Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models

Abstract

Prolonged activation of vascular endothelial growth factor receptor-2 (VEGFR-2) due to mis-regulation of the VEGF pathway induces aberrant blood vessel expansion, which supports growth and survival of solid tumors. Therapeutic interventions that inhibit the VEGFR-2 pathway have therefore become a mainstay of cancer treatment. Non-clinical studies have recently revealed that blockade of angiogenesis can modulate the tumor microenvironment and enhance the efficacy of concurrent immune therapies. Ramucirumab is an FDA-approved anti-angiogenic antibody that inhibits VEGFR-2 and is currently being evaluated in clinical studies in combination with anti-programmed cell death (PD-1) axis checkpoint inhibitors (pembrolizumab, durvalumab, or sintilimab) across several cancer types. The purpose of this study is to establish a mechanistic basis for the enhanced activity observed in the combined blockade of VEGFR-2 and PD-1-axis pathways. Pre-clinical studies were conducted in murine tumor models known to be responsive to anti-PD-1 axis therapy, using monoclonal antibodies that block mouse VEGFR-2 and programmed death-ligand 1 (PD-L1). Combination therapy resulted in enhanced anti-tumor activity compared to anti-PD-L1 monotherapy. VEGFR-2 blockade at early timepoints post-anti-PD-L1 therapy resulted in a dose-dependent and transient enhanced infiltration of T cells, and establishment of immunological memory. VEGFR-2 blockade at later timepoints resulted in enhancement of anti-PD-L1-driven immune cell infiltration. VEGFR-2 and PD-L1 monotherapies induced both unique and overlapping patterns of immune gene expression, and combination therapy resulted in an enhanced immune activation signature. Collectively, these results provide new and actionable insights into the mechanisms by which concurrent VEGFR-2 and PD-L1 antibody therapy leads to enhanced anti-tumor efficacy.

Fig 1. VEGFR-2 blockade induces anti-tumor effects and increases T cell infiltration in tumors.

(A) Mean MC38 tumor volumes in BALB/c mice during 5 mg/kg (green), 20 mg/kg (blue), and 40 mg/kg (red) DC101 treatment (measured twice per week for 3 weeks). (B) Representative IHC images of CD31 (green), α-smooth muscle actin NG2 (red), and cell nuclei (Hoechst) immunostaining on tumor sections from day 15 (scale bars: 100 μM). (C) Percentage of CD3+ cell infiltrates into tumor by IHC on day 15 by IHC (n = 5 for each group). (D) Percentage of CD3+ cells in tumor measured by flow cytometry analysis on day 15 and day 19 (n = 5 for each group). (E) Representative IHC images of CD31 (green), Mega 79 (red), and cell nuclei (Hoechst) immunostaining on tumor sections from day 13. Right bottom graph represents the 60x amplification (scale bar: 10 μM) of inset in left bottom graph (scale bar: 100 μM). Bar graph showed the quantification of Meca 79+ cell area in total tumor area among the treatment groups (n = 4 to 5 for each group). Statistical significance: *P ≤0.05, **P ≤0.01, ***P ≤0.001. Error bars indicate SEM.

Fig 2. Combination of VEGFR-2 blockade with anti-PD-L1 antibodies improves antitumor efficacy and induces immunological memory in MC38 and EMT6-LM2 syngeneic murine model.

(A) Schema of MC38 tumor-bearing mice treatment regimen. Dosing started at day 3. Subsequently, DC101 and Rat IgG were given i.p. 40 mg/kg, twice per week for 3 weeks. Anti-PD-L1 (178G7) was given 500 μg/mouse, i.p., once per week, 3 times in total. (B) Individual tumor growth curves of the DC101 treatment group are superimposed to Rat IgG control group. (C) Individual tumor growth curves of the anti-PD-L1 treatment group are superimposed to Rat IgG control group. (D) Individual tumor growth curves of the combination treatment group (DC101 with anti-PD-L1) are superimposed to Rat IgG control group. (E) Schema of EMT6-LM2 tumor-bearing mice treatment regimen. (F) Individual tumor growth curves for DC101. Dosing started at day 6. Subsequently, DC101 and Rat IgG were given i.p. 40 mg/kg, twice per week for 3 weeks. Anti-PD-L1 (178G7) was given 500 μg/mouse, i.p., once per week, 3 times in total. (G) Individual tumor growth curves for anti-PD-L1 (178G7). (H) Individual tumor growth curves for the combination treatment group. (I) Individual tumor growth curves for the 2 CR mice from (D) (red lines) and 2 naïve mice (black lines), which were re-challenged with MC38 tumor cells on the opposite flank on day 60. (J) Individual tumor growth curves for 7 CR mice from (H) (red lines), which were re-challenged with EMT6-LM2 tumor cells on the opposite flank on day 60. CR, complete response. *P ≤0.05, **P ≤0.01, ***P ≤0.001. Error bars indicate SEM.

Fig 3. Combination of VEGFR-2 blockade with anti-PD-L1Ab results in antitumor effects, increased T cell infiltration, and unique intra-tumor gene upregulation signature in MC38 syngeneic murine model.

(A) Mean MC38 tumor volumes in C57B16 mice for mechanism of action study, according to treatment. *P ≤0.05, **P ≤0.01, ***P ≤0.001. Error bars indicate SEM. (B) Absolute leukocyte subset numbers in tumor collected on day 20. *P ≤0.05, **P ≤0.01 (C) nCounter analysis using PanCancer Immune Profiling in MC38 tumors at day 20; volcano plots show log2-fold change of gene expression in the treatment groups, compared with the control group. Differentially expressed genes (DEGs) that display greater or less than 2-fold differences are indicated by red and green arrows, respectively. Red data points indicate p<0.05 (one-way ANOVA). (D) Venn diagrams show the number of shared and treatment-specific DEGs across experimental groups from PanCancer Immune Profiling (left) and PanCancer Pathways panel (right).

Fig 4. Increased T cell activation and antigen presentation in response to anti-VEGFR-2 and anti-PD-L1 treatment in murine EMT6-LM2 tumor model by flow cytometry.

EMT6-LM2 tumor samples for each group (n = 5) were collected on day 20 from study shown in Fig 2E. (A) The expression of activation markers CD137, CD25, GITR, Ki67, Lag3, and PD1 on intra-tumor CD8 cell were assessed for DC101, anti-PD-L1, or combination treatment. (B) Percentage of MHCII+ cells and MHCII expression level on tumor macrophages and Ly6CG Neg population were assessed for DC101, anti-PD-L1, or combination treatment. (C) Percentages of double positive expression for CD86+ and MHCII+ on tumor macrophages were analyzed and one set of representative plots is shown. (D) PD-L1 expression on tumor infiltrating macrophages was analyzed and one set of representative plots is shown. *P ≤0.05, **P ≤0.01, ***P ≤0.001. Error bars indicate SEM.