肿瘤细胞治疗研究
英文原题：Serpin B9 controls tumor cell killing by CAR T cells.
这些数据表明，serpin B9 是 CAR T 细胞介导的肿瘤细胞杀伤的耐药介质，应通过抑制或绕过它来改善 CAR T 细胞应答。
背景：癌症患者接受基因工程改造嵌合抗原受体（CAR）T 细胞治疗后，初始临床应答令人鼓舞；然而，相当一部分患者会出现原发耐药或复发，阻碍持久缓解。其原因之一是癌细胞的耐药机制限制了 CAR T 细胞有效杀伤。CAR T 细胞通过分泌颗粒酶和穿孔素发挥细胞毒功能。颗粒酶 B（GrB）抑制可导致 T 细胞介导的杀伤耐药；已有研究显示丝氨酸蛋白酶抑制剂 serpin B9 可有效抑制 GrB。本研究旨在确定癌细胞表达 serpin B9 是否会导致 CAR T 细胞耐药。 方法：通过 R2 或 DepMap 数据库挖掘检测 serpin B9 基因表达，并通过 Western blot 或流式细胞术检测蛋白表达。开展共培养杀伤实验，以黑色素瘤细胞系 MeWo、弥漫大 B 细胞淋巴瘤（DLBCL）细胞系 OCI-Ly7 或原代慢性淋巴细胞白血病（CLL）细胞为靶细胞，以 YT-Indy NK 细胞系、CD20 CAR T 或 CD19 CAR T 为效应细胞，并通过流式细胞术分析。 结果：既往研究显示黑色素瘤患者 serpin B9 蛋白表达与临床结局相关；与此一致，我们发现强制提高黑色素瘤细胞 serpin B9 表达会降低其对 GrB 介导杀伤的敏感性。随后在多种原发肿瘤组织和人细胞系中检测 serpin B9，发现其在 B 细胞淋巴瘤中普遍表达，尤其在 DLBCL 和 CLL 中表达最高。使用小干扰 RNA 沉默 DLBCL 细胞中的 serpin B9 后，其对 CD20 CAR T 细胞介导杀伤的敏感性提高。此外，原代 CLL 细胞与 CD20 CAR T 细胞共培养后，serpin B9 高表达的 CLL 细胞被选择性保留，提示这类细胞可抵抗 CAR T 细胞杀伤。 结论：总体而言，数据表明 serpin B9 是 CAR T 细胞介导肿瘤杀伤的耐药因子；抑制或绕过该因子有望改善 CAR T 细胞疗效。
BACKGROUND: Initial clinical responses with gene engineered chimeric antigen receptor (CAR) T cells in cancer patients are highly encouraging; however, primary resistance and also relapse may prevent durable remission in a substantial part of the patients. One of the underlying causes is the resistance mechanisms in cancer cells that limit effective killing by CAR T cells. CAR T cells exert their cytotoxic function through secretion of granzymes and perforin. Inhibition of granzyme B (GrB) can underlie resistance to T cell-mediated killing, and it has been shown that serine proteinase inhibitor serpin B9 can effectively inhibit GrB. We aimed to determine whether expression of serpin B9 by cancer cells can lead to resistance toward CAR T cells. METHODS: Serpin B9 gene and protein expression were examined by R2 or DepMap database mining and by western blot or flow cytometric analysis, respectively. Coculture killing experiments were performed with melanoma cell line MeWo, diffuse large B cell lymphoma (DLBCL) cell line OCI-Ly7 or primary chronic lymphocytic leukemia (CLL) cells as target cells and natural killer cell line YT-Indy, CD20 CAR T cells or CD19 CAR T cells as effector cells and analyzed by flow cytometry. RESULTS: Serpin B9 protein expression was previously shown to be associated with clinical outcome in melanoma patients and in line with these observations we demonstrate that enforced serpin B9 expression in melanoma cells reduces sensitivity to GrB-mediated killing. Next, we examined serpin B9 expression in a wide array of primary tumor tissues and human cell lines to find that serpin B9 is uniformly expressed in B-cell lymphomas and most prominently in DLBCL and CLL. Subsequently, using small interfering RNA, we silenced serpin B9 expression in DLBCL cells, which increased their sensitivity to CD20 CAR T cell-mediated killing. In addition, we showed that co-ulture of primary CLL cells with CD20 CAR T cells results in selection of serpin B9-high CLL cells, suggesting these cells resist CAR T-cell killing. CONCLUSIONS: Overall, the data indicate that serpin B9 is a resistance mediator for CAR T cell-mediated tumor cell killing that should be inhibited or bypassed to improve CAR T-cell responses.
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