Cancer Immune-Interception for Lynch Syndrome

摘要 林奇综合征(LS)是遗传性结直肠癌(CRC)最常见的原因，影响100万人 美国人。LS是由DNA错配修复(MMR)基因的胚系突变引起的。正常结直肠 LS患者的上皮细胞在获得第二次体细胞打击后变得MMR缺陷 含有生殖系突变的同一个MMR基因的等位基因，从而引发数百个 与微卫星序列中数千个碱基错配和插入-缺失突变(INDELs)有关。 这些突变会产生移码多肽(FSP)，这些FSP会变成新抗原(New Ag)，并刺激 适应性免疫系统。我们已经报道了LS癌前病变是免疫激活的，并且显著地存在 适应性免疫基因的高水平表达。因此，LS患者构成了一个明确的和 有可能从免疫拦截策略中受益以预防结直肠癌的流行人群。我们 已经从LS结直肠癌前期和肿瘤中获得了大量的基因组数据，以进行编目和 以确定在这些皮损中出现的最常见的复发肿瘤抗原。此外，我们一直在研究 化学预防策略，可以增强免疫反应，并观察到增加激活 非类固醇抗炎药--萘普生对大肠黏膜中常驻免疫细胞的影响 药物(NSAID)，来自我们对NCI赞助的LS患者Ib期临床的生物标记物分析。此外， 我们在人源化LS小鼠模型上进行了一项联合临床试验，观察到多肽疫苗接种 用新抗原预防LS-CRC非常有效，其联合应用可进一步增强其活性 使用萘普生，从而为这项赠款提案奠定了基础。这项提议的中心假设是 萘普生是一种免疫调节剂，可以激活大肠粘膜中的常驻免疫细胞，这些细胞 会增加对NeoAg的识别和激活驻留的T细胞，从而引发肿瘤细胞的杀伤。要探索这一点 假设，我们提出了三个具体的目标：1.表征受调控的免疫细胞类型 应用单细胞基因组学方法对LS患者进行化学预防的纳普生和阿司匹林 随机II期临床试验中的成像质量细胞术；2.评估候选细胞的免疫原性 共享的新抗原识别LS癌前患者和肿瘤，用于使用四聚体进行个性化免疫预防 与磁珠结合的ELISpots、四聚体染色和共培养患者来源的细胞毒性检测 器官和自体CD8+T细胞；3.新抗原特异性CD8+T细胞的T细胞受体(TCR) 用于追踪LS患者肿瘤免疫原性的细胞克隆。拟议的研究将显著影响 开发多肽疫苗和非甾体抗炎药的组合用于遗传性免疫拦截 第一次患上癌症。该方案结合了一项使用成像技术的化学预防试验，具有很高的创新性 使用四聚体结合的质量细胞术、单细胞基因组学和系统生物学来评估试验终点 在免疫学实验中对克隆进行阳性选择的磁珠。

ABSTRACT Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC), affecting >1 million Americans. LS is caused by germline mutations in the DNA mismatch repair (MMR) genes. Normal colorectal epithelial cells in LS patients become MMR deficient upon acquisition of a ‘second’ somatic hit in the alternative allele of the same MMR gene that harbors the germline mutation, thus triggering the accumulation of hundreds to thousands of base-to-base mismatches and insertion-deletion mutations (indels) in microsatellite sequences. These mutations generate frameshift peptides (FSP) that become neoantigens (neoAg) and stimulate the adaptive immune system. We have reported that LS pre-cancers are immune activated and present strikingly high levels of expression of adaptive immune genes. Therefore, LS patients constitute a well-defined and prevalent population that has the potential to benefit from immune-interception strategies to prevent CRC. We have acquired a substantial amount of genomic data from LS colorectal pre-cancers and tumors to catalog and to identify the most frequent recurrent neoAg present in these lesions. In addition, we have been studying chemopreventive strategies that could augment the immune response and observed increased activation of the resident immune cells in the colorectal mucosa upon exposure to naproxen, a non-steroidal anti-inflammatory drug (NSAID), from our biomarker analysis of our NCI-sponsored Phase Ib clinical in LS patients. Furthermore, we have performed a co-clinical trial in a humanized LS mouse model that has observed that peptide vaccination with neoAg is highly effective in preventing LS CRC with the activity that is further enhanced by its combination with naproxen, thus laying the foundations for this grant proposal. The central hypothesis of this proposal is that naproxen is an immune-modulator that activates resident immune cells in the colorectal mucosa, and these will increase the recognition of NeoAg and activation of resident T-cells eliciting tumor cell killing. To explore this hypothesis, we propose three specific aims: 1. To characterize the immune cell types that are regulated after the administration of chemopreventive naproxen and aspirin in LS patients using single-cell genomics and imaging mass cytometry within a randomized phase II clinical trial; 2. To assess the immunogenicity of candidate shared neoAg identified LS patients pre-cancers and tumors for personalized immunoprevention using tetramer bound to magnetic beads in ELISpots, Tetramer stain, and cytotoxicity assays of co-cultured patient-derived organoids and autologous CD8+ T cells; 3. To profile the T cell Receptor (TCR) of neoantigen-specific CD8+ T cell clones for tracking tumor immunogenicity in LS patients. The proposed research will significantly impact the field by developing a combination of a peptide vaccination and an NSAID for immune-interception in hereditary cancers for the first time. The proposal is highly innovative by combining a chemoprevention trial using imaging mass cytometry, single-cell genomics, and systems biology to assess trial endpoints, and using tetramers bound to magnetic beads for positive selections of clones in immunology experiments.