Engineered Jurkat Cells for Targeting Prostate-Specific Membrane Antigen on Prostate Cancer Cells by Nanobody-Based Chimeric Antigen Receptor

Abstract

Background: Recently, modification of T cells with CAR has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a 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.

Nanobody-based CAR-T cells for cancer immunotherapy

“…Even though these researchers suggested that these findings demonstrate the potential of VHH-based CAR-Ts for CAR-T therapy of prostate cancer, broader investigations including preclinical assessments are required for such conclusions since the mentioned study only includes in vitro assessments [ 84 ]. In 2020, Hassani et al reported the findings of a similar study assessing the antitumor activity of Jurkat cells engineered to express VHH-based PSMA-redirected CARs [ 85 ]. According to this study, these CAR-Ts mediated PSMA-triggered antitumor activity and IL-2 secretion, and upregulated the surface expression of CD25 activation marker upon co-culturing with LNCaP cells [ 85 ].…”

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…The efficacy of chimeric antigen receptor (CAR) T cells has been established in blood-based malignancies; however, their solid tumor implementation has been limited by their inherent immunogenicity and large size of CARs. Various studies have demonstrated the efficacy of utilizing MUC-1 ( 104 ), CD7 ( 105 ), CD38 ( 106 ), VEGFR2 ( 107 ), prostate-specific membrane antigen (PSMA) ( 108 , 109 ), glypican-2 (GPC2) ( 110 ), and T cell receptor (TCR)-like nanobody-CARs ( 111 ) in various tumor models. Bispecific nanobody-CARs targeting CD20 and HER2 have also been developed; however, experiments have yet to be performed in vivo ( 112 ).…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…Also mammalian cell display of nanobodies has found interesting applications, such as their use for the modification of T cells with chimeric antigen receptors (CARs) suitable for cancer immunotherapy. VHHs are preferred to scFvs because are considered less immunogenic, more stable and compact (nanoCARs) [19,100,101].…”