`US 20070190058Al
`
`c19) United States
`c12) Patent Application Publication
`Shams
`
`c10) Pub. No.: US 2007 /0190058 Al
`Aug. 16, 2007
`(43) Pub. Date:
`
`(54) METHOD FOR TREATING INTRAOCULAR
`NEOVASCULAR DISEASES
`
`(75)
`
`Inventor: Naveed Shams, Danville, CA (US)
`
`Correspondence Address:
`GENENTECH, INC.
`1 DNA WAY
`SOUTH SAN FRANCISCO, CA 94080 (US)
`
`Related U.S. Application Data
`
`(63) Continuation of application No. PCT/US05/38006,
`filed on Oct. 21, 2005.
`
`(60) Provisional application No. 60/621,209, filed on Oct.
`21, 2004.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 39/395
`(2006.01)
`(52) U.S. Cl. .......................................................... 424/145.1
`
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA (US)
`
`(57)
`
`ABSTRACT
`
`(21) Appl. No.:
`
`11/738,284
`
`(22) Filed:
`
`Apr. 20, 2007
`
`A method is provided for administering to a mammal
`suffering from, or at risk for, an intraocular neovascular
`disorder with regular dosing of a therapeutically effective
`amount of VEGF antagonist, followed by less frequent
`dosing of a therapeutically effective amount of VEGF
`antagonist.
`
`Mylan Exhibit 1135
`Mylan v. Regeneron, IPR2021-00881
`Page 1
`
`
`
`Patent Application Publication Aug. 16, 2007 Sheet 1 of 2
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`Mylan Exhibit 1135
`Mylan v. Regeneron, IPR2021-00881
`Page 2
`
`
`
`Patent Application Publication Aug. 16, 2007 Sheet 2 of 2
`
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`Mylan Exhibit 1135
`Mylan v. Regeneron, IPR2021-00881
`Page 3
`
`
`
`US 2007/0190058 Al
`
`Aug. 16, 2007
`
`1
`
`METHOD FOR TREATING INTRAOCULAR
`NEOVASCULAR DISEASES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is a continuation of PCT/US2005/
`038006 filed 21 Oct. 2005 which claims priority to U.S.
`Provisional Application No. 60/621,209, filed 21 Oct. 2004,
`the contents of which applications are incorporated herein
`by reference.
`
`FIELD OF THE INVENTION
`
`[0002] This invention relates to methods for treating an
`intraocular neovascular disorder with a VEGF antagonist.
`Methods for administering to a mammal suffering from, or
`at risk for, an intraocular neovascular disorder include
`monthly dosing of a therapeutically effective amount of
`VEGF antagonist, followed by less frequent dosing of a
`therapeutically effective amount of VEGF antagonist.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Angiogenesis is implicated in the pathogenesis of
`intraocular neovascular diseases, e.g., proliferative retino(cid:173)
`pathies, age-related macular degeneration (AMD), etc., as
`well as a variety of other disorders. These include solid
`tumors, rheumatoid arthritis, and psoriasis (Folkman et 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. Gamer A, Klintworth G K, Eds. 2nd
`Edition Marcel Dekker, NY, pp 1625-1710 (1994)).
`
`[0004] The search for positive regulators of angiogenesis
`has yielded many candidates, including aFGF, bFGF, TGF(cid:173)
`a, TGF-~ HGF, TNF-a, angiogenin, IL-8, etc. (Folkman et
`al. and Klagsbrun et al). The negative regulators so far
`identified include thrombospondin (Good et al. Proc. Natl.
`Acad. Sci. USA. 87:6624-6628 (1990)), the 16-kilodalton
`N-terminal fragment of prolactin (Clapp et al. Endocrinol(cid:173)
`ogy, 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)).
`
`[0005] Work done over the last several years has estab(cid:173)
`lished the key role of vascular endothelial growth factor
`(VEGF) in the regulation of normal and abnormal angio(cid:173)
`genesis (Ferrara et al. Endocr. Rev. 18:4-25 (1997)). The
`finding that the loss of even a single VEGF allele results in
`embryonic lethality points to an irreplaceable role played by
`this factor in the development and differentiation of the
`vascular system (Ferrara et al.).
`
`[0006] Human VEGF exists as at least six isoforms
`(VEGF 121 , VEGF 145 , VEGF 165 , VEGF 183 , VEGF 189 , and
`VEGF 206) that arise from alternative splicing of mRNA of a
`single gene (Ferrara N, Davis Smyth T. Endocr Rev 18:1-22
`(1997)). VEGF 165 , the most abundant isoform, is a basic,
`heparin binding, dimeric glycoprotein with a molecular
`mass of -45,000 daltons (Id). Two VEGF receptor tyrosine
`kinases, VEGFRl and VEGFR2, have been identified
`(Shibuya et al. Oncogene 5:519-24 (1990); Matthews et al.,
`Proc Natl Acad Sci USA 88:9026-30 (1991); Terman et al.,
`Oncogene 6: 1677-83 (1991); Terman et al. Biochem Biophys
`Res Commun 187:1579-86 (1992); de Vries et al., Science
`
`255:989-91 (1992); Millauer et al. Cell 72:835-46 (1993);
`and, Quinnetal.ProcNatlAcadSci USA 90:7533-7 (1993)).
`VEGFRl has the highest affinity for VEGF, with a Kd of
`-10-20 pM (de Vries et al., Science 255:989-91 (1992)), and
`VEGFR2 has a somewhat lower affinity for VEGF, with a
`Kd of 75-125 pM (Terman et al., Oncogene 6:1677-83
`(1991); Millauer et al. Cell 72:835-46 (1993); and, Quinn et
`al. Proc Natl Acad Sci USA 90:7533-7 (1993)).
`
`[0007] VEGF has several biologic functions, including
`regulation ofVEGF gene expression under hypoxic condi(cid:173)
`tions (Ferrara N, Davis Smyth T. Endocr Rev 18:1-22
`(1997)), mitogenic activity for micro and macrovascular
`endothelial cells (Ferrara N, Henzel W J. Biochem Biophys
`Res Commun 161:851-8 (1989); Leung et al., Science
`246:1306-9 (1989); Connolly et al. J Clin Invest 84:1470-8
`(1989a); Keck et al. Science 246:1309-12 (1989); Plouet et
`al., EMBO J8:3801-6 (1989); Conn et al. Proc Natl Acad Sci
`USA 87:2628-32 (1990); and, Pepper et al., Exp Cell Res
`210:298-305 (1994)), and induction of expression of plas(cid:173)
`minogen activators and collagenase (Pepper et al., Biochem
`Biophys Res Commun 181:902-6 (1991)).
`
`[0008] Furthermore, VEGF has been shown to be a key
`mediator of neovascularization associated with tumors and
`intraocular disorders (Ferrara et al.). The VEGF mRNA is
`overexpressed by the majority of human tumors examined.
`Berkman et al. 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 VEGF in eye fluids
`are highly correlated to the presence of active proliferation
`of blood vessels in patients with diabetic and other ischemia(cid:173)
`related retinopathies. Aiello et al., N. Engl. J. Med.
`331:1480-1487 (1994). Furthermore, recent studies have
`demonstrated the localization ofVEGF in choroidal neovas(cid:173)
`cular membranes in patients affected by AMD. Lopez et al.,
`Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996); Kvanta et al.,
`Invest Ophthalmol Vis Sci 37:1929-34 (1996).
`
`[0009] Age related macular degeneration (AMD) is a
`leading cause of severe, irreversible vision loss among the
`elderly. Bressler, JAMA 291:1900-1 (2004). It is character(cid:173)
`ized by a broad spectrum of clinical and pathologic findings,
`such as pale yellow spots known as drusen, disruption of the
`retinal pigment epithelium (RPE), choroidal neovasculariza(cid:173)
`tion (CNV), and disciform macular degeneration. The mani(cid:173)
`festations 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 neovas(cid:173)
`cularization characterizes the wet form. Disciform AMD is
`the fibrotic stage of the neovascular lesion.
`
`[0010] 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 much less common, it is responsible for 80%-90%
`of the severe visual loss associated withAMD (Ferris et al.,
`Arch Ophthamol 102:1640-2 (1984)). There is an estimated
`1-1.2 million prevalent cases of wet AMD. The cause of
`AMD is unknown; however, it is clear that the risk of
`developing AMD increases with advancing age. Other
`known risk factors include family history and cigarette
`smoking. Postulated risk factors also include oxidative
`
`Mylan Exhibit 1135
`Mylan v. Regeneron, IPR2021-00881
`Page 4
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`Aug. 16, 2007
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`2
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`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 (1996).
`
`[0011] Dry AMD is characterized by changes in the RPE
`and Bruch's membrane. It is thought that the RPE, compro(cid:173)
`mised by age and other risk factors, deposits lipofuscin and
`cellular debris on Bruch' s membrane. These changes may be
`seen ophthalmoscopically as drusen, which are scattered
`throughout the macula and posterior retinal pole. There are
`also variable degrees of atrophy and pigmentation of the
`RPE. Dry AMD may be asymptomatic or accompanied by
`variable and usually minimal visual loss and is considered to
`be a prelude to development of wet AMD.
`
`[0012] WetAMD is typically characterized by CNV of the
`macular region. The choroidal capillaries proliferate and
`penetrate Bruch' s membrane to reach the RPE and may
`extend into the subretinal space. The increased permeability
`of the newly formed capillaries leads to accumulation of
`serous fluid or blood under the RPE and/or the neurosensory
`retina or within the neurosensory retina. When the fovea
`becomes swollen or detached, decreases in vision occur.
`Fibrous metaplasia and organization may ensue, resulting in
`an elevated subretinal mass called a disciform scar that
`constitutes end-stageAMD and is associated with permanent
`vision loss (D'Amico D J. N Engl J Med 331:95-106
`(1994)).
`
`[0013] The neovascularization in AMD can be classified
`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 occult and are
`associated with different degrees of aggressiveness, vision
`losses, and response to different treatment options.
`
`[0014] The diffusible nature ofVEGF and its specificity of
`action for endothelial cells support a key role in the process
`of abnormal blood vessel growth and vascular leakage.
`Increased expression of VEGF in retinal photoreceptors or
`RPE of transgenic mice stimulates neovascularization
`within the retina, and VEGF antagonists partially inhibit
`retinal neovascularization in animal models (Okamoto et al.
`Am J Pathol 151:281-91 (1997); Schwesinger et al., AM J.
`Pathol. Mar; 158(3):1161-72 (2001)). Anti-VEGF neutral(cid:173)
`izing antibodies inhibit intraocular angiogenesis in models
`of ischemic retinal disorders (Adamis et al. Arch. Ophthal(cid:173)
`mol. 114:66-71 (1996)), and also suppress the growth of a
`variety of human tumor cell lines in nude mice (Kim et al.
`Nature 362:841-844 (1993); Warren et al. J. Clin. Invest.
`95:1789-1797 (1995); Borgstrom 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 VEGF antagonists are promising candi(cid:173)
`dates for use in treatments of intraocular neovascular dis(cid:173)
`orders, and new methods of administering therapeutic com(cid:173)
`pounds, which increases the effectiveness of the therapeutic
`compound, are needed.
`
`SUMMARY OF THE INVENTION
`
`[0015] 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.
`
`[0016] Methods for treating intraocular neovascular dis(cid:173)
`ease are provided. For example, methods include adminis(cid:173)
`tering to a mammal a number of first individual doses of a
`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.
`
`[0017]
`In one embodiment of the invention, a method for
`treating wet form age-related macular degeneration is pro(cid:173)
`vided, which comprises administering to a mammal a num(cid:173)
`ber of first individual doses of an VEGF antagonist, fol(cid:173)
`lowed 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.
`
`[0018]
`In one embodiment, the mammal is in need of
`treatment. Typically, the mammal is a human.
`
`[0019]
`In one embodiment, the administration of the
`VEGF antagonist is ocular. In one aspect, the administration
`is intraocular. In another aspect, the administration is intra(cid:173)
`vitreal.
`
`[0020] A VEGF antagonist is administered in the methods
`of the invention. In one aspect, the VEGF antagonist is an
`anti-VEGF antibody, 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.
`
`[0021]
`In one embodiment of the invention, the first indi(cid:173)
`vidual doses are administered at one month intervals (e.g.,
`about 3 individual doses). Typically, there is more than one
`first 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 inven(cid:173)
`tion, the second individual doses are administered beginning
`three months after the number of first individual doses. In
`one embodiment, a number of second individual doses are
`administered to the mammal during a period of at least 22
`months following the number of first individual doses.
`
`[0022]
`In one embodiment of the invention, the number of
`first individual doses and the number of second individual
`doses are administered over a time period of about 2 years.
`In one aspect, 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, the first individual dose is administered at month
`0, 1, and 2 and the second individual dose is administered at
`month 5, 8, 11, 14, 17, 20 and 23. In one embodiment, the
`VEGF antagonist is administered over less than 2 years, or
`optionally, administered over greater than 2 years.
`
`[0023] Other aspects of the invention will become appar(cid:173)
`ent from the following description of the embodiments
`which are not intended to be limiting of the invention.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0024] FIG. 1 schematically illustrates the study m
`Example 1.
`
`[0025] FIG. 2 schematically illustrates a dosing regimen
`for treating, e.g., age-related macular degeneration (AMD)
`with a VEGF antagonist.
`
`Mylan Exhibit 1135
`Mylan v. Regeneron, IPR2021-00881
`Page 5
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`US 2007/0190058 Al
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`Aug. 16, 2007
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`3
`
`DETAILED DESCRIPTION
`
`Definitions
`
`[0026] 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 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 the like.
`
`[0027] 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 Leung et al., Science 246: 1306 (1989), and
`Houck et al., Mal. Endocrin. 5:1806 (1991) together with the
`naturally occurring allelic and processed forms of those
`growth factors.
`
`[0028] A "VEGF antagonist" refers to a molecule capable
`of neutralizing, blocking, inhibiting, abrogating, reducing or
`interfering with VEGF activities including its binding to one
`or more VEGF receptors. VEGF antagonists include anti(cid:173)
`VEGF antibodies and antigen-binding fragments thereof,
`receptor molecules and derivatives which bind specifically
`to VEGF thereby sequestering its binding to one or more
`receptors, anti-VEGF receptor antibodies and VEGF recep(cid:173)
`tor antagonists such as small molecule inhibitors of the
`VEGFR tyrosine kinases, and fusions proteins, e.g., VEGF(cid:173)
`Trap (Regeneron), VEGF 121 -gelonin (Peregrine). VEGF
`antagonists also include antagonist variants of VEGF, anti(cid:173)
`sense molecules directed to VEGF, RNA aptamers specific
`to VEGF, and ribozymes against VEGF or VEGF receptors.
`Antagonists ofVEGF act by interfering with the binding of
`VEGF to a cellular receptor, by incapacitating or killing cells
`which have been activated by VEGF, or by interfering with
`vascular endothelial cell activation after VEGF binding to a
`cellular receptor. All such points of intervention by a VEGF
`antagonist shall be considered equivalent for purposes of
`this invention. Preferred VEGF antagonists are anti-VEGF
`antagonistic antibodies capable of inhibiting one or more of
`the biological activities of VEGF, for example, its mitoge(cid:173)
`nic, angiogenic or vascular permeability activity. Anti(cid:173)
`VEGF antagonistic antibodies include, but not limited to,
`antibodies A4.6.1, rhuMab VEGF (bevacizumab), Y0317
`(ranibizumab ), G6, B20, 2C3, and others as described in, for
`example, WO98/45331, US2003/0190317, U.S. Pat. Nos.
`6,582,959 and 6,703,020; WO98/45332; WO 96/30046;
`WO94/10202; WO2005/044853; EP 0666868Bl; and Pop(cid:173)
`kov et al., Journal of Immunological Methods 288:149-164
`(2004). More preferably, the anti-VEGF antagonistic anti(cid:173)
`body of the invention is ranibizumab, which is a humanized,
`affinity matured anti-human VEGF antibody Fab fragment
`having the light and heavy chain variable domain sequences
`ofY0317 as described in WO98/45331 and Chen et al J Mal
`Biol 293:865-881 (1999).
`
`[0029] 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 species to be treated, and more prefer-
`
`ably the antibody is human or humanized and the host is
`human. While the antibody can be a polyclonal or mono(cid:173)
`clonal 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 Fe domain
`capable of an effector function or may not be capable of
`binding complement or participating in ADCC.
`
`[0030] The term "VEGF receptor" 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 which retain the ability to bind
`hVEGF. One example of a VEGF receptor is the fms-like
`tyrosine kinase (flt), a transmembrane receptor in the
`tyrosine kinase family. DeVries et al., Science 255:989
`(1992); Shibuya et al., Oncogene 5:519 (1990). The flt
`receptor comprises an extracellular domain, a transmem(cid:173)
`brane domain, and an intracellular domain with tyrosine
`kinase activity. The extracellular domain is involved in the
`binding of VEGF, whereas the intracellular domain is
`involved in signal transduction. Another example of a VEGF
`receptor is the flk-1 receptor (also referred to as KDR).
`Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991);
`Terman et al., Oncogene 6:1677 (1991); Terman et al.,
`Biochem. Biophys. Res. Commun. 187:1579 (1992). Binding
`ofVEGF to the flt 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 compris(cid:173)
`ing two receptor molecules bound to a single molecule of
`VEGF.
`
`[0031] The term "epitope A4.6.1" when used herein,
`unless indicated otherwise, refers to the region of human
`VEGF to which the A4.6.1 antibody disclosed in Kim et al.,
`Growth Factors 7:53 (1992) and Kim et al. Nature 362:841
`(1993), binds.
`
`[0032]
`"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 disorder is to be prevented.
`
`[0033]
`"Mammal" for purposes of treatment refers 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, etc. Typically, the mammal is
`human.
`[0034] The term "antibody" is used in the broadest sense
`and includes monoclonal antibodies (including full length or
`intact monoclonal antibodies), polyclonal antibodies, mul(cid:173)
`tivalent antibodies, multispecific antibodies ( e.g., bi specific
`antibodies), and antibody fragments (see below) so long as
`they exhibit the desired biological activity.
`
`[0035] Unless indicated otherwise, the expression "multi(cid:173)
`valent antibody" is used throughout this specification to
`denote an antibody comprising three or more antigen bind(cid:173)
`ing 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.
`
`[0036]
`"Native antibodies" and "native immunoglobulins"
`are usually heterotetrameric glycoproteins of about 150,000
`daltons, composed of two identical light (L) chains and two
`
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`identical heavy (H) chains. Each light chain is linked to a
`heavy chain by one covalent disulfide bond, while the
`number of disulfide linkages varies among the heavy chains
`of different immunoglobulin isotypes. Each heavy and light
`chain also has regularly spaced intrachain disulfide bridges.
`Each heavy chain has at one end a variable domain (V H)
`followed by a number of constant domains. Each light chain
`has a variable domain at one end (V L) and a constant domain
`at its other end; the constant domain of the light chain is
`aligned with the first constant domain of the heavy chain,
`and the light-chain variable domain is aligned with the
`variable domain of the heavy chain. Particular amino acid
`residues are believed to form an interface between the light(cid:173)
`and heavy-chain variable domains.
`[0037] The term "variable" refers to the fact that certain
`portions of the variable domains differ extensively in
`sequence among antibodies and are used in the binding and
`specificity of each particular antibody for its particular
`antigen. However, the variability is not evenly distributed
`throughout the variable domains of antibodies. It is concen(cid:173)
`trated in three segments called hypervariable regions both in
`the light chain and the heavy chain variable domains. The
`more highly conserved portions of variable domains are
`called the framework region (FR). The variable domains of
`native heavy and light chains each comprise four FRs (FRI,
`FR2, FR3 and FR4, respectively), largely adopting a I-sheet
`configuration, connected by three hypervariable regions,
`which form loops connecting, and in some cases forming
`part of, the ~-sheet structure. The hypervariable regions in
`each chain are held together in close proximity by the FRs
`and, with the hypervariable regions from the other chain,
`contribute to the formation of the antigen-binding site of
`antibodies (see Kabat et al., Sequences of Proteins of Immu(cid:173)
`nological Interest, 5th Ed. Public Health Service, National
`Institutes of Health, Bethesda, Md. (1991), pages 647-669).
`The constant domains are not involved directly in binding an
`antibody to an antigen, but exhibit various effector func(cid:173)
`tions, such as participation of the antibody in antibody(cid:173)
`dependent cellular toxicity (ADCC).
`
`[0038] The term "hypervariable region" when used herein
`refers to the amino acid residues of an antibody which are
`responsible for antigen-binding. The hypervariable region
`comprises amino acid residues from a "complementarity
`determining region" or "CDR" (i.e. residues 24-34 (Ll),
`50-56 (L2) and 89-97 (L3) in the light chain variable domain
`and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy
`chain variable domain; Kabat et al., Sequences of Proteins of
`Immunological Interest, 5th Ed. Public Health Service,
`National Institutes of Health, Bethesda, Md. (1991 )) and/or
`those residues from a "hypervariable loop" (i.e. residues
`26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain
`variable domain and 26-32 (Hl), 53-55 (H2) and 96-101
`(H3) in the heavy chain variable domain; Chothia and Lesk
`J. Mal. Biol. 196:901-917 (1987)). "Framework" or "FR"
`residues are those variable domain residues other than the
`hypervariable region residues as herein defined.
`
`[0039] Papain digestion of antibodies produces two iden(cid:173)
`tical antigen-binding fragments, called "Fab" fragments,
`each with a single antigen-binding site, and a residual "Fe"
`fragment, whose name reflects its ability to crystallize
`readily. Pepsin treatment yields an F(ab')2 fragment that has
`two antigen-combining sites and is still capable of cross(cid:173)
`linking antigen.
`
`[0040]
`"Fv" is the mm1mum antibody fragment which
`contains a complete antigen-recognition and-binding site.
`This region consists of a dimer of one heavy chain and one
`light chain variable domain in tight, non-covalent associa(cid:173)
`tion. It is in this configuration that the three hypervariable
`regions of each variable domain interact to define an anti(cid:173)
`gen-binding site on the surface of the V
`-VL dimer. Collec(cid:173)
`tively, the six hypervariable regions confer antigen-binding
`specificity to the antibody. However, even a single variable
`domain (or half of an Fv comprising only three hypervari(cid:173)
`able regions specific for an antigen) has the ability to
`recognize and bind antigen, although at a lower affinity than
`the entire binding site.
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`[0041] The Fab fragment also contains the constant
`domain of the light chain and the first constant domain
`(CHI) of the heavy chain. Fab' fragments differ from Fab
`fragments by the addition of a few residues at the carboxyl
`terminus of the heavy chain CHI domain including one or
`more cysteine(s) from the antibody hinge region. Fab'-SH is
`the designation herein for Fab' in which the cysteine resi(cid:173)
`due(s) of the constant domains bear a free thiol group.
`F(ab'), antibody fragments originally were produced as pairs
`ofFab' fragments which have hinge cysteines between them.
`Other chemical couplings of antibody fragments are also
`known.
`
`[0042] The "light chains" of antibodies (immunoglobu(cid:173)
`lins) from any vertebrate species can be assigned to one of
`two clearly distinct types, called kappa (K) and lambda (X),
`based on the amino acid sequences of their constant
`domains.
`
`[0043] Depending on the amino acid sequence of the
`constant domain of their heavy chains, immunoglobulins
`can be assigned to different classes. There are five major
`classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,
`and several of these may be further divided into subclasses
`(isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, andigA2. The
`heavy-chain constant domains that correspond to the differ(cid:173)
`ent classes of immunoglobulins are called a, Ii, E, y, and µ,
`respectively. The subunit structures and three-dimensional
`configurations of different classes of immunoglobulins are
`well known.
`
`[0044]
`"Antibody fragments" comprise only a portion of
`an intact antibody, generally including an antigen binding
`site of the intact antibody and thus retaining the ability to
`bind antigen. Examples of antibody fragments encompassed
`by the present definition include: (i) the Fab fragment,
`having VL, CL, VH and CHI domains; (ii) the Fab' frag(cid:173)
`ment, which is a Fab fragment having one or more cysteine
`residues at the C-terminus of the CHI domain; (iii) the Fd
`fragment having VH and CHI domains; (iv) the Fd' frag(cid:173)
`ment having VH and CHI domains and one or more cysteine
`residues at the C-terminus of the CHI domain; (v) the Fv
`fragment having the VL and VH domains of a single arm of
`an antibody; (vi) the dAb fragment (Ward et al., Nature 341,
`544-546 (1989)) which consists of a VH domain; (vii)
`isolated CDR regions; (viii) F(ab')2 fragments, a bivalent
`fragment including two Fab' fragments linked by a disul(cid:173)
`phide bridge at the hinge region; (ix) single chain antibody
`molecules (e.g. single chain Fv; scFv) (Bird et al., Science
`242:423-426 (1988); and Huston et al., PNAS (USA)
`85:5879-5883 (1988)); (x) "diabodies" with two antigen
`binding sites, comprising a heavy chain variable domain
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`(VH) connected to a light chain variable domain (VL) in the
`same polypeptide chain (see, e.g., EP 404,097; WO
`93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
`90:6444-6448 (1993)); (xi) "linear antibodies" comprising a
`pair of tandem Fd segments (VH-CHl-VH-CHl) which,
`together with complementary light chain polypeptides, form
`a pair of antigen binding regions (Zapata et al. Protein Eng.
`8(10):1057 1062 (1995); and U.S. Pat. No. 5,641,870).
`[0045] The term "monoclonal antibody" as used herein
`refers to an antibody obtained from a population of substan(cid:173)
`tially homogeneous antibodies, i.e., the individual antibod(cid:173)
`ies comprising the population are identical except for pos(cid:173)
`sible naturally occurring mutations that may be present in
`minor amounts. Monoclonal antibodies are highly specific,
`being directed against a single antigenic site. Furthermore,
`in contrast to conventional (polyclonal) antibody prepara(cid:173)
`tions which typically include different antibodies directed
`against different 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 monoclonal antibodies
`to be used in accordance with the present invention may be
`made by the hybridoma method first described by Kohler et
`al., Nature 256:495 (1975), or may be made by recombinant
`DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
`"monoclonal antibodies" may also be isolated from phage
`antibody libraries using the techniques described in Clack(cid:173)
`son et al., Nature 352:624-628 (1991) and Marks et al., J.
`Mal. Biol. 222:581-597 (1991), for example.
`
`[0046] The monoclonal antibodies herein specifically
`include "chimeric" antibodies (immunoglobulins) in which
`a portion of the heavy and/or light chain is identical with or
`homologous to corresponding sequences in antibodies
`derived from a particular species or belonging to a parti