(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
` (10) International Publication Number
`
`WO 2006/047325 Al
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`4 May 2006 (04.05.2006)
`
`(51) International Patent Classification:
`A6IK 39/00 (2006.01)
`
`(21) International Application Number:
`PCT/US2005/038006
`
`(22) International Filing Date: 21 October 2005 (21.10.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/621,209
`
`21 October 2004 (21.10.2004)
`
`US
`
`(71) Applicant (for all designated States except US): GENEN-
`TECH, INC. [US/US]; 1 Dna Way, South San Francisco,
`CA 94080-4990 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): SHAMS, Naveed
`[US/US]; 216 Dove Creek Lane, Danville, CA 94506
`(US).
`
`(74) Agent: CUI, Steven X.; GENENTECIL, INC., | Dna Way,
`South San Francisco, CA 94080-4990, (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`
`AT, AU, AZ, BA, BB, BG,BR, BW,BY, BZ, CA, CH, CN,
`co, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, BS, FT,
`GB, GD, GE, GH, GM, HR, HU,ID,IL, IN,IS, JP, KE,
`KG, KM,KP, KR, KZ, LC, LK, LR, LS, LT, LU, LY, LY,
`MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG,NI, NO,
`NZ, OM,PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK,
`SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(84~ Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW,GII,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,ES,FI,
`FR, GB, GR, HU,IE,IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SL SK, TR), OAPI (BE, BJ, CE, CG, Cl, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`Fortwo-letter codes and otherabbreviations, referto the "Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`2006/047325A1IIIMINIMITI
`
`wo
`
`(54) Title: METHOD TOR TREATING INTRAOCULAR NEOVASCULAR DISEASES
`
`Investigator
`determines
`eligibility
`
`Subfoveal CNV +/- Classic Component
`
`Randomized 1:1:1
`
`Trial Design
`
`
` Sham
`
`Injection
`
`0.3 mg
`ranibizumab
`
`0.5mg
`ranibizumab
`
`
`
`
`
`(57) Abstract: A method is provided for administering to a mammal suffering from, orat risk for, an intraocular neovascular
`disorder with regular dosing of a therapeutically effective amount of VEGFantagonist, followed by less frequent dosing of a thera-
`peutically effective amount of VEGF antagonist.
`
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`WO 2006/047325
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`METHOD FOR TREATING INTRAOCULAR NEOVASCULAR
`
`DISEASES
`
`FIELD OF THE INVENTION
`
`This invention relates to methods fortreating an intraocular neovascular disorder with a VEGF
`
`antagonist. Methods for administering to a mammalsuffering from, or at risk for, an
`
`intraocular neovascular disorder include monthly dosing of a therapeutically effective amount
`
`of VEGFantagonist, followed by less frequent dosing of a therapeutically effective amount of
`
`VEGFantagonist.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`Angiogenesis is implicated in the pathogenesis of intraocular neovascular diseases,e.g.,
`
`proliferative retinopathies, age-related macular degeneration (AMD), etc., as well as a variety
`
`of other disorders. These include solid tumors, rheumatoid arthritis, and psoriasis (Folkman et
`
`15
`
`al. J. Biol. Chem. 267:10931-10934 (1992); Klagsbrun et al. Annu. Rev. Physiol. 53:217-239
`
`(1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic
`
`approach. Garner A, Klintworth GK, Eds. 2nd Edition Marcel Dekker, NY, pp 1625-1710
`
`(1994)).
`
`20
`
`The search for positive regulators of angiogenesis has yielded many candidates, including
`
`aFGF, bFGF, TGF-a, TGF-B HGF, TNF-a, angiogenin, IL-8, etc. (Folkmanefal. and
`
`Klagsbrun et al). The negative regulators so far identified include thrombospondin (Good et
`
`al. Proc. Natl.
`
`25
`
`Acad. Sci. USA. 87:6624-6628 (1990)), the 16-kilodalton N-terminal fragmentof prolactin
`(Clapp et al. Endocrinology, 133:1292-1299 (1993), angiostatin (O’Reilly et al. Cell, 79:315-
`328 (1994)) and endostatin (O’Reilly et al. Cell, 88:277-285 (1996)).
`
`Workdone overthe last several years has established the key role of vascular endothelial
`
`growth factor (VEGF)in the regulation of normal and abnormal angiogenesis (Ferrara et al.
`
`30
`
`Endocr. Rev. 18:4-25 (1997)). The finding that the loss of even a single VEGFallele results in
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`embryoniclethality points to an irreplaceable role played by this factor in the development and
`
`differentiation of the vascular system (Ferrara etal.).
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`Human VEGFexists as at least six isoforms (VEGF}21, VEGF\4s, VEGFi6s, VEGF33,
`
`VEGFigo, and VEGF2g) that arise from alternative splicing of mRNAofa single gene (Ferrara
`
`N, Davis Smyth T. Endocr Rev 18:1—22 (1997)). VEGF\¢s, the most abundant isoform,is a
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`basic, heparin binding, dimeric glycoprotein with a molecular mass of ~45,000 daltons(/d).
`
`Two VEGFreceptor tyrosine kinases, VEGFRland VEGFR2,have been identified (Shibuya
`et al. Oncogene 5:519-24 (1990); Matthewset al., Proc Natl Acad Sci U S A 88:9026-30
`(1991); Termanetal., Oncogene 6:1677-83 (1991); Termanet al. Biochem Biophys Res
`Commun 187:1579-86 (1992); de Vrieset al., Science 255:989-91 (1992); Millauer etal. Cell
`
`72:835—-46 (1993); and, Quinn et al. Proc Natl Acad Sci USA 90:7533-7 (1993)). VEGFR1
`
`10
`
`has the highest affinity for VEGF, with a Kd of ~10—20 pM (de Vrieset al., Science 255:989-
`91 (1992)), and VEGFR2 has a somewhat loweraffinity for VEGF, with a Kd of ~75-125 pM
`(Termanet al., Oncogene 6:1677-83 (1991); Millauer et al. Celi 72:835-46 (1993); and,
`
`Quinnet al. Prac Natl Acad Sci USA 90:7533-7 (1993)).
`
`15
`
`VEGFhasseveral biologic functions, including regulation of VEGF gene expression under
`
`hypoxic conditions (Ferrara N, Davis Smyth T. Endocr Rev 18:1—22 (1997)), mitogenic
`
`activity for micro and macrovascular endothelial cells (Ferrara N, Henzel WJ. Biochem
`
`Biophys Res Commun 161:851-8 (1989); Leunget al., Science 246:1306-9 (1989); Connolly
`
`et al. J Clin Invest 84:1470-8 (1989a); Kecket al. Science 246:1309—12 (1989); Plouet etal.,
`
`20
`
`EMBO J 8:3801-6 (1989); Connet al. Prac Natl Acad Sci USA 87:2628-32 (1990); and,
`
`Pepperet al., Exp Cell Res 210:298-305 (1994)), and induction of expression of plasminogen
`
`activators and collagenase (Pepperet al., Biochem Biophys Res Commun 181:902-6 (1991)).
`
`Furthermore, VEGFhas been shownto be a key mediator of neovascularization associated
`
`25
`
`with tumors and intraocular disorders (Ferrara et al.). The VEGF mRNAis overexpressed by
`
`the majority of human tumors examined. Berkmanetal. J Clin Invest 91:153-159 (1993);
`
`Brown et al. Human Pathol 26:86-91 (1995); Brown et al. Cancer Res 53:4727-4735 (1993);
`
`Mattern et al. Brit J Cancer. 73:931-934 (1996); and Dvorak et al. Am J Pathol 146:1029-
`
`1039 (1995). Also, the concentration of VEGFin eye fluids are highly correlated to the
`
`30
`
`presence of active proliferation of blood vessels in patients with diabetic and other ischemia-
`
`related retinopathies. Aiello et al., N. Engl. J. Med. 331:1480-1487 (1994). Furthermore,
`
`recent studies have demonstrated the localization of VEGF in choroidal neovascular
`
`membranesin patients affected by AMD. Lopezetal., Invest. Ophtalmo. Vis. Sci. 37:855-868
`
`(1996); Kvantaet al., Invest Ophthalmol Vis Sci 37:1929-34 (1996).
`2
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`Age related macular degeneration (AMD)is a leading cause of severe, irreversible vision loss
`
`amongthe elderly. Bressler, JAMA 291:1900-1 (2004), It is characterized by a broad
`
`spectrum of clinical and pathologic findings, such as pale yellow spots knownas drusen,
`
`disruption of the retinal pigment epithelium (RPE), choroidal neovascularization (CNV), and
`
`disciform macular degeneration. The manifestations of the disease are classified into two
`forms: non exudative (dry) and exudative (wet or neovascular). Drusen are the characteristic
`lesions of the dry form, and neovascularization characterizes the wet form. Disciform AMD is
`
`the fibrotic stage of the neovascular lesion.
`
`10
`
`There is a dramatic increase in the prevalence of AMD with advancing age. See, e.g.,
`
`Leibowitz et al., Surv Ophthalmol 24(Suppl):335-610 (1980) and Klein et al., Ophthalmology
`
`99:933-43 (1992). Although the wet form of AMD is muchless common,it is responsible for
`
`80%-90% of the severe visual loss associated with AMD (Ferris et al., Arch Ophthamol
`
`15
`
`102:1640-2 (1984)). There is an estimated 1-1.2 million prevalent cases of wet AMD. The
`
`cause of AMDis unknown; however,it is clear that the risk of developing AMDincreases
`
`with advancing age. Other knownrisk factors include family history and cigarette smoking.
`
`Postulated risk factors also include oxidative stress, diabetes, alcohol intake, and sunlight
`
`exposure. D’ Amico, N Engl J Med 331:95-106 (1994) and Christen et al., JAMA 276:1147-51
`
`20
`
`(1996).
`
`Dry AMDis characterized by changes in the RPE and Bruch’s membrane.
`
`It is thought that
`
`the RPE, compromised by age andotherrisk factors, deposits lipofuscin and cellular debris on
`
`Bruch’s membrane. These changes may be seen ophthalmoscopically as drusen, which are
`
`25
`
`scattered throughout the macula and posteriorretinal pole. There are also variable degrees of
`
`atrophy and pigmentation of the RPE. Dry AMD maybe asymptomatic or accompanied by
`
`variable and usually minimal visual loss and is considered to be a prelude to developmentof
`
`wet AMD.
`
`30
`
`Wet AMDis typically characterized by CNV of the macular region. The choroidal capillaries
`
`proliferate and penetrate Bruch’s membrane to reach the RPE and mayextendinto the
`
`subretinal space. The increased permeability of the newly formed capillaries leads to
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`accumulation of serous fluid or blood under the RPE and/or the neurosensory retina or within
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`the neurosensory retina. When the fovea becomes swollen or detached, decreases in vision
`3
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`occur. Fibrous metaplasia and organization may ensue,resulting in an elevated subretinal
`
`masscalled a disciform scar that constitutes end-stage AMDandis associated with permanent
`
`vision loss (D’ Amico DJ. N Engl J Med 331:95-106 (1994)).
`
`The neovascularization in AMD canbeclassified into different patterns based on fluorescein
`
`angiography of subfoveal chorodial neovascular lesions. TAP and VIP Study Groups, Arch
`
`Ophthalmol 121:1253-68 (2003). The major angiographic patterns are termed classic and
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`occult and are associated with different degrees of aggressiveness, vision losses, and response
`
`to different treatment options.
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`10
`
`The diffusible nature of VEGFandits specificity of action for endothelial cells support a key
`
`role in the process of abnormal blood vessel growth and vascular leakage. Increased
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`expression of VEGFin retinal photoreceptors or RPE of transgenic mice stimulates
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`neovascularization within the retina, and VEGFantagonists partially inhibitretinal
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`15
`
`neovascularization in animal models (Okamotoet al. Am J Pathol 151:281-91 (1997);
`
`Schwesingeret al., AM J Pathol. Mar;158(3):1161-72 (2001)). Anti-VEGF neutralizing
`
`antibodies inhibit intraocular angiogenesis in models of ischemic retinal disorders (Adamis et
`
`al. Arch. Ophthalmol. 114:66-71 (1996)), and also suppress the growth of a variety of human
`
`tumorcell lines in nude mice (Kim et al. Nature 362:841-844 (1993); Warrenet al. J. Clin.
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`20
`
`Invest. 95:1789-1797 (1995); Borgstrém et al. Cancer Res. 56:4032-4039 (1996); and Melnyk
`
`et al. Cancer Res. 56:921-924 (1996)). Therefore, anti-VEGF monoclonal antibodies or other
`
`VEGFantagonists are promising candidates for use in treatments of intraocular neovascular
`
`disorders, and new methods of administering therapeutic compounds, which increases the
`effectiveness of the therapeutic compound,are needed.
`
`25
`
`30
`
`SUMMARYOF THE INVENTION
`
`One object of the present invention is to provide an improved method of administering a
`
`therapeutic compound. This and other objects will become apparent from the following
`
`description.
`
`Methodsfor treating intraocular neovascular disease are provided. For example, methods
`
`include administering to a mammal a numberoffirst individual doses of a VEGFantagonist,
`
`followed by administering to the mammal a numberof second individual doses of the
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`antagonist, wherein the second individual doses are administered less frequently than thefirst
`
`individual doses.
`
`In one embodimentof the invention, a method for treating wet form age-related macular
`
`degeneration is provided, which comprises administering to a mammal a numberoffirst
`
`individual doses of an VEGF antagonist, followed by administering to the mammal a number
`
`of second individual doses of the antagonist, wherein the second individual doses are
`
`administered less frequently than the first individual doses.
`
`10
`
`In one embodiment, the mammalis in need of treatment. Typically, the mammalis a human.
`
`In one embodiment, the administration of the VEGF antagonistis ocular. In one aspect, the
`
`administration is intraocular.
`
`In another aspect, the administration is intravitreal.
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`15
`
`A VEGFantagonist is administered in the methods of the invention. In one aspect, the VEGF
`
`antagonist is an anti-VEGFantibody,e.g., a full length anti-VEGF antibody or an antibody
`
`fragment.
`
`In one embodiment, the anti-VEGF antibody is a Fab antibody fragment.
`
`In one
`
`embodiment, the antibody fragment is Y0317.
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`20
`
`In one embodiment of the invention, the first individual doses are administered at one month
`
`intervals (e.g., about 3 individual doses). Typically, there is more than onefirst individual
`
`dose. In another embodiment, the second individual doses are administered at three month
`
`intervals (e.g., about 6 individual doses). In one aspect of the invention, the second individual
`
`doses are administered beginning three months after the numberoffirst individual doses. In
`
`25
`
`one embodiment, a numberof second individual doses are administered to the mammal during
`
`a period of at least 22 months following the numberoffirst individual doses.
`
`In one embodimentof the invention, the numberoffirst individual doses and the numberof
`
`second individual doses are administered over a time period of about 2 years. In one aspect,
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`30
`
`the first individual dose is administered at month 0, 1 and 2.
`
`In another aspect, the second
`
`individual dose is administered at month 5, 8, 11, 14, 17, 20 and 23. For example,thefirst
`
`individual dose is administered at month 0, 1, and 2 and the second individual doseis
`
`administered at month 5, 8, 11, 14, 17, 20 and 23. In one embodiment, the VEGF antagonistis
`
`administered over less than 2 years, or optionally, administered over greater than 2 years.
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`Other aspects of the invention will become apparent from the following description of the
`
`embodiments whichare not intended to be limiting of the invention.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`Figure 1 schematically illustrates the study in Example 1.
`
`Figure 2 schematically illustrates a dosing regimenfor treating, e.g., age-related macular
`
`degeneration (AMD)with a VEGFantagonist.
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`10
`
`Definitions
`
`DETAILED DESCRIPTION
`
`Before describing the present invention in detail, it is to be understood that this invention is
`
`. not limited to particular compositions or biological systems, which can, of course, vary.It is
`
`1S, 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. As used in this specification
`
`and the appended claims, the singular forms "a", "an" and "the" include plural referents unless
`
`the content clearly dictates otherwise. Thus, for example, reference to "a molecule” optionally
`
`includes a combination of two or more such molecules, and thelike.
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`20
`
`The term “human VEGF"as used herein refers to the 165-amino acid human vascular
`
`endothelial cell growth factor, and related 121-, 189-, and 206-, (and other isoforms) amino
`
`acid vascular endothelial cell growth factors, as described by Leunget al., Science 246:1306
`
`(1989), and Houck et al., Mol. Endocrin. 5:1806 (1991) together with the naturally occurring
`
`25
`
`allelic and processed forms of those growth factors.
`
`A “VEGFantagonist” refers to a molecule capable of neutralizing, blocking, inhibiting,
`
`abrogating, reducing or interfering with VEGFactivities including its binding to one or more
`
`VEGFreceptors. VEGF antagonists include anti-VEGFantibodies and antigen-binding
`
`30
`
`fragments thereof, receptor molecules and derivatives which bind specifically to VEGF
`
`thereby sequestering its binding to one or more receptors, anti-VEGFreceptor antibodies and
`
`VEGFreceptor antagonists such as small molecule inhibitors of the VEGFR tyrosine kinases,
`
`and fusions proteins, e.g., VEGF-Trap (Regeneron), VEGF)2:-gelonin (Peregrine). VEGF
`
`antagonists also include antagonist variants of VEGF, antisense molecules directed to VEGF,
`6
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`RNAaptamersspecific to VEGF, and ribozymes against VEGF or VEGFreceptors.
`
`Antagonists of VEGFact byinterfering with the binding of VEGFto a cellular receptor, by
`
`incapacitating or killing cells which have been activated by VEGF,orby interfering with
`
`vascular endothelial cell activation after VEGF bindingto a cellular receptor. All such points
`
`of intervention by a VEGFantagonist shall be considered equivalent for purposesofthis
`
`invention. Preferred VEGFantagonists are anti-VEGF antagonistic antibodies capable of
`
`inhibiting one or more of the biologicalactivities of VEGF, for example,its mitogenic,
`
`angiogenic or vascular permeability activity. Anti-VEGF antagonistic antibodies include, but
`
`not limited to, antibodies A4.6.1, rhuMab VEGF (bevacizumab), Y0317 (ranibizumab), G6,
`
`10
`
`B20, 2C3, and others as described in, for example, WO98/45331, US2003/0190317, U.S.
`
`Patents 6,582,959 and 6,703,020; WO98/45332; WO 96/30046; WO094/10202;
`
`W02005/044853; EP 0666868B 1; and Popkovet al., Journal ofImmunological Methods
`
`288:149-164 (2004). More preferably, the anti-VEGF antagonistic antibody of the invention
`
`is ranibizumab, which is a humanized,affinity matured anti-human VEGFantibody Fab
`
`15
`
`fragment having the light and heavy chain variable domain sequences of Y0317 as described
`
`in WO98/45331 and Chen et al J Mol Biol 293:865-881 (1999).
`
`The antibody is appropriately from any source, including chicken and mammalian such as
`
`rodent, goat, primate, and human. Typically, the antibody is from the same species as the
`
`20
`
`species to be treated, and more preferably the antibody is human or humanized and the hostis
`
`human. While the antibody can be a polyclonal or monoclonal antibody,typically it is a
`
`monoclonal antibody, which can be prepared by conventional technology. The antibody is an
`
`IgG-1, -2, -3, or -4, IgE, IgA, IgM,IgD, or an intraclass chimera in which Fv or a CDR from
`
`one class is substituted into another class. The antibody may have an Fc domain capable of an
`
`25
`
`effector function or may not be capable of binding complementorparticipating in ADCC.
`
`The term "VEGFreceptor” or "VEGFr”as used herein refers to a cellular receptor for VEGF,
`
`ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants
`thereof whichretain the ability to bind hVEGF. One example of a VEGF receptor is thefims-
`
`30
`
`like tyrosine kinase (f7#), a transmembrane receptor in the tyrosine kinase family. DeVries et
`
`al., Science 255:989 (1992); Shibuya et al., Oncogene 5:519 (1990). The fl receptor
`
`comprises an extracellular domain, a transmembrane domain, and an intracellular domain with
`
`tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF,
`
`whereasthe intracellular domain is involvedin signal transduction. Another example of a
`7
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`VEGFreceptoris the flk-/ receptor (also referred to as KDR). Matthewset al., Proc. Nat.
`
`Acad. Sci. 88:9026 (1991); Terman et al., Oncogene 6:1677 (1991); Termanet al., Biochem.
`
`Biophys. Res. Commun. 187:1579 (1992). Binding of VEGFtothe fit receptor results in the
`
`formation of at least two high molecular weight complexes, having apparent molecular weight
`
`of 205,000 and 300,000 Daltons. The 300,000 Dalton complex is believed to be a dimer
`
`comprising two receptor molecules boundto a single molecule of VEGF.
`
`The term “epitope A4.6.1" when used herein, unless indicated otherwise, refers to the region
`
`of human VEGFto which the A4.6.1 antibody disclosed in Kim et al., Growth Factors 7:53
`
`10
`
`(1992) and Kim et al. Nature 362:841 (1993), binds.
`
`"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures.
`
`Those in need of treatment include those already with the disorder as well as those in which
`
`the disorderis to be prevented.
`
`15
`
`"Mammal"for purposes of treatmentrefers to any animal classified as a mammal, including
`
`humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses,cats,
`
`cows, efc. Typically, the mammal is human.
`
`20
`
`The term "antibody" is used in the broadest sense and includes monoclonal antibodies
`
`(including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent
`
`antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see
`
`below) so long as they exhibit the desired biological activity.
`
`25
`
`Unless indicated otherwise, the expression “multivalent antibody” is used throughoutthis
`
`specification to denote an antibody comprising three or more antigen binding sites. The
`
`multivalent antibody is typically engineered to have the three or more antigen binding sites
`
`and is generally not a native sequence IgM or IgA antibody.
`
`30
`
`"Native antibodies” and "native immunoglobulins" are usually heterotetrameric glycoproteins
`
`of about 150,000 daltons, composed of two identical light (L) chains and twoidentical heavy
`
`(H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while
`
`the numberof disulfide linkages varies among the heavy chainsof different immunoglobulin
`
`isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges.
`8
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`Each heavy chain hasat oneend a variable domain (Vy ) followed by a numberof constant
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`domains. Each light chain has a variable domain at one end (V]_) and a constant domain atits
`
`other end; the constant domain of the light chain is aligned with thefirst constant domain of
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`the heavy chain, and the light- chain variable domain is aligned with the variable domain of
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`the heavy chain. Particular amino acid residues are believed to form an interface between the
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`light- and heavy-chain variable domains.
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`The term "variable" refers to the fact that certain portions of the variable domainsdiffer
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`extensively in sequence among antibodies and are used in the binding and specificity of each
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`particular antibody forits particular antigen. However,the variability is not evenly distributed
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`throughout the variable domainsof antibodies. It is concentrated in three segmentscalled
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`hypervariable regions both in the light chain and the heavy chain variable domains. The more
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`highly conserved portions of variable domainsare called the framework region (FR). The
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`variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3
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`and FR4,respectively), largely adopting a B-sheet configuration, connected by three
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`hypervariable regions, which form loops connecting, and in some cases formingpart of, the B-
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`sheet structure. The hypervariable regions in each chain are held together in close proximity
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`by the FRs and, with the hypervariable regions from the other chain, contribute to the
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`formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of
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`20
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`Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda,
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`MD. (1991), pages 647-669). The constant domainsare not involved directly in binding an
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`antibody to an antigen, but exhibit various effector functions, such as participation of the
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`antibody in antibody-dependentcellular toxicity (ADCC).
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`25
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`The term “hypervariable region” when used herein refers to the amino acid residues of an
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`antibody which are responsible for antigen-binding. The hypervariable region comprises
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`aminoacid residues from a “complementarity determining region” or “CDR”(i.e. residues 24-
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`34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65
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`(H2) and 95-102 (H3) in the heavy chain variable domain; Kabatet al., Sequences of Proteins
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`30
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`ofImmunological Interest, Sth Ed. Public Health Service, National Institutes of Health,
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`Bethesda, MD. (1991)) and/or those residues from a “hypervariable loop”(i.e. residues 26-32
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`(L1), 50-52 (L2) and 91-96 (L3)in the light chain variable domain and 26-32 (H1), 53-55
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`(H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J, Mol. Biol.
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`196:901-917 (1987)). "Framework" or "FR"residues are those variable domain residues other
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`than the hypervariable region residues as herein defined.
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`Papain digestion of antibodies produces two identical antigen-binding fragments,called "Fab"
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`fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name
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`reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragmentthat has
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`two antigen-combiningsites andis still capable of cross-linking antigen.
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`"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -
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`binding site. This region consists of a dimer of one heavy chain and one light chain variable
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`domainin tight, non-covalent association. It is in this configuration that the three
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`hypervariable regions of each variable domain interact to define an antigen-binding site on the
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`surface of the Vy4-V], dimer. Collectively, the six hypervariable regions confer antigen-
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`binding specificity to the antibody. However, even a single variable domain (or half of an Fv
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`15
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`comprising only three hypervariable regions specific for an antigen) has the ability to
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`recognize and bind antigen, although at a loweraffinity than the entire binding site.
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`The Fab fragmentalso contains the constant domain ofthe light chain and the first constant
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`domain (CH1) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition
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`20
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`of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or
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`more cysteine(s) from the antibody hinge region. Fab'-SH is the designation herein for Fab’ in
`
`which the cysteine residue(s) of the constant domains bear a free thio] group. F(ab‘)9 antibody
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`fragments originally were producedas pairs of Fab' fragments which have hinge cysteines
`
`between them. Other chemical couplings of antibody fragments are also known.
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`25
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`The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be
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`assigned to one of two clearly distinct types, called kappa («) and lambda (A), based on the
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`amino acid sequencesof their constant domains.
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`30
`
`Depending on the amino acid sequence of the constant domain of their heavy chains,
`
`immunoglobulins can be assignedto different classes. There are five major classes of
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`immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided
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`into subclasses(isotypes), ¢.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain
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`constant domainsthat correspondto the different classes of immunoglobulinsare called a, 6,
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`€, y, and u, respectively. The subunit structures and three-dimensional configurations of
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`different classes of immunoglobulins are well known.
`
`"Antibody fragments” comprise only a portion of an intact antibody, generally including an
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`antigen bindingsite of the intact antibody and thus retaining the ability to bind antigen.
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`Examples of antibody fragments encompassedbythe present definition include: (i) the Fab
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`fragment, having VL, CL, VH and CH1 domains; (ii) the Fab’ fragment, which is a Fab
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`fragment having one or more cysteine residues at the C-terminus of the CH1 domain;(iii) the
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`Fd fragment having VH and CH1 domains;(iv) the Fd’ fragment having VH and CH1
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`domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv
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`fragment having the VL and VH domainsofa single arm of an antibody; (vi) the dAb
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`fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)
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`isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab’
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`15
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`fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody
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`molecules (e.g. single chain Fv; scFv) (Birdet al., Science 242:423-426 (1988); and Huston et
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`al., PNAS (USA) 85:5879-5883 (1988)); (x) “diabodies” with two antigen bindingsites,
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`comprising a heavy chain variable domain (VH) connected to a light chain variable domain
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`(VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollingeret al.,
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`20
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`Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) “linear antibodies” comprising a pair
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`of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain
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`polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10):1057
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`1062 (1995); and US Patent No. 5,641,870).
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`25
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`The term "monoclonal antibody" as used herein refers to an antibody obtained from a
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`population of substantially homogeneousantibodies, i.e., the individual antibodies comprising
`
`the population are identical except for possible naturally occurring mutations that may be
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`present in minor amounts. Monoclonalantibodies are highly specific, being directed against a
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`single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody
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`30
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`preparations whichtypically include different antibodies directed against different
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`determinants (epitopes), each monoclonal antibody is directed against a single determinant on
`the antigen. The modifier “monoclonal” indicates the character of the antibody as being
`obtained from a substantially homogeneous population of antibodies, and is not to be
`
`construed as requiring production of the antibody by any particular method. For example, the
`i
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`monoclonal antibodies to be used in accordance with the present invention may be made by
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`the hybridoma methodfirst described by Kohler etal., Nature 256:495 (1975), or may be
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`made by recombinant DNA methods(see,e.g., U.S. Patent No. 4,816,567). The "monoclonal
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`antibodies" may also be isolated from phage antibodylibraries using the techniques described
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`in Clacksonet al., Nature 352:624-628 (1991) and Markset al., J. Mol. Biol. 222:581-597
`
`(1991), for example.
`
`The monoclonal antibodies herein specifically include "chimeric" antibodies
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`(immunoglobulins) in which a portion of the heavy and/or light chain is identical with or
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`10
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`homologousto corresponding sequencesin antibodies derived from a particular species or
`belongingto a particular antibodyclass or subclass, while the remainder of the chain(s)is
`identical with or homologousto corresponding sequencesin antibodies derived from another
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`species or belonging to another antibody class or subclass, as well as fragments of such
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`antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567;
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`15
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`and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
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`"Humanized" forms of non-