For reprint orders, please contact: reprints@futuremedicine.corn
`
`Afiibercept in epithelial
`ovarian carcinoma
`
`John W Moroney, Anil K Sood & Robert L Colemant
`tAuthor for correspondence: Department of Gynecologic Oncology, University of Texas MD Anderson
`Cancer Center, 1155 Herman Pressler Drive CPE36.3244, Houston, TX 77030-3721, USA
`• rcoleman@mdandersonorg
`
`Angiogenesis is a hallmark of malignant transformation. With improved
`understanding of angiogenic signaling in both the normal and malignant state,
`there have been a number of agents developed that target VEGF signaling.
`These targeted agents can affect downstream VEGF signal transduction via
`unique mechanisms at different cellular and extracellular locations. The
`aflibercept, or VEGF-Trap, molecule is the subject of this article. Its molecular
`structure, pharmacokinetic and pharmacodynamic profile, and preclinical and
`early clinical data in epithelial ovarian carcinoma Is reviewed. For comparison,
`other anti-angiogenic agents that have been or are currently being studied in
`epithelial ovarian carcinoma are also summarized. Finally, the anticipated role
`of aflibercept in the treatment of epithelial ovarian carcinoma is also discussed.
`
`Epithelial ovarian cancer (EOC) is a malig-
`nancy that afflicts one in 70 women. It occurs
`most frequently at approximately 60 years of
`age, and is more common in Caucasian women
`(45%) than in women of other races (Black:
`38%; all others: 17%). In the USA, an esti-
`mated 22,000 women are diagnosed with EOC
`annually [1]. In Europe, the incidence is substan-
`tially higher, with an estimated 44,000 cases
`identified per year. Most women who present
`with EOC are found with advanced disease;
`approximately 75% of patients are stage III—IV
`at diagnosis. Surgical cytoreduction and cyto-
`toxic chemotherapy are the cornerstones of pri-
`mary therapy for advanced disease, and most
`patients achieve a complete clinical remission
`in response to primary therapy. Unfortunately,
`recurrence is both common and lethal; 5-year
`survival for stage IIIC and IV patients is 29 and
`13%, respectively [2].
`Like most other solid tumors, EOCs exhibit
`a large number of molecular abnormalities that
`are thought to be central to carcinogenesis and
`metastatic spread. Among these, increased
`VEGF signal transduction, an important step
`in tumor-associated angiogenesis, appears to be
`one of the primary means by which malignant
`ovarian cells grow and disseminate.
`Targeted inhibition of VEGF signaling has
`been shown to be an effective way to treat many
`different solid tumors, including EOC [3]. Since
`the introduction of monoclonal antibodies such
`as bevacizumab (AvastinTM, Genentech, CA,
`USA), significant emphasis has been placed on
`the development of other VEGF pathway inhibi-
`tors that have the potential to provide improved
`
`efficacy in the treatment of malignancy. One
`of these, the subject of this review, is the drug
`aflibercept, also known as VEGF-Trap.
`Contemporary adjuvant chemotherapy for
`EOC in the primary treatment setting is platinum
`and taxane based. Efforts to improve response
`rate and progression-free survival by adding
`cytotoxic agents to platinum and taxane therapy
`have not been successful [4]. In general, EOC is
`a relatively chemosensitive disease, as up to 80%
`of patients achieve a complete clinical remission.
`Unfortunately, the recurrence rate for EOC in
`patients who achieve this status is approximately
`80% [5]. Treatment in the recurrent setting may
`include surgery, radiotherapy and chemotherapy,
`or any combination thereof. In nearly all cases, the
`approach is considered palliative. As such, there
`is no clear management algorithm and treatment
`options are, in large part, individualized. The
`manner in which they are individualized depends
`upon a number of factors, such as the location
`and estimated number of recurrent implants, the
`patient's previous response to platinum- and tax-
`ane-based chemotherapy and functional status.
`the typical treatment and sur-
`FIGURE iillustrates
`vival timeline for patients with EOC. TABLE 1 lists
`agents offered by the National Comprehensive
`Cancer Network (NCCN) as acceptable for use
`in patients with recurrent EOC [6]. Of note, only
`one targeted biologic agent is listed, bevacizumab.
`This agent has become more widely used in the
`past few years after multiple Phase II studies have
`demonstrated its activity in EOC [7-9] .
`The VEGF family is composed of five glyco-
`proteins referred to as VEGF-A, VEGF-B,
`VEGF-C, VEGF-D and PIGF. Expression
`
`Keywords
`
`aflibercept
`• antiangiogenesls
`. epithelial ovarian
`carcinoma • vascular
`endothelial growth factor
`• VEGF-Trap
`
`-Future
`'medicine fsg
`
`part of
`
`10.2217/FON.09.35 2009 Future Medicine Ltd
`
`Future Once,. (2009) 5(5), 591-600
`
`ISSN 1479-6694
`
`591
`
`Regeneron Exhibit 2014
`Page 01 of 10
`
`

`

`Drug Evaluation Moroney, Sood & Coleman
`
`Diagnosis
`
`Surgical
`evaluation?
`
`Progression
`
`Death
`
`Secondary
`surgery?
`
`7
`
`Symptoms
`
`Chemotherapy #1
`
`Maintenance?
`
`Chemo #2
`
`Chemo #3+
`
`Staging
`
`Supportive
`care
`
`Figure 1. Epithelial ovarian cancer: natural history.
`
`of the different VEGF ligands are associated,
`somewhat uniquely, with key events in physi-
`ologic vasculogenesis and angiogenesis from
`embryonic life forward [to]. The most well
`described of these ligands is VEGF-A (VEGF).
`In mice, homozygous deletion of the VEGF is
`embryologically lethal, resulting in defects in
`vasculogenesis and cardiovascular abnormali-
`ties. VEGF-A is also important to a number
`of postnatal angiogenic processes, including
`wound healing, ovulation, menstruation, main-
`tenance of blood pressure and pregnancy. P1GF
`and VEGF-B, in contrast to VEGF-A, do not
`appear to be essential to organogenesis or early
`development in mice. P1GF knockout mice,
`however, show a reduced ability to respond to
`ischemic damage through angiogenesis and
`adaptive arteriogenesis, suggesting a role for
`
`P1GF in pathologic states in the adult [is]. VEGF
`is alternatively spliced after translation, form-
`ing numerous isoforms of varying lengths (e.g.,
`121-, 145-, 165- and 206-amino acid proteins).
`Of these isoforms, VEGF165 is predominant, and
`is overexpressed in a number of solid tumors.
`The predominant isoform of P1GF (P1GF-1) has
`close structural similarity to VEGF-A [11].
`VEGF ligands as well as P1GF bind to and
`activate three closely related transmembrane
`receptor tyrosine kinases, referred to as VEGF
`receptor (VEGFR)1 (FLTI), VEGFR2 (KDR)
`and VEGFR3 (FLT4). The multiple different
`VEGF ligands have unique binding specificities
`for each VEGFR tyrosine kinase receptor, con-
`tributing to their diversity of function. After fig-
`and binding to VEGF receptors, each tyrosine
`kinase activates a complex network of unique
`
`Hormonal
`therapy
`
`Targeted therapy
`
`Anastrozole
`Letrozole
`Tamoxifen
`
`Bevacizumab
`
`Cytotoxic therapy
`
`Cisplatin (if platinum-sensitive)
`Carboplatin (if platinum-sensitive)
`Gemcitabine
`Carboplatin/paclitaxel (category 1)
`(if platinum-sensitive)
`Gemcitabine/carboplatin
`Liposomal doxorubicin
`Topotecan
`Altretamine
`Capecitabine
`Cyclophosphamide
`Docetaxel
`Etoposide, oral
`Ifosfamide
`Irinotecan
`Melphalan
`Oxaliplatin
`Paclitaxel
`Vinorelbine
`
`Preferred agents
`
`Other potentially
`active agents
`
`Data taken from [6].
`
`592
`
`Future Oncol. (2009) 5(5)
`
`future science group EM
`
`Regeneron Exhibit 2014
`Page 02 of 10
`
`

`

`Aflibercept in epithelial ovarian carcinoma Drug Evaluation
`
`downstream signaling pathways. VEGFR2 is
`expressed primarily within the endothelium,
`and is considered the primary mediator of
`VEGF-induced angiogenesis [12]. VEGFR1 is
`expressed in multiple tissues, including vas-
`cular endothelium; however, its exact role in
`signal transduction-mediated angiogenesis is
`unclear. It has a tenfold higher binding affinity
`to VEGF than VEGFR2, but VEGFR1 is much
`less involved with subsequent signal transduc-
`tion activity than VEGFR2 [12]. VEGFR3 pref-
`erentially binds VEGF-C and VEGF-D, and
`is found primarily with lymphatic endothe-
`lial cells. Recent data has demonstrated that
`VEGFR3 is also located within the vascular
`endothelium. Targeting VEGFR3 leads to
`tumor regression in some mouse xenograft
`studies [13] .
`Neuropilins are a group of VEGFRs that
`were described subsequent to initial characteri-
`zation of the VEGF receptor family. They were
`originally named based on their role in axonal
`guidance in the developing nervous system,
`and were then found expressed on endothelial
`cells as well as overexpressed on tumor cells.
`Neuropilins have since been found to be impor-
`tant co-receptors for VEGFR signaling, and
`also possess intrinsic pro-angiogenic signaling
`capability. They are promising targets for anti-
`angiogenic therapy, particularly in combination
`with cytotoxics and/or other vascular-focused
`therapy. Development of neuropilin inhibi-
`tors is currently underway and awaits clinical
`investigation [12].
`
`Overview of the market
`Despite the promise of anti-angiogenic agents in
`the treatment of ROC, there are relatively few
`clinically available drugs available at the current
`time. There are, however, multiple drugs under
`study (see below). Each of these agents can be
`categorized mechanistically. A brief description
`of each follows (TABLE 2).
`
`Monoclonal antibodies targeting the
`VEGF pathway
`VEGF monoclonal antibodies (Mabs) act by
`antagonizing the VEGF receptors (prima-
`rily KDR and FLTI) or their respective lig-
`ands. Currently, the most widely used VEGF
`Mab is bevacizumab, which is a chimeric
`murine/human antibody targeting the VEGF
`ligand. It currently has US FDA approval in
`combination with 5-fluorouracil-based therapy
`in metastatic colorectal carcinoma, metastatic
`breast carcinoma in combination with paclitaxel
`and in nonsquamous, non-small-cell lung cancer
`patients in combination with carboplatin and
`paclitaxel [3] .
`Although not formally approved for EOC,
`bevacizumab has been widely studied in multiple
`Phase II trials as both a single agent and in com-
`binations with both cytotoxic and other targeted
`therapies (TABLE 3) [7-9] . Several EOC Phase III
`trials in both the primary and recurrent settings
`are underway (TABLE 4).
`
`Tyrosine kinase inhibitors targeting the
`VEGF pathway
`VEGF tyrosine kinase inhibitors (TKIs) are
`small molecules that bind specific intracellular
`domains of VEGFR receptor tyrosine kinases
`(RTKs), inhibiting phosphorylation and subse-
`quent signal transduction. Most RTK inhibitors
`are relatively selective, however, not specific for
`a unique RTK domain. As a result, they func-
`tion, to varying degrees, as multikinase inhibi-
`tors. Commonly, other RTK systems affected are
`within the EGF and PDGF receptor families. In
`addition, small-molecule TKIs frequently act on
`downstream signaling effectors such as Raf, Src
`and Met [10].
`There are no VEGF TKIs currently approved
`in EOC; however, several have been studied in
`the Phase II setting and have shown some activ-
`ity in combination with other agents in heavily
`pretreated patients [14-23] (TABLE 5).
`
`Trial name
`GOG-218
`ICON-7
`ICON-6
`GOG-213
`OCEANS
`*Platinum sensitive.
`'VEGFR-2 tyrosine kinase inhibitor.
`GOG: Gynecologic Oncology Group; ICON: Intemationational Collaborative Ovarian Neoplasm.
`
`Combination
`Carboplatin, paclitaxel, bevacizumab
`Carboplatin, paclitaxel, bevacizumab
`Carboplatin, paclitaxel, cediranib*
`Carboplatin, paclitaxel, bevacizumab
`Carboplatin, paclitaxel, bevacizumab
`
`Treatment setting
`Primary
`Primary
`Recurrent
`Recurrent*
`Recurrent*
`
`Anticipated accrual
`2000
`1444
`2000
`660
`440
`
`fsq future science group
`
`www.futuremedicine.com
`
`593
`
`Regeneron Exhibit 2014
`Page 03 of 10
`
`

`

`Drug Evaluation Moroney, Sood & Coleman
`
`Variable
`
`Burger
`Cannistra Garcia*
`(n = 44) (%) (n = 70) (%) (n = 62) (%)
`
`52
`48
`
`Previous regimens
`1
`2
`3
`Response rate
`CR
`PR
`GI perforations
`Arterial thrombosis
`Bevacizumab-related deaths
`Ref.
`'Combined with oral cyclophosphamide.
`CR: Complete response; GI: Gastrointestinal; PR: Partial response.
`
`0
`16
`11
`7
`7
`[81
`
`100
`
`0
`24
`6
`4
`4
`[9]
`
`34
`
`3
`18
`0
`0
`0
`[7]
`
`Fusion proteins
`Both monoclonal antibodies and TKIs act via
`direct interaction with binding domains on
`either soluble VEGF ligands or membrane-
`bound receptors. Another, unique method to
`inhibit VEGF-mediated angiogenic signaling
`involves the use of a soluble fusion protein com-
`prised of truncated VEGFRI and VEGFR2
`binding domains combined with the Fc portion
`of IgGl. This molecule, via a mechanism similar
`to that of VEGF Mabs, serves to function as a
`decoy receptor, binding with high affinity to the
`VEGF-A ligand and thus preventing VEGFRI
`and VEGFR2 binding and subsequent stimu-
`lation [24] . Despite the mechanistic similarity
`between Mabs and fusion proteins, there are
`clear structural and pharmacokinetic differences
`
`between them, the functional significance of
`which are incompletely understood. The only
`VEGF fusion protein currently in clinical use
`is aflibercept.
`
`I Introduction to the compound
`The aflibercept molecule (parental VEGF -Trap)
`was originally synthesized as a fusion protein
`combining the constant region (Fc) of IgG1 with
`the first three domains of VEGFRI (VEGF881).
`It was found to have impressive, picomolar
`binding affinity to VEGF ligand and promis-
`ing anti-tumor activity in transformed cancer
`cell lines. Unfortunately, it was also found in
`its parental form to have a significant positive
`charge, and as a result, to bind nonspecifically
`to negatively charged extracellular matrix pro-
`teins, causing its systemic half-life to be poor. It
`was then modified to include the Fc region of
`IgG1 fused with domain two of VEGFRI and
`domain three ofVEGFR2 (VEGF8RiR2) (FIGUHE2).
`This modification served to maintain its high
`VEGF-A ligand affinity and relative specificity,
`and to significantly prolong its in vivo half-life,
`making it clinically useful. It also has strong,
`picomolar binding affinity for P1GF. All of the
`aflibercept variants were produced and purified
`from Chinese hamster ovary cells [24] .
`Aflibercept has undergone extensive pre-
`clinical testing to establish it as an effective
`inhibitor of angiogenesis and tumor growth in
`animal models. Subcutaneous mouse tumor
`xenograft studies using cancer cell lines from
`multiple species (murine melanoma, human
`rhabdomyosarcoma and rat glioma) were
`
`‘_
`
`AF doses
`(mg/kg)
`
`Combination
`agent (s)
`
`G2 events (%)
`
`G3—G4 events (%)
`
`Ref.
`
`Year Publication Author
`form
`Phase 1
`2008 Abstract
`
`Rixe
`
`2008 Abstract
`
`Freyer
`
`2-6
`
`4, 6
`
`2008 Absract
`
`Limentani 2, 3, 4, 5
`
`2008 Abstract
`
`Coleman
`
`2-6
`
`Dysphonia (24),
`lrinotecan, 5-FU,
`epistaxis (18)
`leucovorin
`Docetaxel, cisplatin Epistaxis (19), proteinuria
`(17) dysphonia (13)
`Not reported
`
`Oxaliplatin, 5-FU,
`leucovorin
`(FOLFOX-4)
`Docetaxel 75 mg/kg Fatigue (33),
`mucositis (11)
`
`Hypertension (32),
`proteinuria (11)
`Hypertension (13)
`
`Hypertension (13), proteinuria
`(13), hemorrhagic events (3),
`deep vein thrombosis (3)
`Hypertension (22),
`neutropenia (89)
`
`Hypertension (9), proteinuria
`(4), encephalopathy (2),
`renal failure (2)
`Study in progress
`
`[31]
`
`[36]
`
`[35]
`
`[37]
`
`[32]
`
`Phase 11
`2007 Abstract
`
`Tew
`
`2, 4
`
`None
`
`Not reported
`
`2008 Unpublished Coleman
`5-FU: 5-fluorouracil; AF: Aflibercept.
`
`6
`
`Docetaxel 75 mg/kg Study in progress
`
`594
`
`Future Oncol. (2009) 5(5)
`
`future science group in
`
`Regeneron Exhibit 2014
`Page 04 of 10
`
`

`

`Aflibercept in epithelial ovarian carcinoma Drug Evaluation
`
`Drug
`
`Generic name
`
`Target
`
`SU11248
`ZD6474
`Sorafenib
`
`AZD2171
`GW786034
`
`PTK787
`BIBF 1120
`BMS-582664
`
`Sutent
`Vandetanib
`Sorafenib
`
`Cediranib
`Pazopanib
`
`Vatalanib
`Vargatef
`Brivanib
`
`VEGFR, EGFR, PDGFR, c-KIT
`VEGFR, EGER
`RAF-1, VEGFR-2, EGFR-3
`PDGFR-I3, FLT-3, c-KIT
`VEGFR-1-3, PDGFR, c-KIT
`VEGFR1-3, PDGFR-cdp,
`c-KIT
`VEGFR-1-3, PDGFR, c-KIT
`VEGFR, PDGFR, FGFR
`VEGFR-2, FGFR
`
`Current trials in
`ovarian carcinoma
`Phase II/III
`Phase II
`Phase I/II/III
`
`US FDA approval
`(indication)
`Yes (renal)
`Yes
`Yes (renal)
`
`Phase I/11/111
`Phase II
`
`Phase I
`Phase I
`Phase I
`
`No
`No
`
`No
`No
`No
`
`Ref.
`
`[14]
`[21]
`[IC
`
`[22]
`[15]
`
`[20]
`[17]
`[Is]
`
`performed. Aflibercept at high (25 mg/kg) and
`low (2.5 mg/kg) doses was administered subcu-
`taneously twice-weekly. Measurement of tumor
`volumes at multiple time points from initiation
`of therapy demonstrated significant regression in
`tumor in two of the three models. Examination
`of tumor sections from sacrificed treatment and
`control mice demonstrated substantial decreases
`in tumor vascularity in treated mice, particularly
`those who received high doses [24].
`With the knowledge that VEGF plays an
`important role in the development of malig-
`nant ascites, a mouse model of ovarian cancer
`using VEGF overexpressing SKOV-3 cells was
`created. Mice were administered aflibercept
`25 mg/kg twice-weekly or placebo. Aflibercept
`treated mice developed minimal to no ascites in
`comparison to placebo-treated mice. In a similar
`experiment using OVCAR-3 cells, both inhi-
`bition of ascites as well as tumor growth were
`significantly improved with aflibercept relative
`to placebo [25].
`Initial anti-angiogenic strategies targeting
`VEGF were tested in preclinical models and in
`humans who were known to have minimal dis-
`ease (e.g., early stage or immediately following
`surgical cytoreduction). It has been postulated
`that pro-angiogenic growth factors secreted by
`pericytes found in established or bulky tumors
`would make them refractory to VEGF antago-
`nism. To test this in the preclinical setting,
`mice with bulky, orthotopic, SK-NEP-1 (Wilms
`tumor) xenografts were injected subcutaneously
`with aflibercept 500 pg or an equivalent placebo
`volume twice-weekly. Mice were then sacrificed
`at multiple time points and their tumors were
`weighed. Significant differences between treated
`mice and controls were observed at multiple time
`points, with a 79.3% decrease noted in VEGF
`treated mice at the longest tested treatment
`duration (36 days). Terminal deoxynucleotidyl
`
`Si future science group
`
`transferase-mediated dUTP-biotin nick-end labe-
`ling (TUNEL) and platelet endothelial cell adhe-
`sion molecule-1 (PECAM-1) staining strongly
`indicated that apoptosis was the mechanism
`responsible for the loss of tumor cells [26].
`Aflibercept was also evaluated in an orthotopic
`murine renal cancer cell model. Aflibercept's
`ability to prevent tumor formation after ortho-
`topic injection of luciferase-tagged renal cancer
`cells, as well as its effect on measurable tumor
`burden, was assessed. In the prevention assay,
`twice-weekly injections of aflibercept 10 mg/kg
`versus control were performed starting day 3 or 4
`after injection of cancer cells. Luciferase assay at
`30 days demonstrated an 87% decrease in tumor
`
`141
`
`7
`
`Fc
`
`Aflibercept molecule
`
`Kinase
`
`Kinase
`
`VEGFR1
`
`VEGFR2
`
`Figure 2. Aflibercept/VEGF., structure. Domain 2 of VEGFR1 and domain 3
`of VEGFR2 complexed with the Fc portion of human IgG1.
`
`www.futuremedicine.com
`
`595
`
`Regeneron Exhibit 2014
`Page 05 of 10
`
`

`

`Drug Evaluation Moroney, Sood & Coleman
`
`growth (p = 0.007), and survival in these mice
`was increased by 27 days (p < 0.0001). In the
`intervention assay, aflibercept 10 and 25 mg/kg
`versus control was administered subcutaneously
`twice-weekly for up to 30 days. Aflibercept
`inhibited primary tumor growth by 74 and
`78%, respectively by luciferase expression, and
`this was confirmed by tumor weight analysis.
`Formation of detectable lung metastases was
`inhibited in 98% of cases (p = 0.004) [27].
`
`I Chemistry
`As mentioned previously, aflibercept is a solu-
`ble fusion protein consisting of the Fc portion
`of IgG1 combined with the second domain of
`VEGFR1 and the third domain of VEGFR2. Its
`molecular weight is 115 kDa, and it is produced
`from Chinese hamster ovaries [24]. Aflibercept is
`depicted in FIGURE 2.
`
`Pharmacokinetics & metabolism
`Published data regarding the pharmacodynamic
`and pharmacokinetic qualities of aflibercept were
`performed using subcutaneous injection of the
`drug. Since that time, additional experiments
`have been performed comparing intravenous
`dosing with subcutaneous dosing, demonstrat-
`ing nearly identical bioavailability between the
`two dosing methods [28].
`The pharmacokinetic qualities of aflibercept
`were obtained by injecting VEGFR,R, (4 mg/kg)
`and AUC were found to be
`into mice. C
`16 pg/ml and 36.28 pg x days/ml, respectively.
`Equilibrium binding assays were also performed,
`demonstrating a K, = 1 pM for the predominant
`VEGF isoform in humans, VEGF165 [24].
`Pharmacokinetic studies performed during
`development of the aflibercept molecule iden-
`tified that, in comparison with VEGF target-
`ing Mabs, which forms a multimeric immune
`complex, the Trap molecule forms a 1:1 stable
`and inert complex with VEGF-A ligand that is
`measurable and persists systemically for up to
`14 days after a single subcutaneous dose. Holash
`et al. described experiments in murine cancer
`models where the attainment of an optimal ratio
`of free VEGF-Trap to complexed VEGF Trap in
`measured serum samples correlated with maxi-
`mal tumor response, allowing the Trap complex
`to serve as its own biomarker for the achievement
`of optimal dosing [13].
`
`Pharmacodynamics
`Pharmacodynamic performance in vivo was
`assessed by examining the ability of aflibercept
`to block dose-dependent hypotension caused by
`
`VEGF infusion in mice. A single dose of afliber-
`cept (25 mg/kg) 24 h prior to VEGF admin-
`istration completely blocked VEGF-induced
`hypotension. Its in vivo durability was tested
`by measuring the blockage of hypotension at
`1, 3 and 7 days after aflibercept administration
`(5 mg/kg x 1). It was completely effective at
`blocking hypotension through day 3, indicating
`in vivo activity through this duration [24].
`One of the potential differences between
`aflibercept and bevacizumab stems from their
`different pharmacodynamic properties. As men-
`tioned above, the aflibercept molecule forms a
`1:1, relatively low molecular weight complex
`with the VEGF-A ligand [28]. Bevacizumab,
`in contrast, is known to form larger molecular
`weight multimeric immune complexes. Some
`authors have hypothesized that some of the
`class effects of VEGF targeted therapies, such
`as hypertension, proteinuria and arterial throm-
`bosis, are largely a result of immune complex
`deposition and immunogenicity. For this rea-
`son, some authors question whether the smaller
`molecular weight and presumably less immuno-
`genic aflibercept IC will result in fewer such
`adverse effects [28]. Interestingly, Meyer et al.
`recently reported that bevacizumab immune
`complexes were able to induce platelet aggrega-
`tion via activation of the FcyRIIa receptor found
`on human platelets [29].
`
`Clinical efficacy
`To date there is only one peer-reviewable pub-
`lished trial regarding aflibercept; however, there
`have been numerous published abstracts that
`describe its clinical efficacy in a variety of solid
`tumors [30]. While it has shown some activity as
`a single agent, its use in combination with other
`agents has been more impressive.
`A Phase I study of single-agent aflibercept
`administered intravenously every 2 weeks explored
`doses ranging between 0.3 and 7.0 mg/kg. Free
`aflibercept concentrations remained in excess of
`bound aflibercept concentration during the entire
`2-week dosing interval for dose levels at 2.0 mg/
`kg and above, suggesting that enough aflibercept
`was present to bind all endogenous and tumor-
`produced VEGF [31]. Thus, a 4.0 mg/kg every
`2 weeks dose of aflibercept has been utilized in
`several Phase II studies.
`The results of a randomized, double-blind,
`Phase II study of 215 women with advanced
`ovarian cancer (predominantly third- and fourth-
`line therapy) who were treated with single-agent
`aflibercept at a dose of either 2 mg/kg or 4 mg/
`kg every 2 weeks were reported in 2007 [32].
`
`596
`
`Future Oncol. (2009) 5(5)
`
`future science group El
`
`Regeneron Exhibit 2014
`Page 06 of 10
`
`

`

`Aflibercept in epithelial ovarian carcinoma Drug Evaluation
`
`Response to treatment was assessed both by the
`clinical investigators and an independent review
`committee (IRC). As assessed by the investi-
`gators, Response Evaluation In Solid Tumors
`(RECIST) response rates were 7.3 and 3.8% for
`the 4 mg/kg and 2 mg/kg dose, respectively. The
`response rates as assessed by the IRC were 4.6 and
`0.9% in the 4 mg/kg and 2 mg/kg arms, respec-
`tively. The CA-125 response rates were defined as
`at least a 50% reduction in CA-125 protein levels,
`and were approximately 11% in both arms. A
`total of 18 (13.8%) of the 130 patients evaluable
`for CA-125 response from the combined groups
`had either a RECIST (as assessed by the IRC)
`or CA-125 response. The median progression-
`free survival was 13.3 and 13.0 weeks with the
`4 mg/kg and 2 mg/kg doses, respectively. The
`median overall survival was 49.3 and 55.4 weeks
`with the 4 mg/kg and 2 mg/kg doses, respec-
`tively. Of the 40 patients in both dose groups
`who had evaluable ascites at baseline, 77.5% had
`either a complete disappearance or stabilization
`of their ascites [32].
`Aflibercept is also being evaluated in com-
`bination with a variety of chemotherapeutic
`agents. A Phase I study evaluating aflibercept
`with docetaxel revealed that it could be safely
`combined with docetaxel with no increase in
`docetaxel-related toxicity. The recommended
`dose was aflibercept 6.0 mg/kg intravenously
`and docetaxel 75 mg/m2 intravenously every
`21 days [33]. There is also a Phase I/II clinical
`trial in recurrent EOC evaluating aflibercept
`and docetaxel (in progress). The completed
`Phase I trial did not identify a maximum toler-
`ated dose, and recommended that the Phase II
`dose for aflibercept was 6 mg/kg intravenously
`every 21 days in this combination regimen.
`There were two out of nine confirmed partial
`responders in the Phase I; the two-stage designed
`Phase II has suspended accrual after complet-
`ing first-stage enrollment and is evaluating for
`responses in order to determine whether second-
`stage accrual is warranted [COLEMAN RC: Ur DATE ON
`DATA ANALYSIS REGARDING VEGF-TRAP PLUS DOCETAXEL IN
`
`EOC. PERS. COMMUN.].
`Also notable is a Phase I trial performed in
`patients with advanced ovarian carcinoma
`and symptomatic ascites. Based on preclinical
`data describing a dramatic decrease in ascites
`in a murine model, the goal of this trial was
`to achieve a repeat paracentesis response rate,
`defined as a doubling in time between repeat
`paracentesis procedures compared with a base-
`line average. Aflibercept 4 mg/m2 was adminis-
`tered every 2 weeks. Eight out of ten evaluable
`
`patients achieved a repeat paracentesis response
`rate. Significant adverse events were notable as
`well; four out of ten patients experienced a bowel
`obstruction, one patient experienced a bowel
`perforation and three out of ten had grade 3/4
`nausea and vomiting. Apparent from the abstract
`is that the majority of enrolled patients were in
`the terminal phase of their disease, making it less
`likely that aflibercept was primarily responsible
`for the adverse events [34] .
`The current development strategy for afliber-
`cept includes four randomized Phase III trials in
`combination with standard chemotherapy regi-
`mens in patients with prostate, non-small-cell
`lung, colorectal and pancreatic cancers. These
`trials are currently underway.
`
`(Safety & tolerability
`In general, aflibercept has been well-tolerated.
`A Phase I study in patients with advanced
`cancer (multiple tumor types) with afliber-
`cept administered intravenously every 2 weeks
`explored doses ranging between 0.3 and 7.0 mg/
`kg. Dose-dependent adverse events, particu-
`larly hypertension and proteinuria, increased
`in frequency and severity at doses of 4.0 mg/
`kg and above [33]. Combination Phase I and
`II studies have validated these as the primary
`adverse effects of aflibercept, without significant
`potentiation when given in combination with
`docetaxel, irinotecan, 5-fluououracil, cisplatin,
`gemcitabine and oxaliplatin [31,3235]. Grade 3/4
`hypertension has been described in 9-32% of
`patients, and proteinuria in 4-13%. Although
`rare, other well-described anti-angiogenic associ-
`ated adverse events, such as bowel perforations,
`bowel obstructions, hemorrhagic events, revers-
`ible posterior leukoencephalopathy syndrome
`and pulmonary emboli, have been described
`in association with the drug. A summary of
`adverse effects is illustrated in TABLE 4 [31,32,35,36] .
`Overall, it is not clear based on published data
`whether there is any significant difference in the
`frequency or severity of the off-target side effects
`mentioned above when comparing aflibercept
`and bevacizumab.
`
`Regulatory affairs
`Aflibercept has also been identified as VEGF-
`Trap and AVE0005. (NSC #724770, IND
`#BB100137). It was developed by Regeneron
`(NY, USA), and is being developed in collabora-
`tion with Sanofi-Aventis Pharmaceuticals (Paris,
`France). The drug is also distributed by the
`Clinical Trials Evaluation Program, the Division
`of Cancer Treatment and Diagnosis and the
`
`fsg future science group
`
`wwwfuturemedlcine.com
`
`597
`
`Regeneron Exhibit 2014
`Page 07 of 10
`
`

`

`Drug Evaluation Moroney, Sood & Coleman
`
`■
`
`Epithelial ovarian cancer (EOC) is the predominant form of ovarian cancer, and it is most frequently diagnosed after becoming widely
`disseminated. Despite its initial chemosensitivity, it is an aggressive disease with few effective treatment options for patients in whom
`it recurs.
`There is consensus in the setting of primary treatment regarding cytotoxic chemotherapy agents; however, there is little or no
`consensus regarding the utility of adjuvant targeted agents in combination with platinum and taxanes in both the setting of primary
`treatment and recurrence.
`• Clinically available anti-angiogenic agents have shown activity in EOC and can be classified mechanistically as monoclonal antibodies,
`receptor tyrosine kinase inhibitors, and decoy receptor (Trap) molecules.
`• Aflibercept is a synthetic, soluble decoy receptor with high affinity for the predominant VEGF-ligand. It has impressive anti-tumor
`activity in murine xenograft models and early clinical trials based on preliminary data. It is well-tolerated and active as monotherapy and
`in combination therapies in EOC; however, it has shown better clinical activity in combination with cytotoxic agents.
`• The market for VEGF-targeted therapy in EOC is currently dominated by the monoclonal antibody, bevacizumab, which is undergoing
`multiple Phase III trials in both the primary and recurrent treatment setting. Future competitors, including aflibercept, must compare
`favorably with regard to clinical efficacy or side effect profile to supplant this agent. Data to support a valid comparison between these
`two agents as well as other VEGF-targeted therapies has yet to be reported.
`National Cancer Institute. In addition to the
`primary tumors mentioned above, ovarian can-
`cer is the subject of a future randomized clinical
`trial evaluating chemotherapy in combination
`with aflibercept [COLEMAN RC: UPDATE ON DATA ANA-
`LYSIS REGARDING VEGF-TRAP PLUS DOCETAXEL IN EOC. PERS.
`
`it has been US FDA labeled and used extensively
`in multiple cancers, bevacizumab has emerged
`as the default anti-angiogenic agent by which
`all future drugs of this nature will be compared.
`Aflibercept appears to be similar to bevacizumab
`with regard to safety and tolerability; however, it
`is not possible at this time to offer an adequate,
`evidence-based comparison in terms of clinical
`efficacy. Because of its affinity for PIGF, afliber-
`cept does offer the potential to bind more ligands
`in the tumor microenvironment, but whether this
`capacity will translate into better tumor control is
`yet to be elucidated. It is also unclear whether or
`not the pharmacodynamic properties of afliber-
`cept will translate into an improved adverse effect
`profile in comparison with other anti-angiogenic
`therapies, particularly bevacizumab.
`Another important clinical question for
`aflibercept that remains is whether it would be
`better suited for EOC patients in the primary
`or recurrent setting. Bevacizumab, as the first
`available anti-angiogenic agent, is currently
`being evaluated in both groups of patients. It is
`likely that future clinical development will posi-
`tion aflibercept in similar settings, parti