PO.CL05.05 · 临床研究
靶向胸腺癌的新型CAR T细胞疗法的开发
Development of a novel CAR T-cell therapy targeting thymic carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言:胸腺癌是一种罕见、高度侵袭性的纵隔恶性肿瘤,有效疗法有限,且免疫检查点抑制剂伴有相当大的毒性。尽管CAR T细胞疗法已彻底改变了血液系统恶性肿瘤的治疗格局,但由于疾病罕见、缺乏经过验证的表面靶点以及靶向、脱瘤自身免疫毒性的风险,其在包括胸腺上皮肿瘤在内的实体瘤中的应用仍具挑战。在本研究中,我们试图通过表征胸腺癌相关细胞系的抗原表达谱、针对优先候选抗原设计新型CAR构建体并评估抗原特异性细胞毒性来应对这些障碍。这些工作旨在为开发针对这一侵袭性且研究不足的肿瘤类型独特生物学特性的新一代细胞疗法提供依据。
方法:使用流式细胞术定量候选细胞系上TROP-2、c-KIT和CD70的表面表达。CAR T细胞由健康供者PBMC经慢病毒转导产生并离体扩增。通过检测CD3⁺ T细胞上的Fab片段确认CAR表达。使用与选定抗原阳性细胞系的共培养实验在体外评估抗原特异性细胞毒性,并通过7-AAD染色和流式细胞分析定量靶细胞杀伤。
结果:流式细胞术显示在T1889、Kasumi和U266细胞系上分别有稳健的TROP-2(83%)、c-KIT(88%)和CD70(99%)表面表达。慢病毒转导产生的CAR⁺ T细胞群体分别为48.2%(TROP-2)、24.7%(c-KIT)和47.5%(CD70)。所有三种构建体均表现出强效的抗原特异性细胞毒性,大幅超过GFP⁺ CD3⁺对照T细胞,证实了对靶细胞的选择性识别和杀伤。这些发现提供了支持TROP-2、c-KIT和CD70作为胸腺癌可行免疫治疗靶点的首批体外证据之一,并证明了有效的CAR表达和选择性细胞毒性。这项工作为在这一历史上研究不足的实体瘤中进行合理的多抗原CAR T细胞设计奠定了基础。正在进行的研究包括基于FACS的CAR富集、耗竭标志物的表征、开发双抗原synNotch CAR以减轻脱瘤毒性,以及在小鼠模型中进行体内验证以支持未来的临床转化。
查看英文原文 English abstract
Introduction: Thymic carcinoma is a rare, highly aggressive mediastinal malignancy with limited effective therapies and considerable toxicity associated with immune checkpoint inhibitors. Although CAR T-cell therapy has revolutionized the treatment landscape for hematologic malignancies, its application to solid tumors, including thymic epithelial tumors, remains challenging due to disease rarity, a paucity of validated surface targets, and the risk of on-target, off-tumor autoimmune toxicity. In this study, we sought to address these barriers by characterizing antigen expression profiles across thymic carcinoma-relevant cell lines, engineering novel CAR constructs against prioritized candidate antigens, and evaluating antigen-specific cytotoxicity. These efforts aim to inform the development of next-generation cellular therapies tailored to the unique biology of this aggressive and understudied tumor type.
Methods: Surface expression of TROP-2, c-KIT, and CD70 was quantified on candidate cell lines using flow cytometry. CAR T-cells were produced from healthy donor PBMCs via lentiviral transduction and expanded ex vivo. CAR expression was confirmed by detection of Fab fragments on CD3⁺ T cells. Antigen-specific cytotoxicity was evaluated in vitro using co-culture assays with selected antigen-positive cell lines, and target cell killing was quantified by 7-AAD staining and flow cytometric analysis.
Results: Flow cytometry demonstrated robust surface expression of TROP-2 (83%), c-KIT (88%), and CD70 (99%) across the T1889, Kasumi, and U266 cell lines, respectively. Lentiviral transduction yielded CAR⁺ T cell populations of 48.2% (TROP-2), 24.7% (c-KIT), and 47.5% (CD70). All three constructs exhibited potent antigen-specific cytotoxicity substantially exceeding GFP⁺ CD3⁺ control T cells, confirming selective recognition and killing of target cells. These findings provide some of the first in vitro evidence supporting TROP-2, c-KIT, and CD70 as viable immunotherapeutic targets for thymic carcinoma and demonstrate effective CAR expression and selective cytotoxicity. This work establishes a foundation for rational multi-antigen CAR T cell design in a historically understudied solid tumor. Ongoing studies include FACS-based CAR enrichment, characterization of exhaustion markers, development of a dual-antigen synNotch CAR to mitigate off-tumor toxicity, and in vivo validation in murine models to support future clinical translation.
利益披露 Disclosure
L. Ahmadi, None..
M. Khalifeh, None..
D. Donovski, None..
D. Ceppa, None..
P. Loehrer, None..
R. Maniar, None..
H. Salman, None.