Cancer Immune-Interception in a Spontaneous Non-Human Primate Model of 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是由四个DNA错配修复（MMR）基因之一的种系突变引起的。正常 LS患者的结直肠上皮细胞在体细胞“第二次”击中后成为MMR缺陷， 在微卫星序列中模拟数百个插入-缺失突变（indels）。这些插入缺失产生 移码肽（FSP），其成为新抗原（neoAg）并刺激适应性免疫系统。我们 以前曾报道过适应性免疫基因在LS癌前病变中高度表达， 生成了一个详细的neoAg目录，其中> 1，000 FSP neoAg来自LS癌前和早期队列 CRC使用下一代测序工具，结合最先进的生物信息学管道。此外，本发明还提供了一种方法， 我们已经发表了一项在LS患者中进行的Ib期化学预防临床试验的结果， 结直肠粘膜驻留细胞的免疫活化。总之，这些结果强烈指向 开发一种“复发性”和“共享性”LS相关肿瘤neoAg疫苗， LS人群的泛癌症预防。然而，主要的知识差距仍然是选择最佳的， 并在可靠的动物模型中建立疫苗接种的有效性和安全性， 允许即时人工翻译。恒河猴是一种很有前途的非人灵长类动物（NHP）模型， 与人类的基因组最为相似我们的研究小组报告了第一批海绵- 携带MLH 1生殖系突变的恒河猴的皮肤LS。此外，我们还与行业合作伙伴-- AMAL Therapeutics的演讲者开发了一个名为KISIMA的创新疫苗接种平台， 几种选择的FSP与促进细胞渗透的细胞渗透性肽和Toll样受体串联 作为自身佐剂的激动剂。我们的核心假设是我们最先进的生物信息学管道 对于neoAg预测将导致识别最具免疫原性的，跨肿瘤复发的，和共享的 在LS相关肿瘤类型中，FSP neoAg将被整合到KISIMA自佐剂疫苗平台中， 其与萘普生组合将产生强免疫原性。为了探索这一假设，我们建议 三个具体目标：1.在体外验证LS共有的前150种复发性neoAg的免疫原性 使用来自健康人的PBMC和CD 8 + T细胞，使用ELISpot、ELISA和细胞因子测定， 人类供体; 2.开发表达人LS neoAg的人工抗原呈递细胞（aAPC），以验证 neoAg富集的T细胞的细胞毒性;和3.为了评估neoAg组合的免疫原性，使用 在一项联合临床试验中， LS恒河猴。该提案具有高度创新性，因为它正在开发一种新的自佐剂疫苗平台， LS的独特自发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 开发第一阶段人体临床试验的垫脚石，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.