TCR-T Cell Recognition of an NY-ESO-1 Epitope Presented by HLA-A2 Supertype: Implications for Cancer Immunotherapy

Abstract

Background: T-cell receptor (TCR)-engineered T-cell therapy (TCR-T) has become a promising anticancer therapy. Recognition of tumor cells by TCR-T cells requires matched human leukocyte antigen (HLA) alleles and tumor antigens, which seriously limits their population coverage. One strategy to expand the population coverage of a specific TCR-T cell therapy is to enable TCR-T cells to recognize target peptides presented by more HLA alleles.

Methods: In this study, HLA alleles were selected based on the Chinese population frequency and HLA supertype classification. Then, COS-7 and two tumor cell lines (586 mel and 5637) were transduced with selected HLA alleles for functional evaluation of TCR-T cells. HLA-A2 alleles capable of both exogenously and endogenously presenting the NY-ESO-1-derived epitope and thereby being recognized by TCR-T cells were tested.

Results: We demonstrated that a given TCR-T cell product can recognize the NY-ESO-1 peptide exogenously and endogenously presented not only by HLA-A*02:01 but also by HLA-A*02:03, HLA-A*02:06, and HLA-A*02:10, almost doubling the population coverage in the Chinese population from 12.01% to 21.05%.

Conclusions: Our study suggests that cancer patients expressing members of the HLA-A2 supertype may benefit from the TCR-T cell product, and other TCR-T cell products could similarly expand their population coverage even within the non-Chinese population through an analogous approach.

Keywords: HLA-A2 alleles; HLA-A2 supertype; NY-ESO-1; TCR-T; cancer immunotherapy.

Figure 1

Transfection efficiency of transfectants. FACS and RT-PCR were performed to analyze HLA-A2 expression in COS-7-A2 cells (a) and 586 mel-A2 cells (b). Among all the selected HLA-A2 alleles, the expression of all except HLA-A*02:10 and HLA-A*02:53N were detectable by FACS. Specifically, HLA-A*02:10 and HLA-A*02:53N could not be detected using the FACS antibody; however, RT-PCR results confirmed the successful introduction and expression of these two alleles in the corresponding transfected cells.

Figure 2

TCR-T cells restricted by HLA-A*02:01 demonstrated the ability to recognize the target antigen peptide presented by members of the HLA-A2 supertype in COS-7 cells. IFN-γ secretion results of incubating TCR-T cells with COS-7-A2 cells loaded with different peptides (a) or COS-7-NY-A2 cells (b). The experiment was performed in triplicate wells, and data are representative of three independent experiments (n = 3). Data are represented as mean ± SD. ** p < 0.01, and **** p < 0.0001.

Figure 3

Functional avidity of target antigen peptide presented by members of the HLA-A2 supertype. TCR-T cells were co-cultured with T2 cells (a) and COS-7 cells expressing one of 5 active HLA-A2 alleles (A*02:01, A*02:03, A*02:05, A*02:06, and A*02:10) (b–f) loaded with antigen peptide (SLLMWITQC) at different concentrations. IFN-γ secretion was detected by ELISA, and EC50 was calculated using nonlinear regression analysis, specifically a four-parameter logistic model, to fit the concentration–response curves to evaluate functional avidity. The experiment was performed in triplicate wells, and data are representative of four independent experiments (n = 4). Data are represented as mean ± SD.

Figure 4

HLA-A*02:01-restricted TCR-T cells could recognize target antigen peptide presented by members of the HLA-A2 supertype in 586 mel cells. (a) IFN-γ secretion results of incubating TCR-T cells and control T cells with 586 mel-A2 cells, 586 mel WT cells, and 624 mel cells. The experiment was performed in triplicate wells, and data are representative of four independent experiments (n = 4). Data are represented as mean ± SD. The difference between TCR-T and Control-T cells was calculated by using multiple t-tests—one per row with GraphPad Prism 6.01 software. (b,c) Detection of activation markers on T cells by FACS. Data are representative of two independent experiments (n = 2). (d) Correlation between IFN-γ secretion and 4-1BB upregulation. The statistical analysis was performed using the Pearson r test of correlation with GraphPad Prism 6.01 software, and p < 0.05 was considered to indicate a statistically significant difference.