Abstract
BackgroundWe have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.MethodsThe current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.ResultsBy application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.ConclusionWe have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.
Highlights
We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients
A native fully human autoantibody to breast cancer identifies a cancer-associated antigen that localizes to the cytoplasm and membrane We previously described the construction of a unique fusion Partner cell line, MFP-2, and its use for the immortalization of both human peripheral blood and lymph node B-lymphocytes [24,25]
27.B1 antibody was preincubated with a bacterial expressed and refolded recombinant GIPC1 protein prior to blotting (Figure 2-B, lane 1) and compared to non-preincubated control (Figure 2-B, lane 2). These results demonstrate that fully human monoclonal antibodies (fhMAb) 27.B1 binding to the same 42 kDa band was inhibited by the recombinant protein and this confirmed the specificity of this antibody to the GIPC1 antigen
Summary
We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. The body mounts an immune response following the onset of malignant disease since the new cells are recognized as non-self. It is composed of both immune cells that mediate innate, non-specific immunity, and adaptive, antigen-specific immunity [1,2,3]. The potential utility of these antibodies to identify TAAs, to discriminate between neoplastic and normal tissues and potentially act as anti-cancer therapeutics has been the impetus for this work
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