Antiangiogenesis beyond VEGF inhibition: A journey from antiangiogenic single-target to...

Cancer Treatment Reviews xxx (2013) xxx–xxx

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Cancer Treatment Reviews

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Anti-Tumour Treatment

Antiangiogenesis beyond VEGF inhibition: A journeyfrom antiangiogenic single-target to broad-spectrum agents

0305-7372/$ - see front matter � 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.ctrv.2013.11.009

⇑ Corresponding author. Address: Coordenação de Pesquisa Clínica e Incor-poração Tecnológica, Instituto Nacional de Câncer – INCA, Rua André Cavalcanti, 37– 2� andar, sala 40, Rio de Janeiro, RJ – CEP 20231-050, Brazil. Tel.: +55 21 32076544; fax: +55 21 3207 6566.

E-mail address: [email protected] (G. Limaverde-Sousa).

Please cite this article in press as: Limaverde-Sousa G et al. Antiangiogenesis beyond VEGF inhibition: A journey from antiangiogenic single-tabroad-spectrum agents. Cancer Treat Rev (2013), http://dx.doi.org/10.1016/j.ctrv.2013.11.009

Gabriel Limaverde-Sousa ⇑, Cinthya Sternberg, Carlos Gil FerreiraCoordination of Clinical Research and Technology Incorporation, National Cancer Institute of Brazil (INCA), Rio de Janeiro, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 July 2013Received in revised form 22 November 2013Accepted 25 November 2013Available online xxxx

Keywords:Angiogenesis inhibitorsBevacizumabDrug therapyEndostatinMonoclonal antibodiesProtein-tyrosine kinasesSunitinibVascular endothelial growth factor

Although the inhibition of angiogenesis is an established modality of cancer treatment, concerns regard-ing toxicity and drug resistance still constitute barriers to be overcome. For almost a decade since theapproval of bevacizumab in 2004, the efforts on antiangiogenic therapeutics have been mainly focusedin inhibiting the VEGF pathway. The ongoing understanding of the complexity of the angiogenic processhas broadened the spotlight to include concurrent and downstream players to the list of targeted inhib-itors. In this review, we summarize the currently existing and the promising antiangiogenic treatments,envisioning an apparent evolutionary trend towards the development of angiogenesis inhibitors of threemodalities: single-target, multi-target, and broad-spectrum agents. The clinical efficacy and some struc-tural aspects of monoclonal antibodies, small molecules, endogenous and synthetic angiogenesis inhibi-tors and their molecular targets are discussed, and the targeting of endothelial cells with the use ofcytotoxic drugs in a metronomic schedule is appraised. The reader is invited to revisit current expecta-tions about antiangiogenic therapy in an attempt to set consistent clinical endpoints from which patientscould gain real and lasting clinical benefits.

� 2013 Published by Elsevier Ltd.

Introduction

Given the premise that tumor growth, progression and metasta-sis depend on angiogenesis [1], targeting of tumor blood vesselshas been considered a logical approach to the treatment of avariety of malignancies over the past decades [2]. Indeed, there isenough support to the notion that targeting angiogenesis ingeneral, and the vascular endothelial growth factor (VEGF) in par-ticular, constitutes a viable and effective therapeutic strategy inoncology [3–6]. On the other hand, angiogenesis comprises a seriesof complex processes, many of which offer opportunities and chal-lenges for therapeutic intervention beyond inhibition of the VEGFpathway. Because angiogenesis is regulated by different factors,often displaying redundant actions, the inhibition of a single agentis likely to increase the dependency of alternative pathways or toinduce the selection of cells whose angiogenesis is driven by otherfactors [7].

Many antiangiogenic agents directed to molecules other thanVEGF have undergone preclinical and clinical assessment of their

safety and efficacy. These targets include VEGF receptors (VEGFR),platelet-derived growth factor receptors (PDGFRs), and fibroblastgrowth factor receptors (FGFRs), among others. Additionally,endogenous and synthetic broad-spectrum antiangiogenic mole-cules have been developed, providing an attractive alternative tospecific inhibitors.

Although many promising avenues for investigation have beenidentified, and notwithstanding the approval of active and valuableagents, the initial enthusiasm with some target-specific antiangio-genic drugs has been hampered by unimpressive clinical results,and by the fact that angiogenesis inhibitors currently approvedby the FDA display similar issues to cytotoxic therapy: (i) theacquired or innate resistance to the treatment and (ii) significanttoxicity [8].

In this review, a brief discussion of successful and failed antian-giogenic strategies is presented, and an attempt is made to providean overview of currently existing antiangiogenic modalities be-yond the specific inhibition of the VEGF pathway, which are likelyto become the greatest promise for clinical use in the near future(Table 1).

Angiogenesis promoting molecules

In a nutrition deficient microenvironment, a small aggregateformed by a few number of tumor cells can migrate and manage

rget to

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Table 1Antiangiogenic agents, their respective molecular targets and current indications for cancer therapy (approved or in trial).

Anti-angiogenicspectrum

Agent Target Cancer type Phase of development

Single-target Bevacizumab (avastin) VEGF-A Metastatic colorectalcancer, non-squamous non-small cell lung cancer,glioblastoma, metastaticrenal cell carcinoma

Approved (U.S./E.U.)

Cetuximab (erbitux) EGFR Squamous cell carcinoma ofthe head and neck,colorectal cancer


Panitumumab (vectibix) EGFR Metastatic colorectal cancer Approved (U.S./E.U.)Trastuzumab (herceptin) HER-2 HER2-overexpressing breast

and gastric cancerApproved (U.S./E.U.)

Multi-targeted Aflibercept (VEGF-trap; AVE0005;zaltrap)

VEGF-A, VEGF-B, PlGF Metastatic colorectal cancer Approved (U.S.), pending (E.U.)

Axitinib (AG013736; inlyta) VEGFR-1, -2, -3, PDGFR, KIT Advanced renal cellcarcinoma


Cediranib (AZD2171; recentin) VEGFR-1, -2, -3, PDGFR, KIT Metastatic colorectalcancer, recurrentglioblastoma, ovariancancer, lung cancer

Phase III

Indetanib (BIBF1120; vargatef) VEGFR, PDGFR, FGFR Non-small cell lung cancerand ovarian cancer

Phase III

Pazopanib (GW786034; votrient) VEGFR-1, -2, -3, PDGFR, KIT Advanced renal cellcarcinoma, advanced softtissue sarcoma


Sorafenib (BAY439006; nexavar) VEGFR-2, -3, PDGFR, Raf, KIT Hepatocellular carcinoma,advanced renal cellcarcinoma


Sunitinib (SU11248; sutent) VEGFR-1, -2, -3, PDGFR, KIT,FLT3, CSF-1R, RET

Gastrointestinal stromaltumor (GIST), advancedrenal cell carcinoma,advanced or metastaticpancreatic neuroendocrinetumors


Vandetanib (ZD6474; caprelsa) VEGFR-2, EGFR, KIT, RET Medullary thyroid cancer Approved (U.S./E.U.)Vatalanib (PTK787; ZK222584) VEGFR-1, -2, -3, PDGFR, KIT Colorectal cancer Phase III

Broad-spectrum ABT-510 CD36 Head and neck cancer,melanoma, lymphoma,renal cell carcinoma, softtissue sarcoma, non-smallcell carcinoma

Phase II

ATN-161 avb3 and avb1 integrins Brain and central nervoussystem tumors, renal cellcarcinoma

Phase II

Angiostatin ATP synthase, NG2,angiomotin, avb3 integrin,c-met, annexin II

Non-small cell lung cancer Phase II

Cilengitide avb3 and avb5 integrins Glioma, glioblastoma Orphan designation (E.U.), PhaseIII

Endostatin (endostar) a5b1 and avb3 integrins,surface nucleolin, caveolin-1, glypicans-1 and -4, MMP-2, VEGFR-2

Non-small cell lung cancer,breast cancer

Approved (China), phase III/IV

Lenalidomide (CC-5013; revlimid) CRBN Multiple myeloma Approved (U.S./E.U.)pomalidomide (CC-4047) CRBN Multiple myeloma Orphan designation (E.U.), Phase

IIIThalidomide (thalomid) CRBN Multiple myeloma Approved (U.S./E.U.)TNP-470 MetAp2 Pancreatic cancer Phase II

2 G. Limaverde-Sousa et al. / Cancer Treatment Reviews xxx (2013) xxx–xxx

to co-opt pre-existing vessels. Following this stage, angiogenesis isa crucial step for tumor exponential growth beyond 1–2 mm andfor the development of metastasis. During tumorigenesis, a chronicunregulated angiogenic state is installed as cells within the tumorbecome depleted of nutrients and oxygen, leading to an increaseddemand for these substances and the concurrent release of angio-genic factors that will promote the expansion of the vascularnetwork.

The key stimulators of angiogenesis are the vascular endothelialgrowth factor (VEGF, also known as VEGF-A), fibroblast growth fac-tor 2 (FGF2, also known as bFGF), angiopoietins 1 and 2, hepatocytegrowth factor (HGF) and ephrin. Several of these molecules are

Please cite this article in press as: Limaverde-Sousa G et al. Antiangiogenesisbroad-spectrum agents. Cancer Treat Rev (2013), http://dx.doi.org/10.1016/j.ct

either current therapeutic targets or are under investigation to be-come one.

Single-target angiogenesis inhibitors: the first generation

Conventional chemotherapy affects cycling cells, and is associ-ated with significant side effects such as hair loss, oral mucositis,and immunosuppression. To reduce such overall off-target toxicity,engineered small-molecule inhibitors and monoclonal antibodies(mAbs) directed to pathways that are overstimulated mainly inthe pathological context of malignant cells were developed for can-cer therapy.

beyond VEGF inhibition: A journey from antiangiogenic single-target torv.2013.11.009


G. Limaverde-Sousa et al. / Cancer Treatment Reviews xxx (2013) xxx–xxx 3

In this context, instead of targeting nonspecific cell prolifera-tion, antiangiogenic mAbs were developed to inhibit endothelialcells (EC) that are overstimulated to proliferate and migrate to-wards the tumor. Thus, the specific blockage of VEGF would, inprinciple, have a primary effect on pathological angiogenesis, thusbeing a potentially useful strategy against a broad group of malig-nancies. In preclinical studies, VEGF blockers led to significant tu-mor suppression in several types of cancer [9]. Therefore, thedevelopment of bevacizumab (Avastin) was pursued by Genentech,which aggressively invested in dozens of clinical trials across dif-ferent types of cancers and other angiogenesis-related diseases.

Bevacizumab (Avastin)

Bevacizumab is a recombinant humanized mAb against VEGF,one of the most important proangiogenic factors. This mAb con-tains the consensus human IgG1 framework and antigen-bindingregions (93%) and six complementarity-determining regions froma murine mAb (7%). Initially, the affinity of the humanized anti-body for VEGF was significantly weaker than that of the originalmurine antibody. To restore its binding affinity, the humanizedantibody was further engineered, replacing seven framework resi-dues in the variable heavy domain and one in the variable light do-main from human to murine. These modifications restored theaffinity of the humanized antibody to a similar value to that ofthe original murine antibody (Kd�0.5 nM) [9].

In phase III clinical trials, patients with metastatic colorectalcancer showed significant benefits when bevacizumab was com-bined with chemotherapy. These trials were followed by the FDAapproval of bevacizumab in 2004 for treatment of metastatic colo-rectal cancer in combination with 5-FU-based regimens.

Curiously, bevacizumab as a single agent has little activity invarious clinical settings, with response rates of 10% or less [10].Historically, such low response rates have been seldom followedby regulatory approval in the development of cytotoxic agents inoncology. Higher response rates for bevacizumab monotherapyhave been recorded in glioblastoma multiforme [11], in whichtreatment with VEGF inhibitors has been associated with pseu-doresponse, a decrease in contrast-enhancement of brain tumorswithout a decrease in tumor activity [12]. Nevertheless, the addi-tion of bevacizumab to cytotoxic agents leads to marked improve-ments in efficacy, as indicated by consistently increased responserates and progression-free survival (PFS) times [4,5,13]. At leasttwo hypotheses may be raised to explain such contradictory find-ings of low responses to bevacizumab monotherapy and high clin-ical activity when it is combined with cytotoxics. The first is thatalthough bevacizumab therapy alone is unlikely to induce tumorregression, its activity may be sufficient to control tumor progres-sion [7]. Second, bevacizumab has been found to initially reducetumor perfusion, vascular volume, and microvascular density, aconstellation of findings referred to as vascular normalization. Thisphenomenon has been proposed as a plausible explanation for theimproved activity of radiotherapy and cytotoxic agents given incombination with bevacizumab, as tumor normalization improvestissue oxygen levels and drug delivery [14].

Other mAbs with antiangiogenic action

Besides bevacizumab, other mAbs initially designed to blockepithelial growth factor (EGF/HER) receptors, such as cetuximab(Erbitux), trastuzumab (Herceptin) and panitumumab (Vectibix),also exhibit antiangiogenic functions due to crosstalk betweenthe signaling pathways that involve VEGF and EGF/HER. Cetuximabdownregulates VEGF expression in a human colorectal carcinomacell line and in xenograft models [15], which indicates that EGF sig-naling mediates VEGF stimulation and tumor angiogenesis. On the


other hand, HER2 expression was found to correlate with VEGF-Cexpression and lymphangiogenesis in lymph-node-positive breastcancer [16].

Second generation multi-targeted angiogenesis inhibitors:VEGFR and related targets

The downside of the concept of ‘‘smart drugs’’, which are usu-ally designed with a very specific target as an attempt to avoid sideeffects, is that it often leads to drug resistance. In fact, bevacizumabmonotherapy induces a rapid development of resistance in meta-static patients [8]. It is thought that the targeted inhibition of theVEGF pathway and consequent decrease in proliferation and sur-vival of tumor ECs is initially beneficial, but continued treatmentmay lead to increased hypoxia and trigger alternative pathwaysthat drive disease progression, such as PDGFR and FGFR pathways,which are therefore promising targets for optimized antiangio-genic therapy.

Aflibercept (VEGF-Trap, AVE0005, Zaltrap), another VEGF liganddisplaying a wider binding spectrum when compared with bev-acizumab, has been found to improve clinical results in random-ized trials. In advanced colorectal cancer, aflibercept significantlyimproved PFS and overall survival (OS) [17], while in advancedor metastatic non-small-cell lung cancer (NSCLC) it was shown toimprove response rates and PFS, but not OS [18]. This agent is a re-combinant protein containing the VEGF-binding domain fused tothe Fc portion of IgG, and functions as a soluble ‘‘decoy’’ receptor,preventing the binding of VEGF-A, VEFG-B and placental growthfactor (PIGF) to the cell surface receptors.

Tyrosine kinase inhibitors (TKIs)

Unlike antibodies, TKIs cross the cell membrane due to theirsmall size and hydrophobicity, interacting directly with the intra-cellular domain of receptors and/or other signaling molecules. Sev-eral multi-target TKIs display activity as single agents, producingresponse rates in the range of 30–40% in phase III trials [6], andsometimes improving survival [19]. TKIs have also been investi-gated in combination with chemotherapy, especially in advancedNSCLC, showing clinical benefits in some studies, but failing to pro-long OS in others [20,21].

Most currently available VEGFR inhibitors are also able to blockother receptor targets such as PDGFR, c-Kit, the epidermal growthfactor receptor (EGFR), or RET. This is the case of axitinib, pazopa-nib, sunitinib, vandetanib, and vatalanib. It was further demon-strated that sunitinib also inhibits FLT3 and CSF-1R. Moreover,the VEGFR inhibitor sorafenib also targets BRAF, a non-receptorkinase, in addition to PDGFR, c-Kit, and Flt-3. Agents that blockVEGFRs or other angiogenesis modulators are also under develop-ment, such as nintedanib (BIBF1120), an inhibitor of VEGFR,PDGFR, and FGFR signaling. Nintedanib has shown promising re-sults in early clinical trials and is currently in phase III for NSCLCand ovarian cancer [22,23], with some studies evaluating its effi-cacy in combination with chemotherapy [24,25]. In advancedNSCLC, combination of nintedanib with docetaxel as second-linetreatment has been associated with PFS gains and, in certain sub-groups, improved OS [24]. Prolonged PFS was also reported withnintedanib plus pemetrexed after failure of first-line chemother-apy [25]. Given the role of PDGFR and FGFR in tumor biology, espe-cially regarding pericytes and tumor-cell function, it is difficult toassess the contribution of individual targets to the clinical activityof the multi-target agents. However, the preclinical evidencepoints to multi-target inhibition as a sound strategy [26] and theresults of the ongoing phase III clinical trials are eagerly awaited.




Binding modes of antiangiogenic TKIs

Tyrosine kinase receptors (TKRs) transduce extracellular signalsinto the cell via dimerization and auto-phosphorylation of specificintracellular tyrosine residues. The extracellular N-terminal regionof TKRs has a ligand-binding domain, while the kinase domain islocated in the intracellular C-terminal region. The kinase domainhas a bi-lobar structure, with the ATP binding site located betweenthe N- and the C-terminal lobes. In the C-terminal lobe, an activa-tion loop contains a combination of amino acid residues (asparticacid, phenylalanine and glycine), called DFG motif. When inactive(in the so-called DFG-out conformation), this loop creates a hydro-phobic pocket nearby the ATP-binding cleft that is targeted bysome TKIs. When inhibitors are bound to this pocket, the activationloop is unable to switch to the ‘‘active’’ (DFG-in) conformation,blocking signaling activation.

Most kinase inhibitors function by competing with the adeno-sine triphosphate (ATP) molecule, which establishes hydrogenbonds between the adenine ring and the amino acid residues atthe binding cleft of the kinase. Small-molecule inhibitors usuallytarget a site nearby the ATP-binding cleft, mimicking the interac-tions of ATP with the enzyme. Inhibitors that act by this kind ofinteraction, such as sunitinib, are classified as type I TKIs [27].

In contrast to type I TKIs, type II inhibitors, like sorafenib, haveaffinity to the inactive form of the protein kinase. Thus, instead ofdirectly blocking the ATP site, they bind to a hydrophobic pocketthat forms when the activation loop of the kinase acquires theDFG-out conformation. Such interaction with this allosteric siteimpairs conversion of the inactive to the active DFG-in conforma-tion, thus inhibiting its kinase activity [28].

Type III TKIs are called irreversible inhibitors, since they harbora warhead chemical group that covalently links through a disulfidebond to specific cysteine residues in the kinase binding site. Vand-etanib (ZD6474) is a quinazoline-based inhibitor that irreversiblyblocks ATP binding in VEGFR and EGFR, rendering these receptorsinactive [29].

In an attempt to elucidate the structural basis of TKIs action, re-cent results correlated in vitro potencies, whole-cell assays andclinical effects of the VEGF blockade with crystallographic data ofcomplexes of several inhibitors with VEGFR2. A VEGFR constructcontaining a regulatory juxtamembrane domain provided a morefunctionally accurate target for a systematic discovery of VEGFRinhibitors. This strategy enabled a better comparison betweenin vitro results and clinically observed outcomes, opening perspec-tives for design and selection of potentially better drug candidates[30]. This detailed structural observation with such significantfunctional implications highlights the importance of a multi-disci-plinary team ranging from structural biologists and biochemists tophysicians in order to obtain a more comprehensive picture of thenature of the therapeutic target.

Clinical efficacy of TKIs compared with bevacizumab

Unfortunately, very few head-to-head comparisons betweenbevacizumab and a VEGFR inhibitor have been undertaken to date.In colorectal cancer, a phase III trial compared cediranib versusbevacizumab, both combined with oxaliplatin-based chemother-apy, in first-line therapy for advanced disease [31]. Since therewas no significant difference in PFS between cediranib and bev-acizumab, the study failed to meet its primary end-point of non-inferiority of the VEGFR inhibitor vis-à-vis the mAb. In advancedbreast cancer, bevacizumab was superior to sunitinib when bothwere combined with weekly paclitaxel, leading to early stoppingof a phase III trial [32]. In a randomized phase II trial in patientswith advanced renal-cell carcinoma (RCC), the combination ofinterferon-alpha and bevacizumab produced better efficacy results


than monotherapy with sunitinib; nonetheless, these results donot allow the determination of superiority of interferon-alpha plusbevacizumab over sunitinib, as the trial was not statistically pow-ered to address this issue [33].

mAbs plus TKIs: sequential and combination strategies

In spite of the availability of angiogenic inhibitors for testing,the literature is scarce regarding therapeutic strategies that entailsequential inhibition of different targets in the VEGF pathway.Most of evidence comes from retrospective studies or single-armclinical trials. It should be noted though that these agents weretested after disease progression on previous therapies, not beforedrug resistance was clinically evident. Arguably, the latter ap-proach would be more adequate for strategies that aim at prevent-ing or circumventing pharmacodynamic resistance. In RCC, amodel disease for the pathogenic role of VEGF pathway, studiessuggest that VEGFR inhibitors are active in a small proportion ofpatients initially treated with bevacizumab. Likewise, sequentialuse of VEGFR TKIs appears to be effective in some patients,although no phase III studies have been conducted to address theissue [34]. In colorectal cancer, very few responses were notedwhen sunitinib was administered to patients with prior use of bev-acizumab [35]. Thus, the extent to which cross-resistance existsbetween different inhibitors of the VEGF pathway is still incom-pletely understood. Moreover, the value of alternating differentagents before the clinical progression has not been investigatedyet.

Combined inhibition of VEGF and VEGFR has already been at-tempted, and bevacizumab with either sorafenib or sunitinib treat-ment is feasible in patients with advanced solid tumors, althoughdose reductions were frequently needed for sunitinib [36], whichwas also found to be too toxic for further development whencombined with bevacizumab in RCC patients [37]. Likewise, theaddition of sunitinib to standard doses of bevacizumab pluschemotherapy was not considered feasible in advanced breastcancer and NSCLC [38].Common toxicities associated with theuse of antiangiogenic therapy targeting the VEGF pathway are fati-gue, hypertension, diarrhea, nausea, vomiting, severe bleeding,gastrointestinal perforation, proteinuria and myelosuppresion[39], which might be due to disturbance of growth factor signalingin blood vessels, impacting the physiological maintenance and sur-vival of ECs and compromising vascular integrity.

Broad-spectrum angiogenesis inhibitors: the next generation

Pan-VEGF or multi-target TKIs are sometimes being consideredas broad-spectrum agents due to their capacity of binding to sev-eral growth factor receptors. However, some molecules have con-siderably broader activities in the physiology of ECs. Instead ofacting by a single mechanism of inhibition, like mAbs or TKIs,broad-spectrum agents make use of intricate mechanisms thatcounteract several processes in order to inhibit the downstreamangiogenic phenotype, regardless of which angiogenic factor hasinitiated the stimulus (Fig. 1).

Endogenous and peptidic antiangiogenic agents

The concern about side effects of multi-targeted TKIs is justifi-able, since their systemic administration affects several cell types.Therefore, the use of TKIs in tandem with chemotherapy should beavoided due to severe adverse effects. However, for certain broad-spectrum angiogenesis inhibitors, like angiostatin and endostatin,antiangiogenic activity does not seem to correlate to toxicity.




Angiostatin is a 38 kDa amino-terminal fragment of plasmino-gen that has been shown to block angiogenesis and suppressmetastasis in preclinical studies. Several cell surface bindingpartners were identified for angiostatin, such as ATP synthase,chondroitin sulfate proteoglycan NG2, angiomotin, avb3 integrin,c-met and annexin II. These ligands were shown to mediate its vastantiangiogenic activity in vitro, which includes induction of ECsapoptosis, inhibition of filopodial extension, migration and tubeformation, and the in vivo inhibition of tumor growth and metasta-sis [40,41].

Additionally, angiostatin has demonstrated antitumor activitiesthat are not restricted to its antiangiogenic properties, as it exertsdirect effect on tumor cells, such as increased apoptosis and reduc-tion of the proliferation rate and anti-inflammatory effects, includ-ing the inhibition of macrophage infiltration by disruption of itsmotility, as shown in in vitro and in vivo studies. A phase I studyshowed that angiostatin administered subcutaneously is safe forprolonged use [42]. In a phase II study, combination of angiostatinwith paclitaxel and carboplatin in advanced NSCLC resulted in highdisease control rate, compared with historical controls [43].

Endostatin is the 20 kDa C-terminal fragment of collagen XVIII[44], a proteoglycan mainly found in the basement membranearound blood vessels. A unified mechanism of action for endostatinactivity as antiangiogenic agent is still under debate. However,based on the data available to date, endostatin seems to act in bothextracellular matrix–independent and –dependent ways. In its yetincomplete list of mechanisms of action, endostatin is known toblock the response to growth factors, such as VEGF and FGF, affect-ing ECs survival, proliferation and migration. The overall angio-genic phenotype is inhibited by altering the expression of 12% ofthe EC genome, concomitantly affecting cytoskeleton, perivascularcell recruitment and extracellular matrix remodeling [45–49].

Fig. 1. Mechanisms of angiogenesis inhibition by single-target, multi-target and broad sinhibit angiogenesis by blocking the interaction between the ligand (e.g., VEGF) and receact through direct competition with ATP or by stabilizing the inactive conformation of thTyrosine kinase inhibitors are usually able to block more than one growth factor receptPDGFR in pericytes, axitinib may also block pericyte recruitment by angiogenic endotheangiogenic endothelial cell: besides blocking the signaling of angiogenic factors, endostaand downregulates many signaling pathways associated with proangiogenic activity, signmigratory phenotype by several mechanisms, such as: reduction of the extension of pseudavb3 integrins; and inhibition of matrix degradation by metalloproteinases.


Endostatin was shown in cell line studies and murine models toassociate with a5b1 integrin and caveolin-1 in ECs, and its antian-giogenic activity requires the presence of E-selectin [49–51]. Alow-affinity cell surface receptor was described for endostatin asbeing the heparan sulfate proteoglycans glypicans-1 and -4,although this receptor is not specific to endothelial cells [52].Surface nucleolin was described as a high-affinity receptor forendostatin [53], mediating its internalization and translocation tothe EC nucleus through a complex that also includes urokinaseplasminogen activator receptor (uPAR) and integrin a5b1 [54].Endostatin was also shown to associate to VEGFR-2 (KDR/Flk-1),avb3 integrin, and MMP-2, among other molecules [55].

Endostatin was first described in 1997 by Judah Folkman’sgroup and rapidly drove media attention and Wall Street enthusi-asm to EntreMed, the company that licensed its rights. When re-combinant endostatin expressed in bacteria was administeredsubcutaneously in mice as an insoluble preparation, it inducedregression of several types of tumors without inducing resistanceand showing virtually no signs of toxicity [44,56]. Endostatin be-came one of the fastest compounds to enter clinical trials, only3 years after its discovery. As the original administration formwas considered unsuitable for patients, a soluble formulation ofyeast-expressed recombinant human endostatin was used forintravenous infusion in clinical trials. A heterogeneous cohort ofheavily pre-treated patients with advanced solid tumors with met-astatic lesions refractory to standard therapy was submitted to dai-ly injections with soluble endostatin. No significant drug-relatedtoxicity was reported, even for the highest dose tested (600 mg/m2/day) [57]. In a phase I study, clinical benefit was observed inthree of 15 patients of whom two (13.3%) showed stable diseaseand one had a minor response [58]. Although stable disease wasobserved in phase I trials [58,59] and in 80% of patients in a phase

pectrum agents. Single-targets agents, like the monoclonal antibody bevacizumab,ptor (e.g., VEGFR). Multi-target agents, like tyrosine kinase inhibitors (e.g., axitinib),e kinase domain, thus blocking the angiogenic signaling downstream the receptor.or due to the degree of conservation among the kinase domains. By inhibiting the

lial cells. Broad-spectrum receptors, like endostatin, inhibit several pathways in thetin upregulates the expression of anti-angiogenic genes, such as thrombospondin-1,ificantly reducing the expression of HIF1-a. It was shown that endostatin affects theopods through inhibition of assembly of actin polymers; direct binding to a5b1 and




II trial, with a median duration of 10.8 months [60], with somecases exhibiting minor response, tumor regression was not consis-tently observed [57–61]. These results were not considered expres-sive enough when compared with the antitumor activity exhibitedby bevacizumab and the emerging TKIs. The unfulfilled expecta-tions hindered further investments, and clinical trials were haltedby EntreMed, alleging high manufacturing costs of the recombi-nant protein [62].

The use of a recombinant protein whose mechanism of action isnot completely known in clinical trials primes it to potential trans-lational problems. In fact, soluble recombinant endostatin showedactivity by inhibiting EC migration and tumor growth in mice mod-els. However, it did not induce the complete tumor regression ob-served with the insoluble preparation, unless when continuouslyadministrated by an implanted osmotic pump to maintain optimalcirculating levels. Curiously, the authors also speculate that the en-hanced activity may be due to some form of aggregation that mayoccur during incubation at 37oC while endostatin is residing in theimplanted osmotic pump [63]. Despite its halted progress in thewestern world, endostatin development was pursued in Chinaand has been approved by the Chinese SFDA since 2005 (underthe trade name of Endostar) for use in combination with chemo-therapy in patients with NSCLC [64].

To minimize the costs of production of recombinant proteins inlarge scale for therapeutic use, peptides that retain the activity ofthe parent molecule are under development. Peptides have lowtoxicity, high specificity for their targets and usually have a successrate in clinical trials that is two- to threefold higher than thosereported for small molecules [65]. In addition, advances in the so-lid-phase synthesis technology have enabled higher yields andreduction of manufacturing costs. Furthermore, peptides havegood tissue penetration due to their small size, hence facilitatingthe access to their targets.

Some peptides as cilengitide, derived from fibronectin, mimicthe RGD motif recognized by avb3 and avb5 integrins. These tar-gets are present in endothelium and some tumor cells, exhibitingboth antiangiogenic and direct antitumor effects in glioma cells[66]. Cilengitide is being tested in phase II trials in patients withprostate [67], renal, melanoma and colorectal cancers [68] andglioblastoma [69], showing a 6-month PFS of 15% and median OSof 9.9 months in the latter. ATN-161, a peptide derived from fibro-nectin, binds to avb1 and avb3 and has been shown to inhibit thegrowth of metastasis in a breast cancer model [70]. In a phase Itrial in patients with solid tumors, ATN-161 was well tolerated,and 23% of the patients showed stable disease for more than4 months [71].

In a systematic Structure–Activity-Relationship (SAR) study,ABT-510, a 9 amino acid long, synthetic analog of the endogenousantiangiogenic protein thrombospondin-1, was effective in block-ing neovascularization in mouse Matrigel plug assays and inhibitedtumor growth in the murine Lewis lung carcinoma tumor model[72]. Its antiangiogenic activity requires the expression of thethrombospondin receptor CD36 in the ECs [73]. Clinical studiesin soft tissue sarcoma [74], renal cell carcinoma [75] and glioblas-toma [76] showed that although ABT-510 is well tolerated and in-duces disease stabilization in some patients, its efficacy as singleagent was not demonstrated, and a combination with cytotoxictherapy should be considered.

Synthetic broad-spectrum antiangiogenic agents

Thalidomide (Thalomid) is an example of synthetic broad-spec-trum agent that has immunomodulatory, anti-inflammatory andantiangiogenic activities [77]. Once used as anti-emetic drug, itssevere teratogenic birth defects led to its rapid withdrawal fromthe clinic. Later on, the antiangiogenic activity of thalidomide


was demonstrated [78], and its antitumor properties were testedin patients, showing durable responses in refractory multiple mye-loma patients [79]. Thalidomide was approved in 2003 in Australiaand in 2006 in the U.S. in combination with dexamethasone formultiple myeloma, but its use is associated with dose-limiting tox-icities including neuropathy, fatigue, constipation [80] and an in-creased risk of venous thromboembolism, especially whencombined with dexamethasone [81]. The development of thalido-mide derivatives, like lenalidomide and pomalidomide, is beingpursued as an attempt to achieve therapeutic benefits with fewerside effects. Lenalidomide was FDA approved for patients with re-lapsed or refractory multiple myeloma, showing lower rates ofconstipation and neuropathy than thalidomide, but an increasedmyelosuppresion was observed [82]. Pomalidomide is currentlyon clinical trials, displaying encouraging activity with manageabletoxicity (42% of the patients achieved minimal response, 21% a par-tial response and 3% exhibited complete response) in patients withrelapsed and refractory multiple myeloma [83].

The mechanism by which immunomodulatory drugs exert theireffects is not well known, although direct and indirect mechanismsare attributed to its anti-myeloma activity. Lenalidomide andpomalidomide, for example, are capable of inducing apoptosis viacaspase 8 activation on multiple myeloma cells [84]. Thalidomideis able to modulate the TNFa-induced expression of cell adhesionmolecules on the surface of HUVECs and L-selectin on leukocytes[85], and to down-regulate bone marrow levels of VEGF in patientswith myeloma, significantly reducing the microvascular density inthe bone marrow of responding patients [86]. The protein cereblon(CRBN), which plays important roles in protein ubiquitination, cellsurvival, memory and learning, energy balance and fetal develop-ment [87], was recently described as a direct target for thalidomideand its derivatives. CRBN expression was shown to be required forthe activity of lenalidomide and pomalidomide and is possibly abiomarker for the anti-myeloma efficacy of these immunomodula-tory drugs [88].

Fumagillin, an antibiotic produced by the fungus Aspergillusfumigatus fresenius, was found to inhibit EC proliferation and angi-ogenesis in chorioallantoic membrane assay, but purified fumagil-lin causes severe weight loss in mice. To circumvent this problem,synthetic analogs were produced and the analog called TNP-470had comparable effects without the weight loss induction [89].TNP-470 irreversibly inactivates the enzyme methionine amino-peptidase-2 (MetAP2), causing EC cycle arrest in G1 phase viap53 and p21 and inhibition of tumor angiogenesis [90]. In clinicaltesting, TNP-470 showed evidence of anti-tumor activity, but pa-tients presented signs of neurotoxicity [91]. Further developmentof this agent led to a conjugated form of TNP-470 with an HPMApolymer, rendering the drug (then called caplostatin) unable tocross the blood–brain barrier, therefore reducing its potentialneurotoxicity [92]. Another formulation of TNP-470, lodamin, isable to form nanopolymeric micelles that are absorbed by theintestine, has longer half-life and accumulates in the tumor, allow-ing the drug to be taken orally instead of through continuousparenteral administration [93]. Clinical trials with lodamin areexpected.

Antiangiogenic (or Metronomic) Chemotherapy

Conventional chemotherapy is based on the concept of maxi-mum tolerated doses (MTD), according to which regimens usedin late phases of drug development and in clinical practice arethe higher dose levels that were safely administered to patientsin phase I and II trials. Since the MTD is usually toxic for both tu-mor and normally dividing cells, repeated treatment administra-tions must be separated in time (e.g., 3 weeks) to allow recoveryof normal cells, such as the hematopoietic progenitor cells. In the




year 2000, Browder and colleagues from Judah Folkman’s groupproposed a different schedule and dose regimens for currentlyused cytotoxic drugs, such as cyclophosphamide, with the aim oftargeting the endothelial component of tumors. Their hypothesiswas based on the possibility that, similarly to the bone marrowcells, tumor ECs may also be resuming growth during the treat-ment-free period, supporting tumor regrowth and increasing therisk of the emergence of drug-resistant tumor cells. Using thisrationale, they showed that cyclophosphamide administered atlower doses and with shorter interval periods was able to eradicatedrug-resistant tumors by inducing EC apoptosis [94]. The additionof TNP-470 to the regimen potentiated the antitumor effects,regressing drug-resistant tumors in more than 80% of the mice.This concept was confirmed when xenografts of neuroblastomacell lines were treated with low doses of vinblastine and a mAbagainst VEGFR, as sustained tumor regression without apparenttoxicity was observed [95]. The translation of this concept intothe clinic was first shown by the administration of a continuouslylow-dose cyclophosphamide and methotrexate, displaying mini-mal toxicity but effectiveness in heavily pretreated metastaticbreast cancer patients [96].

Further analysis showed that cyclophosphamide administeredat low doses affects different aspects of tumor microenvironment,such as ECs, pericytes, circulating ECs and infiltrating immune cells[97]. It was also demonstrated that low concentration of a deriva-tive of cyclophosphamide (4-hydroxy-cyclophosphamide) upregu-lates thrombospondin-1, an endogenous angiogenesis inhibitor, inECs [98]. Interestingly, animals treated with MTD-level cyclophos-phamide present a high mobilization of circulating ECs a few daysafter drug administration, associated with emergence of resistance.In contrast, administration of cyclophosphamide at low doses cor-relates with a decrease in the number of circulating EC progenitors,longer tumor growth inhibition, and better survival rates [99].

Drug combinations that are not feasible in MTD regimens due tohigh toxicity are well tolerated in metronomic dosing and yieldconsiderable therapeutic benefits. In animal models of end-stagepancreatic islet tumors, a therapeutic regimen was established asa sequential MTD followed by metronomic chemotherapy withcyclophosphamide combined to inhibition of PDGFR and VEGFRusing imatinib and SU10944. Such approach allowed complete re-sponses and remarkable survival advantages through the disrup-tion of pericyte support and destabilization of pre-existing tumorvasculature [97]. In a recent clinical study for metastatic breastcancer, metronomic use of daily oral capecitabine and cyclophos-phamide plus bevacizumab every 3 weeks and daily erlotinib al-lowed for one complete response, 14 partial responses and fivestable disease out of 24 patients, associated with mild toxicity[100].

Conclusions and perspectives

Currently available VEGFR inhibitors are useful in the treatmentof advanced RCC, hepatocellular carcinoma, gastrointestinal stro-mal tumors, and medullary thyroid cancer. Interestingly, all theseagents have been approved for use in monotherapy producing neg-ative results when combined with chemotherapy. Bevacizumab, onthe other hand, is typically combined with cytotoxic agents to im-prove results, despite the rapid onset of resistance developed bymetastatic patients, reinforcing the need for alternative antiangio-genics that can be combined to chemotherapy in order to achievesustained response over time.

There is a continuous search for new targets and agents for can-cer therapy aiming at developing more selective inhibitors as anattempt to overcome acquired resistance to specific inhibitors ofVEGF/VEGFR and reducing negative off-target effects. In this


context, inhibitors selective targeting the tumor endothelial mar-ker-8 (TEM8), a cell surface molecule overexpressed in tumorvasculature that seems to correlate with angiogenesis under path-ological, but not physiological conditions, have emerged as novelpromising anti-angiogenesis drugs. In vitro and in vivo studiesshow that blockage of TEM8 by blocking antibodies display anti-tumor activity, inhibit tumor-induced angiogenesis and increasethe activity of other therapeutic agents without augmenting toxic-ity [101]. This is also the case of MNRP1685A, a human mAb direc-ted to neuropilin-1 (NRP1), a nontyrosine kinase VEGF receptorthat exerts an important role in angiogenesis, being involved inthe regulation of endothelial cell migration and sprouting. Afterpromising results in preclinical settings, dose-escalating doses ofthis mAb as a single agent and in combination with bevacizumabwere evaluated in phase I trials in patient with advanced solid tu-mors and were shown to be well tolerated [102,103]. Targeting ofPIGF and its binding to VEGFR-1 by the humanized mAb TB-403(RO 5323441) has also been proposed as a new treatment strategy,as significant inhibition of tumor growth and metastasis have beensuggested by some preclinical studies [104]. However, controversystill exists regarding the impact of this cytokine in angiogenesisand tumor growth as studies point to opposite directions [105].In a phase I trial conducted in patients with advanced solid tumors,TB-403 was shown to be well tolerated and to exhibit a safety pro-file different from the one reported for inhibitors of VEGF-axis. Sta-ble disease was observed in six out of 23 patients at week 8 [106].

Targeting molecules involved in the interaction of tumor cells totheir microenvironment has emerged as another alternative. Pre-clinical studies showed that CXCR4 and CXCL12 exert a crucial rolein tumor growth and metastasis. AMD3100 (Mozobil, plerixafor), ainhibitor of CXCR4 approved by the FDA as a mobilizer of hemato-poietic CD34+ cells from the bone marrow to the circulation, wasevaluated in a phase I trial combined with chemotherapy in AMLpatients. Given its chemosensitizing properties and well toleratedprofile, plerixafor was later evaluated in a phase II study in whichrelapsed AML patients received the CXCR4 inhibitor prior to thechemotherapy regimen, with promising results. In solid tumor,monotherapy with CTCE-9908, an analogue of CXCL12, was shownto be well tolerated, with 20% of patients experiencing stable dis-ease [107]. Similarly, prokinecticin 2 (PROK2/PK2/Bv8), a factorproduced by myeloid cells, has become a therapeutic target. In arecent study, PKRA7, a small molecule antagonist of PK2, sup-pressed tumor growth in murine xenograft models of glioblastomaand pancreatic cancer [108].

Arguably, the expectations and clinical endpoints may not bethe same for cytotoxic and antiangiogenic agents. While for theformer the reduction of the tumor mass is a requirement for re-sponse, for the latter tumor arrest and growth inhibition may al-ready be a valuable achievement. In addition, the maintenance of‘‘stable disease’’ for patients with non-symptomatic cancer usinga non-toxic drug like endostatin is a significant benefit. Recent dataalso suggest that administration of endostatin plus bevacizumabprevents the hypertension observed in patients receiving thismAb, as endostatin was shown to increase nitric oxide productionby endothelial and smooth muscle cells. Meta-analysis of endo-statin clinical trials revealed a small but significant reduction inblood pressure in patients [109]. Such considerations open per-spectives for the launching of new clinical trials with endostatinbased on the knowledge gathered over the last decade. Patientsin which delaying disease relapse is the major goal or those withindolent tumors are perhaps the group that can derive most benefitfrom endostatin therapy in the future.

The long-term metronomic therapy combining cytotoxic andantiangiogenic agents may become a promising option for patientswith highly refractory and heavily pretreated cancers. Optimiza-tion is still necessary to overcome the empiricism related to drug




choice and combination, as well as to their dose and schedule. Reli-able biomarkers, advanced imaging techniques allied to multicen-ter clinical studies will be needed to validate the best metronomiccombination for each tumor type and patient population.

Tumor angiogenenesis is a complex process and we should notexpect that the therapeutic reversion of this phenomena to be lessintricate. Nonetheless, considerable advances were made in thelast decade with the approval of several antiangiogenic agents thatare being used as adjuncts to chemotherapy for a variety of can-cers. A fundamental interdisciplinary effort, which is needed topromote the translation between basic and clinical research, willprovide a basis for the development of solid antiangiogenic strate-gies that may, in the future, turn cancer into a chronic and manage-able disease.

Conflict of Interest

Authors declare no conflict of interest.Dendrix Ltd. provided editorial service in a preliminary version

of the article with financial support by Boehringer Ingelheim.

Acknowledgements

The authors wish to acknowledge Dr. Bruno K. Robbs for criticalreading of the manuscript. The authors also wish to acknowledgethe financial support provided by the Programa Nacional de Pós-Doutorado (CAPES/PNPD), Brazil; Grant number: PNPD 02954/09-5.

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Antiangiogenesis beyond VEGF inhibition: A journey from antiangiogenic single-target to...

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