肿瘤细胞治疗研究
英文原题：Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.
我们的研究为生成纯度高且活性强的同种异体CAR NK细胞提供了重要见解，从而进一步推动了利用CAR NK细胞进行过继性细胞免疫治疗人类恶性肿瘤的进展。
嵌合抗原受体（CAR）T细胞的巨大临床成功为血液系统恶性肿瘤的免疫治疗开辟了新的高度。将自然杀伤（NK）细胞进行基因修饰作为替代性CAR免疫效应细胞也极具前景，因为NK细胞可以跨越HLA屏障进行移植而不引起移植物抗宿主病。因此，CAR NK细胞产品的现货使用可能有助于广泛扩展临床适应症并限制每位患者的治疗成本。然而，与T细胞不同，制造合适的CAR NK细胞产品具有挑战性，因为对NK细胞进行基因工程改造的标准技术仍在确立之中。在本研究中，我们通过系统测试慢病毒CAR载体的不同内部启动子，并比较慢病毒假型与病毒进入增强子，建立了原代人NK细胞的最佳慢病毒转导方案。我们还额外修饰了识别急性淋巴细胞白血病（ALL）和急性髓系白血病（AML）治疗标准靶抗原——CD19、CD33和CD123——的CAR构建体，使其携带CD34来源的铰链区，从而能够在体外和体内高效检测转导的NK细胞，并便于通过CD34微珠辅助选择将CAR NK细胞产品纯化至>95%纯度以供潜在临床使用。重要的是，由于大多数白血病原始细胞对活化的原代人NK细胞具有先天的免疫原性，我们开发了一种体外系统，阻断这些细胞上的活化受体NKG2D、DNAM-1、NKp30、NKp44、NKp46和NKp80，从而允许系统测试CAR NK细胞对ALL和AML细胞系及原代AML原始细胞的特异性杀伤。最后，我们在NOD/SCID-gamma（NSG）小鼠的ALL异种移植模型中评估了针对CD19+原始细胞的人CD19 CAR NK细胞是否依赖可溶性或膜结合IL15的产生来维持NK细胞持久性以及体内白血病控制。因此，我们的研究为生成纯度高且高活性的同种异体CAR NK细胞提供了重要见解，从而进一步推进了用于人类恶性肿瘤的CAR NK细胞过继性细胞免疫治疗。
The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.
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