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`VEGF Trap as a Novel Antiangiogenic Treatment Currently in
`Clinical Trials for Cancer and Eye Diseases, and VelociGene®-
`based Discovery of the Next Generation of Angiogenesis Targets
`
`J.S. RUDGE, G. THURSTON, S. DAVIS, N. PAPADOPOULOS, N. GALE,
`S.J. WIEGAND, AND G.D. YANCOPOULOS
`Regneron Pharmaceuticals, Inc., Tarrytown, New York 10591
`
`The concept that tumors can be controlled by directly targeting their vascular supply has finally come of age, because clini-
`cal trials using a humanized monoclonal antibody that blocks VEGF have demonstrated exciting efficacy in cancer patients,
`as well as in vascular eye diseases that can lead to blindness. However, data suggest that these current regimens may not pro-
`vide complete VEGF inhibition and, thus, that the maximum therapeutic potential of VEGF blockade has not yet been
`achieved. We describe the status of a very potent and high-affinity VEGF blocker, termed the VEGF Trap, that may provide
`the opportunity to maximize the potential of VEGF blockade in cancer as well as in vascular eye diseases. We also describe
`use of the VEGF Trap as a research tool, when coupled to high-throughput mouse genetics approaches such as VelociGene®
`that can be exploited in strategies to discover and validate the next generation of angiogenesis targets.
`
`The concept that tumors can be controlled by directly
`targeting their vascular supply has finally come of age.
`The first antiangiogenesis approach to be validated in
`human cancer patients involves blocking vascular en-
`dothelial growth factor (VEGF-A). In this regard, the
`most advanced clinical data have been generated with a
`humanized monoclonal antibody termed bevacizumab
`(Avastin) that directly binds and blocks all isoforms of
`VEGF-A (Ferrara et al. 2004). Despite the promising
`data achieved to date, dose-response studies suggest that
`higher doses of bevacizumab may provide even greater
`benefit (Yang et al. 2003; Yang 2004), implying that cur-
`rent bevacizumab regimens may not provide optimal
`VEGF inhibition and thus may not have yet demon-
`strated the maximum potential of VEGF blockade in
`cancer. In addition to the promise of anti-VEGF ap-
`proaches in cancer, blocking VEGF-A has also been im-
`pressive in maintaining and improving vision in wet age-
`related macular degeneration (AMD), a disease marked
`by leaky and proliferating vessels which distort the
`retina, and these data suggest that VEGF blockade may
`provide benefit in other eye diseases involving vascular
`leak and proliferation (Bergsland 2004). Efficacy in wet
`AMD has most notably been achieved using a modified
`fragment of
`the bevacizumab antibody,
`termed
`ranibizumab (Lucentis), delivered via monthly intraocu-
`lar injections (Brown et al. 2006; Heier et al. 2006).
`In this paper, we focus on the development and status
`of a novel VEGF-blocking agent, termed the VEGF Trap,
`that retains many of the advantages of a blocking anti-
`body but may offer further potential (Holash et al. 2002).
`The VEGF Trap consists of portions of VEGF receptors
`that have been fused to the constant region of an antibody,
`resulting in a fully human biologic with exceedingly high
`affinity that blocks not only all isoforms of VEGF-A, but
`also related VEGF family members such as placental
`
`growth factor (PlGF). The VEGF Trap also displays ex-
`tended pharmacological half-life, allowing long-term as
`well as very high affinity blockade. The VEGF Trap has
`performed impressively in extensive animal studies of
`cancer and eye diseases, and initial clinical trials appear
`promising. The VEGF Trap may provide the opportunity
`to explore the potential of more complete VEGF block-
`ade in cancer, as well as the opportunity for more com-
`plete blockade and even longer-interval dosing regimens
`in eye diseases. To conclude this paper, we describe how
`the VEGF Trap can be used as a research tool in efforts to
`discover and validate the next generation of targets in the
`field of angiogenesis.
`
`DISCOVERY OF VEGF AND ITS REQUISITE
`ROLES DURING NORMAL DEVELOPMENT
`AND IN DISEASE SETTINGS
`
`Initial studies by Dvorak and his colleagues (Senger et
`al. 1986; Dvorak et al. 1999) identified a protein in tumor
`ascites fluid that was capable of inducing vascular leak and
`permeability, which they termed vascular permeability fac-
`tor (VPF). Independent efforts by Ferrara and his col-
`leagues to identify secreted factors that could promote
`tumor angiogenesis led to the discovery of a protein in
`bovine pituitary follicular cell conditioned medium with
`mitogenic properties for endothelial cells which they
`termed vascular endothelial cell growth factor (VEGF)
`(Ferrara and Henzel 1989; Leung et al. 1989). Upon se-
`quencing and further studies, this VEGF protein was unex-
`pectedly found to correspond to the VPF previously iden-
`tified by the Dvorak lab. These findings set the stage for a
`concerted effort to define the role of VEGF/VPF (hereon
`VEGF) in cancer angiogenesis as well as other settings of
`vascular disease, which have led to the realization that both
`of its initially realized actions—i.e., promoting vascular
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`permeability and vascular growth—appear critical to un-
`derstanding its roles during normal biology and in disease.
`Approximately two decades of intensive investigation
`by numerous laboratories has revealed a great deal about
`VEGF and its actions. It is now clear that VEGF is per-
`haps the most critical vascular regulator during normal
`development as well as in many disease states, and more-
`over, that its dosage must be exquisitely regulated in a
`spatial and temporal manner to avoid vascular disaster.
`Disruption of both VEGF alleles in developing mice,
`which ablates all VEGF production, results in complete
`failure to develop even a primordial vasculature, demon-
`strating that VEGF is absolutely essential for the earliest
`stages of blood vessel development. Still more remark-
`ably, disruption of even a single VEGF allele in develop-
`ing mice, which decreases VEGF levels by half, also re-
`sults in embryonic lethality due to severe vascular
`abnormalities (Carmeliet et al. 1996; Ferrara et al. 1996),
`demonstrating the need for exquisite regulation of VEGF
`levels to form normal vessels. Reciprocally, modest in-
`creases in VEGF levels during development also lead to
`vascular disaster and lethality (Miquerol et al. 2000).
`VEGF continues to be critical during early postnatal
`growth and development, as evidenced by the lethality
`and major growth disturbances caused by conditional dis-
`ruption of the VEGF gene or by administration of VEGF
`blockers (Ravindranath et al. 1992; Carmeliet et al. 1996;
`Ferrara et al. 1996, 1998; Gerber et al. 1999a; Ryan et al.
`1999; Fraser et al. 2000; Zimmermann et al. 2001; Haz-
`zard et al. 2002; Eremina et al. 2003). However, VEGF
`blockade in older animals is much less traumatic, affect-
`ing only those structures that continue to depend on on-
`going vascular remodeling, such as occurs in bone
`growth plates or during remodeling of the female repro-
`ductive organs (Ferrara et al. 1998; Gerber et al. 1999a,b).
`As discussed in greater detail below, vascular remodeling
`is absolutely required in a variety of pathological settings,
`such as during tumor growth, providing major therapeu-
`tic opportunities for VEGF blockade in the adult setting
`in which such blockade can be tolerated.
`
`VEGF ISOFORMS, VEGF FAMILY MEMBERS,
`AND VEGF RECEPTORS
`
`Further study of the gene encoding human VEGF re-
`vealed eight exons separated by seven introns, which re-
`sults in the generation of four isoforms of increasing
`size—VEGF121, VEGF165, VEGF189, and VEGF206 (sub-
`scripts refer to number of amino acids comprising the iso-
`form, with the VEGF isoforms varying in length at their
`carboxyl termini). The main purpose of these isoforms
`appears to relate to their bioavailability such that the 121
`isoform is diffusible, whereas the higher-molecular-
`weight isoforms remain bound to the extracellular matrix,
`requiring cleavage to be released (Houck et al. 1992; Park
`et al. 1993; Keyt et al. 1996).
`Because of the discovery of additional members of
`the VEGF family, VEGF is now often referred to as
`VEGF-A. Other members of the VEGF family were
`
`identified based on their homology with VEGF, as well
`as their ability to interact with a related set of cell-sur-
`face receptors (Eriksson and Alitalo 1999; see below).
`The first VEGF relative to be identified is PlGF, and un-
`til recently, little was known about its normal function
`(Maglione et al. 1991). Whereas mice lacking PlGF ap-
`pear to undergo normal vascular development, recent
`findings indicate that adult mice lacking PlGF exhibit
`deficiencies in certain models of adult vascular remod-
`eling, including in tumors and eye disease models, rais-
`ing the interesting possibility that the activity of PlGF
`may be limited to these settings and that blockade of
`PlGF may provide enhanced efficacy when combined
`with VEGF blockade (Persico et al. 1999; Carmeliet
`2000). Little is known about VEGF-B, and mice lacking
`VEGF-B are overtly healthy and fertile. VEGF-C and D
`seem to play more critical roles in the lymphatic vascu-
`lature than in the blood vasculature, showing specificity
`for a VEGF receptor (see below) expressed on this vas-
`culature; administration of both of these factors leads to
`lymphatic vessel hyperplasia (Joukov et al. 1996; Orlan-
`dini et al. 1996; Olofsson et al. 1999).
`Following rapidly on the heels of the discovery of
`VEGF came the identification of two closely related
`high-affinity receptors for VEGF—FLT1 (FMS-like ty-
`rosine kinase) now termed VEGFR1 (de Vries et al.
`1992), and KDR or Flk1, now termed VEGFR2 (Shibuya
`et al. 1990; Terman et al. 1992; Millauer et al. 1993).
`These high-affinity receptors share features of many
`other growth factor receptors, in that they contain an ex-
`tracellular domain which binds and is dimerized by lig-
`and, and a cytoplasmic tyrosine kinase domain that can be
`regulated upon binding of ligand to the extracellular do-
`main. VEGFR2 seems to be the receptor which mediates
`the major growth and permeability actions of VEGF,
`whereas VEGFR1 may have a negative role, either by
`acting as a decoy receptor or by suppressing signaling
`through VEGFR2. Thus, mice engineered to lack
`VEGFR2 fail to develop a vasculature and have very few
`endothelial cells (Shalaby et al. 1995), phenocopying
`mice lacking VEGF, whereas mice lacking VEGFR1
`seem to have excess formation of endothelial cells that
`abnormally coalesce into disorganized tubules (Fong et
`al. 1995). Mice engineered to express only a truncated
`form of VEGFR1, lacking its kinase domain, appear
`rather normal, consistent with the notion that the primary
`role of VEGFR1 may be that of a decoy receptor (Hirat-
`suka et al. 1998), and supporting only a minor role for the
`cytoplasmic kinase domain. The third member of this re-
`ceptor family, initially called Flt-4 and now termed
`VEGFR3, does not bind to VEGF-A nor PlGF, and in-
`stead binds to VEGF-C and VEGF-D and seems to medi-
`ate the actions of these latter two factors on the lymphatic
`vasculature (Taipale et al. 1999).
`In addition to these primary receptors, a number of po-
`tential accessory receptors for the VEGFs have been
`identified, although the requisite roles of these receptors
`in mediating VEGF responses have not been clearly elu-
`cidated. These potential accessory receptors include the
`neuropilins (Soker et al. 1998).
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`ROLE OF VEGF IN TUMOR ANGIOGENESIS
`SUPPORTS CONCEPT OF BLOCKING VEGF AS
`AN ANTITUMOR STRATEGY
`
`One area of intense study after the discovery of VEGF
`was the analysis of VEGF expression levels in different
`tumor types using in situ hybridization. VEGF was found
`to be expressed in a number of different tumors such as
`renal, gastrointestinal, breast, ovarian, pancreatic, and
`lung, but the variability in expression across the tumor
`and between different tumor types made the simple cor-
`relation between VEGF and severity of the cancer impos-
`sible (Yoshiji et al. 1996; Sowter et al. 1997; Volm et al.
`1997; Ellis et al. 1998; Tomisawa et al. 1999). However,
`out of these studies came the interesting finding that one
`tumor type, renal cell carcinoma, had particularly high
`VEGF expression which correlated with inactivation of
`the von Hippel Lindau locus, resulting in loss of control
`of the tumor’s oxygen sensor, hypoxia-inducible factor
`(HIF) (Iliopoulos et al. 1996; Lonser et al. 2003). The up-
`regulation of VEGF in an attempt to reoxygenate the tu-
`mor through revascularization led to the belief that this
`tumor may either be highly sensitive to anti-VEGF ther-
`apy or highly refractory. Fortunately, the former seems to
`be the case (Yang et al. 2003).
`Concomitant with the analysis of human tumors for
`VEGF expression came the development of animal mod-
`els of cancer where the hypothesis that VEGF was re-
`quired for tumor vasculature, and thus tumor growth,
`could be tested. In 1993, 4 years after their discovery of
`VEGF, Ferrara and colleagues demonstrated that a mouse
`monoclonal antibody to human VEGF (A.4.6.1) could in-
`hibit the growth of several human tumor types in nude
`mice with inhibition ranging from 70% to more than 90%
`(Kim et al. 1993). Subsequent to this observation, a num-
`ber of laboratories using different strategies to inhibit
`VEGF signaling have shown to a greater or lesser extent
`that inhibition of VEGF can have a major impact on tumor
`growth in mice. In addition to numerous studies using the
`VEGF-blocking antibody, other strategies to block VEGF
`in tumor models included blocking antibodies targeting
`VEGFR2 (Prewett et al. 1999), soluble VEGF receptors
`acting as circulating decoys to capture VEGF and prevent-
`ing it from binding cell-surface receptors (Ferrara et al.
`1998; Gerber et al. 1999a,b; Liang et al. 2006), dominant-
`negative VEGF receptors expressed at high levels on tu-
`mor surfaces, small-molecule inhibitors of VEGF receptor
`kinases and other kinases (Smith et al. 2004), antisense
`oligonucleotides targeting VEGF, and VEGF siRNA
`(Grunweller and Hartmann 2005; Lu et al. 2005).
`As the number of studies increased comparing the dif-
`ferent modes of inhibiting VEGF, it became apparent that
`blocking tumor-derived VEGF without blocking stromal
`VEGF was not as efficacious, implicating stromal VEGF
`as a crucial player in tumor growth and angiogenesis.
`Thus, antibodies such as A.4.6.1 which only block human
`VEGF did not fare as well in blocking human tumor
`growth in immunocompromised mice as reagents block-
`ing both tumor and host stroma-derived VEGF (Gerber et
`al. 2000; Liang et al. 2006).
`
`DEVELOPMENT OF VEGF TRAP
`
`The clinical promise of initial anti-VEGF approaches
`highlighted the need to optimize blockade of this path-
`way. Early studies indicated that one of the most effec-
`tive ways to block the VEGF signaling pathway is to
`prevent VEGF from binding to its endogenous receptors
`by administering soluble decoy receptors (Ferrara et al.
`1998; Gerber et al. 1999b). In particular, a soluble decoy
`receptor created by fusing the first three immunoglobu-
`lin-like (Ig) domains of VEGFR1 to the constant region
`(Fc portion) of human IgG1 resulted in a forced homo-
`dimer that acted as a very high affinity blocking reagent
`with 5–20 picomolar binding affinity for VEGF, and in
`tumor experiments this VEGFR1-Fc reagent was effica-
`cious at approximately 500-fold lower concentration
`than a similar VEGFR2-Fc construct (Kuo et al. 2001).
`Despite its high affinity, the VEGFR1-Fc was not a fea-
`sible clinical candidate because of its poor pharmaco-
`kinetic profile; in rodent studies, this protein had to be
`administered frequently and at very high doses to
`achieve efficacious levels. In addition, this agent ap-
`peared to have nonspecific toxicity effects that did not
`seem to be accounted for by its blocking of VEGF (Kuo
`et al. 2001). We decided to exploit our Trap technology
`platform (Economides et al. 2003), which involves
`defining and fusing minimal binding units from differ-
`ent receptor components to generate chimeric fusion
`proteins that act as high-affinity soluble blockers, in an
`attempt to create a potent and well-behaved Trap for
`VEGF. The result was a chimeric fusion protein con-
`taining a modified domain 2 of VEGFR1 and the third
`Ig domain of VEGFR2 fused to the Fc region of human
`IgG1, resulting in a fully human protein that we term
`VEGF Trap (Holash et al. 2002). This reagent has the
`advantage of being fully human and thus potentially
`non-immunogenic, as well as being substantially
`smaller than previous fusion proteins and antibodies,
`raising the possibility that it might allow improved tis-
`sue and tumor penetration. In addition, this VEGF Trap
`had greatly improved pharmacological bioavailability
`as compared to the initial VEGFR1-Fc reagent, exhibit-
`ing about a 300-fold increase in the maximum concen-
`tration achieved in the circulation (i.e., Cmax), as well as
`about a 1000-fold increase in total circulation exposure
`(i.e., AUC) (Holash et al. 2002). Importantly, the affin-
`ity of VEGF Trap binding to both mouse and human
`VEGF isoforms (0.58 pM, 0.46 pM) was superior to that
`of the parental VEGFR1-Fc (~ 5–20 pM) (Holash et al.
`2002). In addition, the VEGF Trap also bound PlGF
`with high affinity (1.8 pM).
`To determine whether the improved pharmacological
`bioavailabity and high-affinity binding of VEGF Trap
`translated into superior performance in vivo, we first
`used a short-term and quantitative in vivo model of
`VEGF activity in which a single dose of VEGF induces
`a stereotypic reduction in blood pressure. In this acute
`assay model, we found that equivalent doses of VEGF
`Trap were indeed far superior to that of the parental
`VEGFR1-Fc (Holash et al. 2002).
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`RUDGE ET AL.
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`VEGF TRAP EXHIBITS IMPRESSIVE
`EFFICACY IN PRECLINICAL TUMOR MODELS
`
`On the basis of the above evidence suggesting that the
`VEGF Trap was a potent VEGF blocker that was effica-
`cious in vivo, we moved to evaluate the VEGF Trap in tu-
`mor models. Initial studies confirmed the remarkable ef-
`ficacy of the VEGF Trap. Not only did the VEGF Trap
`blunt tumor growth in the early models, but it could com-
`pletely block tumor angiogenesis in many cases, resulting
`in completely avascular tumors (Holash et al. 2002).
`These initial studies inspired exploration of the VEGF
`Trap in multiple tumor models, in the laboratories of
`many different investigators, with impressive results in
`almost every case. In addition to its activity in multiple
`subcutaneous models of melanoma, glioma, and rhab-
`domyosarcoma tumors (Holash et al. 2002), the VEGF
`Trap has been shown to work in multiple pancreatic can-
`cer models (Fukasawa and Korc 2004), Wilms’ tumor
`(Huang et al. 2003), Ewing’s sarcoma (Dalal et al. 2005),
`glioblastoma (Wachsberger et al. 2005), and models of
`ovarian cancer as well as associated malignant ascites
`(Hu et al. 2005).
`In addition to the above published studies, recent un-
`published temporal studies indicate that vascular regres-
`sion can be seen in most tumors within hours of VEGF
`Trap treatment, resulting in marked and widespread hy-
`poxia within the tumors. In addition, transcription profil-
`ing studies during these temporal studies have revealed a
`set of endothelial-specific genes that are rapidly and pro-
`foundly regulated in response to VEGF Trap treatment.
`Further studies on some of these genes have led to their
`identification as potential targets for new antiangiogene-
`sis therapies (see below).
`In summary, animal tumor studies have indicated that
`treatment with VEGF Trap effectively inhibited tumor
`growth of a wide variety of murine, rat, and human tumor
`cell lines implanted either subcutaneously or orthotopi-
`cally in mice. VEGF Trap treatment inhibited the growth
`of tumors representing a variety of tumor types, including
`melanoma, glioma, rhabdomyosarcoma, ovarian, pancre-
`atic, renal, and mammary tumor tissue, with a broad ther-
`apeutic index. Growth of small established tumors was
`also inhibited. Histological analysis indicated that treat-
`ment with VEGF Trap resulted in the formation of largely
`avascular and necrotic tumors, demonstrating that tumor-
`induced angiogenesis was blocked. VEGF Trap was also
`active in blocking tumor growth in similar animal tumor
`models in combination with paclitaxel, docetaxel, or ra-
`diation, and was synergistic with 5-fluorouracil. VEGF
`Trap as a single agent and in combination with paclitaxel
`also prevented the formation of ascites in mouse tumor
`models (Byrne et al. 2003; Hu et al. 2005).
`
`VEGF TRAP IN CLINICAL
`TRIALS FOR CANCER
`
`The above results in animal tumor models supported
`the exploration of the VEGF Trap in human studies. Ini-
`tial clinical studies are promising (Dupont et al. 2005;
`
`Mulay et al. 2006; Rixe et al. 2006) in that the VEGF
`Trap as a single agent has resulted in objective radio-
`graphic responses in several advanced cancer patients
`suffering from multiply treated chemotherapy-refractory
`disease, as well as long-term stable disease in patients.
`Similar data are being generated in patients treated with
`VEGF Trap in combination with various chemotherapeu-
`tic agents. The VEGF Trap is now entering an assortment
`of additional exploratory as well as potentially pivotal ef-
`ficacy studies, both as a single agent and in combination
`with chemotherapy.
`
`VEGF TRAP EXHIBITS IMPRESSIVE
`EFFICACY IN PRECLINICAL MODELS OF
`VASCULAR EYE DISEASES
`
`In addition to the role for VEGF in tumor angiogenesis,
`a variety of studies have indicated that VEGF may play a
`key pathological role in vascular eye diseases, in particu-
`lar in diabetic edema and retinopathy settings, and in age-
`related macular degeneration (AMD), which are leading
`causes of vision loss and blindness. In these diseases, ex-
`cess VEGF is thought to result in vascular leak that con-
`tributes to abnormal swelling of the retina and resulting
`vision impairment, as well as in the abnormal growth of
`choroidal and retinal vessels that can destroy normal reti-
`nal architecture. Consistent with these possibilities, the
`VEGF Trap has demonstrated impressive efficacy in an
`assortment of animal models of these eye diseases.
`Preclinical studies in rodents have shown that VEGF
`Trap can inhibit choroidal (Saishin et al. 2003) and
`corneal (Wiegand et al. 2003) neovascularization, as well
`as suppress vascular leak into the retina (Qaum et al.
`2001), and that the VEGF Trap can also promote the sur-
`vival of corneal transplants by inhibiting associated neo-
`vascularization (Cursiefen et al. 2004). In addition, in a
`primate model of AMD, in which choroidal neovascular
`lesions and vascular leak are induced by using a laser to
`create small lesions in the retinas of adult cynomolgus
`macaques, both systemically and intravitreally delivered
`VEGF Trap not only prevented development of vascular
`leak and neovascular membranes when administered
`prior to laser lesion, but also induced regression when ad-
`ministered after lesions had developed (Wiegand et al.
`2005). These preclinical results support a role for VEGF
`blockade, and in particular for local delivery of the VEGF
`Trap, in multiple vascular eye diseases ranging from
`AMD and diabetic eye diseases to corneal injury and
`transplantation.
`
`VEGF TRAP IN CLINICAL TRIALS FOR
`VASCULAR EYE DISEASES
`
`The above results in animal models have supported the
`exploration of the VEGF Trap in human studies of vascu-
`lar eye diseases. Initial clinical studies in human patients
`suffering from both AMD and diabetic edema and
`retinopathy appear quite promising, with evidence in
`early trials that the VEGF Trap can rapidly and impres-
`sively decrease retinal swelling, and that these changes
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`can be associated with improvement in visual acuity
`(Nguygen et al. 2006; Shah et al. 2006). The VEGF Trap
`is now entering more advanced clinical trials in vascular
`eye diseases.
`
`In addition, the success with this screen in terms of yield-
`ing Dll4 as an exciting new antiangiogenesis target has led
`us to rigorously pursue several additional similarly identi-
`fied targets.
`
`THE NEXT GENERATION OF ANGIOGENESIS
`TARGETS: ANGIOPOIETINS AND Dll4
`
`Despite the promise of anti-VEGF approaches in gen-
`eral, and that of the VEGF Trap in particular, it is clear
`both from animal studies and from the emerging human
`trials that tumors display varying degrees of responsivity
`to VEGF blockade. Whereas some tumors might show
`marked regression and/or very long term stabilization,
`other tumors can continue to grow even in the face of anti-
`VEGF treatments. The realization that some tumors can
`be relatively resistant to anti-VEGF approaches raises the
`need for additional antiangiogenesis approaches that
`might be useful in such settings. Toward this end, as noted
`above, we performed transcriptional profiling screens to
`identify endothelial-specific targets that are markedly reg-
`ulated either by VEGF blockade or by excess VEGF activ-
`ity, reasoning that such targets might prove interesting as
`new antiangiogenesis targets. Confirming the potential of
`such a screen, one target that was “rediscovered” via such
`screens was Angiopoietin-2. We had previously indepen-
`dently identified the Angiopoietins as key new angiogenic
`regulators that seemed to work in tandem with the VEGFs
`(Davis et al. 1996; Suri et al. 1996; Maisonpierre et al.
`1997; Valenzuela et al. 1999; Yancopoulos et al. 2000;
`Gale et al. 2002), and moreover, obtained substantial data
`that Angiopoietin-2 in particular was specifically induced
`in tumor vasculature and that it was important for tumor
`angiogenesis (Holash et al. 1999); a recent study employ-
`ing Angiopoietin-2-blocking antibodies confirmed no-
`table antitumor effects (Oliner et al. 2004). On the basis of
`the confidence in these transcriptional profiling screens
`engendered by the reidentification of Angiopoietin-2, we
`explored additional potential targets identified by the
`screens. Among these targets we have reported the identi-
`fication of Delta-like ligand 4 (Dll4) (a ligand for the
`Notch family of receptors) as a gene that is markedly and
`specifically induced in tumor vasculature (Gale et al.
`2004). Moreover, Dll4 is strikingly up-regulated in
`VEGF-overexpressing tumors and down-regulated in tu-
`mors by VEGF blockade. Using VelociGene® technol-
`ogy, which provides a high-throughput approach to create
`mouse mutants for genes of interest (Valenzuela et al.
`2003), we found that mice lacking Dll4 exhibit profound
`vascular defects early in development (Gale et al. 2004).
`Remarkably, and as previously seen only for VEGF (see
`above), deletion of even just one of the two Dll4 alleles in
`developing embryos resulted in embryonic lethality due to
`vascular defects (Gale et al. 2004). All this evidence for a
`critical role for Dll4 in normal as well as tumor angiogen-
`esis provided a rationale to develop blockers for Dll4. Re-
`cent testing in tumor models indicates that Dll4 may in-
`deed prove to be an important new antiangiogenesis
`target, either alone or in combination with the VEGF Trap,
`or in settings of relative resistance to anti-VEGF therapies.
`
`SUMMARY
`
`In summary, using our Trap technology platform
`(Economides et al. 2003), we have created the VEGF
`Trap, a very potent VEGF blocking agent with excellent
`pharmacological properties. This drug has proven to be
`highly efficacious in a number of diverse preclinical
`models. It dramatically inhibits the growth of a variety of
`types of tumors and can even cause frank tumor regres-
`sion in some settings. In other preclinical cancer models,
`we have found that combination of VEGF Trap with a cy-
`totoxic agent can result in potency far greater than that of
`either single agent. Furthermore, the VEGF Trap is also
`very effective in animal models of vascular eye diseases.
`The impressive efficacy in preclinical models of cancer
`and eye diseases provided a rationale for advancement of
`the VEGF Trap into clinical trials, where it is producing
`promising initial results in both cancer and eye diseases.
`In addition to its potential therapeutic value in cancer
`and vascular eye diseases, the VEGF Trap is also an in-
`valuable research tool. Transcription profiling screens us-
`ing VEGF Trap have allowed a number of strategies de-
`signed to identify new antiangiogenesis targets. It is
`hoped that these strategies are helping to identify the next
`generation of antiangiogenesis targets, which may work
`either alone or in combination with the VEGF Trap, or in
`settings of relative resistance to anti-VEGF therapies.
`
`ACKNOWLEDGMENTS
`
`The authors gratefully acknowledge the substantial
`contributions of our colleagues at Regeneron, in particu-
`lar, Jocelyn Holash, Susan D. Croll, Lillian Ho, Michelle
`Russell, Patricia Boland, Ray Leidich, Donna Hylton,
`Shelly Jiang, Sarah Nandor, Alexander Adler, Hua Jiang,
`Elena Burova, Irene Noguera, Ella Ioffe, Tammy Huang,
`Czeslaw Radziejewski, Calvin Lin, Jingtai Cao, Kevin
`Bailey, James Fandl, Tom Daly, Eric Furfine, Jesse
`Cedarbaum, and Neil Stahl. In addition, we acknowledge
`our collaborators on VEGF Trap studies, including Jes-
`sica Kandel, Darrell Yamashiro, Robert Jaffe, Donald
`McDonald, Murray Korc, Phyllis Wachsberger, Adam
`Dicker, Tony Adamis, C. Cursiefen, J.W. Streilein, and
`Peter Campochiaro. We regret if we have omitted key
`contributors.
`
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