(12) United States Patent
`Daly et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,396,664 B2
`Jul. 8, 2008
`
`USOO7396664B2
`
`(54) VEGF-BINDING FUSION PROTEINS AND
`NUCLECACDS ENCOOING THE SAME
`
`(75) Inventors: Thomas J. Daly, New City, NY (US);
`Nicholas J. Papadopoulos,
`LaGrangeville, NY (US); Margaret
`Karow, Putnam Valley, NY (US)
`
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 297 days.
`(21) Appl. No.: 11/204,709
`
`y x- - -
`
`9
`
`(22) Filed:
`
`Aug. 16, 2005
`
`(65)
`
`Prior Publication Data
`
`US 2006/OO58234 A1
`
`Mar. 16, 2006
`O
`O
`
`(2006.01)
`A638/18
`(2006.01)
`C07K I4/00
`(2006.01)
`C07K I4/7
`(52) U.S. Cl. ................ 435/69.7; 424/134.1; 424/192.1;
`435/71. 1; 435/252.3:435/320.1; 435/325:
`435/358: 514/2: 514/12:530/350; 530/387.3;
`536/234
`(58) Field of Classification Search ....................... None
`See application file for complete search history.
`References Cited
`U.S. PATENT DOCUMENTS
`6,897.294 B2
`5/2005 Davis-Smyth et al.
`FOREIGN PATENT DOCUMENTS
`WOOOf75319
`12/2000
`
`(56)
`
`WO
`
`OTHER PUBLICATIONS
`Davis-Smyth 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.*
`
`Davis-Smyth, T., et al. (1998) J. Bio. Chem. 273(6):3216-3222.
`Related U.S. Application Data
`Heidaran, M., et al., (1990).J. Bio. Chem. 265(31): 18741-18744.
`(60) Continuation-in-part of application No. 1 1/016,097,
`filed on Dec. 17, 2004, and a continuation-in-part of R J N (NSEE, 98.
`application No. 11/016,503, filed on Dec. 17, 2004,
`eldaran, IVT., et al.,
`in
`5707.
`which is a division of application No. 10/009,852, filed
`Wulff, C., et al., (2002) Endocrinology 143(7):2797-2807.
`as application No. PCT/US00/14142 on May 23, 2000,
`* cited by examiner
`now Pat. No. 7,070,959, application No. 11/204,709,
`Primary Examiner Christine J Saoud
`and a continuation-in-part of application No. 10/880,
`Assistant Examiner—Jon M Lockard
`E
`021, filed on Jun. 29, 2004, now Pat. No. 7,279,159,
`74). Att
`Agent, or Fi
`Valeta G
`which is a continuation-in-part of application No.
`(74) Attorney, Agent, or Firm Valeta Gregg, Esq.
`10,609.775, filed on Jun. 30, 2003, OW Pat No. 7,087,
`(57)
`ABSTRACT
`411, which is a continuation-in-part of application No.
`10/009,852, filed on Dec. 6, 2001, now Pat. No. 7,070,
`Nucleic acid molecules and multimeric proteins capable of
`959.
`binding vascular endothelial growth factor (VEGF). VEGF
`(60) Provisional application No. 60/138,133, filed on Jun.
`traps are disclosed which are therapeutically useful for treat
`8, 1999.
`s u. -- s
`ing VEGF-associated conditions and diseases, and are spe
`cifically designed for local administration to specific organs,
`tissues, and/or cells.
`
`(51) Int. Cl.
`CI2N 5/62
`CI2N 15/63
`
`(2006.01)
`(2006.01)
`
`11 Claims, 3 Drawing Sheets
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 1
`
`Joining Petitioner: Apotex
`
`

`

`U.S. Patent
`
`Jul. 8, 2008
`
`Sheet 1 of 3
`
`US 7,396,664 B2
`
`
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`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 2
`Joining Petitioner: Apotex
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 2
`
`Joining Petitioner: Apotex
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`
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`
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`

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`U.S. Patent
`
`Jul. 8, 2008
`
`Sheet 2 of 3
`
`US 7,396,664 B2
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`
`
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`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 3
`Joining Petitioner: Apotex
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 3
`
`Joining Petitioner: Apotex
`
`
`
`
`
`
`
`

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`U.S. Patent
`
`Jul. 8, 2008
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`Sheet 3 of 3
`
`US 7,396,664 B2
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`
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`
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`
`
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`OTansty
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 4
`Joining Petitioner: Apotex
`
`cise|s7sc7|
`
`SOSSEAPIAIOSGO
`OosNOOS
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 4
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`Joining Petitioner: Apotex
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`

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`US 7,396,664 B2
`
`1.
`VEGF-BINDING FUSION PROTEINS AND
`NUCLECACDS ENCOOING THE SAME
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation-in-part of application
`Ser. Nos. 11/016,097 filed 17 Dec. 2004 and 11/016,503 filed
`17 Dec. 2004, which are divisionals of 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 application No. 60/138,133
`filed 8 Jun. 1999; and Application Ser. No. 10/880,021 filed
`29 Jun. 2004, now U.S. Pat. No. 7,279,159 which is a con
`tinuation-in-part of Ser. No. 10/609,775 filed 30 Jun. 2003
`now U.S. Pat. No. 7,087,411, which is a continuation-in-part
`of Ser. No. 10/009,852 filed 6 Dec. 2001, now U.S. Pat. No.
`7,070,959, which is the National Stage of International Appli
`cation 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 appli
`cations are herein specifically incorporated by reference in
`their entireties.
`
`10
`
`15
`
`25
`
`BACKGROUND OF THE INVENTION
`
`2
`The receptor components may be arranged in different
`orders, for example, R1R2F; R2R1F; R1 FR2: R2FR1;
`FR1R2; FR2R1, etc. The components of the fusion polypep
`tide may be connected directly to each other, or connected via
`a Spacer Sequence.
`In a third aspect, the invention features a multimeric
`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 protein
`of the invention is capable of binding VEGF with an affinity
`(Kd) of at least 1x10' M, preferably at least 1x10' M,
`even more preferably at least 1x10' M, as measured by
`BIACORETM based assays.
`The C-region may be created in the multimerizing compo
`nent by insertion, deletion, or mutation, Such that an enzy
`matically or chemically cleavable site is created. The C-re
`gion 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 CuCl2.
`In all embodiments of the VEGF-binding fusion polypep
`tides 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.
`The components of the fusion polypeptide may be con
`nected 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 embodiments,
`one or more receptor and/or fusion partner components are
`connected with spacers.
`In a fourth aspect, the invention encompasses vectors com
`prising 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 polypeptide 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 aspectare VEGF-binding
`fusion polypeptides of the invention 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.
`In a related seventh aspect, the invention features a 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 pro
`duced.
`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. A non-exhaustive list of
`
`30
`
`35
`
`40
`
`1. Field of the Invention
`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
`SC.
`2. Brief Summary of the Invention
`In a first aspect, the invention features an isolated nucleic
`acid molecule encoding a fusion polypeptide comprising
`receptor components R1-R2-F, wherein R1 is vascular endot
`helial cell growth factor (VEGF) receptor component 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.
`In a related second aspect, the invention features a VEGF
`binding fusion polypeptide comprising VEGF receptor com
`ponents R1-R2-F, wherein R1,R2, and Fareas defined above.
`45
`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.
`The fusion component F is selected from the group con
`sisting 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 compris
`ing a cleavable region (C-region), (ii) a truncated multimer
`izing 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 multimerizing 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 SEQID NO:8.
`
`50
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`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 5
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`Joining Petitioner: Apotex
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`3
`specific conditions improved by inhibition or reduction 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 dis
`orders such as psoriasis or arthritis, including rheumatoid
`arthritis; asthma; generalized edema associated with burns;
`ascites and pleural effusion associated with tumors, inflam
`mation or trauma; chronic airway inflammation; asthma; cap
`illary leak syndrome; sepsis: kidney disease associated with
`increased leakage of protein; pancreatic ductal adenocarci
`noma (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.
`Accordingly, in an eighth aspect, the invention features a
`therapeutic method for the treatment of a VEGF-related dis
`ease 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
`25
`binding fusion polypeptide of the invention.
`In a ninth aspect, the invention features pharmaceutical
`compositions comprising a VEGF-binding fusion polypep
`tide of the invention with a pharmaceutically acceptable car
`rier. Such pharmaceutical compositions may comprise a
`dimeric fusion polypeptide trap, or nucleic acids encoding the
`fusion polypeptide. The mini-traps of the invention find spe
`cific 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.
`Other objects and advantages will become apparent from a
`review of the ensuing detailed description.
`
`40
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIGS. 1A-C provides Sugar chain mass assignment for the
`oligosaccharides of two batches of VEGF trap protein (SEQ
`ID NO:8) (P3=VGT C04003M500; P3.5=C04008M500).
`
`45
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Before the present methods are described, it is to be under
`stood 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 limit
`ing, since the scope of the present invention will be limited
`only the appended claims.
`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.
`Unless defined otherwise, all technical and scientific terms
`used herein have the same meaning as commonly understood
`
`50
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`
`4
`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 prac
`tice 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 connection with which the pub
`lications are cited.
`General Description
`The invention encompasses multimeric VEGF-binding
`proteins capable of binding and inhibiting VEGF activity
`with a Kd of at least xx10' M. The molecules of the inven
`tion bind and inhibit the biological action of VEGF and/or the
`physiological reaction or response. For a description 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
`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 compo
`nents, 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, deletions from, or inser
`tions 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 abil
`ity to bind and inhibit VEGF.
`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 vector may be used to
`construct expression vectors encoding the fusion polypep
`tides of the invention under control of transcriptional/trans
`lational control signals.
`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 transformed 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 lim
`ited to, a long terminal repeat (Squinto et al. (1991) Cell
`65:1-20): SV40 early promoter region, CMV, M-MuDV, thy
`midine kinase promoter, the regulatory sequences of the met
`allothionine gene; prokaryotic 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.
`Expression vectors capable of being replicated in a bacte
`rial 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
`
`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 6
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`Joining Petitioner: Apotex
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`binding to VEGF. Transfected cells may transiently or, pref
`erably, constitutively and permanently express the VEGF
`traps of the invention.
`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, hydrophobic interaction chro
`matography, reverse phase chromatography or gel filtration
`may be used.
`VEGF Receptor Components
`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 charac
`teristics as the wild-type domain.
`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 appreci
`ated 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 determined by any Suitable screening
`assay known to the art for measuring the desired characteris
`tic. Examples of Such assays are described in the experimen
`tal section below which allow determination of binding affin
`ity 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. Modifications of protein properties such as thermal
`stability, hydrophobicity, Susceptibility to proteolytic degra
`dation, or tendency to aggregate may be measured by meth
`ods known to those of skill in the art.
`The components of the fusion polypeptide may be con
`nected 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 desirable site of
`interest between components for ease of manipulation. A
`spacer may also be provided to enhance expression of the
`fusion polypeptide from a host cell, to decrease steric hin
`drance such that the component may assume its optimal ter
`tiary 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 Engi
`neering 15:871-879, herein specifically incorporated by ref
`erence. 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
`
`6
`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 compo
`nents which is (are) between 1-100 amino acids, preferably
`1-25.
`In the most specific embodiments, R1 is amino acids
`27-126 of SEQID NO:6, or 1-126 of SEQID NO:6 (includ
`ing 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 SEQID 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.com
`ponent(s) attached to the multimerizing component may fur
`ther comprise a spacer component, for example, the GPG
`sequence of amino acids 229-231 of SEQID NO:5.
`Fusion and Multimerizing Components
`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 immungenic
`ity, or stability. In preferred embodiments, the fusion partner
`is selected from the group consisting of a multimerizing com
`ponent, a serum protein, or a molecule capable of binding a
`serum protein.
`When the fusion partner is a serum protein or fragment
`thereof, it is selected from the group consisting of C-1-micro
`globulin, AGP-1, orosomuciod, C-1-acid glycoprotein, Vita
`min D binding protein (DBP), hemopexin, human serum
`albumin (hSA), transferrin, ferritin, afamin, haptoglobin,
`C.-fetoprotein thyroglobulin, C-2-HS-glycoprotein, B-2-gly
`coprotein, hyaluronan-binding protein, syntaxin, C1R, C1q a
`chain, galectin3-Mac2 binding protein, fibrinogen, polymeric
`Ig receptor (PIGR), C-2-macroglobulin, urea transport pro
`tein, haptoglobin, IGFBPs, macrophage scavenger receptors,
`fibronectin, giantin, Fc, C-1-antichyromotrypsin, C.-1-antit
`rypsin, antithrombin III, apolipoprotein A-I, apolipoprotein
`B, B-2-microglobulin, ceruloplasmin, complement compo
`nent C3 or C4, CI esterase inhibitor, C-reactive protein, cys
`tatin C, and protein C. In a more specific embodiment, fusion
`partner is selected from the group consisting of C-1-micro
`globulin, AGP-1, orosomuciod, C-1-acid glycoprotein, Vita
`min 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: Perga
`mon Press; pp. 255-275).
`When a fusion partner is a molecule capable of binding a
`serum protein, the molecule may be a synthetic Small mol
`ecule, a lipid or liposome, a nucleic acid, including a synthetic
`nucleic acid such as an aptomer, a peptide, or an oligosaccha
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`Mylan Exhibit 1009
`Mylan v. Regeneron, IPR2021-00880
`Page 7
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`Joining Petitioner: Apotex
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`US 7,396,664 B2
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`7
`ride. The molecule may further be a protein, such as, for
`example, FcyR1, FcyR2, Fcy R3, polymeric Ig receptor
`(PIGR), Sclv, and other antibody fragments specific for a
`serum protein.
`When the fusion partner is a multimerizing component
`(MC), it is any natural or synthetic sequence capable of inter
`acting 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 (Müller et al. (1998) FEBS Lett. 432:
`45-49), coil-coil motifs, etc., or other generally accepted mul
`timerizing 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 spe
`cific embodiments, the immunoglobulin-derived domain may
`be selected from the group consisting 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 inven
`tion, the multimerizing component is an IgG4 Fc domain
`(SEQ ID NO:24).
`The fusion polypeptides of the invention may comprise 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 SEQ ID NO:8) (Larson et al. 2005 J Mol Biol 348:
`1177-90, which publication is herein specifically incorpo
`rated by reference in its entirety).
`In specific embodiments, the Fc from IgG1 may be modi
`fied to reduce effector functions. For example, the Fc may
`replace Asn297 (using cabot numbering) with a different
`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 incorpo
`rated by reference in its entirety). Glycosylation may also be
`modified using mutant CHO cell lines, which either eliminate
`glycosylation or modify the extent of glycosylation (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/
`01.00965; 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 publica
`tion is herein specifically 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 incor
`porated 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 spe
`cifically incorporated by reference in its entirety).
`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
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`8
`intact Ig Fc region (U.S. Pat. No. 6,121,022; U.S. Pat. No.
`6,194.551, which publications are herein specifically incor
`porated 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
`Thrat Phe256 (U.S. Pat. No. 6,277.375, which publication is
`herein specifically incorporated by reference in its entirety).
`Therapetic Uses
`The VEGF-binding traps of the invention are therapeuti
`cally 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
`with tumors, inflammation or trauma; chronic airway inflam
`mation; capillary leak syndrome; sepsis: kid