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

微卫星不稳定性阳性结直肠癌（MSI+CRC）是由DNA错配修复功能障碍引起的，具有独特的特点，包括t细胞浸润预后良好，提示免疫系统对移码突变产生的肿瘤特异性肽有应答。在本研究中，我们试图鉴定参与MSI+CRC的肿瘤抗原。我们首先应用SEREX技术鉴定MSI+CRC患者中诱导IgG的肿瘤抗原，然后通过检测不同癌症患者和健康人血清中的IgG来评价抗原的免疫原性。共鉴定出150种抗原，其中IgG仅在MSI+CRC患者中检测到75种抗原。在antrCDX2抗体患者的肿瘤组织中发现分离的CDX2抗原重复序列发生移码突变。利用重组码移CDX2蛋白，发现该抗体能够识别由码移突变引起的c端独特肽。该抗体在治愈性切除7年后消失，提示免疫反应可能作为肿瘤标志物有用。虽然尝试在体外诱导针对突变CDX2的特异性T细胞，但可能由于使用的是来自7年无病患者的淋巴细胞，因此未能诱导特异性T细胞。我们还评估了从已知的MSI靶基因中是否检测到针对可能的移码肽的IgG。然而，没有检测到任何重组肽的IgG。总之，我们首次证明MSI+CRC患者可以诱导针对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。在进一步的研究中，应使用自体肿瘤进行SEREX，并使用患者的t细胞分析对移码肽的免疫反应。

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.