(12) United States Patent
`Wiegand et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,521,049 B2
`Apr. 21, 2009
`
`USOO752.1049B2
`
`(54) USE OF VEGF INHIBITORS FOR
`TREATMENT OF EYE DISORDERS
`(75) Inventors: Stanley J. Wiegand, Croton-on-Hudson,
`NY (US); Nicholas J. Papadopoulos,
`LaGrangeville, NY (US); George D.
`Yancopoulos, Yorktown Heights, NY
`(US); James P. Fandl, LaGrangeville,
`NY (US); Thomas J. Daly, New City,
`NY (US)
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`(21) Appl. No.: 11/998,709
`
`(*) Notice:
`
`(22) Filed:
`(65)
`
`Nov.30, 2007
`Prior Publication Data
`US 2008/022OOO4A1
`Sep. 11, 2008
`O
`O
`Related U.S. Application Data
`(60) Division of application No. 1 1/218.234, filed on Sep.
`1, 2005, now Pat. No. 7,303,747, which is a continua-
`tion-in-part of application No. 11/089,803, filed on
`Mar. 25, 2005, now Pat. No. 7,306,799, which is a
`continuation-in-part of application No. 10/988,243,
`
`(52) U.S. Cl. ..................... 424/134.1; 424/192.1; 514/2:
`514/12:530/350,536/23.4
`(58) Field of Classification Search ............... ... ... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`6,100,071 A
`8/2000 Davis-Smyth et al.
`6,897.294 B2
`5/2005 Davis-Smyth et al.
`2005/0281831 A1 12/2005 Davis-Smyth et al.
`
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`WO97/.44453
`11, 1997
`WO98, 13071
`4f1998
`WO99/03996
`1, 1999
`
`OTHER PUBLICATIONS
`
`Terman, B.I., et al., (1991) Oncogene 6: 1677-1683.
`Terman, B.I., et al., (1992) Biochem. Biophys. Res. Comm.
`187(3): 1579-1586.
`Davis-Smyth, T., et al., (1996) The EMBO Journal 15(18):4919
`4927.
`Holash, J., et al. (2002) PNAS 99(17); 11393-11398.
`Heidaran, M.A., et al., (1990).J. Bio, Chem. 265(31): 18741-18744.
`Cunningham, S.A., et al., (1997) Biochem. Biophys. Res. Comm.
`E.
`(1998) J. Bio. Chem. 273(18): 11 197-11204
`un, J., et al.,
`SO C.
`:
`
`ENE'S Winancischisis.
`Barleon, B., et al., (1997) J. Bio. Chem. 272(16):10382-10388.
`852. filed as application No PCT/US00/442 on Ma s
`s
`pp
`"'Y
`Davis-Smyth, T., et al., (1998) J. Bio. Chem. 273(6):3216-3222.
`23, 2000, now Pat. No. 7,070,959, said application No.
`1 1/218.234 is a continuation-in-part of application No.
`Primary Examiner Christine J. Saoud
`10/880,021, filed on Jun. 29, 2004, now Pat. No. 7,279,
`Assistant Examiner—Jon M Lockard
`159, which is a continuation-in-part of application No.
`(74) Attorney, Agent, or Firm Valeta Gregg, Esq.
`10/609,775, filed on Jun. 30, 2003, now Pat. No. 7,087,
`411.
`(60) Provisional application No. 60/138,133, filed on Jun.
`8, 1999.
`s
`(51) Int. Cl.
`A6 IK 38/18
`C07K I4/7
`CI2N 5/62
`
`(57)
`
`ABSTRACT
`-
`Modified chimeric polypeptides with improved pharmacoki
`netics and improved tissue penetration are disclosed useful
`for treating eye disorders, including age-related macular
`degeneration and diabetic retinopathy.
`
`15 Claims, 11 Drawing Sheets
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`Regeneron Exhibit 2035
`Page 01 of 33
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`

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`U.S. Patent
`
`Apr. 21, 2009
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`Regeneron Exhibit 2035
`Page 02 of 33
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`
`Apr.21, 2009
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`Sheet 11 of 11
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`US 7,521,049 B2
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`LogMTrapor
`
`Figure11
`
`Regeneron Exhibit 2035
`Page 12 of 33
`
`Regeneron Exhibit 2035
`Page 12 of 33
`
`

`

`1.
`USE OF VEGF INHIBITORS FOR
`TREATMENT OF EYE DISORDERS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a divisional of U.S. patent application
`Ser. No. 1 1/218,234 filed 1 Sep. 2005, now U.S. Pat. No.
`7.303,747, which is a continuation-in-part of application Ser.
`No. 11/089,803 filed 25 Mar. 2005, now U.S. Pat. No. 7,306,
`799, which is a continuation-in-part of application Ser. No.
`10/988,243 filed 12 Nov. 2004, now U.S. Pat. No. 7,303,746,
`which is a continuation-in-part of application Ser. No.
`10/009,852 filed 6 Dec. 2001, now U.S. Pat. No. 7,070,959,
`which is the National Stage of International Application No.
`PCT/US00/14142 filed 23 May 2000, which claims the ben
`efit under 35 USC S 119(e) of U.S. Provisional 60/138,133
`filed 8 Jun. 1999, and U.S. patent application Ser. No. 1 1/218,
`234 filed 1 Sep. 2005, now U.S. Pat. No. 7,303,747 is a
`continuation-in-part of application Ser. No. 10/880,021 filed
`29 Jun. 2004, now U.S. Pat. No. 7,279,159, which is a con
`tinuation-in-part of application Ser. No. 10/609.775 filed 30
`Jun. 2003, now U.S. Pat. No. 7,087,411, which applications
`are herein specifically incorporated by reference in their
`entireties.
`
`10
`
`15
`
`25
`
`BACKGROUND
`
`Statement Regarding Related Art
`
`30
`
`35
`
`A class of cell-derived dimeric mitogens with selectivity
`for vascular endothelial cells has been identified and desig
`nated vascular endothelial cell growth factor (VEGF). VEGF
`is a dimer with an apparent molecular mass of about 46 kDa
`with each subunit having an apparent molecular mass of
`about 23 kDa. The membrane-bound tyrosine kinase recep
`tor, known as Flt (also known as VEGFR2), was shown to be
`a VEGF receptor (DeVries et al. (1992) Science 255:989
`991). Anotherform of the VEGF receptor, designated KDR or
`Flk-1 (also known as VEGFR3), is also known to bind VEGF
`40
`and induce mitogenesis (Terman et al. (1991) Oncogene
`6:1677-1683; Terman et al. (1992) Biochem. Biophys. Res.
`Comm. 187: 1579-1586).
`U.S. Pat. No. 6,011,003 describes an altered, soluble form
`of Flt polypeptide capable of binding to VEGF comprising
`45
`five or fewer complete immunoglobulin domains. WO
`97/.44453 describes chimeric VEGF receptor proteins com
`prising amino acid sequences derived from VEGF receptors
`Flt1 and KDR.
`
`BRIEF SUMMARY OF THE INVENTION
`
`50
`
`55
`
`The invention features a therapeutic method for treating or
`ameliorating an eye disorder, comprising administering a vas
`cular endothelial growth factor (VEGF) inhibitor to a patient
`in need thereof. In one embodiment, the eye disorder treated
`is age related macular degeneration. In another embodiment,
`the eye disorder treated is diabetic retinopathy.
`Preferably, the VEGF inhibitor used in the method of the
`invention comprises an immunoglobulin-like (Ig) domain 2
`60
`of a first VEGF receptor and Ig domain 3 of a second VEGF
`receptor, and a multimerizing component, wherein the first
`VEGF receptor is Flt1, the second VEGF receptor is Flk1 or
`Flt4, and the multimerizing component is selected from the
`group consisting of (i) an amino acid sequence between 1 to
`about 200 amino acids in length having at least one cysteine
`residue, and (ii) an immunoglobulin domain, or fragment of
`
`65
`
`US 7,521,049 B2
`
`2
`an immunoglobulin domain. In specific embodiments, the
`VEGF inhibitor is a fusion polypeptide “VEGF trap' selected
`from the group consisting of SEQ ID NO:2
`(Flt1D2. Flk1D3FcAC1(a)),
`SEQ
`ID
`NO:4
`(Flt1D2.VEGFR3D3.FcAC1(a)),
`SEQ
`ID
`NO:6
`(VEGFR1R2 FcAC1(a)), and SEQ ID NO:23. In another
`embodiment, the VEGF inhibitor is a fusion polypeptide
`encoded by a nucleotide sequence selected from the group
`consisting of SEQ ID NO:1, 3, 5, 22, and a nucleotide
`sequence which, as a result of the degeneracy of the genetic
`code, differs from the nucleotide sequence of SEQID NO:1,
`3, 5, and 22.
`In a second aspect, the invention features a method for the
`treatment of a human Subject diagnosed with an eye disorder,
`comprising administering an effective amount of a vascular
`endothelial growth factor (VEGF) inhibitor to the human
`Subject, the method comprising administering to the Subject
`an initial dose of at least approximately 25-4000 ug VEGF
`inhibitor protein to an affected eye, and administering to the
`subject a plurality of subsequent doses of the VEGF inhibitor
`protein in an amount that is approximately the same or less
`than the initial dose, wherein the Subsequent doses are sepa
`rated in time from each other by at least two weeks. The eye
`disorder is one of age-related macular degeneration or dia
`betic retinopathy. In various embodiments, the initial dose is
`at least approximately 25 to 4000 ug of VEGF inhibitor pro
`tein. In various embodiments, the Subsequent doses are sepa
`rated in time from each other by at least two weeks to 12
`months; more preferably, the Subsequent doses are separated
`in time from each other by at least 3-6 months. The VEGF
`inhibitor protein is administered directly to the affected eye,
`including by use of eye drops or intravitreal injection. Pref
`erably, the VEGF inhibitor is a dimer having two fusion
`polypeptides consisting essentially of an immunoglobulin
`like (Ig) domain 2 of Flt1 and Ig domain 3 of Flk1 or Flt4, and
`a multimerizing component. In specific embodiments, the
`VEGF inhibitor is a dimer comprising the fusion polypeptide
`of SEQID NO:2, 4, 6, or 23.
`Other objects and advantages will become apparent from a
`review of the ensuing detailed description.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG.1. Biacore analysis of binding stoichiometry. Binding
`Stoichiometry was calculated as a molar ratio of bound
`VEGF165 to the immobilized Flt1D2Flk1D3.FcAC1(a) or
`VEGFR1R2-FcAC1(a), using the conversion factor of 1000
`RU equivalent to 1 ng/ml.
`FIG. 2. Pharmacokinetics of Flt1(1-3)-Fc (A40),
`Flt1D2. Flk1D3.FcAC1(a) and VEGFR1R2-FcAC1(a).
`FIG. 3. Pharmacokinetics of Flt1(1-3)-Fc (A40),
`Flt1D2. Flk1D3.FcAC1(a) and Flt1D2.VEGFR3D3.FcAC1
`(a).
`FIG. 4. VEGFR1R2-FcAC1(a) prevents neovasculariza
`tion induced by retinal ischemia. Serial 10um cross sections
`were collected and stained with hematoxylin and eosin. For
`each animal, nuclei in preretinal neoVessels were counted in a
`series often sections within 300 microns of the optic nerve
`head and averaged. Counts were obtained in three indepen
`dent experiments, n24 for each treatment group in each
`study.
`FIG. 5. Effect of subcutaneous VEGFR1R2-FcAC1(a)
`injections on choroidal neovascularization area. The size of
`CNV lesions was measured in choroidal flat mounts. The
`images were digitized using an Axioskop microscope
`equipped with a video camera, and the total area of choroidal
`
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`US 7,521,049 B2
`
`3
`neovascularization associated with each laser burn was mea
`Sured using Image-Pro Plus Software.
`FIG. 6. VEGFR1R2-FcAC1(a) inhibits subretinal neovas
`cularization in Rho/VEGF transgenic mice.
`FIGS. 7A-B. VEGF-Induced breakdown of the blood reti
`nal barrier. A. Following intravitreal injections of VEGF,
`adult mice (C57BL/6) treated withinjections of VEGFR1R2
`FcAC1(a) had a significantly smaller retina to lung leakage
`ratio than mice treated with Fc fragment, indicating less
`breakdown of BRB. B. Double transgenic mice treated with
`injections of VEGFR1R2-FcAC1(a) had a significant reduc
`tion in the retina to lung leakage ratio compared to mice
`treated with Fc fragment.
`FIG.8. Effect of VEGFR1R2-FcAC1(a) administration on
`corneal thickness in Suture and alkaliburn models of corneal
`trauma. Corneas were injured by Suture placement or appli
`cation of NaOH as described, and a single dose of
`VEGFR1R2-FcAC1(a) (25 mg/kg, ip) or saline (n=5 per
`group) was administered immediately following injury. The
`contralateral cornea served as normal, undamaged controls.
`Corneas were collected 7 days later and cross-sections were
`cut and stained with hematoxylin and eosin. Corneal thick
`ness was measured as an index of corneal edema.
`FIG. 9. System or intravitreal VEGF trap protein adminis
`tration prevents laser-induced choroidal neovascularization
`(CNV) and reverses vascular leak in established lesions.
`FIG. 10. Dose response curve of Baf/Flt cells grown in
`VEGF.
`FIG. 11. Inhibition of VEGF growth response by VEGF
`trap VEGFR1R2-FcAC1(a) or anti-VEGF antibody.
`
`10
`
`15
`
`25
`
`30
`
`4
`Nucleic Acid Constructs and Encoded Fusion Polypeptides
`The present invention provides for the construction of
`nucleic acid molecules encoding chimeric polypeptide mol
`ecules that are inserted into a vector that is able to express the
`chimeric polypeptide molecules when introduced into an
`appropriate host cell. Appropriate host cells include, but are
`not limited to, bacterial cells, yeast cells, insect cells, and
`mammaliancells. 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 the chimeric
`polypeptide molecules under control of transcriptional/trans
`lational control signals. These methods may include in vitro
`recombinant DNA and synthetic techniques and in vivo
`recombinations (See Sambrook, et al., Molecular Cloning. A
`Laboratory Manual, Cold Spring Harbor Laboratory; Current
`Protocols in Molecular Biology, Eds. Ausubel, et al., Greene
`Publ. Assoc., Wiley-Interscience, N.Y.).
`Expression of nucleic acid molecules encoding the chi
`meric polypeptide molecules may be regulated by a second
`nucleic acid sequence so that the chimeric polypeptide mol
`ecule is expressed in a host transformed with the recombinant
`DNA molecule. For example, expression of the chimeric
`polypeptide molecules described herein may be controlled by
`any promoter/enhancer element known in the art.
`Thus, according to the invention, expression vectors
`capable of being replicated in a bacterial or eukaryotic host
`comprising chimeric polypeptide molecule-encoding nucleic
`acids as described herein, are used to transfect the host and
`thereby direct expression of such nucleic acids to produce the
`chimeric polypeptide molecules, which may then be recov
`ered in a biologically active form. As used herein, a biologi
`cally active form includes aform capable of binding to VEGF.
`Expression vectors containing the chimeric nucleic acid mol
`ecules described herein can be identified by three general
`approaches: (a) DNA-DNA hybridization, (b) presence or
`absence of “marker gene functions, and (c) expression of
`inserted sequences. In the first approach, the presence of a
`foreign gene inserted in an expression vector can be detected
`by DNA-DNA hybridization using probes comprising
`sequences that are homologous to the inserted chimeric
`polypeptide molecule sequences. In the second approach, the
`recombinant vector/host system can be identified and
`selected based upon the presence or absence of certain
`“marker gene functions (e.g., thymidine kinase activity,
`resistance to antibiotics, transformation phenotype, occlu
`sion body formation in baculovirus, etc.) caused by the inser
`tion of foreign genes in the vector. For example, if the chi
`meric polypeptide molecule DNA sequence is inserted within
`the marker gene sequence of the vector, recombinants con
`taining the insert can be identified by the absence of the
`marker gene function. In the third approach, recombinant
`expression vectors can be identified by assaying the foreign
`gene product expressed by the recombinant. Such assays can
`be based, for example, on the physical or functional proper
`ties of the chimeric polypeptide molecules.
`Cells of the present invention may transiently or, prefer
`ably, constitutively and permanently express the chimeric
`polypeptide molecules.
`The chimeric polypeptide molecules may be purified by
`any technique which allows for the Subsequent formation of a
`stable, biologically active chimeric polypeptide molecule.
`For example, and not by way of limitation, the factors may be
`recovered from cells either as soluble proteins or as inclusion
`bodies, from which they may be extracted quantitatively by
`8M guanidinium hydrochloride and dialysis (see, for
`example, U.S. Pat. No. 5,663,304). In order to further purify
`the factors, conventional ion exchange chromatography,
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`35
`
`45
`
`It has been a longstanding problem in the art to produce a
`receptor-based VEGFantagonist that has a pharmacokinetic
`profile that is appropriate for consideration of the antagonist
`as a therapeutic candidate. Applicants describe herein, for the
`first time, a chimeric polypeptide molecule, capable of
`antagonizing VEGF activity, that exhibits improved pharma
`40
`cokinetic properties as compared to other known receptor
`based VEGF antagonists. The chimeric polypeptide mol
`ecules described herein thus provide appropriate molecules
`for use in therapies in which antagonism of VEGF is a desired
`result.
`The extracellular ligand binding domain is defined as the
`portion of a receptor that, in its native conformation in the cell
`membrane, is oriented extracellularly where it can contact
`with its cognate ligand. The extracellular ligand binding
`domain does not include the hydrophobic amino acids asso
`ciated with the receptor's transmembrane domain or any
`amino acids associated with the receptors intracellular
`domain. Generally, the intracellular or cytoplasmic domain of
`a receptor is usually composed of positively charged or polar
`amino acids (i.e. lysine, arginine, histidine, glutamic acid,
`aspartic acid). The preceding 15-30, predominantly hydro
`phobic or apolar amino acids (i.e. leucine, Valine, isoleucine,
`and phenylalanine) comprise the transmembrane domain.
`The extracellular domain comprises the amino acids that
`precede the hydrophobic transmembrane stretch of amino
`acids. Usually the transmembrane domain is flanked by posi
`tively charged or polar amino acids such as lysine or arginine.
`von Heijne has published detailed rules that are commonly
`referred to by skilled artisans when determining which amino
`acids of a given receptor belong to the extracellular, trans
`membrane, or intracellular domains (See, von Heijne (1995)
`BioEssays 17:25.
`
`50
`
`55
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`60
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`5
`hydrophobic interaction chromatography, reverse phase
`chromatography or gel filtration may be used.
`The method of the invention encompasses the use of a
`fusion protein consisting essentially of first and second vas
`cular endothelial growth factor (VEGF) receptor components
`and a multimerizing component, wherein the first VEGF
`receptor component is an immunoglobulin-like (Ig) domain 2
`of Flt1, the second VEGF receptor component is an Ig domain
`3 of a Flk1 or Flt4, and the multimerizing component is
`selected from the group consisting of (i) a multimerizing
`component comprising a cleavable region (C-region), (ii) a
`truncated multimerizing component, (iii) an amino acid
`sequence between 1 to about 200 amino acids in length hav
`ing at least one cysteine residue, (iv) a leucine Zipper, (v) a
`helix loop motif (vi) a coil-coil motif, and (vii) an immuno
`globulin domain. Examples of the VEGF inhibitors useful in
`the method of the invention include fusion proteins encoded
`by a nucleotide sequence selected from the group consisting
`of the nucleotide sequence of SEQID NO:1, 3, 5, 22, and a
`nucleotide sequence which, as a result of the degeneracy of
`the genetic code, differs from the nucleotide sequence of SEQ
`ID NO:1, 3, 5, or 22, and fusion protein selected from the
`group consisting of SEQ ID NO:2 (Flt1D2. Flk1D3FcAC1
`(a)), SEQID NO:4 (Flt1D2.VEGFR3D3.FcAC1(a)), SEQID
`NO:6 (VEGFR1R2 FcAC1(a)) and (SEQID NO:23).
`25
`Therapeutic Methods
`The present invention also has diagnostic and therapeutic
`utilities. In particular embodiments of the invention, methods
`of detecting aberrancies in the function or expression of the
`chimeric polypeptide molecules described herein may be
`used in the diagnosis of disorders. In other embodiments,
`manipulation of the chimeric polypeptide molecules or ago
`nists orantagonists which bind the chimeric polypeptide mol
`ecules may be used in the treatment of diseases. In further
`embodiments, the chimeric polypeptide molecule is utilized
`as an agent to block the binding of a binding agent to its target.
`By way of example, but not limitation, the method of the
`invention may be useful in treating clinical conditions that are
`characterized by vascular permeability, edema or inflamma
`tion Such as brain edema associated with injury, stroke or
`tumor, edema associated with inflammatory disorders such as
`psoriasis or arthritis, including rheumatoid arthritis; asthma,
`generalized edema associated with burns; ascites and pleural
`effusion associated with tumors, inflammation or trauma;
`chronic airway inflammation; capillary leak syndrome; sep
`sis; kidney disease associated with increased leakage of pro
`tein; and eye disorders such as age related macular degenera
`tion and diabetic retinopathy.
`Combination Therapies
`In numerous embodiments, a VEGF inhibitor may be
`administered in combination with one or more additional
`compounds or therapies, including a second VEGF inhibitor.
`Combination therapy includes administration of a single
`pharmaceutical dosage formulation which contains a VEGF
`55
`inhibitor molecule and one or more additional agents; as well
`as administration of a VEGF inhibitor and one or more addi
`tional agent(s) in its own separate pharmaceutical dosage
`formulation. For example, a VEGF inhibitor 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-specific
`fusion protein of the invention and one or more additional
`agents can be administered concurrently, or at separately
`staggered times, i.e., sequentially. The therapeutic methods of
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`the invention may also be combined with other agents or
`medical procedures used for treatment of eye disorders.
`Treatment Population
`The eye comprises several structurally and functionally
`distinct vascular beds, which Supply ocular components criti
`cal to the maintenance of vision. These include the retinal and
`choroidal vasculatures, which Supply the inner and outer por
`tions of the retina, respectively, and the limbal vasculature
`located at the periphery of the cornea. Injuries and diseases
`that impair the normal structure or function of these vascular
`beds are among the leading causes of visual impairment and
`blindness. For example, diabetic retinopathy is the most com
`mon disease affecting the retinal vasculature, and is the lead
`ing cause of vision loss among the working age population in
`the United States. Vascularization of the cornea secondary to
`injury or disease is yet another category of ocular vascular
`disease that can lead to severe impairment of vision.
`"Macular degeneration is a medical term that applies to
`any of several disease syndromes which involve a gradual loss
`or impairment of eyesight due to cell and tissue degeneration
`of the yellow macular region in the center of the retina.
`Macular degeneration is often characterized as one of two
`types, non-exudative (dry form) or exudative (wet form).
`Although both types are bilateral and progressive, each type
`may reflect different pathological processes. The wet form of
`age-related macular degeneration (AMD) is the most com
`mon form of choroidal neovascularization and a leading
`cause of blindness in the elderly. AMD affects millions of
`Americans over the age of 60, and is the leading cause of new
`blindness among the elderly. It is characterized and usually
`diagnosed by the presence of elevated levels of two types of
`cellular debris within the retina, called drusen and lipofuscin.
`There are several types of symptomatic treatment, how
`ever, that have been used with limited and isolated success,
`depending on the particular condition of the patient, to treat
`exudative (wet form) macular degeneration. Laser photoco
`agulation therapy may benefit certain patients with macular
`degeneration. However, there are high recurrence rates for
`selected choroidal neovascular membranes which may ini
`tially respond to laser therapy. Vision loss may also result
`from the laser therapy. Low dose radiation (teletherapy) has
`also been hypothesized as a possible treatment to induce
`regression of choroidal neovascularization. Surgical removal
`of neovascular membranes is another possible treatment, but
`it is a highly specialized procedure and reportedly has not had
`promising results to date. There is presently no effective
`treatment for non-exudative (dry form) macular degenera
`tion. Management of non-exudative macular degeneration is
`limited to early diagnosis and careful follow-up to determine
`if the patient develops choroidal neovascularization. Protec
`tion against exposure to ultraviolet light and prescribed dos
`ages of anti-oxidant vitamins (e.g., vitamin A, 3-carotene,
`lutein, Zeaxanthin, vitamin C and vitamin E) and Zinc may
`also be of some benefit, but as yet these treatments remain
`unproven.
`Accordingly, the population to be treated by the method of
`the invention is preferably one of (i) a human Subject diag
`nosed as Suffering from macular degeneration, (ii) a human
`Subject diagnosed as Suffering from diabetes-related retin
`opathy, and (iii) a human Subject Suffering from pathological
`vascularization of the cornea secondary to injury or disease.
`Methods of Administration and Compositions
`Preferably, administration of the VEGF inhibitor will be
`directly to the eye, e.g., topical. Topical methods of adminis
`tration include, for example, by eye drops, Subconjunctival
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`injections or implants, intravitreal injections or implants, Sub
`Tenon's injections or implants, incorporation in Surgical irri
`gating Solutions, etc.
`Compositions suitable for topical administration are
`known to the art (see, for example, US Patent Application
`2005/0059639). In various embodiments, compositions of
`the invention can comprise a liquid comprising an active
`agent in Solution, in Suspension, or both. As used herein,
`liquid compositions include gels. Preferably the liquid com
`position is aqueous. Alternatively, the composition can take
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`form of an ointment. In a preferred embodiment, the compo
`sition is an in situ gellable aqueous composition, more pref
`erably an in situ gellable aqueous solution. Such a composi
`tion can comprise agelling agent in a concentration effective
`to promote gelling upon contact with the eye or lacrimal fluid
`in the exterior of the eye. Aqueous compositions of the inven
`tion have ophthalmically compatible pH and osmolality. The
`composition can comprise an ophthalmic depot formulation
`comprising an active agent for Subconjunctival administra
`tion. The microparticles comprising active agent can be
`embedded in a biocompatible pharmaceutically acceptable
`polymer or a lipid encapsulating agent. The depot formula
`tions may be adapted to release all or substantially all the
`active material over an extended period of time. The polymer
`or lipid matrix, if present, may be adapted to degrade Suffi
`ciently to be transported from the site of administration after
`release of all or substantially all the active agent. The depot
`formulation can be a liquid formulation, comprising a phar
`maceutical acceptable polymer and a dissolved or dispersed
`active agent. Upon injection, the polymer forms a depot at the
`injections site, e.g. by gelifying or precipitating. The compo
`sition can comprise a solid article that can be inserted in a
`Suitable location in the eye, Such as between the eye and
`eyelid or in the conjuctival sac, where the article releases the
`active agent. Solid articles suitable for implantation in the eye
`in Such fashion generally comprise polymers and can be
`bioerodible or non-bioerodible.
`In one embodiment of the method of the invention, a
`human Subject with at least one visually impaired eye is
`treated with 25-4000 ug of a VEGF inhibitor protein via
`intravitreal injection. Improvement of clinical symptoms are
`monitored by one or more methods known to the art, for
`example, indirect ophthalmoscopy, fundus photography,
`fluorescein angiopathy, electroretinography, external eye
`examination, slit lamp biomicroscopy, applanation tonom
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`etry, pachymetry, and autorefaction. Subsequent doses may
`be administered weekly or monthly, e.g., with a frequency of
`2-8 weeks or 1-12 months apart.
`Other features of the invention will become apparent in the
`course of the following descriptions of exemplary embodi
`ments which are given for illustration of the invention and are
`not intended to be limiting thereof.
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`EXAMPLES
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`Example 1
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`Modified Flt1 Receptor Vector Construction
`
`Chimeric molecules were constructed, denoted R1R2
`(Flt1...D2. Flk1D3.FcAC1(a) and VEGFR1R2-FcAC1(a) and
`R1R3 (Flt1D2.VEGFR3D3-FcAC1(a) and VEGFR1R3
`FcAC1(a) respectively, wherein R1 and Flt1D2=Ig domain 2
`of Flt1 (VEGFR1); R2 and Flk1D3–Ig domain 3 of Flk1
`(VEGFR2); and R3 and VEGFR3D3–Ig domain 3 of Flt4
`(VEGFR3)) were much less sticky to ECM, as judged by an
`in vitro ECM binding assay and had greatly improved PK as
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`described herein. In addition, these molecules were able to
`bind VEGF tightly and block phosphorylation of the native
`Flk1 receptor expressed in endothelial cells.
`plasmid
`Construction
`of
`the
`expression
`plasmids
`pFlt1D2. Flk1D3.FcAC1(a).
`Expression
`pMT21. Flt1(1-3).Fc (6519 bp) and pMT21.Flk-1 (1-3).Fc
`(5230 bp) are plasmids that encode amplicillin resistance and
`Fc-tagged versions of Ig domains 1-3 of human Flt1 and
`human Flk1, respectively. These plasmids were used to con
`struct a DNA fragment consisting of a fusion of Ig domain 2
`of Flt1 with Ig domain 3 of Flk1, using PCR amplification of
`the respective Ig domains followed by further rounds of PCR
`to achieve fusion of the two domains into a single fragment.
`For Ig domain 2 of Flt1, the 5' and 3' amplification primers
`were as follows: 5': bsp/flt1D2 (5'-GACTAGCAGTCCGG
`AGGTAGACCTTTCGTAGAGATG-3') (SEQ ID NO:8), 3':
`Flt1D2-Flk1D3.as (5'-CGGACTCAGAACCACATCTAT
`GATTGTATTGGT3') (SEQID NO:9). The 5' amplification
`primer encodes a BspE1 restriction enzyme site upstream of
`Ig domain 2 of Flt1, defined by the amino acid sequence
`GRPFVEM (SEQID NO:10) corresponding to amino acids
`27-33 of SEQ ID NO:2. The 3' primer encodes the reverse
`complement of the 3' end of Flt1 Ig domain 2 fused directly to
`the 5' beginning of Flk1 Ig domain 3, with the fusion point
`defined as TIID of Flt1 (corresponding to amino acids 123
`126 of SEQ ID NO:2) and continuing into VVLS (SEQ ID
`NO:7) (corresponding to amino acids 127-130 of SEQ ID
`NO:2) of Flk1.
`For Ig domain 3 of Flk1, the 5' and 3' amplification primers
`were as follows:5': Flt1D2-Flk1D3.s (5'-ACAATCATAGAT
`GTGGTTCTGAGTCCGTCTC