Genome Engineered Natural Killer Cell Immunotherapy against Human Osteosarcoma

摘要 在过去的十年中，基于嵌合抗原受体的T细胞治疗(CAR-T)已经发展成为一种有效的 针对某些癌症的免疫疗法。然而，CAR-T细胞疗法有几个缺点和临床 成功主要局限于血液病癌症。CAR-T细胞治疗面临的挑战包括肿瘤 肿瘤细胞靶抗原表达缺失和CAR-T细胞功能抑制导致的免疫逃逸 由肿瘤表达的抑制分子。自然杀伤(NK)细胞是T细胞的替代品，可能是 更有效，因为他们有能力进行抗原依赖和独立的杀戮。NK细胞有 当被设计为表达T细胞CARS和NK细胞时，证明了抗原特异性杀伤作用 直接杀伤MHC表达减少或缺失的转化细胞。事实上，NK细胞携带抗体 依赖细胞介导的细胞毒性(ACDD)，通过NK细胞CD16A受体结合抗体的细胞。到期 由于杀伤癌细胞的方式多种多样，人们对NK细胞用于癌症的兴趣也越来越大 免疫疗法。由于NK细胞与移植物抗宿主疾病、神经毒性、长期 自身免疫，也不是细胞因子释放综合征，它们比T细胞更适合用于异基因环境 并具有作为现成产品使用的巨大临床潜力。然而，以前的出版物和 临床试验表明，使用未经处理的NK细胞治疗癌症的效果微乎其微， 可能是由于有限的植入，很少的体内扩张，以及肿瘤微环境的抑制。NK 表达膜结合白介素21(mBIL-21)的饲养细胞激活和扩增的细胞 在高危髓系恶性肿瘤和几种实体瘤的临床前研究中显示了有希望的结果 模特们。因此，我们假设，经过基因编辑的激活/扩增的NK细胞可以是 用于成功治疗骨肉瘤，这种疾病患者的预后在30多年内没有改善 好几年了。我们提出的目标是评估静息和激活的NK细胞对 各种骨肉瘤细胞系，敲除NK细胞功能的负调控因子(特别是c-CBL，IL-1R8， 和SMAD3)，并实施一种特定的CAR，该CAR最佳地激活NK细胞抗原特异性杀伤。从基因上讲 工程NK细胞将在骨肉瘤模型中进行增强治疗效果和安全性的评估。 我们的初步数据有力地支持了以NK细胞为基础的癌症免疫治疗完全可以 使用激活的、基因组工程的NK细胞实现。

Abstract Over the last decade Chimeric Antigen Receptor based T cell therapy (CAR-T) has developed into an effective immunotherapy for some cancers. However, CAR-T cell therapies have several shortcomings and clinical success has primarily been limited to hematological cancers. Challenges of CAR-T cell therapy include tumor immune evasion through loss of target antigen expression by tumor cells and inhibition of CAR-T cell function by tumor expressed inhibitory molecules. Natural killer (NK) cells present an alternative to T cells that could be more effective due to their ability to perform both antigen dependent and independent killing. NK cells have demonstrated antigen specific killing when engineered to express T cell CARs and NK cells also mediate the direct killing of transformed cells with reduced or absent MHC expression. In fact, NK cells carry out antibody dependent cell mediated cytotoxicity (ACDD) of cells that bind antibodies via the NK cell CD16A receptor. Due to the multiple modalities for cancer cell killing, there is an increased interest in NK cells for cancer immunotherapy. As NK cells are not associated with graft versus host disease, neurotoxicity, long-term autoimmunity, nor cytokine release syndrome, they are more suited for use in allogeneic settings than T cells and have significant clinical potential for use as off-the-shelf products. However, previous publications and clinical trials have demonstrated that the use of unmanipulated NK cells to treat cancer is minimally effective, likely due to limited engraftment, little in vivo expansion, and suppression by the tumor microenvironment. NK cells activated and expanded with feeder cells expressing membrane bound interleukin-21 (mbIL-21) have shown promising results clinically with high-risk myeloid malignancies and preclinically in several solid tumor models. Therefore, we hypothesize that activated/expanded NK cells that have be genetically edited can be used to successfully treat osteosarcoma, a disease for which patient outcome has not improved in over thirty years. Our proposed objectives are to evaluate the baseline response of rested- and activated-NK cells against various osteosarcoma cell lines, knockout negative regulators of NK cell function (specifically, c-CBL, IL-1R8, and SMAD3), and implement a specific CAR that optimally activates NK cell antigen-specific killing. Genetically engineered NK cells will be evaluated for enhanced therapeutic efficacy and safety in osteosarcoma models. Our preliminary data strongly supports the hypothesis that NK cell-based cancer immunotherapy can be fully realized using activated, genome engineered NK cells.