Cancer Immune-Interception for Lynch Syndrome

摘要 Lynch综合征（LS）是遗传性结直肠癌（CRC）的最常见原因，影响超过100万人 美国人LS是由DNA错配修复（MMR）基因中的种系突变引起的。正常大肠 LS患者的上皮细胞在获得“第二次”体细胞命中后变得MMR缺陷， 携带种系突变的同一MMR基因的等位基因，从而引发数百个 到微卫星序列中数以千计的碱基错配和插入缺失突变（indels）。 这些突变产生移码肽（FSP），其成为新抗原（neoAg）并刺激免疫应答。 适应性免疫系统我们已经报道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患者构成了一个定义明确且 有可能受益于免疫拦截策略以预防CRC的流行人群。我们 已经从LS结直肠癌前病变和肿瘤中获得了大量的基因组数据， 以确定这些病变中最常见的复发性neoAg。此外，我们一直在研究 化学预防策略，可以增强免疫反应，并观察到增加的激活， 暴露于非甾体抗炎药萘普生后，结肠直肠粘膜中的常驻免疫细胞 非甾体类抗炎药（NSAID），来自我们在LS患者中开展的NCI申办的Ib期临床的生物标志物分析。此外，委员会认为， 我们在人源化LS小鼠模型中进行了一项共同临床试验，观察到肽疫苗接种 与neoAg联合使用可高度有效预防LS CRC，其联合使用可进一步增强其活性 与纳波利塔诺，从而奠定了基础，这项赠款建议。这一提议的核心假设是 萘普生是一种免疫调节剂，可以激活结肠直肠粘膜中的常驻免疫细胞， 将增加NeoAg的识别和引发肿瘤细胞杀伤的驻留T细胞的活化。探索这个 假设，我们提出了三个具体目标：1。为了表征免疫细胞类型， 使用单细胞基因组学在LS患者中给予化学预防性那普利生和阿司匹林， 随机II期临床试验中的成像质谱细胞术; 2.评估候选疫苗的免疫原性 共享neoAg鉴定LS患者癌前病变和肿瘤，用于使用四聚体的个性化免疫预防 在ELISpot、四聚体染色和共培养的患者来源的细胞的细胞毒性测定中结合到磁珠 类器官和自体CD 8 + T细胞; 3.分析新抗原特异性CD 8 + T细胞的T细胞受体（TCR）， 用于追踪LS患者中的肿瘤免疫原性的细胞克隆。这项研究将对 通过开发肽疫苗接种和NSAID的组合用于遗传性免疫拦截， 癌症首次。该提案是高度创新的结合使用成像的化学预防试验 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，并使用四聚体结合 涉及免疫学实验中克隆阳性选择的磁珠。

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.