Immunostimulatory CKb11 gene combined with immune checkpoint PD-1/PD-L1 blockade activates immune response and simultaneously overcomes the immunosuppression of cancer

Abstract

Immunosuppression tumor microenvironment (TME) seriously impedes anti-tumor immune response, resulting in poor immunotherapy effect of cancer. This study develops a folate-modified delivery system to transport the plasmids encoding immune stimulatory chemokine CKb11 and PD-L1 inhibitors to tumor cells, resulting in high CKb11 secretion from tumor cells, successfully activating immune cells and increasing cytokine secretion to reshape the TME, and ultimately delaying tumor progression. The chemokine CKb11 enhances the effectiveness of tumor immunotherapy by increasing the infiltration of immune cells in TME. It can cause high expression of IFN-γ, which is a double-edged sword that inhibits tumor growth while causing an increase in the expression of PD-L1 on tumor cells. Therefore, combining CKb11 with PD-L1 inhibitors can counterbalance the suppressive impact of PD-L1 on anti-cancer defense, leading to a collaborative anti-tumor outcome. Thus, utilizing nanotechnology to achieve targeted delivery of immune stimulatory chemokines and immune checkpoint inhibitors to tumor sites, thereby reshaping immunosuppressive TME for cancer treatment, has great potential as an immunogene therapy in clinical applications.

Keywords: CKb11; Immune checkpoint PD-1/PD-L1; Immunogene therapy; Nanomedicine; Ovarian cancer.

Graphical abstract

Scheme 1

Schematic illustration of the anti-tumor mechanisms of the combined immunogene therapy by Fa-PCD delivering pCKb11 and iPD-L1. After administration, the delivery of pCKb11 by Fa-PCD achieved tumor-specific secretion of CKb11. The delivery of iPD-L1 by Fa-PCD blocked the immunosuppressive role of PD-L1 overexpression in tumor cells induced by higher IFN-γ concentration. The codelivery of pCKb11 and iPD-L1 by Fa-PCD significantly reshapes the immunosuppressive TME, inducing proliferation and activation of lymphocytes, macrophage repolarization, and DC maturation in TME, and inhibits tumor progression.

Fig. 1

Characteristics of the nanocomposites. (A) A model of self-assembled nanocomposite Fa-PCD/pCKb11/iPD-L1. (B) TEM of PCD/pCkb11/iPD-L1 (left) and Fa-PCD/pCkb11/iPD-L1 (right). (C) Zeta potential of PCD/pCkb11/iPD-L1 (left) and Fa-PCD/pCkb11/iPD-L1 (right). (D) Sizes of PCD/pCkb11/iPD-L1 (left) and Fa-PCD/pCkb11/iPD-L1 (right). (E) Agarose gel electrophoresis of Fa-PCD/pCkb11/iPD-L1. Lane 1 marker; 2–4, naked pCKb11; lane 5–13: different mass ratios of composites with pCKb11 (pCKb11: Fa-PCD, lane 5–7, 1:25; lane 8–10, 1:50; and lane 11–13, 1:100), (scale bar, 50 nm; n = 3).

Fig. 2

The transfection efficiency of the nanoparticles. (A) Fluorescent images of ID8 cells treated with Fa-PCD/pEGFP and PCD/pEGFP for 48 h. (B) Transfection efficiency by FCM. (C) Quantification of CKb11 released by ID8 cells by Elisa. (n = 3, **p < 0.01, ****p < 0.0001; One-way ANOVA).

Fig. 3

The treatment of Fa-PCD/pCKb11/iPD-L1 enhanced cytokines secretion from lymphocytes to promote apoptosis of tumor cells. (A) The cell viability of ID8 cells was evaluated by CCK8 assay. (B–F) Co-incubation of CFSE-labeling ID8 cells treated with different nanocomposites and lymphocytes. TNF-α (B) and IFN-γ (C) secreted from lymphocytes by Elisa. (D) PD-L1 expression of CFSE-labeling ID8 cells by flow cytometry. (E) Representative images of ID8 cells co-cultured with lymphocytes at a ratio of 1:20 (ID8 cells: lymphocytes). Statistical results showed an average number of lymphocytes surrounding a tumor cell. (F) The apoptosis of CFSE-labeling ID8 cells was assessed by FCM. (n = 3, ***p < 0.001, ****p < 0.0001; One-way ANOVA; scale bar, 50 μm).

Fig. 4

Fa-PCD/pCKb11/iPD-L1 treatment promotes proliferation and activation of lymphocytes. ID8 cells were treated with different nanocomposites for 48 h. Then the cell supernatants were collected for treating lymphocytes. (A) Cell viability of lymphocytes assessed by CCK8. (B) Activated T lymphocytes detected by FCM, which were defined as CD8⁺CD69⁺, CD8⁺IFN-γ⁺, CD4⁺CD69⁺, CD4⁺IFN-γ⁺ lymphocytes. (n = 3, *p < 0.05, **p < 0.01, ****p < 0.0001, n. s. , no significance; One-way ANOVA).

Fig. 5

Fa-PCD/pCKb11/iPD-L1 treatment inhibits M2-polarization. ID8 cells were treated with different nanocomposites for 48h. The cell supernatants were collected to treat macrophages for 48 h. Then the supernatants from macrophages were collected for Elisa. The macrophages were collected for flow cytometry, PCR and WB. (A) Polarization status of macrophages was assessed by flow cytometry. Polarized M2 cells were defined as CD45⁺CD11b⁺F480⁺ CD206⁺ macrophages. (B) TNF-α and (C) IFN-γ expression in supernatant from macrophages was detected by Elisa. (D) Relative mRNA expression of macrophage-related genes by RT-PCR. (E) Expression of macrophages-related proteins by WB. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; One-way ANOVA).

Fig. 6

Mature DCs are increased after treatment of Fa-PCD/pCKb11/iPD-L1. ID8 cells were treated with different nanocomposites for 48 h. The cell supernatants were collected to treat DCs for 48 h. Then the DCs were collected and detected by FCM. CD11c⁺CD80⁺, CD11c⁺CD86⁺ and CD11c⁺MHC II⁺ were defined as mature DCs. (n = 3, **p < 0.01, ****p < 0.0001, n. s. , no significance; One-way ANOVA).

Fig. 7

Fa-PCD/pCKb11/iPD-L1 inhibited tumor growth. The abdominal ID8 tumor-bearing mice were intraperitoneally injected according to their groups for 42 days. Then mice were sacrificed and tumors were collected. (A) Representative images of tumor-bearing mice and their corresponding abdominal cavity. (B) Images of tumors. (C) Tumor weight. (D) Volume of ascites. (E) Body weight. (n = 6, *p < 0.05, **p < 0.01, ****p < 0.0001; One-way ANOVA).

Fig. 8

Fa-PCD/pCKb11/iPD-L1 inhibited tumor proliferation and angiogenesis. (A) CD31 and (B) Ki67 immunohistochemistry of ID8 tumors. (scale bar, 50 μm, n = 5, *p < 0.05, **p < 0.01, ****p < 0.0001; One-way ANOVA).

Fig. 9

Fa-PCD/pCKb11/iPD-L1 increased CKb11, IFN-γ and TNF-α expression in vivo. Mice-bearing tumors were treated for 42 days. Then mice were sacrificed and their blood, ascites and tumors were collected. (A) CKb11, (B) IFN-γ and (C) TNF-α levels in tumor, ascites and serum. (D) CKb11 level in different organs by Elisa. (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n. s. , no significance; One-way ANOVA).