Detection and Functional Analysis of CD8+ T Cells Specific for PRAME: a Target for T-Cell Therapy

Abstract

Purpose: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape fromT-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancerT-cell therapies. Experimental Design: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8 + T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-g enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specificT-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. Results: In 30% to 40% of healthy individuals and patients, PRA 100-108 -specific CD8 + T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA À memory PRA 100-108 -specific T cells were found in some individuals, the majority of PRA 100-108 -tetramer + Tcells expressed CD45RA, suggesting a naive phenotype. PRA 100-108 -tetramer + T-cell clones were shown to recognize and lyse HLA-A*0201 + and PRAME + melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA 100-108 -tetramer + Tcells. Conclusion: These data support the usefulness of PRAME and in particular the PRA [100][101][102][103][104][105][106][107][108] epitope as target forT-cell therapy of PRAME-overexpressing cancers.Tumor antigens that are used as targets in clinical studies belong to the melanocyte differentiation antigens, cancer-testis antigens, or antigens overexpressed in tumors (1 -5). Cancertestis antigens are expressed in different tumors but not in normal tissues, except for testis, and are therefore useful targets for T-cell therapy. Most cancer-testis antigens, however, are expressed at low frequencies (30-50%). Preferentially expressed antigen on melanomas (PRAME) has been identified as an antigen recognized by an HLA-A24-restricted CTL isolated from a melanoma patient (6). Semiquantitative reverse transcription-PCR (RT-PCR) analysis showed frequent PRAME expression in melanomas (95%) and other tumor types, including lung and breast tumors and leukemias but not in healthy tissues, except for testis and low expression in endometrium, ovaries, and adrenals. DNA microarray analysis revealed the gene encoding PRAME as one of the genes of an expression profile for poor prognosis in breast carcinoma (7). Recently, the function of PRAME has been elucidated by Epping et al. (8). PRAME binds to retinoic acid receptor a, thereby inhibiting retinoic acid -induced differ...

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells

“…21,22,24 These findings are further supported by HLA-peptide multimer data in ex vivo PBMC showing most precursors of LAA-reactive CD8 T cells in the naïve subset of healthy individuals. 25 In contrast, other groups detected HLA-peptide multimer binding leukemiareactive CD8 T cells also in the antigen-experienced memory pool. This was demonstrated for LAA specificities in healthy donors 26 as well as for mHag specificities in donors with a history of alloantigen priming in vivo (e.g.…”

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells

“…(A) Specificity of the generated CTL lines using PRAME-PepMix as assessed by INF␥ ELISpot assay against control PepMix, PRAME PepMix, and a pool of the 4 previously identified HLA-A*02-restricted peptides (P4). 8,18 Medium was used to evaluate background production of IFN␥ by nonstimulated CTL. Each symbol represents 1 of the 23 individual PRAME-CTLs; horizontal lines represent the mean group value.…”

“…15 Indeed, it has recently been demonstrated that PRAME overexpression contributes to leukemogenesis by inhibiting myeloid differentiation through blockage of the retinoic acid receptor-␣-signaling pathway. 14,16 We 17 and others 18 have generated CTLs targeting PRAMEderived peptides from healthy donors and leukemic patients using antigen-presenting cells (APCs) loaded with specific peptides 17 selected by in vitro digestion of long peptides 8 or by mass spectrometry of acid elutes obtained from tumor cells. 19 Unfortunately, these approaches have produced PRAME epitopes that have preferentially expanded low-avidity CTLs, whose modest functional activity would likely be suboptimal for clinical benefit.…”

A Causal Role for the Human Tumor Antigen Preferentially Expressed Antigen of Melanoma in Cancer

“…Furthermore, the low expression of PRAME in other normal tissues may not be sufficient for CTL recognition. PRAME-specific lysis of multiple tumor cell lines by CTL in an HLA-restricted manner has been shown (23). However, whether patients can be stimulated to consistently mount a robust immunologic response to this antigen has yet to be established.…”