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Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR)
Leila Jafarzadeh1, Elham Masoumi2, Khadijeh Alishah3
1Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. jafarzade.leila@yahoo.com.
Abstract:
Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3ζ and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) α, and Interferon-(IFN) γ production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting.
Insights
This study developed a fully human mesothelin (MSLN)-targeted CAR T cell therapy. The novel CAR T cells demonstrated potent and specific anti-tumor activity against MSLN-expressing solid tumors in vitro.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) T cell therapy shows promise for hematological malignancies but faces challenges in solid tumors due to immunosuppressive microenvironments and limited target antigens.
- Mesothelin (MSLN), a tumor-associated antigen (TAA), is highly expressed in various solid tumors (e.g., gastrointestinal, ovarian, pancreatic) with low expression in normal tissues, making it an attractive therapeutic target.
- Previous MSLN-targeted therapies, including monoclonal antibodies, have been developed, highlighting the antigen's therapeutic potential.
Purpose of the Study:
- To develop and functionally evaluate a second-generation, fully human anti-MSLN CAR T cell construct for solid tumor treatment.
- To assess the in vitro efficacy, specificity, and cytokine production of the MSLN-CAR T cells against MSLN-expressing tumor cells.
Main Methods:
- Production of a second-generation CAR T cell construct featuring a fully human anti-MSLN scFv, CD3ζ, and 4-1BB signaling domains.
- In vitro functional evaluation of MSLN-CAR T cells against an MSLN-expressing cell line, assessing proliferation, cytotoxicity, and cytokine release (IL-2, TNF-α, IFN-γ).
- Determination of optimal effector-to-target ratios and assessment of specificity against MSLN-negative cell lines.
Main Results:
- The fully human MSLN-CAR T cells exhibited potent and antigen-dependent proliferation and cytotoxic activity against MSLN-expressing cell lines.
- Significant production of IL-2, TNF-α, and IFN-γ was observed in an antigen-dependent manner.
- Optimal cytotoxic efficacy was noted at an effector-to-target ratio of 10:1, with no non-specific activity against MSLN-negative cells.
Conclusions:
- Primary human T cells engineered with the fully human MSLN-CAR construct demonstrate effective in vitro anti-tumor activity.
- This MSLN-CAR construct holds potential as a therapeutic agent for MSLN-expressing solid tumors in future clinical applications.
