嵌合抗原受体（CAR）T细胞是癌症免疫治疗中的突破。成功的CAR T细胞疗法的主要步骤是设计特定的单链抗体变量（scFv）。本研究旨在利用生物信息学技术验证设计的抗BCMA（B细胞成熟抗原）CAR，并进行以下实验评估。在设计了第二代抗BCMA CAR之后，使用不同的建模和对接服务器（包括Expasy、I-TASSER、HDock和PyMOL软件）确认了抗BCMA CAR构建物的蛋白质结构、功能预测、配体-受体界面的物理化学互补性以及结合位点分析。为生成CAR T细胞，分离的T细胞进行了转导。然后，采用实时-PCR和流式细胞术方法分别确认了抗BCMA CAR mRNA及其表面表达。使用抗（Fab’）2和抗CD8抗体评估抗BCMA CAR的表面表达。最后，将抗BCMA CAR T细胞与BCMA +/-细胞系共培养，以评估CD69和CD107a作为激活和细胞毒性标志物的表达。体外结果证实了scFv（89 ± 1.15％）和CD8α（54 ± 2.88％）的高表达。 CD69（91.97 ±1.7％）和CD107a（92.05 ± 1.29％）的表达显著增加，表明适当的激活和细胞毒性。体内研究结果证实了受体-配体结合位点上的功能区域的正确定位和完美取向。高激活和细胞毒性的抗BCMA CAR T细胞表明，我们的CAR构建方法将适用于定义CAR T细胞疗法的路线图。 ©2023 Elsevier GmbH出版。

Chimeric Antigen Receptor (CAR) T-cell is a breakthrough in cancer immunotherapy. The primary step of successful CAR T cell therapy is designing a specific single-chain fragment variable (scFv). This study aims to verify the designed anti-BCMA (B cell maturation antigen) CAR using bioinformatic techniques with the following experimental evaluations.Following the second generation of anti-BCMA CAR designing, the protein structure, function prediction, physicochemical complementarity at the ligand-receptor interface, and biding sites analysis of anti-BCMA CAR construct were confirmed using different modeling and docking server, including Expasy, I-TASSER, HDock, and PyMOL software. To generate CAR T-cells, isolated T cells were transduced. Then, anti-BCMA CAR mRNA and its surface expression were confirmed by real-time -PCR and flow cytometry methods, respectively. To evaluate the surface expression of anti-BCMA CAR, anti-(Fab')2 and anti-CD8 antibodies were employed. Finally, anti-BCMA CAR T cells were co-cultured with BCMA+/- cell lines to assess the expression of CD69 and CD107a as activation and cytotoxicity markers.In-silico results approved the suitable protein folding, perfect orientation, and correct locating of functional domains at the receptor-ligand binding site. The in-vitro results confirmed high expression of scFv (89 ± 1.15% (and CD8α (54 ± 2.88%). The expression of CD69 (91.97 ± 1.7%) and CD107a (92.05 ± 1.29%) were significantly increased, indicating appropriate activation and cytotoxicity.In-silico studies before experimental assessments are crucial for state-of-art CAR designing. Highly activation and cytotoxicity of anti-BCMA CAR T-cell revealed that our CAR construct methodology would be applicable to define the road map of CAR T cell therapy.Copyright © 2023. Published by Elsevier GmbH.