Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer

Abstract

Adoptive cell transfer (ACT)-based immunotherapies can mediate objective cancer regression in animal models and in up to 70% of patients with metastatic melanoma; however, it remains unclear whether the tumor vasculature impedes the egress of tumor-specific T cells, thus hindering this immunotherapy. Disruption of the proangiogenic interaction of vascular endothelial growth factor (VEGF) with its receptor (VEGFR-2) has been reported to "normalize" tumor vasculature, enhancing the efficacy of chemotherapeutic agents by increasing their delivery to the tumor intersitium. We thus sought to determine whether disrupting VEGF/VEGFR-2 signaling could enhance the effectiveness of ACT in a murine cancer model. The administration of an antibody against mouse VEGF synergized with ACT to enhance inhibition of established, vascularized, B16 melanoma (P = 0.009) and improve survival (P = 0.003). Additive effects of an antibody against VEGFR-2 in conjunction with ACT were seen in this model (P = 0.013). Anti-VEGF, but not anti-VEGFR-2, antibody significantly increased infiltration of transferred cells into the tumor. Thus, normalization of tumor vasculature through disruption of the VEGF/VEGFR-2 axis can increase extravasation of adoptively transferred T cells into the tumor and improve ACT-based immunotherapy. These studies provide a rationale for the exploration of combining antiangiogenic agents with ACT for the treatment of patients with cancer.

Conflict of interest statement

Authors declare no potential conflict of interest.

Figures

Figure 1. Anti-tumor effect of ant-VEGF antibody alone and in combination with ACT

(A) Six doses of 200μg anti-VEGF antibody had a substantial growth inhibitory effect on small (50 mm2) established B16 tumors (p=0.009) and a lesser effect on large (100 mm2) tumors (p=0.028). Closed symbols are groups receiving α-VEGF and open symbols are groups receiving rat IgG. (B) Significant impact was also seen on the survival of mice. (C) Four doses of 200μg α-VEGF exhibited a substantial antitumor effect when given alone or in combination with ACT on small B16 tumors (50 mm2). (D) Significant impact was also seen on the survival of mice.

Figure 2. Combined therapeutic effect of anti-VEGF antibody with ACT on large B16 tumor treatment

(A) The administration of four, six and eight doses of 100μg of anti-VEGF antibody alone had no impact on the growth of these large established B16 melanomas. A synergistic anti-tumor effect was seen when 6 and 8 doses of anti-VEGF antibody was given in conjunction with ACT (p=0.009). (B) Survival of mice was also improved (p=0.003) (C) Eight doses of 100 μg of α-VEGF alone had no impact on tumor growth but synergized with ACT (p=0.01). The impact of ACT with and without antibody administration was dependent on the administration of 500cGy total body irradiation prior to the cell transfer. (D) Survival of mice correlated with the anti-tumor growth effect.

Figure 3. Effect of anti-VEGF antibody on pmel-1-Ly5.1 cell infiltration into B16 tumor following ACT

(A) The percent of infiltrating Ly5.1+PI- cells into B16 melanoma on day 4 post cell transfer was significantly increased in mice administered with 200μg or 500μg anti-VEGF antibody compared to mice receiving 500μg rat IgG. (B) The total number of infiltrating Ly5.1+PI- cells was also increased in mice receiving 200μg and 500μg α-VEGF compared to mice receiving 500μg rat IgG. (C) and (D) A repeat experiment evaluating the infiltration of transferred Ly5.1 pmel-1 cells on days 3, 4, 5 and 6 gave a similar result. Thus, prior administration of α-VEGF to B16 tumor bearing mice receiving ACT significantly enhances tumor infiltration of the adoptively transferred pmel-1 cells.

Figure 4. Impact of rat anti-mouse VEGFR-2 antibody (DC101) on tumor treatment and cell infiltration in B16 tumor bearing mice following ACT

(A and B) Three doses of 800μg DC101 antibody mediated a modest growth inhibitory effect on small established B16 melanoma compared to rat IgG alone. An additive effect of the antibody was seen when combined with ACT (p=0.013). Open symbols are groups receiving antibody alone while closed symbols are groups receiving antibody with ACT (PVI group). (C and D) Six doses of 800μg DC101 had no impact on the growth of large (100 mm2) B16 melanoma but did mediate a modest tumor inhibitory effect when added to ACT. In accord with our previous results vaccination with recombinant vaccinia virus expressing hgp10025-33 peptide was required to see the tumor inhibitory impact of ACT. Mice receiving PI (without vaccination) exhibited no anti-tumor effect with or without anti-VEGFR-2 antibody administration.

Figure 5. Anti-vascular effect of a single dose of anti-VEGFR-2 antibody (DC101) on B16 tumor vasculature

(A and B) Mean tumor vessel area of B16 tumors in mice receiving a single dose of 800μg DC101 was decreased compared to mice receiving rat IgG on various days post antibody administration. Results for each time point are the mean of 5 images for each section/mice and 2 or 5 mice in each group (A and B respectively). Two separate experiments are shown. (C) Representative microscopic sections from mice receiving DC101 antibody or control rat IgG. Vessel area was determined by blinded measurement of CD31 expression. At 3 and 4 days after DC101 administration a significant decrease in tumor vessels was seen compared to mice receiving rat IgG.