Cancer Immune-Interception in a Spontaneous Non-Human Primate Model of Lynch Syndrome

摘要 林奇综合征(LS)是影响100万人的遗传性结直肠癌(CRC)的最常见原因 美国人。LS是由四个DNA错配修复(MMR)基因之一的胚系突变引起的。正常 LS患者的结肠上皮细胞在第二次体细胞撞击后变得MMR缺陷，从而产生Acc- 模拟微卫星序列中数百个插入-缺失突变(INDELs)。这些Indels产生了 移码多肽(FSP)，成为新的抗原(新抗原)，刺激适应性免疫系统。我们 之前曾报道过获得性免疫基因在LS癌前病变中高度表达，我们已经 用&gt；1,000 FSP新抗原生成了详细的新抗原目录，这些新抗原来自一组LS癌前和早期患者 使用下一代测序工具的CRC与最先进的生物信息学管道相结合。此外, 我们已经公布了使用萘普生对LS患者进行Ib期化学预防临床试验的结果，显示 大肠粘膜驻留细胞的免疫激活。综上所述，这些结果强烈地指向 复发性和共享性LS相关肿瘤新抗原联合萘普生疫苗的研制 LS人群中的泛癌预防。然而，主要的知识差距仍然是选择最合适的- 并在可靠的动物模型中建立疫苗的有效性和安全性 允许立即进行人工翻译。猕猴是一种很有前途的非人类灵长类(NHP)模式动物。 扮演着与人类最接近的基因组相似性。我们的研究小组报告了第一个鱼群- 在MLH1中携带胚系突变的恒河猴的自发性LS。此外，我们还与行业合作伙伴合作- Amal Treeutics的演讲者开发了一个名为Kisima的创新疫苗接种平台，将 几种选择的FSP与促进细胞穿透的细胞通透性多肽和Toll样受体结合 作为一种自我佐剂的激动剂。我们的中心假设是我们最先进的生物信息学管道 对于新抗原的预测将导致确定最具免疫原性的、跨肿瘤复发和共享的 在将整合到Kisima自佐剂疫苗平台中的LS相关肿瘤类型FSP新抗原中， 与萘普生联合使用具有很强的免疫原性。为了探索这一假设，我们提出了 三个特定目的：1.体外验证LS共有的前150个复发肿瘤抗原的免疫原性 用ELISpot、ELISAT和细胞因子检测非结直肠肿瘤患者的PBMC和CD8+T细胞 建立表达人LS新抗原的人工抗原提呈细胞(AAPC) 富含新抗原的T细胞的细胞毒性；以及3.评估新抗原组合的免疫原性 新型自佐剂疫苗平台Kisima单独以及与萘普生联合进行的联合临床试验 都是恒河猴。这一提议具有很高的创新性，因为它正在开发一种新的自佐剂疫苗平台 LS独特的自发NHP模型。拟议的研究将对该领域产生重大影响，因为它是一项 开发第一阶段人类临床试验的垫脚石，以测试用于LS患者的新型CRC疫苗。

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 one of four DNA mismatch repair (MMR) genes. Normal colorectal epithelial cells in LS patients become MMR deficient after a somatic ‘second’ hit generating the accu- mulation of hundreds of insertion-deletion mutations (indels) in microsatellite sequences. These indels generate frameshift peptides (FSP) that become neoantigens (neoAg) and stimulate the adaptive immune system. We have previously reported that adaptive immune genes are highly expressed in LS pre-cancers, and we have generated a detailed neoAg catalog with >1,000 FSP neoAg from a cohort of LS pre-cancers and early-stage CRCs using next-generation sequencing tools coupled with a state-of-the-art bioinformatics pipeline. In addition, we have published the results of a phase Ib chemoprevention clinical trial in LS patients using naproxen showing immune-activation of colorectal mucosa resident cells. Taken together, these results point strongly towards the development of a vaccine for ‘recurrent’ and ‘shared’ LS-associated tumor neoAg combined with naproxen for pan-cancer prevention in the LS population. However, the main knowledge gap remains to select the most opti- mal neoAg peptides and to establish the efficacy and safety of the vaccination in a reliable animal model that allows immediate human translation. Rhesus macaques are a promising non-human primate (NHP) model dis- playing the closest genomic resemblance to humans. Our research team has reported the first colony of spon- taneous LS in rhesus carrying a germline mutation in MLH1. In addition, we are partnering with industry collab- orators in AMAL Therapeutics that have developed an innovative vaccination platform called KISIMA, integrating several selected FSP in tandem with a cell-permeable peptide fostering cell penetration, and a toll-like receptor agonist that acts as a self-adjuvant. Our central hypothesis is that our state-of-the-art bioinformatics pipeline for neoAg prediction will lead to the identification of the most immunogenic, recurrent across tumors, and shared among LS-associated tumor types FSP neoAg to be integrated in the KISIMA self-adjuvant vaccine platform, which will render a strong immunogenicity in combination with naproxen. To explore this hypothesis, we propose three specific aims: 1. To validate in vitro the immunogenicity of the top 150 recurrent neoAg shared by LS non-colorectal tumors using ELISpot, ELISA, and cytokine assays using PBMCs and CD8+ T cells from healthy human donors; 2. To develop artificial antigen-presenting cells (aAPC) expressing human LS neoAg to validate the cytotoxicity of neoAg-enriched T cells; and 3. To assess the immunogenicity of a neoAg combination using the novel self-adjuvant vaccine platform KISIMA alone and in combination with naproxen in a co-clinical trial in LS rhesus. The proposal is highly innovative because is developing a novel self-adjuavnt vaccine platform in a unique spontaneous NHP model of LS. The proposed research will significantly impact the field because it is a stepping stone to develop a Phase I first-in-human clinical trial to test a novel CRC vaccine for LS patients.