The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region VH-JH, Vκ-Jκ, and Vλ-Jλ genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected VH and Vκ clones and tested for binding activity. Our data analysis revealed that some of the VH-JH, Vκ-Jκ, and Vλ-Jλ region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment κ minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the VH of which proved to be the overexpressed VH3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

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.

The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum-free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum-free formulations and selection rules for the serum-free ingredients. In general, serum-free media which are "lipid rich" can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum-free media, irrespective of the lipid content.

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