`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, IPR2020-01318
`
`
`
`US 6,884.879 B1
`Page 2
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`Regeneron v. Novartis, IPR2020-01318
`
`
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`Page 3
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`* cited by examiner
`
`Novartis Exhibit 2012.003
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 1 of 16
`
`US 6,884,879 B1
`
`Varisble Heavy
`PIOLVCSGPELKQPGETVRISCKASGYTFINYGMNWVKQAPGKGLKWMG
`A4.6.3
`*
`ti oe
`tee e
`+
`et
`F(ab)-12 *VOLVESGGGLVQPGGSLRLSCAASGYTFINYGNNWVROAPGKGLEWVG
`+
`tf *& +
`*
`humtIT
`SVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVS
`=
`10
`20
`30
`40
`
`WINTYTGEPTYAADFKRRFTFSLETSASTAYLOQISNLKNDDTATYFCAK
`A4d.6.1
`*
`*
`kee kek
`** *
`Fig. 1A
`P(ab)-12 WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLOQMNSLRAEDTAVYYCAK
`& KXEZE Fae
`tee kt * €
`¥ €
`*
`RumIII—_VISGDGGSTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAR
`50
`a
`60
`70
`80
`abc
`90
`
`44.6.1
`
`XPHYYGSSHWYFDVWGAGTTVTVSS (SEQ 1p NO:%)
`*
`*
`F(ab)-12 YPHYYGSSHWwyFrpvWwGoGTLVTVss (SE@ \m Now)
`*
`*
`G-~-------- FDYWGOGTLVTVsS CSEQ ID NO: by)
`110
`
`humzII
`
`Variable Light
`DLOMTOTTSSLSASLGDRVIISCSSASODISNYLNWYQQKPDGTVKVLIY
`A4.6.1
`F(ab)-12 DIOMTOSPSSLSASVGDRVTITCSSASODISNYLNWYOQOKPGKAPKVLIY
`*
`*
`x
`*
`humkKI
`DIQMTQSPSSLSASVGDRVTITCRASOS ISNYLAWYOOKEGKAPKLLIY
`1
`10
`20
`30
`40
`
`EKKX
`
`kk
`
`**
`
`*&
`
`“DSSLESGV?SRFSGSGSGIDYSLTISNLEPEDIATYYCOOYSTVPWEF
`A4d.6.1
`tk
`* &€
`*
`F(ab)-12 ETESL"SGVPSRFSGSGSGIDFTLTISSLOPEDF:"“ATYYCOOYSTVEWTF
`kk
`*
`kKkKe
`humKI
`_HASSLESWPSR?SGSCSGTDFTLTISSLOPEDFATYYCOQYNSLPWTF
`Ze:
`466
`70
`80
`90
`
`Fig. 1B
`
`GGGTKLEIKR (SEG@ io No: to)
`A4.6.1
`t
`t-
`F(ab)-12 GQGTKVEIKR CSEQ 1p NO:8)
`humkI
`GOGTKVEIKR (SEQ 1D NO: ID
`100
`
`Novartis Exhibit 2012.004
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.004
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 2 of 16
`
`US 6,884,879 B1
`
`
`
`Novartis Exhibit 2012.005
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr.26, 2005
`
`Sheet 3 of 16
`
`US 6,884,879 B1
`
`(ng/ml) 0.1
`
`MAbConcentration
`
`
`
`100010000
`
`100
`
`10
`
`Fig.3
`
`MRNoaddition
`
`@®VEGF
`
`+VEGF+rhuMAbVEGF
`
`oe
`oO
`uJ
`
`>2 < =2 E+
`
`uw
`
`Ou
`
`w >
`
`180000
`
`140000
`
`100000
`
`60000
`
`20000
`
`8AM Jad siiao feyeuopuy
`
`Novartis Exhibit 2012.006
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.006
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`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, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr.26, 2005
`
`Sheet 5 of 16
`
`US 6,884,879 B1
`
`Vi domain
`
`10
`20
`30
`40
`A4.6.1 DIQMTOTTSSLSASLGDRVIISCSASQDISNYLNWYQQOKP
`hu2.0
`DIQMTQSPSSLSASVGDRVTETCSASQDISNYLNWYOOKP
`hu2.10 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP
`
`«x «
`
`kt
`
`60
`70
`60
`50
`A4.6.1 DGIVKVLIYFTSSLHUSGVPSRFSGSGSGTDYSLTISNIEP
`ekKkKe
`«et
`* *
`GKAPKLLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLOQP
`
`hu2.0
`
`hu2.10 GKAPKLLIYFITSSLHSGVPSRFSGSGSGTDYTLTISSLOP
`
`Fig. 5A
`
`90
`100
`A4.6.1 EDIATYYCQQYSTVPWIFGGGTKLEIK (SEQ 1p NO:16)
`*
`*
`*
`hu2.0
`EDFATYYCQQYSTVPWIFGQGTKVEIK (SEQ \o NO:12)
`
`hu2.10 EDFATYYCQQYSTVPWTFGOGTKVELK CSEQI\DNO: iS)
`
`- Vy domain
`
`10
`20
`30
`40
`Ad.6.1 BTOLVOSGPELKOPGETVRISCKASGYTFINYGMNWVKQA
`hu2.0
`EVOLVESGGGLVOPGGSLRLSCAASGYTFINYGHNWVROA
`hu2.10 EVQLVESGGGLVOQPGGSLRLSCAASGYTFTNYGMNWIROA
`
`*
`
`zeke «
`
`kkk «&
`
`*
`
`50a
`60
`70
`80
`A4.6.1 PGRGLEWMGWINTYTGEPTYAADFRRRFTFSLETSASTAYL
`hu2.0
`PGKGLEWVGWINTYTGEPTYAADFRRRFTISRDNSKNTLYL
`
`re
`
`«€
`
`k k&Kk&k xe
`
`Fig. 58
`
`hu2.10 PGKGLEWVGWINTYTGEPTYAADFRKRRFTISLDTSASTVYL
`
`kee «&£KE
`
`abe
`90
`100abcdef
`110
`A4.6.1 OPSULKNDDTATYPCAKYPEYYGSSHWYFDVWGAGTTVTVSS (SEQ OND:a)
`hu2.0
`OMNSLRAEDTAVYYCARYPHYYGSSHWYFDVWGOGTLVTVSS CSE@ \oNo* ie)
`hu2.10 QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGOGTLVTVss (SEQ (p NO: \6)
`
`* «€
`
`«
`
`Novartis Exhibit 2012.008
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.008
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 6 of 16
`
`US 6,884,879 B1
`
`
`
`Novartis Exhibit 2012.009
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`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, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 8 of 16
`
`US 6,884,879 B1
`
`OVGYSODYSOY
`SLODLLIYOSY
`ONdLWS9905
`WWWLOSW5OV
`1519959599
`SWOWLSOVOL
`WOLIVOLISS
`OOWOVWOOWS
`LVWWWO5955
`IVIWWOSOLI
`
`SIVLODLOOL
`OWDOWWLOOL
`SSQWVLOS999
`LILILIWOOLIOL
`WOWIDDOOOD
`DLOLWOOLSY
`LOWWLOWWOO
`
`SOLSLISSLO
`WILLLS9O999
`VWIVILSOOVY
`
`LOWOYDO999
`OWOLOLIOVY
`VIWOLOLOOV
`OWWOLLLIOL
`WLLOVWWWW
`LOVOLDOLOO
`LVOOWWOLIYV
`LLOVWOVWWWL
`SOWDLWS099
`SLOOLTOOWSS
`
`VSIDIB99959
`SLOVOVWOLL
`IVLOWOWSOL
`OLLOWWWWoW
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`Novartis Exhibit 2012.011
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.011
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 9 of 16
`
`US 6,884,879 B1
`
`TOTT,
`
`Novartis Exhibit 2012.012
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 10 of 16
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`US 6,884,879 B1
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`Novartis Exhibit 2012.013
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 11 of 16
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`US 6,884,879 B1
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`Novartis Exhibit 2012.014
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 12 of 16
`
`US 6,884,879 B1
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`Novartis Exhibit 2012.015
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 13 of 16
`
`US 6,884,879 B1
`
`= differences from F(ab)-12
`
`F(ab)-12
`MB1.6
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`Novartis Exhibit 2012.016
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.016
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 14 of 16
`
`US 6,884,879 B1
`
`F(ab)-
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`
`Novartis Exhibit 2012.017
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.017
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`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, IPR2020-01318
`
`
`
`U.S. Patent
`
`Apr. 26, 2005
`
`Sheet 16 of 16
`
`US 6,884,879 B1
`
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`Novartis Exhibit 2012.019
`Regeneron v. Novartis, IPR2020-01318
`
`Novartis Exhibit 2012.019
`Regeneron v. Novartis, IPR2020-01318
`
`
`
`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. Nova