Inhibiting Vascular Endothelial Growth Factor Therapeutic Implications, Perspectives and Limitations

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1 Inhibiting Vascular Endothelial Growth Factor Therapeutic Implications, Perspectives and Limitations a report by Christian Fischer and Peter Carmeliet Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Catholic University of Leuven Blood vessels deliver oxygen and nutrients to all tissues in the body and thus ensure normal tissue function. The walls of blood vessels are composed of endothelial cells, pericytes and smooth muscle cells that are embedded in an extracellular matrix. Vessels can grow in several ways. Vasculogenesis refers to the formation of blood vessels through in situ differentiation of endothelial progenitors to endothelial cells that assemble into an immature vasculature. Angiogenesis denotes the growth and remodelling process of the nascent primitive network into a complex network involving sprouting and bridging from pre-existing vessels, while arteriogenesis refers to the stabilisation of these sprouts by mural cells. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, while new vessels in adults mainly arise through angiogenesis, although vasculogenesis by bone marrow-derived endothelial progenitors also may occur. 1,2 In 1971, Judah Folkman first described the fundamental points of tumour angiogenesis: 3 most primary solid tumours go through a prolonged state of avascular growth in which the maximum size attainable is 1 2mm in diameter. Up to this size, simple passive diffusion enables tumour cells to obtain the necessary oxygen and nutrient supplies that they require for growth and survival. However, beyond this diffusion limit, tumours need to switch on angiogenesis by recruiting and inducing surrounding mature host blood vessels to sprout new capillaries that grow towards and finally infiltrate the tumour mass, thus enabling the expansion of the tumour itself and haematogeneous metastatic spread. 4 Therefore, inhibiting tumour growth by blocking tumour angiogenesis represents a therapeutic approach that is not curative in the usual sense as it would not eradicate all tumour cells, but would, instead, either prevent any new expansion of tumour mass or cause sustained regression of established solid tumours to a size where survival is possible without any blood supply. 5 Physiological vessel growth differs greatly from vessels arising in cancer. 1 Tumour vessels are structurally and functionally abnormal, as well as tortuous and enlarged with uneven diameter. They also have excessive brunching and shunts. Due to numerous openings (endothelial fenestrae, vesicles and trans-cellular holes), widened inter-endothelial junctions and a discontinuous or absent basement membrane, tumour vessels are also leaky. 6 In addition, they often lack protective mechanisms that normal vessels acquire during maturation. For example, tumour vessels lack an organised coat of perivascular cells that normally protect against vessel regression and provide vessels with necessary vasoactive control. 7,8 Consequently, blood flow in tumour vessels is chaotic, slow and not sufficient in meeting metabolic demands, 9 leading to hypoxic and acidic regions in tumours, conditions that may lower the efficacy of anti-cancer treatments. 9,10 Hence, normalisation of the chaotic tumour vasculature by angiogenic inhibitors may facilitate intra-tumoural drug delivery. 11 Pathological vessel formation is a common feature of nearly all gastrointestinal (GI) tumours, including colon, hepatocellular and gastric cancer Another major finding is that the degree of angiogenesis detected in a primary tumour correlates with the prognosis of many tumour entities: the greater the degree of tumour angiogenesis, the shorter the survival In line with these observations, plasma expression levels of angiogenic factors were found to directly correlate with disease prognosis Therapeutic Inhibition of Angiogenesis Since it was first appreciated that tumours secrete angiogenic factors to secure their own vascularisation and nourishment, several of these molecules have been identified. Numerous studies have established vascular endothelial growth factor (VEGF) as a key angiogenic player in cancer because VEGF expression is induced in almost all tumours upon hypoxia, hypoglycaemia, growth factors or oncogenes Plasma levels of VEGF are often elevated in cancer patients and are correlated with tumour progression and a marker of poor clinical outcome. 25,32 34 Recognition of the VEGF pathway as an important regulator of angiogenesis has led to considerable efforts to exploit its potential for Christian Fischer Peter Carmeliet Christian Fischer is carrying out his postdoctoral training at the Center for Transgene Technology and Gene Therapy at Flanders Interuniversity Institute for Biotechnology, the Catholic University of Leuven. Previously, he was a resident in internal medicine, gastroenterology, in the Charité-Hospital at Humboldt University, and a resident in internal medicine, oncology, at the Memorial Sloan-Kettering Cancer Center at Cornell University. Dr Fischer is a member of the German Society of Gastroenterology and has co-authored several articles. Peter Carmeliet is Adjunct-Director of the Center for Transgene Technology and Gene Therapy at Flanders Interuniversity Institute for Biotechnology, the Catholic University of Leuven. He is also Professor at the Cardiovascular Research Institute Maastricht and Full Professor at the Faculty of Medicine at the Catholic University of Leuven. Dr Carmeliet is a member of the International Society on Thrombosis and Haemostasis, the International Society of Fibrinolysis, the Council of the European Vascular Biology Association (invited member) and the North American Vascular Biology Association, and Secretary of the European Vascular Biology Association. 1

2 Table 1: VEGF/VEGFR Inhibitors Under Clinical Investigation Drug Class Target Company Development Bevacizumab MAB VEGF-A Genentech FDA-approved IMC-1121B MAB VEGFR-2 ImClone Systems Phase 1 VEG-trap Soluble hybrid VEGF-A, PIGF Regeneron Phase1/2 receptor VEGF-B PTK-787/ZK TKI VEGFR-1,-2,-3 Novartis/Schering Phase 1,2, PDGFR, c-kit AEE788 TKI VEGFR-2, Novartis Phase 1,2 EGFR AZD2171 TKI VEGFR-1, -2 AstraZeneca Phase 1 CEP-7055 TKI VEGFR-1,-2,-3 Cephelon Phase 1 GW TKI VEGFR-1,-2,-3 GlaxoSmithKline Phase 1 Bay TKI VEGFR-1,-2,-3 Bayer/Onyx Phase 1,2,3 Raf SU11248 TKI VEGFR-1,-2 Pfizer Phase 1 PDGFR AG TKI VEGFR-1,-2 Pfizer Phase 2 ZD6474 TKI VEGFR-1,-2,-3 AstraZeneca Phase 1,2 EGFR VEGF=vascular endothelial growth factor; VEGFR = VEGF receptor; MAB = monoclonal antibody; TKI = tyrosine kinase inhibitor; EGFR = epidermal growth factor receptor; PDGFR = platelet-derived growth factor receptor; PIGF = placental growth factor. Table 2: Selection of VEGF/VEGFR Inhibitory Trials in Colorectal Cancer Title Bevacizumab in treating patients with metastatic breast or colorectal cancer who recieved bevacizumab on a phase 2 or phase 3 clinical trial Phase 2 randomised study of bevacizumab and cetuximab with or without irinotecan in patients with irinotecan-refractory metastatic colorectal cancer Phase 1 study of neoadjuvent bevacizumab, fluorouracil and external beam radiotherapy in patients with stage 2 or 3 rectal cancer CONFIRM 1 study colorectal oral novel therapy for the inhibition of angiogenesis and retarding of metastases in first line. (PTK787/ZK /Oxaliplatin/ 5-FU/Leukovorin) CONFIRM 2 study colorectal oral novel therapy for the inhibition of angiogenesis and retarding of metastases in second line. (PTK787/ZK /Oxaliplatin/ 5-FU/Leukovorin) Website /2005/pi/Q1/050321_ptkzk.php?n=mep&c1 =press_releases _confirm2.php?n=mep 2 therapy in cancer diseases. It is therefore not surprising that most of the anti-angiogenic therapeutic strategies currently in pre-clinical and clinical development focus on inhibiting the VEGF/VEGF receptor (VEGFR)-2 pathway. Several anti-vegf strategies have been developed, including neutralising antibodies to VEGF or VEGFR-2, soluble VEGF/VEGFR hybrids and tyrosine kinase (TK) inhibitors to VEGFRs (see Table 1). 35,36 Some of the most advanced clinical trials to study the efficacy of VEGF/VEGFR pathway inhibition are discussed below. Inhibition of VEGF Using Monoclonal Anti-VEGF Antibodies One of the earliest strategies used to inhibit VEGF activity was the development of a humanised neutralising antibody to VEGF after pre-clinical studies that confirmed that a murine anti-vegf

3 Inhibiting Vascular Endothelial Growth Factor monoclonal antibody inhibited angiogenesis and growth of human tumour xenografts. 37,38 However, the most encouraging efficacy results in initial clinical trials were seen when bevacizumab was combined with chemotherapy. 39,40 It was therefore anticipated that anti-vegf therapy would require combinatorial cytotoxic therapy directly targeting neoplastic cells to achieve the highest frequency of tumour regressions and optimal clinical benefit in these tumour types. In the continuation of a phase 2 clinical trial for the treatment of colorectal cancer, 39 a phase 3 clinical trial published in 2004 finally proved, for the first time, that inhibiting VEGF by using the anti-vegf monoclonal antibody bevacizumab in combination with chemotherapy prolonged the survival of patients with previously untreated metastatic colorectal cancer for longer periods than expected: 813 patients were randomised to receive treatment with either irinotecan, fluorouracil and leucovorin (IFL) plus bevacizumab, or IFL plus placebo. The addition of bevacizumab to IFL resulted in a significant increase of overall survival, with a 34% reduction in the hazard of death (p<0.001). Median survival was increased from 15.6 months in the IFL plus placebo arm to 20.3 months in the IFL plus bevacizumab arm. Similar increases were seen in progression-free survival (6.2 months versus 10.6 months, p<0.001), response rate (34% versus 44.8%, p=0.004) and duration of response (7.1 months versus 10.4 months, p<0.001). 41 The clinical benefit of bevacizumab was accompanied by an absolute increase of approximately 10% in the overall incidence of grade 3 and 4 adverse effects that were mainly caused by induction of grade 3 hypertension (2.3% versus 11.0%) requiring antihypertensive medication. 41 Arterial thromboembolic events (ATEs) represented the most serious side effects. In a pooled analysis of five randomised controlled trials of bevacizumab, the addition of bevacizumab to chemotherapy increased the risk of ATEs compared with chemotherapy alone (3.8% versus 1.7%). This incidence of ATEs was even more increased in patients older than 65 years (7.1% versus 2.5%) and in patients with a prior history of atherosclerosis (15.7% versus 3.4%). 42 Other common side effects were epistaxis and proteinuria. One new potential bevacizumab-related toxicity was GI perforation, which was uncommon and had variable clinical presentations, ranging from a perforated stomach ulcer to bowel obstruction due to colonic perforation. Haemoptysis was also uncommon, but resulted in fatality in patients with non-small cell lung cancer. 41 In the meantime, clinical trials have been initiated to test the efficacy of bevacizumab in colorectal cancer as a second-line or adjuvant therapy in combination with chemotherapy (see Table 2). In addition, various other GI tumours (such as hepatocellular, pancreatic and gastric cancer) and non-gi tumours (such as renal cell, breast and lung cancer) are being investigated in current phase 3 trials for their responsiveness to bevacizumab. The Eastern Cooperative Oncology Group (ECOG) and the National Institutes of Health (NIH) provide an overview of these trials on their websites. 43 An alternative anti-vegf approach under earlystage clinical investigation represents the VEGF-trap. This fusion protein between an immunoglobulin G (IgG)-constant region and the extracellular domains of VEGFR-1 and -2 neutralises VEGF with extremely high affinity and effectively suppresses tumour growth and vascularisation in a variety of pre-clinical xenograft tumour models. 44,45 Small-molecule VEGFR TK Inhibitors A variety of small-molecule receptor (R) TK inhibitors targeting VEGF receptors have been developed. However, the course towards the development of an effective VEGF RTK inhibitor has not been straightforward, and it has been challenging to find a balance between efficacy, pharmacokinetic characteristics and safety. A firstgeneration molecule, SU5416, failed to show any survival benefit or prolongation of time-toprogression in a controlled phase 3 trial in colorectal cancer patients in combination with chemotherapy, 46 in spite of encouraging previous results. 47 Inhibitors targeting several RTKs have also been developed, such as SU11248 and AG , inhibiting VEGFRs, platelet-derived growth factor receptor (PDGFR) and c-kit. The most advanced RTK inhibitor is PTK787/ZK (PTK/ZK), an orally available molecule that is investigated in current phase 3 trials as first- (CONFIRM 1) and second-line (CONFIRM 2) treatment of metastatic colorectal cancer in combination with oxaliplatin, leucovorin and fluorouracil (FOLFOX4). 48,49 PTK/ZK is characterised by its high-affinity binding to all three VEGFR-1, -2 and -3. In addition, it inhibits the activity of TKs of PDGFR and c-kit. 48 Although clinical data show convincing anti-tumour activity, first announcements indicated that the early primary end-point progression-free survival favoured PTK/ZK, but did not achieve significance between the 583 patients who received FOLFOX4 and placebo and the 585 patients who received PTK/ZK in combination with FOLFOX4 when assessed by central radiologic review. However, when determined by investigator review, progression-free survival reached significance. Complete results, including overall survival, will be needed to fully assess the benefit of PTK/ZK in the treatment of metastatic colorectal cancer. 3

4 The development of a series of other RTK inhibitors (see Table 1) and their convincing anti-tumoural effects in pre-clinical and clinical trials raises the hope that these agents might significantly help to enhance the efficacy of cancer treatment in the future. Limitations and Perspectives of Anti-angiogenic Therapies With increasing knowledge about the molecular mechanisms of vessel growth, the aim to efficiently inhibit tumour angiogenesis has become more realistic. The recent milestone that an anti-vegf antibody prolonged life of colorectal cancer patients with unsurpassed efficiency proves the overall feasibility of angiogenesis inhibition strategies. However, many questions are still unanswered. For example, it still remains to be established how the efficacy of anti-angiogenic therapies can be clinically assessed, whether administration of a single angiogenic molecule is sufficient, whether tumours easily find escape routes to switch on alternative angiogenic programmes and whether long-term antiangiogenic treatment will cause delayed toxicity. Difficulties Associated with Clinical Evaluation of Anti-angiogenic Drug Efficacy delivery of chemotherapeutic drugs. Therefore, the use of various non-invasive radiologic techniques, such as dynamic contrast-enhanced magnetic resonance imaging (DCE MRI), spectral imaging, [ 15 O]H 2 O positron emission tomography (PET) and [ 18 F] fluorodeoxyglucose (FDG) PET to monitor changes in tumour blood flow, vascular structure, permeability and tumour metabolism might represent a successful approach They might also be effective in tracking the normalisation window of anti-angiogenic therapies. 58 Although serum levels of VEGF have been found at higher levels in many tumours and in metastatic cancers and have long been regarded as potential surrogate markers of cancer growth and anti-angiogenic drug efficacy, many studies of their use as surrogate markers have not proven evidence of their reliability. 59 Meanwhile, new research on tumour growth markers is shifting away from VEGF to the cells on which it exerts its effects: haematopoietic stem cells, circulating endothelial cells and, specifically, circulating endothelial progenitor cells from the bone marrow to the tumour that contribute to the formation of tumour vasculature. Indeed, recent pre-clinical and clinical studies indicate that circulating endothelial progenitor cells might represent a reliable surrogate marker of anti-angiogenic therapy. 58,60,61 4 Traditional chemotherapy drugs are used at the maximum tolerated dose, which is determined by toxicity. 50 Successful chemotherapy will typically result in a reduction of the cross-sectional diameter of a tumour when measured on serial computed tomography (CT) scans. However, measuring the efficacy of angiogenesis inhibitors is more difficult; these agents may not cause rapid involution of tumours and may simply slow or stop tumour growth; thus, the goal of tumour regression may not be a realistic endpoint for angiogenesis inhibitors. This provides a problem for phase 1 studies the biologically active dose may be difficult to predict in humans, because it may be considerably lower than the maximumtolerated dose having a safe toxicology profile. Therefore, determining new surrogate markers and new end-points that do not rely on toxicity or tumour regression is both complex and necessary in order to judge efficacy of anti-angiogenic compounds. In the experimental animal model, tumours can be removed and examined for the therapeutic changes of the extent of vascularisation, vascular structure and endothelial cell viability or apoptosis and for growth factor expression. However, taking serial biopsies of metastatic tumours may not be a particularly practical approach in the clinical situation, considering the additional risk for patients. The flow of blood and nutrient to tumours is a key principle in angiogenesis, but also provides the intra-tumoural Escape from Anti-angiogenic Therapy and Acquisition of Drug Resistance In light of the fact that the cellular target of antiangiogenic drugs is a non-transformed, and, hence, genetically stable, host vascular endothelial cell, it was first proposed that anti-angiogenic therapy might bypass acquired drug resistance encountered in virtually all chemotherapy-based strategies used to treat cancer. 62 To date, experimental evidence for acquired resistance to VEGF-targeted agents is limited. However, some pre-clinical studies have described recurrent tumour growth after an apparent initial period of suppression during chronic VEGF/VEGFR blockade. 63 Furthermore, on-going clinical trials reveal that treatments with a single antiangiogenic molecule is more challenging than anticipated, and may not suffice in order to combat the wide array of angiogenic factors produced by cancer cells and the growing tumour. Redundancy of alternative proangiogenic factors in anti-angiogenic monotherapies, upregulation of proangiogenic or anti-apoptotic factors, or heterogeneity of vascular dependency among tumour sub-populations might be possible mechanisms to contribute to the development of acquired drug resistance during antiangiogenic treatment. 62,64 Recent pre-clinical data support this hypothesis. Upon prolonged anti-vegf treatment of Wilms s tumour xenografts, tumour and stromal cells switched to other proangiogenic factors

5 Inhibiting Vascular Endothelial Growth Factor (PDGF-B and ephrinb), resulting in remodelling and increased efficiency of tumour vasculature and recurrent tumour growth. 65 Furthermore, the PDGFR-α signalling pathway has recently been identified as an important mechanism for stromal fibroblast recruitment, representing an essential route for tumour angiogenesis when tumour cells are deficient in VEGF production. 66 End-stage tumours might contain more pericyte-coated vessels, explaining why endothelial-targeted therapies have been reported to fail to induce regression of established (old) tumours, while targeting pericytes in addition of endothelial cells have been more effective. Therefore, destabilisation of tumour vessels by targeting smooth muscle cells may be an attractive means to combat established tumours. 67,68 The Prospect of Delayed Toxicity Associated with Long-term Anti-angiogenic Therapy The knowledge of long-term side effects of many anti-angiogenic therapies on normal tissues and physiological angiogenesis is insufficient. VEGF and VEGFR-2 are not only involved in pathological angiogenesis, but they significantly regulate normal adult vessel growth and maintenance, 69 and even affect other processes, such as neurogenesis, motor neuron survival, 70 lung function, 71 pulmonary hypertension, 72 liver regeneration 73 and control of blood pressure. 74 Mice expressing a mutant VEGF gene in which the hypoxia-response element was deleted suffer adult-onset motor neuron degeneration similar to that of patients with amyotrophic lateral sclerosis. 70 Considering the link between angiogenesis and neurogenesis, the question is whether long-term anti-angiogenic treatment will cause undesired neuronal effects. Along with desired response in tumours, long-term interference with VEGF signalling may therefore cause tumourdependent normal tissue toxicity. The development of effective, yet safe angiogenesis inhibitors will become an increasingly important issue in the future. Moreover, optimal cancer treatment may require the targeting of multiple angiogenic pathways, and the challenge for the oncologist will be to formulate cocktails of anti-angiogenic agents specifically tailored to the angiogenic profile of individual tumours. In this context, placental growth factor (PlGF), a homologue of VEGF, might offer novel therapeutic opportunities. Gene targeting studies in mice have elucidated a functional role of PlGF as key regulator of pathological angiogenesis. Loss of PlGF, while not causing any vascular defects during development, reproduction or normal adult life, impaired angiogenesis in cancer and reduced tumour growth. 75 These conclusions are further supported by the findings that PlGF expression levels are significantly increased in a wide array of different tumours Recently, it has been shown that PlGF expression levels not only increased in colorectal cancer, but correlated with disease stage and survival, whereas no correlation was observed between patient survival and elevated VEGF expression. 79 Given that VEGF/VEGFR-2 targeting agents may provoke unwanted short- and long-term toxicities, 80 the improved understanding of why the PlGF/VEGFR- 1 pathway is effective only in pathological and not in physiological angiogenesis could therefore be relevant for the design of safe and effective therapeutic strategies. 75,81,82 References 1. Carmeliet P, Angiogenesis in health and disease, Nat. Med. (2003);9: pp Carmeliet P, Manipulating angiogenesis in medicine, J. Intern. Med. (2004);255: pp Folkman J, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. (1971);285: pp. 1,182 1, Fidler I J, Ellis L M, The implications of angiogenesis for the biology and therapy of cancer metastasis, Cell (1994);79: pp Gimbrone M A, Jr, Leapman S B, Cotran R S, Folkman J, Tumor dormancy in vivo by prevention of neovascularization, J. Exp. Med. (1972);136: pp Hashizume H et al., Openings between defective endothelial cells explain tumor vessel leakiness, Am. J. Pathol. (2000);156: pp. 1,363 1, Morikawa S et al., Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors, Am. J. Pathol. (2002);160: pp , Benjamin L E, Golijanin D, Itin A, Pode D, Keshet E, Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal, J. Clin. Invest. (1999);103: pp Helmlinger G, Yuan F, Dellian M, Jain R K, Interstitial ph and po2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation, Nat. Med. (1997);3: pp Baish J W, Jain R K, Fractals and cancer, Cancer Res. (2000);60: pp. 3,683 3, Jain R K, Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy, Science (2005);307: pp

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