A Human Monoclonal Anti-ANG2 Antibody Leads to Broad Antitumor Activity in Combination with VEGF Inhibitors and Chemotherapy Agents in Preclinical Models

Abstract
Localized angiopoietin-2 (Ang2) expression functions as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches due to reduced immunogenicity and increased half-life. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti-vascular endothelial growth factor (VEGF) agents showed significant improvements in antitumor activity over single-agent treatments alone, with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or VEGF inhibitors for the treatment of cancer.

Introduction
Angiopoietin-2 (Ang2) is a context-dependent regulator of vascular development and tumor-associated blood vessel formation, pointing to its potential as a next-generation antiangiogenesis therapy. Ang2-Tie2 signaling plays a significant role in the tumor microenvironment, influencing blood vessel plasticity, recruitment of Tie2-expressing monocytes, and regulation of extracellular matrix interactions in the vascular endothelium. While Ang1 and Ang2 share about 55% sequence identity and bind Tie2 with similar affinity, they have distinct functional roles. Ang2 acts as either an agonist or antagonist to Tie2 signaling, increasing vascular permeability and promoting endothelial cell migration and angiogenesis in response to VEGF and other inducers. Ang2 expression is dynamic and heterogeneous in tumors, correlating with regions of high angiogenic activity, while Ang1 is constitutively expressed and maintains blood vessel stability.

Blocking Ang2-Tie2 interactions is hypothesized to be an effective antiangiogenic therapy for solid tumors. Deletion of Ang2 in mice leads to vascular defects and lethality. Previous studies with anti-Ang2 antibodies, peptide-Fc fragments, and Ang2-specific RNA aptamers have shown in vivo efficacy and antiangiogenic activity in tumor models. Elevated Ang2 levels have been reported in various human tumors, including breast, colon, lung, prostate, liver, gastric, glioma, renal, ovarian, and melanoma. Ang2 may also promote early tumor growth by recruiting Tie2-expressing monocytes and stimulating tumor cell motility and invasion via α5β1 integrin interactions.

This study describes the generation and characterization of the first fully human Ang2-specific monoclonal antibody, 3.19.3, and its broad antitumor activity as a single agent and in combination with VEGF inhibitors and cytotoxic chemotherapy in preclinical models.

Materials and Methods
Antibody Generation:
Human anti-Ang2 antibodies were generated by immunizing Abgenix XenoMouse strains with recombinant human Ang2. Hybridomas were screened for Ang2 binding, lack of cross-reactivity with Ang1, and inhibition of Ang2 binding to Tie2. The top 27 neutralizing antibodies were selected, and 10 monoclonal lineages were further characterized. Clone 3.19.3 was chosen for its cross-reactivity with mouse Ang2 and potent inhibition of Ang2-induced Tie2 phosphorylation.

Binding Affinity and Selectivity:
The affinity of 3.19.3 for human Ang2 was determined using KinExA and Biacore technology. The antibody showed a dissociation constant (K_D) of 86 pmol/L for Ang2 and >500-fold selectivity over Ang1.

Functional Assays:
Competitive ELISA and cell-based assays were used to confirm the ability of 3.19.3 to block Ang2-Tie2 interactions and inhibit Tie2 phosphorylation. The antibody inhibited Ang2-induced Tie2 phosphorylation with an IC₅₀ of 580 pmol/L and showed minimal activity against Ang1-induced Tie2 phosphorylation.

In Vivo Angiogenesis and Tumor Models:
Matrigel plug assays and a variety of human tumor xenograft models (subcutaneous and orthotopic) were used to evaluate the antiangiogenic and antitumor efficacy of 3.19.3. Tumor growth inhibition was measured, and vessel density was assessed by CD31 immunohistochemistry.

Combination Studies:
3.19.3 was combined with VEGF inhibitors (vandetanib, cediranib, AZ10167514, DC101) and chemotherapy agents (docetaxel, 5-fluorouracil, irinotecan, oxaliplatin, gemcitabine) to assess additive or synergistic effects.

Pharmacokinetics:
Pharmacokinetic profiles were determined in mice and cynomolgus monkeys, with serum concentrations measured after single or multiple doses.

Results
Antibody Characterization:
3.19.3 binds Ang2 with high affinity (K_D = 86 pmol/L) and shows >500-fold selectivity over Ang1. It blocks Ang2-Tie2 binding and inhibits Ang2-induced Tie2 phosphorylation in cell-based assays.

Inhibition of Angiogenesis:
In Matrigel plug assays, 3.19.3 treatment resulted in an 86% reduction in vessel length and nodes, and an 89% reduction in functional blood vessel formation compared to controls.

Antitumor Activity:
As a single agent, 3.19.3 inhibited tumor growth by 33% to 71% across multiple xenograft models, including LoVo, SW480, A431, Colo205, HT29, Calu6, HCT116, MCF7, and MDA-MB-231. In orthotopic models, inhibition reached up to 73%. No significant toxicity was observed, as judged by stable body weights.

Reduction in Tumor Vasculature:
CD31 staining showed a 64% reduction in blood vessel density in Colo205 tumors and a 40% reduction in MDA-MB-231 tumors after 3.19.3 treatment. Treated tumors also exhibited increased necrosis and restricted growth to regions near surviving vasculature.

Combination Therapy:
Combination of 3.19.3 with VEGF inhibitors or chemotherapy agents resulted in significantly greater tumor growth inhibition than either agent alone. For example, in LoVo xenografts, the combination of 3.19.3 and vandetanib led to 83% inhibition, compared to 46% and 48% for the single agents, respectively (P<0.0001). Similar additive effects were seen with other VEGF inhibitors and chemotherapy agents across different models, with no increased toxicity.

Pharmacokinetics:
In mice, the terminal elimination half-life of 3.19.3 was 11–12 days. In cynomolgus monkeys, the half-life was 3–4 days. No immunogenicity was detected in monkeys. The predicted human half-life is 10–14 days, similar to other therapeutic antibodies.

Discussion
The Ang2-Tie2 system is crucial for blood vessel formation during development and in pathological angiogenesis. The fully human monoclonal antibody 3.19.3 selectively binds Ang2, blocks its interaction with Tie2, and inhibits downstream signaling. In vivo, 3.19.3 potently inhibits angiogenesis and tumor growth as a single agent and enhances the efficacy of VEGF inhibitors and chemotherapy when used in combination, without additional toxicity.

The antiangiogenic effect of 3.19.3 is supported by reductions in vessel density and increased tumor necrosis. The antibody’s high selectivity for Ang2 over Ang1 minimizes interference with normal vessel maintenance. The pharmacokinetic profile supports dosing schedules compatible with clinical use.

Combination therapy with 3.19.3 and VEGF inhibitors or chemotherapy may offer improved clinical outcomes by providing a more comprehensive blockade of angiogenesis. These preclinical results support further clinical investigation of 3.19.3 as a novel antiangiogenic therapy for solid tumors, both as a single agent and in combination regimens.

Conclusion
3.19.3 is a potent, fully human anti-Ang2 monoclonal antibody that inhibits angiogenesis and tumor growth in preclinical models. It shows broad antitumor activity as a single agent and significantly enhances the efficacy of VEGF inhibitors and chemotherapy agents when used in combination, without increasing toxicity. The antibody’s favorable pharmacokinetic and immunogenicity profiles support its potential for clinical development as a novel antiangiogenic Tie2 kinase inhibitor 1 therapy for cancer.