Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

中文翻译：

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成人和脐带血衍生的高亲和力 gB-CAR-T 细胞有效对抗人类巨细胞病毒感染。

人类巨细胞病毒 (HCMV) 再激活与移植后总体存活率降低有关。HCMV 反应性扩增或选择的 T 细胞的过继转移可用作同情用途，但需要人类白细胞抗原匹配的供体提供针对 HCMV 的记忆细胞。为了克服这个问题，我们开发了表达嵌合抗原受体 (CAR) 的工程化 T 细胞，该受体针对病毒再激活时表达的 HCMV 糖蛋白 B (gB)。源自人类高亲和力 gB 特异性中和单克隆抗体 (SM5-1) 的单链可变片段 (scFvs) 与具有 4-1BB (BBL) 或 CD28 (28S) 共刺激结构域的 CAR 融合并亚克隆到逆转录病毒载体中。从用载体转导的 HCMV 血清阴性成人血液或脐带血 (CB) 中获得的 CD4+ 和 CD8+ T 细胞有效表达 gB-CAR。gB-CAR-T 细胞的特异性和效力在体外使用以下物质进行了证明和比较：表达 gB 的 293T 细胞，以及感染了表达 Gaussia 荧光素酶 (HCMV/GLuc) 的 HCMV TB40 菌株的间充质干细胞。与 28-gB-CAR-T 细胞相比，用成人或 CB 产生的 BBL-gB-CAR-T 细胞表现出显着更高的体外活化和细胞毒性性能。Nod.Rag.Gamma (NRG) 小鼠移植了具有长期人类免疫重建的人类 CB CD34+ 细胞，用于通过光学成像分析模拟体内 HCMV/GLuc 感染。给药 1 周后，观察到 5/8 只小鼠对 BBL-gB-CAR-T 细胞疗法的反应，定义为与未处理对照相比生物发光信号显着降低。对治疗的反应偶尔与脾脏中的 CAR 检测相关。因此，探索源自高亲和力 gB 抗体 SM5-1 和 4-1BB 信号结构域的 scFv 用于 CAR 设计，实现了体外高靶向效应和细胞毒性，并在体内取得了令人鼓舞的结果。因此，gB-CAR-T 细胞可以成为治疗 HCMV 再激活的未来临床选择，特别是当来自供体的记忆 T 细胞不可用时。探索源自高亲和力 gB 抗体 SM5-1 和 4-1BB 信号域的 scFv 用于 CAR 设计，实现了体外高靶向效应和细胞毒性，并在体内取得了令人鼓舞的结果。因此，gB-CAR-T 细胞可以成为治疗 HCMV 再激活的未来临床选择，特别是当来自供体的记忆 T 细胞不可用时。探索源自高亲和力 gB 抗体 SM5-1 和 4-1BB 信号域的 scFv 用于 CAR 设计，实现了体外高靶向效应和细胞毒性，并在体内取得了令人鼓舞的结果。因此，gB-CAR-T 细胞可以成为治疗 HCMV 再激活的未来临床选择，特别是当来自供体的记忆 T 细胞不可用时。