肿瘤细胞治疗研究
英文原题：Universal high-sensitivity CAR T-cell monitoring by targeting linker sequences.
Universal high-sensitivity CAR T-cell monitoring by targeting linker sequences.
这些发现确立了抗Whitlow/218和抗G4S mAb作为敏感、特异且通用的试剂，可用于跨多个靶点、构建体和物种的CAR检测，为在临床前、临床试验和诊断环境中统一CAR T细胞监测提供了标准化平台。
嵌合抗原受体（CAR）T细胞疗法已成为肿瘤学的标准治疗，然而由于CAR构建体多样且检测试剂可及性有限，循环CAR T细胞的标准化监测仍是一大挑战。CAR的抗体结构域通常由通过连接肽相连的重链和轻链组成，连接肽通常为4个甘氨酸和1个丝氨酸（G4S）或一段确定的氨基酸序列（Whitlow/218）。在此，我们评估了靶向连接肽序列的新型单克隆抗体（mAbs）作为CAR检测通用工具的价值。
我们使用流式细胞术，将抗连接子mAb与常规试剂进行了比较，包括抗独特型CD19.FMC63 mAb、基于CD19和BCMA抗原的检测试剂（Ag）以及抗F(ab')2 mAb。分析在商业化CD19和BCMA靶向的CAR T细胞产品以及一种研究性Claudin-6（CLDN6）CAR T细胞产品上进行。进一步在多种用于临床监测的实验平台以及小鼠模型中评估了性能，以评价灵敏度和转化相关性。
Linker-mAbs以高特异性和高灵敏度检测了所有测试的CAR构建体，其靶向特异性结合与传统Ag试剂相当。抗linker mAbs显示出极低的背景和较低的定量下限，均与Ag试剂相当。在淋巴瘤和骨髓瘤患者中的纵向监测显示，抗linker mAbs与Ag试剂之间的CAR T细胞动力学一致。基于linker的CAR检测在高维度、多参数流式细胞术、免疫荧光成像以及抗CD19 CAR T细胞的小鼠模型中均表现出高性能。
INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy has become a standard-of-care in oncology, yet standardized monitoring of circulating CAR T cells remains a major challenge due to diverse CAR constructs and limited availability of detection reagents. The antibody domain of the CAR commonly consists of the heavy and light chain connected through a linker, typically either 4x glycine and 1x serine (G4S) or a defined amino acid sequence (Whitlow/218). Here, we evaluated the novel monoclonal antibodies (mAbs) targeting the linker sequence as a universal tool for CAR detection. METHODS: Using flow cytometry, we compared anti-linker mAbs with conventional reagents, including anti-idiotype CD19.FMC63 mAb, CD19 and BCMA antigen-based detection reagents (Ag), and anti-F(ab') 2 mAb. Analyses were performed on commercial CD19- and BCMA-directed CAR T-cell products and an investigational Claudin-6 (CLDN6) CAR T-cell product. Performance was further assessed across diverse experimental platforms for clinical monitoring and in a murine model to evaluate sensitivity and translational relevance. RESULTS: Linker-mAbs detected all tested CAR constructs with high specificity and sensitivity, matching target-specific binding with conventional Ag reagents. Anti-linker mAbs demonstrated minimal background and low limits of quantification both comparable to Ag reagents. Longitudinal monitoring in lymphoma and myeloma patients revealed consistent CAR T-cell kinetics between anti-linker mAbs and Ag reagents. High performance of linker-based CAR-detection was demonstrated in high-dimensional, multi-parameter flow cytometry, in immunofluorescence imaging and in a murine model of anti-CD19 CAR T cells. CONCLUSION: These findings establish anti-Whitlow/218 and anti-G4S mAbs as sensitive, specific, and universal reagents for CAR detection across multiple targets, constructs, and species, providing a standardized platform for harmonization of CAR T-cell monitoring in preclinical, clinical trial, and diagnostic settings.
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