Induction of PRAME-specific immunity by overlapping pentadecapeptides in patients with hematologic malignancies

Our group provided evidence that the cancer testis antigen PRAME is a potential target for adoptive T-cell or vaccine therapy of many hematologic malignancies and solid tumors. PRAME-specific T cells can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using artificial antigen presenting cells (aAPC)1. So far, four PRAME-derived epitopes have been identified by a proteosome digestion, assay that may fail to identify of putative peptides in vivo generated. We have now adopted an alternative method that uses a library consisting of 135 synthetic pentadecapeptides, spanning PRAME protein, to evaluate if multiple immunogenic epitopes can be identified and used to generate polyclonal PRAME-CTL lines. CD8 + cells from 21 healthy donors and 7 patients with Chronic Myelogenus Leukemia (CML) were primed with dendritic cells loaded with PRAME-peptide library, and then expanded by stimulation with aAPC. We consistently generated PRAME-CTLs in 19/21 healthy donors and all 7 CML patients. These PRAME-CTLs were also able to target autologous CML cells when cultured with PRAME+CML blasts, demonstrating that the same peptides were presented physiologically. A Cr51 release assay confirmed that the PRAME-CTLs were cytotoxic, lysing autologous-PHA blasts loaded with the peptides derived from the PRAME-library. Using pentadecapeptides sub-pools, we found that the responses of our expanded PRAME-CTLs were polyclonal, since they consistently released IFN gamma in response to 1 to 6 pentadecapeptides pools. In conclusion, this novel approach should facilitate expansion of polyclonal PRAME-CTLs for adoptive transfer or after vaccine administration to patients with PRAME+hematological malignancy.