目的：开发针对结直肠癌（CRC）个性化治疗的新抗原肽疫苗，探讨新抗原肽及其诱导的新抗原反应性T（NRT）细胞 治疗CRC的可行性和有效性。方法：提取小鼠结肠癌CT26细胞的DNA和RNA，采用全外显子和转录组测序分析肿瘤基因的 突变及表达。通过基于机器学习的新抗原预测体系，筛选、合成具有高免疫原性多肽。用合成的多肽经皮下注射免疫小鼠，通过 流式细胞术检测免疫鼠脾细胞的IFN-γ分泌水平，筛选具有强免疫原性多肽。用免疫原性多肽负载小鼠骨髓来源的树突状细胞 （BMDC）免疫结肠癌建模小鼠，通过ELISPOT检测效应细胞分泌IFN-γ的能力，时间分辨荧光免疫分析法检测免疫鼠脾细胞对 相应靶细胞的杀伤力，观察荷瘤小鼠肿瘤生长情况和小鼠存活期。结果：新抗原肽Glud1-V546I具有更强的诱导NRT细胞分泌 IFN-γ的能力（P < 0.000 1）。与野生肽（Glud1-WT）相比，Glud1-V546I 在荷瘤鼠体内诱导的 NRT 细胞有更高的IFN-γ分泌能 力（P ）和细胞毒作用（P < 0.000 1）。同时，Glud1-V546I能明显抑制小鼠肿瘤生长（P < 0.001）并延长荷瘤鼠的生存期 （P < 0.01）。结论：小鼠CT26细胞的新抗原肽Glud1-V546I能够显著促进小鼠NRT细胞的IFN-γ的分泌，用其制备的DC疫苗在 结肠癌荷瘤鼠体内显示出有效的抗肿瘤反应，提示开发基于新抗原的CRC个性化免疫治疗是可能的。

Objective: To develop a neoantigen peptide vaccine for personalized treatment of colorectal cancer (CRC), and to explore the feasibility and effectiveness of neoantigen peptide and its induced neoantigen reactive T cells (NRT) therapy for CRC. Methods: DNA and RNA were extracted from mouse CRC cell line CT26, followed by whole-exome and transcriptome sequencing to analyze tumor gene mutations and expression. Peptides with high immunogenicity were screened and synthesized through a machine learning based neoantigen prediction system. Mice were subcutaneously immunized with synthesized peptides, and the interferon (IFN)-γ level of splenocytes from immunized mice was determined using flow cytometry to screen peptides with strong immunogenicity. Afterwards, bone marrow-derived dendritic cells (BMDCs) loaded with immunogenic peptides were used to immunize mice bearing CRC model. The IFN-γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of γ secretion ability by effector cells was determined by ELISPOT assay, and the cytotoxicity of splenocytes from immunized mice was examined by time-resolved fluorescence immunoassay. In addition, the tumor growth and survival period of tumor-bearing mice were observed. Results: The neoantigen Glud1-V546I induced stronger IFN-γ secretion by NRT cells (P < 0.000 1). Compared with the wild peptide (Glud1-WT), Glud1-V546I induced higher IFN-γ secretion by NRT cells (P < 0.000 1) and stronger cytotoxicity (P < 0.000 1) in tumor-bearing mice. Meanwhile, Glud1-V546I significantly inhibited tumor growth (P < 0.001) and prolonged the survival of tumor-bearing mice (P < 0.01). Conclusion: The neoantigen peptide Glud1-V546I from mouse CT26 cells demonstrates effective anti-tumor responses in tumor-bearing mice, suggesting the potential for developing neoantigenbased personalized immunotherapies in CRC.

国家自然科学基金青年基金项目（No. 82072927）；青岛市2022年度医药卫生科研指导项目（No. 2022-WJZD065）