目的：构建并优化靶向唾液酸结合免疫球蛋白样凝集素6（Siglec-6）的CAR-T 细胞，评价其对急性髓系白血病（AML）细胞的靶向杀伤作用、初步体内疗效及短期安全性，为Siglec-6作为AML免疫治疗靶点提供实验依据。方法：设计靶向Siglec-6 的CAR 重组基因，筛选3 种人源化scFv，并在二代CAR 架构下比较4-1BB、CD28、ICOS、HVEM、TMIGD2 和OX40 等共刺激结构域对CAR-T细胞功能的影响。采用慢病毒转导人原代T细胞制备Siglec-6 CAR-T细胞，流式细胞术检测CAR阳性率及细胞表型。检测U937、MV4-11和K562细胞表面Siglec-6表达，并构建293T-Siglec-6过表达（OE）细胞、U937-Siglec-6敲除（KO）细胞及U937-Luc/U937-Siglec-6 KO-Luc细胞模型。采用流式杀伤实验、ELISA、实时无标记细胞分析（RTCA）及萤光素酶生物发光法评价CAR-T细胞的体外杀伤活性与靶点特异性；建立U937-Luc异种移植白血病模型，评价其体内抗肿瘤效应，并结合体质量、活体成像、外周血T细胞水平、主要器官H-E染色、器官指数及血清生化指标进行短期安全性评估。结果：3种候选CAR-T细胞中，CAR-T-1在CAR表达水平、短期杀伤活性及后续构建可行性方面表现较优。不同共刺激结构域比较显示，4-1BB-CAR-T细胞CAR阳性率较稳定，并在多种效靶比条件下表现出较强体外杀伤活性。Siglec-6 CAR-T细胞可有效杀伤Siglec-6阳性U937和MV4-11细胞，并显著促进IFN-γ和TNF-α释放（P < 0.000 1），而对Siglec-6阴性K562细胞无明显杀伤作用。293T-Siglec-6过表达模型和U937-Siglec-6 KO模型进一步证实，该杀伤效应具有明确的Siglec-6靶点依赖性。抗原刺激后，Siglec-6 CAR-T细胞向效应表型分化并显著活化（P < 0.000 1），并伴随部分耗竭相关标志物表达水平升高（P < 0.01）。体内实验显示，Siglec-6 CAR-T治疗可降低U937-Luc荷瘤小鼠肿瘤负荷并延长生存期；短期观察中，两组小鼠体质量变化无明显差异，主要器官H-E染色未见明显形态学损伤，部分器官指数及血清生化指标存在组间差异（P < 0.01）。结论：Siglec-6 CAR-T 细胞可特异性杀伤Siglec-6 阳性AML细胞，并在AML异种移植模型中发挥初步抗白血病作用，其机制可能与Siglec-6靶点依赖性识别、CAR-T细胞活化及IFN-γ/TNF-α分泌水平增加有关。

Objective: To construct and optimize chimeric antigen receptor T (CAR-T) cells targeting sialic acid-binding immunoglobulin- like lectin 6 (Siglec-6), and to evaluate their targeted cytotoxicity against acute myeloid leukemia (AML) cells, preliminary in vivo efficacy and short-term safety, thereby providing experimental evidence for Siglec-6 as an immunotherapeutic target for AML. Methods: A recombinant CAR gene targeting Siglec-6 was designed. Three humanized single-chain variable fragments (scFvs) were screened, and the effects of different co-stimulatory domains, including 4-1BB, CD28, ICOS, HVEM, TMIGD2 and OX40, on CAR-T cell function were compared within a second-generation CAR framework. Siglec-6 CAR-T cells were generated by lentiviral transduction of primary human T cells, and CAR positivity and cellular phenotypes were detected by flow cytometry. Surface expression of Siglec-6 on U937, MV4-11 and K562 cells was examined, and 293T-Siglec-6 overexpression (OE) cells, U937-Siglec-6 knockout (KO) cells, and U937-Luc/U937-Siglec-6 KO-Luc cell models were established. Flow cytometry-based cytotoxicity assays, ELISA, real-time cell analysis (RTCA) and luciferase bioluminescence assays were used to evaluate the in vitro cytotoxic activity and target specificity of CAR-T cells. A U937-Luc xenograft leukemia model was established to assess in vivo antitumor efficacy, and short-term safety was evaluated based on body weight, bioluminescence imaging, peripheral blood T-cell levels, hematoxylin-eosin staining of major organs, organ indices and serum biochemical parameters. Results: Among the three candidate CAR-T cells, CAR-T-1 showed superior performance in terms of CAR expression level, short-term cytotoxic activity and feasibility for subsequent construct development. Comparison of different co-stimulatory domains showed that 4-1BB-CAR-T cells exhibited relatively stable CAR positivity and stronger in vitro cytotoxic activity under multiple effector-to-target ratios. Siglec-6 CAR-T cells effectively killed Siglec-6-positive U937 and MV4-11 cells and significantly promoted the release of IFN-γ and TNF-α (P < 0.000 1), whereas no obvious cytotoxicity was observed against Siglec-6-negative K562 cells. The 293T-Siglec-6 overexpression model and U937-Siglec-6 KO model further confirmed that this cytotoxic effect was clearly dependent on Siglec-6 targeting. After antigen stimulation, Siglec-6 CAR-T cells differentiated into effector phenotypes and were significantly activated (P < 0.000 1), accompanied by increased expression of some exhaustion-related markers (P < 0.01). In vivo experiments showed that Siglec-6 CAR-T treatment reduced tumor burden and prolonged survival in U937-Luc tumor-bearing mice. During short-term observation, no obvious difference in body weight change was observed between the two groups, and hematoxylin-eosin staining of major organs revealed no apparent morphological injury, although some organ indices and serum biochemical parameters differed between groups (P < 0.01). Conclusion: Siglec-6 CAR-T cells specifically killed Siglec-6-positive AML cells and exerted preliminary anti-leukemic activity in an AML xenograft model, and the underlying mechanism may be associated with the target- dependent recognition of Siglec-6, the activation of CAR-T cells and the increased secretion level of IFN-γ/TNF-α.

国家自然科学基金（82273207）