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Engineered Jurkat Cells for Targeting Prostate-Specific Membrane Antigen on Prostate Cancer Cells by Nanobody-Based
Mahmoud Hassani1, Fatemeh Hajari Taheri2, Zahra Sharifzadeh2
1Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Background:
Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct.
Methods:
In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation.
Results:
Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-).
Conclusion:
Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.
Insights
This study developed a novel nanobody-based chimeric antigen receptor (CAR) for prostate cancer therapy. The engineered CAR demonstrated specific recognition of prostate cancer cells, leading to T-cell activation and proliferation.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) T-cell therapy is a promising cancer immunotherapy.
- Traditional CARs use murine-derived single-chain variable fragments (scFv), which can trigger immune responses.
- Nanobodies offer a low-immunogenic alternative for CAR construction due to their camelid origin and human VH homology.
Purpose of the Study:
- To develop and evaluate a novel CAR construct utilizing a nanobody targeting Prostate-Specific Membrane Antigen (PSMA).
- To assess the efficacy of nanobody-based CARs in activating T-cells against prostate cancer cells.
Main Methods:
- A CAR construct (NBPII-CAR) was engineered using a PSMA-targeting nanobody.
- Jurkat cells were transfected with NBPII-CAR and analyzed for expression via flow cytometry.
- Functional assays involved co-culturing engineered Jurkat cells with PSMA-positive and PSMA-negative prostate cancer cells to measure IL-2 secretion, CD25 expression, and proliferation.
Main Results:
- NBPII-CAR expression was confirmed on transfected Jurkat cells.
- Engineered Jurkat cells showed specific activation against PSMA-positive prostate cancer cells, evidenced by increased IL-2 secretion (370 pg/ml) and CD25 expression (30%).
- A significant increase in Jurkat cell proliferation (nearly 60%) was observed when co-cultured with PSMA+ LNCaP cells compared to PSMA- DU145 cells.
Conclusions:
- Nanobody-based CARs can effectively recognize PSMA and activate T-cells.
- This nanobody-based CAR construct shows potential for clinical applications in prostate cancer immunotherapy.
