US007303746B2
`
`United States Patent
`(12)
`(10) Patent No.:
`US 7,303,746 B2
`Wiegandetal.
`(45) Date of Patent:
`Dec. 4, 2007
`
`
`(54) METHODS OF TREATING EYE DISORDERS
`WITH MODIFIED CHIMERIC
`POLYPEPTIDES
`
`6,011,003 A
`2005/0260203 AL”
`2006/0030529 AL*
`2006/0172944 Al*
`
`1/2000 Charnock-Joneset al.
`11/2005 Wiegand et al... 424/145.1
`2/2006 Wiegandet al.
`$14/12
`
`............. SI4/12
`8/2006 Wiegandet al.
`
`FOREIGN PATENT DOCUMENTS
`a
`ve
`ttaonbeneeey
`aes
`W099/03996
`1/1999
`
`.
`Ne
`Wo
`
`(75)
`
`(*) Notice:
`
`(21) Appl. No.: 10/988,243
`(22) Filed
`Nov:
`12.2008
`“ied:
`Nov.
`12,
`
`Inventors: Stanley J. Wiegand, Croton on
`Hudson, NY (US); Nicholas
`Papadopoulos, LaGrangeville, NY
`(US); George D. Yancopoulos,
`Yorktown Heights, NY (US)
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`OTHER PUBLICATIONS
`Tarrytown, NY (US)
`Subject to any disclaimer, the termofthis wae ete ue scarad oeare Sage an
`stent
`3
`ded or adjusted under 35
`angiogenesis in eye disease.
`Prog. Retin.
`Eye
`Res. 22:1-29.
`patent 3 extende
`cms
`ag
`°
`Terman, B. [., et al, “Identification of a newendothelial cell growth
`U.S.C. 154(b) by 425 days.
`factor receptor tyrosine kinase”, Oncogene (1991) 6:1677-1683.
`Terman, B.L., et al, “Identification of the KDR tyrosine kinase as a
`receptor
`for vascular endothelial cell growth factor”, Biochem
`Biophys Res Comm (1992) 187(3): 1579-1586,
`Davis-Smyth, T., et al, 1996, “The second tmmunoglobulin-like
`domain of the VEGFtyrosine kinase receptor Flt-| determines
`ligand binding and may initiate a signal transduction cascade”, The
`EMBOJournal 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, 8.A., et al., (1997) Biochem. Biophys. Res. Comm.
`231:596-599.
`.
`Fuh, G., et al., (1998)J. Bio. Chem. 273(18):11197-11204.
`Wiesmann, C., et al., (L997) Cell 91:695-704.
`Barleon, B., et al., (1997) J. Bio. Chem. 272(16): 10382-10388,
`Davis-Smyth, T., et al., (1998) J. Bio. Chem. 273(6):3216-3222.
`* cited by examiner
`7
`Bie
`;
`6
`Fhicaonpy
`Snag
`Srna noainanae Christine J. er
`Assistant Examiner—Jon M. Lockard
`(74) Attorney, Agent, or Firm—Valeta Gregg, Esq.
`oes
`(57)
`ABSTRACT
`
`(65)
`
`Prior Publication Data
`:
`US 2005/0175610 Al
`Aug. 11, 2005
`:
`“
`Releied U5. Application Dota
`(63) Continuation-in-part of application No. 10/009.852,
`filed as application No. PCT/US00/14142 on May 23,
`2000, now Pat, No. 7,070,959,
`io
`—
`Provisional application No. 60/138,133, filed on Jun.
`8, 1999.
`
`(60)
`
`(51)
`
`Int. Cl.
`(2006.01)
`AGIK 38/18
`3006.01
`CO7K 14/71
`(
`01)
`(2006.01)
`CI2N 15/62
`we
`424/134.1; 424/192.1;
`(52) U.S. Cl.
`...
`§14/2; 514/12; 530/350; 530/387.3; 536/23.4
`(58) in tativebs sani ser4|bhiste None
`See PPPUABON Te
`Lor Comp leis sear msary:
`(56)
`References Cited
`U.S. PATENT DOCUMENTS
`5,712,380 A
`1/1998 Kendall et al.
`
`Modified chimeric polypeptides with improved pharmaco-
`kinetics are disclosed useful
`for
`treating eye disorders.
`including age-related macular degeneration and diabetic
`retinopathy.
`
`5 Claims, 21 Drawing Sheets
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 1 of 155
`Page 1 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 1 of 21
`
`US 7,303,746 B2
`
`—ais2i
`
`(0.5 pg/ml
`
`»
`
`10.25 pg/ml
`
`
`94-(€-L)k-dxpeylpowun
`o4-(€-b)b-s+paye|Ajaoe
`
`VEGF
`(0.1 g/m!
`
`)
`
`+
`
`9-4-(€-1)b-A
`
`ON
`
`paying
`
`peyind
`
`94-(€-b)bs+peljjpowun
`
`
`
`ednsSOO
`
`
`
`04-(8V¢-1)ta:LNW
`
`
`
`ednsSOO
`
`+I
`
`(NCHe-1)aig:pan
`
`adnsSoo
`
`Fig.
`
`1
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 2 of 155
`Page 2 of 155
`
`
`
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 2 of 21
`
`US 7,303,746 B2
`
`-Lt Unmodified Fit-1(1-3)-Fe
`“Mutt : Fit-1(1-3 ,p)-Fe
`~-O- Mut2 : Fit-1(2-3 ,p)-Fe
`
`--Zs- Mut: Fit-1(2-3)-Fe
`
`0.01
`
`0.1
`
`1
`
`10
`
`100
`
`1000
`
`Fig.
`
`2
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 3 of 155
`Page 3 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 3 of 21
`
`US 7,303,746 B2
`
`0.5 [5 Fit-1(1-3)-Fe COS supe
`Mutt : Fit-1(1-3,p)-Fe COS supe --
`--O- Mut2 : Fit-1(2-3,p)-Fe COS supe
`0.4 --/,- Mut: Fit-1(2-3)-Fe COS supe
`
`OD
`
`0.1
`
`0.3
`
`0.2
`
`Fig. 3
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 4 of 155
`Page 4 of 155
`
`

`

`
`
`
`
`——AcetylatedFit-1(1-3)-Fe(40X)
`
`Dec. 4, 2007
`
`Sheet 4 of 21
`
`US 7,303,746 B2
`
`
`
`——Mutt:Fit-1(1-3,pR)-Fe
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 5 of 155
`Page 5 of 155
`
`U.S. Patent
`
`°
`LL
`
`—©f
`
`s—
`
`= =i
`
`Days
`
`L
`3D
`==
`
`o° &
`
`ee
`
`

`

`U.S. Patent
`
`Dec.4, 2007
`
`Sheet 5 of 21
`
`US 7,303,746 B2
`
`25
`
`7.5
`5.0
`[Modified Fit Receptor] (nM)
`
`10.0
`
`12.5
`
`@ Fit1D2FIkiD3.FcdeltaC1 (a)
`/\FitiD2VEGFR3D3.FedeltaC1 (a)
`V TIE2-Fe
`
`= Fiti(1-3)-Fe
`
`Fig. 5
`
`4
`
`3
`
`Si.
`
`i X
`
`o
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 6 of 155
`Page 6 of 155
`
`

`

`Dec. 4, 2007
`
`Sheet 6 of 21
`
`US 7,303,746 B2
`
`100
`
`LO
`©
`_-
`
`is
`
`e
`
`1000
`
`[pM]
`
`WU0ZS/OSp Bs9URqIOSgY
`
`Fig.
`
`6
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 7 of 155
`Page 7 of 155
`
`U.S. Patent
`
`e= s
`Oo
`
`ic
`a
`cD
`OQ
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`LL Oo
`NASu
`Ae Sa
`= 2
`LL Le
`
`
`
`—s—FitiD2VEGFR3D3.FcdeltaC1(a)s-
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 7 of 21
`
`US 7,303,746 B2
`
`
`
`(2)LOV94-ZHLHADSA8(28)LOV94'ECPAZLid©}S9LADAAY40AewoiysioysBuipulg
`
`
`
`
`
`
`(8)LOV94-ZHLHADSA/ADSA
`
`(8)LOV94EQMNAZOHISs/49DSA|(WY)S9L4D35Au
`
`
`
`Fig,
`
`7
`
`20°0*460€0'0*960ABCIS*abeIeAY
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 8 of 155
`Page 8 of 155
`
`
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 8 of 21
`
`US 7,303,746 B2
`
`100
`
`—— Fiti(1-3)-Fc(A40)
`— st FitiD2FIk1D3.FcdeltaC1(a)
`—— t FitiD2FikiD3.FedeltaC1(a)
`—— t VEGFR1R2-FcdeltaC1 (a)
`
`ie
`
`SS
`
`
`
`.
`f
`10 ne
`if
`
`——
`
`
`
`ModifiedFitReceptor[ug/ml]
`
`Fig. 8
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 9 of 155
`Page 9 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 9 of 21
`
`US 7,303,746 B2
`
`100
`
`10
`
`—— Fit1(1-3)-Fc(A40)
`—— FitiD2VEGFR3D3.FedeltaC1(a)
`—— FitiD2Fik1D3.FcdeltaC1 (a)
`
`[pg/ml] 0.01 0
`
`ModifiedFitReceptor
`
`0.1
`
`Days
`
`Fig. 9
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 10 of 155
`Page 10 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 10 of 21
`
`US 7,303,746 B2
`
`
`
`
`
`(eww)auNjOAJowWN|
`
`BplysA
`
`4(OvV)O4-(€-+)Hd
`
`6y/Gwgz
`
`MMW/XZ
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`4+yM/xz25yy6wsz2
`
`eahlazaris
`
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`(ov)94-(€-1)Ls
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`
`Fig.
`
`10
`
`4(2)}oeyepoy
`
`
`
`
`41dO04byy/bw¢z2
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 11 of 155
`Page 11 of 155
`
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 11 of 21
`
`US 7,303,746 B2
`
`300
`
`
`
`
`
`(GWU)SUUNJOAJON
`
`a}1YaA
`
`
`
`(ory)04-(€-b)Ls
`
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`
`MWXZ
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`
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`
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`=MW/xzBy/bwige?
`
`
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`
`
`
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`
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`
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`10045y/Sw¢z
`
`Fig.
`
`ll
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 12 of 155
`Page 12 of 155
`
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 12 of 21
`
`US 7,303,746 B2
`
`PMSG-
`
`.FcAC1(a)
`
`FItiD2VEGFR3D3
`
`
`PMSG-
`
`
`
`150
`
`100
`
`°S
`
`o
`Nn
`
`350
`
`300
`
`250
`
`(Bw) yyBiem auuain
`
`Fig. 12
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 13 of 155
`Page 13 of 155
`
`fe]Dessicated
`
`=
`
`FcAC1(a)PMSGBSFit1(1-3)-FittD2FIk1Fe(A40)D3.
`
`PMSG-
`
`PBS-PBS
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 13 of 21
`
`US 7,303,746 B2
`
`
`
`
`
`SerumProgesteroneLevels(ng/ml)
`
`sie PMSG-
`
`PMSG-
`PBS
`
`Fit1(1-3)-
`Fc (A40)
`25mg/kg
`
`FItiD2-
`FIk1D3.
`FcACi (a)
`25mg/kg
`
`Fig.
`
`134A
`
`PBS
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 14 of 155
`Page 14 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 14 of 21
`
`US 7,303,746 B2
`
`Levels 10 SerumProgesterone(ng/ml)
`
`30
`
`25
`
`20
`
`15
`
`PMSG-
`Fiti(1-3)-
`Fo (A40)
`
`PMSG-
`-FitiD2
`—~FIK1D3.
`FcAC1(a)
`
`PMSG-
`PBS
`
`
`
`
`Fig.
`
`1383
`
`PBS
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 15 of 155
`Page 15 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 15 of 21
`
`US 7,303,746 B2
`
`
`
`
`
`Evansblue-albuminpermeation
`
`plasmaxgretina-1xhr7-1)
`(ul
`
`
`
`Non-Diabetic
`
`Diabetic
`
`Diabetic with VGT
`
`FIG. 14
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 16 of 155
`Page 16 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet16 of 21
`
`US 7,303,746 B2
`
`nuclei/section
`preretinalendothelial
`
`
`VGT 25mg/kg
`
`control
`
`FIG. 15
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 17 of 155
`Page 17 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 17 of 21
`
`US 7,303,746 B2
`
`Area 19
`
`CNV
`
`Control
`
`VEGF Trap
`
`FIG. 16
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 18 of 155
`Page 18 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
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`Sheet 18 of 21
`
`US 7,303,746 B2
`
`(mm?x10°3 )
`
`
`
`TotalareaofNeovascularization 8 P<0.0001
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`VEGF-TRAP
`( n=17)
`
`( n=19)
`
`FIG.17
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 19 of 155
`Page 19 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 19 of 21
`
`US 7,303,746 B2
`
`
`
`
`
`RetinatoLungLeakageRatio
`
`lyf
`
`P=0.045191
`
`ml po
`
`
`
`Fe
`
`VEGF-TRAP
`
`(n=18)
`
`(n=18)
`
`FIG. 18A
`
`P=0.040595
`
`1
`0.9
`0.8
`
`0.7
`
`0.6
`
`0.5
`
`0.47
`
`0.3
`
`0.2
`
`0.1
`
`
`
`RetinatoLungLeakageRatio
`
`
`
`|
`|
`
`|
`
`Fe
`(n=20)
`
`VEGF-TRAP
`(n=20)
`
`FIG. 18B
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 20 of 155
`Page 20 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
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`Sheet 20 of 21
`
`US 7,303,746 B2
`
`HB
`
`Suture Injury
`
`BB Chemicalinjury
`
`P<0.01
`e—__
`
`P< 0.01
`
`1
`
`F
`
`400
`
`P<0.01
`
`P <0.01
`eed
`
`Thickness
`
`(um)
`
`Control
`
`Suture
`
`Suture-VGT
`
`Control
`
`ChemicalInjury
`
`Chem-VGT
`
`FIG. 19
`
`
`
`
`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 21 of 155
`Page 21 of 155
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
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`Sheet 21 of 21
`
`US 7,303,746 B2
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`Apotex Exhibit 1012
`Apotex Exhibit 1012
`Page 22 of 155
`Page 22 of 155
`
`

`

`US 7,303,746 B2
`
`1
`METHODS OF TREATING EYE DISORDERS
`WITH MODIFIED CHIMERIC
`POLYPEPTIDES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application 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 benefit under 35 USC § 119(e) of ULS.
`Provisional 60/138, 133 filed 8 Jun. 1999, which applications
`are herein specifically incorporated by reference in their
`entireties.
`
`BACKGROUND
`
`STATEMENT REGARDING RELATED ART
`
`A class ofcell-derived dimeric mitogens with selectivity
`for vascular endothelial cells has been identified and desig-
`nated vascular endothelial cell growth factor
`(VEGF).
`VEGFis 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-boundtyrosine kinase
`receptor, known as F'lt (also known as VEGFR2), was shown
`to be a VEGFreceptor (DeVries et al. (1992) Science
`255:989991), Another form of the VEGF receptor, desig-
`nated KDR or FIk-1 (also known as VEGFR3),
`is also
`knownto bind VEGF and induce mitogenesis (Termanet al,
`(1991) Oncogene 6:1677-1683;, Terman et al. (1992) Bio-
`chem. Biophys. Res. Comm. 187:1579-1586).
`U.S. Pat. No. 6,011,003 describesan altered, soluble form
`of Flt polypeptide capable of binding to VEGF comprising
`five or
`fewer complete immunoglobulin domains. WO
`97/44453 describes chimeric VEGF receptor proteins com-
`prising aminoacid sequences derived from VEGF receptors
`Flt] and KDR.
`
`BRIEF SUMMARY OF THE INVENTION
`
`In a first aspect, the inventionprovides an isolated nucleic
`acid molecule, comprising (a) a nucleotide sequence encod-
`ing a vascular endothelial growth factor (VEGF) receptor
`component consisting essentially ofan immunoglobulin-like
`(Ig) domain 2 ofa first VEGF receptor and Ig domain 3 of
`a second VEGF receptor, and (b) a nucleotide sequence
`encoding a multimerizing component, wherein the first
`VEGFreceptor is Flt], the second VEGF receptor is FIk] or
`Flt4, and the VEGFreceptor componentis the only VEGF
`receptor component of the fusion polypeptide.
`In one
`embodiment, the nucleotide sequence encoding Ig domain 2
`of the extracellular domain of the first VEGF receptor is
`upstream of the nucleotide sequence encoding Ig domain 3
`ofthe extracellular domain ofthe second VEGFreceptor.In
`another embodiment, the nucleotide sequence encoding Ig
`domain 2 of the extracellular domain of the first VEGF
`receptor is downstreamofthe nucleotide sequence encoding
`Ig domain 3 ofthe extracellular domain of the second VEGF
`receptor. In one embodiment, the multimerizing component
`comprises an immunoglobulin domain. Preferably,
`the
`immunoglobulin domain is the Fe domain of IgG or the
`heavychain of IgG. In specific embodiments, the nucleotide
`sequenceis selected from the group consisting ofthe nucle-
`otide sequence of SEQ ID NO:11, 13, and 15, ora nucleotide
`
`5
`
`40
`
`45
`
`2
`sequence which, as a result of the degeneracy ofthe genetic
`code, differs from the nucleotide sequence of SEQ ID
`NO:11, 13, and 15.
`The componentsofthe fusion polypeptide encoded by the
`nucleic acid molecule of the invention are arranged as 1,2,3;
`1,3,2; 21,3; 2,3,1; 3,1,2; or 3,2,1, wherein 1
`is the first
`VEGF receptor component, 2 is the second VEGF receptor
`component, and 3 is the mullimerizing component.
`In a second aspect, the invention features a vector com-
`prises a nucleic acid molecule ofthe invention. In a more
`specific embodiment,
`the vector is an expression vector
`comprising the nucleic acid molecule of the invention opera-
`tively linked to an expression control sequence.
`In a third aspect.
`the invention features a host-vector
`; system for the production of a fusion polypeptide which
`comprises the expression vector of the invention in a suit-
`able hostcell. The suitable host cell may be a bacterial cell,
`yeast cell, insect cell, or mammalian cell.
`In a preferred
`embodiment, the host cell is an £. coli cell or a CHO cell.
`In a fourth aspect,
`the invention features a method of
`producing a fusion polypeptide which comprises growing
`cells of the host-vector system of the invention under
`conditions permitting production ofthe fusion polypeptide
`and recovering the fusion polypeptide so produced.
`Ina fifth aspect, the invention features a dimeric vascular
`° endothelial growth factor (VEGF) antagonist, comprising
`two fusion polypeptides, each fusion polypeptide compris-
`ing (a)a VEGF receptor component consisting essentially of
`an immunoglobulin-like (Ig) domain 2 of an Flt-1 WEGF
`receptor and Iz domain 3 of an FIk-1 or Flt-4 VEGF
`receptor; and (b) a multimerizing component, wherein the
`VEGF receptor component
`is
`the only VEGF receptor
`component of each fusion protein. In specific embodiments,
`the dimeric VEGFantagonist is modified by acetylation or
`pegylation.
`Ina sixth aspect, the invention features a fusion polypep-
`tide, comprising (a) a WEGF receptor component consisting
`essentially of an immunoglobulin-like (lg) domain 2 of an
`Flt-1 VEGF receptor and Ig domain 3 of an FIk-1 or Flt-4
`VEGF receptor; and (b) a multimerizing component,
`wherein the VEGF receptor component is the only VEGF
`receptor component of the fusion polypeptide.
`In one
`embodiment,
`the multimerizing component comprises an
`immunoglobulin domain. More specifically, the multimer-
`izing component is an immunoglobulin domain whichis one
`of the Fe domain of IgG or the heavy chain of IgG.
`In
`specific embodiments, the fusion polypeptide comprises an
`amino acid sequence selected from the group consisting of
`SEQ ID NO:12 (FItID2. FIkID3FcACI(a)), SEQ ID NO:14
`(FILID2VEGFR3D3FeAC1(a)),
`and
`SEQ ID NO:16
`(VEGFRIR2 FeACI(a)).
`In a seventh aspect, the invention features a pharmaceu-
`tical composition comprising the fusion polypeptide ofthe
`invention and a pharmaceutically acceptable carrier,
`In an eighth aspect, the invention features a therapeutic
`method for treating or ameliorating an eye disorder, com-
`prising administering the pharmaceutical compositionof the
`invention to a patient in need thereof. In one embodiment,
`the eye disordertreated is age related macular degeneration.
`In another embodiment, the eye disorder treated is diabetic
`retinopathy
`Other objects and advantages will become apparent from
`a reviewof the ensuing detailed description.
`BRIEF DESCRIPTION OF THE FIGURES
`
`5
`
`5
`
`FIG. 1. Binding of unmodified Fltl(1-3)-Fe, basic region
`deletion mutant FItl(1-3)-Fe, and FItl(-*),_.,, mutant pro-
`teins in a Biacore-based assay.
`
`
`
`
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`Apotex Exhibit 1012
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`

`US 7,303,746 B2
`
`4
`Cormeas 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. 20. System or intravitreal VEGFtrap administration
`prevents laser-induced choroidal neovascularization (CNV)
`and reverses vascular leak in established lesions.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`o
`
`It has been a long standing probleminthe art to produce
`a receptor based VEGF antagonist that has a pharmacoki-
`netic profile that
`is appropriate for consideration of the
`antagonist as a therapeutic candidate. Applicants describe
`ur
`5 herein, for the first time, a chimeric polypeptide molecule,
`capable of antagonizing WEGF activity,
`that exhibits
`improved pharmacokinetic properties as compared to other
`known receptor-based VEGF antagonists. The chimeric
`polypeptide molecules described herein thus provide for the
`first time 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 associated with the receptor’s transmembrane domain
`or any amino acids associated with the receptor’s intracel-
`lular domain. Generally,
`the intracellular or cytoplasmic
`domain of a receptor is usually composed ofpositively
`charged or polar aminoacids(i.e. lysine, arginine,histidine,
`glutamicacid, aspartic acid). The preceding 15-30, predomi-
`nantly hydrophobic or apolar amino acids (i.e.
`leucine,
`valine,
`isoleucine, and phenylalanine) comprise the trans-
`> membrane domain. The extracellular domain comprises the
`amino acids that precede the hydrophobic transmembrane
`stretch of amino acids. Usually the transmembrane domain
`is flanked by positively 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, transmembrane, or intracellular domains
`(See, von Heijne (1993) BioEssays 17:25.
`
`3
`FIG. 2. Binding of unmodified Fltl(1-3)-Fe, Muth: Fltl
`(1-3,.4)-Fe, Mut2: Fltl(2-3,.,)-Fe, and Fit] (2-3) mutant pro-
`teins to Matrigel® coated plates.
`FIG. 3. Binding of unmodified Fltl(1-3)-Fe, Muth: Fltl
`(1-3,,5)-Fe, Mut2: Fit] (2-3,)-Fe, and Fit] (2-3) mutant pro-
`teins in an ELISA-based assay.
`FIG, 4. Pharmacokinetic profiles of unmodified Fltl(1-
`3)-Fe, Mutl: FItl(1-3,,)-Fe, Mut2: Fitl(2-3,,)-Fe, and
`Fitl(2-3) mutant proteins.
`FIG. 5. Extra cellular matrix (ECM) assay of FltlD2.
`FIkKID3, FeACI(a) and FltLD2, VEGFR3D3. FeAC 1 (a).
`FIG. 6. MG/R2 Cell proliferation assay. Modified Flt
`receptors Fitl(1-3)-Fe, FitID2. FIkID3. FeACI(a) and
`FItLD2. VEGFR3D3. FeAC1 (a), plus an irrelevant receptor
`termed Tie2-Fc as a negative control, were titrated from 40
`nM to 20 pM and incubated onthe cells for 1 hr at 37° C,
`FIG. 6. Biacore analysis of binding stoichiometry. Bind-
`ing stoichiometry was calculated as a molar ratio of bound
`VEGF165 to the immobilized Flt!D2FIKID3. FeACI(a) or
`VEGFRIR2-FeAC1 (a), using the conversion factor of 1000 2
`RU equivalent to 1 ng/ml.
`FIG. 8. Pharmacokinetics of Flt1(1-3)-Fe (A40), Fln1D2.
`FIkID3. FeAC1(a) and VEGERIR2-FcACI(a).
`FIG. 9. Pharmacokinetics of Fltl(1-3)-Fe (A40), Flt D2.
`FIKID3, FeAC1(a) and FltlD2. VEGFR3D3. FeAC1(a).
`FIG. 10. The ability of FitlD2. FIKID3. FeACI(a) to
`inhibit HT-1080 fibrosarcoma tumor growth in vive.
`FIG, 11. The ability of FitlD2. FIk1D3. FeAC1(a) to
`inhibit C6 glioma tumor growth in vivo.
`FIG, 12. VEGF-induced uterine hyperpermeability.
`FIGS. 13.4-B. Assessment of corpus luteum angiogenesis
`using progesterone as a readout.
`FIG. 14. VEGFRIR2-FeACI{a) prevents Evans Blue
`leakage in streptozotocin-treated rats.
`FIG. 15. VEGFRIR2-FeAC1(a) prevents neovasculariza-
`tion induced by retinal ischemia. Serial 10 «um 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 micronsofthe optic nerve
`head and averaged. Counts were obtained in three indepen-
`dent experiments, n=4 for each treatment group in each
`study.
`FIG. 16. Effect of subcutaneous VWEGFRIR2-FeACI (a)
`injections on choroidal neovascularization area. ‘The size of
`CNVlesions was measured in choroidal
`flat mounts. The
`images were digitized using an Axioskop microscope
`equipped with a video camera, and the total area ofchoroidal
`neovascularization associated with each laser burn was
`measured using Image-Pro Plus software.
`subretinal
`FIG.
`17. VEGFRIR2-FeAC1(a)
`inhibits
`neovascularization in Rho/VEGF transgenic mice.
`FIGS. 18A-B. VEGF-Induced breakdown of the blood
`retinal barrier. A. Following intravitreal injections of VEGF,
`adult mice
`(CS7BL/6)
`treated with
`injections of
`Fi on
`VEGFRIR2-FeACI (a) had a significantly smaller retina to 5
`lung leakage ration than mice treated with Fe fragment,
`indicating less breakdown of BRB. B. Double transgenic
`mice treated with injections of VEGFRIR2-FeACI (a) had a
`significant reduction in the retina to lung leakage ration
`compared to mice treated with Fe fragment.
`FIG. 19. Effect of VEGFRIR2-FeAC1(a) administration
`on comeal thickness in suture and alkali burn models of
`corneal trauma. Corneas were injured by suture placement or
`application of NaOH as described, and a single dose of
`VEGPRIR2-FeAC I (a) (25 mg/kg,
`ip) or saline (n=5 per
`group) was administered immediately following injury. The
`contralateral cornea served as normal, undamaged controls.
`
`§
`
`60
`
`s Nucleic Acid Constructs
`The present
`invention provides for the construction of
`nucleic acid molecules encoding chimeric polypeptide mol-
`ecules that are inserted into a vectorthat is able to express
`the chimeric polypeptide molecules whenintroduced into an
`appropriate hostcell. Appropriate host cells include, but are
`not limited to, bacterial cells, yeast cells, insect cells, and
`mammalian cells. Any of the methods knownto 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 oftranscrip-
`tional/translational 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 Labo-
`ratory; Current Protocols
`in Molecular Biology, Eds.
`Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience,
`NEY.)
`Expression of nucleic acid molecules encoding the chi-
`meric polypeptide molecules may be regulated by a second
`5 nucleic acid sequence so that
`the chimeric polypeptide
`molecule is expressed in a host transformed with the recom-
`binant DNA molecule. For example, expression of the
`
`
`
`
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`Page 24 of 155
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`

`US 7,303,746 B2
`
`6
`notype, occlusion body formation in baculovirus, etc.)
`caused by the insertion of foreign genes in the vector. For
`example,
`if the chimeric polypeptide molecule DNA
`sequenceis inserted within the marker gene sequenceofthe
`vector, recombinants containing 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 properties of the chimeric polypeptide
`molecules,
`Cells of the present invention maytransiently 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 oflimitation, the factors may
`be recovered from cells either as soluble proteins or as
`inclusion bodies, from which they may be extracted quan-
`titatively 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 chro-
`matography.
`hydrophobic
`interaction
`chromatography.
`reverse phase chromatography orgel filtration may be used.
`
`5
`
`5
`
`a
`chimeric polypeptide molecules described herein 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
`limited to, the long terminal repeat as described in Squinto
`et al., (1991, Cell 65:1-20); the 8SV40 early promoter region
`(Bernoist et al.
`(1981) Nature 290:304-310),
`the CMV
`promoter,
`the M-MuL¥5' terminal repeat
`the promoter
`contained in the 3' long terminal repeat of Rous sarcoma
`virus (Yamamoto et al. (1980) Cell 22:787-797), the herpes
`thymidine kinase promoter (Wagneret al. (1981) Proc, Natl.
`Acad. Sci, U.S.A. 78: 144-1445), the regulatory sequences of
`the metallothionine gene (Brinster et al. (1982) Nature
`296:39-42); prokaryotic expression vectors such as the
`f-lactamase promoter (Villa-Kamaroff et al. (1978) Proc.
`Natl. Acad. Sci. U.S.A, 75:3727-3731), or the tac promoter
`(DeBoeret al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-
`25); promoter elements fromyeast or other fungi such as the
`Gal 4 promoter,
`the ADH (alcohol dehydrogenase) pro-
`moter, PGK (phosphoglycerol kinase) promoter, alkaline
`phosphatase promoter, and the following animal transcrip-
`tional control regions, which exhibit tissue specificity and
`have been utilized in transgenic animals: elastase | gene
`control region whichis active in pancreatic acinarcells (see
`for example, Swifi et al. (1984) Cell 38:639-646); insulin
`gene control region whichis active in pancreatic beta cells
`(Hanahan (1985) Nature 315:115-122),
`immunoglobulin
`gene control
`region which is active in lymphoid cells
`(Grosschedl et al. (1984) Cell 38:647-658), mouse mam-
`mary tumorvirus control region which is active in testicular,
`breast, lymphoid and mast cells (Leder et al. (1986) Cell
`45:485-495), albumin gene control region which is active in
`liver (Pinkert et al. (1987) Genes Devel, 1:268-276), alpha-
`fetoprotein gene control region which is active in liver
`(Krumlaufet al. (1985) Mol. Cell. Biol. 5:1639-1648); alpha
`l-antitrypsin gene control region whichis active in the liver
`(Kelsey et al. (1987) Genes Devel. 1:161-171), beta-globin
`gene control region which is active in myeloid cells (Mo-
`gram et al. (1985) Nature 315:338-340); myelin basic pro-
`tein gene control region whichis active in oligodendrocyte
`cells in the brain (Readhead et al. (1987) Cell 48:703-712);
`myosin light chain-2 gene control region which is active in
`skeletal muscle (Shani (1985) Nature 314:283-286), and
`gonadotropic releasing hormone gene control region which
`is active in the hypothalamus (Masonet al. (1986) Science
`234:1372-1378).
`Thus, according to the invention, expression vectors
`capable of being replicated in a bacterial or eukaryotic host
`comprising
`chimeric
`polypeptide molecule-encoding
`nucleic acid as described herein, are used to transfect the 5
`host and thereby direct expression of such nucleic acids to
`produce the chimeric polypeptide molecules, which may
`then be recovered in a biologically active form. As used
`herein, a biologically active form includes a form capable of
`binding to VEGF. Expression vectors containing the chi-
`meric nucleic acid molecules described herein can be iden-
`tified by three general approaches: (a) DNA-DNA hybrid-
`ization, (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-DNAhybridiza-
`tion using probes comprising sequencesthat 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 ofcertain “marker” gene functions (e.g., thymidine
`kinaseactivity, resistance to antibiotics, transformation phe-
`
`Therapeutic Methods
`The presentinvention also has diagnostic and therapeutic
`utilities. In particular embodiments of the invention, meth-
`ods ofdetecting 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
`agonists or antagonists which bind the chimeric polypeptide
`molecules may be used in the treatment of diseases.
`In
`further embodiments, the chimeric polypeptide moleculeis
`utilized as an agent to block the binding of a binding agent
`to its target.
`By way of example, but notlimitation, the method ofthe
`invention may be useful in treating clinical conditions that
`are characterized by vascular permeability, edema or inflam-
`mation such as brain edemaassociated withinjury, stroke or
`tumor; edema associated with inflammatory disorders such
`as psoriasis or arthritis,
`including rheumatoid arthritis:
`5 asthma; generalized edema associated with burns; ascites
`and pleural effusion associated with tumors, inflammation or
`trauma; chronic airway inflammation; capillary leak syn-
`drome: sepsis: kidney disease associated with increased
`leakage of protein; and eye disorders such as age related
`macular degeneration and diabetic retinopathy.
`
`Specific Embodiments
`
`Pi on
`5
`
`The eye comprises several structurally and functionally
`distinct vascular beds, which supply ocular components
`critical
`to the maintenance of vision. These include the
`retinal and choroidal vasculatures, which supply the inner
`and outer portions ofthe retina, respectively, and the limbal
`vasculature located at the periphery ofthe 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 retinopa-
`thy is the most commondisease affecting the retinal vascu-
`lature, and is the leading cause of vision loss among the
`5 working age population in the United States, while the wet
`form of age-related macular degeneration (AMD) is the
`most commonform of choroidal neovascularization and a
`
`
`
`
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`US 7,303,746 B2
`
`8
`cells/mL, and a final Fltl(1-3)-Fe concentration at harvest
`was 95 mg/L. At harvest the cells were removed by tangen-
`tial flow filtration using 0.45 jum Prostak Filters (Millipore,
`Inc., Bedford, Mass. ).
`
`Example 2
`
`Purification of Fltl(1-3)-Fe Protein Obtained from
`CHOK1 Cells
`
`o
`
`4
`leading cause ofblindness in the elderly. 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.
`Each of the above conditions is characterized by patho-
`logical neovascularization, associated with or preceded by
`abnormal, excessive vascular permeability that often leads
`to pronounced edema in the affected tissue (cornea or
`retina). The production of abnormally high levels of VEGF
`has been implicated as a principal cause ofthe increased
`vascular permeability, as well as pathological angiogenesis
`(Aiello et al. (1994) N. Engl. J. Med. 331:1480-1487).
`Moreover, both the edema associated with abnormal vascu-
`lar permeability and pathological neovascularization con-
`tribute directly to the impairments of vision. Therefore,
`inhibition of VEGF action is one strategy now being
`explored for the treatment of ocular vascular diseases such
`as diabetic retinopathy and AMD.
`Other features of the invention will become apparent in
`the course of the following descriptions of exemplary
`embodiments which are given forillustration of the inven-
`tion and are not intended to be limiting thereof.
`
`EXAMPLES
`
`Example 1
`
`Expression of Fltl(1-3)-Fe Protein in CHO K1
`Cells
`
`Fitl(1-3)-Fe protein was initially purified by affinity chro-
`matography. A Protein A column wasused to bind, with high
`specificity,
`the Fe portion of the molecule. This affinity-
`purified protein was then concentrated and passed over a
`5 SEC column. The protein was then eluted into the formu-
`lation buffer. The following describes these procedures in
`detail.
`Materials and Methods. All chemicals were obtained from
`J.T. Baker, Phillipsburg, N.J. with the exception of PBS,
`which was obtained as a 10x concentrate from Life Tech-
`nologies, Gaithersburg, Md. Protein A Fast Flow and Super-
`dex 200 preparation