(12) United States Patent
`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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`Novartis Exhibit 2012.002
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`US 6,884.879 B1
`Page 3
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`* cited by examiner
`
`Novartis Exhibit 2012.003
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet 1 0f 16
`
`US 6,884,879 B1
`
`Variable Heavy
`
`A$.6.1
`
`F(6b)-12
`
`humIII
`
`EIQLVQSGPELKQPGETVRISCKASQXIEIEXQMHWVKQAPGKGLKWMG
`*
`t
`«4 t
`1*. t
`t
`t
`t *
`bVQLVESGGGLVQPGGSLRLSCAASGIIEINXQMEWVRQAPGKGLEWVG
`*
`it * i
`*
`PVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS
`‘
`10
`20
`30
`40
`
`A4 . 6 .1 WWRFTFSLETSASTAYLQISNLKNDDTATYFCAK
`*
`t
`*** ***
`f *
`F(ab)- 12 WRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAK
`f t *
`* **** *’*
`*** i
`i *
`*
`VISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
`so
`a
`60
`70
`so
`abc
`9o
`
`humIII
`
`1A
`
`“3’
`
`A4.6.1
`
`xgflxxcgsammvwcacmvrvss (SEQ \D mom)
`i
`*
`
`F(ab) ~12 Wmcocmwvss (SEQ. m Nor-D
`*
`i
`
`humIII
`
`G ---------- FDYWGQGTLVTVSS (9661 \D N01 ‘5
`110
`
`Variable Light
`
`A4.6.l
`
`DIQMTQTTSSLSASLGDRVIISCSSAS9218 NZLEWYQQKPDGTVKVLIY
`t*
`t t
`****
`F(ab)-12 DIQMTQSPSSLSASVGDRVTITCSSASQDISEZLEWYQQKPGKAPKVLIY
`*
`*
`t
`t
`
`humKI
`
`DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIY
`1
`10
`20
`30
`40
`
`Fig.
`
`IB
`
`A4.6.l
`
`,EISSLHEGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQIEIEEHIF
`Qt
`* t
`*
`F(ab) - 12 msg'gcvpsapscSGSGTDFTLTISSLQPED‘E-'ATYYCWF
`*1
`*
`*ti
`AASSLESHVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPWTF
`36
`66
`70
`80
`90
`
`humKI
`
`A4.6.1
`
`GGGTKIEIKR (SEQ m Not 103
`k
`t
`
`F(ab)-12 GQGTm-EIKR (SEQ. \D N028)
`
`humKI
`
`GQGTKVEIKR (SEQ \D N021?)
`100
`
`Novartis Exhibit 2012.004
`
`Regeneron V. Novartis, IPR2020-013 18
`
`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
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet 3 0f 16
`
`US 6,884,879 B1
`
`INoaddition
`
`OVEGF
`
`
`
`100010000
`
`100
`
`
`
`
`
`MAbConcentration(ng/ml)
`
`Fig.3
`
`>.
`
`D < Z3 E+ U
`
`.
`(9
`UJ
`
`0.1
`
`180000
`
`140000
`
`100000
`
`60000
`
`20000
`
`”8M Jed suao IBHGUIODUE!
`
`Novartis Exhibit 2012.006
`
`Regeneron V. Novartis, IPR2020-013 18
`
`U.
`(5
`Lu
`
`>‘D‘VEGF+rhuMAbVEGF
`
`10
`
`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
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet 5 0f 16
`
`US 6,884,879 B1
`
`Fig. 5A
`
`VL domain
`
`4 0
`3 0
`20
`10
`A4 . 6 . 1 DIQMTQTTSSLSASLGDRVIISCSASQDISNYLNWYQQKP
`t l'
`t
`t t
`
`11112 . 0
`
`DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP
`
`hu2 . 10 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP
`
`60
`7 0
`60
`5 o
`A4 . 6 . 1 DGTVKVLIYETSSLHSGVPSRFSGSGSGTDYSLTISNLEP
`* t t l- t
`t t
`I
`t
`
`hu2 . o
`
`GKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQP
`
`hu2 . 10 GKAPKLLIYETSSLESGVPSRFSGSGSGTDYTLTISSLQP
`
`A4 . 6 . 1
`
`9 0
`100
`EDIATYYCQQYSTVPWTFGGGTKLEIK (SECS \D No: \63
`t
`i-
`t
`
`hu2 . o
`
`EDFATYYCQQYSTVPWTFGQGTKVEIK ($63 \0 N O \37
`
`hu2. 10
`
`EDPATYYCQQYSTVPW‘I‘FGQGTKVEIK (.SEQ \D N 0'. IS}
`
`- VH domain
`
`A4.6. 1
`
`4 0
`3 0
`20
`10
`EIQLVQSGPELKQPGETVRISCKASGYTFTNYGMNWVKQA
`*
`t
`t t t
`i- t t t
`t
`
`hu2 . 0
`
`EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGWVRQA
`
`hu2 . 10 BVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGWWIRQA
`
`so
`70
`60
`so a
`A4. 6.1 PGKGLKWMGWINTYTGEPTYAADFKRRFTFSLETSASTAYL
`‘-
`i- t t
`i- i
`t
`
`Fig~ SB
`
`hu2 . 0
`
`PGKGLEWVGWINTY‘I‘GEPTYAADFKRRFTISRDNSKNTLYL
`
`hu2 . 10
`
`PGKGLEWVGWINTYTGEPTYAADPKRRPTISLDTSASTVYL
`
`110
`100abcdef
`90
`abc
`A4. 6.1 QISNLKNDDTATYFCAKYPEYYGSSHWYFDVWGAGTTVTVSS LS EQ \D No: a.)
`l- t t l- t t
`i I
`*
`
`hu2. 0
`
`QMNSLRAEDTAVYYCARYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ \D No- “Q
`
`hu2.10 QMNSLRAEDTAWicaxpoyrcssmmvwcocTLv'rvss (SEQ (D NOz‘Q
`
`Novartis Exhibit 2012.008
`
`Regeneron V. Novartis, IPR2020-013 18
`
`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
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet8 0f16
`
`US 6,884,879 B1
`
`un4m>nwhquw
`
`2
`
`0484004004
`0800884004
`0008400000
`4448004004
`8080000000
`0404800408
`4088408800
`0040440040
`8444400000
`8484400088
`
`0848008008
`0400448008
`0004800000
`8884008008
`4040000000
`0808400804
`8044804400
`0080880080
`4888800000
`4848800044
`
`8040400000
`0408088044
`4840808004
`0440888808
`4488044444
`8040800800
`8400440884
`8804404448
`0048840000
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`R3genenn1vfiNOvafim,IPR20204H318
`
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`
`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
`
`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
`
`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
`
`JEVOVALY,5)5O5).
`
`I089
`
`I069
`
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`
`Novartis Exhibit 2012.015
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet 13 0f 16
`
`US 6,884,879 B1
`
`= ditferences from F(ab)-12
`
`F(ab)-12
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`KPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDEELTIs
`KPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTIS
`
`Fig. 9A
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`CDR-L2
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`80
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`
`Novartis Exhibit 2012.016
`
`R3genenn1vfiNOvafim,IPR20204H318
`
`Novartis Exhibit 2012.016
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet 14 0f 16
`
`US 6,884,879 B1
`
`= differences from F(ab)-12
`F(ab)-12 DIQMTQSPSgiSASVGDRV%ETCSASQDIghYLNWYQQ
`Y0243-l DIQZEQSPSSLSASVGDRVTIT0P‘WWJ‘
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`
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`1713, 103
`
`Novartis Exhibit 2012.017
`
`Regeneron V. Novartis, IPR2020-013 18
`
`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
`
`

`

`US. Patent
`
`Apr. 26, 2005
`
`Sheet16 0f16
`
`US 6,884,879 B1
`
`
`
`
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`Novartis Exhibit 2012.019
`
`Regeneron V. Novartis, IPR2020-013 18
`
`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. Novartis, IPR2020-01318
`
`

`

`3
`identified heavy chain variable domain Sequences or with
`other heavy chain variable domain Sequences, p