Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions

Abstract

Purpose: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear.

Experimental design: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors.

Results: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation.

Conclusions: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis.

Figure 1

New non-mutated T-cell antigens are identified by cDNA library screening. (A) COS-7 cells were transfected with HLA cDNA constructs, together with full-length SERPINE2 transcription variant 1 (TV1), variant 2 (TV2) or clone 12E1 isolated from Mel 2359 cDNA library. These transfected cells were co-cultured with TIL 2359 T cells overnight. (B) COS-7 cells were transfected with HLA cDNA constructs, together with full-length WT DUSP12 or its transcription variant (DUSP12 TV). These transfected cells were co-cultured overnight with TIL 2359 T cells, and the release of IFN-γ was determined by ELISA. (C) COS-7 cells were transfected with HLA cDNA constructs, together with full-length WT SLC24A5 or its transcription variant (SLC24A5 TV). These transfected cells were co-cultured with TIL 2591 T cells overnight. (D) The copy numbers of SLC24A5 WT or SLC24A5 TV in normal melanocyte samples or melanoma lines were determined by quantitative PCR. (E) Healthy donor T cells were transduced with SLC24A5 TCR isolated from TIL 2591. These T cells were co-cultured with COS-7 cells transfected with HLA cDNA constructs, together with SLC24A5 WT or SLC24A5 TV. (F) SLC24A5 TCR transduced T cells were co-cultured overnight with various melanoma cells lines. The secretion of IFN-γ was determined by ELISA. The types of HLA-C are indicated below.

Figure 2

TIL 2359 T cells recognize mutated KIF2C gene product. (A) An example of tandem minigene construct, which encoded polypeptides containing 6 identified mutated amino acid residues flanked on their N- and C- termini, 12 amino acids on both sides. These tandem minigenes were synthesized and cloned into pcDNA3.1 vector. (B) A screening assay was carried out to co-transfect HLA-A*0205 with individual tandem minigene construct into COS-7 cells. TIL 2359 T cells were co-cultured with these transfected COS-7 cells overnight, and the release of IFN-γ from TIL 2359 was evaluated by ELISA. The structure of the tandem minigene construct RJ-1 has been shown in (A). (C) Each point mutation site at the minigenes of RJ-1 construct was converted back to WT sequence individually, indicated on the table. TIL 2359 were co-cultured with COS-7 cells transfected with individual RJ-1 variant and HLA-A*0205. The secretion of IFN-γ by TIL 2359 was determined by ELISA. (D) COS-7 cells were transfected with WT or mutated KIF2C cDNA construct, together with HLA cDNA constructs. These transfected cells were co-cultured with TIL 2359 T cells overnight. (E) HEK293 cells stably expressed HLA-A*0205 were pulsed with WT or mutated KIF2C peptides, followed by co-cultured with TIL 2359 T cells overnight. The release of IFN-γ was determined by ELISA.

Figure 3

Mutated POLA2 gene product is recognized by TIL 2591 T cells. (A) Individual tandem minigene construct was transfected in HEK293 cells stably expressed HLA-C*0701. TIL 2591 T cells were co-cultured with these transfected cells overnight, and the release of IFN-γ was determined by ELISA. (B) The structure of tandem minigene construct DW-6. (C) Each point mutation site at the minigenes of DW-6 construct was converted back to WT sequence individually, indicated on the table. TIL 2591 T cells were co-cultured with COS-7 cells transfected with individual DW-6 variant and HLA-C*0701. The secretion of IFN-γ by TIL 2591 was determined by ELISA. (D) COS-7 cells were transfected with WT or mutated POLA2 cDNA construct, together with HLA cDNA constructs. These transfected cells were co-cultured with TIL 2359 T cells overnight. (E) HEK293 cells stably expressed HLA-C*0701 were pulsed with WT or mutated POLA2 peptides, followed by co-cultured with TIL 2591 T cells overnight. The release of IFN-γ was determined by ELISA.

Figure 4

IFN-γ ELISPOT responses of TILs. (A) IFN-γ spots per 10⁵ cells of TIL 2359. TIL2359 T cells were co-cultured with autologous Mel 2359 or HLA-A*0205-positive HEK293 cells pulsed with peptides. Additionally, TIL 2591 T cells were cultured with autologous Mel 2591 in the ELISPOT assay. TIL 2359 T cells stimulated with the polyclonal activator PMA plus ionomycin generated 59,385 spots per 10⁵ T cells in this assay. (B) IFN-γ spots per 10⁵ cells of TIL 2591 T cells. These T cells were co-cultured HLA-A*02-positive or HLA-C*07-positive HEK293 cells pulsed with peptides. In addition, TIL 2591 T cells were cultured with autologous Mel 2591 in the ELISPOT assay. TIL 2591 T cells stimulated with the polyclonal activator PMA plus ionomycin generated 60,162 spots per 10⁵ T cells in this assay.