Identification of Class II epitopes specific for IGFBP-2, a tumor antigen in breast and ovarian cancer patients

Abstract

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Insulin like growth factor binding proteins are involved in the induction and progression of both breast and ovarian cancers. Studies from our group have shown that patients with cancer can have endogenous IgG antibody immunity directed against IGFBP-2. We questioned whether IGFBP-2 specific CD4+ T cell responses could be identified in cancer patients. Recent studies have demonstrated that CD4+ T cells are necessary to achieve sustained survival and enhanced cytotoxic function of antigen-specific CD8+ T cells. In addition, the generation of an antigen specific CD4+ T cell response, in and of itself, can elicit the generation of CD8+ T cells recognizing the same antigen. Since the generation of antigen-specific CD4+ T lymphocytes depends on specific stimulation by MHC class II-presented antigens, several algorithms for predicting the binding of peptides and HLA-DR have been generated. Some studies have suggested that combining a variety of algorithms may be better than using a single algorithm for prediction of the binding affinity, thus, we developed a scoring system which combines data from 5 publicly available algorithms and identified 14 potential HLA-DR binding peptides derived from IGFBP2. The five algorithms used in this study were SYFPEITHI, Propred, MHC-Thread, Average Binding matrix method, and Rankpep. Twenty IGFBP-2 peptides were identified for each of the 15 most common MHC class II molecules. To predict best binding, we summed the scores that were assigned for different MHC II molecules from the five algorithms (S), and gave a score for the number of MHC class II molecules that the peptide sequence could be predicted to bind to, i.e. promiscuity (N). The final rank scores were assigned by multiplying S and N, and the top 14 peptides were selected. The peptides were then synthesized, and their immunogenicity was interrogated by IFN-γ ELISPOT utilizing peripheral blood mononuclear cells (PBMCs) from volunteer donors and breast cancer patients. Peptide specific T cells could be identified in both volunteer donors and breast cancer patients using 10 of the 14 peptides. Studies are ongoing demonstrating the specificity of the peptide specific T cells for IGFBP-2 protein. The definition of class II epitopes specific for IGFBP-2 will contribute to the development of a vaccine targeting that antigen.