`Baca et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,884,879 B1
`Apr. 26, 2005
`
`USOO6884879B1
`
`(54) ANTI-VEGF ANTIBODIES
`(75) Inventors: Manuel Baca, Foster City, CA (US);
`James A. Wells, Burlingame, CA (US)
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA (US)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(*) Notice:
`
`(21) Appl. No.: 08/908,469
`(22) Filed:
`Aug. 6, 1997
`Related U.S. Application Data
`(63) Continuation-in-part of application No. 08/833,504, filed on
`Apr. 7, 1997, now abandoned.
`(51) Int. Cl." ................................................ C07H 21/04
`(52) U.S. Cl. ................................ 536/23.53; 435/320.1;
`530/387.3; 530/388.85
`(58) Field of Search ................................. 435/327, 252,
`435/1, 320.1, 252.3, 69.1, 536/23.1, 23.53;
`530/382.1, 387.3,388, 388.85
`
`(56)
`
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`Primary Examiner-Larry R. Helms
`(74) Attorney, Agent, or Firm-Genentech, Inc.
`(57)
`ABSTRACT
`Humanized and variant anti-VEGF antibodies and various
`uses therefor are disclosed. The anti-VEGF antibodies have
`strong binding affinities for VEGF; inhibit VEGF-induced
`proliferation of endothelial cells in vitro; and inhibit tumor
`growth in Vivo.
`
`14 Claims, 16 Drawing Sheets
`
`Novartis Exhibit 2012.001
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`US 6,884.879 B1
`Page 2
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`
`
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`Page 3
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`* cited by examiner
`
`Novartis Exhibit 2012.003
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 1 of 16
`
`US 6,884,879 B1
`
`Varisble Heavy
`PIQLVCSGPELKQPGETVRISCKASGYTPINYGMNWVKQAPGKGLKWMG
`Aq.6.3
`*
`ee
`t
`*
`tte «
`t
`«
`© tft
`F(ab)-12 tVQLVESGGGLVQPGGSLRLSCAASGYTFINYGMNWVROAPGKGLEWVG
`:
`+*
`cece © ©
`*
`numTIT
`SVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS
`i
`10
`20
`30
`40
`
`WINTYTGEPTYAADFKRRFTFSLETSASTAYLOISNLKNDDTATYFCAK
`A4@.6.1
`*
`Cd
`kkk kek
`*«
`t&
`F(ab)-12 WINTYTGEPTYAADPKRRFTFSLDTSKSTAYLOMNSLRAEDTAVYYCAK
`te KKEE £2% tke &
`k«
`*€
`*&
`* oo
`.
`humIII
`VISGDGGSTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAR
`50
`a
`60
`70
`80
`abc
`90
`
`1A
`
`Fig.
`
`24.6.1
`
`F(ab)-12
`
`humZII
`
`PHYYGS
`
`XPHYYGSSHWYFDVWGAGTTVTVSS (SEQ 1H WO!D
`*
`*
`VWGOGTLYTVSS (SEQ@ 1b NO.)
`*
`*
`Gre----- eee FDYWGacTLYTVss CSE iD NO: )
`110
`
`Variable Light
`DIQMTOTTSSLSASLGDRVI ISCSASODISNYLNWYQQKPDGTVKVLIY
`A4.6.1
`nk
`*
`**
`*
`KKK
`F(ab)-12 DIQMTOSPSSLSASVGDRVTITCSASODISNYLNWYOQOKPGKAPKVLIY
`*
`*
`x
`*
`humKI
`DIQMTOSPSSLSASVGDRVTITCRASOSISNYLAWYOOKEGKAPKLLIY
`1
`10
`20
`30
`40
`
`“DSSLUSGV?SRFSGSGSGIDYSLTISNLEPEDIATYYCOOYETVPWEF
`A4.6.1
`tt
`hook
`t
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`kk
`*
`kee
`humKr
`AASSLESWPSRTEGSCSGIDFTLTISSLQOPEDFATYYCQOYNSLPWTP
`Th
`66
`70
`80
`90
`
`Fig. 1B
`
`GGGTKLEIKR (SEQ@ io No: (05
`A4.6.1
`‘t
`t
`F(ab)-12 GQGTKVEIKR CSEQ@ ip NO:8)
`humkr
`GOGTKVEIKR (SEQ 1D NO. ID)
`100
`
`Novartis Exhibit 2012.004
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.004
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 2 of 16
`
`US 6,884,879 B1
`
`
`
`Novartis Exhibit 2012.005
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 3 of 16
`
`US 6,884,879 B1
`
`(ng/ml) 0.1
`
`MAbConcentration
`
`
`
`w o
`
`O
`uJ
`
`>+VEGF+rhuMAbVEGF
`
`MNoaddition
`
`®VEGF
`
`
`
`100010000
`
`100
`
`10
`
`Fig.3
`
`>2< =~ E+
`
`aw
`oO
`uJ
`
`180000
`
`140000
`
`100000
`
`60000
`
`20000
`
`IAM Jad sijaQ yeyauopuz
`
`Novartis Exhibit 2012.006
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.006
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 4 of 16
`
`US 6,884,879 B1
`
`Tumor Weight (gm)
`
`
`
`Control MAb (5)
`muMAb VEGF (0.5)
`
`muMAb VEGF (5)
`
`rhuMAb VEGF (0.5)
`
`rhuMAb VEGF (5)
`
`Fig. 4
`
`Novartis Exhibit 2012.007
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 5 of 16
`
`US 6,884,879 BL
`
`VL domain
`
`A4.6.1
`
`hu2.0
`
`hu2.10
`
`A4.6.1
`
`hu2.0
`
`hu2.10
`
`40
`30
`20
`10
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`rt
`t
`k t
`
`DIQMTQS PSSLSASVGDRVTITCSASQDISNYLNWYQOKP
`
`DIQMTOQS PSSLSASVGDRVTI TCSASQDISNYLNWYOOKP
`
`60
`70
`60
`50
`DGTVKVLIYFISSLHSGVPSRFSGSGSGTDYSLTISNI-EP
`tt.
`Kkkt «
`«tk
`«
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`
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`
`Ad.6.1
`
`hu2.0
`
`100
`90
`EDIATYYCQOQYSTVPWIFGGGTKLEIK (SEQ \D NO110)
`*
`*
`*
`EDFATYYCQQYSTVPWIFGOGTKVEIK (SEQ \o NO:I2)
`
`hu2.10
`
`EDFATYYCQQYSTVPWIFGQGTKVEIK CSEQ.\D NO:IS)
`
`Fig. 5A
`
`- Vy domain
`
`40
`30
`20
`10
`EIQLVOSGPELKOPGETVRISCKASGYTFTNYGMNWVKOA
`«
`«
`tk €
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`
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`
`80
`70
`60
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`x
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`
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`
`Fig. 58
`
`A4.6.1
`
`hu2.0
`
`hu2.10
`
`A4.6.1
`
`hu2.0
`
`hu2.10
`
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`
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`
`hu2.10
`
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`*
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`OMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGOGTLVTVss (SEQ (Dp NO:16)
`
`Novartis Exhibit 2012.008
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.008
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 6 of 16
`
`US 6,884,879 B1
`
`
`
`Novartis Exhibit 2012.009
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 7 of 16
`
`US 6,884,879 B1
`
`CL
`
`St.
`
`VH
`
`CH1
`
`g|II (249-406)
`
`CS fl ori
`
`OpBR322 ori
`
`VL
`
`St.
`
`phoA W
`
`Amp
`
`Transform E. coli
`
`+M13KO7 helper phage
`
`
`
`Fab-pII fusion
`
`s
`
`Fig. 7
`
`Novartis Exhibit 2012.010
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 8 of 16
`
`US 6,884,879 BL
`
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`Novartis Exhibit 2012.011
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.011
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 9 of 16
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`US 6,884,879 B1
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`Novartis Exhibit 2012.012
`Regeneron v. Novartis, IPR2020-01317
`
`
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`U.S. Patent
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`Apr. 26, 2005
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`Novartis Exhibit 2012.013
`Regeneron v. Novartis, IPR2020-01317
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`
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`U.S. Patent
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`Apr. 26, 2005
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`Apr. 26, 2005
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`US 6,884,879 B1
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`Regeneron v. Novartis, IPR2020-01317
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`
`
`U.S. Patent
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`Apr. 26, 2005
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`Sheet 13 of16
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`Novartis Exhibit 2012.016
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.016
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 14 of16
`
`US 6,884,879 BL
`
`= differences from F(ab)-12
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`
`Novartis Exhibit 2012.017
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.017
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 15 of 16
`
`US 6,884,879 B1
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`Novartis Exhibit 2012.018
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 16 of 16
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`US 6,884,879 BL
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`Novartis Exhibit 2012.019
`Regeneron v. Novartis, IPR2020-01317
`
`Novartis Exhibit 2012.019
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`1
`ANTI-VEGF ANTIBODES
`
`US 6,884,879 B1
`
`CROSS REFERENCES
`This application is a continuation-in-part of U.S. Appli
`cation Ser. No. 08/833,504, now abandoned filed Apr. 7,
`1997, converted to a provisional application Apr. 6, 1998
`which application is incorporated herein by reference and to
`which application priority is claimed under 35 USC S120.
`
`BACKGROUND OF THE INVENTION
`
`15
`
`35
`
`40
`
`1. Field of the Invention
`This invention relates generally to anti-VEGF antibodies
`and, in particular, to humanized anti-VEGF antibodies and
`variant anti-VEGF antibodies.
`2. Description of Related Art
`It is now well established that angiogenesis is implicated
`in the pathogenesis of a variety of disorders. These include
`Solid tumors, intraocular neovascular Syndromes Such as
`proliferative retinopathies or age-related macular degenera
`tion (AMD), rheumatoid arthritis, and psoriasis (Folkman et
`al. J. Biol. Chem. 267:10931–10934 (1992); Klagsbrun et al.
`Annu. Re: Physiol. 53:217-239 (1991); and Garner A,
`Vascular diseases. In: Pathobiology of Ocular disease. A
`25
`dynamic approach. Garner A, Klintworth GK, Eds. 2nd
`Edition Marcel Dekker, NY, pp 1625–1710 (1994)). In the
`case of Solid tumors, the neovascularization allows the
`tumor cells to acquire a growth advantage and proliferative
`autonomy compared to the normal cells. Accordingly, a
`correlation has been observed between density of microves
`Sels in tumor Sections and patient Survival in breast cancer
`as well as in several other tumors (Weidner et al. N Engl J
`Med 324:1–6 (1991); Horak et al. Lancet 340:1120–1124
`(1992); and Macchiarini et al. Lancet 340: 145-146 (1992)).
`The Search for positive regulators of angiogenesis has
`yielded many candidates, including aFGF, bFGF, TGF-C.,
`TGF-B, HGF, TNF-C, angiogenin, IL-8, etc. (Folkman et al.
`and Klagsbrun et al). The negative regulators So far identi
`fied 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.
`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)).
`Work done over the 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. 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.,). Furthermore, VEGF has been shown to be
`a key mediator of neovascularization associated with tumors
`and intraocular disorders (Ferrara et al.). The VEGF mRNA
`55
`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. 53:47274735 (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-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 membranes in patients affected by AMD (Lopez
`
`45
`
`50
`
`60
`
`65
`
`2
`et al. Invest. Ophtalmo. Vis. Sci. 37:855–868 (1996)). Anti
`VEGF neutralizing antibodies 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); Borgström et al. Cancer Res.
`56:4032-4039 (1996); and Melnyk et al., Cancer Res.
`56:921-924 (1996)) and also inhibit intraocular angiogen
`esis in models of ischemic retinal disorders (Adamis et al.
`Arch. Ophthalmol. 114:66–71 (1996)). Therefore, anti
`VEGF monoclonal antibodies or other inhibitors of VEGF
`action are promising candidates for the treatment of Solid
`tumors and various intraocular neovascular disorders.
`
`SUMMARY OF THE INVENTION
`This application describes humanized anti-VEGF anti
`bodies and anti-VEGF antibody variants with desirable
`properties from a therapeutic perspective, including Strong
`binding affinity for VEGF; the ability to inhibit VEGF
`induced proliferation of endothelial cells in vitro; and the
`ability to inhibit VEGF-induced angiogenesis in vivo.
`The preferred humanized anti-VEGF antibody or variant
`anti-VEGF antibody herein binds human VEGF with a K.
`value of no more than about 1x10 M and preferably no
`more than about 5x10M. In addition, the humanized or
`variant anti-VEGF antibody may have an ED50 value of no
`more than about 5 nM for inhibiting VEGF-induced prolif
`eration of endothelial cells in vitro. The humanized or
`variant anti-VEGF antibodies of particular interest herein
`are those which inhibit at least about 50% of tumor growth
`in an A673 in vivo tumor model, at an antibody dose of 5
`mg/kg.
`In one embodiment, the anti-VEGF antibody has a heavy
`and light chain variable domain, wherein the heavy chain
`variable domain comprises hyperVariable regions with the
`following a mino acid sequences: CD RH1
`(GYXFTX-YGMN, wherein X is T or D and X is N or
`H; SEQ ID NO:128), CDRH (WINTYTGEPTYMDFKR;
`SEQ ID NO:2) and CDRH3 (YPXYYGXSHWYFDV,
`wherein X is Y or H and X is S or T, SEQ ID NO:129).
`For example, the heavy chain variable domain may com
`prise the amino acid sequences of CDRH1
`(GYTFTNY GMN; SEQ ID NO: 1), CDR H2
`(WINTYTGEPTYMDFKR, SEQ ID NO:2) and CDRH3
`(YPHYYGSSHWYFDV; SEQ ID NO:3). Preferably, the
`three heavy chain hyperVariable regions are provided in a
`human framework region, e.g., as a contiguous Sequence
`represented by the following formula: FR1-CDRH1-FR2
`CDRH2-FR3-CDRH3-FR4.
`The invention further provides an anti-VEGF antibody
`heavy chain variable domain comprising the amino acid
`Sequence:
`EVOLVESGGGLVOPGGSLRLSCAASGYX FTX,YGM
`NWVROAPGKGLEWVG WINTYTGEPTYMDFKRRF
`TFSLDTSKSTAYLOMNSLRAEDTAVYYCAKYPXYY
`GXSHWYFDVWGQGTLV TVSS (SEQ ID NO:125),
`wherein X is T or D; X is N or H; X is Y or H and X is
`S or T. One particularly useful heavy chain variable domain
`sequence is that of the F(ab)-12 humanized antibody of
`Example 1 and comprises the heavy chain variable domain
`sequence of SEQ ID NO:7. Such preferred heavy chain
`variable domain Sequences may be combined with the
`following preferred light chain variable domain Sequences
`or with other light chain variable domain Sequences, pro
`vided that the antibody so produced binds human VEGF.
`The invention also provides preferred light chain variable
`domain Sequences which may be combined with the above
`
`Novartis Exhibit 2012.020
`Regeneron v. Novartis, IPR2020-01317
`
`
`
`3
`identified heavy chain variable doma