Enhanced efficacy of dual chimeric antigen receptor-T cells targeting programmed death-ligand 1 and cancer-associated fibroblasts in colorectal cancer in vitro

Abstract

Objective: Colorectal cancer (CRC) presents significant treatment challenges, including immune evasion and tumor microenvironment (TME) suppression. Chimeric antigen receptor (CAR) T-cell therapy has shown promise in hematologic malignancies, but its effectiveness against solid tumors is hampered by the detrimental effects of the TME. This article aims to explore the potential of bispecific CAR T cells targeting programmed death-ligand 1 (PD-L1) and cancer-associated fibroblasts (CAFs) in CRC treatment.

Material and methods: Dual-targeted CAR-T cells against PD-L1 and CAF were engineered using the GV400 lentiviral vector. Programmed death-1 (PD-1)/nanobody (Nb) and fibroblast activation protein (FAP)/Nb-encoding lentiviral vectors were generated, and CAR T cells were produced through a three-plasmid system in 293T cells. Human peripheral blood mononuclear cells (PBMCs) were separated, transduced with these vectors, and then expanded. Functional characterization of CAR-T cells was performed through enzyme-linked immunosorbent assay (ELISA), Western blot analysis, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and cell counting kit-8 (CCK-8) assay. Migration and invasion assays were conducted using Transwell chambers to assess the ability of FAP-PD-1/Nb CAR-T cells to migrate toward tumor cells and invade the extracellular matrix.

Results: We developed dual-targeted CAR-T cells incorporating PD-L1 and CAF Nbs, which continuously secreted PD-1/Nb. Western blot confirmed PD-1/Nb expression in PD-1/Nb and FAP-PD-1/Nb CAR-T cells, with no expression in the untreated (UTD) group (P < 0.01). Flow cytometry showed a significantly higher cluster of differentiation (CD)25 and CD69 expression in FAP-PD-1/Nb CAR-T cells upon stimulation with FAP-positive target cells compared with the other groups (P < 0.01). TUNEL, flow cytometry, and CCK-8 assays revealed that FAP-PD-1/Nb CAR-T cells exhibited superior cytotoxicity and proliferation inhibition against FAP-positive HCT116 cells (P < 0.01). ELISA demonstrated increased interferon-gamma and tumor necrosis factor-alpha levels and reduced interleukin-10 (P < 0.01), suggesting enhanced cytokine modulation and antitumor immunity. Compared with single-target CAR-T cells and UTD, FAP-PD-1/Nb CAR-T cells showed notably enhanced Matrigel penetration and invasion (P < 0.01). Safety tests confirmed minimal cytotoxicity to normal PBMCs, indicating favorable safety.

Conclusion: This study successfully developed dual-targeted CAR-T cells against PD-L1 and CAF and demonstrated their superior antitumor activity and immunomodulatory effects on CRC treatment. This novel therapeutic strategy was established using CAR T-cell technology for the treatment of CRC.

Keywords: Chimeric antigen receptor-T cells; Colorectal cancer; Fibroblast activation protein; Programmed death-ligand 1; Tumor microenvironment.

Figure 1:

Construction of dual-targeting CAR-T cells targeting PD-L1 Nb and CAF. (a) ELISA demonstrated sustained PD-1/Nb secretion from PD-1/Nb and FAP-PD-1/Nb CAR-T cells. (b and c) Western blot confirmed PD-1/Nb expression (^✶✶P < 0.01, vs. UTD control). (d and e) Flow cytometry analysis of FAP expression in HCT116 and SW480 target cells following co-culture with FAP-PD-1/Nb CAR-T cells (^✶✶P < 0.01, vs. HCT116 or SW480 group). n = 3. CAR-T: Chimeric antigen receptor T cell, ELISA: Enzyme-linked immunosorbent assay, CAF: Cancer-associated fibroblast, PD-L1: Programmed death-ligand 1, Nb: Nanobody, SD: Standard deviation, UTD: Untreated, FAP: fibroblast activation protein.

Figure 2:

Flow cytometry analysis of CD25⁺CD69⁺ level on the surface of CAR-T cells. (a) Representative flow cytometry plots showing the expression of CD25+CD69+ on the surface of CAR-T cells. (b) Quantification of CD25+CD69+ levels. n = 3. ^✶P < 0.05, ^✶✶P < 0.01 (vs. UTD control). ##P < 0.01 (vs. PD-1/Nb CAR-T). ^^P < 0.01 (vs. FAP/Nb CAR-T). CAR-T: Chimeric antigen receptor T cell, CD: Cluster of differentiation, UTD: Untreated, Nb: Nanobody, PD-1: Programmed cell death protein 1, FAP: Fibroblast activation protein.

Figure 3:

Effects of FAP-PD-1/Nb CAR-T cells on FAP-positive CRC cells. (a and b) TUNEL assay assessing apoptosis in HCT116 cells. (c and d) Flow cytometry analysis determining apoptosis. (e) CCK-8 assay evaluating proliferation in HCT116 cells (n = 3; ^✶✶P < 0.01 vs. UTD; ##P < 0.01 vs. PD-1/Nb CAR-T; ^^P < 0.01 vs. FAP/Nb CAR-T). n = 3. CAR-T: Chimeric antigen receptor T cell, TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling, CCK-8: Cell counting kit-8, FAP: Fibroblast activation protein, UTD: Untreated, Nb: Nanobody, PD-1: Programmed cell death protein 1, CRC: Colorectal cancer.

Figure 4:

Evaluation of cell migration, invasion capacities, and cytokine in different CAR-T Treatment Groups. (a and b) Transwell migration assay results for different CAR-T cell types. (c and d) Matrigel invasion assay results for different CAR-T cell types. (e-g) ELISA analysis of IFN-γ, TNF-α, and IL-10 levels (^✶✶P < 0.01 vs. UTD; ^##P < 0.01 vs. PD-1/Nb CAR-T; ^^P < 0.01 vs. FAP/Nb CAR-T). n = 3. CAR-T: Chimeric antigen receptor T cell, ELISA: Enzyme-linked immunosorbent assay, UTD: Untreated, Nb: Nanobody, PD-1: Programmed cell death protein 1, FAP: Fibroblast activation protein, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, IL-10: Interleukin-10.

Figure 5:

FAP-PD-1/Nb CAR-T Cells safety evaluation. (a and b) Flow cytometry analysis of apoptosis in PBMCs co-cultured with different concentrations of different CAR-T cell types. (c and d) TUNEL assay for detecting cell apoptosis. (e) CCK-8 assay for detecting cell proliferation. Data are representative of three independent experiments. n = 3. CAR-T: Chimeric antigen receptor T cell, PBMC: Peripheral blood mononuclear cell, TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling, CCK-8: Cell counting kit-8, Nb: Nanobody, FAP: Fibroblast activation protein, PD-1: Programmed cell death protein 1.