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`Old's‘n9963L
`'no/Lz/nIIIIIIliIIIIIIIIIIIIIiIII
`
`
`
`
`
`Attorney Docket P1093P1D1
`PATENT
`
`
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BOX PATENT APPLICATION
`Assistant Commissioner of Patents
`Washington, DC. 20231
`
`NON-PROVISIONAL APPLICATION TRANSMITTAL UNDER 37 CFR 1.53 b
`
`E E
`3“: Es
`«$5.7, ES
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`
`
`
`Transmitted herewith for filing is a non—provisional patent application:
`
`Inventor(s) (or Application "Identifier"):
`
`Manuel Baca, Foster City, CA
`James A. Wells, Burlingame, CA
`Leonard G. Presta, San Francisco, CA
`
`Henry B. Lowman, El Granada, CA
`Yvonne Man-yee Chen, San Mateo, CA
`
`TMB:
`
`ANTI-VEGF ANTIBODIES
`
`Type of Application
`
`This application is for an original, non-provisional application.
`
`This is a non-provisional application claiming priority to provisional application no. _ , filed
`_, the entire disclosure of which is hereby incorporated by reference.
`
`application
`[X ] divisional
`] continuation
`[
`] continuation-in-part
`[
`This is a
`
`claiming priority to application Serial Number 08/908 469 , filed
`August 6, 1997 , the
`entire disclosure of which is hereby incorporated by reference.
`
`Papers Enclosed Which Are Required For Filing Date Under 37 CFR 1.53(b)
`(Non-provisional )
`
`80
`pages of specification
`5
`pages of claims
`1
`page(s) of abstract
`16
`sheet(s) of drawings
`[] formal
`[X] informal
`
`1.
`
`[
`
`[
`
`]
`
`]
`
`[X]
`
`2.
`
`#83589
`
`
`Regeneron Exhibit 1024.0001
`
`

`

`P1093P1D1
`
`Page 2 of3
`
`3.
`
`Declaration or Oath
`
`(for new and CIP applications; also for Cont/Div. where inventor(s) are being added)
`An executed declaration of the inventor(s) [] is enclosed
`[]will follow.
`
`(for Cont/Div. where inventorship is the same or inventor(s) being deleted)
`X A copy of the executed declaration/oath filed in the prior application is enclosed
`— (37 CFR1.63(d)).
`
`(for Cont/Div. where inventor(s) being deleted)
`A signed statement
`is attached deleting inventor(s) named in the prior
`application (see 37 CFR 1.63(d)(2) and 1.33(b)).
`
`4.
`
`Assignment
`
`(for new and CIP applications)
`An Assignment of the invention to GENENTECH, INC.
`attached Recordation Form Cover Sheet []wiil follow.
`
`[]
`
`is enclosed with
`
`(for cont/div.)
`X The prior application is assigned of record to Genentech, Inc.
`
`5.
`
`Amendments
`
`(for continuation and divisional applications)
`
`X
`
`Cancel in this application original claims 1-38 of the prior application before calculating the
`filing fee.
`(At least one original independent claim must be retained for filing purposes.)
`
`(Claims added by this amendment have been
`A preliminary amendment is enclosed.
`properly numbered consecutively beginning with the number next following the highest
`numbered original claim in the prior application.)
`
`Relate Back -- 35 U.S.C. 120 or 35 U.S.C. 119
`
`X
`
`Amend the specification by inserting before the first line the sentence:
`
`
`
`
`--This is a
`
`non-provisional application
`continuation
`divisional
`
`continuation-in—part
`
`of co-pending application(s)
`
`lit
`
`
`Serial No. 08/908 469 filed on August 6, 1997 , which application(s) is(are) incorporated
`herein by reference and to which application(s) priority is claimed under 35 USC §120. --
`International Application _ filed on _which designated the US, which application(s) is(are)
`incorporated herein by reference and to which application(s) priority is claimed under 35 USC
`§120.——
`
`#83589
`
`
`Regeneron Exhibit 1024.0002
`
`

`

`P1093P1D1
`
`Page 3 of3
`
`provisional application No. _filed _, the entire disclosure of which is hereby incorporated by
`reference and to which application(s) priority is claimed under 35 USC §119.--.
`
`6.
`
`Payment of Fees
`
`X
`
`Applicants request deferral of payment of the filing fee until submission of the missing parts
`of application. DO NOT CHARGE THE FILING FEE AT THIS TIME.
`
`7.
`
`Additional Papers Enclosed
`[]
`Information Disclosure Statement (37 CFR §1.98)w/ PTO-1449 and citations
`I]
`Submission of "Sequence Listing", computer readable copy, certificate re: sequence listing,
`and/or amendment pertaining thereto for biological invention containing nucleotide and/or amino
`acid sequence.
`[X] Associate Power of Attorney.
`[] Other:
`
`Maintenance of Copendency of Prior Application
`8.
`applications)
`[This item must be completed and the necessary papers filed in the prior application if the period set
`in the prior application has run]
`
`(for continuation and divisional
`
`A petition, fee and/or response has been filed to extend the term in
`the pending prior application until
`A copy of the petition for extension of time in the prior application
`is attached.
`
`9.
`
`Correspondence Address:
`
`X
`
`Address all future communications to:
`
`Attn: Steven X. Cui
`GENENTECH, INC.
`1 DNA Way
`South San Francisco, CA 94080-4990
`
`(650) 225-8674
`
`
`
`
`
`
`Respectfully submitted,
`GENENTECH, INC.
`
`By:
`
`1 gig Q X
`
`Steven X. Cui
`
`( Lag
`
`Reg. No. 44,637
`Telephone No. (650) 225-8674
`
`Date: November 27, 2000
`
`lllllll|||l|lllllllllllllllllllllll
`09157
`PATENT TRADEMARK OFFICE
`
`#83589
`
`
`Regeneron Exhibit 1024.0003
`
`

`

`Express Mail No.: EL599584388US
`Atty. Docket No.: P1093P1D1
`Date Mailed: November 27, 2000
`
`ANTI-VEGF ANTIBODIES
`
`CROSS REFERENCES
`
`This application is a continuation-in-part of co-pending US. Application Serial No.
`
`10
`
`08/833,504, filed April 7, 1997, which application is incorporated herein by reference and to which
`
`application priority is claimed under 35 USC §120.
`
`BACKGROUND OF THE INVENTION
`
`
`
`
`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.
`
`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 degeneration (AMD), 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: Pathobio/ogy of ocular disease. A
`
`25
`
`dynamic approach. GarnerA, 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 microvessels 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
`
`30
`
`(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-or, TGF-B, HGF, TNF-a, angiogenin, lL-8, etc. (Folkman et al. and Klagsbrun
`
`
`
`Regeneron Exhibit 1024.0004
`
`

`

`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. 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)).
`
`5
`
`'
`
`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. 1814-25 (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
`
`10
`
`a key mediator of neovascularization associated with tumors and intraocular disorders (Ferrara
`
`
`
`
`
`
`5,; 3
`.20
`
`
`
`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. 732931-934 (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 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); Borgstrom et al. Cancer Res. 56:4032—4039 (1996); and Melnyk et al.
`
`Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesis 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
`
`25
`
`treatment of solid tumors and various intraocular neovascular disorders.
`
`SUMMARY OF THE INVENTION
`
`This application describes humanized anti-VEGF antibodies and anti-VEGF antibody variants
`
`with desirable properties from a therapeutic perspective, including strong binding affinity for
`
`30
`
`VEGF; the ability to inhibit VEGF-induced proliferation of endothelial cells in vitro; and the ability
`
`
`Regeneron Exhibit 1024.0005
`
`

`

`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,1 value of no more than about 1 x 10‘3M and preferably no more than about
`5 x 10'9M. In addition, the humanized or variant anti—VEGF antibody may have an ED50 value of
`
`5
`
`no more than about 5nM for inhibiting VEGF-induced proliferation 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
`
`5mg/kg.
`In one embodiment, the anti-VEGF antibody has a heavy and light chain variable domain,
`
`10
`
`wherein the heavy chain variable domain comprises hypervariable regions with the following
`amino acid sequences: CDRH1 (GYX1FTX2YGMN, wherein X1 is T or D and X2 is N or H; SEQ
`
`
`
`
`
`
`ID NO:128),
`CDRH2
`(WINTYTGEPTYAADFKR;
`SEQ ID N022)
`and
`CDRH3
`(YPX1YYGXZSHWYFDV, wherein X1 is Y or H and X2 is S or T; SEQ ID NO:129). For example,
`the heavy chain variable domain may comprise the amino acid sequences of CDRH1
`(GYTFTNYGMN; SEQ iD N021), CDRH2 (WINTYTGEPTYAADFKR;SEQ ID N02) and CDRH3
`
`(YPHYYGSSHWYFDV;SEQ lD N013). 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:
`
`EVQLVESGGGLVQPGGSLRLSCAASGYX1FTX2YGMNVWRQAPGKGLEWVGWlNTYTGEPT
`
`YAADFKRRFTFSLDTSK STAYLQMNSLRAEDTAVYYCAKYPX3YYGX4SHWYFDVWGQGTLV
`
`TVSS (SEQ ID NO:125), wherein X1 is T or D; X2 is N or H; X3 is Y or H and X4 is S or T. One
`
`particularly useful heavy chain variable domain sequence is that of the F(ab)-12 humanized
`antibody of Example 11 and comprises the heavy chain variable domain sequence of SEQ ID
`
`25
`
`N027. 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, provided that the antibody so produced binds human VEGF.
`
`The invention also provides preferred light chain variable domain sequences which may be
`
`30
`
`combined with the above-identified heavy chain variable domain sequences or with other heavy
`
`
`Regeneron Exhibit 1024.0006
`
`

`

`chain variable domain sequences, provided that the antibody so produced retains the ability to
`
`bind to human VEGF. For example, the light chain variable domain may comprise hypervariable
`
`regions with the following amino acid sequences: CDRL1 (SASQDISNYLN; SEQ ID N024),
`
`CDRL2 (FTSSLHS; SEQ lD N015) and CDRL3 (QQYSTVPWT; SEQ ID NO:6). Preferably, the
`
`5
`
`three light chain hypervariable regions are provided in a human framework region, 6.9., as a
`
`contiguous sequence represented by the following formula: FR1-CDRL1-FR2-CDRL2-FR3-
`
`CDRL3-FR4.
`
`In one embodiment, the invention provides a humanized anti-VEGF antibody light chain
`
`variable domain comprising the amino acid sequence:
`
`10
`
`DIQX1TQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFS
`
`GSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID N02124), wherein
`
`X1 is M or L. One particularly useful light chain variable domain sequence is that of the F(ab)-12
`
`humanized antibody of Example 1 and comprises the light chain variable domain sequence of
`
`SEQ lD N028.
`
`The invention also provides a variant of a parent anti-VEGF antibody (which parent antibody
`
`is preferably a humanized or human anti-VEGF antibody), Wherein the variant binds human
`
` VEGF and comprises an amino acid substitution in a hypervariable region of the heavy or light
` amino acid subsition(s) may be in the CDRH1 and/or CDRH3 of the heavy chain variable domain.
`
`chain variable domain of the parent anti-VEGF antibody. The variant preferably has one or more
`
`substitution(s)in one or more hypervariable region(s) of the anti-VEGF antibody. Preferably, the
`
`substitution(s)are in the heavy chain variable domain of the parent antibody. For example, the
`
`Preferably, there are substitutions in both these hypervariable regions. Such “affinity matured”
`
`variants are demonstrated herein to bind human VEGF more strongly than the parent anti-VEGF
`
`antibody from which they are generated, i.e., they have a K<1 value which is significantly less than
`
`25
`
`that of the parent anti-VEGF antibody. Preferably, the variant has an E050 value for inhibiting
`
`VEGF-induced proliferation of endothelial cells in vitro which is at least about 10 fold lower,
`
`preferably at least about 20 fold lower, and most preferably at least about 50 fold lower, than that
`
`of the parent anti-VEGF antibody. One particularly prefered variant is the Y0317 variant of
`
`Example 3, which has a CDRH1 comprising the amino acid sequencezGYDFTHYGMN (SEQ D
`
`30
`
`NO:126) and a CDRH3 comprising the amino acid sequencezYPYYYGTSHWYFDV (SEQ ID
`
`
`Regeneron Exhibit 1024.0007
`
`

`

`NO:127). These hypervariable regions and CDRH2 are generally provided in a human framework
`region, e.g., resulting in a heavy chain variable domain comprising the amino acid sequence of
`SEQ ID NO:116. Such heavy chain variable domain sequences are optionally combined with a
`
`light chain variable domain comprising the amino acid sequence of SEQ ID NO:124, and
`
`5
`
`preferably the light chain variable domain amino acid sequence of SEQ ID NO:115.
`
`Various forms of the antibody are contemplated herein. For example, the anti-VEGF
`
`antibody may be a full length antibody (e.g. having an intact human Fc region) or an antibody
`
`fragment (e.g. a Fab, Fab‘ or F(ab')2). Furthermore, the antibody may be labeled with a
`
`detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound
`
`10
`
`(such as a cytotoxic agent).
`
`Diagnostic and therapeutic uses for the antibody are contemplated.
`
`In one diagnostic
`
`application, the invention provides a method for determining the presence of VEGF protein
`
`comprising exposing a sample suspected of containing the VEGF protein to the anti-VEGF
`
`antibody and determining binding of the antibody to the sample. For this use, the invention
`
` provides a kit comprising the antibody and instructionsfor using the antibody to detect the VEGF
`
`
`protein.
`
`The invention further provides:
`
`isolated nucleic acid encoding the antibody; a vector
`
`comprising that nucleic acid, optionally operably linked to control sequences recognized by a host
`
` cell transformed with the vector; a host cell comprising that vector; a process for producing the
`
`
`
`antibody comprising culturing the host cell so that the nucleic acid is expressed and, optionally,
`
`recovering the antibody from the host cell culture (9.9. from the host cell culture medium). The
`
`invention also provides a composition comprising the anti-VEGF antibody and a pharmaceutically
`
`acceptable carrier or diluent. The composition for therapeutic use is sterile and may be
`
`lyophilized. The invention further provides a method for treating a mammal suffering from a
`
`2 5
`
`tumor or retinal disorder, comprising administering a therapeutically effective amount of the anti-
`
`VEGF antibody to the mammal.
`
`Figs. 1A and 1B depict the amino acid sequences of variable heavy domain (SEQ ID N029)
`
`3 0
`
`and light domain (SEQ lD NO:10) of muMAbVEGFA.4.6.1 , variable heavy domain (SEQ ID NO:7)
`
`Brief Description of the Drawings
`
`
`Regeneron Exhibit 1024.0008
`
`

`

`and light domain (SEQ ID N018) of humanized F(ab) (F(ab)-12) and human consensus
`frameworks (hum III for heavy subgroup III (SEQ ID NO:11); humK1 for light K subgroup | (SEQ
`ID NO:12)). Fig. 1A aligns variable heavy domain sequences and Fig. 1B aligns variable light
`domain sequences. Asterisks indicate differences between humanized F(ab)-12 and the murine
`
`5
`
`MAb or between F(ab)—12 and the human framework. Complementarity Determining Regions
`
`(CDRs) are underlined.
`Fig. 2 is a ribbon diagram of the model of humanized F(ab)-12 VL and VH domains. VL
`domain is shown in brown with CDRs in tan. The sidechain of residue L46 is shown in yellow. VH
`
`domain is shown in purple with CDRs in pink. Sidechains of VH residues changed from human
`
`10
`
`to murine are shown in yellow.
`
`Fig. 3 depicts inhibition of VEGF-induced mitogenesis by humanized anti-VEGF F(ab)—12from
`Example 1. Bovine adrenal cortex-derived capillary endothelial cells were seeded at the density
`of 6 X 103 cells/well in six well plates, as described in Example 1. Either muMAb VEGF A4.6.1
`
`or rhuMAb VEGF (IgG1; F(ab)-12) was added at the indicated concentrations. After 2-3 hours,
`
`rhVEGF165 was added at the final concentration of 3 ng/ml. After five or six days, cells were
`
`trypsinized and counted. Values shown are means of duplicate determinations. The variation from
`
`the mean did not exceed 10%.
`Fig. 4 shows inhibition of tumor growth in vivo by humanized anti-VEGF F(ab)-12 from
`
`
`
`
`
`..
`-
`
` 2 x 106 per mouse. Starting 24 hours after tumor cell inoculation, animals were injected with a
`
`Example 1. A673 rhabdomyosarcoma cells were injected in BALB/c nude mice at the density of
`
`
`
`control MAb, muMAb VEGF A4.6.1 or rhuVEGF MAb (lgG1; F(ab)—12) twice weekly, intra
`
`peritoneally.The dose of the control Mab was 5 mg/kg; the anti—VEGF MAbs were given at 0.5 or
`
`5 mg/kg, as indicated (n = 10). Four weeks after tumor cell injection, animals were euthanized and
`
`tumors were removed and weighed. *: significantdifferencewhen compared to the control group
`
`25
`
`by ANOVA (p < 0.05).
`
`Figs. 5A and SB show the acid sequences of the light and heavy variable domains
`
`respectively of murine antibody A4.6.1 (SEQ ID NO:10 for the VL and SEQ ID NO:9 for the VH)
`
`and humanized A4.6.1 variants hu2.0 (SEQ ID NO:13 for the VL and SEQ ID NO:14 for the VH)
`
`and hu2.10 (SEQ ID NO:1 5 for the VL and SEQ ID NO:16 for the VH) from Example 2. Sequence
`
`3 o
`
`numbering is according to Kabat et 3]., Sequences of Proteins of Immunological Interest, 5th Ed.
`
`
`Regeneron Exhibit 1024.0009
`
`

`

`Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and mismatches are
`
`indicated by asterisks (murine A4.6.1 vs hu2.0) or bullets (hu2.0 vs hu2.10). Variant hu2.0
`
`contains only the CDR sequences (bold) from the murine antibody grafted onto a human light
`
`chain K subgroup I consensus framework (SEQ ID NO:12) and heavy chain subgroup Ill
`
`5
`
`consensus framework (SEQ ID N0111). hu2.10 was the consensus humanized clone obtained
`
`from phage sorting experiments described herein.
`
`Fig. 6 depicts framework residues targeted for randomization in Example 2.
`
`Fig. 7 depicts the phagemid construct for surface display of Fab-pill fusions on phage. The
`
`phagemid encodes a humanized version of the Fab fragment for antibody A4.6.1 fused to a
`
`10
`
`portion of the M13 gene lll coat protein. The fusion protein consists of the Fab joined at the
`
`carboxyl terminus of the heavy chain to a single glutamine residue (from suppression of an amber
`
`codon in supE E. coli), then the C-terminal region of the gene Ill protein (residues 249-406).
`
`Transformation into F" E. coli, followed by superinfection with M13KO7 helper phage, produces
`
`phagemid particles in which a small proportion of these display a single copy of the fusion protein.
`
`Figs. 8A-E depict the double stranded nucleotide sequence (SEQ ID NO:99) for phage-
`
`display antibody vector phMB4—19—1.6 in Example 3 and the amino acid sequence encoded
`
`
` CDRs are underlined and designated by L, light, or H, heavy chain, and numbers 1-3. Residues
`
`thereby (SEQ ID N01100).
`
`Figs. 9A and SB depict an alignment of the amino acid sequences for the light and heavy
`
`variable domains respectively of affinity matured anti-VEGF variants in Example 3, compared to
`
`F(ab)-12 of Example 1 (SEQ ID NOS 8 and 7 for light and heavy variable domains, respectively).
`
`are numbered sequentially in the VL and VH domains, as opposed to the Kabat numbering
`
`scheme. The template molecule, MB1.6 (SEQ ID NO’s 101 and 102 for light and heavy variable
`
`domains, respectively) is shown, along with variants: H2305.6 (SEQ ID N05 103 and 104 for light
`
`25
`
`and heavy variable domains, respectively),Y0101 (SEQ ID N05 105 and 106 for light and heavy
`
`variable domains, respectively), and Y0192 (SEQ ID NO’s 107 and 108 for light and heavy
`
`variable domains, respectively). Differences from F(ab)-12 are shown in shaded boxes.
`
`Figs. 10A and 1GB depict an alignment of the amino acid sequences for the light and heavy
`
`variable domains respectively of affinity matured anti-VEGF variants from Example 3 compared
`
`30
`
`to F(ab)—12 of Example 1
`
`(SEQ ID NOS 8 and 7 for light and heavy variable domains,
`
`Regeneron Exhibit 1024.0010
`
`

`

`respectively). CDRs are underlined and designated by L, light, or H, heavy chain. and numbers
`1-3. The variants are designated Y0243-1 (SEQ lD N05 109 and 110 for light and heavy variable
`
`domains, respectively),Y0238—3 (SEQ ID N03 111 and 112 for light and heavy variable domains,
`
`respectively), Y0313-1 (SEQ ID NO’s 113 and 114 for light and heavy variable domains,
`respectively), and Y0317 (SEQ ID NO’s 115 and 116 for light and heavy variable domains,
`
`5
`
`respectively). Differences from F(ab)-12 are shown in shaded boxes.
`Fig. 11 depicts the results of the HuVEC activity assay in Example 3 for variants Y0238-3,
`
`YO192 and Y0313-1 as well as full length F(ab)—12 from Example 1.
`
`Fig. 12 depicts inhibition of VEGF-induced mitogenesis by full length F(ab)-12 from Example
`1 (rhuMAb VEGF), a Fab fragment of F(ab)-12 from Example 1 (rhuFab VEGF), and a Fab
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`10
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`fragment of affinity matured variant Y0317 from Example 3 (rhuFab VEGF (affinity matured».
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`Detailed Description of the Preferred Embodiments
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`The term "human VEGF" as used herein refers to the 165-amino acid human vascular
`
` I. Definitions
`
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`endothelial cell growth factor, and related 121-, 189-, and 206-amino acid vascular endothelial cell
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`growth factors, as described by Leung et al., Science 24611306 (1989), and Houck et al., Mo].
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`Endocn‘n. 5: 1 806 (1991) together with the naturally occurring allelic and processed forms of those
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`growth factors.
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` The present invention provides anti-VEGF antagonistic antibodies which are capable of
`inhibiting one or more of the biological activities of VEGF, for example, its mitogenic or angiogenic
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`activity. Antagonists of VEGF act by interfering with the binding of VEGF to a cellular receptor,
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`by incapacitating or killing cells which have been activated by VEGF, or by interfering with vascular
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`endothelial cell activation after VEGF binding to a cellular receptor. All such points of intervention
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`25
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`by a VEGF antagonist shall be considered equivalent for purposes of this invention.
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`The term "VEGF receptor" or "VEGFr" as used herein refers to a cellular receptor for VEGF,
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`ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants thereof
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`which retain the ability to bind hVEGF. One example of a VEGF receptor is the fms-like tyrosine
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`kinase (flt), a transmembrane receptor in the tyrosine kinase family. DeVries et al., Science
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`30
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`255:989 (1992); Shibuya et al., Oncogene 5:519 (1990). The fit receptor comprises an
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`Regeneron Exhibit 1024.0011
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`extracellulardomain, a transmembrane domain, and an intracellular domain with tyrosine kinase
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`activity. The extracellular domain is involved in the binding of VEGF, whereas the intracellular
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`domain is involved in signal transduction. Another example of a VEGF receptor is the flk—1
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`receptor (also referred to as KDR). Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991);
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`5
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`Terman et al., Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys. Res. Commun.
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`18721579 (1992). Binding of VEGF to the fit receptor results in the formation of at least two high
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`molecularweight complexes, having apparent molecular weight of 205,000 and 300,000 Daltons.
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`The 300,000 Dalton complex is believed to be a dimer comprising two receptor molecules bound
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`to a single molecule of VEGF.
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`10
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`The term "epitope A4.6.1" when used herein, unless indicated otherwise, refers to the region
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`of human VEGF to which the A4.6.1 antibody disclosed in Kim et al., Growth Factors 7:53 (1992)
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`and Kim et al. Nature 3622841 (1993), binds.
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`"Treatment" refers to both therapeutictreatment and prophylactic or preventative measures.
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`Those in need of treatment include those already with the disorder as well as those in which the
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`"Mammal" for purposes of treatment refers to any animal classified as a mammal, including
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`humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats,
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`cows, etc. Preferably, the mammal is human.
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` disorder is to be prevented.
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`"Antibodies" (Abs) and "immunoglobulins"(lgs) are glycoproteins having the same structural
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`characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins
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`include both antibodies and other antibody-like molecules which lack antigen specificity.
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`Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and
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`at increased levels by myelomas.
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`"Native antibodies" and "native immunoglobulins"are usually heterotetrameric glycoproteins
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`2 S
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`of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H)
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`chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the
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`number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes.
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`Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy
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`chain has at one end a variable domain (VH) followed by a number of constant domains. Each
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`30
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`light chain has a variable domain at one end (VL) and a constant domain at its other end; the
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`constant domain of the light chain is aligned with the first constant domain of the heavy chain, and
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`the light— chain variable domain is aligned with the variable domain of the heavy chain. Particular
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`amino acid residues are believed to form an interface between the light- and heavy-chain variable
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`domains.
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`5
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`The term "variable“ refers to the fact that certain portions of the variable domains differ
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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 for its particular antigen. However, the variability is not evenly distributed
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`throughout the variable domains of antibodies.
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`It is concentrated in three segments called
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`hypervariable regions both in the light chain and the heavy chain variable domains. The more
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`10
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`highly conserved portions of variable domains are called the framework region (FR). The variable
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`domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,
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`respectively), largely adopting a B—sheet configuration, connected by three hypervariable regions,
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`which form loops connecting, and in some cases forming part of, the B-sheet structure. The
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`hypervariable regions in each chain are held togetherin close proximity by the FRs and, with the
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` of antibodies (see Kabat et aI., Sequences of Proteins of Immunological Interest, 5th Ed. Public
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`hypervariable regions from the other chain, contribute to the formation of the antigen—binding site
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` 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
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`effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
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` antibody which are responsible for antigen—binding. The hypervariable region comprises amino
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`The term “hypervariable region” when used herein refers to the amino acid residues of an
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`acid residues from a “complementaritydetermining region” or “CDR” (i.e. residues 24-34 (L1 ), 50-
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`56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
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`(H3) in the heavy chain variable domain; Kabat et a/., Sequences of Proteins of Immunological
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`25
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`Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or
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`those residues from a “hypervariableloop” (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in
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`the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
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`variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
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`"Framework" or "FR"
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`residues are those variable domain residues other than the hypervariable region residues as
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`3 0
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`herein defined.
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`10
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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 fragment that has two
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`antigen-combining sites and is still capable of cross-linking antigen.
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`5
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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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`domain in tight, non-covalent association.
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`It is in this configuration that the three hypervariable
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`regions of each variable domain interact to define an antigen-binding site on the surface of the VH-
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`VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the
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`10
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`antibody. However, even a single variable domain (or half of an Fv comprising only three
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`hypervariable regions specific for an antigen) has the ability to recognize and bind antigen,
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`although at a lower affinity than the entire binding site.
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`The Fab fragment also contains the constant domain of the 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 of a
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`few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more
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`cysteine(s) from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which
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`the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments
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`originally were produced as pairs of Fab' fragments which have hinge cysteines between them.
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`Other chemical couplings of antibody fragments are also known.
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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 (K) and lambda (A), based on the amino
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`acid sequences of their constant domains.
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`Depending on the amino acid sequence of the constant domain of their heavy chains,
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`immunoglobulins can be assigned to different classes. There are five major classes of
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`25
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`immunoglobulins: lgA, lgD, lgE, lgG, and lgM, and several of these may be further divided into
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`subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA, and IgA2. The heavy-chain constant
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`domains that correspond to the different classes of immunoglobulins are called or, 6, e, v, and u,
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`respectively. The subunit structu