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.

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A human IgG1.k monoclonal antibody (MAb) designated GMA1 was developed by fusing pooled lymph node lymphocytes from cancer patients with the human lymphoblastoid cell line, SHFP-1. The GMA1 MAb reacted with several melanoma and neuroblastoma cell lines. Normal tissue derived from human brain and tumor-cell lines derived from colon, ovary, and breast were not reactive. FACS analysis performed using live cells demonstrated that the antibody recognizes a cell-surface antigen. Enzyme immunoassay (EIA) and thin layer chromatography (TLC) immunostaining with purified gangliosides indicated that the antibody has specificity for the major tumor associated gangliosides GD3, GM3, and GD2. GMA1 heavy and light chain genes were isolated by RT-PCR and a recombinant derivative of this human antibody was expressed in Chinese hamster ovary (CHO) cells. High-level antibody synthesis and secretion was achieved using a vector designed to maximize expression. FACS analysis and TLC immunostaining indicated recombinant GMA1 reacted with human tumor cell lines and gangliosides GD3, GM3, GD2 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

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