Polyclonal PRAME-Specific Cytotoxic T Lymphocytes Generated Using Protein-Spanning Pools of Overlapping Pentadecapeptides Target Chronic Myeloid Leukemia
Authors: Quintarelli Concetta , Dotti Gianpietro , De Angelis Biagio , Hoyos Valentina , Pane Fabrizio , Mims Martha P. , Heslop Helen E. , Rooney Cliona M , Brenner Malcolm K , Savoldo Barbara 
Published: October 2019

Abstract
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 cytotoxic T lymphocytes (PRAME-CTLs) can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using an optimized combination of artificial antigen presenting cells (aAPC) (K562 cell line genetically modified to express the HLA-A*02, CD80, CD40L and OX40L molecules) and cytokines (IL12, IL7 and IL15).
Four HLA-A*02 PRAME-derived epitopes (P100, P142, P300 and P425) have previously been identified using a proteosome-mediated digestion analysis. However, this strategy, since relying only on the major cleavage site targeted by the immune-proteosome machinery for epitopes generation, may limit the potential clinical value of the identified peptides. We have now adopted an alternative method that uses a peptide-library consisting of 125 synthetic pentadecapeptides, overlapping by 11 aminoacids, spanning the entire PRAME protein. We evaluated whether novel HLA-A*02 restricted CD8+ T-cell responses to multiple immunogenic epitopes can be identified and used to consistently generate polyclonal PRAME-CTL lines from healthy donors and patients with hematologic malignancies.
CD8+ T lymphocytes from 14 HLA-A*02 healthy donors and 3 patients with chronic myelogenenous leukemia (CML) were primed with autologous CD40L-activated B blasts loaded with the PRAME-peptide library in the presence of low doses of IL12, IL7 and IL15, and then expanded by weekly re-stimulation with peptide loaded aAPC and IL-2. The frequency and specificity of PRAME-CTLs were evaluated using IFNg Elispot and 51Cr release assays against PHA-blasts loaded with the PRAME-library. Using this approach we consistently generated PRAME-CTLs in 12 of the 14 HLA-A*02 healthy donors (526±101 SFC/105 cells as assessed by IFNg Elispot assay) compared to an irrelevant peptide-library (7±2 SFC/105). Similarly, PRAME-CTLs were generated from all 3 CML patients (441±250 SFC/105 cells vs 22±10 SFC/105 against an irrelevant peptide-library). These PRAME-CTLs were also able to target autologous tumor blasts (57±6 IFNg SFC/105), demonstrating that the same peptides were processed and presented physiologically. A Cr51 release assay confirmed that the PRAME-reactive T cells were cytotoxic, lysing autologous-PHA blasts loaded with the peptides derived from the PRAME-library (63±14% at a 20:1 E: T ratio), but not with irrelevant peptides (<15%). MHC class-I blocking experiments using specific antibodies showed that both IFNg release and cytotoxic activity were HLA-restricted. Using pentadecapeptides sub-pools, we found that the responses of our expanded PRAME-CTLs were polyclonal, since they consistently released IFNg in response to 1 to 6 pentadecapeptides pools (59% were specific for 1 or 2 pools, 25% to 3 pools, and 16% to 6 pools). Moreover, the approach we describe has allowed us to identify 6 potential new immunogenic 15-mer peptides that are processed and presented by tumor cells, and should facilitate expansion of polyclonal PRAME-CTLs for adoptive transfer or after vaccine administration to patients with PRAME+ hematological malignancy.