Th-1 Cytokine and Antibody Mediated Immunity against HER Family Expressing Breast Cancer Cells.

摘要

A recent neoadjuvant vaccine trial to treat early breast cancer demonstrated powerful induction of Th1 immunity against the HER-2, complete pathologic responses in over 18% of subjects, and for many subjects, evidence of down-regulated HER-2 expression on residual disease. To explain these observations, we investigated the action of archetypical Th1 cytokines (TNF-alpha + IFN-gamma) on both murine and human breast cancer cell lines that varied in the surface expression of HER-family receptor tyrosine kinases. We found that most tumor cell lines were sensitive to dual Th1 cytokines as evidenced by lower metabolic activity (alamar blue assay), lower proliferation, and enhanced apoptosis (AnnexinV/PI staining and TUNEL assay) as well as a reversible inhibition of surface expression of HER proteins. Apoptotic cell death was accompanied by demonstrated increases in activated caspase-3. Furthermore, the pharmacologic caspase-3 activator, procaspase-activating compound (PAC-1), mimicked both the killing effects and HER-2 suppressive activities of Th1 cytokines, while the caspase 3/7 inhibitor, prevented cytokine-induced HER-2 downregulation.;These studies therefore demonstrated that many of the in vivo effects of vaccination (apparent tumor cell death and loss of HER-2 expression) could be replicated in vitro using only the principle Th1 cytokines. These findings are consistent with the notion that IFN-gamma and TNF-alpha work in concert to mediate some of the biological effects of therapeutic Th1-polarizing vaccination through the induction of a caspase 3-dependent cellular death mechanism. The secondary aim of the study was to identify linear B-cell (antibody) epitopes within the extracellular region of human HER-3 and to characterize the in vitro biological activity of antisera from mice vaccinated with HER-3 library peptides. It was revealed that the antisera against peptide 27 and antisera against peptide 55 exhibited high ability to recognize whole HER-3 across the various ELISA and flow cytometry assays. Although we did not see any significant decrease in metabolic activity of HER-3 positive cells treated with serum 27 and 55 only as compared to the cells treated with control sera, an apparent biological activity for antisera directed against either peptide 27 or peptide 55 was observed when target cells were also treated with IFN-gamma and TNF-alpha.