A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells

Alireza Rajabzadeh; Fatemeh Rahbarizadeh; Davoud Ahmadvand; Maryam Kabir Salmani; Amir Ali Hamidieh

doi:10.22074/cellj.2021.6917

A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells

Cell J. 2021 Jan;22(4):502-513. doi: 10.22074/cellj.2021.6917. Epub 2020 Apr 22.

Authors

Alireza Rajabzadeh¹, Fatemeh Rahbarizadeh², Davoud Ahmadvand³, Maryam Kabir Salmani⁴, Amir Ali Hamidieh^{1

5}

Affiliations

¹ Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
² Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Electronic Address: rahbarif@modares.ac.ir.
³ Department of Biochemistry, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran.
⁴ Department of Stem Cell and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
⁵ Pediatric Cell Therapy Research Centre, Tehran University of Medical Sciences, Tehran, Iran. Electronic Address: aahamidieh@sina.tums.ac.ir.

Abstract

Objective: Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety, which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and cytotoxicity of redirected human T cells.

Materials and methods: In this experimental study, we established a second generation novel CAR (VHH-based anti- MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane domain and signalling endodomains of CD28 and CD3ζ. Next, we constructed lentiviral vectors that contained this CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3⁺ T cells.

Results: The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared with A431 (MUC1-negative) or untransduced T cells.

Conclusion: Our results suggested that, given the unique properties of VHHs to prevent immunogenic responses and tonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy.

Keywords: Immunotherapy; Single-Chain Antibodies; T-Lymphocytes.