BiTE® Molecule vs. CAR-T targeting FLT3 in AML: How Positive Co-Stimulation Tips the Scale

T-cell-based immunotherapies have revolutionized treatment paradigms in B-cell malignancies, yet their translation to acute myeloid leukemia (AML) has been hindered by a scarcity of tumor-restricted antigens and the risk of on-target off-leukemia toxicity. FLT3 has emerged as a promising therapeutic target with limited expression in healthy hematopoietic tissues. Here, we perform a head-to-head preclinical comparison of a FLT3-directed bispecific T-cell engager (BiTE® molecule) and second-generation FLT3-specific chimeric antigen receptor (CAR) T cells. Both approaches induced potent cytotoxicity against AML cell lines and primary patient-derived blasts while sparing healthy hematopoietic stem and progenitor cells. Despite similar short-term efficacy, prolonged antigen exposure demonstrated progressive functional decline and metabolic exhaustion; however, CAR T cells maintained cytotoxic capacity and proliferative potential over time. In AML xenograft models, CAR T cells achieved superior tumor control, prolonged survival, and greater T-cell infiltration than BiTE® molecule-treated counterparts. Transcriptomic profiling of T cells recovered from bone marrow further revealed a distinct exhaustion-associated gene signature in BiTE®-redirected T cells. Importantly, provision of CD86-mediated co-stimulation significantly enhanced the antitumor activity of BiTE®-redirected T cells in vitro and in vivo. These findings establish FLT3 as a viable and selective immunotherapeutic target in AML and underscore the functional and transcriptional differences between BiTE® molecule-redirected T cells and CAR T cells. Moreover, they reveal the critical role of co-stimulatory signaling in sustaining the efficacy of T-cell-based therapies in vivo, offering a rational path for improving T-cell redirection strategies in myeloid malignancies. Overall design: RNA bulk sequecing profile of human BiTE or CART-redirected T cells after a 18 day in vivo AML xenograft model

基于T细胞的免疫疗法已彻底革新B细胞恶性肿瘤的治疗范式，然而将其应用于急性髓系白血病（acute myeloid leukemia, AML）却受限于肿瘤限制性抗原匮乏以及靶标相关非白血病毒性风险。FLT3作为极具潜力的治疗靶点，在健康造血组织中的表达水平有限。本研究针对FLT3靶向双特异性T细胞衔接器（bispecific T-cell engager, BiTE®分子）与第二代FLT3特异性嵌合抗原受体（chimeric antigen receptor, CAR）T细胞开展了头对头临床前对比研究。两种疗法均可诱导AML细胞系及患者原代白血病母细胞产生强效细胞毒性，且不会损伤健康造血干细胞与祖细胞。尽管二者短期疗效相近，但延长抗原暴露时间后，二者均出现进行性功能衰退与代谢耗竭；然而CAR T细胞可随时间推移维持细胞毒性与增殖潜能。在AML异种移植模型中，相较于BiTE®分子治疗组，CAR T细胞可实现更优的肿瘤控制、更长的存活期以及更高的T细胞浸润水平。对从骨髓中回收的T细胞进行转录组测序分析进一步揭示，BiTE®重定向T细胞存在独特的耗竭相关基因特征。值得注意的是，CD86介导的共刺激可显著增强BiTE®重定向T细胞在体外与体内的抗肿瘤活性。本研究证实FLT3是AML中一种可行且具有选择性的免疫治疗靶点，并明确了BiTE®分子重定向T细胞与CAR T细胞之间的功能及转录差异。此外，本研究揭示了共刺激信号在维持体内基于T细胞疗法疗效中的关键作用，为髓系恶性肿瘤中优化T细胞重定向策略提供了合理路径。总体实验设计：经18天体内AML异种移植模型造模后，人类BiTE®或CAR T细胞重定向T细胞的批量RNA测序谱。