Neoantigen-reactive T cells exhibit effective anti-tumor activity against colorectal cancer

Abstract

Neoantigens play a crucial role in cancer immunotherapy. However, the effectiveness and safety of neoantigen-based immunotherapies in patients with colorectal cancer (CRC), particularly in the Chinese population, have not been well studied. This study explored the feasibility and effectiveness of neoantigens in the treatment of CRC. Whole-exome sequencing (WES) and transcriptome sequencing were used to identify somatic mutations, RNA expression, and human leukocyte antigen (HLA) alleles. Neoantigen candidates were predicted, and immunogenicity was assessed. The neoantigens TSHZ3-L523P, RARA-R83H, TP53-R248W, EYA2-V333I, and NRAS-G12D from Patient 4 (PW4); TASP1-P161L, RAP1GAP-S215R, MOSPD1-V63I, and NAV2-D1973N from Patient 10 (PW10); and HAVCR2-F39V, SEC11A-R11L, SMPDL3B-T452M, LRFN3-R118Q, and ULK1-S248L from Patient 11 (HLA-A0201⁺PW11) induced a heightened neoantigen-reactive T cell (NRT) response as compared with the controls in peripheral blood lymphocytes (PBLs) isolated from patients with CRC. In addition, we identified neoantigen-containing peptides SEC11A-R11L and ULK1-S248L from HLA-A0201⁺PW11, which more effectively elicited specific CTL responses than the corresponding native peptides in PBLs isolated from HLA-A0201⁺PW11 as well as in HLA-A2.1/K^b transgenic mice. Importantly, adoptive transfer of NRTs induced by vaccination with two mutant peptides could effectively inhibit tumor growth in tumor-bearing mouse models. These data indicate that neoantigen-containing peptides with high immunogenicity represent promising candidates for peptide-mediated personalized therapy.Abbreviations: CRC: colorectal cancer; DCs: dendritic cells; ELISPOT: enzyme-linked immunosorbent spot; E:T: effector:target; HLA: human leukocyte antigen; MHC: major histocompatibility complex; Mut: mutant type; NGS: next-generation sequencing; NRTs: neoantigen-reactive T cells; PBMCs: peripheral blood mononuclear cells; STR: short tandem repeat; PBLs: peripheral blood lymphocytes; PBS: phosphate-buffered saline; PD-1: programmed cell death protein 1; TILs: tumor-infiltrating lymphocytes; RNA-seq: RNA sequencing; Tg: transgenic; TMGs: tandem minigenes; WES: whole-exome sequencing; WT: wild-type.

Keywords: Colorectal cancer; cancer vaccine; immunotherapy; neoantigens; tumor.

Figure 1.

Number of somatic mutations and predicted neoantigens in 10 patients with CRC (a) WES and RNA-seq were performed in 10 patients with CRC. Tumor-specific non-synonymous somatic mutations were identified. The number of somatic mutations of each patient is shown. (b) Neoantigens were predicted for each patient. The number of neoantigens as well as strong (%rank <0.5) and weak binders (0.5< %rank <2) of each patient is shown.

Figure 2.

Immunogenicity testing of neoantigens from patients with CRC. Autologous PBMCs were stimulated with candidate mutated peptides every 3 days in the presence of IL-2. On day 10, T-cell responses to each antigen were measured by an IFN-γ ELISPOT assay. The PBMCs in a, b, and c were obtained from PW4, PW10, and PW11 with CRC, respectively. No peptide (medium alone) or VSV-NP_43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) stimulation was used as a control. Data are presented as mean ± S.D. of three independent experiments. **p< .01 and *p< .05 compared with IFN-γ production by PBMCs stimulated without peptide or with VSV-NP_43-69. NRTs, neoantigen-reactive T cells; SFC, spot-forming cell; VSV-NP, nucleoprotein of vesicular stomatitis virus; S.D., standard deviation.

Figure 3.

Cytotoxicity of NRTs raised by in vitro stimulation of PBLs of HLA-A0201⁺PW11. NRTs were induced with autologous HAVCR2-F39V, SEC11A-R11L, or ULK1-S248L-pulsed DCs derived from the PBLs of PW11. On day 7, after the third stimulation, the NRTs were harvested for analysis. (a) IFN-γ secretion by neoantigen-reactive T-cell lines in response to mutated and WT peptides. WT-HAVCR2-F39V, WT-SEC11A-R11L, and WT-ULK1-S248L represent wild type peptides, and HAVCR2-F39V, SEC11A-R11L, and ULK1-S248L represent mutant peptides. IFN-γ-positive SFCs/10⁵ NRTs were detected by cytokine-specific ELISPOT (b, c, and d). Cytotoxicity measured by a CCK8 kit at the specified E:T ratio. Peptide-specific targets were mutated protein-pulsed T2 cells and minimally nucleated SW480 cells (HLA-A2.1⁺, minigene expression), whereas VSV-NP_43-69-pulsed T2 cells, T2 cells alone, and SW480 cells alone were used as controls. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. **p< .01. NRTs, neoantigen-reactive T cells; WT, wild-type; E:T, effector: target; PBL, peripheral blood lymphocyte; SFC, spot-forming cells; SEM, standard error of the mean.

Figure 4.

SEC11A-R11L and ULK1-S248L induce more efficient NRT responses than WT epitopes in HLA-A2.1/K^b-Tg mice. (a) Splenocytes of mice (n= 5) vaccinated with mutated peptides were tested by ELISPOT for the recognition of mutated peptides compared to the recognition of the corresponding WT sequences. Data are presented as mean ± standard deviation (SD) of three independent experiments. **p< .01 compared with IFN-γ production by splenocytes stimulated without peptide or with VSV-NP_43-69 (b, c, and d). Splenocytes from HLA-A2.1/K^b-Tg mice immunized with mutated peptides were restimulated in vitro with the same mutated peptide for 7 days. Ex vivo cytotoxicity against corresponding mutated peptide-pulsed T2 cells and minigene-nucleofected SW480 cells were examined by CCK8 kit assays at the indicated E:T ratio. VSV-NP_43-69-pulsed T2 cells, T2 cells alone, and SW480 cells alone were used as controls. Data are presented as the mean ± S.E.M. of three independent experiments. **p< .01. E:T, effector:target; SFC, spot-forming cell.

Figure 5.

Adoptive immunotherapy of minigene-nucleofected SW480 tumor-bearing nude mice. Small gene-nuclear transfected SW480 tumor cells (5 × 10⁶ cells/mouse) were injected into the flanks of C57BL/6^nu/nu mice, and 3 days later, HLA-matched immunization with SEC11A-R11L and ULK1-S248L was performed. Splenocytes (1 × 10⁸ cells/mouse) from HLA-A2.1/K^b-Tg mice were prepared as described in the materials and methods. The control group received only IL-2 or did not receive any treatment. (a) Tumor growth was observed every 3 days and recorded as the mean tumor size (mm²). (b) Mouse survival after tumor inoculation was monitored (n= 10 mice per group).