Background/Objectives:Chimeric Antigen Receptor (CAR)-T cell therapy has demonstrated impressive clinical results against hematological malignancies. However, currently commercialized CAR-T therapies are designed for autologous use, which entails some disadvantages, including high costs, manufacturing delays, complex standardization, and frequent production failures due to patient T cell dysfunction. Moreover, their CARs target one specific antigen, increasing the probability of antigen-negative tumor relapses. To overcome these limitations, we developed a novel NKG2D CAR-T cell therapy for allogeneic use with broad target specificity, as this CAR targets eight different ligands commonly upregulated in both solid and hematological tumors. Additionally, the manufacturing process was optimized to improve the phenotypic characteristics of the final product.Methods:Multiplex CRISPR/Cas9 technology was applied to eliminate the expression of TCR and HLA class I complexes in healthy donor T cells to reduce the risk of graft-versus-host disease and immune rejection, respectively, as well as lentiviral transduction for introducing the second-generation NKG2D-CAR. Moreover, we sought to optimize this manufacturing process by comparing the effect of different culture interleukin supplementations (IL-2, IL-7/IL-15 or IL-7/IL-15/IL-21) on the phenotypic and functional characteristics of the product obtained.Results: Our results showed that the novel CAR-T cells effectively targeted cervicouterine and colorectal cancer cells, and that those manufactured with IL-7/IL-15/IL-21 supplementation showed the most suitable characteristics among the conditions tested, considering genetic modification efficiency, cell proliferation, antitumor activity and proportion of the stem cell memory T cell subset, which is associated with enhanced in vivo CAR-T cell survival, expansion and long-term persistence.Conclusions:In summary, this new prototype of NKG2D CAR-T cell therapy for allogeneic use represents a promising universal treatment for a wide range of tumor types.
背景/目的：嵌合抗原受体（CAR）-T细胞疗法在治疗血液系统恶性肿瘤方面已展现出显著的临床疗效。然而，目前商业化的CAR-T疗法均为自体疗法设计，存在成本高昂、制备周期长、标准化复杂以及因患者T细胞功能障碍导致生产失败率高等局限性。此外，现有CAR仅靶向单一特定抗原，增加了抗原阴性肿瘤复发的风险。为突破这些限制，我们开发了一种新型通用型NKG2D CAR-T细胞疗法，该疗法具有广泛的靶向特异性——其CAR可识别八种在实体瘤和血液肿瘤中普遍高表达的配体。同时，我们通过优化制备工艺以提升终产物的表型特征。 方法：采用多重CRISPR/Cas9技术敲除健康供体T细胞的TCR和HLA I类复合物表达，以分别降低移植物抗宿主病和免疫排斥风险，并通过慢病毒转导导入第二代NKG2D-CAR。此外，通过比较不同白细胞介素培养补充方案（IL-2、IL-7/IL-15或IL-7/IL-15/IL-21）对产物表型及功能特征的影响，系统优化了制备工艺。 结果：研究显示新型CAR-T细胞能有效靶向宫颈癌和结直肠癌细胞。在基因修饰效率、细胞增殖能力、抗肿瘤活性及干细胞记忆T细胞亚群比例等关键指标上，采用IL-7/IL-15/IL-21联合补充方案制备的细胞展现出最优特性，该亚群比例与体内CAR-T细胞存活、扩增及长期持久性增强密切相关。 结论：综上所述，这种新型通用型NKG2D CAR-T细胞疗法为广谱肿瘤治疗提供了具有前景的通用型治疗方案。
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