Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

中文翻译：

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慢病毒和α-逆转录病毒工程的CD19特异性嵌合抗原受体天然杀伤细胞对急性淋巴细胞白血病的高细胞毒性效率。

对CD19有特异性的自体嵌合抗原受体修饰（CAR）T细胞在针对复发和难治性B细胞急性淋巴细胞白血病（B-ALL）的临床试验中显示出强大的抗肿瘤功效。与T细胞相反，天然杀伤（NK）细胞以非抗原特异性方式杀死其靶标，并且不承担诱发移植物抗宿主病（GvHD）的风险，从而允许将供体来源的细胞应用于同种异体环境。因此，与自体CAR-T细胞不同，治疗性CD19-CAR-NK细胞可以作为来自健康供体的现成产物而产生。然而，外周血（PB）衍生的NK细胞的基因工程仍然具有挑战性，需要优化的方案。在我们的研究中 我们旨在通过逆转录病毒转导来优化CD19-CAR-NK细胞的生成，以提高NK细胞的高抗白血病能力。我们比较了两种不同的逆转录病毒载体平台，即慢病毒和α逆转录病毒，两者均与两种不同的转导增强子（Retroectin和Vectofusin-1）结合。我们进一步探索了不同的NK细胞分离技术（NK细胞富集和CD3 / CD19耗竭），以确定用于NK细胞基因工程的最有效方法。我们的结果表明，用RD114-TR假型逆转录病毒载体与Vectofusin-1结合转导NK细胞是生成CD19-CAR-NK细胞的最有效方法。Retronectin在增强慢病毒/ VSV-G基因向NK细胞的传递方面很有效，但对α逆转录病毒/ RD114-TR无效。此外，由α逆转录病毒/ RD114-TR和慢病毒/ RD114-TR载体递送的CD19-CAR介导的基于Vofofusin的NK细胞转导优于慢病毒/ VSV-G载体。与未转导的NK细胞相比，最终产生的CD19-CAR-NK细胞对表达CD19的靶细胞表现出优异的细胞毒活性，达到高达90％的特异性杀伤活性。总而言之，我们的发现提出了将RD114-TR假型逆转录病毒颗粒与Vectofusin-1结合使用作为遗传修饰PB衍生NK细胞以获得高细胞毒性CD19-CAR-NK细胞的成功策略。与未转导的NK细胞相比，最终产生的CD19-CAR-NK细胞对表达CD19的靶细胞表现出优异的细胞毒活性，达到高达90％的特异性杀伤活性。总而言之，我们的发现提出了将RD114-TR假型逆转录病毒颗粒与Vectofusin-1结合使用作为遗传修饰PB衍生NK细胞以获得高细胞毒性CD19-CAR-NK细胞的成功策略。与未转导的NK细胞相比，最终产生的CD19-CAR-NK细胞对表达CD19的靶细胞表现出优异的细胞毒活性，达到高达90％的特异性杀伤活性。总而言之，我们的发现提出了将RD114-TR假型逆转录病毒颗粒与Vectofusin-1结合使用作为遗传修饰PB衍生NK细胞以获得高细胞毒性CD19-CAR-NK细胞的成功策略。