A comparative analysis of human and canine iNKT cells for ACT

项目概述（见说明）：

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