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

• Published in: Immunobiology (1979) 2023-05, Vol.228 (3), p.152376-152376, Article 152376
• Main Authors: Moazzeni, Ali, Kheirandish, Maryam, Khamisipour, Gholamreza, Rahbarizadeh, Fatemeh
• Format: Article
• Language: English
• Subjects: B-cell maturation antigen; B-Cell Maturation Antigen - genetics; B-Cell Maturation Antigen - metabolism; CAR T-cell; Chimeric antigen receptor; Humans; Immunotherapy, Adoptive - methods; In-silico; Ligands; Multiple Myeloma - genetics; Multiple Myeloma - therapy; Receptors, Chimeric Antigen - genetics; Receptors, Chimeric Antigen - metabolism; T-Lymphocytes
• ISSN: 0171-2985; 1878-3279
• DOI: 10.1016/j.imbio.2023.152376

•Targeting BCMA-specific CAR T Cells by bioinformatic approaches.•The IL-2 treatment, polyberen, and spinoculation significantly increase the T-cell transduction rate (90%) during the CAR T cell generation process.•Besides the scFv domain, the detection of the hinge domain (CD8) could be introduced as a good choice for confirmation of CAR construct surface expression to be detected.•In silico studies before experimental studies seem necessary for designing CAR T cells. 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.