USOO7300653B2
`
`(12) United States Patent
`Wiegand et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,300,653 B2
`Nov. 27, 2007
`
`(54) METHOD OF TREATING CORNEAL
`TRANSPLANT REUECTION
`
`(75) Inventors: Stanley Wiegand, Croton on Hudon,
`NY (US); Jingtai Cao, Chappaqua, NY
`(US); Claus Cursiefen, Erlangen (DE)
`(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 544 days.
`(21) Appl. No.: 10/830,902
`(22) Filed:
`Apr. 23, 2004
`
`(65)
`
`Prior Publication Data
`US 2005/OOO4O27 A1
`Jan. 6, 2005
`
`Related U.S. Application Data
`(60) Provisional application No. 60/492.865, filed on Aug.
`6, 2003, provisional application No. 60/473,734, filed
`on May 28, 2003.
`
`(51) Int. Cl.
`(2006.01)
`A638/8
`(2006.01)
`C07K I4/7
`(2006.01)
`CI2N 5/62
`(52) U.S. Cl. ............................... 424/134.1; 424/192.1;
`514/2; 514/12:530/350,536/23.4
`(58) Field of Classification Search ..................... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`2005/O1972.91 A1*
`2006/0172944 A1*
`
`U.S. PATENT DOCUMENTS
`9/2005 Wiegand et al. .............. 514/12
`8/2006 Wiegand et al. .............. 514/12
`
`FOREIGN PATENT DOCUMENTS
`
`WO
`WO
`WO
`
`3, 1999
`WO99/13909 A1
`WOOO,75319 A1 12/2000
`WOO3,O72O29 A2
`9, 2003
`
`OTHER PUBLICATIONS
`Phillips, A.J. (2001). The challenge of gene therapy and DNA
`delivery. J. Pharm. Pharmacol. 53:1169-1174.*
`Stagner et al. (2004). Beta-cell sparing in transplanted islets by
`vascular endothelial growth factor. Transplant. Proc. 36:1178
`1180.*
`Yietal. (2007). VEGF gene therapy for the survival of transplanted
`fat tissue in nude mice. J. Plast. Reconstr. Aesthet. Surg. 60:272
`2.78.*
`Paluet al. (1999). In pursuit of new developments for gene therapy.
`J. Biotechnol. 68:1–13.
`de Freitas et al. (2006). Causes and risk factors for graft failure in
`Surgeries performed by physicians in fellowship training. Cornea.
`25(3):251-256.*
`Yatoh, et al., TRANSPLANTATION, Vol. 66, No. 11, pp. 1519
`1524, (1998).
`Lai et al. Inhibition of Corneal Neovascularization by Recombinant
`Adenovirus Mediated Antisene VEGF RNA. (2002) Experimental
`Eye Research 75:625-634.
`* cited by examiner
`Primary Examiner Christine J. Saoud
`Assistant Examiner—Jon M Lockard
`(74) Attorney, Agent, or Firm—Valeta Gregg, Esq.
`
`(57)
`
`ABSTRACT
`
`Methods of preventing, reducing, or treating corneal trans
`plant rejection to improve transplant Survival in a subject in
`need thereof comprising administering an agent capable of
`blocking or inhibiting vascular endothelial growth factor
`(VEGF) are provided. The methods are useful for inhibiting
`or preventing corneal transplant rejection in a human Subject
`who is the recipient of a transplanted cornea.
`
`8 Claims, No Drawings
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`Mylan Exhibit 1145
`Mylan v. Regeneron, IPR2021-00881
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`US 7,300,653 B2
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`1.
`METHOD OF TREATING CORNEAL
`TRANSPLANT REUECTION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims the benefit under 35 USC S 119(e)
`of U.S. Provisionals 60/473.734 filed 28 May 2003 and
`60/492.865 filed 6 Aug. 2003, which applications are herein
`specifically incorporated by reference in their entirety.
`
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`
`BACKGROUND
`
`1. Field of the Invention
`The field of the invention is related to methods of using
`VEGF antagonists to reduce, prevent, or treat corneal trans
`plant rejection, thus improving long-term transplant Sur
`vival.
`2. Description of Related Art
`It has previously been reported that topical application of
`an anti-VEGF neutralizing antibody Suppresses acute
`allograft rejection in a rat corneal transplant model (Yatoh et
`al. (1998) Transplantation 66(11):1519-24).
`
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`
`BRIEF SUMMARY OF THE INVENTION
`
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`Subject in need of the agent is topical administration to the
`eye or Subconjunctival administration. Administration may
`occur prior to or following corneal transplantation, prefer
`ably following Surgery. Administration may also include a
`Second agent, such as an immunosuppressive agent.
`The subject to be treated is preferably a human subject
`who has or will receive a corneal transplant.
`In a second aspect, the invention features a method of
`preventing corneal transplant rejection in a Subject in need
`thereof, comprising administering to the Subject an agent
`capable of blocking, inhibiting, or ameliorating vascular
`endothelial growth factor (VEGF)-mediated activity, such
`that corneal transplant rejection is prevented.
`In a third aspect, the invention features a method of
`reducing the incidence of corneal transplant rejection in a
`Subject in need thereof, comprising administering to the
`Subject an agent capable of blocking, inhibiting, or amelio
`rating vascular endothelial growth factor (VEGF)-mediated
`activity, Such that the incidence of corneal transplant rejec
`tion is reduced.
`In a fourth related aspect, the invention features a method
`of treating corneal transplant rejection in a subject in need
`thereof, comprising administering to the Subject an agent
`capable of blocking, inhibiting, or ameliorating vascular
`endothelial growth factor (VEGF)-mediated activity, such
`that corneal transplant rejection is treated.
`In a fifth aspect, the invention features a pharmaceutical
`composition comprising a VEGF antagonist, for example
`the VEGF trap VEGFR1R2-FcAC1(a), in a pharmaceuti
`cally acceptable carrier. Such pharmaceutical compositions
`may be liquid, gel, ointment, salve, slow release formula
`tions or other formulations suitable for ophthalmic admin
`istration.
`Other objects and advantages will become apparent from
`a review of the ensuing detailed description.
`
`DETAILED DESCRIPTION
`
`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 by 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 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 in their entirety.
`General Description
`Experiments were undertaken to evaluate occurrence and
`time course of hem- and lymphangiogenesis after normal
`risk corneal transplantation and to test whether pharmaco
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`The invention is based in part on the finding that admin
`istration of an agent capable of blocking or inhibiting
`vascular endothelial growth factor (VEGF) prevents corneas
`transplant rejection. The experiments, described below, con
`ducted in an animal model of corneal transplantation show
`that long-term transplant survival is promoted by blocking
`VEGF-mediated activity.
`In a first aspect, the invention features a method of
`improving transplant Survival in a subject in need thereof,
`comprising administering to the Subject an agent capable of
`blocking, inhibiting, or ameliorating vascular endothelial
`growth factor (VEGF)-mediated activity, such that trans
`plant Survival is improved.
`In specific embodiments, the agent capable of blocking,
`inhibiting, or ameliorating VEGF-mediated activity is a
`VEGF antagonist. The VEGFantagonist may be a polypep
`tide, an antibody, a small molecule, or a nucleic acid. More
`specifically, the VEGF antagonist includes a VEGF trap
`selected from the group consisting of acetylated Flt-1 (1-3)-
`Fc, Flt-1 (1-3-)-Fc, Flt-1 (1-3A)-Fc, Flt-1 (2-3A)-Fc, Flt
`1(2-3)-Fc, Flt-1D2-VEGFR3D3-FcAC1(a), Flt-1D2-Flk
`1D3-FcAC1(a), and VEGFR1R2-FcAC1(a). In a specific
`and preferred embodiment, the VEGF trap is VEGFR1R2
`FcAC1(a) (also termed VEGF trap) having the nucle
`50
`otide sequence set forth in SEQID NO: 1 and the amino acid
`sequence set forth in SEQID NO: 2. The invention encom
`passes the use of a VEGF trap that is at least 90%. 95%,
`98%, or at least 99% homologous with the nucleotide
`sequence set forth in SEQ ID NO: 1 and/or the amino acid
`sequence set forth in SEQ ID NO:2.
`In other embodiments, the agent capable of blocking,
`inhibiting, or ameliorating vascular endothelial growth fac
`tor (VEGF)-mediated activity is a nucleic acid-based
`antagonist capable of interfering with the expression of
`VEGF. A specific example of this embodiment is one in
`which the nucleic acid-based antagonist is an aptamer, an
`siRNA, or an antisense molecule.
`Administration of the agent may be by any method known
`in the art, including Subcutaneous, intramuscular, intrader
`mal, intraperitoneal, intravenous, intranasal, oral, or topical
`routes of administration. Preferable, administration to the
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`logic strategies inhibiting both processes improve long-term
`graft Survival. As described in the experimental section
`below, normal-risk allogeneic (C57BL/6 to BALB/c) and
`syngeneic (BALB/c to BALB/c) corneal transplantations
`were performed and occurrence and time course of hem- and
`lymphangiogenesis after keratoplasty was observed using
`double immunofluorescence of corneal flatmounts (with
`CD31 as panendothelial and LYVE-1 as lymphatic vascular
`endothelial specific marker). A molecular trap designed to
`eliminate VEGF-A (“VEGF Trap; 12.5 mg/kg) was
`tested for its ability to inhibit both processes after kerato
`plasty and to promote long-term graft Survival (intraperito
`neal injections on the day of Surgery and 3, 7, and 14 days
`later). The results show that no blood or lymph vessels were
`detectable immediately after normal-risk transplantation in
`either donor or host cornea, but hem- and lymphangiogen
`esis were clearly visible at day 3 after transplantation. Both
`vessel types reached donor tissue at one week after allo- and
`similarly after Syngeneic grafting. Early postoperative trap
`ping of VEGF-A significantly reduced both hem- and lym
`phangiogenesis and significantly improved long-term graft
`survival (78% versus 40%, p<0.05). There is concurrent,
`VEGF-A-dependent hem- and lymphangiogenesis after nor
`mal-risk keratoplasty within the preoperatively avascular
`recipient bed. Inhibition of hem- and lymphangiogenesis
`(which mediate the efferent and afferent arms of an immune
`response) after normal-risk corneal transplantation improves
`long-term graft Survival, establishing that early postopera
`tive hem- and lymphangiogenesis are risk factors for graft
`rejection even in low-risk eyes.
`Definitions
`By the term “therapeutically effective dose' is meant a
`dose that produces the desired effect for which it is admin
`istered. The exact dose will depend on the purpose of the
`treatment, and will be ascertainable by one skilled in the art
`using known techniques (see, for example, Lloyd (1999)
`The Art, Science and Technology of Pharmaceutical Com
`pounding).
`40
`By the term “blocker”, “inhibitor', or “antagonist' is
`meant a Substance that retards or prevents a chemical or
`physiological reaction or response. Common blockers or
`inhibitors include but are not limited to antisense molecules,
`antibodies, antagonists and their derivatives. More specifi
`45
`cally, an example of a VEGF blocker or inhibitor is a VEGF
`receptor-based antagonist including, for example, an anti
`VEGF antibody, or a VEGF trap such as VEGFR1R2
`FcAC1(a) (SEQID NOS:1-2). For a complete description of
`VEGF-receptor based antagonists including VEGFR1R2
`50
`FcAC1(a), see PCT publication WO/00/75319, the contents
`of which is incorporated in its entirety herein by reference.
`A “small molecule' is defined herein to have a molecular
`weight below about 500 Daltons, and may include chemical
`as well as peptide molecules.
`VEGF Antagonists
`In one aspect of the invention, VEGF-mediated activity is
`blocked or inhibited by the use of VEGF receptor-based
`blockers of VEGF-mediated activity. A non-limiting
`example of a VEGF receptor-based blocker includes, but is
`not limited to, VEGFR1R2-FcAC1(a). Other suitable recep
`tor-based blockers include acetylated Flt-1(1-3)-Fc, Flt-1 (1-
`3-)-Fc, Flt-1 (1-3A)-Fc, Flt-1 (2-3A)-Fc, Flt-1 (2-3)-Fc.
`Flt-1D2-VEGFR3D3-FcAC1(a), Flt-1D2-Flk-1D3-FcAC1
`65
`(a). For a complete description of these and other VEGF
`receptor-based blockers, including pegylated receptor-based
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`blockers, see PCT Publication No. WO/00/75319, the con
`tents of which is incorporated in its entirety herein by
`reference.
`In addition to the VEGF receptor-based blockers
`described in PCT Publication No. WO/00/75319, variants
`and derivatives of such VEGF receptor-based blockers are
`also contemplated by the invention. The sequence of the
`variants or derivatives may differ by a change which is one
`or more additions, insertions, deletions and/or Substitutions
`of one or more nucleotides of the sequence set forth in SEQ
`ID NO:1. Changes to a nucleotide sequence may result in an
`amino acid change at the protein level, or not, as determined
`by the genetic code. Thus, nucleic acid according to the
`present invention may include a sequence different from the
`sequence shown in SEQID NO:1, yet encode a polypeptide
`with the same amino acid sequence as SEQID NO:2. On the
`other hand, the encoded polypeptide may comprise an amino
`acid sequence which differs by one or more amino acid
`residues from the amino acid sequence shown in SEQ ID
`NO:2. Nucleic acid encoding a polypeptide which is an
`amino acid sequence variant or derivative of the sequence
`shown in SEQ ID NO:2 is further provided by the present
`invention. Nucleic acid encoding Such a polypeptide may
`show at the nucleotide sequence and/or encoded amino acid
`level greater than about 90%. 95%, 98%, or 99% homology
`with the coding sequence shown in SEQID NO:1 and/or the
`amino acid sequence shown in SEQ ID NO:2. For amino
`acid “homology', this may be understood to be similarity
`(according to the established principles of amino acid simi
`larity, e.g. as determined using the algorithm GAP (Genetics
`Computer Group, Madison, Wis.)) or identity. GAP uses the
`Needleman and Wunsch algorithm to align two complete
`sequences that maximizes the number of matches and mini
`mizes the number of gaps. Generally, the default parameters
`are used, with a gap creation penalty=12 and gap extension
`penalty-4.
`Individual components of the VEGF-specific fusion pro
`teins of the invention may be constructed by molecular
`biological methods known to the art with the instructions
`provided by the instant specification. These components are
`selected from a first cellular receptor protein, such as, for
`example, VEGFR1; a second cellular receptor protein, such
`as, for example, VEGFR2; a multimerizing component. Such
`as an Fc.
`Specific embodiments of the VEGF-specific fusion pro
`teins useful in the methods of the invention comprise a
`multimerizing component which allows the fusion proteins
`to associate, e.g., as multimers, preferably dimers. Prefer
`ably, the multimerizing component comprises an immuno
`globulin derived domain. Suitable multimerizing compo
`nents are sequences encoding an immunoglobulin heavy
`chain hinge region (Takahashi et al. 1982 Cell 29:671-679);
`immunoglobulin gene sequences, and portions thereof.
`The nucleic acid constructs encoding the fusion proteins
`useful in the methods of the invention are inserted into an
`expression vector by methods known to the art, wherein the
`nucleic acid molecule is operatively linked to an expression
`control sequence. Host-vector systems for the production of
`proteins comprising an expression vector introduced into a
`host cell suitable for expression of the protein are known in
`the art. The suitable host cell may be a bacterial cell such as
`E. coli, a yeast cell. Such as Pichia pastoris, an insect cell,
`Such as Spodoptera frugiperda, or a mammalian cell. Such as
`a COS, CHO, 293, BHK or NS0 cell.
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`Antisense Nucleic Acids
`In one aspect of the invention, VEGF-mediated activity is
`blocked or inhibited by the use of VEGF antisense nucleic
`acids. The present invention provides the therapeutic or
`prophylactic use of nucleic acids comprising at least six
`nucleotides that are antisense to a gene or cDNA encoding
`VEGF or a portion thereof. As used herein, a VEGF “anti
`sense' nucleic acid refers to a nucleic acid capable of
`hybridizing by virtue of some sequence complementarity to
`a portion of an RNA (preferably mRNA) encoding VEGF.
`The antisense nucleic acid may be complementary to a
`coding and/or noncoding region of an mRNA encoding
`VEGF. Such antisense nucleic acids have utility as com
`pounds that prevent VEGF expression, and can be used in
`the treatment or prevention of corneal transplant rejection.
`The antisense nucleic acids of the invention are double
`stranded or single-stranded oligonucleotides, RNA or DNA
`or a modification or derivative thereof, and can be directly
`administered to a cell or produced intracellularly by tran
`Scription of exogenous, introduced sequences.
`The VEGF antisense nucleic acids are of at least six
`nucleotides and are preferably oligonucleotides ranging
`from 6 to about 50 oligonucleotides. In specific aspects, the
`oligonucleotide is at least 10 nucleotides, at least 15 nucle
`otides, at least 100 nucleotides, or at least 200 nucleotides.
`The oligonucleotides can be DNA or RNA or chimeric
`mixtures or derivatives or modified versions thereof and can
`be single-stranded or double-stranded. In addition, the anti
`sense molecules may be polymers that are nucleic acid
`mimics, such as PNA, morpholino oligos, and LNA. Other
`types of antisence molecules include short double-stranded
`RNAs, known as siRNAs, and short hairpin RNAs, and long
`dsRNA (>50 bp but usually 2500 bp).
`Short Interfering RNAs
`In another embodiment, VEGF-mediated activity is
`blocked by blocking VEGF expression. One method for
`inhibiting VEGF expression is the use of short interfering
`RNA (siRNA) through RNA interference (RNAi) or post
`transcriptional gene silencing (PTGS) (see, for example,
`40
`Ketting et al. (2001) Genes Develop. 15:2654-2659). siRNA
`molecules can target homologous mRNA molecules for
`destruction by cleaving the mRNA molecule within the
`region spanned by the siRNA molecule. Accordingly, siR
`NAS capable of targeting and cleaving homologous VEGF
`45
`mRNA are useful for treating, reducing or preventing cor
`neal transplant rejection.
`Inhibitory Ribozymes
`In aspect of the invention, corneal transplant rejection
`may be treated or prevented in a Subject Suffering from Such
`disease by decreasing the level of VEGF activity by using
`ribozyme molecules designed to catalytically cleave gene
`mRNA transcripts encoding VEGF, preventing translation of
`target gene mRNA and, therefore, expression of the gene
`product.
`55
`Ribozymes are enzymatic RNA molecules capable of
`catalyzing the specific cleavage of RNA. The mechanism of
`ribozyme action involves sequence-specific hybridization of
`the ribozyme molecule to complementary target RNA, fol
`lowed by an endonucleolytic cleavage event. The composi
`tion of ribozyme molecules must include one or more
`sequences complementary to the target gene mRNA, and
`must include the well known catalytic sequence responsible
`for mRNA cleavage. For this sequence, see, e.g., U.S. Pat.
`No. 5,093,246. While ribozymes that cleave mRNA at
`site-specific recognition sequences can be used to destroy
`mRNAs encoding VEGF, the use of hammerhead ribozymes
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`is preferred. Hammerhead ribozymes cleave mRNAs at
`locations dictated by flanking regions that form complemen
`tary base pairs with the target mRNA. The sole requirement
`is that the target mRNA has the following sequence of two
`bases: 5'-UG-3'. The construction and production of ham
`merhead ribozymes is well known in the art. The ribozymes
`of the present invention also include RNA endoribonu
`cleases (hereinafter “Cech-type ribozymes') such as the one
`that occurs naturally in Tetrahymena thermophila (known as
`the IVS, or L-19 IVS RNA). The Cech-type ribozymes have
`an eight base pair active site that hybridizes to a target RNA
`sequence where after cleavage of the target RNA takes
`place. The invention encompasses those Cech-type
`ribozymes that target eight base-pair active site sequences
`that are present in the gene encoding VEGF.
`Generation of Antibodies to VEGF Proteins
`In another aspect of the invention, the invention may be
`practiced with an anti-VEGF antibody or antibody fragment
`capable of binding and blocking VEGF activity. Anti-VEGF
`antibodies are disclosed, for example, in U.S. Pat. No.
`6,121,230, herein specifically incorporated by reference.
`The term “antibody” as used herein refers to a polypeptide
`comprising a framework region from an immunoglobulin
`gene or fragments thereof that specifically binds and recog
`nizes an antigen. The recognized immunoglobulin genes
`include the kappa, lambda, alpha, gamma, delta, epsilon, and
`mu constant regions, as well as the myriad immunoglobulin
`variable region genes. Light chains are classified as either
`kappa or lambda. Heavy chains are classified as gamma, mu,
`alpha, delta, or epsilon, which in turn define the immuno
`globulin classes, IgG, IgM, IgA, Ig), and IgE, respectively.
`Within each IgG class, there are different isotypes (eg. IgG,
`IgG, etc.). Typically, the antigen-binding region of an
`antibody will be the most critical in determining specificity
`and affinity of binding.
`Antibodies exist as intact immunoglobulins, or as a num
`ber of well-characterized fragments produced by digestion
`with various peptidases. For example, pepsin digests an
`antibody below the disulfide linkages in the hinge region to
`produce F(ab)', a dimer of Fab which itself is a light chain
`joined to V-C1 by a disulfide bond. The F(ab)' may be
`reduced under mild conditions to break the disulfide linkage
`in the hinge region, thereby converting the F(ab)' dimer into
`an Fab' monomer. The Fab' monomer is essentially Fab with
`part of the hinge region. While various antibody fragments
`are defined in terms of the digestion of an intact antibody,
`one of skill will appreciate that such fragments may be
`synthesized de novo either chemically or by using recom
`binant DNA methodology. Thus, the terms antibody, as used
`herein, also includes antibody fragments either produced by
`the modification of whole antibodies, or those synthesized
`de novo using recombinant DNA methodologies (e.g., single
`chain Fv)(scFv) or those identified using phase display
`libraries (see, for example, McCafferty et al. (1990) Nature
`348:552-554).
`Methods for preparing antibodies are known to the art.
`See, for example, Kohler & Milstein (1975) Nature 256:
`495-497; Harlow & Lane (1988) Antibodies: a Laboratory
`Manual, Cold Spring Harbor Lab., Cold Spring Harbor,
`N.Y.). The genes encoding the heavy and light chains of an
`antibody of interest can be cloned from a cell, e.g., the genes
`encoding a monoclonal antibody can be cloned from a
`hybridoma and used to produce a recombinant monoclonal
`antibody. Gene libraries encoding heavy and light chains of
`monoclonal antibodies can also be made from hybridoma or
`plasma cells. Random combinations of the heavy and light
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`chain gene products generate a large pool of antibodies with
`different antigenic specificity. Techniques for the production
`of single chain antibodies or recombinant antibodies (U.S.
`Pat. No. 4,946,778; U.S. Pat. No. 4,816,567) can be adapted
`to produce antibodies used in the fusion proteins and meth
`ods of the instant invention. Also, transgenic mice, or other
`organisms such as other mammals, may be used to express
`human or humanized antibodies. Alternatively, phage dis
`play technology can be used to identify antibodies and
`heteromeric Fab fragments that specifically bind to selected
`antigens.
`Antibody Screening and Selection
`Screening and selection of preferred antibodies can be
`conducted by a variety of methods known to the art. Initial
`screening for the presence of monoclonal antibodies specific
`to a target antigen may be conducted through the use of
`ELISA-based methods, for example. A secondary screen is
`preferably conducted to identify and select a desired mono
`clonal antibody for use in construction of the multi-specific
`fusion proteins of the invention. Secondary screening may
`be conducted with any suitable method known to the art.
`One preferred method, termed “Biosensor Modification
`Assisted Profiling” (“BiaMAP) is described in co-pending
`U.S. Ser. No. 60/423,017 filed 1 Nov. 2002, herein specifi
`25
`cally incorporated by reference in its entirety. BiaMAP
`allows rapid identification of hybridoma clones producing
`monoclonal antibodies with desired characteristics. More
`specifically, monoclonal antibodies are sorted into distinct
`epitope-related groups based on evaluation of antibody:
`antigen interactions.
`Treatment Population
`A suitable subject for treatment by the method of the
`invention is a human who has received or will receive a
`corneal transplant. Corneal transplantation is the oldest,
`most successful and most commonly performed tissue trans
`plantation, with nearly 40,000 transplantations a year alone
`in the US. When corneal grafts are placed into an avascular
`recipient bed (so-called normal-risk keratoplasty), 2-year
`graft survival rates approach 90% under cover of topical
`steroids, even without HLA-matching. This very successful
`outcome is attributed to corneal immune privilege, i.e. the
`phenomenon of Suppressed corneal inflammation induced
`by an array of endogenous mechanisms downregulating
`alloimmune and inflammatory responses in the cornea and
`its bed. These mechanisms include the lack of both afferent
`lymphatic and efferent blood vessels in the normal-risk
`recipient cornea, lack of MHC II antigen presenting cells
`(APCs), FASL-expression on corneal epithelium and endot
`helium, and the anterior chamber associated immune privi
`lege (ACAID) directed at graft antigens etc. (Streilein et al.
`(1999) Transplant Proc. 31:1472-1475).
`In contrast, Survival rates of cornea grafts placed into
`vascularized, not immune-privileged recipient beds (so
`called high-risk keratoplasty) drop significantly to below
`50% (even with local and systemic immune Suppression).
`Pre-existing corneal stromal blood vessels have been iden
`tified as strong risk factors for immune rejection after
`corneal transplantation, both in the clinical setting as well as
`in the well-defined mouse model of corneal transplantation
`(Sano et al. (1995) Invest. Ophthalmol. Vis. Sci. 36:2176
`85). Recently, in addition to blood vessels, biomicroscopi
`cally undetectable lymphatic vessels have been found in
`association with blood vessels in vascularized high-risk
`human corneas (Cursiefen et al. (2003) Cornea. 22:273-81)
`and it is likely that corneal lymphatic vessels enable effec
`tive access of donor and host APCs and antigenic material to
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`regional lymph nodes where accelerated sensitisation to
`graft antigens occurs (Liu et al. (2002) J. Exp. Med. 195:
`259-68) even in the normal-risk setting (with a preopera
`tively avascular recipient bed), where mild corneal heman
`giogenesis develops after keratoplasty. Outgrowth of new
`blood vessels from the limbal arcade towards the graft can
`be observed within the first postoperative year in about 50%
`of patients undergoing normal-risk keratoplasty, and in 10%
`of patients these new blood vessels even reach the interface
`or invade donor tissue (Cursiefen et al. (2001) Graefes Arch.
`clin. Exp. Ophthalmol. 39:514-21) at corneal suture sites,
`and then proceed centrally.
`Methods of Administration
`The invention provides methods of treatment comprising
`administering to a subject an effective amount of an agent of
`the invention. In a preferred aspect, the agent is substantially
`purified (e.g., Substantially free from Substances that limit its
`effect or produce undesired side-effects). The subject is
`preferably an animal, e.g., Such as cows, pigs, horses,
`chickens, cats, dogs, etc., and is preferably a mammal, and
`most preferably human.
`Various delivery systems are known and can be used to
`administer an active agent of the invention, e.g., delivery
`systems Suitable for topical administration, preferably topi
`cal administration directly to the eye, or Subconjunctival
`administration, as well as other delivery systems such as
`those that utilize encapsulation in liposomes, microparticles,
`microcapsules, recombinant cells capable of expressing the
`compound, receptor-mediated endocytosis (see, e.g., Wu
`and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
`of a nucleic acid as part of a retroviral or other vector, etc.
`Methods of introduction are preferably topical or subcon
`junctival, but may be enteral or parenteral including but are
`not limited to intradermal, intramuscular, intraperitoneal,
`intravenous, Subcutaneous, intranasal, and oral routes. The
`active agents may be administered by any convenient route,
`for example by absorption through epithelial (e.g. topical
`administration to the eye) or mucocutaneous linings (e.g.,
`oral mucosa, intestinal mucosa, etc.) or infusion or bolus
`injection, and may be administered together with other
`biologically active agents. Administration can be systemic
`or local. Administration can be acute or chronic (e.g. daily,
`weekly, monthly, etc.) or in combination or alteration with
`other agents. Pulmonary administration can also be
`employed, e.g., by use of an inhaler or nebulizer, and
`formulation with an aerosolizing agent.
`In another embodiment, the active agent can be delivered
`in a vesicle, in particular a liposome (see Langer (1990)
`Science 249:1527-1533). In yet another embodiment, the
`active agent can be delivered in a controlled release system.
`In one embodiment, a pump may be used (see Langer (1990)
`Supra). In another embodiment, polymeric materials can be
`used (see Howard et al. (1989) J. Neurosurg. 71:105). In
`another embodiment where the active agent of the invention
`is a nucleic acid encoding a protein, the nucleic acid can be
`administered in vivo to promote expression of its encoded
`protein, by constructing it as part of an appropriate nucleic
`acid expression vector and administering it so that it
`becomes intracellular, e.g., by use of a retroviral vector (see,
`for example, U.S. Pat. No. 4,980.286), or by direct injection,
`or by use of microparticle bombardment (e.g., a gene gun;
`Biolistic, Dupont), or coating with lipids or cell-surface
`receptors or transfecting agents, or by administering it in
`linkage to a homeobox-like peptide which is known to enter
`the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.
`Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid
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`Mylan Exhibit 1145
`Mylan v. Regeneron, IPR2021-00881
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`can be introduced intracellularly and incorporated within
`host cell DNA for expression, by homologous recombina
`tion.
`In a specific embodiment, it may be desirable to admin
`ister the pharmaceutical compositions of the invention
`locally to the area in need of treatment; this may be
`achieved, for example, and not by way of limitation, by
`topical administration, Subconjunctival administration, local
`infusion during Surgery, e.g., by injection, by means of a
`catheter, or by means of an implant, said implant being of a
`10
`porous, non-porous, or gelatinous material, includin