Combining Antiangiogenics to Overcome Resistance: Rationale and Clinical Experience

Published OnlineFirst April 30, 2012; DOI: 10.1158/1078-0432.CCR-11-1275

Clinical Cancer Review Research

Victor Moreno Garcia, Bristi Basu, L. Rhoda Molife, and Stan B. Kaye

Abstract Antiangiogenic therapies are now well established in oncology clinical practice; however, despite initial optimism, the results of late-phase trials, especially in the adjuvant setting, have largely proved disappointing. In the context of metastatic disease, resistance to antiangiogenic agents arises through a range of mechanisms, including the development of alternative angiogenic pathways. One of the proposed strategies to overcome this resistance is to combine antiangiogenic agents with different mechanisms of action. Early- phase clinical trials assessing the tolerability and efﬁcacy of different combinations of antiangiogenic drugs, including those that target the VEGF pathway or the angiopoietins, as well as vascular disrupting agents, are increasing in number. An example of this strategy is the combination of sorafenib and bevacizumab, which has elicited major responses in different tumor types, including ovarian carcinoma and glioblastoma. However, overlapping and cumulative toxicities pose a real challenge. This review summarizes the preclinical rationale for this approach and current clinical experience in combining antiangiogenic therapies. Clin Cancer Res; 18(14); 3750–61. 2012 AACR.

Introduction mab was also shown to prolong the time to progression as The importance of tumor angiogenesis has been recog- monotherapy (10) and in combination with interferon (13). nized for almost 80 years (1), and 4 decades ago, Folkman Tyrosine kinase inhibitors (TKI) targeting VEGFR, such as (2) proposed it as a therapeutic target, recognizing its critical sunitinib (8) and sorafenib (5), as monotherapies have also role in the growth and survival of tumors larger than 1 mm3. produced an OS benefit for patients with mRCC. Similarly, However, it is only in the last 10 years that this knowledge has sorafenib has now become the standard of care for advanced successfully translated to inhibitory strategies in clinical hepatocellular carcinoma because it was shown to increase practice. Endothelial cells (EC) have been assumed to show OS compared with placebo (14). However, in other tumors, genomic stability in comparison with the multiple somatic despite the initial benefit seen in some patients treated with mutations acquired within tumors during their growth, VEGF pathway inhibitors, complete responses have not leading to the belief that ECs would avoid resistance to generally been documented, and most patients will experi- therapy (3, 4). Targeting of angiogenesis has been validated ence tumor progression and succumb to their disease. More- by evidence of efficacy from several agents directed against over, attempts to use these drugs in the adjuvant setting (e.g., components of the main proangiogenic VEGF signaling in colorectal cancer) have thus far been disappointing, pathway at the level of the ligand (VEGF) and its receptors yielding the same results as chemotherapy alone (15). [VEGFR (5–10)]. Bevacizumab, a monoclonal antibody Lessons could be learned from the paradigm of mRCC (mAb) targeting VEGF, was shown to result in improved management, in which antiangiogenic strategies have been overall survival (OS) in metastatic colorectal and lung cancer most successfully adopted in routine clinical practice. when combined with chemotherapy (6, 9), and progression- Acquired resistance to anti-VEGF therapy may mean that free survival (PFS) in metastatic breast and ovarian cancer the order in which targeted agents are introduced to patients (11, 12). In metastatic renal cell cancer (mRCC), bevacizu- is important, such that a survival benefit may be derived from sequential monotherapy with multitargeted TKIs (16). An analysis of a prospective trial (17), numerous retrospec- Authors' Affiliation: Division of Clinical Sciences, Institute of Cancer tive studies, and a subgroup analysis of expanded-access Research and Royal Marsden NHS Trust, Sutton, Surrey, United Kingdom programs for sorafenib suggested that there is an increase in Note: Victor Moreno Garcia and Bristi Basu contributed equally to this PFS for a TKI switch in mRCC (18). These data support a lack article. of cross-resistance between multitargeted TKIs; however, Corresponding Author: Victor Moreno, Drug Development Unit, Division ongoing sorafenib/sunitinib crossover trials, such as of Clinical Sciences, Institute of Cancer Research/The Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: SWITCH (www.ClinicalTrials.gov; NCT00732914), may 44-020-8642-3539; Fax: 44-020-8642-6011; E-mail: address these issues more definitively. [email protected] An alternative to using VEGF-targeting agents sequent- doi: 10.1158/1078-0432.CCR-11-1275 ially is to focus on combining agents that affect different 2012 American Association for Cancer Research. aspects of angiogenesis. Our knowledge about the

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complexity of angiogenesis is growing rapidly, and it is Translational Relevance recognized that VEGF-independent drivers include multiple Targeting angiogenesis in cancer has been shown to interactions among diverse growth factors and receptors increase overall and/or progression-free survival for sev- involving ECs, NOTCH/d-like ligand 4 (DLL4), angiopoie- eral tumor types, including renal, ovarian, colon, lung, tin (Ang)-Tie, placental growth factor (PlGF), tumoral cells and breast cancers. However, many tumors are intrinsi- (SDF1/CXCR4), pericytes [platelet-derived growth factor cally resistant to antiangiogenic therapies. Other tumors, (PDGF) and transforming growth factor (TGF-b)], extracel- after an initial response, acquire secondary resistance lular matrix (ECM) components (integrins and cadherins), leading to further tumor growth. There is an urgent need inﬂammatory cells (tumor-associated macrophages and to identify mechanisms to overcome this resistance, and Tie-2–expressing monocytes), and bone-marrow–derived preclinical studies suggest that combining different anti- cells (Fig. 1; reviewed in refs. 19 and 20). A new paradigm angiogenics could be a suitable strategy. However, over- for the development of malignant angiogenesis was recently lapping toxicities are an issue of major concern. Early proposed based on the ﬁnding in glioblastoma multiforme clinical experience shows that trials combining different (GBM) that tumor ECs can arise directly through differen- þ þ antiangiogenics are complex, and schedules cannot be tiation of CD133 /CD144 tumor cells (21). In addition, easily extrapolated from single-agent studies. Careful preclinical work is providing insights into mechanisms clinical development of these combinations will be of resistance to antiangiogenic therapy. These mechanisms crucial to assess the relevance of positive data coming are described as being intrinsic (preexisting) or adaptive from preclinical models. (acquired), and they may explain why some tumors do not respond from the outset and why others progress after initial

Indirect inhibition

Vertical Horizontal

VEGF VEGF ANG

VEGFR NRP TIE2

Proliferation NOTCH Survival DLL4 Endothelial cell

PDGFR Integrin

ECM MMP Invasion

Direct inhibition VDA

Figure 1. Schematic diagram showing theoretical combinations targeting angiogenesis. MMP, matrix metalloproteinase; NRP, neuropilin.

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Table 1. Angiogenesis inhibitors

Group Name Action MTD/phase II dose as single agent

VEGFR and PDGFR AEE788 VEGFR-2, EGFR 550 mg qd TKIs Axitinib (AG-013736) VEGFR-1,2,3; PDGFR-b; c-KIT 5 mg b.i.d. Intedanib (BIBF 1120) VEGFR-1,2,3; PDGFR-a,b 250 mg b.i.d. Brivanib (BMS-540215) VEGFR-2,3; FGFR1,2 800 mg qd Cediranib (AZD2171) VEGFR-1,2,3 45 mg qd CP 547,632 VEGFR-2 200 mg qd Dovitinib (TKI258) VEGFR-1,2,3; PDGFR-b; c-KIT; 125 mg qd FLT3; FGFR1,2,3 Motesanib (AMG706) VEGFR-1,2,3; PDGFR-b; c-KIT 125 mg qd OSI 930 VEGFR-2; c-KIT 500 mg b.i.d. Pazopanib (GW786034) VEGFR-1,2,3; PDGFR; c-KIT 800 mg qd Semaxinib (SU5416) VEGFR-2, RET 145 mg/m2 q1w Sorafenib (BAY 43-9006) VEGFR-2,3; PDGFR-b, RET, c-KIT, 400 mg b.i.d. RAF SU6668 VEGFR-2; FGFR1, c-KIT, PDGFR-b 100 mg/m2 Sunitinib (SU11248) VEGFR-1,2; PDGFR-b; c-KIT; FLT3; 50 mg qd 4w/6w PDGFR-a,b Telatinib (BAY 57-9352) VEGFR-2,3; PDGFR-b 900 mg b.i.d. Vandetanib (AZD6474) VEGFR-2, EGFR 300 mg qd Vatalanib (PTK/ZK) VEGFR-1,2,3; PDGFR; c-KIT 1200 mg qd Anti-VEGFR Ramucirumab (IMC-1121B) mAb against VEGFR-2 13 mg/kg q1w Anti-VEGF Aﬂibercept (VEGF-trap) Soluble VEGFR-1,2 4 mg/kg q2w i.v. 1600 mg/kg q1w s.c. Bevacizumab mAb against VEGF-A 5–15 mg/kg q2w VEGF-AS (Veglin) VEGF antisense oligonucleotide 85 mg/m2 days 1–5 q2w VDA ABT-751 Tubulin binding 250 mg qd; 150 mg b.i.d. ASA404 (DMXAA) Flavonoid 1000–2000 mg/m2 q1w CYT 997 Tubulin binding 202 mg/m2 q3w EPC2407 Tubulin binding 13 mg/m2 days 1–3 q3w Fosbretabulin (CA4P) Tubulin binding 52 mg/m2 days q3w MN-029 Tubulin binding 180 mg/m2 q1w (3w/4w) MPC-6827 Tubulin binding 3.3 mg/m2 q1w NPI-2358 Tubulin binding 30 mg/m2 q1w (3w/4w) Ombrabulin (AVE8062) Tubulin binding 22 mg/m2 q3w OXi4503 (CA1P) Tubulin binding ND TZT 1027 Tubulin binding 2.7 mg/m2 q3w ZD 6126 Tubulin binding 80 mg/m2 q2w Integrin inhibitors ATN-161 Anti-a5b1 16 mg/kg 3x/q1w Cilengitide Anti-avb3 and anti-avb5 600 mg/m2 2x/q1w (EMD 121974) CNTO95 Anti-av 10 mg/kg days 0, 28, 35, and 42, and q3w thereafter Etaracizumab Anti-avb3 6 mg/kg 2x/q1w Volociximab Anti-a5b1 15 mg/kg q1w MMP inhibitors BMS-275291 MMP-1, -2, -7, -9, -14 1200 mg qd Marimastat (BB-2516) Pan-MMP inhibitor 50 mg b.i.d. Metastat (COL-3) MMP-2, -9 50 mg/m2 qd MMI270 (CGS27023A) MMP-1, -2, -3, -9, -13 300 mg b.i.d. Neovastat (AE-941) MMP-2, -9, -12 240 mL qd Prinomastat (AG3340) MMP -2, -3, -9, -13, -14 5–10 mg b.i.d. Tanomastat (BAY 129566) MMP-2, -3, -9 800 mg b.i.d. (Continued on the following page)

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Table 1. Angiogenesis inhibitors (Cont'd )

Group Name Action MTD/phase II dose as single agent

Anti-NOTCH-DLL4 MK-0752 NOTCH inhibitor ND PF-03084014 g Secretase inhibitor ND RO4929097 g Secretase inhibitor ND REGN421 Anti-DLL4 ND OMP-21M18 Anti-DLL4 ND Anti-EGFL-7 MEGF0444A mAb against EGFL-7 15 mg/kg Targeting AMG 386 ANGPT-1, 2 inhibitor ND (>30 mg/kg q1w) ANG-Tie pathway CVX-060 ANGPT-2 inhibitor 15 mg/kg

Abbreviations: b.i.d., twice a day; MMP, matrix metalloproteinase; MTD, maximum tolerated dose; ND, not determined; q1w, once a week; q2w, every 2 weeks; q3w, every 3 weeks; qd, daily. shrinkage (22). This information has driven the clinical apy (27). Similarly, the combination of bevacizumab and drug development of a number of compounds that target the VDA combretastatin A1 phosphate (OXi4503) resulted various components of tumor angiogenesis (Table 1). In in significantly enhanced tumor growth delay in a renal this review, we discuss the progress that has been made and cancer xenograft (27 days; P < 0.05) compared with bev- the pitfalls involved in combining different antiangiogenic acizumab (8 days) or OXi4503 (18 days) as monotherapy strategies with the aim of overcoming resistance. (28). The rationale and preclinical results for these combinations have encouraged clinical trials of different VDAs with VEGF inhibitors, especially bevacizumab. Preclinical Experience in Combining Antiangiogenics Combining 2 indirect inhibitors Strategies that have been devised to overcome some of the Indirect inhibitors target receptors/ligands that stimulate known mechanisms of resistance to antiangiogenic thera- EC growth/survival. Horizontal blockade encompasses pies may conceptually be divided into 2 groups: (i) com- the targeting of different classes of receptors. The intimate bining direct (targeting ECs) and indirect (targeting growth relationship between ECs and pericytes is associated with factors/receptors) inhibitors; and (ii) combining 2 indirect increased expression of PDGF receptor (PDGFR)-b, Tie-2, inhibitors, resulting in either horizontal blockade from 2 and Ang-1 (29). These factors may well contribute to the different inhibitors targeting proteins across separate sig- relative lack of efficacy seen with anti-VEGFR-2 (semaxinib) naling pathways or vertical blockade from targeting mole- treatment in tumor xenografts compared with SU6668, an cules within the same pathway (Fig. 1; ref. 23). inhibitor of PDFGR, VEGFR, and fibroblast growth factor receptor [FGFR (30, 31)]. Of interest, although greater Combining indirect and direct inhibition benefit was observed from the combination in a Rip1-Tag2 Vascular disrupting agents (VDA) are direct inhibitors of mouse model of pancreatic neuroendocrine tumors, when angiogenesis that target established vasculature. The selec- used as monotherapy, the VEGFR-2 inhibitor (semaxinib) tivity of VDAs for tumoral vessels results from structural was more efficacious at blocking initial angiogenic switch- differences, with tumor blood vessels being immature and ing, whereas the PDGFR inhibitor (SU6668) was more therefore inadequately covered by pericytes. This makes effective against end-stage bulky disease (32). In this in vivo them more susceptible to microtubule depolarization, lead- model, adaptive resistance to anti-VEGFR-2 therapy was ing to disruption of the actin cytoskeleton and subsequent also abrogated with the use of a dual FGF/VEGFR-2 TKI cell reshaping that causes blood flow reduction and tumoral [brivanib (33)]. Similar strategies targeting both ECs and necrosis (24, 25). However, regrowth is characteristically pericytes with TKIs that target VEGFR-2 (AEE788) and observed from a peripheral rim of residual tumor cells via PDGFR (imatinib), respectively, showed that the reduced rapid recruitment of circulating endothelial progenitor cells pericyte coverage as a result of PDGFR blockade with (EPC). Pretreatment with VEGFR-2 inhibitors was associ- imatinib monotherapy was ineffective at controlling tumor ated with the disruption of EPC recruitment and enhance- growth; however, when imatinib was used in combination ment of VDA-mediated blood flow reduction in tumor- with AEE788, tumor control was superior to that seen with bearing mice (26). Combinations of VDA with agents AEE788 alone (34). inhibiting the VEGF pathway have displayed synergy in The finding that tumors that show intrinsic resistance xenograft models. For example, the VDA ZD6126 and the to anti-VEGF therapy display sensitivity to blockade of VEGFR-2 TKI vandetanib (ZD6474), when used in combi- DLL4-NOTCH signaling led to interest in targeting the nation, produced 3.5-fold and 6-fold greater delays in tumor NOTCH-DLL4 and VEGF pathways together (35–37). growth compared with that observed with either monother- NOTCH inhibition using g secretase inhibitors may alter

www.aacrjournals.org Clin Cancer Res; 18(14) July 15, 2012 3753

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Table 2. Clinical trials of combination of antiangiogenics

Drug 1 Drug 2 Phase Cancer MTD/phase II dose Grade 3/4 toxicities Reference

Bevacizumab ABT-510 I Solid tumors B: BV 10 mg/kg Gastrointestinal bleed (4%), headache (73) clincancerres.aacrjournals.org q14d (4%) Published OnlineFirstApril30,2012;DOI:10.1158/1078-0432.CCR-11-1275 ABT-510: 100 mg b.i.d. Bevacizumab Axitinib (þFOLFOX) I Solid tumors B: 2 mg/kg q14d Hypertension (18%), neutropenia (12%) (71) A: 5 mg b.i.d. Bevacizumab Combretastatin I Solid tumors B: 10 mg/kg q14d Atrial ﬁbrillation (7%), hemorrhage (7%) (54) C: 63 mg/m2 q14d Bevacizumab Cediranib I Solid tumors B: 5 mg/kg q14d CNS bleed (8%), nausea (6%) and (76) hypertension (6%) C: 20 mg/day Bevacizumab Sorafenib I Solid tumors B: 5 mg/kg q14d Hypertension: (33%), diarrhea (12%), (63)

Research. S: 200 mg b.i.d. Bevacizumab Sorafenib I Solid tumors B: 5 mg/kg q14d Hypertension: (18%), diarrhea (12%), (64) transaminitis (12%) S: 200 mg b.i.d. days 1–5/q1w Bevacizumab Sorafenib (þpaclitaxel) I Solid tumors B: 5 mg/kg q14d Hypertension (30%), hfs (22%), (70) neutropenia (17%), neuropathic pain, (8%), mucositis (8%) S: 200 mg b.i.d. days 1–5/q1w (þP: 80 mg/m2 q1w) Bevacizumab Sorafenib (þpaclitaxel) I Solid tumors B: 0–5 mg/kg q14d HFS 25%, rash 8%, stomatitis 8%, (69) (dose escalation diarrhea 16%, granulocytopenia 8% ongoing) hoarseness 8% S: 200 mg b.i.d. 2 lnclCne Research Cancer Clinical (þP: 90 mg/m d1,8,15 q4w) Bevacizumab Sunitinib I Solid tumors B: 5 mg/kg q14d Hypertension (47%), fatigue (24%), (59) thrombocytopenia (18%), proteinuria (13%), HFS (13%) S: 50 mg/qd Bevacizumab Sunitinib I RCC B: 10 mg/kg q14d (60) (Continued on the following page) www.aacrjournals.org Downloaded from

Table 2. Clinical trials of combination of antiangiogenics (Cont'd )

Drug 1 Drug 2 Phase Cancer MTD/phase II dose Grade 3/4 toxicities Reference clincancerres.aacrjournals.org Published OnlineFirstApril30,2012;DOI:10.1158/1078-0432.CCR-11-1275 Hypertension (65%), proteinuria (39%), thrombocytopenia (29%), HFS (22%), fatigue (13%); 2 cases of severe MAHA S: 50 mg/qd Bevacizumab Vandetanib I Solid tumors B: 5 mg/kg q21d Diarrhea, elevated creatinine, anemia (57) thrombocytopenia, hemorrhage, prolonged QTc interval, proteinuria, rash V: 200 mg qd Bevacizumab Sorafenib II GBM B: 5 mg/kg q14d fatigue, thrombosis, hypophosphatemia, (65) muscle weakness S: 200 mg b.i.d. days on September 30, 2021. © 2012American Association for Cancer 1–5/q1w Research. Bevacizumab Sorafenib II Melanoma B: 5 mg/kg q14d Hypertension (14.2%), HFS (7%), (67) proteinuria (7%), thrombocytopenia (7%) S: 200 mg b.i.d. days 1–5/q1w Bevacizumab Sorafenib II Colorectal B: 5 mg/kg q14d Fatigue (18%), hypertension (14%), rash (68) (8%), elevated lipase (7%) S: 200 mg b.i.d. days 1–5/q1w Bevacizumab Sorafenib II NET B: 5 mg/kg q14d HFS (20%) asthenia (16%) (66) S: 200 mg/qd Sorafenib AMG-386 II Renal Arm A: S: 400 m Arm A: diarrhea (8%), HFS (12%), and (75) lnCne e;1(4 uy1,2012 15, July 18(14) Res; Cancer Clin

b.i.d. þ A: 10 mg/kg hypertension (18%). Therapies Antiangiogenic Combining Arm B: S: S: 400 m Arm B: diarrhea (10%), HFS (16%), b.i.d. þ A: 3 mg/kg hypertension (20%) Arm C: S: 400 m Arm C: diarrhea (8%), HFS (28%), and b.i.d. þ placebo hypertension (14%)

Abbreviations: b.i.d., twice a day; CNS, central nervous system; MAHA, microangiopathic hemolytic anemia; MTD, maximum tolerated dose; NET, neuroendocrine tumor; P, paclitaxel; q1w, once a week; q14d, every 14 days; q21d, every 21 days; qd, once a day; S, sorafenib. 3755 Published OnlineFirst April 30, 2012; DOI: 10.1158/1078-0432.CCR-11-1275

Moreno Garcia et al.

the self-renewing capacity of intestinal crypt cells; however, pathwayarenowinformingahighlyactivefocusfor DLL4 seems to be restricted to the vascular compartment research in the clinical setting. (38, 39). Combination therapy using antibodies against DLL4 and VEGF substantially inhibited tumor growth in Clinical Experience in Combining non–small cell lung cancer xenografts [Calu6 and MV522 Antiangiogenics (37)]. However, recent studies showed that chronic DLL4 Current antiangiogenic strategies in clinical oncology in vivo blockade can produce more vascular tumors , thus practice either use single-agent, multitargeted TKIs with raising concerns over its clinical applicability (40). For this activity against different proangiogenic receptors (e.g., reason, regulators implicated in attenuation of NOTCH VEGFRs, PDGFR-b, FLT3, and FGFRs) or involve sequenc- signaling, such as epidermal growth factor–like domain 7 ing of drugs that work within the same vertical angiogenic (EGFL-7), are now being investigated as potential alterna- axis. In addition, combination (horizontal) antiangiogenic tive targets (41). strategies are now emerging in early clinical trials, and it will The angiopoietins Ang-1, -2, -3, and -4 regulate EC be important to establish their tolerability and determine homeostasis via interactions with their receptors Tie-1/ whether additional gains in efficacy are offset by challenges Tie-2. This results in vascular quiescence/angiogenesis in administration due to toxicity. Because bevacizumab is (Ang-1) or vessel regression and angiogenic sprouting the best-established antiangiogenic agent, many combina- (Ang-2), depending on the environmental cues present tions use this antibody as the backbone (Tables 2 and 3). (42, 43). Ang-2–directed therapies have been assessed Treatments targeting the VEGF pathway share well-recog- along with anti-VEGF strategies (44). Reduction of endo- nized class-adverse effects related to VEGF blockade, name- thelial sprout formation and colon cancer xenograft ly, hypertension, proteinuria, hemorrhage, arterial throm- growth following treatment with an Ang-2 binding pepti- boembolic events, and poor wound healing. These effects body, L1-7 (N), was even more significant when VEGF are typically downstream consequences of suppression of antibodies were added (45). Similarly, addition of an Ang- cellular signaling pathways important in the regulation and 2 antibody, 3.19.3, to the VEGFR-1,2,3 TKI cediranib, the maintenance of the microvasculature, and they highlight VEGFR-2 and epidermal growth factor receptor (EGFR) the properties shared by tumor vessels and the vasculature TKI vandetanib, AZ10167514 (VEGFR TKI), or DC101 of normal organs (51). In addition to the adverse effects (VEGFR-2 antibody) doubled the rate of tumor growth related to VEGF-axis blockade, off-target effects result in inhibition over that seen with each single agent (46). frequently observed toxicities, such as nausea, diarrhea, Other signaling pathways and molecules implicated in hand–foot syndrome (HFS), and mucositis (52). A key resistance to antiangiogenics, such as integrins, PI3K- issue in the development of antiangiogenic drugs is the AKT-mTOR, cMET/HGF, and TGF-b (endoglin, Alk1), are lack of reliable predictive biomarkers for sensitivity to this being targeted in combination with classic antiangiogenics strategy. A retrospective study on genetic polymorphisms in to expand the options of novel combinations to overcome the VEGF gene showed correlations with OS and toxicity primary and secondary resistance. (53). Prospective confirmatory studies are needed, and Vertical blockade of the VEGF pathway at different many rationally selected candidates, such as serum levels levels has been extensively investigated in preclinical of VEGF, FGF, PlGF, and other cytokines, have failed to studies. Mouse models with the human head and neck show any ability to select patients who are more likely to cancer cell line CAL33 treated with cediranib (VEGFR- benefit (54). The importance of establishing pharmacody- 1,2,3 TKI) and bevacizumab showed a significant tumor namic biomarkers early in the development of antiangio- growth reduction compared with either agent alone. Mice genic combinations cannot be overstated. Hitherto, the treated with single-agent therapy showed increased most common approach was to use imaging studies to tumoral secretion of VEGF; however, this adaptive effect identify changes in tumor blood flow or vascular perme- was abrogated with the combination therapy, suggesting ability (i.e., changes in blood flow by contrast-enhanced that inhibition of both VEGF ligand and its receptor ultrasound or changes in Ktrans by MRI). Serum levels of (VEGFR) can provide additional benefit over inhibition circulating markers (VEGF, sVEGFR-2, sVEGFR-3, and of either alone (47). The transmembrane glycoproteins PlGF) are also usually modified by antiangiogenic thera- neuropilins (NRP)-1,2 act as cofactors to enhance the pies, but their value as early markers of activity remains to be binding of VEGF to VEGFR-2 (48). Recently, 2 high- determined. Current efforts are focused on measuring the affinity mAbs that bind discrete Sema3A and VEGF bind- soluble isoforms of circulating VEGFA-VEGFA121; howev- ing domains of NRP-1 were tested in combination with a er, at present, the lack of a reliable intermediate biomarker VEGF antibody with good preclinical activity (49). Fur- of efficacy in early-phase studies means that most combi- thermore, blocking the same receptor at 2 different levels nation antiangiogenic therapies will still require larger was recently observed to enhance receptor inactivation, phase II/III trials to prove clinical benefit. and combining antibodies that separately inhibit ligand binding and VEGFR-3 dimerization more effectively inhibited tumor angiogenesis and lymphangiogenesis in Combining direct and indirect inhibitors vivo (50). The data gained from these preclinical studies Investigators in clinical trials have pursued the rationale combining inhibition at multiple points within the VEGF for combining VDAs with VEGF(-R) inhibitors to abrogate

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Table 3. Ongoing clinical trials combining antiangiogenics

ClinicalTrials.gov Drug 1 Drug 2 Phase Cancer identiﬁer

ABT-510 (TSP-1 analog) Bevacizumab I Advanced solid cancer, solid tumors NCT00276562 AMG-386 (ANGPT1/2 antibody) AMG-706 I Advanced solid cancer, solid tumors NCT00861419 Bevacizumab Sorafenib Sunitinib Bevacizumab Pazopanib I Renal NCT00992121 CVX-060 (ANGP2 binding peptide) Sunitinib I Renal NCT00982657 Fosbretabulin Bevacizumab I High-grade gliomas NCT01052363 Ombrabulin Bevacizumab I Advanced solid cancer, solid tumors NCT01193595 Vandetanib Bevacizumab (þCapOx) I Colorectal NCT00532909 Vandetanib Bevacizumab I Advanced solid cancer, solid tumors, NCT00734890 or lymphoma Etaracizumab (anti-a5b3 integrin) Bevacizumab I/II Renal NCT00684996 Bevacizumab Sorafenib (þoxaliplatin) I/II Melanoma NCT00538005 Sorafenib Thalidomide I/II Hepatocellular NCT00971126 AMG-386 Bevacizumab (þpaclitaxel) II Breast NCT00511459 AMG-386 (ANGPT1/2 antibody) Sunitinib II Renal NCT00853372 Bevacizumab Sorafenib II Renal NCT00126503 Bevacizumab Thalidomide II Multiple myeloma NCT00022607 Combretastatin (CA4P) Bevacizumab II Ovarian NCT01305213 Combretastatin (CA4P) Bevacizumab II Lung NCT00653939 Sorafenib Bevacizumab II Colorectal NCT00826540 Sorafenib Bevacizumab II Breast NCT00632541 Sorafenib Bevacizumab II Liver NCT00881751 Sorafenib Bevacizumab II GBM NCT00621686

Abbreviations: CapOx, capecitabine and oxaliplatin. malignant progression at the viable tumor rim. To date, Combining 2 indirect inhibitors however, the schedules used have not followed the preclin- Vertical combinations. Bevacizumab was assessed in ical studies, in which abrogation of the circulating EPC combination with vandetanib in a phase I study in which spike was maximal when a VEGFR-2 inhibitor was admin- hypertension was frequent and the dose-limiting toxicity istered prior to the VDA. Instead, in clinical trials conducted (DLT) was allergic reaction (58). Despite 2 partial responses to date, the VDA was administered before the VEGF inhib- observed in patients with carcinomas of the small bowel itor for the purpose of monitoring safety. Combretastatin and pancreas, chronic dosing was associated with a number A4 phosphate (CA4P) added to 10 mg/kg of bevacizumab of grade 2–3 toxicities, including myelosuppression, rash, was well tolerated at doses up to 63 mg/m2 CA4P every 2 prolonged QTc interval, and diarrhea, such that dose esca- weeks in a phase I study of 14 patients, with 2 grade 3 toxi- lation was limited to 2 dose levels. The selected dose for cities (asymptomatic self-limiting atrial fibrillation and further studies was vandetanib, 200 mg orally once daily hemorrhage) observed (55). One third of the patients had with bevacizumab, 5 mg/kg every 21 days. Pharmacody- disease stabilization for more than 4 months (I. Judson, namic studies supported further exploration of this dual personal communication), and dynamic contrast-enhanced antiangiogenic approach. Although vandetanib alone (DCE)-MRI showed statistically significant reductions in would be expected to produce a 20% reduction in blood tumor perfusion and vascular permeability (56). The benefit flow, DCE-MRI studies in the combination phase I trial of adding CA4P and bevacizumab to carboplatin and pac- revealed a 31% reduction in mean Ktrans in all 7 patients litaxel is currently being tested in a randomized phase II trial following 2 cycles of therapy, with decreased levels of EPC in 60 patients with non–small cell lung cancer, with prelim- in 5 patients and increased numbers of apoptotic mature inary results showing an increase in response rate (RR) from ECs in 3 patients (59). 38% to 55%, favoring the CA4P arm, and a similar PFS The combination of bevacizumab with sunitinib has [median, 8.6 months vs. 8.9 in the control arm (57)]. A shown clinical activity, albeit with significant toxicity, with newer VDA, ombrabulin (AVE8062), is currently being high RRs in early-phase clinical trials, including all histo- tested in combination with bevacizumab in a phase I dose logic subtypes of mRCC and other typically chemoresis- escalation study (ClinicalTrials.gov; NCT01193595). tant tumors, such as urothelial cancers and melanoma. The

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maximum tolerated dose (MTD) was judged to be 50 mg Horizontal combinations. Theoretically, angiogenic daily of sunitinib in a 4-weeks-on/2-weeks-off schedule, blockade at different receptors could overcome the draw- with 10 mg/kg bevacizumab every 2 weeks. However, this back of overlapping toxicity, and horizontal combinations dose was considered too toxic for chronic therapy, with 87% are now being explored in early clinical trials. Thrombos- of patients developing grade 3 adverse events [mainly pondin-1 (TSP-1) is an endogenous inhibitor of angiogen- hypertension, fatigue, thrombocytopenia, proteinuria, and esis that mediates its effects through interactions with HFS (60)]. In the phase I study in mRCC, 48% of the different receptors (av and b integrins, CD47, and CD36), patients were withdrawn because of adverse events (61). and it can also inhibit nitric oxide signaling (73). A synthetic The development of microangiopathic hemolytic anemia in analogue of the N-terminal region of TSP-1, ABT-510, in 5 of 12 patients treated at the highest dose level was of major combination with bevacizumab (10 mg/kg) has shown no concern. Two further patients developed severe symptoms DLT at doses up to 100 mg s.c. twice daily in a phase I with hypertension, thrombocytopenia, renal insufficiency, study, suggesting a lack of overlapping toxicities clinically; proteinuria, and reversible posterior leukoencephalopathy however, efficacy data are yet to be published (74). Pro- syndrome, leading to a U.S. Food and Drug Administration angiogenic interactions between Ang-1,2 and the Tie-2 warning (62) and discontinuation of this combination in receptor can be suppressed therapeutically with Fc-peptide other clinical trials (63). fusion proteins, such as AMG-386, that comprise the Fc The combination of bevacizumab with sorafenib seems domain of immunoglobulin G1 fused to a synthetic pep- to be safer, although toxicity remains a concern. A phase I tide with potent binding to Ang-1,2 and result in inhibition combination trial established an MTD at dose level 1, using of Tie-2 receptor activation (75). The results of a random- sorafenib, 200 mg twice daily and bevacizumab, 5 mg/kg ized, double-blind, placebo-controlled phase II combina- every 2 weeks (64). Dose-limiting toxicities were protein- tion study of AMG-386 and sorafenib in 152 patients with uria and thrombocytopenia; however, 74% of the patients mRCC were recently reported (76). Patients were random- required dose reductions, mainly due to HFS, fatigue, and ized to sorafenib, 400 mg twice daily, with AMG-386, hypertension. A high rate of antitumor activity (43% RR for 10 mg/kg; AMG-386, 3 mg/kg; or placebo. The combina- heavily pretreated patients with ovarian cancer, with a tion was well tolerated, and although an increase in the median of 5 lines of prior chemotherapy) encouraged the incidence of diarrhea was reported (70%, 67%, and 56%, development of better-tolerated regimens. An intermittent respectively), other common toxicities, such as HFS and schedule of sorafenib, 200 mg twice daily 5 days per week, hypertension, were similar among the 3 arms. Despite a and bevacizumab, 5 mg/kg every 2 weeks, was associated higher RR with the combination of sorafenib þ AMG-386 with fewer dose reductions (41%) while maintaining a high compared with the sorafenib þ placebo combination (37% RR of 47% in ovarian cancer (65). This schedule was also vs. 24%), the primary endpoint was not met, as the median tested in 39 patients with GBM, resulting in a 37% RR, PFS of 9 months was similar in all 3 arms (P ¼ 0.52). although poor tolerance led to one third of the patients It therefore remains to be determined whether, in addi- discontinuing treatment. Dose reduction to sorafenib, tion to their greater tolerability, these combinations can 200 mg daily, significantly improved tolerability, with provide a benefit beyond increased RRs. 15% of the patients experiencing grade 3 toxicities (66). The same schedule was tested in a phase II trial in 44 patients with advanced neuroendocrine tumors, with an RR of 9.8% Conclusions reported; however, 20% of patients developed grade 3 Although some encouraging examples of activity from HFS and 15% developed grade 3 asthenia (67). No antiangiogenics in a number of tumors have been objective responses were observed when this schedule was reported, resistance to these agents is a major hurdle in used in phase II trials in advanced melanoma and colorectal the further development of this class of cancer therapy. cancer (68, 69). Two phase I studies (70, 71) are investi- Mechanisms of resistance are still being defined, and it is gating the safety and tolerability of this combination with anticipated that the knowledge gained will give rise to paclitaxel in solid tumors, and preliminary reports suggest increasingly rational attempts to overcome them. Active some activity, with 3 complete responses (ovarian, endo- efforts are under way to address the optimal scheduling metrial, and urachal cancers) and 8 partial responses (6 and combinations of antiangiogenics. It remains to be ovarian, 1 endometrial, and 1 prostate) among 19 evaluable determined whether vertical inhibition at numerous cancer patients. levels within the VEGF pathway will improve clinical Following a similar rationale, investigators combined outcomes. Likewise, despite positive data from preclinical bevacizumab with axitinib (AG-013736) in a phase I study studies, the anticancer efficacy of horizontally combining with FOLFOX chemotherapy. The DLT was hypertension, agents that act across different antiangiogenic pathways observed in 81% of patients on combination treatment remains to be established in clinical practice. Although compared with 26% on axitinib monotherapy. The MTD these preclinical studies were frequently performed using was bevacizumab 2 mg/kg q14d with axitinib 5 mg twice different agents compared with those tested in clinical daily. A phase II study in advanced CRC patients (Clini- trials, they generally involved the same mechanisms of calTrials.gov; NCT00460603) leading on from this study is action. Other explanations, such as a lack of specificity or ongoing, and an RR of 33% has been reported (72). different potency for the target, might therefore explain

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some of the discrepancies seen so far between preclinical ment of rational combinations, such as VEGFR/C-MET and clinical studies. combinations. Recent evidence from early clinical trials raises concerns A key area under investigation in this arena is the devel- that overlapping toxicities from antiangiogenic combina- opment of relevant predictive biomarkers and pharmaco- tions may limit feasibility in the long term due to cumu- dynamic endpoints. This would permit appropriate patient lative toxicity. It will be important to determine whether selection and advance the concept of treating to a biolog- sequencing these agents can provide similar benefit while ically effective dose rather than the MTD and thus accelerate sparing the toxicities from the combinations. This is impor- the therapeutic development of better-tolerated combina- tant because, unlike conventional cytotoxics, antiangio- tions and schedules for future use in clinical practice. genic treatments may require chronic dosing, and therefore traditional DLT endpoints within the first cycle of treatment Disclosure of Potential Conflicts of Interest will underestimate the severity of adverse events from these S.B. Kaye is a consultant/advisory board member of Roche. No other potential conflicts of interest were disclosed. agents together. Nevertheless, interest in pursuing alternative doses and schedules of a combined antiangiogenic Authors' Contributions approach remains, largely due to the encouraging efficacy Conception and design: V.M. Garcia, B. Basu, L.R. Molife, S.B. Kaye signals in terms of increased tumor response, especially in Development of methodology: V.M. Garcia, L.R. Molife Analysis and interpretation of data: B. Basu, L.R. Molife, S.B. Kaye traditionally chemoresistant tumors and in patients who Writing, review, and/or revision of the manuscript: V.M. Garcia, B. Basu, have been heavily pretreated. L.R. Molife, S.B. Kaye As more antiangiogenic agents that target pathways other than VEGF enter clinical trials, the opportunities for testing Grant Support The Drug Development Unit of the Royal Marsden NHS Foundation Trust novel antiangiogenic combinations will continue to and the Institute of Cancer Research is supported in part by a program grant expand. For new drugs that target the VEGF pathway itself, from Cancer Research UK. Support was also provided by the Experimental this could increase the potential for achieving successful Cancer Medicine Centre (to the Institute of Cancer Research) and the National Institute for Health Research Biomedical Research Centre (jointly combinations if they show a different safety profile. How- to the Royal Marsden NHS Foundation Trust and the Institute of Cancer ever, the list of available agents (see Table 1) is already long, Research). V.M. Garcia was supported by a grant from the Fundacion Para la and a key goal now is to gain an improved understanding Investigacion del Hospital Universitario La Paz (REX-09). of the mechanisms that underlie clinical resistance to Received May 18, 2011; revised February 29, 2012; accepted April 10, VEGFR inhibitors. This should lead to the further develop- 2012; published OnlineFirst April 30, 2012.

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Combining Antiangiogenics to Overcome Resistance: Rationale and Clinical Experience

Victor Moreno Garcia, Bristi Basu, L. Rhoda Molife, et al.

Clin Cancer Res 2012;18:3750-3761. Published OnlineFirst April 30, 2012.

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