(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0058234 A1
`Daly et al.
`(43) Pub. Date:
`Mar. 16, 2006
`
`US 20060058234A1
`
`(54) VEGF TRAPS AND THERAPEUTIC USES
`THEREOF
`(76) Inventors: Thomas J. Daly, New City, NY (US);
`Nicholas J. Papadopoulos,
`LaGrangeville, NY (US); Margaret
`Karow, Putnam Vally, NY (US)
`Correspondence Address:
`REGENERON PHARMACEUTICALS, INC
`777 OLD SAW MILL RIVER ROAD
`TARRYTOWN, NY 10591 (US)
`
`(21) Appl. No.:
`
`11/204,709
`
`(22) Filed:
`
`Aug. 16, 2005
`
`Related U.S. Application Data
`(60) Continuation-in-part of application No. 11/016,097,
`filed on Dec. 17, 2004.
`Continuation-in-part of application No. 11/016,503,
`filed on Dec. 17, 2004, which is a division of appli
`cation No. 10/009,852, filed on Dec. 6, 2001, filed as
`371 of international application No. PCT/US00/
`14142, filed on May 23, 2000.
`Continuation-in-part of application No. 10/880,021,
`
`filed on Jun. 29, 2004, which is a continuation-in-part
`of application No. 10/609,775, filed on Jun. 30, 2003,
`which is a continuation-in-part of application No.
`10/009,852, filed on Dec. 6, 2001.
`(60) Provisional application No. 60/138,133, filed on Jun.
`8, 1999.
`
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`C07K 14/71
`(2006.01)
`A6IK 38/17
`(2006.01)
`C07H 21/04
`(2006.01)
`CI2P 21/06
`(2006.01)
`CI2N 5/06
`(52) U.S. Cl. .......................... 514/12; 530/350; 435/69.1;
`435/320.1; 435/358; 536/23.5
`ABSTRACT
`(57)
`Nucleic acid molecules and multimeric proteins capable of
`binding vascular endothelial growth factor (VEGF). VEGF
`traps are disclosed which are therapeutically useful for
`treating VEGF-associated conditions and diseases, and are
`Specifically designed for local administration to specific
`organs, tissues, and/or cells.
`
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`US 2006/0058234 A1
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`Mar. 16, 2006
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`VEGF TRAPS AND THERAPEUTIC USES
`THEREOF
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001. This application is a continuation-in-part of appli
`cation Ser. No. 11/016,097 filed 17 Dec. 2004 and Ser. No.
`11/016,503 filed 17 Dec. 2004, which are divisionals of Ser.
`No. 10/009,852 filed 6 Dec. 2001, 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 Application No. 60/138,133
`filed 8 Jun. 1999; and application Ser. No. 10/880,021 filed
`29 Jun. 2004, which is a continuation-in-part of Ser. No.
`10/609,775 filed 30 Jun. 2003, which is a continuation-in
`part of Ser. No. 10/009,852 filed 6 Dec. 2001, 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 Application No. 60/138,
`133 filed 8 Jun. 1999, which applications are herein spe
`cifically incorporated by reference in their entireties.
`
`BACKGROUND OF THE INVENTION
`0002) 1. Field of the Invention
`0003. The invention encompasses fusion polypeptides
`capable of binding vascular endothelial cell growth factor
`(VEGF), VEGF family members, and splice variants with
`Specifically desirable characteristics, as well as therapeutic
`methods of use.
`0004 2. Brief Summary of the Invention
`0005. In a first aspect, the invention features an isolated
`nucleic acid molecule encoding a fusion polypeptide com
`prising receptor components R1-R2-F, wherein R1 is vas
`cular endothelial cell growth factor (VEGF) receptor com
`ponent Ig domain 2 of Flt-1 (Flt1D2), R2 is VEGF receptor
`component Ig domain 3 of Flk-1 (Flk1D3) (also known as
`KDR), and F is a fusion component.
`0006. In a related second aspect, the invention features a
`VEGF-binding fusion polypeptide comprising VEGF recep
`tor components R1-R2-F, wherein R1, R2, and F are as
`defined above. The components may be connected directly
`to each other or connected via one or more Spacer Sequences.
`In a preferred embodiment, R1 and R2 are the only receptor
`components present. In a specific embodiment, the VEGF
`binding fusion polypeptide is amino acids 27-129 (R1) and
`130-231 (R2) of SEQ ID NO:8, or a variant thereof.
`0007. The fusion component F is selected from the group
`consisting of a multimerizing component, a Serum protein,
`or a molecule capable of binding a Serum protein. In a
`preferred embodiment, F is a multimerizing component
`capable of interacting with a multimerizing component on
`another fusion polypeptide to form a multimeric structure,
`e.g., a dimer or trimer. Most preferably, the F is Selected
`from the group consisting of (i) a multimerizing component
`comprising a cleavable region (C-region), (ii) a truncated
`multimerizing component, (iii) an amino acid sequence
`between 1 to about 200 amino acids in length having at least
`one cysteine residue, (iv) a leucine Zipper, (v) a helix loop
`motif and (vi) a coil-coil motif. Preferably, the multimeriz
`ing component is an immunoglobulin domain. In one
`
`embodiment, F is a full-length or truncated immunoglobulin
`domain consisting of amino acids 232-458, 232-457, or
`352-458 of SEO ID NO:8.
`0008. The receptor components may be arranged in dif
`ferent orders, for example, R1R2F; R2R1F, R1FR2; R2FR1;
`FR1R2; FR2R1, etc. The components of the fusion polypep
`tide may be connected directly to each other, or connected
`Via a Spacer Sequence.
`0009. In a third aspect, the invention features a multim
`eric VEGF-binding protein, comprising two or more fusion
`polypeptides of the invention (also called a VEGF“trap"). A
`VEGF trap composed of two fusion polypeptides having at
`least one truncated multimerizing component is termed a
`“truncated mini-trap.” The multimeric VEGF-binding pro
`tein of the invention is capable of binding VEGF with an
`affinity (Kd) of at least 1x10
`M, preferably at least
`1x11 M, even more preferably at least 1x10' M, as
`measured by Biacore-based assayS.
`0010. The C-region may be created in the multimerizing
`component by insertion, deletion, or mutation, Such that an
`enzymatically or chemically cleavable site is created. The
`C-region may be created in any multimerizing component
`and at any position within; preferably, the C-region is
`created in a full-length Fc domain, or a fragment thereof, or
`a C3 domain. The C-region may be a site cleavable by an
`enzyme, Such as, thrombin, ficin, pepsin, matrilysin, or
`prolidase or cleavable chemically by, for example, formic
`acid or CUC1.
`0011. In all embodiments of the VEGF-binding fusion
`polypeptides of the invention (including full length VEGF
`binding fusion polypeptides, truncated VEGF-binding
`fusion polypeptides, etc.), a signal Sequence (S) may be
`included at the beginning (or N-terminus) of the fusion
`polypeptide of the invention. The Signal Sequence may be
`native to the cell, recombinant, or Synthetic.
`0012. The components of the fusion polypeptide may be
`connected directly to each other or be connected via Spacers.
`In Specific embodiments, one or more receptor and/or fusion
`partner components of the fusion polypeptide are connected
`directly to each other without Spacers. In other embodi
`ments, one or more receptor and/or fusion partner compo
`nents are connected with Spacers.
`0013 In a fourth aspect, the invention encompasses vec
`tors comprising the nucleic acid molecules of the invention,
`including expression vectors comprising the nucleic acid
`molecule operatively linked to an expression control
`Sequence. In a fifth aspect, the invention encompasses
`host-vector Systems for the production of a fusion polypep
`tide which comprise the expression vector, in a Suitable host
`cell; host-vector Systems wherein the Suitable host cell is a
`bacterial, yeast, insect, mammalian cell; an E. Coli cell, or
`a COS or CHO cell. Additional encompassed in a sixth
`aspect are VEGF-binding fusion polypeptides of the inven
`tion modified by acetylation or pegylation, and other post
`translational modifications resulting from expression in a
`mammalian cell line. Methods for acetylating or pegylating
`a protein are well known in the art. In Specific embodiments,
`the fusion polypeptide of the invention expressed in a
`mammalian cell line Such as a CHO cell comprises amino
`acids 27-457 of SEQ ID NO:8 and is glycosylated at ASn
`residues 62, 94, 149, 222 and 308.
`
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`In a related seventh aspect, the invention features a
`0.014.
`method of producing a VEGF-binding fusion polypeptides
`of the invention, comprising culturing a host cell transfected
`with a vector comprising a nucleic acid Sequence of the
`invention, under conditions Suitable for expression of the
`protein from the host cell, and recovering the fusion
`polypeptides So produced.
`0015 The VEGF-binding fusion polypeptides of the
`invention are therapeutically useful for treating any disease
`or condition which is improved, ameliorated, or inhibited by
`removal, inhibition, or reduction of VEGF. Anon-exhaustive
`list of specific conditions improved by inhibition or reduc
`tion of VEGF include, for example, undesirable plasma
`leakage or vascular permeability, undesirable blood vessel
`growth, e.g., Such as in a tumor, edema associated with
`inflammatory disorderS Such as psoriasis or arthritis, includ
`ing rheumatoid arthritis, asthma, generalized edema associ
`ated with bums, ascites and pleural effusion associated with
`tumors, inflammation or trauma; chronic airway inflamma
`tion; asthma, capillary leak Syndrome; Sepsis, kidney disease
`asSociated with increased leakage of protein; pancreatic
`ductal adenocarcinoma (PDAC) and eye disorderS Such as
`age related macular degeneration and diabetic retinopathy.
`The VEGF mini-trap is particularly useful in treatment of
`eye disorders, and as an adjuvant to eye Surgeries, including
`glaucoma Surgery; and the treatment of intra-ocular tumors,
`Such as for example, uveal melanoma, retinoblastoma, Via
`intravitreal delivery.
`0016. Accordingly, in an eighth aspect, the invention
`features a therapeutic method for the treatment of a VEGF
`related disease or condition, comprising administering a
`VEGF-binding fusion polypeptide of the invention to a
`subject suffering from a VEGF-related disease or condition.
`Although any mammal can be treated by the therapeutic
`methods of the invention, the Subject is preferably a human
`patient Suffering from or at risk of Suffering from a condition
`or disease which can be improved, ameliorated, inhibited or
`treated with a VEGF-binding fusion polypeptide of the
`invention.
`0.017. In a ninth aspect, the invention features pharma
`ceutical compositions comprising a VEGF-binding fusion
`polypeptide of the invention with a pharmaceutically accept
`able carrier. Such pharmaceutical compositions may com
`prise a dimeric fusion polypeptide trap, or nucleic acids
`encoding the fusion polypeptide. The mini-traps of the
`invention find specific uses in conditions in which a VEGF
`inhibitor with reduced Serum half life (e.g., faster clearance),
`and/or increased tissue penetration due to Smaller Size is
`desirable. Specific applications for the VEGF mini-trap
`include, for example, diseases where local administration to
`a specific tissue or cell is desirable. Examples of Such a
`condition or disease are ocular diseases of the eye.
`0.018. Other objects and advantages will become apparent
`from a review of the ensuing detailed description.
`
`BRIEF DESCRIPTION OF THE FIGURES
`FIGS. 1A-C provides sugar chain mass assignment
`0.019
`for the oligosaccharides of two batches of VEGF trap protein
`(SEQ ID NO:8) (P3=VGT CO4003M500; P3.5-
`C04008M500).
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`0020. Before the present methods are described, it is to be
`understood that this invention is not limited to particular
`methods, and experimental conditions described, as Such
`methods and conditions may vary. It is also to be understood
`that the terminology used herein is for the purpose of
`describing particular embodiments only, and is not intended
`to be limiting, Since the Scope of the present invention will
`be limited only the appended claims.
`0021 AS used in this specification and the appended
`claims, the singular forms “a”, “an', and “the' include plural
`references unless the context clearly dictates otherwise.
`Thus for example, a reference to “a method’ includes one or
`more methods, and/or Steps of the type described herein
`and/or which will become apparent to those perSons skilled
`in the art upon reading this disclosure and So forth.
`0022 Unless defined otherwise, all technical and scien
`tific terms used herein have the same meaning as commonly
`understood by one of ordinary skill in the art to which this
`invention belongs. Although any methods and materials
`Similar or equivalent to those described herein can be used
`in the practice or testing of the present invention, the
`preferred methods and materials are now described. All
`publications mentioned herein are incorporated herein by
`reference to describe the methods and/or materials in con
`nection with which the publications are cited.
`General Description
`0023 The invention encompasses multimeric VEGF
`binding proteins capable of binding and inhibiting VEGF
`activity with a Kd of at least X x 10' M. The molecules of
`the invention bind and inhibit the biological action of VEGF
`and/or the physiological reaction or response. For a descrip
`tion of VEGF-receptor-based antagonist VEGF traps
`Flt1D2. Flk1D3.FcAC1(a) (SEQ ID NOS:5-6) and
`VEGFR1R2-FcAC1(a) (SEQ ID NOs:7-8), see PCT
`WO/0075319, the contents of which is incorporated in its
`entirety herein by reference.
`Nucleic Acid Constructs and Expression
`0024. The present invention provides for the construction
`of nucleic acid molecules encoding fusion polypeptides
`capable of multimerizing to form VEGF traps capable of
`binding VEGF with high affinity. The nucleic acid molecules
`of the invention may encode wild-type R1 and R2 receptor
`components, or may encode functionally equivalent variants
`thereof. Amino acid Sequence variants of the R1 and R2
`receptor components of the traps of the invention may also
`be prepared by creating mutations in the encoding nucleic
`acid molecules. Such variants include, for example, dele
`tions from, or insertions or Substitutions of, amino acid
`residues within the amino acid Sequence of R1 and R2. Any
`combination of deletion, insertion, and Substitution may be
`made to arrive at a final construct, provided that the final
`construct possesses the ability to bind and inhibit VEGF.
`0025 These nucleic acid molecules are inserted into a
`vector that is able to express the fusion polypeptides when
`introduced into an appropriate host cell. Appropriate host
`cells include, but are not limited to, bacterial, yeast, insect,
`and mammalian cells. Any of the methods known to one
`skilled in the art for the insertion of DNA fragments into a
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`vector may be used to construct expression vectors encoding
`the fusion polypeptides of the invention under control of
`transcriptional/translational control Signals.
`0.026
`Expression of the nucleic acid molecules of the
`invention may be regulated by a Second nucleic acid
`Sequence So that the molecule is expressed in a host trans
`formed with the recombinant DNA molecule. For example,
`expression may be controlled by any promoter/enhancer
`element known in the art. Promoters which may be used to
`control expression of the chimeric polypeptide molecules
`include, but are not limited to, a long terminal repeat
`(Squinto et al. (1991) Cell 65:1-20); SV40 early promoter
`region, CMV, M-Mul V, thymidine kinase promoter, the
`regulatory Sequences of the metallothionine gene, prokary
`otic expression vectorS Such as the B-lactamase promoter, or
`the tac promoter (see also Scientific American (1980)
`242:74–94); promoter elements from yeast or other fungi
`Such as Gal 4 promoter, ADH, PGK, alkaline phosphatase,
`and tissue-specific transcriptional control regions derived
`from genes Such as elastase I.
`0.027
`Expression vectors capable of being replicated in a
`bacterial or eukaryotic host comprising the nucleic acid
`molecules of the invention are used to transfect the host and
`thereby direct expression of Such nucleic acids to produce
`the fusion polypeptides of the invention, which form traps
`capable of binding to VEGF. Transfected cells may tran
`Siently or, preferably, constitutively and permanently
`express the VEGF traps of the invention.
`0028. The fusion polypeptides of the invention may be
`purified by any technique which allows for the Subsequent
`formation of a stable, biologically active trap. For example,
`and not by way of limitation, the factors may be recovered
`from cells either as Soluble proteins or as inclusion bodies,
`from which they may be extracted quantitatively by 8M
`guanidinium hydrochloride and dialysis (see, for example,
`U.S. Pat. No. 5,663,304). In order to further purify the
`factors, conventional ion exchange chromatography, hydro
`phobic interaction chromatography, reverse phase chroma
`tography or gel filtration may be used.
`VEGF Receptor Components
`0029. The VEGF receptor components of the VEGF mini
`trap consist of the Ig domain 2 of Flt-1 (Flt1D2) (R1), the Ig
`domain 3 of Flk-1 (Flk1D3) (R2) (together, R1R2. The term
`“Ig domain” of Flt-1 or Flk-1 is intended to encompass not
`only the complete wild-type domain, but also insertional,
`deletional, and/or Substitutional variants thereof which Sub
`Stantially retain the functional characteristics of the intact
`domain. It will be readily apparent to one of skill in the art
`that numerous variants of the above Ig domains can be
`obtained which will retains substantially the same functional
`characteristics as the wild-type domain.
`0030 The term “functional equivalents” when used in
`reference to R1 and R2, is intended to encompass an R1 and
`R2 domain with at least one alteration, e.g., a deletion,
`addition, and/or Substitution, which retains Substantially the
`Same functional characteristics as does the wild type R1 and
`R2, that is, a substantially equivalent binding to VEGF. It
`will be appreciated that various amino acid Substitutions can
`be made in R1 and R2 without departing from the spirit of
`the invention with respect to the ability of these receptor
`components to bind and inactivate VEGF. The functional
`
`characteristics of the traps of the invention may be deter
`mined by any Suitable Screening assay known to the art for
`measuring the desired characteristic. Examples of Such
`assays are described in the experimental Section below
`which allow determination of binding affinity of the traps for
`VEGF (Kd), as well as their half-life of dissociation of the
`trap-ligand complex (T2). Other assays, for example, a
`change in the ability to specifically bind to VEGF can be
`measured by a competition-type VEGF binding assay. Modi
`fications of protein properties Such as thermal Stability,
`hydrophobicity, Susceptibility to proteolytic degradation, or
`tendency to aggregate may be measured by methods known
`to those of skill in the art.
`0031. The components of the fusion polypeptide may be
`connected directly to each other or be connected via Spacers.
`Generally, the term “spacer” (or linker) means one or more
`molecules, e.g., nucleic acids or amino acids, or non-peptide
`moieties, Such as polyethylene glycol, which may be
`inserted between one or more component domains. For
`example, Spacer Sequences may be used to provide a desir
`able site of interest between components for ease of manipu
`lation. A Spacer may also be provided to enhance expression
`of the fusion polypeptide from a host cell, to decrease Steric
`hindrance Such that the component may assume its optimal
`tertiary Structure and/or interact appropriately with its target
`molecule. For Spacers and methods of identifying desirable
`Spacers, See, for example, George et al. (2003) Protein
`Engineering 15:871-879, herein specifically incorporated by
`reference. A Spacer Sequence may include one or more
`amino acids naturally connected to a receptor component, or
`may be an added Sequence used to enhance expression of the
`fusion polypeptides, provide Specifically desired Sites of
`interest, allow component domains to form optimal tertiary
`Structures and/or to enhance the interaction of a component
`with its target molecule. In one embodiment, the Spacer
`comprises one or more peptide Sequences between one or
`more components which is (are) between 1-100 amino acids,
`preferably 1-25.
`0032. In the most specific embodiments, R1 is amino
`acids 27-126 of SEQ ID NO:6, or 1-126 of SEQ ID NO:6
`(including the Signal Sequence 1-26); or amino acids 27-129
`of SEQ ID NO:8, or 1-129 of SEQ ID NO:8 (including the
`Signal sequence at 1-26). In the most specific embodiments,
`R2 is amino acids 127-228 of SEQ ID NO:6, or amino acids
`130-231 of SEQID NO:8. When, for example, R2 is placed
`at the N-terminus of the fusion polypeptide, a signal
`Sequence may desirably precede the receptor component.
`The receptor component(s) attached to the multimerizing
`component may further comprise a spacer component, for
`example, the GPG sequence of amino acids 229-231 of SEQ
`ID NO:5.
`Fusion and Multimerizing Components
`0033. The fusion partner is any component that enhances
`the functionality of the fusion polypeptide. Thus, for
`example, an fusion partner may enhance the biological
`activity of the fusion polypeptide, aid in its production
`and/or recovery, or enhance a pharmacological property or
`the pharmacokinetic profile of the fusion polypeptide by, for
`example, enhancing its Serum half-life, tissue penetrability,
`lack of immungenicity, or Stability. In preferred embodi
`ments, the fusion partner is Selected from the group con
`Sisting of a multimerizing component, a Serum protein, or a
`molecule capable of binding a Serum protein.
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`0034. When the fusion partner is a serum protein or
`fragment thereof, it is Selected from the group consisting of
`C-1-microglobulin, AGP-1, orosomuciod, C-1-acid glyco
`protein, vitamin D binding protein (DBP), hemopexin,
`human Serum albumin (hSA), transferrin, ferritin, afamin,
`haptoglobin, C.-fetoprotein thyroglobulin, C-2-HS-glycopro
`tein, B-2-glycoprotein, hyaluronan-binding protein, Syn
`taxin, C1R, C1q a chain, galectin3-Mac2 binding protein,
`fibrinogen, polymeric Ig receptor (PIGR), O-2-macroglobu
`lin, urea transport protein, haptoglobin, IGFBPS, macroph
`age Scavenger receptors, fibronectin, giantin, Fc, C-1-anti
`chyromotrypsin,
`C-1-antitrypsin,
`antithrombin
`III,
`apolipoprotein A-I, apolipoprotein B, B-2-microglobulin,
`ceruloplasmin, complement component C3 or C4, CI
`esterase inhibitor, C-reactive protein, cyStatin C, and protein
`C. In a more Specific embodiment, fusion partner is Selected
`from the group consisting of C-1-microglobulin, AGP-1,
`orosomuciod, C-1-acid glycoprotein, Vitamin D binding
`protein (DBP), hemopexin, human serum albumin (hSA),
`afamin, and haptoglobin. The inclusion of a fusion partner
`component may extend the Serum half-life of the fusion
`polypeptide of the invention when desired. See, for example,
`U.S. Pat. Nos. 6,423,512, 5,876,969, 6.593,295, and 6,548,
`653, herein specifically incorporated by reference in their
`entirety, for examples of Serum albumin fusion polypeptides.
`hSA is widely distributed throughout the body, particularly
`in the intestinal and blood components, and has an important
`role in the maintenance of OSmolarity and plasma Volume. It
`is slowly cleared in the liver, and typically has an in vivo
`half-life of 14-20 days in humans (Waldmann et al. (1977)
`Albumin, Structure Function and Uses; Pergamon Press; pp.
`255-275).
`0035. When a fusion partner is a molecule capable of
`binding a Serum protein, the molecule may be a Synthetic
`Small molecule, a lipid or liposome, a nucleic acid, including
`a Synthetic nucleic acid Such as an aptomer, a peptide, or an
`oligosaccharide. The molecule may further be a protein,
`such as, for example, Fcy R1, FcyR2, Fcy R3, polymeric Ig
`receptor (PIGR), ScFv, and other antibody fragments spe
`cific for a Serum protein.
`0.036 When the fusion partner is a multimerizing com
`ponent (MC), it is any natural or synthetic sequence capable
`of interacting with another MC to form a higher order
`Structure, e.g., a dimer, a trimer, etc. Suitable MCS may
`include a leucine Zipper, including leucine Zipper domains
`derived from c-jun or c-fos, Sequences derived from the
`constant regions of kappa or lambda light chains, Synthetic
`Sequences Such as helix-loop-helix motifs (Miller et al.
`(1998) FEBS Lett. 432:45-49), coil-coil motifs, etc., or other
`generally accepted multimerizing domains known to the art.
`In Some embodiments, the fusion component comprises an
`immunoglobulin-derived domain from, for example, human
`IgG, IgM or IgA. In Specific embodiments, the immunoglo
`bulin-derived domain may be Selected from the group con
`Sisting of the Fc domain of IgG, the heavy chain of IgG, and
`the light chain of IgG. The Fc domain of IgG may be
`Selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as
`well as any allotype within each isotype group. In one
`example of the VEGF trap of the invention, the multimer
`izing component is an IgG4 Fc domain (SEQ ID NO:24).
`0037. The fusion polypeptides of the invention may com
`prise a truncated multimerizing component. For example,
`when the multimerizing component is a human Fc, in a
`
`particular embodiment the C2 component of Fc may be
`deleted (239-351 of SEQID NO:8) (Larson et al. 2005 J Mol
`Biol 348: 1177-90, which publication is herein specifically
`incorporated by reference in its entirety).
`0038. In specific embodiments, the Fc from IgG1 may be
`modified to reduce effector functions. For example, the Fc
`may replace ASn297 (using cabot numbering) with a differ
`ent amino acid, for example, Ala or Gln, Asp, Met, or Tyr,
`to eliminate glycosylation (Tao et al. 1989 J. Immunol 143:
`2595-2601, which publication is herein specifically incor
`porated by reference in its entirety). Glycosylation may also
`be modified using mutant CHO cell lines, which either
`eliminate glycosylation or modify the extent of glycosyla
`tion (Wright et al. 1998 J. Immunol. 160:3393-3402, which
`publication is herein specifically incorporated by reference
`in its entirety). Mutations known by the art to eliminate
`effector functions are Leu234Ala, Leu235Ala/Glu (U.S.
`patent publication 2005/0100965; Reddy et al. 2000 J.
`Immunol. 164:1925-1933, which publications are herein
`Specifically incorporated by reference in their entirety), and
`the class 1 mutations described in Sheilds et al 2001 JBC
`276:6591-6604, which publication is herein specifically
`incorporated by reference in its entirety), Such as Arg265Ala
`and Phe329Ala. An IgG4 or IgG2 Fc can also be used to
`reduce effector functions. In the case of IgG4, mutations that
`Stabilize the molecule may be desirable, for example,
`Ser228Pro to stabilize covalent dimer formation (1993 Mol.
`Immunol. 30:105-108, which publication is herein specifi
`cally incorporated by reference in its entirety). Alternatively,
`the molecule may be enhanced for effector function activity
`by including mutations such as Ser298Ala, Glu33Ala,
`Lys334Ala (Sheilds et al Supra; U.S. 2005/0054832, which
`publication is herein specifically incorporated by reference
`in its entirety, or by decreasing the fucosylation of the Sugar
`side chain (Niwa et al. 2005 Clin Cancer Res 11:2327-2336,
`which publication is herein Specifically incorporated by
`reference in its entirety).
`0039. The in vivo half-life of the antibodies can also be
`improved by modifying the Salvage receptor epitope of the
`Ig constant domain or an Ig-like constant domain Such that
`the molecule does not comprise an intact CH2 domain
`(239-351) or an intact Ig Fc region (U.S. Pat. No. 6,121,022;
`U.S. Pat. No. 6,194.551, which publications are herein
`Specifically incorporated by reference in their entirety). The
`in vivo half-life can furthermore be increased by making
`mutations in the Fc region, e.g. by Substituting Thr at
`Leu252, Thr at Ser254, or Thr at Phe256 (U.S. Pat. No.
`6,277.375, which publication is herein specifically incorpo
`rated by reference in its entirety).
`Therapetic Uses
`0040. The VEGF-binding traps of the invention are thera
`peutically useful for treating any disease or condition which
`is improved, ameliorated, inhibited or prevented by removal,
`inhibition, or reduction of VEGF. A non-exhaustive list of
`Specific conditions improved by inhibition or reduction of
`VEGF include, clinical conditions that are characterized by
`excessive vascular endothelial cell proliferation, Vascular
`permeability, edema or inflammation 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
`
`Regeneron Exhibit 2009
`Page 08 of 30
`
`

`

`US 2006/0058234 A1
`
`Mar. 16, 2006
`
`with tumors, inflammation or trauma; chronic airway
`inflammation; capillary leak Syndrome; Sepsis, kidney dis
`ease associated with increased leakage of protein; and eye
`disorderS Such as age related macular degeneration and
`diabetic retinopathy.
`0041. The compositions of the invention are therapeuti
`cally useful for treating a wide variety of diseases associated
`with increased VEGF levels. For example, exaggerated Th2
`inflammation and airway remodeling are characteristic in the
`pathogenesis of asthma (see, for example, Elias et al. (1999)
`J. Clin. Invest. 104:1001-6). Elevated VEGF levels have
`been detected in tissues and biologic Samples from patients
`with asthma, which correlate directly with disease activity
`(Lee et al. (2001).J. Allergy Clin. Immunol. 107:1106-1108)
`and inversely with airway caliber and airway responsive
`ness. Further, VEGF has been postulated to contribute to
`asthmatic tissue edema.
`0.042 Another disease associated with increased VEGF is
`pancreatic ductal adenocarcinoma (PDAC). This malig
`nancy often exhibits enhanced foci of endothelial cell pro
`liferation and frequently overexpresses VEGF (Ferrara
`(1999) J. Mol. Med. 77:527-543). PDAC is responsible for
`over 20% of deaths due to gastrointestinal malignancies,
`making it the fourth most common cause of cancer-related
`mortality in the U.S. and other industrialized countries.
`Experimental evidence supports an important role for VEGF
`in pancreatic cancer, thus a VEGF inhibitor has promise as
`a therapeutic to attenuate intrapancreatic tumor growth and
`regional and distal metastasis.
`0.043 A smaller, non-glycosylated mini-trap expressed in
`E. coli (Example 4), a glycosylated mini-trap expressed in
`CHO cells (Example 5), or a receptor-based monomeric trap
`(Example 6) has optimized characteristics for local/intra
`vitreal delivery, ie. a shorter serum half life for faster
`clearance and minimizing unwanted Systemic exposure. In
`addition due to its Smaller size, the mini-trap has the ability
`to penetrate through the inner-limiting membrane (ILM) in
`the eye, and diffuse through the vitreous to the retina/retinal
`pigment epithelial (RPE) layer which will help to treat
`retinal disease. Additionally, the mini-trap can be used for
`local administration for the treatment of ocular disease Such
`as choroidal neovascularization, diabetic macular edema,
`proliferative diabetic retinopathy, corneal neovasculariza
`tion/transplant rejection. Still further, the mini-trap can be
`used in any situation where transient (short-term) blocking
`of VEGF is required, e.g., to avoid chronic exposure to
`VEGF blockade, such as, for example, in the treatment of
`pSoriasis.
`0044) A serious problem leading to failure following
`glaucoma Surgery is early