Next Generation Engineered NK Cells for Lymphoma Patients after CD19 CAR-T Cell Failure.

总结： CD 19定向CAR-T细胞改变了B细胞淋巴恶性肿瘤的治疗格局。 然而，尽管初始完全缓解率较高，但复发发生在治疗的第一年内， 约50%的患者接受市售自体CAR 19 T细胞。复发可能是 分为两种模式：与CAR T细胞耗竭和衰老相关的CD 19阳性复发，或与CAR T细胞耗竭和衰老相关的CD 19阳性复发。 阴性复发与靶抗原丢失相关。CAR 19 T细胞治疗后复发的患者， 因此，迫切需要开发下一代CAR工程化的免疫效应子。 靶向肿瘤的细胞具有功效和最小的毒性。人们对自然杀伤（NK）细胞越来越感兴趣 作为CAR治疗的候选者，因为它们可以通过其杀死肿瘤细胞的先天能力来防止抗原逃逸 并且它们是安全的并且非常适合于在同种异体治疗环境中使用。在一项首次人体研究中， 显示了脐带血（CB）衍生的CAR-NK细胞靶向患者CD 19的安全性和有前景的活性 B淋巴恶性肿瘤（Liu et al NEJM 2020）。这项建议旨在建立在这个平台上， 通过最佳共刺激增强NK细胞效力和持久性， 信号传导、细胞因子装甲和检查点抑制。我们已经确定了CD 70作为一种新的治疗靶点， 在CAR 19 T细胞衰竭后的B-NHL患者中，开发了一种新的策略，通过基因治疗靶向CD 70， 用逆转录病毒载体（iC 9-CD 27-DAP 10-CD 3 β-IL-15）修饰CB-NK细胞，所述逆转录病毒载体（iC 9-CD 27-DAP 10-CD 3 β-IL-15）掺入（i）用于以下的基因： 截短的人CD 27（CD 70的天然受体）以重定向它们的特异性;（ii）DAP 10作为NK特异性受体， （iii）IL-15以支持它们的存活和增殖， 和（iv）作为自杀基因的诱导型胱天蛋白酶-9（iC 9）。我们的初步数据显示， 方法在体外和体内，并支持其翻译到临床。此外，我们还开发了一个强大的 冷冻保存CAR-NK细胞的策略，允许生成现成的工程化NK细胞生物库 电池，从而降低成本并增加可及性。最后，我们设计了一种新的策略， 免疫检查点CIS在我们的CAR-NK细胞中调节它们的代谢适应性和效力。我们假设 使用iC 9/CAR 27 D10 NK/IL-15 NK细胞靶向CD 70将大大改善NHL患者的结局， CAR 19 T细胞失败，并且通过靶向免疫检查点CIS，我们可以进一步增强CAR-NK细胞的功能。 代谢健康和潜能。这些概念将在三个具体目标中进行评估： 一项I/II期临床试验，测试iC 9/CAR 27 D10 ζ/IL-15 NK细胞在CD 70+患者中的安全性和有效性 CAR 19 T细胞治疗失败的NHL患者（FDA批准，IND #27757）。在目标2中，我们将应用创新 单细胞蛋白质组学和转录组学研究，以全面表征CAR-NK细胞的命运 并确定功效和抗性的关键机制。在目标3中，我们将进行机理研究， 阐明CIS缺失如何增强CAR-NK细胞的代谢适应性，并将进行IND使能研究 为测试CIS缺陷型iC 9/CAR 27 D10 NK/IL-15 NK细胞的下一代临床研究做准备。

Summary: CD19 directed CAR-T cells have transformed the treatment landscape of B-cell lymphoid malignancies. However, despite high initial complete remission rates, relapses occur within the first year of therapy in approximately 50% of patients who receive commercially available autologous CAR19 T-cells. Relapses can be classified into two patterns: CD19-positive relapse related to CAR T-cell exhaustion and senescence, or CD19- negative relapse related to target antigen loss. Patients who relapse after CAR19 T-cell therapy have poor prognosis; hence, there is an urgent need to develop the next-generation of CAR engineered immune effector cells that target tumors with efficacy and with minimal toxicity. There is growing interest in natural killer (NK) cells as a candidate for CAR therapy as they may prevent antigen escape through their innate ability to kill tumor cells and they are safe and well-suited for use in the allogeneic therapy setting. In a first-in-human study, our group showed the safety and promising activity of cord blood (CB) derived CAR-NK cells targeting CD19 in patients with B-lymphoid malignancies (Liu et al NEJM 2020). This proposal aims to build on this platform to develop the next-generation NK cell therapies by enhancing NK cell potency and persistence through optimal costimulatory signaling, cytokine armoring and checkpoint inhibition. We have identified CD70, as a novel therapeutic target in patients with B-NHL after CAR19 T-cell failure and developed a novel strategy to target CD70 by genetically modifying CB-NK cells with a retroviral vector (iC9-CD27-DAP10-CD3ζ-IL-15) that incorporates (i) the gene for a truncated human CD27 (the natural receptor for CD70) to redirect their specificity; (ii) DAP10 as an NK-specific costimulatory domain linked to a CD3ζ signaling endodomain; (iii) IL-15 to support their survival and proliferation, and (iv) inducible caspase-9 (iC9) as a suicide gene. Our preliminary data show the efficacy and safety of this approach in vitro and in vivo and support its translation to the clinic. In addition, we have developed a robust strategy to cryopreserve CAR-NK cells, allowing for the generation of a biobank of off-the-shelf engineered NK cells, thus reducing cost and increasing accessibility. Finally, we have devised a novel strategy to target the immune checkpoint CIS in our CAR-NK cells to modulate their metabolic fitness and potency. We hypothesize that targeting CD70 with iC9/CAR27D10ζ/IL-15 NK cells will greatly improve outcomes in patients with NHL after CAR19 T-cell failure and that by targeting the immune checkpoint CIS we can further enhance the CAR-NK cells’ metabolic fitness and potency. These concepts will be evaluated in three specific aims: In Aim 1 we will conduct a Phase I/II clinical trial to test the safety and efficacy of iC9/CAR27D10ζ/IL-15 NK cells in patients with CD70+ NHL who have failed CAR19 T-cell therapy (FDA approved, IND #27757). In Aim 2 we will apply innovative single-cell proteomic and transcriptomic studies to comprehensively characterize the fate of the CAR-NK cells and to identify key mechanisms of efficacy and resistance. In Aim 3, we will perform mechanistic studies to elucidate how CIS deletion enhances the metabolic fitness of CAR-NK cells and will perform IND enabling studies in preparation for the next-generation clinical studies testing CIS deficient iC9/CAR27D10ζ/IL-15 NK cells.