Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: The proof of principle concerning the tumor-infiltrating B lymphocytes

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 V-H-J(H), V-kappa-J(kappa), and V lambda-J lambda 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 V-H and V-kappa clones and tested for binding activity. Our data analysis revealed that some of the V-H-J(H), V-kappa-J(kappa), and V lambda-J lambda 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 ic minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V-H of which proved to be the overexpressed V(H)3-1 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.