Tumor-specific CAR-NK cells for adoptive cancer immunotherapy

自然杀伤（NK）细胞有望成为过继性癌症免疫疗法。与T细胞类似，NK细胞的抗肿瘤活性可以通过表达嵌合抗原受体（car）来增强，car有助于选择性识别和杀死恶性细胞。因此，NK细胞的独特特性，如其内源性天然细胞毒性与car介导的抗肿瘤活性互补，以及供体来源细胞应用后移植物抗宿主病的低风险，有利于使用NK细胞作为car工程效应器。然而，诸如NK细胞有限的扩增潜力和CAR基因转移的有效性降低等挑战正在减缓CAR工程原代NK细胞的临床发展。在这个项目中，我们的目标是研究造血干细胞（HSC）的直接离体分化作为产生全功能NK细胞的策略，这将进一步扩大使用特定的细胞因子混合物和基因修饰的饲养细胞，以可靠地产生高效应细胞数量。这种方法将用于生成具有增强抗肿瘤活性的CAR工程NK细胞，其基础是将慢病毒CAR基因转移到造血干细胞中，然后在体外分化为CAR-NK细胞。因此，CAR特异性肿瘤相关表面抗原ErbB2 （HER2）将被用作临床相关模型，通过使用NK特异性启动子将CAR表达限制在NK细胞谱系中。由此产生的体外生成的NK和CAR-NK细胞将进行表型和功能表征，并在体外对癌细胞和体内免疫缺陷小鼠的人类肿瘤异种移植物进行抗肿瘤活性测试。随后，CAR-NK细胞离体分化过程中建立的原理将应用于人源化小鼠模型系统中CAR-NK细胞的体内分化。移植携带在NK细胞中以谱系特异性方式表达的CAR序列的造血干细胞可以促进多克隆CAR-NK细胞在体内的连续繁殖，并允许持久的疾病控制。我们希望这些研究能够建立CAR基因转导的造血干细胞作为产生肿瘤特异性CAR- nk细胞的有用来源，其数量与治疗相关，可以作为目前应用的CAR- t细胞的有希望的替代方案，用于开发有效的癌症免疫疗法。

Natural killer (NK) cells hold promise for adoptive cancer immunotherapy. Similar to T cells, the antitumor activity of NK cells can be enhanced by expression of chimeric antigen receptors (CARs) that facilitate selective recognition and killing of malignant cells. Thereby, characteristics unique to NK cells such as their endogenous natural cytotoxicity complementing CAR-mediated antitumor activity, and the low risk of graft versus host disease upon application of donor-derived cells favor the use of NK cells as CAR-engineered effectors. Nevertheless, challenges such as the limited expansion potential of NK cells and reduced effectiveness of CAR gene transfer are slowing clinical development of CAR-engineered primary NK cells. With the proposed project, we aim to investigate directed ex vivo differentiation of hematopoietic stem cells (HSC) as a strategy for the generation of fully functional NK cells, which will be further expanded using specific cytokine cocktails and gene-modified feeder cells to reliably yield high effector cell numbers. This approach will then be employed to generate CAR-engineered NK cells with enhanced antitumor activity, based on lentiviral CAR gene transfer into HSCs followed by ex vivo differentiation into CAR-NK cells. Thereby, a CAR specific for the tumor-associated surface antigen ErbB2 (HER2) will be employed as a clinically relevant model, with CAR expression restricted to the NK cell lineage through the use of an NK-specific promoter. The resulting ex vivo generated NK and CAR-NK cells will be phenotypically and functionally characterized, and their antitumor activity will be tested against cancer cells in vitro and human tumor xenografts in immunodeficient mice in vivo. Subsequently, the principles established during ex vivo differentiation of CAR-NK cells will be applied for in vivo CAR-NK cell differentiation in humanized mouse model systems. Transplantation of HSCs carrying a CAR sequence expressed in NK cells in a lineage-specific manner may facilitate continuous in vivo repopulation with polyclonal CAR-NK cells and allow durable disease control. We expect these studies to establish CAR gene transduced HSCs as a useful source for the generation of tumor-specific CAR-NK cells in therapeutically relevant numbers, which could serve as a promising alternative to currently applied CAR-T cells for the development of effective cancer immunotherapies.