Directed targeting of B-cell maturation antigen-specific CAR T cells by bioinformatic approaches: From in-silico to in-vitro.

Autor: Moazzeni A; Immunology Department, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine (IBTO), Tehran, Iran., Kheirandish M; Immunology Department, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine (IBTO), Tehran, Iran. Electronic address: M.Kheirandish@ibto.ir., Khamisipour G; Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran. Electronic address: R.Khamisipour@bpums.ac.ir., Rahbarizadeh F; Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Jazyk: angličtina
Zdroj: Immunobiology [Immunobiology] 2023 May; Vol. 228 (3), pp. 152376. Date of Electronic Publication: 2023 Mar 08.
DOI: 10.1016/j.imbio.2023.152376
Abstrakt: Aims: 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.
Main Methods: 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.
Key Findings: 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.
Significance: 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023. Published by Elsevier GmbH.)
Databáze: MEDLINE
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