VEGF Neutralization Plus CTLA-4 Blockade Alters Soluble and Cellular Factors Associated with Enhancing Lymphocyte Infiltration and Humoral Recognition in Melanoma

Abstract

Immune recognition of tumor targets by specific cytotoxic lymphocytes is essential for the effective rejection of tumors. A phase I clinical trial of ipilimumab (an antibody that blocks CTLA-4 function) in combination with bevacizumab (an antibody that inhibits angiogenesis) in patients with metastatic melanoma found favorable clinical outcomes were associated with increased tumor endothelial activation and lymphocyte infiltration. To better understand the underlying mechanisms, we sought features and factors that changed as a function of treatment in patients. Ipilimumab plus bevacizumab (Ipi-Bev) increased tumor vascular expression of ICAM-1 and VCAM-1. Treatment also altered concentrations of many circulating cytokines and chemokines, including increases of IP-10, IL1α, TNFα, GRO, IFNα2, and IL8, with decreases in VEGF-A in most patients. IL1α and TNFα induced expression of E-selectin, ICAM-1, and VCAM-1 on melanoma tumor-associated endothelial cells (TEC) in vitro and promoted adhesion of activated T cells onto TEC. VEGF-A inhibited TNFα-induced expression of ICAM-1 and VCAM-1 and T-cell adhesion, which was blocked by bevacizumab. IP-10 promoted T-cell migration across TEC in vitro, was frequently expressed by melanoma cells, and was upregulated in a subset of tumors in treated patients. Robust upregulation of IP-10 in tumors was accompanied by increased T-cell infiltration. Ipi-Bev also augmented humoral immune responses recognizing targets in melanoma, tumor endothelial, and tumor mesenchymal stem cells. Our findings suggest that Ipi-Bev therapy augments immune recognition in the tumor microenvironment through enhancing lymphocyte infiltration and antibody responses. IL1α, TNFα, and IP-10, together with VEGF neutralization, contribute to Ipi-Bev–induced melanoma immune recognition.

Angiopoietin-2 as a Biomarker and Target for Immune Checkpoint Therapy

“…Tumor associated endothelial cells (TEC) were isolated using Dynabeads CD31 Endothelial Cell as guided by the manufacturer (Life Technologies) and confirmed by surface expression of CD31 and VEGFR2 and tube formation (34). HUVEC were purchased from Lonza.…”

Evolving Roles for Targeting CTLA-4 in Cancer Immunotherapy

“…The combination of ipilimumab and anti-receptor activator of NF-κB (RANK) ligand (RANKL) denosumab in metastatic melanoma was recently reported [113]. A phase I clinical trial of ipilimumab in combination with bevacizumab (an antibody that inhibits angiogenesis) in patients with metastatic melanoma showed augmenting immune recognition in the tumor microenvironment through enhancing lymphocyte infiltration [114]. In addition, tremelimumab in combination with other therapies was also investigated and showed better clinical outcomes [93,115].…”

The Role of the Tumor Vasculature in the Host Immune Response: Implications for Therapeutic Strategies Targeting the Tumor Microenvironment

“…This combination appears safe and well tolerated (109, 117) and warrants further investigation and comparison to current treatment regimens. Tumor endothelial cells isolated from melanoma patients treated with this combination of ipilimumab and bevacizumab showed variable upregulation of adhesion molecules E-selectin, ICAM-1, and VCAM-1, with resulting enhancement of T cell infiltration into the tumor (109, 230). Changes in levels of circulating chemokines, cytokines, and growth factors were seen following treatment, including increased levels of chemoattractant IP-10 (CXCL10) and decreased levels of VEGF-A (230).…”

“…Tumor endothelial cells isolated from melanoma patients treated with this combination of ipilimumab and bevacizumab showed variable upregulation of adhesion molecules E-selectin, ICAM-1, and VCAM-1, with resulting enhancement of T cell infiltration into the tumor (109, 230). Changes in levels of circulating chemokines, cytokines, and growth factors were seen following treatment, including increased levels of chemoattractant IP-10 (CXCL10) and decreased levels of VEGF-A (230). Endothelial anergy induced by VEGF-A could be demonstrated in these samples and reversed by the addition of bevacizumab (230).…”

“…Changes in levels of circulating chemokines, cytokines, and growth factors were seen following treatment, including increased levels of chemoattractant IP-10 (CXCL10) and decreased levels of VEGF-A (230). Endothelial anergy induced by VEGF-A could be demonstrated in these samples and reversed by the addition of bevacizumab (230). A recent report describes results from a phase I study combining bevacizumab and the anti-PD-L1 antibody atezolizumab in the treatment of advanced renal cell carcinoma (110).…”