US007972598B2
`
`(12) United States Patent
`Daly et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,972,598 B2
`*Jul. 5, 2011
`
`(54) VEGF-BINDING FUSION PROTEINS AND
`THERAPEUTIC USES THEREOF
`
`(75) Inventors: Thomas J. Daly, New City, NY (US);
`James P. Fandl, LaGrangeville, NY
`(US); Nicholas J. Papadopoulos,
`LaGrangeville, NY (U S)
`
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (U S)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is subject to a terminal dis
`claimer.
`
`(21)
`
`(22)
`
`(65)
`
`(63)
`
`App1.No.: 12/623,869
`
`Filed:
`
`Nov. 23, 2009
`
`Prior Publication Data
`
`US 2010/0087632 A1
`
`Apr. 8, 2010
`
`Related U.S. Application Data
`
`Continuation of application No. 12/ 135,549, ?led on
`Jun. 9, 2008, now Pat. No. 7,635,474, which is a
`continuation of application No. ll/346,008, ?led on
`Feb. 2, 2006, now Pat. No. 7,399,612, which is a
`continuation-in-part of application No. 10/880,021,
`?led on Jun. 29, 2004, now Pat. No. 7,279,159, which
`is
`a continuation-in-part of application No.
`10/609,775, ?led on Jun. 30, 2003, now Pat. No.
`7,087,411.
`
`(51) Int. Cl.
`(2006.01)
`A61K 38/18
`(2006.01)
`C07K 14/71
`(52) U.S. Cl. ................. .. 424/134.1; 424/192.1; 514/1.1;
`514/81; 530/350
`(58) Field of Classi?cation Search ...................... .. None
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,851,999 A 12/1998 Ullrich et al.
`6,011,003 A
`1/2000 Charnock-Jones et al.
`6,100,071 A
`8/2000 Davis-Smyth et al.
`6,270,993 B1
`8/2001 Shibuya et al.
`6,897,294 B2
`5/2005 Davis-Smyth et al.
`2005/0281831 A1 12/2005 Davis-Smyth et al.
`
`WO
`W0
`W0
`
`FOREIGN PATENT DOCUMENTS
`WO97/44453
`11/1997
`WO 98/13071
`4/1998
`WO 00/75319
`12/2000
`
`OTHER PUBLICATIONS
`
`Holash et al., 2002, “VEGF-Trap: aVEGF blocker with potent anti
`tumor effects.” PNAS Aug. 20;99(17): 11393-8.
`Heidaran et al., 1990, “Chimeric alpha- and beta-platelet-derived
`growth factor (PDGF) receptors de?ne three immunoglobulin-like
`
`domains of the alpha-PDGF receptor that determine PDGF-AA bind
`ing speci?city.” J Biol Chem. Nov. 5;265(31):18741-4.
`Cunningham et al., 1997, “Identi?cation of the extracellular domains
`of Flt-1 that mediate ligand interactions.” Biochem Biophys Res
`Commun. Feb. 24;231(3):596-9.
`Fuh et al., “Requirements for binding and signaling of the kinase
`domain receptor for vascular endothelial growth factor” J Biol Chem.
`May 1;273(18):11197-204, 1998.
`Wiesman et al., 1997, “Crystal structure at 1.7 A resolution of VEGF
`in complex with domain 2 of the Flt-1 receptor” Cell. Nov.
`28;91(5):695-704.
`Barleon et al., 1997, “Mapping of the sites for ligand binding and
`receptor dimeriZation at the extracellular domain of the vascular
`endothelial growth factor receptor FLT-1” J Biol Chem. Apr.
`18;272(16): 10382-8.
`Davis-Smyth et al., 1998, “Mapping the charged residues in the
`second immunoglobulin-like domain of the vascular endothelial
`growth factor/placenta growth factor receptor Flt-1 required for bind
`ing and structural stability.” J Biol Chem. Feb. 6;273(6):3216-22.
`Wulff et al., 2002, “Prevention of thecal angiogenesis, antral fol
`licular growth, and ovulation in the primate by treatment with vas
`cular endothelial growth factor Trap R1R2” Endocrinology.
`Jul;143(7):2797-807.
`Davis-Smyth et al., 1996, “The second immunoglobulin-like domain
`of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding
`and may initiate a signal transduction cascade.” EMBO J. Sep.
`16;15(18):4919-27.
`Palu et al., 1999, “In pursuit of new developments for gene therapy of
`human diseases” J Biotechnol. Feb. 5;68 (1):1-13.
`Wang et al., 1999, “Rapid analysis of gene expression (RAGE) facili
`tates universal expression pro?ling” Nucleic Acids Res. Dec.
`1;27(23):4609-18.
`Kaufman et al. 1999, Transgenc anaysis of a 100-kb human B-globin
`cluster-containing DNA fragment propagated as a bacterial arti?cial
`chromosome. Blood 94(9): 3178-3184.
`Wigley et al., 1994, “Site-speci?c transgene insertion: an approach”
`Reprod. Fertil. Dev. 6:585-588.
`Wells, 1990, “Additivity of mutational effects in proteins” Biochem
`istry 29(37): 8509-8517.
`Ngo et al., 1994, “Computational complexity, protein structure pre
`diction, and the Levinhal paradox. In MerZ and Le Grand (Eds) The
`Protein Folding Problem and Tertiary Structure Prediction”
`Birkhauser: Boston, pp. 491-495.
`Skolnick et al., 2000, “From genes to protein structure and function:
`novel applications of computational appoaches in the genomic era”
`Trends in Biotechnology 18:34-39.
`Phillips, 2001, “The challenge of gene therarpy and DNA delivery”
`Journal of Pharmacy and Pharmacology 53:1169-1174.
`
`Primary Examiner * Christine J Saoud
`Assistant Examiner * Jon M Lockard
`(74) Attorney, Agent, or Firm * Valeta Gregg; Frank R.
`Cottingham
`
`(57)
`
`ABSTRACT
`
`Fusion proteins which bind and inhibit vascular endothelial
`growth factor (V EGF). The VEGF-binding fusion proteins
`are therapeutically useful for treating VEGF-associated con
`ditions and diseases, and are speci?cally designed for local
`administration to speci?c organs, tissues, and/or cells.
`
`1 Claim, No Drawings
`
`Mylan Exhibit 1158
`Mylan v. Regeneron, IPR2021-00881
`Page 1
`
`

`

`US 7,972,598 B2
`
`1
`VEGF-BINDING FUSION PROTEINS AND
`THERAPEUTIC USES THEREOF
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of application Ser. No.
`12/135,549 ?led 9 Jun. 2008, now US. Pat. No. 7,635,474,
`Which is a continuation of application Ser. No. 11/346,008
`?led 2 Feb. 2006, now US. Pat. No. 7,399,612, Which is a
`continuation-in-part of application Ser. No. 10/880,021 ?led
`29 Jun. 2004, now US. Pat. No. 7,279,159, Which is a con
`tinuation-in-part of application Ser. No. 10/ 609,775 ?led 30
`Jun. 2003, now US. Pat. No. 7,087,411, Which applications
`are herein speci?cally incorporated by reference in their
`entirety.
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`The invention encompasses fusion proteins capable of
`binding vascular endothelial cell growth factor (VEGF),
`VEGF family members, and splice variants With speci?cally
`desirable characteristics, as Well as therapeutic methods of
`use.
`
`Sequence Listing
`
`AnASCII compliant text ?le of the sequence listing is ?led
`concurrently With the present speci?cation (37 CFR §1.52(e)
`and 37 CFR §1.821). The contents of the text ?le are herein
`incorporated by reference. The text ?le containing the
`sequence listing is named “VEGFT_SeqList”, Was created on
`18 Nov. 2009, and contains approximately 40 kilobytes.
`
`BRIEF SUMMARY OF THE INVENTION
`
`In a ?rst aspect, the invention features an isolated nucleic
`acid molecule encoding a fusion protein Which binds VEGF,
`comprising receptor components (R1R2)X and/ or (R1R3)Y,
`Wherein R1 is vascular endothelial cell groWth factor (V EGF)
`receptor component Ig domain 2 of Flt-1 (Flt1D2), R2 is
`VEGF receptor component Ig domain 3 of Flk-1 (Flk1D3),
`and R3 is VEGF receptor component Ig domain 3 of Flt-4
`(Flt1D3 or R3), and Wherein X21 and Yil. In a preferred
`embodiment the nucleic acid molecule encodes a fusion pro
`tein comprising (R1R2)2 (SEQ ID NO:24).
`In a related second aspect, the invention features a mono
`meric VEGF-binding fusion protein comprising VEGF
`receptor components (R1 R2) X and/ or (R1 R3) YWherein X; 1,
`Yil, and R1, R2, and R3 are as de?ned above. The VEGF
`receptor components R1, R2, and R3, may be connected
`directly to each other or connected via one or more spacer
`sequences. In one speci?c embodiment, the fusion protein is
`(R1R2)X, Were X:2. In a more speci?c embodiment, the
`fusion protein is SEQ ID NO:24, or a functionally equivalent
`amino acid variant thereof. The invention encompasses a
`fusion protein consisting of VEGF receptor components
`(R1R2)X and/or (R1R3)Y, and functionally equivalent amino
`acid variants thereof.
`The receptor components of the VEGF -binding fusion pro
`tein may be arranged in different orders, for example, R1 R2
`R1R2; R2R1-R2R1; R1R2-R2R1, etc. The components of
`the fusion protein may be connected directly to each other, or
`connected via a spacer sequence. In speci?c embodiments,
`one or more receptor and/ or fusion partner components of the
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`fusion polypeptide are connected directly to each other With
`out spacers. In other embodiments, one or more receptor
`and/or fusion partner components are connected With spac
`ers.
`In all embodiments of the fusion protein of the invention, a
`signal sequence (S) may be included at the beginning (or
`N-terminus) of the fusion polypeptide of the invention. The
`signal sequence may be native to the cell, recombinant, or
`synthetic. When a signal sequence is attached to the N-termi
`nus of a ?rst receptor component, thus a fusion polypeptide
`may be designated as, for example, S-(R1R2)X.
`The invention encompasses vectors comprising the nucleic
`acid molecules of the invention, including expression vectors
`comprising the nucleic acid molecule operatively linked to an
`expression control sequence. The invention further encom
`passes host-vector systems for the production of a fusion
`polypeptide Which comprise the expression vector, in a suit
`able host cell; host-vector systems Wherein the suitable host
`cell is a bacterial, yeast, insect, mammalian cell; an E. coli
`cell, or a COS or CHO cell. Additional encompassed are
`VEGF-binding fusion proteins of the invention modi?ed by
`acetylation or pegylation. Methods for acetylating or pegy
`lating a protein are Well knoWn in the art.
`In a related ninth aspect, the invention features a method of
`producing a fusion protein of the invention, comprising cul
`turing a host cell transfected With a vector comprising a
`nucleic acid sequence of the invention, under conditions suit
`able for expression of the protein from the host cell, and
`recovering the fusion polypeptides so produced.
`The VEG-binding fusion proteins of the invention are
`therapeutically useful for treating any disease or condition
`Which is improved, ameliorated, or inhibited by removal,
`inhibition, or reduction of VEGF. A non-exhaustive list of
`speci?c conditions improved by inhibition or reduction of
`VEGF include, for example, undesirable plasma leakage or
`vascular permeability, undesirable blood vessel groWth, e. g.,
`such as in a tumor, edema associated With in?ammatory dis
`orders such as psoriasis or arthritis, including rheumatoid
`arthritis; asthma; generaliZed edema associated With burns;
`ascites and pleural effusion associated With tumors, in?am
`mation or trauma; chronic airWay in?ammation; asthma; cap
`illary leak syndrome; sepsis; kidney disease associated With
`increased leakage of protein; pancreatic ductal adenocarci
`noma (PDAC) and eye disorders such as age related macular
`degeneration and diabetic retinopathy. The fusion protein of
`the invention is particularly useful in treatment of eye disor
`ders, and as an adjuvant to eye surgeries, including glaucoma
`surgery; and the treatment of intra-ocular tumors, such as for
`example, uveal melanoma, retinoblastoma, via intravitreal
`delivery.
`Accordingly, in a tenth aspect, the invention features a
`therapeutic method for the treatment of a VEGF-related dis
`ease or condition, comprising administering aVEGF-binding
`fusion protein of the invention to a subject suffering from a
`VEGF-related disease or condition. Although any mammal
`can be treated by the therapeutic methods of the invention, the
`subject is preferably a human patient suffering from or at risk
`of suffering from a condition or disease Which can be
`improved, ameliorated, inhibited or treated With a VEGF trap.
`In a eleventh aspect, the invention further features diagnos
`tic and prognostic methods, as Well as kits for detecting,
`quantitating, and/ or monitoring VEGF With the fusion pro
`teins of the invention.
`In a tWelfth aspect, the invention features pharmaceutical
`compositions comprising a VEGF-binding fusion protein of
`the invention With a pharmaceutically acceptable carrier.
`
`Mylan Exhibit 1158
`Mylan v. Regeneron, IPR2021-00881
`Page 2
`
`

`

`US 7,972,598 B2
`
`3
`Such pharmaceutical compositions may comprise a fusion
`protein or a nucleic acid encoding the fusion protein.
`Other objects and advantages Will become apparent from a
`revieW of the ensuing detailed description.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Before the present methods are described, it is to be under
`stood that this invention is not limited to particular methods,
`and experimental conditions described, as such methods and
`conditions may vary. It is also to be understood that the
`terminology used herein is for the purpose of describing
`particular embodiments only, and is not intended to be limit
`ing, since the scope of the present invention Will be limited
`only the appended claims.
`As used in this speci?cation and the appended claims, the
`singular forms “a”, “an”, and “the” include plural references
`unless the context clearly dictates otherwise. Thus for
`example, a reference to “a method” includes one or more
`methods, and/or steps of the type described herein and/or
`Which Will become apparent to those persons skilled in the art
`upon reading this disclosure and so forth.
`Unless de?ned otherWise, all technical and scienti?c terms
`used herein have the same meaning as commonly understood
`by one of ordinary skill in the art to Which this invention
`belongs. Although any methods and materials similar or
`equivalent to those described herein can be used in the prac
`tice or testing of the present invention, the preferred methods
`and materials are noW described. All publications mentioned
`herein are incorporated herein by reference.
`General Description
`The invention encompasses a fusion protein capable of
`binding and inhibiting VEGF activity. The molecules of the
`invention bind and inhibit the biological action of VEGF
`and/ or the physiological reaction or response. For a descrip
`tion of VEGF-receptor-based antagonist VEGF traps
`FltlD2.Flkl D3.FcACl(a) (SEQ ID NOsz7-8) and
`VEGFRlR2-FcACl(a) (SEQ ID NOsz9-l0), see PCT
`WO/00753l9, the contents of Which is incorporated in its
`entirety herein by reference. The fusion proteins of the inven
`tion are smaller than the full siZed trap, e.g., about 50-60 kD
`versus 120 kD of the parent trap, and are monomers. As
`shoWn in the experimental section beloW, the fusion proteins
`of the invention exhibit unique kinetic properties yet retain a
`high binding af?nity to VEGF.
`Nucleic Acid Constructs and Expression
`The present invention provides for the construction of
`nucleic acid molecules encoding fusion proteins capable of
`binding VEGF. The nucleic acid molecules of the invention
`may encode Wild-type R1, R2, and/or R3 receptor compo
`nents, or functionally equivalent variants thereof. Amino acid
`sequence variants of the R1, R2 and/or R3 receptor compo
`nents of the traps of the invention may also be prepared by
`creating mutations in the encoding nucleic acid molecules.
`Such variants include, for example, deletions from, or inser
`tions or substitutions of, amino acid residues Within the amino
`acid sequence of R1, R2 and/or R3. Any combination of
`deletion, insertion, and substitution may be made to arrive at
`a ?nal construct, provided that the ?nal construct possesses
`the ability to bind and inhibit VEGF.
`These nucleic acid molecules are inserted into a vector that
`is able to express the fusion proteins of the invention When
`introduced into an appropriate host cell. Appropriate host
`cells include, but are not limited to, bacterial, yeast, insect,
`and mammalian cells. Any of the methods knoWn to one
`skilled in the art for the insertion of DNA fragments into a
`vector may be used to construct expression vectors encoding
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`the fusion proteins of the invention under control of transcrip
`tional/translational control signals.
`Expression of the nucleic acid molecules of the invention
`may be regulated by a second nucleic acid sequence so that
`the molecule is expressed in a host transformed With the
`recombinant DNA molecule. For example, expression may
`be controlled by any promoter/enhancer element knoWn in
`the art. Promoters Which may be used to control expression of
`the chimeric polypeptide molecules include, but are not lim
`ited to, a long terminal repeat (Squinto et al. (1991) Cell
`65: 1-20); SV40 early promoter region, CMV, M-MuLV, thy
`midine kinase promoter, the regulatory sequences of the met
`allothionine gene; prokaryotic expression vectors such as the
`[3-lactamase promoter, or the tac promoter (see also Scienti?c
`American (1980) 242:74-94); promoter elements from yeast
`or other fungi such as Gal 4 promoter, ADH, PGK, alkaline
`phosphatase, and tissue-speci?c transcriptional control
`regions derived from genes such as elastase I.
`Expression vectors capable of being replicated in a bacte
`rial or eukaryotic host comprising the nucleic acid molecules
`of the invention are used to transfect the host and thereby
`direct expression of such nucleic acids to produce the fusion
`proteins of the invention Which bind and inhibitVEGF. Trans
`fected cells may transiently or, preferably, constitutively and
`permanently express the fusion proteins of the invention.
`VEGF Receptor Components
`The VEGF receptor components of the fusion proteins of
`the invention consist of the Ig domain 2 of Flt-l (FltlD2)
`(R1), the Ig domain 3 of Flk-l (FlklD3) (R2) (together,
`RlR2), and/or R1 and Ig domain 3 of Flt-4 (FltlD3) (R3)
`(together, RlR3). The term “Ig domain” of Flt-l. Flt-4, or
`Flk-l is intended to encompass not only the complete Wild
`type domain, but also insertional, deletional, and/or substitu
`tional variants thereof Which substantially retain the func
`tional characteristics of the intact domain. It Will be readily
`apparent to one of skill in the art that numerous variants of the
`above Ig domains can be obtained Which Will retains substan
`tially the same functional characteristics as the Wild-type
`domain.
`The term “functional equivalents” When used in reference
`to R1, R2, or R3, is intended to encompass an R1, R2, or R3
`domain With at least one alteration, e.g., a deletion, addition,
`and/or substitution, Which retains substantially the same
`functional characteristics as does the Wild type R1, R2, or R3
`domain, that is, a substantially equivalent binding to VEGF. It
`Will be appreciated that various amino acid substitutions can
`be made in R1, R2, or R3 Without departing from the spirit of
`the invention With respect to the ability of these receptor
`components to bind and inactivate VEGF. The functional
`characteristics of the traps of the invention may be deter
`mined by any suitable screening assay knoWn to the art for
`measuring the desired characteristic. Examples of such
`assays are described in the experimental section beloW Which
`alloW determination of binding characteristics of the traps for
`VEGF (Kd), as Well as their half-life of dissociation of the
`trap-ligand complex (Tl/2). Other assays, for example, a
`change in the ability to speci?cally bind to VEGF can be
`measured by a competition-type VEGF binding assay. Modi
`?cations of protein properties such as thermal stability,
`hydrophobicity, susceptibility to proteolytic degradation, or
`tendency to aggregate may be measured by methods knoWn to
`those of skill in the art.
`The components of the fusion polypeptide may be con
`nected directly to each other or be connected via spacers.
`Generally, the term “spacer” (or linker) means one or more
`molecules, e.g., nucleic acids or amino acids, or non-peptide
`moieties, such as polyethylene glycol, Which may be inserted
`
`Mylan Exhibit 1158
`Mylan v. Regeneron, IPR2021-00881
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`US 7,972,598 B2
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`5
`between one or more component domains. For example,
`spacer sequences may be used to provide a desirable site of
`interest betWeen components for ease of manipulation. A
`spacer may also be provided to enhance expression of the
`fusion polypeptide from a host cell, to decrease steric hin
`drance such that the component may assume its optimal ter
`tiary structure and/or interact appropriately With its target
`molecule. For spacers and methods of identifying desirable
`spacers, see, for example, George et al. (2003) Protein Engi
`neering 151871 -879, herein speci?cally incorporated by ref
`erence. A spacer sequence may include one or more amino
`acids naturally connected to a receptor component, or may be
`an added sequence used to enhance expression of the fusion
`polypeptides, provide speci?cally desired sites of interest,
`alloW component domains to form optimal tertiary structures
`and/or to enhance the interaction of a component With its
`target molecule. In one embodiment, the spacer comprises
`one or more peptide sequences betWeen one or more compo
`nents Which is (are) betWeen 1-100 amino acids, preferably
`1-25.
`In the most speci?c embodiments, R1 is amino acids
`27-126 of SEQ ID N018, or 1-126 of SEQ ID N018 (includ
`ing the signal sequence 1-26); or amino acids 27-129 of SEQ
`ID N0110, or 1-129 of SEQ ID N0110 (including the signal
`sequence at 1-26). In the most speci?c embodiments, R2 is
`amino acids 127-228 of SEQ ID N018, or amino acids 130
`231 of SEQ ID N0110. In the most speci?c embodiments, R3
`is amino acids 127-225 of SEQ ID N01 13 (Without a signal
`sequence). When, for example, R2 is placed at the N-terminus
`of the fusion polypeptide, a signal sequence may desirably
`precede the receptor component. The receptor component(s)
`attached to the multimerizing component may further com
`prise a spacer component, for example, the GPG sequence of
`amino acids 229-231 of SEQ ID N017.
`Therapeutic Uses
`The VEGF-binding fusion proteins of the invention are
`therapeutically useful for treating any disease or condition
`Which is improved, ameliorated, inhibited or prevented by
`removal, inhibition, or reduction of VEGF. A non-exhaustive
`list of speci?c conditions improved by inhibition or reduction
`ofVEGF include, clinical conditions that are characterized by
`excessive vascular endothelial cell proliferation, vascular
`permeability, edema or in?ammation such as brain edema
`associated With injury, stroke or tumor; edema associated
`With in?ammatory disorders such as psoriasis or arthritis,
`including rheumatoid arthritis; asthma; generaliZed edema
`associated With burns; ascites and pleural effusion associated
`With tumors, in?ammation or trauma; chronic airWay in?am
`mation; capillary leak syndrome; sepsis; kidney disease asso
`ciated With increased leakage of protein; and eye disorders
`such as age related macular degeneration and diabetic retin
`opathy.
`The compositions of the invention are therapeutically use
`ful for treating a Wide variety of diseases associated With
`increased VEGF levels. For example, exaggerated Th2
`in?ammation and airWay remodeling are characteristic in the
`pathogenesis of asthma (see, for example, Elias et al. (1999)
`J. Clin. Invest. 10411001-6). Elevated VEGF levels have been
`detected in tissues and biologic samples from patients With
`asthma, Which correlate directly With disease activity (Lee et
`al. (2001) J. Allergy Clin. Immunol. 10711106-1108) and
`inversely With airWay caliber and airWay responsiveness. Fur
`ther, VEGF has been postulated to contribute to asthmatic
`tissue edema.
`Another disease associated With increased VEGF is pan
`creatic ductal adenocarcinoma (PDAC). This malignancy
`often exhibits enhanced foci of endothelial cell proliferation
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`and frequently overexpresses VEGF (Ferrara (1999) J. Mol.
`Med. 771527-543). PDAC is responsible for over 20% of
`deaths due to gastrointestinal malignancies, making it the
`fourth most common cause of cancer-related mortality in the
`US. and other industrialized countries. Experimental evi
`dence supports an important role for VEGF in pancreatic
`cancer, thus a VEGF inhibitor has promise as a therapeutic to
`attenuate intrapancreatic tumor groWth and regional and dis
`tal metastasis.
`The fusion proteins of the invention differ from larger
`VEGF antagonists in being optimiZed for local/intra-vitreal
`delivery, ie. a shorter serum half life for faster clearance and
`minimiZing unWanted systemic exposure. In addition due to
`its smaller siZe, the fusion proteins of the invention have the
`ability to penetrate through the inner-limiting membrane
`(ILM) in the eye, and diffuse through the vitreous to the
`retina/retinal pigment epithelial (RPE) layer Which Will help
`to treat retinal disease. Additionally, the fusion proteins of the
`invention can be used for local administration for the treat
`ment of ocular disease such as choroidal neovasculariZation,
`diabetic macular edema, proliferative diabetic retinopathy,
`corneal neovasculariZation/transplant rejection. Still further,
`the mini-trap can be used in any situation Where transient
`(short-term) blocking of VEGF is required, e.g., to avoid
`chronic exposure to VEGF blockade, such as, for example, in
`the treatment of psoriasis.
`A serious problem leading to failure folloWing glaucoma
`surgery is early in?ammation and angiogenesis, as Well as too
`aggressive Wound healing. Accordingly, the VEGF-binding
`fusion proteins of the invention may be usefully employed is
`as an adj uvant to glaucoma surgery to prevent early hem- and
`lymphangio genesis and macrophage recruitment to the ?lter
`ing bleb after glaucoma surgery, and improve surgical out
`come.
`Combination Therapies
`In numerous embodiments, the fusion protein of the inven
`tion may be administered in combination With one or more
`additional compounds or therapies, including a second
`VEGF -binding molecule, a chemotherapeutic agent, surgery,
`catheter devices, and radiation. Combination therapy
`includes administration of a single pharmaceutical dosage
`formulation Which contains the fusion protein of the inven
`tion and one or more additional agents; as Well as adminis
`tration of a the fusion protein of the invention and one or more
`additional agent(s) in its oWn separate pharmaceutical dosage
`formulation. For example, a fusion protein and a cytotoxic
`agent, a chemotherapeutic agent or a groWth inhibitory agent
`can be administered to the patient together in a single dosage
`composition such as a combined formulation, or each agent
`can be administered in a separate dosage formulation. Where
`separate dosage formulations are used, the VEGF-speci?c
`fusion polypeptide of the invention and one or more addi
`tional agents can be administered concurrently, or at sepa
`rately staggered times, ie., sequentially.
`The term “cytotoxic agent” as used herein refers to a sub
`stance that inhibits or prevents the function of cells and/or
`causes destruction of cells. The term is intended to include
`radioactive isotopes (e.g. I131, I125, Y90 and Re186), chemo
`therapeutic agents, and toxins such as enZymatically active
`toxins of bacterial, fungal, plant or animal origin, or frag
`ments thereof.
`A “chemotherapeutic agent” is a chemical compound use
`ful in the treatment of cancer. Examples of chemotherapeutic
`agents include alkylating agents such as thiotepa and cyclos
`phosphamide (CYTOXAN®)); alkyl sulfonates such as
`busulfan, improsulfan and piposulfan; aZiridines such as ben
`Zodopa, carboquone, meturedopa, and uredopa; ethylen
`
`Mylan Exhibit 1158
`Mylan v. Regeneron, IPR2021-00881
`Page 4
`
`

`

`US 7,972,598 B2
`
`7
`imines and methylamelamines including altretamine, trieth
`ylenemelamine,
`trietylenephosphoramide,
`triethylenethiophosphaoramide and trimethylolomelamine;
`nitrogen mustards such as chlorambucil, chlomaphaZine,
`cholophosphamide, estramustine, ifosfamide, mechlore
`thamine, mechlorethamine oxide hydrochloride, melphalan,
`novembichin, phenesterine, prednimustine, trofosfamide,
`uracil mustard; nitrosureas such as carrnustine, chloroZoto
`cin, fotemustine, lomustine, nimustine, ranimustine; antibi
`otics such as aclacinomysins, actinomycin, authramycin, aZa
`serine, bleomycins, cactinomycin, calicheamicin, carabicin,
`carminomycin, carZinophilin, chromomycins, dactinomycin,
`daunorubicin, detorubicin, 6-diaZo-5-oxo-L-norleucine,
`doxorubicin, epirubicin, esorubicin, idarubicin, marcellomy
`cin, mitomycins, mycophenolic acid, nogalamycin, olivomy
`cins, peplomycin, pot?romycin, puromycin, quelamycin,
`rodorubicin, streptonigrin, streptoZocin, tubercidin, uben
`imex, Zinostatin, Zorubicin; anti-metabolites such as methotr
`exate and 5-?uorouracil (5 -FU); folic acid analogues such as
`denopterin, methotrexate, pteropterin, trimetrexate; purine
`analogs such as ?udarabine, 6-mercaptopurine, thiamiprine,
`thioguanine; pyrimidine analogs such as ancitabine, aZaciti
`dine, 6-aZauridine, carmofur, cytarabine, dideoxyuridine,
`doxi?uridine, enocitabine, ?oxuridine; androgens such as
`calusterone, dromostanolone propionate, epitiostanol, mepi
`tiostane, testolactone; anti-adrenals such as aminoglutethim
`ide, mitotane, trilostane; folic acid replenisher such as frolinic
`acid; aceglatone; aldophosphamide glycoside; aminole
`vulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;
`defofamine; demecolcine; diaZiquone; e?ornithine; ellip
`tinium acetate; etoglucid; gallium nitrate; hydroxyurea; len
`tinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
`nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic
`acid; 2-ethylhydraZide; procarbaZine; PSK®; raZoxane; siZo
`furan; spirogermanium; tenuaZonic acid; triaZiquone; 2,2',2"
`trichlorotriethylamine; urethan; vindesine; dacarbaZine;
`mannomustine; mitobronitol; mitolactol; pipobroman; gacy
`tosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;
`taxanes, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb
`Oncology, Princeton, N1.) and docetaxel (TAXOTERE®;
`Aventis Antony, France); chlorambucil; gemcitabine;
`6-thioguanine; mercaptopurine; methotrexate; platinum ana
`logs such as cisplatin and carboplatin; vinblastine; platinum;
`etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
`vincristine; vinorelbine; navelbine; novantrone; teniposide;
`daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
`topoisomerase inhibitor RFS 2000; di?uoromethylomithine
`(DMFO); retinoic acid; esperamicins; capecitabine; and
`pharmaceutically acceptable salts, acids or derivatives of any
`of the above. Also included in this de?nition are anti-hor
`monal agents that act to regulate or inhibit hormone action on
`tumors such as anti-estrogens including for example tamox
`ifen, raloxifene, aromatase inhibiting 4(5)-imidaZoles, 4-hy
`droxytamoxifen, trioxifene, keoxifene, LY 117018, onapris
`tone, and toremifene (Fareston); and anti-androgens such as
`?utamide, nilutamide, bicalutamide, leuprolide, and goser
`elin; and pharmaceutically acceptable salts, acids or deriva
`tives of any of the above.
`A “groWth inhibitory agent” When used herein refers to a
`compound or composition Which inhibits groWth of a cell,
`especially a cancer cell either in vitro or in vivo. Examples of
`groWth inhibitory agents include agents that block cell cycle
`progression (at a place other than S phase), such as agents that
`induce G1 arrest and M-phase arrest. Classical M-phase
`blockers include the vincas (vincristine and vinblastine),
`TAXOL®, and topo II inhibitors such as doxorubicin, epiru
`bicin, daunorubicin, etoposide, and bleomycin. Those agents
`
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`that arrest G1 also spill over into S-phase arrest, for example,
`DNA alkylating agents such as tamoxifen, prednisone, dac
`arbaZine, mechlorethamine, cisplatin, methotrexate, 5-?uo
`rouracil, and ara-C.
`Methods of Administration
`The invention provides methods of treatment comprising
`administering to a subject an effective amount of a VEGF
`binding fusion protein of the invention. In a preferred aspect,
`the trap is substantially puri?ed (e.g., substantially free from
`substances that limit its effect or produce undesired side
`effects). The subject is preferably a mammal, and most pref
`erably a human.
`Various delivery systems are knoWn and can be used to
`administer an agent of the invention, e.g., encapsulation in
`liposomes, microparticles, microcapsules, recombinant cells
`capable of expressing the compound, receptor-mediated
`endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
`262:4429-4432), construction of a nucleic acid as part of a
`retroviral or other vector, etc. Methods of introduction can