Angiopoietins: Novel Targets for Anti-angiogenesis Therapy

Avastin (bevacizumab) is the standard bearer among anti-angiogenic biological drugs currently on the market. It is a humanized monoclonal antibody (mAb) that targets vascular endothelial growth factor (VEGF). In spite of FDA’s recent revoking of the previous approval of the drug for the treatment of breast cancer, Avastin is used worldwide for colorectal, brain, lung, and kidney cancer. The drug generated $6.3 billion in 2012 sales for Roche/Genentech. Avastin was rejected for approval in the US for ovarian cancer although it was approved in the EU. Recently, a new set of data from the US National Institutes of Health showed that the drug increased lifespan in patients with advanced cervical cancer.

Despite such controversy regarding its effectiveness in various cancer types, Avastin has established the field of angiogenesis as a target for discovering and developing potential anti-angiogenic drugs, not only for the treatment of cancer but also for other disease indications. Ranibizumab, for example, is a mAb fragment derived from bevacizumab that has been approved for wet age-related macular degeneration (AMD). Eylea (aflibercept), jointly developed and marketed by Bayer and Regeneron for wet AMD, is a recombinant fusion protein consisting of portions of human VEGF receptors 1 and 2 extracellular domains fused to the Fc portion of human IgG1.

In an effort to generate safer and more efficacious anti-angiogenic therapeutics, the angiopoietin pathway has received increased attention in recent years as an alternative to the VEGF pathway as targets. Several angiopoietin family members have been identified, of which angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been employed as targets along with their tyrosine protein kinase receptor, Tie-2. The angiopoietin-Tie pathway is considered to be an especially attractive system for therapeutic intervention because of its crucial role not only for angiogenesis and vascular homeostasis but also for the important link it provides between angiogenic and inflammatory pathways. This article reviews currently available anti-angiogenic treatments and novel therapies in development that target the angiopoietin pathway.

Background on angiogenesis
The process of angiogenesis, (i.e., the development of new vasculature from pre-existing blood vessels and/or circulating endothelial stem cells) has been known to medical practitioners and scientists for more than 200 years. Angiogenesis plays an important role in many physiological processes such as embryogenesis, wound healing, and menstruation as well as pathological events including solid tumor growth (see Figure 1) and metastasis, arthritis, psoriasis, and diabetic retinopathy (1, 2).

Major steps in angiogenesis, as shown in Figure 2, involve activation of endothelial cells, migration, proliferation, sprout formation, tube formation, and stabilization of the capillary sprout by recruitment of pericytes and deposition of basement membranes. The angiogeneic process is regulated in normal and malignant tissues by the balance of multiple angiogenic factors. Some of these factors are stimulants while others are inhibitors, produced in the target tissue and at distant sites. Angiogenesis occurs when the net balance is tipped more favorably towards the stimulant factors.

Judah Folkman introduced the concept of anti-angiogenesis in 1971 based on the theory that blocking blood vessel formation in tumors will retard or block their growth. Since that pioneering work four decades ago, the anti-angiogenesis field has moved beyond its conceptual stage into a major force in modern medicine by generating several anti-angiogenic drugs for cancer, AMD, diabetic wounds, and inflammation. Some of these drugs are already approved while others are undergoing development. Beneficial effects are generated either by disabling the factors that activate and promote cell growth, or by blocking the multiple targets within the signal transduction pathway of angiogenic stimulators (see Figure 3).

On the basis of their therapeutic targets, FDA-approved drugs can be grouped into four major categories: small-molecule growth factor inhibitors, mAb therapeutics, small-molecule receptor tyrosine kinase (RTK) and its pathway inhibitors, and those with unknown mechanism of action. The most prominent among the targets of currently marketed drugs is VEGF. While Tarceva (erlotinib) and Iressa (gefitinib) block production of angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF), Avastin binds human VEGF and prevents activation of VEGFR1 and VEGFR2. It was first marketed in 2004. Several drugs such as Nexavar (sorafenib), Sutent (sunitinib), and Glivec (imatinib) are approved small-molecule drugs that target any of the three major signal transduction pathways for angiogenic growth factors. These drugs include the Ras/Raf/MAPK pathway, the PI3K/Akt/mTOR pathway, and PKC pathway (see Figure 3).

Afinitor (everolimus) and Torisel (temsirolimus), on the other hand, work by inhibiting intracellular metabolic pathway mTOR, which is frequently impaired in many cancers. Velcade (bortezomib) and Thalomid (thalidomide) inhibit angiogenesis by mechanisms of action that are not fully understood (see Figure 4). Despite the clinical benefits of the current anti-angiogenic drugs, there are multiple concerns, arising from resistance to the drugs as well as other side effects, including hypertension, thromboemboli, hemorrhage, gastrointestinal perforations, and potentially life-threatening events such as wound dehiscence in certain patients.

Angiopoietins as new targets for anti-angiogenic drugs
Ang-1 and Ang-2 are functional ligands of the Tie-2 receptor tyrosine kinase. Ang-1, expressed by many cell types such as pericytes, smooth muscle cells, and fibroblasts, acts as an agonistic ligand of Tie-2. Ang-1 mediated Tie-2 activation leads to decrease of endothelial cell permeability and stabilization of developing blood vessels. On the other hand, Ang-2 expression is carried out by endothelial cells. Ang-2 largely acts as an antagonist of Tie-2 by blocking Ang-1-dependent Tie-2 activation. Upregulation of Ang-2 correlates with metastasis and malignancy of various types of human cancers such as breast, metastatic melanoma, and lung (3-5).

Moreover, this upregulation behavior of Ang-2 is observed in proangiogenic diseases such as macula degeneration, rheumatoid arthritis, osteoarthritis, and psoriasis. Local production of Ang-2 has been identified as an early marker of glioma- and glioblastoma-induced neovascularization. More interestingly, upregulation of Ang-1 and Ang-2 has been shown to be a part of “angiogenic rescue” when VEGF-mediated angiogenesis is blocked during tumor progression, resulting in acceleration of metastasis (6-9).

Given the role of Ang-1 and Ang-2 in angiogenesis, therapeutics against these angiopoietins might provide a benefit to patients with cancer as well as other angiogenesis-prone diseases. Various approaches aimed at developing angiopoietin-targeted drugs include mAbs, bitargeted antibodies, and small molecule kinase inhibitors. Table I lists a selected number of candidates that are either in clinical research or in early stages of development.

Table I: Agents targeting angiopoietin-Tie pathway in development.

Agent

Type

Target

Disease

Status

Company

AMG-386

Peptibody

Ang-1, Ang-2

Various cancers

Phase I-III

Amgen

CVX-060

CovX body

Ang-2

Cancer

Phase I

Pfizer

CEP-11981

Small molecule

Tie-2 and
VEGFR

Cancers

Phase I

Teva

MEDI3617

mAb

Ang-2

Cancers

Phase I

AstraZeneca

Ang-2/VEGC lgG1

Bivalent-cross mAb

Ang-2/VEGF

Cancers

Preclinical I

Roche

Anti-Ang-1/2
mAb

Bitargeted
mAb

Ang-1, Ang-2

Cancers

Clinical

Synergys/NeoPharm

A wealth of new therapeutic agentsAMG-386 (Amgen)
AMG 386 is an anti-angiopioetin peptibody (peptide-Fc fusion protein) developed by Amgen, targeting the angiopoietin axis by blocking the interactions between Ang-1 and Ang-2 and their receptor
Tie-2. Dual inhibition of Ang-1 and Ang-2 by AMG-386 resulted in suppression of tumor xenograft growth and ovarian follicular inhibition in animal models. In dose-escalation studies in patients with solid tumors, AMG-386 was generally well tolerated. It reduced tumor blood flow and displayed modest antitumor activity (10). However, in a Phase I combination trial with a FOLFOX-4 regimen (carboplatin and paclitaxel or docetaxel) for advanced solid tumors, both complete and partial responses were observed (11). In a followup Phase II study, AMG-386 in combination with paclitaxel showed improvement in progression-free survival among recurrent ovarian cancer patients (12). In 2011, Amgen started a Phase III global multicenter, randomized, double-blind trial of paclitaxel and carboplatin plus AMG 386 or placebo in women with stage III-IV epithelial
ovarian, primary peritoneal or fallopian tube cancers. The primary endpoint is progression-free survival.

CVX-060 (Pfizer)
Pfizer scientists created a new class of biotherapeutics called CovX-bodies by chemical fusion of a peptide and a carrier antibody scaffold. Pfizer’s bivalent Ang-2 CovX-bodies such as CVX-060, which is a humanized mAb fused to two Ang-2 binding peptides, selectively blocks the Ang-2-Tie-2 interaction with dramatically improved pharmacokinetics, yielding a half-life of 100 hours. In a preclinical colorectal cancer trial, a significant tumor growth inhibition (TGI of 40-63%) was observed (13). Ang-2 protein levels were reduced by approximately 50% inside tumors, while tumor microvessel and intratumor proangiogenic Tie-2 +/ CD11b+ cells were significantly reduced. When combined with either sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang-2 CovX-bodies produced even greater efficacy (approximately 80% TGI) (13). In a Phase Ib safety trial in patients with advanced solid tumors, CVX-060 was found to be safe when combined with sunitinib at various dosage levels without exacerbation of sunitinib-associated toxicities (14). Combination trials of CVX-060 and other VEGF inhibitors are planned.

CEP-11981 (Cephalon/Teva Pharmaceuticals)
CEP-11981 is an orally bioavailable small molecule inhibitor of the vascular endothelial growth factor receptor and Tie-2 receptor tyrosine kinases. It inhibited the formation of blood vessels and thereby slowed growth and/or induced regressions of a variety of tumors in preclinical models. Consequently, the drug was evaluated in a clinical trial for its anti-angiogenic and antineo-plastic activities. The open-label study was conducted to determine the maximum tolerated oral dose of the kinase inhibitor in patients with advanced cancer. The study has been completed and a publication based on these findings is in preparation.

MEDI3617 (MedImmune/AstraZeneca)
MEDI3617 is a human anti-Ang-2 mAb. The compound demonstrated neutralizing capability of Ang-2 by preventing its binding to the Tie-2 receptor in vitro. It also showed inhibition of angiogenesis and tumor growth in vivo in several mouse models both as a single agent, as well as in combination with chemotherapeutic agents, or the mAb bevacizumab. A Phase 1/1b, open-label, dose-escalation, and expansion study has been initiated to evaluate the safety and antitumor activity of MEDI3617 as a single-agent or in combination therapy in adult subjects with advanced solid tumors.

Bispecific bivalent Ang-2/ VEGF IgG1 cross mAb (Roche)
Roche has generated a novel human bispecific bivalent IgG1 cross mAb that blocks VEGF-A and Ang-2 function simultaneously. A cross mAb format enforces correct chain association in a bispecific heterodimeric antibody. In multiple subcutaneous and orthotopic in-vivo models, the Ang-2-VEGF cross mAb, which is more selective for Ang-2 than Ang-1, effectively reduced angiogenesis, tumor growth and metastasis (15). The cross mAb is expected to exhibit a better side effect profile compared to the respective monotherapies. The drug is positioned to enter a Phase I clinical trial.

Anti-Ang-1/2 mAb (Synergys/NeoPharm)
Synergys Biotherapeutics (Walnut Creek, CA) has in-licensed a therapeutic fully human anti-Ang-1/2 IgG1 antibody from NeoPharm (Daejeon, South Korea) for development as an anti-cancer and anti-inflammatory drug. The antibody was isolated from a human scFv phage display library, affinity matured, and converted to human IgG. This first-in-class mAb showed reduced tumor growth in colon and pancreatic preclinical cancer models. When used in combination with gemcitabine, the mAb reduced tumor size and blood vessel formation.

Future prospects
Significant progress has been made during the past couple of decades in the field of anti-angiogenesis drug discovery for cancer, ocular, and anti-inflammatory disease areas. Consequently, several therapeutics, both small molecules and biologics, have received market approval. Most of the approved drugs, however, are targeting either VEGF or a component of the VEGF pathway for inhibition of angiogenesis. Because of the significant concerns about safety issues involved in some of these drugs, and also due to the fact that they only provide a modest efficacy and survival benefits, the angiopoietin-Tie-2 pathway has become an alternative target area as new anti-angiogenesis drug discovery treatment strategy. Several candidates are in clinical trials with efficacy and potentially better safety profiles, and more are already in the pipeline as next generation anti-angiogenic treatments.

From the early days of cancer chemotherapy, clinicians have been frustrated by the ability of cancer cells to develop resistance towards the antitumor agents. Cancer cells are highly unstable due to the collapse of effective DNA repair systems as well as the intervention of epigenetic variability that may occur at rates that are orders of magnitude greater than that of base pair changes in the responsible genetic loci. This phenomenon has spurred the development of adjuvant chemotherapy protocols, in which two or more agents may be combined, lowering the probability of simultaneous resistance to multiple agents.

Antibody treatments are not immune to drug resistance, given that treatment of metastatic cancers brings prolonged survival but not a cure. Additionally, because of the redundancies in molecular pathways underlying tumor
formation and survival, tumors are able to adapt and generate resistance to such treatments of individual antibody therapeutics. Combination of antibodies as new therapeutic modalities is being increasingly explored for the purpose of overcoming this drawback (16).

While several clinical studies are being conducted with bevacizumab in combination with other antibodies such as rituximab, cetuximab, transtuzumab, pertuzumab, and MetMAb, bispecific or bitargeted antibodies, where a single antibody is engineered to target two antigens, are being widely employed as a variation of the combination antibody strategy.

A few examples of such bitargeted or bispecific antibodies involving angiopoietins as potential anti-angiogenic drugs have been discussed in this article, some of which are already in clinical studies for the treatment of various cancers. Novel bispecific antibodies are being generated for other indications as well, for example, one that targets TNF-α and VEGF for retinopathy and psoriasis (17), and another consisting of an anti-TNF-α antibody
coupled to an Ang-2 targeting peptide that enhanced efficacy in an in-vivo model of arthritis (18). With a substantial toolbox available and a number of newly developed strategies under evaluation, the coming years look promising in terms of advances in anti-angiogenic therapeutic outcome.

References
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About the Authors
Rathin Das, PhD, MBA, is chief executive officer of Synergys Biotherapeutics, 1945 Arbol Grande, Walnut Creek, CA 94595, rathindas2009@gmail.com; and K. John Morrow, Jr., PhD, is president of Newport Biotechnology Consultants, 625 Washington Avenue, Newport, KY 41071, kjohnmorrowjr@insightbb.com.