Background
Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a promising treatment approach for various cancers, particularly hematological malignancies. However, when it comes to solid tumors, the efficacy of CAR-T therapy faces significant challenges due to the presence of immune escape mechanisms. One prominent mechanism involves the upregulation of immune checkpoint molecules, such as PD-L1, PD-1, and TIGIT, which can dampen the anti-tumor immune response and promote tumor immune evasion. Thus, there is a critical need to develop strategies that can address these immune escape mechanisms and enhance the therapeutic efficacy of CAR-T therapy in solid tumors.
Methods
To tackle this formidable challenge, we put forward an innovative approach that capitalizes on our in-house developed mesothelin (MSLN) CAR-T cells, known as OriCAR627. In order to overcome the limitations imposed by immune checkpoint molecules, specifically PD-L1 and TIGIT, we have further engineered these MSLN CAR-T cells to secrete antibodies targeting PD-L1 and/or TIGIT (figure 1). This multifaceted strategy aims to not only enhance the cytotoxic potential of CAR-T cells against tumor cells expressing MSLN but also address the immune escape mechanisms by simultaneously neutralizing the inhibitory effects of PD-L1 and TIGIT in the tumor microenvironment.
Results
In vitro studies investigating the impact of CART cells secreting checkpoint inhibitors yielded unexpected results, as they did not exhibit improved tumor cell killing or cytokine secretion (figure 2). However, when tested in an in vivo tumor model, mesothelin-directed CART cells engineered to secrete a PD-L1 antibody (OriCAR627-PD-L1) demonstrated a remarkable enhancement in tumor killing capabilities (figure 3). In contrast, mesothelin-directed CAR T cells engineered to secrete a PD-L1-TIGIT bispecific antibody (OriCAR627-PD-L1/TIGIT) did not exhibit superior anti-tumor efficacy compared to PD-L1 antibody-secreting CAR-T cells (OriCAR627-PD-L1) (figure 4). These findings suggest that PD-L1 antibody, but not TIGIT antibody, has the potential to improve the antitumor efficacy of MSLN-directed CAR-T therapy. Further experiments confirmed spontaneous PD-L1 Ab secretion from OriCAR627-PD-L1 without target cell stimulation, with enhanced secretion upon target cell stimulation in a time-dependent manner. These results provide further evidence supporting the notion that the PD-L1 antibody holds promise for enhancing the effectiveness of MSLN-directed CAR-T therapy (figure 5).
Conclusions
In conclusion, the integration of PD-L1 blockade with MSLN-directed CAR-T cells (OriCAR627-PD-L1) presents a promising alternative strategy for controlling solid tumors in adoptive transfer therapy.
Ethics Approval
All animal experiments were approved by the Ethical Committee of East China Normal University.
Abstract 333 Figure 1
(A) Illustration of MSLN CAR-T cells engineered to secrete checkpoint inhibitors: A Schematic Overview. (B) MSLN expression in the target ovarian cancer cell line (SK-OV3-MSLN) is confirmed by flow cytometry.
Abstract 333 Figure 2
CART cells and target cells were incubated for 24h at a ratio of 3:1 or 1:1. Cytotoxicity (A,B), IL-2 and IFN-y (C,D) were measured by ELISA.
Abstract 333 Figure 3
(A) Schematic of the ovarian cancer xenograft model for investigating in vivo activity of engineered MSLN CAR-T cells in various groups. (B) Tumor growth was monitored at the indicated time points. (C-D) Peripheral blood was collected once a week, PD-L1 secretion (D) and CD3+ T cell (E) were detected. n=5 mice/group.
Abstract 333 Figure 4
(A) Proliferation of CAR-T cells in each group. (B) Cytotoxicity of CAR-T cells evaluated by luciferase assay during co-culture with SK-OV3-MSLN tumor cells. Concentrations of IFN-y (C) and IL-2 (D) measured by ELISA. (E) Quantitative assessment of PD-L1 concentrations using ELISA in the supernatants of co-cultured SK-OV3 cells (right) or non-co-cultured cells (left).
Abstract 333 Figure 5