Simultaneous blockade of programmed death 1 and vascular endothelial growth factor receptor 2 (VEGFR2) induces synergistic anti-tumour effect in vivo

Abstract

Recent basic and clinical studies have shown that the programmed death ligand (PD-L)/PD-1 pathway has a significant role in tumour immunity, and its blockade has a therapeutic potential against several human cancers. We hypothesized that anti-angiogeneic treatment might augment the efficacy of PD-1 blockade. To this end, we evaluated combining the blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2) in a murine cancer model using Colon-26 adenocarcinoma. Interestingly, simultaneous treatment with anti-PD-1 and anti-VEGFR2 monoclonal antibodies (mAbs) inhibited tumour growth synergistically in vivo without overt toxicity. Blocking VEGFR2 inhibited tumour neovascularization significantly, as demonstrated by the reduced number of microvessels, while PD-1 blockade had no impact on tumour angiogenesis. PD-1 blockade might promote T cell infiltration into tumours and significantly enhanced local immune activation, as shown by the up-regulation of several proinflammatory cytokine expressions. Importantly, VEGFR2 blockade did not interfere with T cell infiltration and immunological activation induced by PD-1 blockade. In conclusion, simultaneous blockade of PD-1 and VEGFR2 induced a synergistic in-vivo anti-tumour effect, possibly through different mechanisms that might not be mutually exclusive. This unique therapeutic strategy may hold significant promise for future clinical application.

Fig. 1

Simultaneous blockade of programmed death (PD)-1 and vascular endothelial growth factor receptor 2 (VEGFR2) induced synergistic anti-tumour effect in vivo. BALB/c mice were inoculated subcutaneously with Colon-26 cells and were given with control rat immunoglobulin (Ig)G, anti-PD-1 monoclonal antibody (mAb), anti-VEGFR2 mAb or both mAbs five times (arrow). Data are presented as mean ±standard error of seven to 10 mice of each group. *P < 0·05; **P < 0·01.

Fig. 2

Programmed death (PD)-1and vascular endothelial growth factor receptor 2 (VEGFR2) blockade did not have any direct effect on cancer cell growth in vitro. A total of 3000 Colon-26 cells were co-cultured with anti-PD-1 monoclonal antibody (mAb), anti-VEGFR2 mAb or control rat immunoglobulin (Ig)G for 72 h, and cell proliferation was determined by MTS assay.

Fig. 3

Treatment with anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody (mAb) inhibited tumour neovascularization. (a) Immunohistochemistry analysis by staining with CD34. Representative tumours from mice treated with control rat IgG, anti-programmed death (PD)-1 mAb, anti-VEGFR2 mAb or both mAbs. (b) Tumour microvessels were counted at ×200 magnification. Data are collected from four to seven mice of each group. *P < 0·01; **P < 0·001.

Fig. 4

(a) Immunohistochemical staining of CD4⁺ and (b) CD8⁺ T cells in tumour tissue at day 11. Representative pictures of mice for each treatment are shown. Programmed death (PD)-1 blockade and combination treatment seemed to induce more CD4⁺ and CD8⁺ T cell infiltration compared to control and vascular endothelial growth factor receptor 2 (VEGFR2) blockade. (c) Quantification of tumour-infiltrating CD4⁺ and (d) CD8⁺ T cells by real-time polymerase chain reaction (PCR). There was a tendency towards increased T cell infiltration by the treatment of anti-PD-1 monoclonal antibody (mAb) and combination treatment. Anti-VEGFR2 mAb treatment did not interfere with T cell infiltration. Data are collected from four to seven mice of each group.

Fig. 5

Expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and granzyme B was up-regulated significantly by anti-programmed death (PD)-1 monoclonal antibody (mAb) or combination mAb treatment compared with control. Treatment of anti-VEGFR2 mAb alone did not increase each cytokine expression. Data are collected from four to seven mice of each group. *P < 0·05.