USOO73O3746B2
`
`(12)
`
`United States Patent
`Wiegand et al.
`
`(10) Patent No.:
`45) Date of Patent:
`
`US 7,303,746 B2
`Dec. 4, 2007
`
`9
`
`(54) METHODS OF TREATING EYE DISORDERS
`WITH MODIFIED CHIMERC
`POLYPEPTIDES
`(75) 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.,
`Tarrytown, NY (US)
`
`- r
`c
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 425 days.
`
`(21) Appl. No.: 10/988,243
`
`(22) Filed:
`
`Nov. 12, 2004
`
`(65)
`
`Prior Publication Data
`US 2005/0175610 A1
`Aug. 11, 2005
`O
`O
`Related U.S. Application Data
`(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.
`(60) Provisional application No. 60/138,133, filed on Jun.
`8, 1999.
`
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/18
`(2006.01)
`C07K I4/7
`(2006.01)
`CI2N 5/62
`(52) U.S. Cl. ............................... 424/134.1; 424/192.1;
`514/2: 514/12:530/350; 530/387.3: 536/23.4
`(58) Field Elisatist Seash 'search hist None
`ee application file for complete search history.
`(56)
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,712.380 A
`
`1/1998 Kendall et al.
`
`1/2000 Charnock-Jones et al.
`6,011,003 A
`2005/0260203 A1* 11/2005 Wiegand et al. ......... 424,145.1
`2006/0030529 A1* 2/2006 Wiegand et al. .............. 514/12
`2006/0172944 A1* 8/2006 Wiegand et al. .............. 514/12
`
`FOREIGN PATENT DOCUMENTS
`WSs
`ty E.
`WO99/03996
`1, 1999
`
`W
`WO
`
`OTHER PUBLICATIONS
`
`Witmer et al. (2003). Vascular endothelial growth factors and
`angiogenesis in eye disease. Prog. Retin. Eye Res. 22:1-29.*
`& 8
`M
`Terman, B.I., et al., “Identification of a new endothelial cell growth
`factor receptor tyrosine kinase”. Oncogene (1991) 6:1677-1683.
`Terman, B.I., 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 immunoglobulin-like
`domain of the VEGF tyrosine kinase receptor Flt-1 determines
`ligand binding and may initiate a signal transduction cascade'. 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.
`231:596-599.
`Fuh, G. et al., (1998).J. Bio. Chem. 273(18): 11197-11204.
`Wiesmann, C., et al., (1997) Cell 91:695-704
`Barleon, B., et al., (1997) J. Bio. Chem. 272(16):10382-10388
`arleOn, S., et al.,
`SO. C.
`:
`Davis-Smyth, T., et al., (1998) J. Bio. Chem. 273(6):3216-3222.
`* cited by examiner
`Primary Examiner Christine J. Saoud
`Assistant Examiner—Jon M. Lockard
`E
`74). Att
`Agent, or Fi
`Valeta G
`(74) Attorney, Agent, or Firm Valeta Gregg, Esq.
`(57)
`ABSTRACT
`
`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
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 1
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 1 of 21
`
`US 7,303,746 B2
`
`
`
`1.0 ug/ml
`
`
`
`Fig.
`
`1.
`
`og (Nºt)
`?dns SOO
`
`?dns SOO
`o+(BVG-1)!-IH : Lùnw
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 2
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 2 of 21
`
`US 7,303,746 B2
`
`- Unmodified Flt-1 (1-3)-Fc
`"K). Mut 1: Flt-1 (1-3AB)-FC
`--O-Mut?: Flt-1 (2-3AB)-Fc
`--A Mut3: Flt-1 (2-3)-Fo
`
`0.4
`
`
`
`
`
`O.3
`
`S
`0.2
`
`O.
`
`O.O1
`
`o
`
`1 O
`
`OO
`
`1OOO
`
`Fig. 2
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 3
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 3 of 21
`
`US 7,303,746 B2
`
`
`
`- Flt-1 (1-3)-Fo COS supe
`rig-Mut1: Flt-1 (1-3AB)-FC COS Supe
`--O-Muta: Flt-1 (2-3AB)-FC COS Supe
`E-A-Muts : Flt-1 (2-3)-Fo COS supe
`
`Fig. 3
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 4
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 4 of 21
`
`S 7,303,746 B2
`
`
`
`—+AcetylatedFit-1(1-3)-Fe(40X)
`
`&io° E> U
`
`
`
`
`
`—e—Mutt:Fit-1(1-3,p)-Fe
`
`Days
`
`©Y
`
`P
`oe
`=™
`
`=L
`
`L
`oD
`
`10
`
`0.01
`
`0.001
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 5
`Joining Petitioner: Apotex
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 5
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 5 of 21
`
`US 7,303,746 B2
`
`O.O
`
`2.5
`
`7.5
`5.0
`Modified Flt Receptor (nM)
`
`1 O.O
`
`12.5
`
`O FittD2FIk1 D3. FodeltaC1(a)
`A Flt1 D2VEGFR3D3.FcdeltaC1(a)
`V TE2-FC
`Fiti (1-3)-Fc
`
`Fig. 5
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 6
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 6 of 21
`
`US 7,303,746 B2
`
`
`
`g01+0.1000||00||01 .
`
`uUO/g/Ogy e3UeCuOSCW
`
`Fig. 6
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 7
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`US 7,303,746 B2
`
`
`
`
`
`Fig.
`
`7
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 8
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 8 of 21
`
`US 7,303,746 B2
`
`- Fiti (1-3)-Fc(A40)
`-- St Fiti D2FIk1 D3.FcdeltaC1(a)
`-- t FittD2FIk1 D3.FcdeltaC1(a)
`S
`as N sh-t VEGFR1R2-FcdeltaC1(a)
`
`100
`
`103
`
`
`
`O.
`
`Fig. 8
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 9
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 9 of 21
`
`US 7,303,746 B2
`
`
`
`10
`
`O.1
`
`O.O1
`
`- Fitt (1-3)-Fc(A40)
`-- FittD2VEGFR3D3.FcdeltaC1(a)
`-- Fiti D2FIk1D3.FcdeltaC1(a)
`
`Days
`
`Fig. 9
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 10
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`US 7,303,746 B2
`
`
`
`150
`
`1 OO
`
`
`
`
`
`(9uuuu) ?Uun?OA JOUun L
`
`5
`
`Fig.
`
`10
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 11
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 11 of 21
`
`US 7,303,746 B2
`
`
`
`
`
`(€uuuu) ?uun?OA JOUun. Li
`
`
`
`300
`
`O
`
`O
`
`Fig.
`
`11
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 12
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 12 of 21
`
`US 7,303,746 B2
`
`
`
`099
`
`00€
`
`092
`
`00Z
`
`00||
`
`09
`
`Fig.
`
`12
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 13
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 13 of 21
`
`US 7,303,746 B2
`
`
`
`PMSG-
`Fitt (1-3)-
`FC (A40)
`25mg/kg
`
`PMSG-
`FltD2-
`Fk1D3.
`FoAC1(a)
`25mg/kg
`
`PMSG-
`PBS
`
`PBS
`PBS
`
`Fig. 13A
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 14
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 14 of 21
`
`US 7,303,746 B2
`
`30
`
`
`
`25
`
`20
`
`15
`
`10
`
`PMSG-
`Fitt (1-3)-
`Fo (A40)
`
`PMSG-
`FitD2
`Fk1D3.
`FoAC1(a)
`
`PMSG-
`PBS
`
`PBS
`PBS
`
`Fig. 13B
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 15
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 15 of 21
`
`US 7,303,746 B2
`
`8
`
`
`
`9 -
`
`6
`
`4
`
`S X
`CD
`-
`.
`.
`E
`is
`9
`X
`(d
`9
`o ()
`is a 2
`it 5
`
`O
`
`Non-Diabetic
`
`Diabetic
`
`Diabetic with VGT
`
`FIG. 14
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 16
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 16 of 21
`
`US 7,303,746 B2
`
`
`
`OO
`
`90 -
`
`80 -
`
`7O -
`
`6O
`
`SO =
`
`3O -
`
`2O -
`
`10
`
`VGT 25mg/kg
`
`Control
`
`FIG. 15
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 17
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 17 of 21
`
`US 7,303,746 B2
`
`
`
`CNV
`Area 15
`
`Control
`
`VEGF Trap
`
`FIG. 16
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 18
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 18 of 21
`
`US 7,303,746 B2
`
`(mmx10)
`60
`
`
`
`P<0.0001
`
`5 O
`
`40
`
`3 O
`
`2O
`
`10
`
`O
`
`VEGF-TRAP
`(n=17)
`
`(n=19)
`
`FIG. 17
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 19
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 19 of 21
`
`US 7,303,746 B2
`
`1
`P=0.04519
`0.9 -
`0.8
`
`0.7
`
`0.6
`
`O.5
`
`0.4
`
`O.3
`
`O.2
`
`O. 1
`
`1
`0.9
`O.8
`O.7
`O.6
`
`O.S
`O.4
`0.3
`O.2
`O.1
`
`FC
`(n = 18)
`
`VEGF-TRAP
`(n = 18)
`
`FIG. 18A
`
`P=0.04.0595
`
`
`
`FC
`(n = 20)
`FIG. 18B
`
`VEGF-TRAP
`(n = 20)
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 20
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 20 of 21
`
`US 7,303,746 B2
`
`Suture injury
`
`Chemical injury
`
`500
`
`
`
`400
`
`300
`
`200
`
`100
`
`Control
`
`Suture
`
`Suture-VGT
`
`Control
`
`Chemical injury Chem-VGT
`
`FIG. 19
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 21
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 21 of 21
`
`US 7,303,746 B2
`
`
`
`Sp
`
`G
`S
`() |g
`2
`25
`SN O
`
`cd CN
`
`O
`
`O)
`u
`
`
`
`(N to 5 E
`
`O
`
`O
`
`1G
`St
`e
`
`r zE
`SS i
`6%
`
`O O O O O O O
`O D
`V
`Y
`GN
`V
`
`Suo Se 7 epei 9
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 22
`
`Joining Petitioner: Apotex
`
`

`

`US 7,303,746 B2
`
`1.
`METHODS OF TREATING EYE DSORDERS
`WITH MODIFIED CHMERC
`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 S 119(e) of U.S.
`Provisional 60/138,133 filed 8 Jun. 1999, which applications
`are herein specifically incorporated by reference in their
`entireties.
`
`10
`
`15
`
`BACKGROUND
`
`STATEMENT REGARDING RELATED ART
`
`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
`receptor, known as Flt (also known as VEGFR2), was shown
`to be a VEGF receptor (DeVries et al. (1992) Science
`255: 98.9991). Another form of the VEGF receptor, desig
`nated KDR or Flk-1 (also known as VEGFR3), is also
`known to bind VEGF and induce mitogenesis (Terman et 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 describes an altered, soluble form
`35
`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 amino acid sequences derived from VEGF receptors
`Flt1 and KDR.
`
`25
`
`30
`
`40
`
`BRIEF SUMMARY OF THE INVENTION
`
`2
`sequence which, as a result of the degeneracy of the genetic
`code, differs from the nucleotide sequence of SEQ ID
`NO:11, 13, and 15.
`The components of the fusion polypeptide encoded by the
`nucleic acid molecule of the invention are arranged as 1.2.3,
`1.3.2; 2,13: 2.3.1, 3,12; or 3.2.1, wherein 1 is the first
`VEGF receptor component, 2 is the second VEGF receptor
`component, and 3 is the multimerizing component.
`In a second aspect, the invention features a vector com
`prises a nucleic acid molecule of the 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 host cell. 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 E. 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 of the fusion polypeptide
`and recovering the fusion polypeptide so produced.
`In a 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 VEGF
`receptor and Ig domain 3 of an Flk-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 VEGF antagonist is modified by acetylation or
`pegylation.
`In a sixth aspect, the invention features a fusion polypep
`tide, comprising (a) a VEGF receptor component consisting
`essentially of an immunoglobulin-like (Ig) domain 2 of an
`Flt-1 VEGF receptor and Ig domain 3 of an Flk-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 which is one
`of the Fc 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
`SEQID NO:12 (Flt1D2. Flk1D3FcAC1(a)), SEQ ID NO:14
`(Flt1D2VEGFR3D3FcAC1(a)), and SEQ ID NO:16
`(VEGFR1R2 FcAC1(a)).
`In a seventh aspect, the invention features a pharmaceu
`tical composition comprising the fusion polypeptide of the
`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 composition of the
`invention 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
`Other objects and advantages will become apparent from
`a review of the ensuing detailed description.
`
`In a first aspect, the invention provides an isolated nucleic
`acid molecule, comprising (a) a nucleotide sequence encod
`ing a vascular endothelial growth factor (VEGF) receptor
`component consisting essentially of an immunoglobulin-like
`(Ig) domain 2 of a first VEGF receptor and Ig domain 3 of
`a second VEGF receptor; and (b) a nucleotide sequence
`encoding a multimerizing component, wherein the first
`VEGF receptor is Flt1, the second VEGF receptor is Flk1 or
`Flt4, and the VEGF receptor component is 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
`of the extracellular domain of the second VEGF receptor. In
`another embodiment, the nucleotide sequence encoding Ig
`domain 2 of the extracellular domain of the first VEGF
`receptor is downstream of the nucleotide sequence encoding
`Ig domain 3 of the extracellular domain of the second VEGF
`receptor. In one embodiment, the multimerizing component
`comprises an immunoglobulin domain. Preferably, the
`immunoglobulin domain is the Fc domain of IgG or the
`heavy chain of IgG. In specific embodiments, the nucleotide
`sequence is selected from the group consisting of the nucle
`otide sequence of SEQID NO:11, 13, and 15, or a nucleotide
`
`45
`
`50
`
`55
`
`60
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`65
`
`FIG. 1. Binding of unmodified Flt1(1-3)-Fc, basic region
`deletion mutant Flt1(1-3)-Fc, and Flt1(1-)- mutant pro
`teins in a Biacore-based assay.
`
`Mylan Exhibit 1016
`Mylan v. Regeneron, IPR2021-00880
`Page 23
`
`Joining Petitioner: Apotex
`
`

`

`3
`FIG. 2. Binding of unmodified Flt1(1-3)-Fc, Mut1: Flt1
`(1-3A)-Fc, Mut2: Flt1 (2-3A)-Fc, and Flt1(2-3) mutant pro
`teins to Matrigel(R) coated plates.
`FIG. 3. Binding of unmodified Flt1(1-3)-Fc, Mut1: Flt1
`(1-3A)-Fc, Mut2: Flt1 (2-3A)-Fc, and Flt1 (2-3) mutant pro
`teins in an ELISA-based assay.
`FIG. 4. Pharmacokinetic profiles of unmodified Flt1(1-
`3)-Fc. Mut1: Flt1(1-3A)-Fc, Mut2: Flt1(2-3A)-Fc, and
`Flt1(2-3) mutant proteins.
`FIG. 5. Extra cellular matrix (ECM) assay of Flt1D2.
`Flk1 D3. FcAC1(a) and Flt1D2. VEGFR3D3. FcAC1(a).
`FIG. 6. MG/R2 Cell proliferation assay. Modified Flt
`receptors Flt1(1-3)-Fc, Flt1D2. Flk1D3. FcAC1(a) and
`Flt1D2. VEGFR3D3. FcAC1(a), plus an irrelevant receptor
`termed Tie2-Fc as a negative control, were titrated from 40
`nM to 20 pM and incubated on the 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 Flt1D2Flk1D3. FcAC1(a) or
`VEGFR1R2-FcAC1(a), using the conversion factor of 1000
`RU equivalent to 1 ng/ml.
`FIG.8. Pharmacokinetics of Flt1(1-3)-Fc (A40), Flt1D2.
`Flk1 D3. FcAC1(a) and VEGFR1R2-FcAC1(a).
`FIG.9. Pharmacokinetics of Flt1(1-3)-Fc (A40), Flt1D2.
`Flk1 D3. FcAC1(a) and Flt1D2. VEGFR3D3. FcAC1(a).
`FIG. 10. The ability of Flt1D2. Flk1 D3. FcAC1(a) to
`inhibit HT-1080 fibrosarcoma tumor growth in vivo.
`FIG. 11. The ability of Flt1D2. Flk1D3. FcAC1(a) to
`inhibit C6 glioma tumor growth in vivo.
`FIG. 12. VEGF-induced uterine hyperpermeability.
`FIGS. 13 A-B. Assessment of corpus luteum angiogenesis
`using progesterone as a readout.
`FIG. 14. VEGFR1R2-FcAC1(a) prevents Evans Blue
`leakage in Streptozotocin-treated rats.
`FIG. 15. 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. 16. Effect of subcutaneous VEGFR1R2-FcAC1(a)
`injections on choroidal neovascularization area. The size of
`CNV lesions was measured in choroidal flat mounts. The
`45
`images were digitized using an Axioskop microscope
`equipped with a video camera, and the total area of choroidal
`neovascularization associated with each laser burn was
`measured using Image-Pro Plus Software.
`FIG. 17. VEGFR1R2-FcAC1(a) inhibits subretinal
`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 (C57BL/6) treated with injections
`of
`VEGFR1R2-FcAC1(a) had a significantly smaller retina to
`55
`lung leakage ration 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 reduction in the retina to lung leakage ration
`compared to mice treated with Fc fragment.
`FIG. 19. Effect of VEGFR1R2-FcAC1(a) administration
`on corneal 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
`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.
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`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. 20. System or intravitreal VEGF trap administration
`prevents laser-induced choroidal neovascularization (CNV)
`and reverses vascular leak in established lesions.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`It has been a long standing problem in the 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
`herein, for the first time, a chimeric polypeptide molecule,
`capable of antagonizing VEGF 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 receptors intracel
`lular 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, 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 (1995) BioEssays 17:25.
`Nucleic Acid Constructs
`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
`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 the
`chimeric polypeptide molecules under control of transcrip
`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,
`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
`molecule is expressed in a host transformed with the recom
`binant DNA molecule. For example, expression of the
`
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`5
`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 SV40 early promoter region
`(Bernoist et al. (1981) Nature 290:304-310), the CMV
`promoter, the M-Mul V 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 (Wagner et 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
`B-lactamase promoter (Villa-Kamaroff et al. (1978) Proc.
`Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter
`(DeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21
`25); promoter elements from yeast 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 I gene
`control region which is active in pancreatic acinar cells (see
`for example, Swift et al. (1984) Cell 38:639-646); insulin
`gene control region which is 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 tumor virus control region which is active in testicular,
`30
`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
`(Krumlaufetal. (1985) Mol. Cell. Biol. 5:1639-1648); alpha
`1-antitrypsin gene control region which is 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 which is 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 (Mason et 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
`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-DNA hybridiza
`tion 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 phe
`
`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
`sequence is inserted within the marker gene sequence of the
`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 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 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 or gel filtration may be used.
`Therapeutic Methods
`The present invention also has diagnostic and therapeutic
`utilities. In particular embodiments of the invention, meth
`ods 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
`agonists or antagonists which bind the chimeric polypeptide
`molecules 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 inflam
`mation 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 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
`
`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 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 retinopa
`thy is the most common disease affecting the retinal vascu
`lature, and is the leading cause of vision loss among the
`working age population in the United States, while the wet
`form of age-related macular degeneration (AMD) is the
`most common form of choroidal neovascularization and a
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`leading cause of blindness 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 of the 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 for illustration of the inven
`tion and are not intended to be limiting thereof.
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`cells/mL, and a final Flt1(1-3)-Fc concentration at harvest
`was 95 mg/L. At harvest the cells were removed by tangen
`tial flow filtration using 0.45 um Prostak Filters (Millipore,
`Inc., Bedford, Mass.).
`
`Example 2
`
`Purification of Flt1(1-3)-Fc Protein Obtained from
`CHO K1 Cells
`
`Flt1(1-3)-Fc protein was initially purified by affinity chro
`matography. A Protein A column was used to bind, with high
`specificity, the Fc portion of the molecule. This affinity
`purified protein was then concentrated and passed over a
`SEC column. The protein was then eluted into the formu
`lation buffer. The following describes these procedures in
`detail.
`Materials and Methods.