Abstract
BackgroundHigh-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor.MethodsA*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR α and β chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2Rγ-deficient (NSG) mice.ResultsA rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8α, and CD8β but not CD56 and recognized A*02+NY-ESO-1+ cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4+ and CD8+ single-positive T cells. Both CD4+ and CD8+ T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02+NY-ESO-1+ cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8+ single-positive T-cell clones functioned only in CD8+ T cells. Both 19305DP-TCR gene-engineered CD4+ and CD8+ T cells eliminated A*02+NY-ESO-1+ tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins.ConclusionTogether, our results indicate that the naturally occurring 19305DP-TCR derived from CD4+CD8+ double-positive αβ T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02+ patients with NY-ESO-1-expressing tumor.
Highlights
High-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients
Generation of a tumor antigen-specific CD4+CD8+ doublepositive T-cell clone In previous studies, we reported that a subset of NY-ESO-1-specific CD4+ T cells directly recognizes NY-ESO-1-expressing cancer targets in a MHC class II-restricted manner [24, 29]
(See figure on previous page.) Fig. 1 Characterization of NY-ESO-1-specific CD4+CD8+ T cells. a IFN-γ production by NY-ESO-1-specific CD4+ T-cell line was determined by intracellular cytokine staining following stimulation with NY-ESO-1157-170 peptide-pulsed, protein-pulsed, mRNA-electroporated or untreated (−) autologous Epstein-Barr virus (EBV)-transformed B cells (EBV-B), or SK-MEL-37. b IFN-γ production by SK-MEL-37-reactive CD4+ T-cell line was determined by flow cytometry. c Purification of CD4+Vβ8+ T cells was confirmed by flow cytometry. d Expression of cell surface molecules on 19305DP was determined by flow cytometry. e Expression of CD4 and CD8α on 19305DP was compared to those in CD4+ single-positive T-cell clone (CD4SP) and CD8+ single-positive T-cell clone (CD8SP) by flow cytometry
Summary
High-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. The feasibility, safety, and therapeutic effects of TCR gene-engineered autologous T cells have been demonstrated in clinical trials Several of these clinical studies have used MHC class I-restricted tumor antigen-specific TCR to manufacture anti-tumor T-cell products [1,2,3]. It is generally accepted that high-affinity TCR is required to provide efficient tumor-recognizing ability to T cells. This is in part because high-affinity MHC class I-restricted TCR confers tumor-reactivity to CD4+ T cells in addition to CD8+ T cells by bypassing the requirement of CD8 co-ligation for activation [4,5,6,7]. In addition to enhanced anti-tumor effects by CD8+ T cells, provision of tumor-reactivity to CD4+ T cells by high-affinity TCR may enhance the efficiency of in vivo tumor destruction by cooperative functions of CD8+ and CD4+ T cells
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