Identification of Novel HLA-Restricted PRAME Peptides to Facilitate “Off-the-shelf” Tumor-Associated Antigen-specific T-cells

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Background & Aim Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is overexpressed in many human malignancies while poorly expressed or absent in healthy tissues, making it a good target for anticancer immunotherapy. Studies have shown that autologous multi tumor-associated antigen (TAA)-specific T-cells can safely induce disease stabilization or maintain durable remissions in patients with relapsed or refractory solid tumors and leukemias/lymphomas expressing PRAME antigen. “Off-the-shelf” third-party T-cell products derived from healthy donors is a potential, readily available alternative to autologous TAA T cell products. Development of an effective “off the shelf” adoptive T-cell therapy however depends on the identification of PRAME antigens recognized by the TAA-T cell product. Hence, our goal was to identify specific HLA-restricted PRAME peptide epitopes which have not been studied extensively. We hypothesized that both TAA-T would recognize a broad number of PRAME epitopes that would be both CD4+ and CD8+ restricted. Methods, Results & Conclusion Peptide libraries of 15mer peptides overlapping by 11 amino acids spanning the entire sequence of PRAME protein were designed to identify HLA class I and class II-restricted epitopes. T cell response to pools of peptide libraries and responses to single PRAME peptides were evaluated by IFN-γ ELISpot assay. HLA-restriction of these peptides was determined by intracellular cytokine staining, measuring IFN-γ and TNF-α release. We further determined the minimal 9-mer epitope recognized by HLA I restricted cell lines. Finally, the restricted HLA allele was tested using autologous and partially HLA-matched 9-mer peptide-pulsed PHA blasts. We successfully generated PRAME-specific T-cell products from PBMCs derived from 12 healthy donors. Ex-vivo expanded T-cells were polyclonal consisting of both CD4+ and CD8+ T-cells. We identified five novel and two previously described MHC class I-restricted PRAME epitopes. Within two previously described epitopes, recognition was restricted by same HLA-allele in all but one case. We also characterized 12 15-mer peptide sequences confirmed as CD4-restricted epitopes. Our findings demonstrate that TAA T-cells derived from healthy donors recognize a broad number of CD4+ and CD8+ HLA-restricted PRAME epitopes which will be critical for the clinical translation in the donor-specific as well as the “off-the-shelf” setting.