Project 4: Off-the-shelf engineered cord blood-derived natural killer cells for the treatment acute lymphoblastic leukemia

摘要： 在过去的几年中，免疫效应细胞的工程化已经取得了巨大的进展， 治疗癌症的方法然而，嵌合抗原受体（CAR）修饰的T细胞具有许多限制。 为每个个体患者生成自体产物在逻辑上是繁琐和限制性的 用于广泛的临床应用。CAR-T细胞的制造通常需要几周时间，这使得它不切实际。 对于病情快速发展的患者。此外，并不总是能够产生临床相关的 剂量的CAR-T细胞，这些细胞来自大量预先治疗的，通常是淋巴细胞减少的患者。以前收集的同种异体 产品可以克服这些局限性;然而，同种异体T细胞（即使HLA匹配）携带 通过其天然αβ T细胞受体抑制介导的移植物抗宿主病（GVHD）的显著风险 它们作为临床产品的用途，而无需进一步操作以消除T细胞受体。自然杀伤细胞（NK） 细胞为CAR工程化提供了T细胞的极具吸引力的替代方案。NK细胞不会引起GVHD 并因此提供了生产用于立即临床使用的现成产品的机会。而且随着 工程NK细胞还应该保留其完整的天然受体阵列，它们具有发挥作用的潜力。 通过除了CAR特异性所决定的机制之外的机制，其原则上可以 降低由CAR靶向抗原丢失介导的复发风险，如CAR-T细胞治疗所报告的。 自体NK细胞可以在体外可重复地产生，但对肿瘤细胞的活性极其有限。 自体肿瘤不能通过CAR工程克服。脐带血（CB）是一种现成的 同种异体NK细胞的来源具有明显的优势。CB是一种现成的冷冻产品， 这一优势已经得到了从NK细胞中产生大量高功能NK细胞的方法的支持。 离体冷冻CB单位。从大规模储存的冷冻CB单位产生CAR转导的NK细胞 全球CB银行库存有望实现广泛的可扩展性，这是个人无法复制的 需要筛查和白细胞去除术的成年捐献者。 在目标1中，我们将进行第一次非人临床试验，以测试CB-NK细胞工程的安全性和有效性。 表达针对CD 19（一种B细胞特异性抗原）的CAR，异位产生IL-15以支持其体内 增殖和持久性，并表达基于IC 9的自杀基因，这将解决安全性问题 与直接毒性的潜在风险相关;在目标2中，我们将应用高度创新的相关研究， 描述临床试验的治疗潜力;目的3在临床前小鼠研究中，我们将保护 使用新型逆转录病毒构建体从TGF-β/SMAD信号传导轴转导NK细胞，除了 CD 19和IL-15，包括人TGFβ受体II的显性负性形式（TGFβ-1）的基因。 DNRII）用于下一代临床研究。

Summary: The past several years have seen tremendous advances in the engineering of immune effector cells as therapy for cancer. However, chimeric antigen receptor (CAR)-modified T-cells have a number of limitations. The generation of an autologous product for each individual patient is logistically cumbersome and restrictive for widespread clinical use. The manufacturing of CAR T-cells often takes several weeks, making it impractical for patients with rapidly advancing disease. Furthermore, it is not always possible to generate clinically relevant doses of CAR T-cells from heavily pre-treated, often lymphopenic patients. A previously collected allogeneic product could overcome these limitations; however, allogeneic T-cells (even if HLA-matched) carry a significant risk of graft-versus-host disease (GVHD) mediated through their native αβ T-cell receptor prohibiting their use as a clinical product without further manipulation to eliminate the T cell receptor. Natural killer (NK) cells provide an extremely attractive alternative to T-cells for CAR engineering. NK cells do not cause GVHD and thus open opportunities to produce an off-the-shelf product for immediate clinical use. Moreover, as engineered NK cells should also retain their full array of native receptors, they have the potential to exert cytotoxicity through mechanisms other than that dictated by the specificity of the CAR, which in principle could reduce the risk of relapse mediated by loss of CAR-targeted antigen, as reported for CAR-T cell therapy. Autologous NK cells can be reproducibly generated in vitro, but have extremely limited activity against autologous tumor which cannot be overcome by CAR engineering. Cord blood (CB) is a readily available source of allogeneic NK cells with clear advantages. CB is available as an off-the-shelf frozen product, an advantage that has been bolstered by methods to generate large numbers of highly functional NK cells from frozen CB units ex vivo. The generation of CAR-transduced NK cells from frozen CB units stored in large global CB bank inventories holds promise for widespread scalability that cannot be replicated with individual adult donors who require screening and leukapheresis. In Aim 1 we will perform the first-inhuman clinical trial to test the safety and efficacy of CB-NK cells engineered to express a CAR against CD19 (a B cell-specific antigen), to ectopically produce IL-15 to support their in vivo proliferation and persistence, and to express a suicide gene, based on IC9, that will address safety concerns related to the potential risk of direct toxicity; In Aim 2 we will apply highly innovative correlative studies to describe the therapeutic potential of the clinical trial; Aim 3 in preclinical murine studies, we will protect the transduced NK cells from the TGF-β/SMAD signaling axis using a novel retroviral construct that, in addition to CAR.CD19 and IL-15, includes the gene for the dominant-negative version of human TGFβ receptor II (TGFβ- DNRII) for next-generation clinical studies.