小鼠结肠癌新抗原Glud1-V546I及其DC疫苗能够在体内和体外诱导有效的抗肿瘤免疫应答

Mouse colon cancer neoantigen Glud1-V546I and its DC vaccine can induce potent anti-tumor immune responses in vivo and in vitro

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<0.0001)。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<0.0001)和细胞毒作用(P<0.0001)。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)-γlevel 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-γsecretion ability by effector cells was determined by ELISPOT assay,and the cytotoxicity ofγsecretion 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-γsecretion by NRT cells(P<0.0001).Compared with the wild peptide(Glud1-WT),Glud1-V546I induced higher IFN-γsecretion by NRT cells(P<0.0001)and stronger cytotoxicity(P<0.0001)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 neoantigen-based personalized immunotherapies in CRC.