Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma

Abstract

Background and aims: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown.

Approach and results: We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8⁺ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4⁺ cells. We also found that under anti-PD-1 therapy, CD4⁺ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody.

Conclusions: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.

Fig. 1:. Efficacy of dual anti-PD-1/DC101 treatment in HCC models.

A) Kaplan-Meier survival distribution in the orthotopic HCA-1 model after immune checkpoint blockade (ICB) with anti-PD-1 antibody, anti-VEGFR-2 blockade with DC101 low-dose (AA-low, 10mg/kg) or high-dose (AA, 40mg/kg) or their combinations compared with Control (C) IgG group. Combination of anti-PD-1 treatment with either low-dose (AA-low/IBC) or high-dose (AA/IBC) of DC101 showed significantly longer survival compared with Control IgG group [AA-low/IBC vs C: Hazard Ratio (HR) = 0.13, 95% confidence interval (CI) 0.04, 0.42, p=0.001; AA/IBC vs C: HR=0.17, 95%CI 0.05, 0.52, p=0.002]. B) Kaplan-Meier survival distribution in the orthotopic RIL-175 model after ICB with anti-PD-1 antibody, anti-VEGFR-2 blockade with DC101 (AA) or their combinations compared with IgG group (C). The AA/ICB combination showed significantly longer survival compared with control, with a HR=0.054, 95%CI 0.006, 0.46, p=0.007. *p<0.05; **p<0.01; ***p<0.001.

Fig. 2:. Effect of dual anti-PD-1/VEGFR-2 blockade on immune stimulation in HCC.

A) Changes in CD8+ cytotoxic T lymphocytes (CTLs) among tumor-infiltrated immune cells (shown as fractions of CD45+ positive cells) in RIL-175 tumors treated with control IgG, anti-PD-1 antibody (ICB), anti-VEGFR-2 antibody (AA), or their combination, measured by flow cytometry at day 8. AA decreases the fraction of CTLs among HCC-infiltrating immune cells. B) All treatments decreased the fraction of infiltrating CD4+FoxP3+ T regulatory cells (Tregs). C) As a result, ICB-treated groups showed favorable ratios of CTLs to Tregs after treatment. D) Similarly, the fraction of interferon gamma (IFN-γ)-positive CD8+ T cells increased in both ICB-treated groups. E, Changes in immune transcriptome from RNA-sequencing analysis (complete dataset in Table S1) showing gene expression patterns consistent with the activation of anti-tumor immunity. *p<0.05; **p<0.01; ***p<0.001.

Fig. 3:. PD-L1 and PD-1 upregulation after VEGFR-2 blockade in HCC.

A) Fraction of PD-L1 positive cells by flow cytometry in RIL-175 HCC tissues. Fraction of PD-L1 positive cells is increased in anti-VEGFR-2 antibody (AA)-treated groups irrespective of dose compared to control or compared to anti-PD-1 therapy (ICB) alone, respectively. B) Representative PD-L1 IHC in spontaneous HCCs from Mst1/2-mutant mice: AA treatment increased PD-L1 expression. C) Double-immunofluorescence for PD-L1 and the TAM marker F4/80; PD-L1 expression is predominantly seen in TAMs, with the exception of high-dose DC101 treatment of mice bearing HCC, where PD-L1 expression is increased in tumor and other stromal cells (arrows). D-F) AA treatment does not significantly change the fraction of CD4+ cells in HCC tissue (D) but increases the fraction of PD1+CD4+ cells (E), and also the PD-1 staining intensity – mean fluorescence intensity or MFI (F) – measured by flow cytometry. **p<0.01; ***p<0.001.

Fig. 4:. VEGFR-2 blockade in endothelial cells increases PD-L1 expression in HCC cells.

A, B) VEGFR-2 blockade with DC101 induces high PD-L1 expression in heterotypic tumor organoids cultured in normoxic (A) as well as hypoxic (B) conditions (see controls in Fig. S4C, D). C, D) Heterotypic organoid culture of primary HCC-derived cells were cultured with or without DC101 and anti-IFN-γ antibody (C). IFN-γ blockade partially prevented the increase in PD-L1 expression induced by DC101 treatment independently of hypoxia (D). Quantification was performed using Image J and in five random fields. ***p<0.001.

Fig. 5:. Effects of dual anti-PD-1/VEGFR-2 blockade on HCC vessel formation, structure and function and the role of CD4+ cells.

A) Representative immunofluorescence (CD31, α-SMA, and DAPI counterstaining) of endothelial cells and pericytes in size-matched HCC tissues after treatment with immune checkpoint blockade (ICB) alone, ICB + anti-VEGFR-2 therapy (AA) and, ICB + AA + anti (a)CD4 antibody therapy. B, C) Dual PD-1/VEGFR-2 blockade significantly increases microvessel density (MVD in B) and pericyte-covered MVD (C) in HCC, and this effect is prevented by CD4 cell depletion. D) Representative immunofluorescence for CA-IX (to detect hypoxia), CD31 (for endothelial cells), and DAPI counterstaining in RIL-175 HCC tissues after treatment. E, ICB treatment decreased tissue hypoxia, both alone and combined with AA, and this effect was prevented by CD4+ cell depletion. *p<0.05; **p<0.01; ***p<0.001.

Fig. 6:. Schematic of the mechanism of interaction between anti-PD-1 and anti-VEGFR-2 therapy in HCC.

The benefit of dual anti-PD-1/VEGFR-2 treatment is due to CD4+ cell-mediated normalized vessel formation; normalized vessels and blockade of PD-1/PD-L1 axis lead to reduction in Tregs and CCR2+ monocytes, shift from M2– to M1-type in tumor-associated macrophages, and promotion of cytotoxic T lymphocyte (CTL) infiltration and activation.