Project 4 Treatment of Advanced Ovarian Cancer Using Gene-Edited NK CAR Cells

摘要--项目4 被诊断为晚期上皮性卵巢癌(EOC)的女性的中位5年存活率约为20%。 而复发性平机会基本上仍是无法治愈的。新的免疫疗法，包括免疫检查点 阻断和嵌合抗原受体(CAR)T细胞疗法已经取得了一些成功，但应答率 通常都在20%以下。同样，对分子靶向治疗的应答率也是 有限，部分原因是缺乏反复突变的靶点和高度的染色体不稳定 具有EoC的特点。通过抑制PARP和阻断血管内皮细胞生长因子的合成致命性已经证明了一些 但这些新疗法的耐药机制已经浮出水面。的长期目标是 我们的研究计划是开发高效的基于免疫细胞的疗法，能够消除 经常性的平等机会委员会。在项目4中，我们将使用我们开发的高级基因编辑技术来制作 功能亢进的NK细胞治疗晚期卵巢癌我们专注于NK细胞，因为i)它们确实如此 不需要抗原启动和ii)副作用，如移植物抗宿主病和细胞因子释放 综合征，与T细胞疗法相比微乎其微。我们建议对人类进行两项根本性的改变 NK细胞取自健康供者。第一个基因改变移除了细胞内的信号蛋白 使用我们开发的一种改进的CRISPR/Cas9方案来抑制NK激活(CISH基因) 对原代人类NK细胞非常有效。第二个基因改变是定点整合 我们开发的针对NK细胞的嵌合抗原受体(CAR)基因。这辆车的目标是NK 间皮蛋白，一种流行的卵巢癌肿瘤抗原。我们的中心假设是，基因编辑可以 用于克服信号抑制，提高NK细胞的靶向性、持久性和功能，从而导致 改善晚期卵巢癌妇女的治疗结果。本项目拟开展的工作包括 通过与卵巢癌细胞共培养产生和测试增强功能的基因编辑的NK细胞 在体外，并在细胞系和患者来源的异种移植模型中进行体内试验。来自这些的数据 临床前试验将用于指导使用这些经过基因编辑的NK细胞的I期临床试验的设计。 该项目将确定基因编辑的CAR NK细胞免疫疗法在人类中是否安全，并具有 在建立经常性的平等机会委员会方面，有可能改善结果。此外，我们从这项研究中学到的将是 对未来应用于其他恶性肿瘤的潜在影响有更广泛的影响，如工程技术 能迅速适应去除不同的负性调节因子，并与NK-1靶向其他肿瘤抗原。 特定的汽车。

ABSTRACT – PROJECT 4 Women diagnosed with advanced epithelial ovarian cancer (EOC) have a median 5-year survival rate of ~20%, and recurrent EOC remains essentially incurable. Novel immunotherapies, including immune checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies have had some success, but response rates have been typically below 20%. Similarly, the response rates to molecularly targeted therapies have been limited, partly due to the lack of recurrently mutated targets and the high level of chromosomal instability characteristic of EOC. Synthetic lethality via PARP inhibition and VEGF blockade have demonstrated some benefit, but mechanisms of resistance have already surfaced for these new therapies. The long-term goal of our research program is to develop highly effective immune cell-based therapies capable of eliminating recurrent EOC. In Project 4, we will use advanced gene editing techniques that we have developed to produce hyperfunctional Natural Killer (NK) cells for treating advanced EOC. We focus on NK cells because i) they do not require antigen priming and ii) side effects, such as graft versus host disease and cytokine release syndrome, are minimal compared to T cell therapy. We propose to make two fundamental alterations to human NK cells obtained from healthy donors. The first genetic alteration removes an intracellular signaling protein that dampens NK activation (the CISH gene) using a modified CRISPR/Cas9 protocol we have developed that is highly effective in primary human NK cells. The second genetic alteration is to site-specifically integrate a chimeric antigen receptor (CAR) gene that we have developed specifically for NK cells. This CAR NK targets the mesothelin protein, a prevalent EOC tumor antigen. Our central hypothesis is that gene editing can be used to overcome signaling inhibition and improve NK cell targeting, persistence and function, resulting in improved therapeutic outcomes for women with advanced EOC. The work proposed in this project includes generating and testing the gene edited NK cells for enhanced function by co-culturing with ovarian cancer cells in vitro and testing in both cell line- and patient-derived xenograft models of EOC in vivo. The data from these preclinical tests will be used to guide the design of the Phase I clinical trial using these gene-edited NK cells. This project will determine whether gene-edited CAR NK cell immunotherapy in humans is safe and has the potential to improve outcomes in the setting of recurrent EOC. In addition, what we learn from this study will have the broader impact of potential future application to other malignancies, as the engineering techniques can be quickly adapted to remove different negative regulators and to target other tumor antigens with NK- specific CARs.