Development of diagnositic and therapeutic methods using frameshift mutated peptides for colorectal cancers with microsatellite instability

由 DNA 错配修复故障引起的微卫星不稳定性阳性结直肠癌 (MSI CRC) 具有独特的特征，包括 T 细胞浸润的良好预后，表明针对移码突变产生的肿瘤特异性肽的免疫反应。在这项研究中，我们试图鉴定与 MSI CRC 相关的肿瘤抗原。我们首先应用 SEREX 来鉴定在 MSI CRC 患者中诱导 IgG 的肿瘤抗原，然后通过检测各种癌症患者和健康个体的血清中的 IgG 来评估抗原的免疫原性。鉴定出 150 种抗原，其中包括 75 种仅在 MSI CRC 患者中检测到的 IgG 抗原。在使用antrCDX2抗体的患者的肿瘤组织中发现了分离的CDX2抗原中重复序列的移码突变。使用重组移码 CDX2 蛋白，发现该抗体能够识别由移码突变引起的 C 端独特肽。这种抗体在治愈性切除后 7 年消失，表明免疫反应可能可用作肿瘤标志物。尽管尝试体外诱导突变CDX2特异性T细胞，但没有诱导出特异性T细胞，可能是因为使用了来自7年无病患者的淋巴细胞。我们还评估了是否针对已知 MSI 目标基因中可能的移码肽检测到 IgG。然而，没有检测到针对任何测试的重组肽的 IgG。总之，我们首次证明在 MSI CRC 患者中可以诱导针对 MSI 引起的移码肽的肿瘤特异性免疫反应。这些肿瘤特异性突变肽可能有助于开发 MSI CRC 患者的诊断和治疗方法。使用自体肿瘤的 SEREX 以及使用患者 T 细胞对移码肽的免疫反应的分析应该在进一步的研究中进行。

Microsatellite instability positive colorectal cancers (MSI+CRC) caused by malfunction of DNA mismatch repair has unique characteristics including good prognosis with T-cell infiltrates, suggesting immune responses against tumor specific peptides generated by frameshift mutations. In this study, we have attempted to identify tumor antigens involved in MSI+CRC. We first applied SEREX to identify tumor antigens that induced IgG in MSI+CRC patients, then, immunogenicity of the antigens was evaluated by detecting IgG among sera from various cancer patients and healthy individuals. 150 antigens including 75 antigens for that IgG were detected only in MSI+CRC patients, were identified. The frameshift mutation in the repetitive sequence in the isolated CDX2 antigen was found in the tumor tissue of the patient with antrCDX2 antibody. Using recombinant frameshift CDX2 proteins, the antibody was found to recognize the C-terminal unique peptide caused by the frameshift mutation. This antibody was disappeared 7 years after the curative resection, suggesting that the immune responses may be useful as tumor markers. Although in vitro induction of T-cells specific for the mutated CDX2 was attempted, specific T cells were not induced possibly because of the use of lymphocytes from the 7 year disease free patient. We have also evaluated whether IgG were detected against possible frameshift peptides from the known MSI target genes. However, no IgG was detected against any recombinant peptides tested. In summary, we demonstrated for the first time that tumor specific immune response against the frameshift peptides caused by MSI could be induced in MSI+CRC patients. These tumor specific mutated peptides may be useful for the development of diagnostic and therapeutic methods for patients with MSI+ CRC. SEREX using autologous tumors and analysis of immune responses against the frameshift peptides using patient's T-cells should be performed in the further study.