Off-The-Shelf Dual Targeted NK Cells For NHL

限制复发是自体移植治疗非霍奇金淋巴瘤后最大的挑战。 抗原导向的CAR T细胞产品和其他靶向疗法可能正在改变这一领域，但对高危人群来说 淋巴瘤，需要新的、更安全的方法。我们将研究抗原导向的， 现成的诱导多能干细胞(IPSC)来源的NK细胞被设计用于双重抗原靶向。我们 优化了IPSC来源的CD34+细胞向高活性NK细胞的分化，在 明尼苏达大学，现在可以用克隆的IPSC在一次生产中生产数百剂 以高亲和力、不可切割的CD16(称为hnCD16)转导。2019年底，我们启动了临床试验 测试这一经过设计的IPSC产品(指定为FT516)。这个项目的首要假设是 使用CAR和通过hnCD16双靶向NK细胞将防止自体移植后复发 淋巴瘤和未来改变新陈代谢的基因编辑将加强过继转移。这一假设符合 本方案和项目2的主题将向本方案介绍现成NK细胞的开发情况， NK Car和膜IL-15的作用，以及临床前是否会操控代谢 加强NK细胞治疗。这些调查得到以下具体目标的支持。目标1将测试DUAL 使用ADCC和NK CAR两种作用机制将NK细胞靶向CD19和CD20，以防止 淋巴瘤自体移植后复发。我们将测试三重基因修饰的IPSC来源的NK细胞 用NK细胞优化的CD19 CAR hnCD16和膜结合15/IL-15ra转导产物(FT596) 融合。FT596将联合利妥昔单抗作为维持治疗，以限制自体红细胞移植后的复发。 这项试验是FDA批准的，将首先在植入后+30天建立MTD，然后，如果单剂 是安全的，移至植入前+7天，目标是在最初100天内接种3剂，以防止复发。 目的2检测ARID5B过表达和CD38基因敲除对NK细胞代谢适应性的增强作用。我们的 初步数据显示，适应性NK细胞和CD38基因敲除的IPSC来源的NK细胞都能抵抗氧化应激- 诱导细胞死亡。ARID5B的过度表达在适应性NK细胞中自然增加，也介导了类似的效应。 我们将产生新的IPSC系，以FT596为基础，转基因表达ARID5B或CRISPR基因敲除 CD38的表达。将对这些克隆进行评估，以探索它们如何推动NK细胞代谢和 增强功能。AIM 3将测试操纵ARID5B和CD38是否会推动新陈代谢适应和 增强FT596在体内的持久性和功能。这些临床前试验将为未来的临床试验提供信息 修饰-ALL通过延长转移的NK细胞持久性和促进增强抗淋巴瘤活性 威力。这些代谢和效力的发现将直接为项目1提供信息，以改善细胞靶向和 新陈代谢，关于细胞持久性的项目3，以及定义自主优势的所有3个项目 IL-15的呈递。

Limiting relapse is the biggest challenge following autologous transplantation for non-Hodgkin’s lymphoma. Antigen-directed CAR T cell products and other targeted therapies may be changing the field, yet for high-risk lymphomas, new and safer approaches are needed. We will study the safety and efficacy of antigen-directed, off-the-shelf, induced pluripotent stem cell (iPSC)-derived NK cells engineered for dual-antigen targeting. We have optimized the differentiation of iPSC-derived CD34+ cells to highly potent NK cells, scaled production at the University of Minnesota, and can now produce hundreds of doses in a manufacturing run with a clonal iPSC transduced with a high-affinity, non-cleavable CD16 (called hnCD16). In late 2019, we initiated clinical trials testing this engineered iPSC product (designated FT516). The overarching hypothesis for this project is that dual targeting of NK cells with a CAR and through hnCD16 will protect against relapse after auto-transplant for lymphoma and that future gene edits to alter metabolism will enhance adoptive transfer. This hypothesis fits with the themes of this Program, and Project 2 will inform the Program on the development of off-the-shelf NK cells, NK CAR, and the role of membrane IL-15, as well as pre-clinically on whether manipulating metabolism will enhance NK cell therapy. These investigations are supported by the following Specific Aims. Aim 1 will test dual targeting of NK cells to CD19 and CD20 using two mechanisms of action, ADCC and an NK CAR, to prevent relapse after autologous transplantation for lymphoma. We will test a triple gene-modified iPSC-derived NK cell product (FT596) transduced with hnCD16, an NK cell-optimized CD19 CAR, and membrane bound 15/IL-15Ra fusions. FT596 will be combined with rituximab as maintenance therapy to limit relapse after autologous HCT. This trial is FDA-approved and will first establish an MTD at day +30 after engraftment and then, if single dosing is safe, move to day +7 before engraftment with the goal of 3 doses in the first hundred days to prevent relapse. Aim 2 will test enhancement of NK cell metabolic fitness by ARID5B overexpression and CD38 knockout. Our preliminary data shows both adaptive NK cells and CD38 knockout iPSC-derived NK cells resist oxidative stress- induced cell death. ARID5B overexpression, naturally increased in adaptive NK cells, mediates similar effects. We will generate new iPSC lines to build on FT596 with transgenic expression of ARID5B or a CRISPR knockout of CD38. These clones will be evaluated to probe the mechanisms on how they drive NK cell metabolism and augment function. Aim 3 will test whether manipulating ARID5B and CD38 will drive metabolic fitness and enhance FT596 persistence and function in vivo. These pre-clinical trials will inform future clinical trial modifications—all to promote enhanced anti-lymphoma activity by extending transferred NK cell persistence and potency. These metabolic and potency findings will directly inform Project 1 to improve cell targeting and metabolism, Project 3 for cell persistence, and all 3 Projects to define the advantages of autonomous presentation of IL-15.