Next-Generation Engineered NK Cell Immunotherapy for Ovarian Cancer

项目2概要/摘要 卵巢癌是第二常见的妇科恶性肿瘤，并且仍然是导致卵巢癌的主要原因。 美国妇科癌症死亡人数。因此，对于新的治疗选择存在着关键的未满足的需求。 嵌合抗原受体（CAR）T细胞疗法已导致一些血液癌症的范式转变，但 对实体瘤的疗效仍然有限，部分原因是缺乏高度特异性的靶点和免疫抑制。 肿瘤微环境（TME）此外，制造自体细胞的时间和高成本 产品，以及与CAR T细胞疗法相关的毒性挑战需要通用的新产品， 安全有效人们对使用自然杀伤（NK）细胞进行CAR工程化越来越感兴趣，因为它们具有 具有杀死肿瘤细胞的先天能力，并且在同种异体环境中是安全的。在一项首次人体研究中， 显示了靶向CD 19的脐带血（CB）衍生CAR-NK细胞在B- 淋巴恶性肿瘤该提案旨在建立在这个平台上开发下一代NK细胞 通过最佳共刺激增强NK细胞的效力和持久性来治疗卵巢癌 信号传导、细胞因子装甲和检查点抑制。我们已经确定TROP 2是一种有前途的治疗药物， 靶向铂耐药卵巢癌，并开发了一种新的策略，靶向TROP 2的基因， 用逆转录病毒载体修饰CB-NK细胞，所述逆转录病毒载体掺入（i）人源化RS7单链 靶向TROP 2的可变片段;（ii）作为NK特异性共刺激结构域的DAP 10;（iii）IL-15，以支持 它们的存活和增殖;和（iv）诱导型半胱天冬酶-9（iC 9）作为安全开关（iC 9/TROP 2CAR/IL-15）。我们 初步数据显示了这种方法在体外和体内的有效性和安全性，并支持其转化为 诊所此外，我们已经开发了一种稳健的策略来冷冻保存CAR-NK细胞， 生成现成的工程NK细胞生物库，可以在床边解冻和输注，因此 降低成本并增加可访问性。最后，我们设计了一种新的策略， 代谢检查点CREM来调节CAR-NK细胞在酸性TME中的代谢适应性和效力。 我们假设用iC 9/TROP 2CAR/IL-15 NK细胞靶向TROP 2将极大地改善治疗的结果。 铂耐药卵巢癌，通过靶向代谢免疫检查点CREM，我们可以进一步 增强NK细胞的适应性和效力。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I/II期临床试验，以测试腹膜内递送的iC 9/TROP 2CAR/IL-1的安全性和有效性。 15 TROP 2+铂耐药卵巢癌患者的NK细胞（方案2022-0687）。在目标2中， 应用创新的单细胞蛋白质组学和转录组学研究，全面表征 过继转移的CAR-NK细胞，它们与腹膜TME的相互作用，以及关键的疗效机制 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细胞的代谢适应性和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.