Angiogenesis
`
`An Integrative Approach From Science to Medicine
`
`4
`
`EXHIBIT
`
`ro
`
`1*
`
`
`
`Edited by
`
`William D. Figg
`Medical Oncology Branch, Center for Cancer Research,
`National Cancer Institute, Bethesda, Maryland, USA
`
`Judah Folkman
`Department of Surgery, Harvard Medical School and
`Vascular Biology Program, Children's Hospital Boston,
`Boston, Massachusetts, USA
`
`I•L Springer
`
`Mylan Exhibit 1113
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`i.
`
`Editors
`William D. Figg
`Medical Oncology Branch
`Center for Cancer Research
`National Cancer Institute
`Bethesda, Maryland
`USA
`
`Judah Folkman
`Department of Surgery
`Harvard Medical School and
`Vascular Biology Program
`Children's Hospital Boston
`Boston, Massachusetts
`USA
`
`ISBN: 978-0-387-71517-9
`DOl: 10.1007/978-0-387-71518-6
`
`e-ISBN: 978-0-387-71518-6
`
`Library of Congress Control Number: 2007934647
`
`© 2008 Springer Science+Business Media, LLC
`All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science +Business
`Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection
`with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
`developed is forbidden.
`The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of
`opinion as to whether or not they are subject to proprietary rights.
`While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the
`publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect
`to the material contained herein.
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`Printed on acid-free paper
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`987654321
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`[This materialmay be protected by Copyright law (Title 17 U.S. Code(
`
`Chapter 36
`Clincal Development of VEGF Trap
`
`John S. Rudge, Ella Joffe, Jingtai Cao, Nick Papadopoulos, Gavin Thurston,
`Stanley J. Wiegand, and George D. Yancopoulos
`
`Keywords: VEGF Trap, vascular eye disease, kinase inhibitors
`
`Abstract: The inhibition of angiogenesis is proving to be
`an effective strategy in treating diseases involving pathologi-
`cal angiogenesis such as cancer and ocular vascular diseases.
`Since its discovery in the 1980s, vascular endothelial cell
`growth factor (VEGF) has been shown to play a vital role in
`both physiological and pathological angiogenesis, resulting in
`the development of numerous approaches to block VEGF and
`VEGF signaling, ranging from small molecule tyrosine kinase
`inhibitors to protein-based and RNA-based therapeutic
`candidates. VEGF Trap is one such protein-based agent that
`has been engineered to bind and sequester VEGF, as well as
`placental growth factor (PlGF), with high affinity. VEGF Trap
`has been shown to effectively inhibit pathological angiogen-
`esis in numerous preclinical models of cancer and eye disease,
`and is now being evaluated in clinical trials in several types of
`cancer, as well as the 'wet' or neovascular form of age-related
`macular degeneration (AMD). This chapter will summarize
`the basic biology of VEGF and the progress of the VEGF Trap
`from the bench to the clinic.
`
`Introduction
`
`Angiogenesis is a vital process not only during development,
`but also in the adult in settings of wound repair and reproduc-
`tion [1, 2]. However, in diseases characterized by uncontrolled,
`pathological angiogenesis, such as solid tumors or vascular
`diseases of the eye, inhibition of angiogenesis would be of
`therapeutic benefit. While diverse factors might be expected
`to regulate angiogenesis in different settings, a clear con-
`sensus is emerging that a single growth factor, VEGF is the
`
`Regeneron Pharmaceuticals, 777 Old Saw Mill River Road
`Tarrytown, NY 10591, USA
`E-mail: John.Rudge@regeneron.com
`
`critical requisite driver of both physiological and pathological
`angiogenesis in most settings [3]. Thus, while blocking VEGF
`signaling during early development can lead to severe growth
`retardation, it can also produce highly beneficial effects when
`applied to disease states characterized by pathological neovas-
`cularization [3]. Recent clinical studies have validated VEGF
`as a bona fide target for therapeutic intervention in cancer
`as well as in vascular diseases affecting the eye, such as wet
`AMID. These studies have led to approval by the U.S. Food
`and Drug Administration (FDA) of drugs that target the VEGF
`pathway, specifically antibodies directed against VEGF, or
`kinase inhibitors which block activation of the VEGF receptors
`[4-7]. Emerging therapeutic candidates that otherwise target
`VEGF signaling have also reported encouraging results [8].
`
`Biology of VEGF and Its Receptors
`
`VEGF is widely known to promote angiogenesis, by stimulat-
`ing vascular endothelial cell proliferation, migration and tube
`formation, and it can also markedly increase vascular perme-
`ability [9]. VEGF-A is the prototypical member of a family of
`factors that also consists of VEGF-B, VEGF-C, VEGF-D and
`P1GF, which bind differentially to VEGF receptors 1, 2 and 3
`and neuropilin with different specificities [10,11]. In addition,
`alternative exon splicing results in the production of four major
`isoforms of human VEGF-A - VEGF 121' VEGF 165' VEGF 189,
`VEGF206 differentiated by their heparin binding affinity [12].
`VEGF 121differs from the other isoforms in that it does not bind
`heparin and thus is freely diffusible. As the isoforms increase
`in molecular weight, they show increasing heparin bind-
`ing affinity and consequently bind extracellular matrix such
`that they tend to be sequestered near their site of production
`[13-15]. Matrix metalloproteases (IvIMPs) appear to play an
`important role in regulating the release of these isoforms from
`matrix [16]. Although endothelial cells are the primary targets
`of VEGF actions, more recently it has been shown that other
`cell types, including monocytes, hematopoietic stem cells and
`neurons, can also respond to VEGF [17-19].
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`J.S. Rudge et al.
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`The VEGF receptor 2 (VEGFR2) appears to be the major
`mediator of the mitogenic, angiogenic and pro-permeability
`actions of VEGF-A. Indeed, genetic deletion of VEGFR2
`results in profound defects in embryonic vasculogenesis,
`including a failure to develop blood islands and organized
`blood vessels [20]. In contrast, the role of VEGFR1, which
`was the first VEGF receptor identified, is more difficult to dis-
`cern [21]. To date, VEGFR1 has been implicated in MMP9
`induction, hematopoiesis, and monocyte chemotaxis, and also
`appears to act as a 'biological sink' which sequesters VEGF
`from binding to the lower affinity receptor, VEGFR2 [11]. A
`further level of complexity was revealed when VEGF was
`shown to bind neuropilin, a molecule previously identified
`as a receptor for the collapsin/semaphorin family of ligands
`involved in axon guidance. The binding of VEGF to neuropi-
`lin is thought to result in presentation of VEGF to VEGFR2,
`augmenting VEGFR2 signal transduction [22].
`
`The Evidence for VEGF as a Key Player
`in Tumor Angiogenesis
`
`In situ hybridization studies have revealed that VEGF is highly
`expressed in a number of human tumors [23-27]. This is most
`apparent in renal cell carcinoma where mutations in the von
`Hippel-Lindau (VHL) tumor suppressor gene result in increased
`transcription of hypoxia-inducible factor (HMI) genes [28,29].
`Numerous preclinical studies have shown that the growth
`of many different tumor types can be inhibited using agents
`that variously inhibit VEGF signaling (small molecule inhibi-
`tors, VEGFR2 antibodies, soluble VEGF receptors including
`the VEGF Trap) [30-35]. These studies have also shown that
`VEGF derived from the stromal compartment, as well as the
`tumor itself, plays an important role in mediating the angio-
`genesis which supports tumor growth [32,36].
`Not surprisingly, antiangiogenic therapy results in a cyto-
`static effect in many tumor types rather than frank regression.
`Thus, in many models, combination of VEGF blockade with
`chemotherapy or radiation therapy results in greater efficacy
`than either approach alone [37,38].
`
`VEGF Pathway Inhibitors Approved
`for the Treatment of Cancer
`
`Bevacizumab (Avastin®)
`
`The positive results from precinical studies have led to the test-
`ing of several VEGF inhibitors in clinical trials. These include
`the humanized anti-VEGF-A monoclonal antibody (bevaci-
`zumab, Avastin), an anti-VEGFR antibody [39], small mole-
`cules that inhibit VEGFR signaling and VEGF Trap [4,40-42].
`The first clinical validation of the anti-VEGF approach came
`with FDA approval of bevacizumab in 2004, based on the
`results of a randomized double-blind phase ifi trial in which
`
`bevacizumab was combined with bolus IFL (irinotecan, SFU,
`leucovorin) chemotherapy as first line therapy for metatstatic
`colorectal cancer. Median survival increased from 15.6 months
`in the IFL alone arm to 20.3 months in the IFL + bevacizumab
`arm. Severe hypertension was observed in about 10% of beva-
`cizumab treated patients, and gastrointestinal perforation was
`noted in —2% of patients. In addition, the incidence of arterial
`thromboembolic complications (stroke, myocardial infarction,
`transient ischemic attacks, unstable angina) was double the
`incidence seen with chemotherapy alone [43].
`
`Kinase Inhibitors
`
`An alternative approach to using antibodies that bind and neu-
`tralize VEGF is the use of tyrosine kinase inhibitors that target
`the VEGF and other receptors. To date, two such kinase inhibi-
`tors have been approved by the FDA: SU1 1248 (sunitinib; Sutent®)
`and Bay 43-9006 (sorafenib; Nexavar®). Sunitinib inhibits tyro-
`sine phosphorylation of VEGFRl, VEGFR2, platelet derived
`growth factor receptor (PDGFR), c-kit and Flt3, and has been
`approved for the treatment of Gleevec-resistant gastrointesti-
`nal stromal tumors (GIST) and metastatic renal cell carcinoma
`[7]. Sorafenib, which inhibits tyrosine phosphorylation of raf,
`VEGFR2/3, PDGFR, kit and Flt3, was approved in 2006 for the
`treatment of metastatic renal cell carcinoma [5,6]. It should be
`pointed out that these kinase inhibitors do not block VEGF sig-
`naling selectively or completely, and much of their clinical ben-
`efit could derive from the inhibition of other kinase pathways.
`
`Development and Application of VEGF
`Trap in Preclinical Animal Models
`
`Despite the important benefits in patient care provided by the
`currently approved VEGF pathway blockers, current evidence
`suggests that optimal VEGF blockade may not have yet been
`achieved (e.g., dose response studies do not indicate that satura-
`tion of benefit has been reached) [44,45], raising the possibility
`that more potent VEGF blockade could provide even more ben-
`efit for patients. The VEGF Trap may provide the opportunity
`to test this hypothesis, as it was designed to bind VEGF with
`exceedingly high-affinity. The VEGF Trap is a soluble chime-
`ric receptor in which key domains of VEGFR1 (domain 2) and
`VEGFR2 (domain 3) are fused to the constant region (Fe por-
`tion) of human IgGi [34]. This fully human protein is capable
`of binding all isoforms of VEGF-A with very high affinity (KD
`<0.5 pM for hVEGF 165). Moreover, and in contrast to antibodies
`directed against VEGF-A, the VEGF Trap also binds PIGF, a
`VEGF family member also implicated in pathological angiogen-
`esis [46] (KD —25 pM for hPlGF2). In addition, the VEGF Trap
`was engineered to exhibit excellent pharmacokinetic properties,
`and has a circulating half-life in humans of approximately 2-3
`weeks, allowing for bi-weekly or even less frequent dosing.
`Once VEGF Trap had been optimized, it was tested and
`shown to have significant antiangiogenic and anti-tumor efficacy in
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`36. Clincal Development of VEGF Trap
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`numerous preclinical tumor models [34,37,38,47-50]. When
`administered in combination with chemotherapy or radiation,
`VEGF Trap also produced a significant additive impact on
`tumor growth. Moreover, in an ovarian cancer model, VEGF
`Trap potently inhibited ascites formation [37,38,50]. The
`impressive efficacy profile of VEGF Trap in preclinical ani-
`mal models justified its progression into clinical trials.
`Interestingly, VEGF Trap also has an additional property that
`distinguishes it from antibodies: while antibodies form multi-
`meric complexes with their antigens that are rapidly cleared from
`the systemic circulation, VEGF Trap forms an inert 1:1 complex
`with VEGF that remains in the circulation and can thus be read-
`ily measured. Thus, when given in doses that saturate VEGF
`binding, this unique property of the VEGF:VEGF Trap complex
`allows for the accurate determination of tumor and whole body
`VEGF production rates [51]. Somewhat surprisingly, when non-
`tumor bearing animals and humans are given the VEGF Trap,
`we find that total body production rates of VEGF are quite high,
`challenging previous claims that systemic VEGF levels can be
`used as a sensitive measure of tumor burden [51]. This find-
`ing has the important corollary that agents designed to bind and
`inactivate tumor-derived VEGF must be provided in amounts
`sufficient to ensure that they are not effectively consumed by the
`large amounts of VEGF normally produced by the body. Build-
`ing on this finding, we have determined that measurement of the
`levels of free and bound VEGF Trap provides a clear index of
`the doses required to capture all available VEGF, offering a use-
`ful guide for dosing of this antiangiogenic agent. Based on these
`assays, the doses currently being used in the clinic for cancer
`appear to be in the therapeutic range.
`
`VEGF Trap in Clinical Trials for Cancer
`
`Multiple Phase 1 trials have been carried out using the VEGF
`Trap as a single agent, or in combination with various
`chemotherapeutic regimens, in patients with advanced can-
`cers, with more than 300 patients treated to date. Numerous
`objective responses as well as cases of prolonged stable dis-
`ease have been noted in these trials. Interestingly, in addition
`to anti-tumor responses, improvements in tumor-associated
`co-morbidities have been observed: in particular, substantial
`or complete resolution of tumor-associated ascites. The major
`adverse events noted in these trials are consistent with those
`seen with other anti-VEGF agents, and include hypertension
`and proteinuria. Importantly, no antibodies to VEGF Trap
`were observed in any patients.
`VEGF Trap is currently in Phase II clinical trials and is about
`to initiate multiple large Phase 3 trials. Preliminary results
`from two of these studies were recently reported at the Ameri-
`can Society of Clinical Oncology 2007 annual meeting [41,42]
`Results were reported from an interim analysis of a random-
`ized, double-blind, multi-center Phase 2 trial comparing two doses
`of VEGF Trap in patients with recurrent, platinum-resistant
`epithelial ovarian cancer. The patients selected for this study
`
`were heavily pre-treated and had failed several other treatment
`regimens. While the study remains blinded with respect to dose,
`the pooled preliminary results from both the high and low dose
`groups demonstrated anti-tumor activity, as evidenced by an
`8% objective tumor response rate, with a 13% response rate
`as measured by a 50% reduction in circulating levels of the
`tumor marker CA-125. Disease was judged to be stable in 77%
`of patients at 4 weeks, and in 41% of patients at 14 weeks. In
`addition, of 24 patients in the study who had tumor-associated
`ascites, 29% experienced complete resolution of their ascites
`while 54% experienced no increase in ascites during treatment.
`Tolerability was similar to other molecules in this class with
`hypertension being the most common grade 3/4 event (16%).
`Two patients (1.2%) experienced bowel perforation, both of
`whom recovered. In a collaboration involving Sanofi-Aventis
`and Regeneron Pharmaceuticals, the ongoing single-agent
`studies of VEGF Trap will be complemented by a large Phase
`3 program combining VEGF Trap with standard chemotherapy
`regimens in at least 5 different advanced solid tumors:
`colorectal, non-small cell lung, prostate, pancreas and gastric
`cancer, with the first of these studies initiating in 2007.
`
`Preclinical Studies with VEGF Trap
`in Vascular Eye Diseases
`
`The finding that VEGF-A was up-regulated in the aqueous
`and vitreous humor of patients with proliferative diabetic
`retinopathy extended the focus of antiangiogenic agents from
`cancer to vascular eye disease [52,53]. Wet age-related macu-
`lar degeneration (AMD) is the most common cause of vision
`loss in the elderly [53,54]. In this disease, VEGF is believed to
`mediate the abnormal growth of choroidal vessels that, together
`with the associated vascular leak and edema, disrupts the nor-
`mal retinal architecture. VEGF Trap has also been tested in
`a number of rodent models of ocular vascular disease, where
`it has been shown to inhibit choroidal [55] and corneal [56]
`neovascularization in addition to suppressing vascular leak in
`the retina [57]. VEGF Trap has also been shown to improve
`the survival of corneal transplants [58]. In a primate model of
`AMID, in which a laser was used to induce choroidal vascular
`lesions, intravitreal administration of VEGF Trap was found
`not only to prevent the development of vascular leak and neo-
`vascularization when administered before the time of injury
`but also completely resolved vascular leak when administration
`was delayed until after the lesions had fully developed [59].
`
`VEGF Trap in Clinical Trials for Vascular
`Eye Disease
`
`The promising results in preclinical models supported the
`introduction of VEGF Trap into the clinic for treatment of
`both wet AMID and diabetic macular edema, using a version of
`VEGF Trap specifically formulated for intra-ocular administration,
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`termed VEGF Trap-Eye. Following successful completion of
`Phase I studies in wet AMD, VEGF Trap-Eye has progressed
`to a Phase 2 trial, for which interim results were recently pre-
`sented at the Association for Research in Vision and Ophthal-
`mology (ARVO) 2007 annual meeting [60,61]. In this trial,
`VEGF Trap-Eye was given at monthly intervals at doses of
`0.5 or 2.0 mg, or as a single injection of 0.5, 2.0 or 4.0 mg. In
`the interim analysis, VEGF Trap-Eye met the pre-specified
`primary endpoint of reducing retinal thickness as measured by
`ocular coherence tomography (OCT) at 12 weeks compared
`with baseline (all groups combined, decrease of 135 gm, P <
`0.0001). Mean change in visual acuity, a key secondary end-
`point of the study, also demonstrated a statistically significant
`improvement (all groups combined, increase of 5.9 letters,
`P < 0.0001). Interestingly, patients in the highest monthly
`dose group achieved an average vision gain of more than 10
`letters at 12 weeks, and even a single injection of VEGF Trap-
`Eye resulted in improved mean visual acuity at 12 weeks (all
`dose levels combined). Thus, the increased potency of VEGF
`Trap-Eye may offer the potential for improved efficacy and/or
`longer dosing intervals compared to the current standard of
`care [62-64]. In the VEGF Trap-Eye Phase 2 trial, there were
`no drug-related serious adverse events, and treatment with the
`VEGF Trap-Eye was generally well-tolerated. The most com-
`mon adverse events were those typically associated with intra-
`vitreal injections. A Phase ifi program for VEGF Trap-Eye in
`wet AMD will be initiated in 2007.
`In addition, as reported at ARVO 2007 [60,65], a Phase 1
`study of VEGF Trap-Eye in diabetic macular edema (DME)
`showed that a single intravitreal injection in patients with
`longstanding diabetes resulted in a marked decrease in mean
`central retinal thickness and mean macular volume through-
`out the 6 week observation period. Additional trials with VEGF
`Trap-Eye in DME are anticipated in the future.
`
`Conclusion
`
`The VEGF Trap is an all human, chimeric receptor-based pro-
`tein engineered to potently bind all forms of VEGF-A and
`PIGF, and to exhibit superior pharmacokinetic properties. The
`promise of the VEGF Trap in preclinical models of cancer and
`vascular eye diseases is being maintained during its initial eval-
`uation in Phase 1 and 2 human trials, offering the hope that this
`therapeutic candidate may offer additional benefit to patients
`suffering from cancer or blinding vascular eye diseases.
`
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