Next-Generation Engineered NK Cell Immunotherapy for Ovarian Cancer

项目2摘要/摘要 卵巢癌是第二常见的妇科恶性肿瘤，仍然是导致 美国妇科癌症死亡人数。因此，对新的治疗方案的迫切需求尚未得到满足。 嵌合抗原受体(CAR)T细胞疗法导致了一些血液病癌症的范式转变，但 实体瘤的疗效仍然有限，部分原因是缺乏高度特异的靶点和免疫抑制。 在肿瘤微环境(TME)中。此外，制造自体细胞的时间和成本很高 产品，以及与CAR T细胞疗法相关的毒性挑战要求具有普遍性的新产品， 安全的，有效的。越来越多的人对使用自然杀伤(NK)细胞进行汽车工程感兴趣，因为它们 一种天生的杀死肿瘤细胞的能力，在同种异体环境中是安全的。在一项首次人类研究中，我们小组 显示针对CD19的脐带血来源的CAR-NK细胞在B细胞白血病患者中的安全性和有效性。 淋巴样恶性肿瘤。这项提议旨在以此为平台开发下一代NK细胞 通过最佳共刺激增强NK细胞的活性和持久性治疗卵巢癌 信号、细胞因子装甲和检查点抑制。我们已经确定TROP2是一种很有前途的治疗方法 靶向铂耐药卵巢癌，并开发了一种通过基因靶向TROP2的新策略 用含有(I)人源化RS7单链基因的逆转录病毒载体修饰CB-NK细胞 靶向TROP2的可变片段；(Ii)作为NK特异性共刺激结构域的DAP10；(Iii)支持IL-15 可诱导的caspase-9(IC9)作为安全开关(iC9/TROP2CAR/IL-15)。我们的 初步数据显示，这种方法在体外和体内的有效性和安全性，并支持其翻译为 诊所。此外，我们已经开发了一种强大的策略来冷冻保存CAR-NK细胞，允许 建立一个现成的工程化NK细胞库，可以在床边解冻和输注，从而 降低成本，提高可及性。最后，我们设计了一种新的策略来瞄准免疫 代谢检查点CREM，以调节酸性TME中CAR-NK细胞的代谢适合性和能力。 我们假设靶向TROP2的iC9/TROP2CAR/IL-15 NK细胞将极大地改善患者的预后 通过靶向代谢免疫检查点CREM，我们可以进一步 增强NK细胞的适合性和效力。我们将在三个具体目标中测试我们的假设：在目标1中，我们将 进行一项I/II期临床试验，以测试iC9/TROP2CAR/IL-2腹膜腔内注射的安全性和有效性 TROP2+铂耐药卵巢癌患者的15个NK细胞(2022-0687方案)。在《目标2》中我们将 应用创新的单细胞蛋白质组学和转录组学研究来全面表征 过继转移的CAR-NK细胞及其与TME的相互作用和疗效的关键机制 和抵抗。在目标3中，我们将进行机制研究，以阐明CREM缺失如何增强 CAR-NK细胞的代谢适合性和IND前研究，为下一代临床研究做准备。

Project 2 SUMMARY/ ABSTRACT Ovarian cancer is the second most common gynecologic malignancy and remains the leading cause of gynecologic cancer deaths in the US. Therefore, there is a critical unmet need for new treatment options. Chimeric antigen receptor (CAR) T-cell therapy has led to a paradigm shift in some hematologic cancers, but efficacy in solid tumors remains limited, partly due to the lack of highly specific targets and immunosuppression in the tumor microenvironment (TME). Moreover, the time and high cost of manufacturing autologous cell products, and the toxicity challenges related to CAR T-cell therapy call for novel products that are universal, safe, and potent. There is growing interest in using natural killer (NK) cells for CAR engineering since they have an innate ability to kill tumor cells and they are safe in the allogeneic setting. In a first-in-human study, our group showed the safety and efficacy of cord blood (CB)-derived CAR-NK cells targeting CD19 in patients with B- lymphoid malignancies. This proposal aims to build on this platform to develop the next-generation NK cell therapies for ovarian cancer by enhancing NK cell potency and persistence through optimal co-stimulatory signaling, cytokine armoring and checkpoint inhibition. We have identified TROP2 as a promising therapeutic target in platinum-resistant ovarian cancer and developed a novel strategy to target TROP2 by genetically modifying CB-NK cells with a retroviral vector that incorporates the genes for (i) the humanized RS7 single chain variable fragment targeting TROP2; (ii) DAP10 as an NK-specific co-stimulatory domain; (iii) IL-15 to support their survival and proliferation; and (iv) inducible caspase-9 (iC9) as a safety switch (iC9/TROP2CAR/IL-15). 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 that could be thawed and infused at bedside, thus reducing cost and increasing accessibility. Finally, we have devised a novel strategy to target the immune metabolic checkpoint CREM to modulate the metabolic fitness and potency of CAR-NK cells in the acidic TME. We hypothesize that targeting TROP2 with iC9/TROP2CAR/IL-15 NK cells will greatly improve outcomes for platinum-resistant ovarian cancer and that by targeting the metabolic immune checkpoint CREM we can further enhance the fitness and potency of NK cells. We will test our hypothesis in three specific aims: In Aim 1 we will conduct a Phase I/II clinical trial to test the safety and efficacy of intraperitoneally delivered iC9/TROP2CAR/IL- 15 NK cells in patients with TROP2+ platinum-resistant ovarian cancer (Protocol 2022-0687). In Aim 2 we will apply innovative single-cell proteomic and transcriptomic studies to comprehensively characterize the fate of the adoptively transferred CAR-NK cells, their interaction with the peritoneal TME, and key mechanisms of efficacy and resistance. In Aim 3 we will perform mechanistic studies to elucidate how CREM deletion enhances the metabolic fitness of CAR-NK cells and pre-IND studies in preparation for the next-generation clinical studies.