Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies

Abstract

Background aims: Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized.

Methods: Peptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes.

Results: PRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes.

Conclusions: TAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.

Keywords: PRAME; T-cell epitope; cancer immunotherapy; off-the-shelf T-cell therapy.

Figure 1.

PRAME-specific T-cells can be expanded from healthy donors. (A) Total cell number of PRAME-specific T-cells generated from 12 healthy donors. PBMCs were primed with PRAME-pulsed DCs on day 0 and restimulated on day 7 and day 14. (B) Phenotyping analysis of PRAME-specific T-cell products, assessed by flow cytometry, shows a mixture of both CD4+ and CD8+ T-cells with a balanced predominance of central memory (CD3⁺CD45RO⁺CD62L⁺) and effector memory (CD3⁺CD45RO⁺CD62L^-) phenotype. There was no outgrowth of NK (CD3^-CD16⁺CD56⁺) or NKT (CD3⁺CD16⁺CD56⁺) cells. (C) The specificity of the T-cell product for the PRAME antigen was measured by IFN-γ ELISpot assay.

Figure 2.

Off-the-Shelf TAA-T cells are cytolytic against Wilms Tumor cell lines (A) Wilms tumor cell lines 17.94 (left) and Wit49 (middle) stained positive for PRAME by immunofluorescence, compared to pancreatic cells, negative control (right). (B) TAA-T products are polyclonal and demonstrate cytokine secretion upon stimulation with PRAME as measured by flow cytometry and intracellular cytokine staining. (C) TAA-T products demonstrate specificity for PRAME as measured by IFN-γ ELISpot assay (background response to actin subtracted from final results). (D)-(I) PRAME specific TAA-T products demonstrate antigen-specific cytotoxicity to Wilms tumor cell lines 17.94 (D)(F)(G) and Wit49 (F)(H) with absent non-specific activity to autologous PHA blasts or PBMCs (I).

Figure 3.

Identification of CD8-restricted T-cell epitopes (A) IFN-γ production by T-cells in response to PRAME peptide pool stimulation. (B)(C) T-cell responses to single PRAME peptides, present in the mini-pools, were evaluated by IFN-γ ELISpot assay. (D) HLA-I-restricted epitopes (36, 37, 43, and 44) were determined by measuring IFN-γ and TNF-α release by CD8+ T-cells. (E) CD4+ T-cells showed no specificity for HLA-I-restricted epitopes (36, 37, 43, 44). (F) The minimal 9-mer peptides FPEPEAAQP and VEVLVDLFL were determined by IFN-γ ELISpot assay. (G) FPEPEAAQP HLA restriction was confirmed using anti-HLA-B*35 antibody.

Figure 4.

Identification of CD4-restricted T-cell epitopes (A) IFN-γ production by T-cells in response to PRAME peptide pool stimulation. (B)(C) T-cell responses to single PRAME peptides, present in the mini-pools 2, 11, 17, and 18, were evaluated by IFN-γ ELISpot assay. (D) HLA-II-restricted epitopes (50 and 71) were determined by measuring IFN-γ and TNF-α release by CD4+ T-cells. (E) CD8+ T-cells did not release IFN-γ and TNF-α in response to class II-restricted epitopes.

Figure 5.

T-cell epitopes elicit both CD4+ and CD8+ T-cell responses Cytotoxic (CD8+) and helper (CD4+) T-cell response to PRAME epitope RLVELAGQSLLKDEA. Results show that this peptide activated both CD8+ and CD4+ T-cells.

Figure 6.

PRAME protein diagram with identified epitopes The locations of CD4 and CD8-restricted epitopes identified within PRAME protein are shown.