10% heat-inactivated FCS, 100 U/ml penicillinstreptomycin . The cells were incubated for 1 h at 37°C and the B cells were poured off.…”

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

“…All 10 rescued colonies from the one-step selection contained plasmid DNAs with a restriction pattern characteristic of EBO-pcD-hprt (data not shown); 5 of the 10 rescued colonies from the two-step selection contained plasmid DNA with a restriction pattern characteristic of EBO-pcD-hprt, 4 contained plasmid DNA with a restriction pattern characteristic of EBO-pSV2-neo DNA, and 1 clone was apparently rearranged and lacked either hprt or neo sequences (data not shown). Five of the 10 clones rescued by one-step selection and the 5 EBO-pcDhprt-like clones rescued from the two-step selection were reintroduced into UC cells; all 10 of the clones yielded HAT-resistant transformants. This set of experiments indicates that the EBO-pcD expression vector can be used to directly select and recover very rare clones A high-molecular-weight form of some copies of EBOpcD-Leit-2 occurred after four rounds of FACS selection (Fig.…”

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets

“…1 For example, accessing the functional successes of in vivo humoral immune system defenses, which have evolved side-by-side with dynamic infectious agents, has allowed the cloning of broadly neutralizing antibodies to complex infectious diseases using a variety of approaches. [2][3][4][5][6][7] A fascinating trend is the discovery of specific Ig germline usage among unrelated and geographically disperse individuals against specific viral antigens.…”