Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Boulevard, Building 421, SPE 8-105, Philadelphia, PA, USA; Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
Glioblastoma Translational Center of Excellence, The Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA; Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Boulevard, Building 421, SPE 8-105, Philadelphia, PA, USA.
Mol Ther. 2022 Mar 2;30(3):1201-1214. doi: 10.1016/j.ymthe.2021.11.014. Epub 2021 Nov 20.
Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.
在过继转移之前，CAR T 细胞被激活，通过慢病毒感染 CAR 转基因，并在 9 到 11 天内扩增。这个过程的一个意外后果是 CAR T 细胞在培养过程中随着时间的推移逐渐分化。分化的 T 细胞植入效果差，限制了它们在血液肿瘤和实体瘤中持续存在和提供持续肿瘤控制的能力。实体瘤包括 CAR T 细胞治疗的其他障碍，包括抑制效应功能和持久性的免疫和代谢检查点。唾液酸是具有已知免疫检查点功能的普遍存在的表面分子。产气荚膜梭菌神经氨酸酶 (CpNA) 可从靶细胞中去除唾液酸残基，在生理条件下具有良好的活性。与半乳糖氧化酶 (GO) 结合，已发现 NA 可刺激 T 细胞有丝分裂和细胞毒性。在这里，我们确定 CpNA 单独使用和与 GO 联合使用是否能促进 CAR T 细胞的抗肿瘤疗效。我们发现 CpNA 在体外培养过程中抑制 CAR T 细胞分化，产生具有增强治疗潜力的后代。表达 CpNA 的 CAR T 细胞在体外具有更好的效应功能和细胞毒性。在 Nalm-6 白血病异种移植模型中，表达 CpNA 的 CAR T 细胞显示出增强的抗肿瘤疗效。用 CpNA 武装 CAR T 细胞也增强了胶质母细胞瘤异种移植模型和黑色素瘤同种异体模型中的肿瘤控制。根据我们的发现，我们假设通过表面糖蛋白的电荷排斥是影响分化的调节参数。当 T 细胞与肿瘤内的靶细胞结合并通过其 CAR 持续激活时，负电荷的关键阈值可能会阻碍突触形成和细胞溶解的细胞间相互作用。用 CpNA 去除致密的负细胞表面电荷是限制 CAR T 细胞分化和增强整体持久性和疗效的有效方法。
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