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`a2) United States Patent
`US 7,396,664 B2
`(10) Patent No.:
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`Dalyet al.
`(45) Date of Patent:
`Jul. 8, 2008
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`US007396664B2
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`(54) VEGF-BINDING FUSION PROTEINS AND
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`NUCLEIC ACIDS ENCODING THE SAME
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`(75)
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`Inventors: Thomas J. Daly, New City, NY (US);
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`Nicholas J. Papadopoulos,
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`LaGrangeville, NY (US); Margaret
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`Karow, Putnam Valley, NY (US)
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`(73) Assignee: Regeneron Pharmaceuticals,Inc.,
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`Tarrytown, NY (US)
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`(*) Notice:
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`Subject to any disclaimer, the term of this
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 297 days.
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`.No.:
`(21) Appl. No.: 11/204,709
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`Aug. 16, 2005
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`Filed:
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`CO7K 14/71
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`(52) US.Ch oe. 435/69.7, 424/134.1; 424/192.1,
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`435/71.1; 435/252.3; 435/320.1; 435/325;
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`435/358; 514/2; 514/12; 530/350; 530/387 .3;
`536/23.4
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`(58) Field of Classification Search .....0.0.00000... None
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`See application file for complete search history.
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`References Cited
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`U.S. PATENT DOCUMENTS
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`6,897,294 B2
`5/2005 Davis-Smyth etal.
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`FOREIGN PATENT DOCUMENTS
`WO 00/75319
`12/2000
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`_OTHER PUBLICATIONS
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`Davis-Smyth et al. (1996). The second immunoglobulin-like domain
`Prior Publication Data
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`:
`ofthe VEGFtyrosine kinase receptor Flt-1 determinesligand binding
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`and mayinitiate a signal transduction cascade. The EMBO Journal.
`US 2006/0058234 Al
`Mar. 16, 2006
`15(18):49 19-4927.
`wo
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`Davis-Smyth,T,, et al, (1998) J. Bio. Chom. 273(6):3216-3222,
`Related U.S. Application Data
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`Heidaran, M.,et al., (1990) J. Bio, Chem. 265(31):18741-18744.
`(60) Continuation-in-part of application No. 11/016,097,
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`filed on Dec. 17, 2004, and a continuation-in-part of ae ie as sr998)EASED49:140-14:
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`application No. 11/016,503, filed on Dec. 17, 2004,
`eidaran,
`Nin Cas
`Senay yIQ7
`98
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`whichisa divisionofapplication No. 10/009,852.filed
`Wulff, C., et al., (2002) Endocrinology 143(7):2797-2807.
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`as application No. PCT/US00/14142 on May 23, 2000,
`* cited by examiner
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`nowPat. No. 7,070,959, application No. 11/204,709,
`Primary Examiner—Christine J Saoud
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`anda continualion-in-part of application No. 10/880,
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`Assistant Examiner—Jon M Lockard
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`021, filed on Jun. 29, 2004, now Pat. No. 7,279,159, . Agent.orFi74) Att Valeta G E
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`Attorney, Agent, or Firm—Valeta Gregg,
`which is a continuation-in-part of application No.
`Esq.
`(VA)
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`10/609,775,filedon Jun. 30, 2003. now Pat. No. 7,087,
`ABSTRACT
`(57)
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`411, which is a continuation-in-part of application No.
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`10/009,852,filed on Dec. 6, 2001, now Pat. No. 7,070,
`Nucleic acid molecules and multimeric proteins capable of
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`binding vascular endothelial growth factor (WEGF). VEGF
`959.
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`(60) Provisional application No. 60/138,133, filed on Jun.
`traps are disclosed whichare therapeutically useful for treat-
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`ing VEGF-associated conditions and diseases, and are spe-
`8. 1999.
`——
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`cifically designed for local administrationto specific organs,
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`tissues, and/orcells.
`Int. Cl.
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`CI2N 15/62
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`CI2QN 15/63
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`(65)
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`(51)
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`(2006.01)
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`11 Claims, 3 Drawing Sheets
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`CELLTRION- EXHIBIT 1009
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`CELLTRION - EXHIBIT 1009
`
`

`

`U.S. Patent
`
`Jul. 8, 2008
`
`Sheet 1 of 3
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`US 7,396,664 B2
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`U.S. Patent
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`Jul. 8, 2008
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`U.S. Patent
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`Jul. 8, 2008
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`US 7,396,664 B2
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`CROSS-REFERENCE TO RELATED
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`APPLICATIONS
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`a
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`1
`VEGF-BINDING FUSION PROTEINS AND
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`NUCLEIC ACIDS ENCODING THE SAME
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`2
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`The receptor components may be arranged in different
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`orders,
`for example, RIR2F; R2R1F; R1FR2; R2FR1;
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`FR1R2; FR2R1, etc. The components of the fusion polypep-
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`tide may be connected directly ta each other, or connected via
`a spacer sequence.
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`In a third aspect, the invention features a multimeric
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`VEGF-binding, protein, comprising two or more fusion
`This application is a continuation-in-part of application
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`polypeptides of the invention(also called a VEGF “trap”). A
`Ser. Nos. 11/016,097 filed 17 Dec. 2004 and 11/016,503filed
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`VEGFtrap composed of two fusion polypeptides having at
`17 Dec. 2004, which are divisionals of Ser. No. 10/009,852
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`least one truncated mullimerizing component is termed a
`filed 6 Dec. 2001, now U.S. Pat No. 7,070,959, which is the
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`“truncated mini-trap.” The multimeric VEGF-binding protein
`National Stage of International Application No. PCT/US00/
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`of the invention is capable of binding VEGFwith anaffinity
`14142filed 23 May 2000, which claims the benefit under 35
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`(Kd) ofat least 1x107'° M,preferably at least 1x107'! M,
`USC § 119(e) ofU.S. provisional application No. 60/138,133
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`even more preferably at least 1x10-'? M, as measured by
`filed 8 Jun. 1999; and Application Ser. No. 10/880,021 filed
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`29 Jun. 2004, now US. Pat. No. 7,279,159 which is a con-
`BIACORE™based assays.
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`The C-region maybecreated in the multimerizing compo-
`tinuation-in-part of Ser. No. 10/609,775 filed 30 Jun. 2003
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`nent by insertion, deletion, or mutation, such that an enzy-
`now USS. Pat. No. 7,087,411, which is a continuation-in-part
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`of Ser. No. 10/009,852 filed 6 Dec. 2001, now U.S. Pat. No.
`matically or chemically cleavable site is created. The C-re-
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`gion may becreated in any multimerizing component and at
`7,070,959, which is the National Stage of International Appli-
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`any position within; preferably, the C-region is created in a
`cation No. PCT/US00/14142 filed 23 May 2000, which
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`full-length Fe domain, or a fragment
`thereof, or a C,,3
`claimsthe benefit under 35 USC § 119(e) of U.S.provisional
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`domain. The C-region may beasite cleavable by an enzyme,
`Application No. 60/138,133 filed 8 Jun. 1999, which appli-
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`such as, thrombin, ficin, pepsin, matrilysin, or prolidase or
`cations are herein specifically incorporated by reference in
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`their entireties.
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`cleavable chemically by, for example, formic acid or CuCl,.
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`In all embodiments of the VEGF-binding fusion polypep-
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`tides of the invention (including full length VEGF-binding
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`fusion polypeptides,
`truncated WEGF-binding
`fusion
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`1. Field of the Invention
`polypeptides, etc.), a signal sequence (S) maybe included at
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`the beginning (or N-terminus) ofthe fusion polypeptide ofthe
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`The invention encompassesfusion polypeptides capable of
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`invention. The signal sequence may be native to the cell,
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`binding vascular endothelial cell growth factor (VEGF),
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`recombinant, or synthetic.
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`VEGFfamily members, and splice variants with specifically
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`The components of the fusion polypeptide may be con-
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`desirable characteristics, as well as therapeutic methods of
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`use.
`nected directly to each other or be connected via spacers. In
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`specific embodiments, one or more receptor and/or fusion
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`2. Brief Summaryofthe Invention
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`partner components ofthe fusion polypeptide are connected
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`Inafirst aspect, the invention features an isolated nucleic
`directly to each other without spacers. In other embodiments,
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`acid molecule encoding a fusion polypeptide comprising
`one or more receptor and/or fusion partner components are
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`receptor components R1-R2-F, wherein R1is vascular endot-
`connected with spacers.
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`helial cell growth factor (VEGF) receptor component Ig
`Ina fourth aspect, the invention encompasses vectors com-
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`domain 2 of Flt-1 (F1t1ID2), R2 is VEGF receptor component
`prising the nucleic acid moleculesofthe invention, including
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`Ig domain3 ofFIk-1 (FIk1D3) (also known as KDR), and F is
`expression vectors comprising the nucleic acid molecule
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`a fusion component.
`operatively linked to an expressioncontrol sequence. Ina fifth
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`Ina related second aspect, the invention features a VEGF-
`aspect, the invention encompasseshost-vector systemsfor the
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`binding fusion polypeptide comprising VEGF receptor com-
`production of a fusion polypeptide which comprise the
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`ponents R1-R2-F, wherein R1, R2, and F areas defined above.
`expression vector, in a suitable host cell; host-vector systems
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`The components may be connected directly to each other or
`insect,
`wherein the suitable host cell
`is a bacterial, yeast,
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`connected via one or more spacer sequences. In a preferred
`mammalian cell; an &. Coli cell, or a COS or CHO cell.
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`embodiment, R1 and R2 are the only receptor components
`Additional encompassed in a sixth aspect are VEGF-binding
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`present. In a specific embodiment, the VEGF-binding fusion
`fusion polypeptides of the invention madified by acetylation
`a 2
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`polypeptide is amino acids 27-129 (R1) and 130-231 (R2) of 5
`or pegylation, and other post-translational modifications
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`SEQ ID NO:8, or a variant thereof.
`resulting from expression in a mammalian cell line. Methods
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`The fusion component F is selected from the group con-
`for acetylating or pegylating a protein are well known in the
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`sisting of a multimerizing component, a serum protein, or a
`art. In specific embodiments, the fusion polypeptide of the
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`molecule capable of binding a serum protein. In a preferred
`invention expressed in a mammalian cell line such as a CHO
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`embodiment, F is a multimerizing component capable of
`cell comprises amino acids 27-457 of SEQ ID NO:8 andis
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`interacting with a multimerizing component on another
`glycosylated at Asn residues 62, 94, 149, 222 and 308.
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`fusion polypeptide to form a multimeric structure, e.g.. a
`Inarelated seventh aspect, the invention features a method
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`dimeror trimer. Most preferably, the F is selected from the
`of producing a VEGF-binding fusion polypeptides of the
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`group consisting of (i) a multimerizing component compris-
`invention, comprising culturing a host cell transfected with a
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`ing a cleavable region (C-region), (ii) a truncated multimer-
`vector comprising a nucleic acid sequence ofthe invention,
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`izing component,(iii) an amino acid sequence between | to
`under conditions suitable for expression of the protein from
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`about 200 aminoacids in length having at least one cysteine
`the host cell, and recovering the fusion polypeptides so pro-
`duced.
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`residue, (iv) a leucine zipper, (v) a helix loop motif and (vi) a
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`coil-coil motif. Preferably, the multimerizing componentis
`The VEGF-binding fusion polypeptides of the invention
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`an immunoglobulin domain. In one embodiment, F is a full-
`are therapeutically useful for treating any disease or condition
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`length or truncated immunoglobulin domain consisting of
`which is improved, ameliorated, or inhibited by removal,
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`inhibition, or reduction of VEGF. A non-exhaustive list of
`amino acids 232-458, 232-457, or 352-458 of SEQ ID NO:8.
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`BACKGROUND OF THE INVENTION
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`mb on
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`specific conditions improved by inhibition or reduction of
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`VEGF include, for example, undesirable plasma leakage or
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`vascular permeability, undesirable blood vessel growth, e.g.,
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`suchas in a tumor, edema associated with inflammatory dis-
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`orders such as psoriasis or arthritis,
`including rheumatoid
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`arthritis; asthma; generalized edema associated with burns;
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`ascites and pleural effusion associated with tumors, inflam-
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`mation or trauma; chronic airway inflammation; asthma; cap-
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`illary leak syndrome; sepsis; kidney disease associated with
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`increased leakage of protein; pancreatic ductal adenocarci-
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`noma (PDAC)and eye disorders suchas age related macular
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`degeneration and diabetic retinopathy. The VEGF mini-trap
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`is particularly useful in treatment of eye disorders, and as an
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`adjuvant to eye surgeries, including glaucomasurgery; and
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`the treatment of intra-ocular tumors, such as for example,
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`uveal melanoma,retinoblastoma,via intravitreal delivery.
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`Accordingly, in an eighth aspect, the invention features a
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`therapeutic method for the treatment of a VEGF -related dis-
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`ease or condition, comprising administering a VEGF-binding
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`fusion polypeptide ofthe invention to a subject suffering from
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`a VEGF-related disease or condition. Although any mammal
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`can be treated by the therapeutic methodsofthe invention,the
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`subject is preferably a human patient suffering from or at risk
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`of suffering from a condition or disease which can be
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`improved, ameliorated, inhibited or treated with a VEGF-
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`binding fusion polypeptide of the invention.
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`In a ninth aspect, the invention features pharmaceutical
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`compositions comprising a VEGF-binding fusion polypep-
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`tide of the invention with a pharmaceutically acceptable car-
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`rier. Such pharmaceutical compositions may comprise a
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`dimeric fusion polypeptide trap, or nucleic acids encoding the
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`fusion polypeptide. The mini-traps of the invention find spe-
`cific uses in conditions in which a VEGF inhibitor with
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`reduced serum half life (c.g.
`faster clearance), and/or
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`increased tissue penctration duc to smaller size is desirable.
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`Specific applications for the VEGF mini-trap include, for
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`example, diseases where local administration to a specific
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`tissue or cell is desirable. Examples of such a condition or
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`disease are ocular diseases ofthe eye.
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`Other objects and advantages will become apparent from a
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`reviewof the ensuing detailed description.
`BRIEF DESCRIPTION OF THE FIGURES
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`ie)°°
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`FIGS. 1A-C provides sugar chain mass assignment for the
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`oligosaccharides of two batches of VEGFtrap protein (SEQ
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`ID NO:8) (P3=VGT C04003MS500; P3.5=C04008M500).
`DETAILED DESCRIPTION OF THE INVENTION
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`Before the present methodsare described,it is to be under-
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`stood that this invention is not limited to particular methods,
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`and experimental conditions described, as such methods and
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`conditions may vary. It is also to be understood that the
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`terminology used herein is for the purpose of describing
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`particular embodimentsonly, and is not intended to be limit-
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`ing, since the scope of the present invention will be limited
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`only the appendedclaims.
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`Asusedin this specification and the appendedclaims, the
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`singular forms“a’’, “an”, and “the” include plural references
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`unless the context clearly dictates otherwise. Thus for
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`example, a reference to “a method” includes one or more
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`methods, and/or steps of the type described herein and/or
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`whichwill become apparentto those personsskilled in the art
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`upon reading this disclosure and so forth.
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`Unless defined otherwise,all technical and scientific terms
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`used herein have the same meaning as commonly understood
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`US 7,396,664 B2
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`4
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`by one of ordinary skill in the art to which this invention
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`belongs. Although any methods and materials similar or
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`equivalent to those described herein can be used inthe prac-
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`tice or testing of the present invention, the preferred methods
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`and materials are now described. All publications mentioned
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`herein are incorporated herein by reference to describe the
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`methods and/or materials in connection with which the pub-
`lications are cited.
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`General Description
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`The invention encompasses multimeric WEGF-binding
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`proteins capable of binding and inhibiting VEGF activity
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`with a Kd of at least xx107'° M. The moleculesofthe inven-
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`tion bind and inhibit the biological action ofVEGFand/or the
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`physiological reaction or response. For a description of
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`VEGF-receptor-based
`antagonist
`VEGF
`traps
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`FItID2.FIkID3.FcACl(a)
`(SEQ ID NOs:5-6)
`and
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`see PCT
`VEGFRIR2-FcACl(a)
`(SEQ ID NOs:7-8),
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`W0O/0075319, the contents of which is incorporated in its
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`entirety herein by reference.
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`Nucleic Acid Constructs and Expression
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`The present invention provides for the construction of
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`nucleic acid molecules encoding fusion polypeptides capable
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`of mulumerizing to form VEGF traps capable of binding
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`VEGFwith high affinity. The nucleic acid molecules of the
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`invention may encode wild-type R1 and R2 receptor compo-
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`nents, or may encodefunctionally equivalentvariants thereof.
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`Amino acid sequence variants of the R1 and R2 receptor
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`components ofthe traps ofthe invention mayalso be prepared
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`by creating mutations in the encoding nucleic acid molecules.
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`Such variants include, for example, deletions from,or inser-
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`tions or substitutions of, amino acid residues within the amino
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`acid sequence of R1 and R2. Any combination of deletion,
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`insertion, and substitution may be made toarriveat a final
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`construct, providedthat the final construct possesses the abil-
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`ity to bind and inhibit VEGF.
`These nucleic acid molecules are inserted into a vector that
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`is able to express the fusion polypeptides when introduced
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`into an appropriate host cell. Appropriate host cells include,
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`but are notlimited to, bacterial, yeast, insect, and mammalian
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`cells. Any of the methods knownto oneskilled in the art for
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`the insertion of DNA fragments into a vector may be used to
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`construct expression vectors encoding the fusion polypep-
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`tides of the invention under control of transcriptional/trans-
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`lational control signals.
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`Expression of the nucleic acid molecules of the invention
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`may be regulated by a second nucleic acid sequence so that
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`the molecule is expressed in a host transformed with the
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`recombinant DNA molecule. For example, expression may
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`be controlled by any promoter/enhancer element known in
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`the art. Promoters which may be used to control expression of
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`the chimeric polypeptide molecules include, but are not lim-
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`ited to, a long terminal repeat (Squinto et al. (1991) Cell
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`65:1-20); SV40 early promoter region, CMV, M-MuLV, thy-
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`midine kinase promoter, the regulatory sequencesof the met-
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`allothionine gene; prokaryotic expression vectors such as the
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`(-lactamase promoter,or the tac promoter(see also Scientific
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`American (1980) 242:74-94); promoter elements from yeast
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`or other fungi such as Gal 4 promoter, ADI], PGK, alkaline
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`phosphatase, and tissue-specific transcriptional control
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`regions derived from genes suchaselastase I.
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`Expression vectors capable of being replicated in a bacte-
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`rial or eukaryotic host comprising the nucleic acid molecules
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`of the invention are used to transfect the host and thereby
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`direct expression of such nucleic acids to producethe fusion
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`polypeptides of the invention, which form traps capable of
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`US 7,396,664 B2
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`binding to VEGF. Transfected cells may transiently or, pref-
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`erably, constitutively and permanently express the VEGF
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`traps ofthe invention.
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`The fusion polypeptides of the invention maybepurified
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`by anytechnique which allows for the subsequent formation
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`of a stable, biologically active trap. For example, and not by
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`way of limitation, the factors may be recovered from cells
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`either as soluble proteins or as inclusion bodies, from which
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`they may be extracted quantitatively by 8M guanidinium
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`hydrochloride and dialysis (see, for example, U.S. Pat. No.
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`5,663,304).In orderto further purify the factors. conventional
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`ion exchange chromatography, hydrophobic interaction chro-
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`matography, reverse phase chromatography orgel filtration
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`may be used.
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`VEGFReceptor Components
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`‘The VEGFreceptor components of the VEGF mini trap
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`consist of the 1g domain 2 of FIt-1 (Flt]D2) (R1), the Ig
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`domain 3 of FIk-1 (FIk1D3) (R2) (together, R1R2. he term
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`“Ig domain” of FIt-1 or FIk-1 is intended to encompass not
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`only the complete wild-type domain, but also insertional,
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`deletional, and/or substitutional variants thereof which sub-
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`stantially retain the functional characteristics of the intact
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`domain.It will be readily apparentto one ofskill in the art that
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`numerous variants of the above 1g domains can be obtained
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`which will retains substantially the same functional charac-
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`teristics as the wild-type domain.
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`The term “functional equivalents” when used in reference
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`to R1 and R2,is intended to encompass an R1 and R2 domain
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`with at least onealteration, e.g., a deletion, addition, and/or
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`substitution, which retains substantially the same functional
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`characteristics as does the wild type R1 and R2, that is, a
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`substantially equivalent binding to VEGF.It will be appreci-
`ated that various amino acid substitutions can be madein R1
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`and R2 without departing from thespirit ofthe invention with
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`respectto the ability ofthese receptor components to bind and
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`inactivate VEGF.The functional characteristics ofthe traps of
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`the invention may be determined by any suitable screening
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`assay knownto the art for measuring the desired characteris-
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`tic. Examples of such assays are described in the experimen-
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`tal section below which allow determination of binding affin-
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`ity of the traps for VEGF (Kd), as well as their half-life of
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`dissociation of the trap-ligand complex (T,,). Other assays,
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`for example. a change in the ability to specifically bind to
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`VEGFcan be measured by a competition-type VEGF binding
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`assay. Modifications of protein propertics such as thermal
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`stability, hydrophobicity, susceptibility to proteolytic degra-
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`dation, or tendency to aggregate may be measured by meth-
`ods knownto those ofskill in the art.
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`The components of the fusion polypeptide may be con-
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`nected directly to each other or be connected via spacers.
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`Generally, the term “spacer” (or linker) means one or more
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`molecules, e.g., nucleic acids or amino acids, or non-peptide
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`moieties, such as polyethylene glycol, which maybe inserted
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`between one or more component domains. For example,
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`spacer sequences maybe used to provide a desirable site of
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`interest between components for ease of manipulation. A
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`spacer may also be provided to enhance expression of the
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`fusion polypeptide from a host cell, to decrease steric hin-
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`drance such that the component may assumeits optimalter-
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`tiary structure and/or interact appropriately with its target
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`molecule. For spacers and methods of identifying desirable
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`spacers, see, for example, George et al. (2003) Protein Engi-
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`neering 15:871-879, herein specifically incorporated by ref-
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`erence. A spacer sequence may include one or more amino
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`acids naturally connected to a receptor component, or may be
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`an added sequence used to enhance expression ofthe fusion
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`6
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`polypeptides, provide specifically desired sites of interest,
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`allow component domains to form optimaltertiary structures
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`and/or to enhance the interaction of a component with its
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`target molecule. In one embodiment, the spacer comprises
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`one or more peptide sequences between one or more compo-
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`nents whichis (are) between 1-100 amino acids, preferably
`1-25.
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`is amino acids
`In the most specific embodiments, Rl
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`27-126 of SEQ ID NO:6, or 1-126 of SEQ ID NO:6 (includ-
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`ing the signal sequence 1-26); or amino acids 27-129 of SEQ
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`ID NO:8, or 1-129 of SEQ ID NO:8 (including the signal
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`sequence at 1-26). In the most specific embodiments, R2 is
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`amino acids 127-228 of SEQ ID NO:6, or aminoacids 130-
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`231 of SEQ ID NO:8. When, for example, R2 is placed at the
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`N-terminusof the fusion polypeptide, a signal sequence may
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`desirably precede the receptor component. The receptor com-
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`ponent(s) attached to the multimerizing component mayfur-
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`ther comprise a spacer component, for example, the GPG
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`sequence of amino acids 229-231 of SEQ ID NO:5.
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`Fusion and Multimerizing Components
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`The fusion partner is any component that enhances the
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`functionality ofthe fusion polypeptide. Thus, for example, an
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`fusion partner may enhance the biological activity of the
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`fusion polypeptide, aid in its production and/or recovery, or
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`enhance a pharmacological propertyor the pharmacokinetic
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`profile of the fusion polypeptide by, for example, enhancing
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`its serumhalf-life, tissue penetrability, lack of immungenic-
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`ity, or stability. In preferred embodiments,the fusion partner
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`is selected fromthe group consisting ofa multimerizing com-
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`ponent, a serumprotein, or a molecule capable of binding, a
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`serumprotein.
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`When the fusion partner is a serum protein or fragment
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`thereof, it is selected from the group consisting ofa-1-micro-
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`globulin, AGP-1, orosomuciod, ct-1-acid glycoprotein, vita-
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`min D binding protein (DBP), hemopexin, human serum
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`albumin (hSA),
`transferrin, ferritin, afamin, haptoglobin,
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`a-fetoprotein thyroglobulin, a-2-HS-glycoprotein, $-2-gly-
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`coprotein, hyaluronan-binding protein, syntaxin, CIR, Clqa
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`chain, galectin3-Mac2 bindingprotein,fibrinogen, polymeric
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`Ig receptor (PIGR), a-2-macroglobulin, urea transport pro-
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`tein, haptoglobin, IGFBPs, macrophage scavengerreceptors,
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`fibronectin, giantin, Fc, a-1l-antichyromotrypsin, o-1-antit-
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`rypsin, antithrombin III, apolipoprotein A-I, apolipoprotein
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`B, B-2-microglobulin, ceruloplasmin, complement compo-
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`nent C3 or C4, Cl esterase inhibitor, C-reactive protein, cys-
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`tatin C, and protein C. Ina more specific embodiment, fusion
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`partner is selected from the group consisting of a-1-micro-
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`globulin, AGP-1, orosomuciod, c-1-acid glycoprotein, vita-
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`min D binding protein (DBP), hemopexin, human serum
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`albumin (hSA), afamin, and haptoglobin. The inclusion ofa
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`fusion partner component mayextend the serum half-life of
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`the fusion polypeptide ofthe invention whendesired. See, for
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`example, U.S. Pat. Nos. 6,423,512, 5,876,969, 6,593,295,
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`and 6,548,653, herein specifically incorporated by reference
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`in their entirety, for examples of serum albumin fusion
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`polypeptides. hSA is widely distributed throughout the body,
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`particularly in the intestinal and blood components, and has
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`an important role in the maintenance of osmolarity and
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`pla