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细胞疗法（CAR-T）已经发展成为一种有效的免疫疗法。 对某些癌症的免疫治疗。然而，CAR-T细胞疗法具有几个缺点和临床应用。 成功主要限于血液癌症。CAR-T细胞疗法的挑战包括肿瘤 通过肿瘤细胞失去靶抗原表达和抑制CAR-T细胞功能的免疫逃避 通过肿瘤表达的抑制分子。自然杀伤（NK）细胞是T细胞的替代品， 由于它们能够进行抗原依赖性和非依赖性杀伤，因此更有效。NK细胞具有 当被工程化以表达T细胞汽车时，证明了抗原特异性杀伤，并且NK细胞也介导抗原特异性杀伤。 直接杀死MHC表达减少或缺失的转化细胞。事实上，NK细胞携带抗体， 通过NK细胞CD 16 A受体结合抗体的细胞的依赖性细胞介导的细胞毒性（ACDD）。由于 由于癌症细胞杀伤的多种方式，人们对NK细胞治疗癌症的兴趣越来越大， 免疫疗法由于NK细胞与移植物抗宿主病、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细胞更适合用于同种异体环境 并且具有用作现成产品的显著临床潜力。然而，以前的出版物和 临床试验已经证明使用未经操作的NK细胞治疗癌症的效果最低， 这可能是由于有限的植入、很少的体内扩增和肿瘤微环境的抑制。NK 用表达膜结合白细胞介素-21（mbIL-21）的饲养细胞活化和扩增的细胞， 在高危骨髓恶性肿瘤的临床和几种实体瘤的临床前研究中显示出有希望的结果 模型因此，我们假设已经被基因编辑的活化/扩增的NK细胞可以被激活。 用于成功治疗骨肉瘤，这种疾病的患者结局在30多年来没有改善， 年我们提出的目标是评估静息和活化NK细胞对 各种骨肉瘤细胞系，敲除NK细胞功能的负调节因子（具体地，c-CBL，IL-1 R8， 和SMAD 3），并实现最佳激活NK细胞抗原特异性杀伤的特异性CAR。基因 将评估工程化NK细胞在骨肉瘤模型中增强的治疗功效和安全性。 我们的初步数据有力地支持了基于NK细胞的癌症免疫疗法可以被完全抑制的假设。 使用激活的基因组工程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.