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Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) has been approved for treating multiple myeloma (MM). Some clinical studies reported suboptimal outcomes, including reduced cytotoxicity of CAR-T cells and tumor evasion through increased expression of programmed death-ligand 1 (PD-L1). To enhance CAR-T cell efficiency and overcome PD-L1-mediated T cell suppression, we developed anti-BCMA-CAR5-T cells equipped with three costimulatory domains and the ability to secrete anti-PD-L1 single-chain variable fragment (scFv) blockade molecules. Anti-BCMA-CAR4-T cells contained a fully human anti-BCMA scFv and three intracellular domains (CD28, 4–1BB, and CD27) joined with CD3ζ. Anti-BCMA-CAR5-T cells were generated by fusing anti-BCMA-CAR4 with anti-PD-L1 scFv. Both anti-BCMA-CAR4-T and anti-BCMA-CAR5-T cells demonstrated comparable antitumor activity against parental MM cells. However, at an effector-to-target ratio of 1:2, only anti-BCMA-CAR5-T cells maintained cytolytic activity against PD-L1 high MM cells, unlike anti-BCMA-CAR4 T cells. Anti-BCMA-CAR5-T cells were specifically activated by BCMA-expressing target cells, resulting in increased CAR-T cell proliferation, release of cytolytic mediators, and pro-inflammatory cytokines. Anti-BCMA-CAR5-T cells demonstrated specific cytotoxicity against BCMA-expressing target cells, leading to decreased target cell numbers, increased CAR-T cell numbers, and preserved CAR expression during antigenic re-stimulation. Interestingly, only anti-BCMA-CAR5-T cells showed reduced PD-1 receptor levels, which correlated with decreased PD-L1 expression on target cells. We successfully generated anti-BCMA-CAR5-T cells capable of secreting anti-PD-L1 scFv. These cells exhibited superior antitumor efficiency, proliferative capacity, and alleviated T-cell exhaustion against MM cells. Further investigation into the antitumor efficacy of anti-BCMA-CAR5-T cells is warranted in ex vivo and clinical research settings. [Display omitted] • Anti-BCMA-CAR5-T cells exhibit heightened antitumor efficacy, attributable to their secretion of anti-PD-L1 scFv. • The presence of anti-PD-L1 scFv enhances the proliferation of anti-BCMA-CAR5-T cells, achieved through the PD-L1 blockade. • Anti-BCMA-CAR5-T cells bolstered with anti-PD-L1 scFv reduce T cell exhaustion and restore their antitumor function. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anti-CD19-CAR5-T cells exerted more effective cytotoxicity than anti-CD19-CAR4-T cells at a low effector-to-target ratio. • Cytotoxicity of anti-CD19-CAR4-T cells was inhibited by upregulations of PD-1 on CAR-T cells and PD-L1 on the target cancer cells. • The secreted anti-PD-L1 scFv from anti-CD19-CAR5-T cells could restore the cytotoxic effect of anti-CD19-CAR4-T cells. • The secreted anti-PD-L1 scFv promoted self-proliferation of anti-CD19-CAR5-T cells. • PD-L1 blockade by anti-PD-L1 scFv secreted from CAR5-T cells is a feasible strategy to improve CAR-T antitumor efficiency. Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anti-CD19-CAR5-T cells exerted more effective cytotoxicity than anti-CD19-CAR4-T cells at a low effector-to-target ratio. • Cytotoxicity of anti-CD19-CAR4-T cells was inhibited by upregulations of PD-1 on CAR-T cells and PD-L1 on the target cancer cells. • The secreted anti-PD-L1 scFv from anti-CD19-CAR5-T cells could restore the cytotoxic effect of anti-CD19-CAR4-T cells. • The secreted anti-PD-L1 scFv promoted self-proliferation of anti-CD19-CAR5-T cells. • PD-L1 blockade by anti-PD-L1 scFv secreted from CAR5-T cells is a feasible strategy to improve CAR-T antitumor efficiency. Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma. [ABSTRACT FROM AUTHOR]

• The superior antitumor function of fourth-generation CAR T compared to second-generation. • We developed a fourth-generation CAR targeting Trop2 (referred to as anti-Trop2 CAR4), which includes three co-stimulatory domains: CD28, 4-1BB, and CD27. • Anti-Trop2 CAR4-T cells exhibited potent cytotoxicity against breast cancer cells in both 2D and 3D cell cultures. • Anti-Trop2 CAR4-T cell proliferation and cytokine secretion were enhanced when exposed to Trop2-expressing cells. • Anti-Trop2 CAR4-T cells hold promise for improving the effectiveness of breast cancer treatment. The treatment of breast cancer (BC) remains a formidable challenge due to the emergence of drug resistance, necessitating the exploration of innovative strategies. Chimeric antigen receptor (CAR)-T cell therapy, a groundbreaking approach in hematologic malignancies, is actively under investigation for its potential application in solid tumors, including BC. Trophoblast cell surface antigen 2 (Trop2) has emerged as a promising immunotherapeutic target in various cancers and is notably overexpressed in BC. To enhance therapeutic efficacy in BC, a fourth-generation CAR (CAR4) construct was developed. This CAR4 design incorporates an anti-Trop2 single-chain variable fragment (scFv) fused with three costimulatory domains –CD28/4-1BB/CD27, and CD3ζ. Comparative analysis with the conventional second-generation CAR (CAR2; 28ζ) revealed that anti-Trop2 CAR4 T cells exhibited heightened cytotoxicity and interferon-gamma (IFN-γ) production against Trop2-expressing MCF-7 cells. Notably, anti-Trop2 CAR4-T cells demonstrated superior long-term cytotoxic functionality and proliferative capacity. Crucially, anti-Trop2 CAR4-T cells displayed specific cytotoxicity against Trop2-positive BC cells (MDA-MB-231, HCC70, and MCF-7) in both two-dimensional (2D) and three-dimensional (3D) culture systems. Following antigen-specific killing, these cells markedly secreted interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), IFN-γ, and Granzyme B compared to non-transduced T cells. This study highlights the therapeutic potential of anti-Trop2 CAR4-T cells in adoptive T cell therapy for BC, offering significant promise for the advancement of BC treatment strategies. [ABSTRACT FROM AUTHOR]