A comparative analysis of human and canine iNKT cells for ACT

项目总结（见说明）： 过继性细胞疗法（ACT）的圣杯是从以下公司生产适合的、即用型的CART产品： 健康的供体，可以安全有效地跨越同种异体屏障转移，以改善 CAR疗法的可用性和有效性。恒定自然杀伤T细胞（iNKT）是一种异质性群体 "非常规“T细胞表达一个不变的TCRα，它识别非多态性的糖脂- 呈递MHC分子CD 1d。因此，未编辑的同种异体iNKT细胞（allo-iNKT）可以被过继性地移植。 不会引起GVHD。此外，iNKT细胞抑制由常规T细胞引起的GVHD， 抑制同种异体细胞的排斥，促进实体器官移植物的终身耐受。此外，iNKT细胞具有 天然肿瘤向性，杀死CD 1d+肿瘤相关巨噬细胞并激活肿瘤中的效应免疫细胞。 鉴于这些特性，我们假设allo-iNKT可以安全有效地用于促进allo-CART 持久性和增强抗肿瘤免疫力。 iNKT功能的复杂性可能归因于不同的iNKT亚群，其表现出细胞毒性与细胞毒性。 监管属性。了解亚群异质性将使最“理想的”iNKT亚群能够 选择用于ACT组合的评价。与小鼠不同，犬iNKT细胞具有相似的表型和 人类iNKT细胞的生物学特征，这表明患有自发性癌症的宠物狗可能代表了 理想的临床前模型来研究使用iNKT细胞的组合allo-ACT。在这里，与目标一致， PRECINCT支持犬免疫治疗临床试验，生产免疫试剂， IOTN的兴趣，以产生一个现成的CART细胞平台，我们将进行比较的方法， 通过整合的单细胞RNAseq/CITEseq方法研究可操作的iNKT细胞异质性， 人iNKT和使用迁移学习从scRNA估算犬iNKT亚群表型标记 seq数据。此外，为了扩大犬NK研究的免疫试剂工具箱， 噬菌体展示文库将用于简单的淘选实验，以产生和验证急需的犬- 针对NK活化和抑制性受体的特异性抗体，这将有助于理解 控制犬NK激活和抑制的机制。总之，这些研究将奠定必要的 为将来产生和体内测试最佳iNKT细胞产物以用于组合同种异体移植奠定基础。 iNKT/CART治疗免疫功能正常的犬癌症患者，目的是加速新型allo-ACT 进入人类诊所。 相关性（参见说明）： iNKT细胞的天然特性有助于安全的同种异体过继转移和抑制 同种异体反应的反应，否则会消除allo-CART产品。在这里，我们将提供 对人和犬iNKT细胞进行必要的比较评价，这将使未来的体内试验成为可能。 用于免疫功能正常患者的allo-iNKT/CART联合治疗的最佳、预防相关的iNKT产品 犬癌症患者，旨在加速新型allo-ACT策略进入人类临床

PROJECT SUMMARY (See instructions): The holy grail of adoptive cell therapies (ACT) is the generation of fit, ready-to-use, CART products from healthy donors that could be safely and effectively transferred across allogeneic barriers to improve the availability and efficacy of CAR-therapies. Invariant Natural Killer T cells (iNKT) are a heterogenous population of `non-conventional' T cells expressing an invariant TCRα which recognizes the non-polymorphic, glycolipid- presenting MHC molecule CD1d. As such, unedited allogeneic iNKT cells (allo-iNKT) can be adoptively transferred without causing GVHD. Further, iNKT cells suppress GVHD caused by conventional T cells and inhibit rejection of allogeneic cells promoting lifelong tolerance of solid organ grafts. In addition, iNKT cells have natural tumor tropism, kill CD1d+ tumor associated macrophages and activate effector immune cells in the tumor. Given these properties, we hypothesize that allo-iNKT could be safely and effectively used to promote allo-CART persistence and enhance anti-tumor immunity. The complexity of iNKT function may be attributed to distinct iNKT subsets that exhibit cytotoxic versus regulatory properties. Understanding subset heterogeneity would enable the most “desirable” iNKT subsets to be selected for evaluation of combination ACT. Unlike mice, canine iNKT cells share similar phenotypic and biological features to human iNKT cells, suggesting that pet dogs with spontaneous cancer may represent the ideal pre-clinical model to investigate combination allo-ACT using iNKT cells. Here, in alignment with the aims of PRECINCT to support canine immunotherapy clinical trials and generate immunological reagents and with the interest of IOTN to generate an off-the-shelf CART cell platform, we will perform a comparative approach to investigate actionable iNKT cell heterogeneity through an integrated, single cell RNAseq/CITEseq approach in human iNKT and employ transfer learning to impute phenotypic markers of canine iNKT subsets from scRNA seq data. Furthermore, to expand the immunoreagent toolbox for canine NK studies, a comprehensive scFv phage display library will be used in simple panning experiments to generate and validate much needed canine- specific antibodies against NK activation and inhibitory receptors that will be of value in understanding the mechanisms governing canine NK activation and inhibition. Together, these studies will lay the necessary groundwork for future generation and in vivo testing of optimal iNKT cell products for combination allo- iNKT/CART therapies in immune competent canine cancer patients, with the goal to accelerate novel allo-ACT strategies into the human clinic. RELEVANCE (See instructions): The natural properties of iNKT cells lend themselves to safe allogeneic adoptive transfer and suppression of alloreactive responses that would otherwise eliminate allo-CART products. Here, we will provide the necessary comparative evaluation of human and canine iNKT cells that will enable future in vivo testing of optimal, translationally relevant iNKT products for combination allo-iNKT/CART therapies in immune competent canine cancer patients, aiming to accelerate novel allo-ACT strategies into the human clinic