High-affinity anti-VEGF antibody

[0001] The present invention relates to a high-affinity anti-VEGF antibody.

[0002] Vascular endothelial growth factor (VEGF) can induce vasculogenesis (angiogenesis) in vivo, and is a heparin-binding growth factor that is specifically expressed in vascular endothelial cells. Human VEGF protein was purified and identified in 1989. The gene thereof has been cloned and thus the gene sequence has been determined.

[0003] VEGF can accelerate vasculogenesis, and all members belonging to the VEGF family bind to receptors (VEGFR) corresponding to respective family molecules existing on cell membrane surfaces and thus to activate cells. As a result of dimerization due to binding of VEGF, the group of these receptors undergoes auto-phosphorylation and is thus activated. VEGFR consists of seven immunoglobulin-like extracellular domains, one transmembrane domain, and one intracellular domain containing a tyrosine kinase region. VEGF-A can bind to VEGF receptor-1 (receptor Flt-1) and VEGF receptor-2 (KDR/Flk-1). Among VEGF receptors, particularly, VEGF receptor-2 mediates almost all known biological functions of VEGF. Regarding VEGF physiological activity and VEGF receptors, Marti et al., (Non Patent Literature 1: Angiogenesis in ischemic disease. Thromb. Haemost. 1999. Supplement 1: 44-52) and Matsumoto et al., (Non Patent Literature 2: VEGF Receptor Signal Transduction. Sci. STKE. 2001. re21) have studied in detail.

[0004] In the U.S. and Europe, Avastin® (bevacizumab) that is a recombinant anti-VEGF humanized monoclonal antibody is used for treatment of colorectal cancer, breast cancer, non-small-cell lung cancer, glioma in the central nervous system, and age-related macular degeneration (AMD). The sales of Avastin in 2013 reached US$6,746,000,000, but Avastin does not have high affinity for VEGF (Non Patent Literature 3: Aflibercept as a Treatment for Age-related Macular Degeneration. US Ophthalimic Rev. 2013. 6:58-63). Furthermore, the exclusive production and exclusive form of selling of Avastin force patients who need to use Avastin to pay high medical expenses. Therefore, development of a new anti-VEGF monoclonal antibody is required also from viewpoints of easing the burden of patients and reducing the costs of treatment.

[0005] Antibodies having affinity for VEGF higher than that of Avastin have been developed to date, and such antibodies exert tumor suppression superior to Avastin (Patent Literature 1: JP Laid-Open Publication No. 2013-502445).

• [0006] Patent Literature 1: JP Laid-Open Publication No. 2013-502445

• [0007] Non Patent Literature 1: Marti et al., Thromb. Haemost. 1999. Supplement 1: 44-52
• [0008] Non Patent Literature 2: Matsumoto et al., VEGF Receptor Signal Transduction. Sci. STKE. 2001. re2l
• [0009] Non Patent Literature 3: US Ophthalimic Rev., 2013. 6:58-63

[0010] The present invention has been achieved under such circumstances. A technical problem to be solved by the present invention is to provide a novel antibody that inhibits the physiological activity of VEGF by binding to a vascular endothelial growth factor (VEGF) with its high affinity for VEGF compared to the prior art to inhibit binding of VEGF to a VEGF receptor.

[0011] As a result of intensive studies to solve the problem, the present inventors have prepared an antibody that binds to VEGF with high affinity, have discovered that the antibody inhibits the physiological action of VEGF, and thus have completed the present invention.

[0012] That is, the present invention is as follows.

(1) A monoclonal antibody against VEGF that binds to a vascular endothelial growth factor (VEGF) with a dissociation constant of 1×10⁻¹¹ mol/L or less.

[0013] (2) The monoclonal antibody according to (1) above, wherein the monoclonal antibody inhibits binding of a vascular endothelial growth factor (VEGF) to at least one receptor selected from the group consisting of vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2).
(3) A monoclonal antibody that binds to a site to which the monoclonal antibody according to (1) or (2) above binds.
(4) The monoclonal antibody according to any one of (1) to (3) above, wherein the monoclonal antibody is a chimeric antibody or a humanized antibody.
(5) The antibody according to any one of (1) to (4) above, comprising CDR-H1 that comprises the amino acid sequence of SEQ ID NO: 14, CDR-H2 that comprises the amino acid sequence of SEQ ID NO: 16, and CDR-H3 that comprises the amino acid sequence of SEQ ID NO: 18.
(6) The antibody according to any one of (1) to (4) above, comprising CDR-L1 that comprises the amino acid sequence of SEQ ID NO: 20, CDR-L2 that comprises the amino acid sequence of Trp-Ala-Ser, and CDR-L3 that comprises the amino acid sequence of SEQ ID NO: 22.
(7) The antibody according to any one of (1) to (4) above, comprising:
CDR-H1 that comprises the amino acid sequence of SEQ ID NO: 14, CDR-112 that comprises the amino acid sequence of SEQ ID NO: 16, and CDR-H3 that comprises the amino acid sequence of SEQ ID NO: 18; and
CDR-L1 that comprises the amino acid sequence of SEQ ID NO: 20, CDR-L2 that comprises the amino acid sequence of Trp-Ala-Ser, and CDR-L3 that comprises the amino acid sequence of SEQ ID NO: 22.
(8) The antibody according to any one of (1) to (4) above, comprising CDR-H1 that comprises the amino acid sequence of SEQ ID NO: 24, CDR-H2 that comprises the amino acid sequence of SEQ ID NO: 26, and CDR-H3 that comprises the amino acid sequence of SEQ ID NO: 28.
(9) The antibody according to any one of (1) to (4) above, comprising CDR-L1 that comprises the amino acid sequence of SEQ ID NO: 30, CDR-L2 that comprises the amino acid sequence of Gly-Thr-Asn, and CDR-L3 that comprises the amino acid sequence of SEQ ID NO: 32.
(10) The antibody according to any one of (1) to (4) above, comprising CDR-H1 that comprises the amino acid sequence of SEQ ID NO: 24, CDR-H2 that comprises the amino acid sequence of SEQ ID NO: 26, and CDR-H3 that comprises the amino acid sequence of SEQ ID NO: 28, and, CDR-L1 that comprises the amino acid sequence of SEQ ID NO: 30, CDR-L2 that comprises the amino acid sequence of Gly-Thr-Asn, and CDR-L3 that comprises the amino acid sequence of SEQ ID NO: 32.
(11) The antibody according to (7) or (10) above, further comprising an amino acid sequence derived from a human IgG1 heavy chain constant region and an amino acid sequence derived from a human IgG1 light chain constant region.
(12) The antibody according to (11) above, wherein the amino acid sequence derived from a human IgG1 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 42, and the amino acid sequence derived from a human IgG1 light chain constant region comprises the amino acid sequence of SEQ ID NO: 44.
(13) The antibody according to (12) above, comprising:
a heavy chain that comprises the amino acid sequence of SEQ ID NO: 34 or 38, and the amino acid sequence of SEQ ID NO: 42; and
a light chain that comprises the amino acid sequence of SEQ ID NO: 36 or 40, and the amino acid sequence of SEQ ID NO: 44.
(14) The antibody according to (7) or (10) above, further comprising an amino acid sequence derived from a canine IgGB heavy chain constant region and an amino acid sequence derived from a canine Ig light chain (κ chain) constant region or a canine Ig light chain (λ chain) constant region.
(15) The antibody according to (14) above, wherein an amino acid sequence derived from a canine IgGB heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 46, an amino acid sequence derived from a canine Ig light chain (κ chain) constant region comprises the amino acid sequence of SEQ ID NO: 48, and an amino acid sequence derived from a canine Ig light chain (λ chain) constant region comprises the amino acid sequence of SEQ ID NO: 50.
(16) The antibody according to (15) above, comprising:
a heavy chain that comprises the amino acid sequence of SEQ ID NO: 34 or 38 and the amino acid sequence of SEQ ID NO: 46; and
a light chain that comprises the amino acid sequence of SEQ ID NO: 36 or 40 and the amino acid sequence of SEQ ID NO: 48 or 50.
(17) A fragment of the monoclonal antibody according to any one of (1) to (16) above.
(18) The fragment according to (17) above, wherein the fragment is an antigen-binding fragment.
(19) The fragment according to (18) above, wherein the antigen-binding fragment is a single-chain antibody or a double-chain antibody.
(20) A hybridoma that produces the monoclonal antibody according to any one of (1) to (3) above.
(21) A pharmaceutical composition comprising the monoclonal antibody or the fragment thereof according to any one of (1) to (19) above.
(22) The pharmaceutical composition according to (21) above, for use in treatment or prevention of a VEGF-mediated disease.
(23) The pharmaceutical composition according to (22) above, wherein the VEGF-mediated disease is a cancer or a VEGF-mediated eye disease.
(24) The pharmaceutical composition according to (23) above, wherein the treatment or prevention of the cancer or the VEGF-mediated eye disease is by inhibiting angiogenesis or vascular hyperpermeability.
(25) The pharmaceutical composition according to (24) above, wherein the angiogenesis is pathological angiogenesis.
(26) The pharmaceutical composition according to (23) or (24) above, wherein the cancer is solid cancer or hematologic neoplasm.
(27) The pharmaceutical composition according to (23) or (24) above, wherein the cancer is selected from the group consisting of colorectal cancer, rectal cancer, breast cancer, non-small-cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreas cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid tumor, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma.
(28) The pharmaceutical composition according to (23) or (24) above, wherein the VEGF-mediated eye disease is at least one selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, neovascular glaucoma, retinal vein occlusion, retinopathy of prematurity, choroidal neovascularization associated with pathological myopia, pterygium, rubeosis, pannus, Stevens-Johnson syndrome, and an immunological rejection in a transplanted tissue of the eye.
(29) An angiogenesis inhibitor comprising the monoclonal antibody or the fragment thereof according to any one of (1) to (19) above.
(30) A reagent comprising the monoclonal antibody or the fragment thereof according to any one of (1) to (19) above.
(31) A kit comprising the monoclonal antibody or the fragment thereof according to any one of (1) to (19) above.
(32) A method for treating or preventing a cancer or a VEGF-mediated eye disease, comprising a step of administering to a subject a therapeutically effective amount of the antibody or the fragment thereof according to any one of (1) to (19) above.
(33) The antibody or the fragment thereof according to any one of (1) to (19) above, for use in a method for treating or preventing a cancer or a VEGF-mediated eye disease.
(34) The antibody or the fragment thereof according to any one of (1) to (19) above, for use in the manufacture of a medicament for treatment or prevention of a cancer or a VEGF-mediated eye disease.

[0014] According to the present invention, a novel antibody against VEGF that binds with significantly high affinity compared to the prior art to a vascular endothelial growth factor (VEGF) to inhibit its binding to receptor.

[0025] The present invention will be described in detail as follows. The following embodiments are merely examples for describing the present invention, and are not intended to limit the present invention only to these embodiments. The present invention can be implemented in various modes without departing from the scope of the invention. Moreover, this description includes the contents as disclosed in the specification and drawings of Japanese Patent Application (Japanese Patent Application No. 2016-001277) filed on Jan. 6, 2016, which is priority document of the present application.

1. Summary

[0026] Currently, Avastin is known as an anti-VEGF antibody effective for treatment of cancer and age-related macular degeneration, however, Avastin is known to have low affinity for VEGF.

[0027] On the other hand, the present inventors have developed an antibody having significantly high affinity for VEGF compared to the prior art, and thus have discovered that the antibody is effective for suppression of angiogenesis and treatment of cancer.

[0028] The present invention has been completed based on these findings.

2. Vascular Endothelial Growth Factor (VEGF)

[0029] VEGFs are proteins playing an important role in angiogenesis. VEGFs involve cell division and migration, induction of differentiation, vascular hyperpermeability, activation of monocytes and macrophages, and the like through binding to their receptors to cause intracellular signal transduction.

[0030] In the present invention, examples of VEGF include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PIGF-1, and PIGF-2, and a preferable example thereof is VEGF-A.

[0031] VEGF in the present invention may be derived from any mammal. Examples of such a mammal include mice, rats, rabbits, dogs, goats, monkeys and humans, preferably, mice, rats, dogs and humans.

[0032] Examples of human VEGF-A include VEGF (VEGF₁₆₅) consisting of 165 amino acid residues, a subtype thereof; that is, VEGF (VEGF₁₂₁) consisting of 121 amino acid residues, VEGF (VEGF₁₄₅) consisting of 145 amino acid residues, VEGF (VEGF₁₈₃) consisting of 183 amino acid residues, VEGF (VEGF₁₈₉) consisting of 189 amino acid residues, VEGF (VEGF₂₀₆) consisting of 206 amino acid residues, VEGF (VEGF_165b) differing from the above VEGF₁₆₅ in the amino acid sequence of the C-terminal region, and naturally-occurring allelic variants thereof and processing variants thereof. In the present invention, as examples of human VEGF-A, VEGF₁₂₁ and VEGF₁₆₅ are preferred. VEGF is encoded on chromosome 6p12, and the mRNA is 16,272 bp long. VEGF is consisting of exons 1 to 5, and 6a, 6b, 7a, 7b, 8a and 8b. VEGF₁₆₅ binds to all of NRP1, VEGFR1 and VEGFR2.

[0033] Furthermore, examples of canine VEGF include, but are not limited to, VEGF (VEGF₁₆₄) consisting of 164 amino acid residues, VEGF (VEGF₁₂₀) consisting of 120 amino acid residues, VEGF (VEGF₁₄₄) consisting of 144 amino acid residues, VEGF (VEGF₁₄₇) consisting of 147 amino acid residues, VEGF (VEGF₁₆₂) consisting of 162 amino acid residues, VEGF (VEGF₁₈₂) consisting of 182 amino acid residues, VEGF (VEGF₁₈₈) consisting of 188 amino acid residues, VEGF (VEGF₂₀₅) consisting of 205 amino acid residues, VEGF (VEGF_164b) differing from the above VEGF₁₆₄ in the amino acid sequence of the C-terminal region, and naturally occurring allelic variants thereof and processing variants thereof.

[0034] In the present invention, the amino acid sequences of mouse, rat, canine and human VEGFs, and VEGF₁₂₁ and VEGF₁₆₅ are represented by SEQ ID NOS: 2, 4, 6, 8, 10, and 12, respectively. Moreover, the base (nucleotide) sequences of DNA encoding mouse, rat, canine and human VEGFs, and VEGF₁₂₁ and VEGF₁₆₅ are represented by SEQ ID NOS: 1, 3, 5, 7, 9 and 11, respectively. These amino acid sequences and nucleotide sequences are each registered in the GenBank database under given Accession Nos.

[0035] Mouse VEGF amino acid sequence: NP_001020421.2 (SEQ ID NO: 2)

Rat VEGF amino acid sequence: NP_114024.2 (SEQ ID NO: 4)

Canine VEGF amino acid sequence: NP_001003175 (SEQ ID NO: 6)

Human VEGF-A amino acid sequence: NP_001020537.2 (SEQ ID NO: 8)

VEGF₁₂₁ amino acid sequence: ABO26344.1 (SEQ ID NO: 10)

VEGF₁₆₅ amino acid sequence: AAM03108.1 (SEQ ID NO: 12)

Mouse VEGF-encoding DNA nucleotide sequence: NM_001025250.3 (SEQ ID NO: 1)

Rat VEGF-encoding DNA nucleotide sequence: NM_031836.2 (SEQ ID NO: 3)

Canine VEGF-encoding DNA nucleotide sequence: NM_001003175.2 (SEQ ID NO: 5)

Human VEGF-A-encoding DNA nucleotide sequence: NM_001025366.2 (SEQ ID NO: 7)

VEGF₁₂₁-encoding DNA nucleotide sequence: EF424789.1 (SEQ ID NO: 9)

VEGF₁₆₅-encoding DNA nucleotide sequence: AF486837.1 (SEQ ID NO: 11)

[0036] VEGFs to be used in the present invention include the following proteins (a) to (c):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12;

[0037] (b) a protein comprising an amino acid sequence on which one or several amino acids are deleted, substituted or added with respect to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and having binding activity to a VEGF receptor; and
(c) a protein comprising an amino acid sequence that has 80% or higher homology (identity) with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and having binding activity to a VEGF receptor.

[0038] In the present invention, examples of “a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12” include a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12.

[0039] Furthermore, examples of “an amino acid sequence on which one or several amino acids are deleted, substituted or added with respect to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12” include:

(i) an amino acid sequence on which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and further preferably 1) amino acids are deleted with respect to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12;

[0040] (ii) an amino acid sequence on which 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and further preferably 1) amino acids are substituted with other amino acids with respect to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12;
(iii) an amino acid sequence on which (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and further preferably 1) amino acids are added with respect to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12; and
(iv) an amino acid sequence mutated as a combination of (i) to (iii) above.

[0041] In the present invention, “VEGF receptor” is at least one VEGF receptor selected from the group consisting of vascular endothelial growth factor receptor-1 (VEGFR1 (another name: Flt-1)), vascular endothelial growth factor receptor-2 (VEGFR2 (another name: KDR)) and vascular endothelial growth factor receptor-3 (VEGFR3), and is preferably vascular endothelial growth factor receptor-1 (VEGFR1) and/or vascular endothelial growth factor receptor-2 (VEGFR2), and more preferably vascular endothelial growth factor receptor-2 (VEGFR2). Moreover, the term “binding activity to a VEGF receptor” means activity of specifically binding to a VEGF receptor. The presence or the absence of the binding activity can be determined using a known method, such as immunological techniques including immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay) and the like or pull-down assay. Furthermore, the term “binding activity to a VEGF receptor” means having at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more activity, when compared to the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, which is set to 100%.

[0042] Furthermore, examples of VEGF in the present invention include, in addition to a protein having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, a protein having an amino acid sequence that has 80% or more homology (identity) with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and having binding activity to a VEGF receptor. Examples of such a protein also include a protein having an amino acid sequence that has about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and having binding activity to a VEGF receptor (an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12). Homology can be determined using a homology search site on the Internet, for example, by performing a homology search program such as FASTA, BLAST, PSI-BLAST or the like on the DNA Data Bank of Japan (DDBJ). Homology search can also be performed using BLAST on the National Center for Biotechnology Information (NCBI).

[0043] Mutagenesis of DNA encoding the relevant protein in order to prepare a protein having the above mutation can be performed using a kit for mutagenesis using site-directed mutagenesis such as Kunkel method, Gapped duplex method or the like, such as QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km and the like: Takara Bio) or the like. Moreover, a method such as a site-directed mutagenesis method described in “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)) can be used.

3. Antibody Against VEGF

[0044] In the present invention, the term “antibody against VEGF” (hereafter, may also be referred to as “anti-VEGF antibody”) refers to an antibody that specifically binds to the above VEGF. The anti-VEGF antibody of the present invention binds to VEGF with high affinity compared to the prior art to inhibit binding of VEGF to a VEGF receptor (VEGFR).

[0045] Note that, bevacizumab (Avastin®) that is well known as an anti-VEGF antibody is known to have a dissociation constant of 47.9 nmol/L for binding to VEGF.

[0046] In the present invention, the term “inhibit binding” does not always means 100% inhibition of the binding of VEGF to the VEGFR. The antibody of the present invention inhibits binding of VEGF to the VEGFR, for example, 50% or more, preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more thereof.

[0047] An inhibiting effect on the binding can be evaluated using a known method for a binding inhibition test, such as a method used in Example 2 of the Description.

[0048] The antibody of the present invention is a neutralizing antibody that neutralizes VEGF activity through its specific binding to VEGF. In the present invention, the term “specifically bind to” means binding to (reacting with) a target molecule, but substantially not binding (not reacting with) molecules other than the target molecule. Furthermore, in the present invention, the term “neutralize” at least means to inhibit (suppress) the activity of VEGF to bind to VEGFR. Whether or not binding is specific can be confirmed by immunological techniques, such as ELISA, Western blot method or immunohistological staining.

[0049] Hereafter, a method for preparing an anti-VEGF antibody is described.

(1) Preparation of Antigen

[0050] VEGF is used as an immunogen for preparing the antibody of the present invention.

[0051] When VEGF is used as an immunogen, a peptide comprising an amino acid sequence that is a portion of the full length sequence of VEGF can also be used. VEGF to be used as an antigen or an immunogen and methods for introducing mutations are as described in the above “2. VEGF”.

[0052] VEGF may be natural VEGF purified from a mouse, rat, dog, or human tissues or cells, for example, or VEGF produced via genetic engineering techniques. For example, a biological sample confirmed to contain VEGF is fractionated into a soluble fraction and an insoluble fraction using various surfactants, such as Triton-X or Sarkosyl. The insoluble fraction is further dissolved in urea, guanidine hydrochloride or the like, and allow to bind to various columns, such as a heparin column or a binding resin, so that VEGF can be obtained. Moreover, VEGF to be used as an antigen can also be synthesized by specifying the amino acid sequence and then using a known protein synthesis method such as a solid phase method or a commercially available protein synthesizer. A synthesized peptide is bound to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or thyroglobulin and thus the resultant can be used as an immunogen.

(2) Preparation of Polyclonal Antibody

[0053] The above-prepared VEGF or a partial peptide alone is administered or the same is administered together with a carrier, a diluent or the like to a non-human mammal, such as a rabbit, a dog, a guinea pig, a mouse, a rat, or a goat for immunization. The dosage of the antigen per animal ranges from 0.1 mg to 10 mg when an adjuvant is used. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and an aluminum hydroxide adjuvant. Immunization is performed by mainly intravenous, subcutaneous, intraperitoneal injection, or the like. Furthermore, the immunization interval is not particularly limited, and immunization is performed at intervals of several days to several weeks, and preferably at intervals of 1 to 2 weeks for 2 to 10 times and preferably 3 to 5 times. The immunization interval can be determined by persons skilled in the art considering the resulting antibody titer. Preferably, blood is sampled when subcutaneous immunization is performed 3 to 4 times and then the antibody titer is measured. Antibody titer in serum can be measured by ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), radioimmunoassay (RIA) or the like. After confirmation of a sufficient increase in antibody titer, whole blood is collected, and then an antibody can be separated and purified by a generally employed method. Regarding separation and purification, a known method such as an ammonium sulfate salting-out method, ion exchange chromatography, gel filtration chromatography, and affinity chromatography is adequately selected or combined, and then purification can be performed. That is, a serum containing a target antibody is applied to a column to which proteins other than VEGF have been bound, a flow-through fraction is collected, and then a polyclonal antibody having improved specificity to VEGF can be obtained.

(3) Preparation of Monoclonal Antibody

(i) Collection of Antibody-Producing Cells

[0054] In a manner similar to that for preparation of a polyclonal antibody, VEGF or a partial peptide alone or the same together with a carrier or a diluent is administered to a non-human mammal for immunization. The dosage of an antigen per animal, the type of an adjuvant to be used, an immunization method, and immunization intervals are similar to those for preparation of a polyclonal antibody. One to 30 days, and preferably 2 to 5 days after the final immunization date, individuals confirmed to have antibody titers are selected, and then antibody-producing cells are collected. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and among them spleen cells or lymph node cells are preferred.

(ii) Cell Fusion

[0055] To obtain hybridomas, cell fusion of antibody-producing cells and myeloma cells is performed. Cell fusion procedures can be implemented according to a known method such as the method of Kohler et al. As myeloma cells to be fused to antibody-producing cells, generally available established cell lines of an animal such as a mouse can be used. Cell lines that can be preferably used herein have drug selectivity and have a property of being unable to survive in HAT selective medium (containing hypoxanthine, aminopterin, and thymidine) when the cells are in an unfused state, but able to survive only in a state fused to antibody-producing cells. Examples of myeloma cells include mouse myeloma cell lines such as P3-x63-Ag8U.1, SP2/O-Ag14, PAI, P3U1, NSI/1-Ag4-1, and NSO/I, and rat myeloma cell lines such as YB2/0.

[0056] The cell fusion of myeloma cells and antibody-producing cells described above is performed by mixing 1×10⁸ to 5×10⁸ antibody-producing cells and 2×10⁷ to 10×10⁷ myeloma cells in animal cell culture medium such as serum-free DMEM and RPMI-1640 medium (the cell ratio of antibody-producing cells to myeloma cells ranges from 10:1 to 1:1), and then performing fusion reaction in the presence of a cell fusion accelerator. As a cell fusion accelerator, polyethylene glycol having an average molecular weight of 1000-6000 daltons or Sendai virus can be used, for example. In addition, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(iii) Hybridoma Selection and Cloning

[0057] A target hybridoma is selected from cells after cell fusion treatment. Such a method is performed by appropriately diluting a cell suspension with 10% to 20% fetal calf serum-containing RPMI-1640 medium or the like, placing the resultant on a microtiter plate at about 0.3 cells/well as calculated by a limiting dilution method, adding selective medium such as HAT medium to each well, and then culturing while appropriately exchanging selective media. As a result, cells that grow around 10 days after the start of culture in selective media can be obtained as hybridomas.

[0058] Next, hybridomas that have grown are further screened. Hybridoma screening may be performed according to a general method and is not particularly limited. For example, a portion of a culture supernatant contained in wells in which hybridomas have been cultured is collected and then can be screened by enzyme immunoassay, radioimmunoassay or the like. As a specific example, an antigen is adsorbed to a 96-well plate, followed by blocking with calf serum. The culture supernatant of hybridoma cells is reacted with an immobilized antigen at 37° C. for 1 hour, and then reacted with peroxidase-labeled anti-mouse IgG at 37° C. for 1 hour, for color development using orthophenylenediamine as a substrate. The reaction is stopped with acid, and then absorbance at a wavelength of 490 nm is measured, so that screening can be performed. Hybridomas producing a monoclonal antibody found positive as a result of the above measurement method is cloned by a limiting dilution method or the like. Finally, hybridomas that are cells producing the monoclonal antibody that specifically binds to VEGF are established.

(iv) Collection of Monoclonal Antibody

[0059] As a method for collecting a monoclonal antibody from the thus established hybridomas, a general cell culture method, an ascites formation method, or the like can be employed. According to a cell culture method, the hybridomas are cultured in animal cell culture medium such as 10% fetal calf serum-containing RPMI-1640 medium, MEM, or serum free medium under general culture conditions (for example, 37° C., 5% CO₂ concentration) for 7 to 14 days, and then an antibody is obtained from the culture supernatant. According to an ascites formation method, about 5×10⁶ to 2×10⁷ hybridomas are administered intraperitoneally to an animal of the same species as that of a mammal from which myeloma cells are derived, such as mouse (BALB/c), so that hybridomas are grown in large amounts. One to 2 weeks later, ascites is collected. When the above method for collecting an antibody requires purification of the antibody, purification can be performed by appropriately selecting a known method such as an ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, and affinity chromatography, or using these methods in combination.

[0060] Examples of the antibody of the present invention include, but are not limited to, an antibody wherein:

[0061] a heavy chain variable region (VH) comprises a heavy chain complementarity determining region (CDR)1 (CDR-H1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 14, heavy chain CDR2 (CDR-112) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 16, and/or heavy chain CDR3 (CDR-113) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 18; and/or
a light chain variable region (VL) comprises light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 20, light chain CDR2 (CDR-L2) comprising or consisting of the amino acid sequence that consists of tryptophan (W)-alanine (A)-serine (S) (may also be referred to as “amino acid sequence WAS”, “amino acid sequence Trp-Ala-Ser”), and/or light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 22.

[0062] In another aspect, examples of the antibody of the present invention include, but are not limited to, an antibody wherein:

[0063] a heavy chain variable region comprises heavy chain CDR1 (CDR-H1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 24, heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 26 and/or heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 28; and/or
a light chain variable region comprises light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 30, light chain CDR2 (CDR-L2) comprising or consisting of the amino acid sequence that consists of glycine (G)-threonine (T)-asparagine (N) (may also be referred to as “amino acid sequence GTN”, “amino acid sequence Gly-Thr-Asn”) and/or light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 32.

[0064] In another aspect, examples of the antibody of the present invention include, but are not limited to, an antibody wherein: the amino acid sequence of a heavy chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 34 or 38; and/or the amino acid sequence of a light chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 36 or 40.

(4) Preparation of Recombinant Antibody

[0065] A preferred aspect of the antibody of the present invention is a recombinant antibody. Examples of the recombinant antibody include, but are not limited to, a chimeric antibody, a humanized antibody, and a caninized antibody.

[0066] A chimeric antibody refers to an antibody prepared by linking immunoglobulin gene fragments of animals of different species. In the present invention, examples of a chimeric antibody include a humanized chimeric antibody, and a caninized chimeric antibody, but the types of animals, from which chimeric antibody variable and constant regions are derived, are not limited. A humanized chimeric antibody is an antibody prepared by linking (joining) a mouse-derived antibody variable region to a human-derived constant region, for example (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), for example). A caninized chimeric antibody is an antibody prepared by linking a mouse-derived antibody variable region to a dog-derived constant region, for example. When a chimera is prepared, this can be easily constructed by gene recombination techniques so that an antibody prepared through such ligation can be obtained. Here, examples of a mouse-derived antibody variable region include, but are not limited to, a heavy chain variable region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 34 or 38, and a light chain variable region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 36 or 40.

[0067] When a humanized antibody is prepared, a technique referred to as, namely, CDR grafting, can be employed. CDR grafting is a method for preparing a reconstructed variable region, whereby a complementarity determining region (CDR) is grafted from a mouse antibody variable region to a human variable region, so that the framework region (FR) is human-derived and CDR is mouse-derived. Next, such humanized reconstructed human variable region is ligated to a human constant region. Such a method for preparing a humanized antibody is known in the art (see e.g., Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); and Japanese Patent No. 2828340).

[0068] Here, examples of the amino acid sequence of mouse-derived CDR, which can be used for the humanized antibody of the present invention include, but are not limited to,

CDR1-3 of a heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3) comprising or consisting of the amino acid sequences represented by SEQ ID NO: 14, 16 and 18, respectively,

[0069] CDR1-3 of a light chain variable region (CDR-L1, CDR-L2 and CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 20, the amino acid sequence that consists of tryptophan (W)-alanine (A)-serine (S) (amino acid sequence Trp-Ala-Ser), and/or the amino acid sequence represented by SEQ ID NO: 22, respectively.

[0070] In another aspect, examples of the amino acid sequences of mouse-derived CDRs that can be used for the humanized antibody of the present invention include, but are not limited to,

CDR1-3 of a heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3) comprising or consisting of the amino acid sequences represented by SEQ ID NO: 24, 26, and 28, respectively,

[0071] CDR1-3 of a light chain variable region (CDR-L1, CDR-L2 and CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 30, the amino acid sequence that consists of glycine (G)-threonine (T)-asparagine (N) (amino acid sequence Gly-Thr-Asn), and/or the amino acid sequence represented by SEQ ID NO: 32, respectively.

[0072] A caninized antibody can also be prepared by a technique similar to the above method for preparing a humanized antibody.

[0073] In the present invention, examples of a human heavy chain constant region that can be used for a chimeric antibody and a humanized antibody include, but are not limited to, a human heavy chain constant region comprising an amino acid sequence derived from a human IgG1 heavy chain constant region, and for example, a human heavy chain constant region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 42, and examples of a human light chain constant region include, but are not limited to, a human light chain constant region comprising an amino acid sequence derived from a human IgG1 light chain constant region, and for example, a human light chain constant region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 44. Furthermore, examples of DNA encoding a human heavy chain constant region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 41. Examples of DNA encoding a human light chain constant region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 43.

[0074] Furthermore, in the present invention, examples of a canine heavy chain constant region that can be used for a chimeric antibody and a caninized antibody include, but are not limited to, a canine heavy chain constant region comprising an amino acid sequence derived from the canine IgGB heavy chain constant region, and for example, a canine heavy chain constant region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 46. Moreover, examples of a canine light chain constant region that can be used for a chimeric antibody and a caninized antibody include, but are not limited to, a canine light chain constant region comprising an amino acid sequence derived from a canine Ig light chain (κ chain) constant region or an amino acid sequence derived from a canine Ig light chain (λ chain) constant region, and for example, a canine light chain (κ chain) constant region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 48, or a canine light chain (λ chain) constant region comprising or consisting of the amino acid sequence represented by SEQ ID NO: 50. Moreover, examples of DNA encoding a canine heavy chain constant region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 45. Examples of DNA encoding a canine light chain (κ chain or λ chain) constant region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 47 or 49.

[0075] In the present invention, a chimeric antibody, a humanized antibody, and a caninized antibody can be prepared according to the above-mentioned known method using a hybridoma or DNA, RNA or the like extracted from the hybridoma as a raw material.

[0076] Furthermore, the antibody of the present invention in the form of a fusion protein can be prepared according to a known gene recombination technique using antibody variable regions and another protein. The fusion protein can also be prepared by cross-linking a monoclonal antibody and another protein using a cross-linker.

(5) Preparation of Antibody Fragment

[0077] A fragment of the antibody against VEGF to be used in the present invention specifically binds to VEGF.

[0078] An antibody fragment refers to a polypeptide containing a partial region of the antibody of the present invention. As an antibody fragment, an antigen-binding fragment is preferred. Examples of the antigen-binding fragment include, but are not limited to, single-chain antibodies (scFv (single chain Fv)), sc (Fv)₂), double-chain antibodies (Fab, Fab′, diabody (diabody (dibodies), dsFv), and F(ab′)₂. The above antibody fragment can be obtained by cleaving the antibody of the present invention with various proteases depending on purposes.

[0079] For example, Fab can be obtained by treating an antibody molecule with papain, and F (ab′)₂ can be obtained by treating an antibody molecule with pepsin. Moreover, Fab′ can be obtained by cleaving the disulfide bond of the above F(ab′)₂ hinge region.

[0080] In the case of scFv, cDNA encoding antibody H chain V region and L chain V region is obtained and then DNA encoding scFv is constructed. The DNA is inserted into an expression vector, and then the expression vector is introduced into a host organism for expression, so that scFv can be produced.

[0081] In the case of a diabody, cDNA encoding antibody H chain V region and L chain V region is obtained, and then DNA encoding scFv is constructed so that a peptide linker has an amino acid sequence with a length of 8 or less residues. The DNA is inserted into an expression vector, and then the expression vector is introduced into a host organism for expression, so that a diabody can be produced.

[0082] In the case of dsFv, cDNA encoding antibody H chain V region and L chain V region is obtained, and then DNA encoding dsFv is constructed. The DNA is inserted into an expression vector, and then the expression vector is introduced into a host organism for expression, so that dsFv can be produced.

[0083] In the present invention, examples of the nucleotide sequence of DNA encoding a heavy chain variable region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 33 or 37, and examples of the nucleotide sequence of DNA encoding a light chain variable region include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 35 or 39.

[0084] Specific examples of the antibody fragment of the present invention include, but are not limited to, an antibody fragment comprising:

[0085] a heavy chain variable region (VH) that comprises heavy chain CDR1 (CDR-H1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 14, heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 16, and/or heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 18; and/or a light chain variable region (VL) that comprises light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 20, light chain CDR2 (CDR-L2) comprising or consisting of the amino acid sequence that consists of tryptophan (W)-alanine (A)-serine (S) (amino acid sequence Trp-Ala-Ser) and/or light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 22.

[0086] In another aspect, specific examples of the antibody fragment of the present invention include, but are not limited to, an antibody fragment comprising:

[0087] a heavy chain variable region comprises heavy chain CDR1 (CDR-H1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 24, heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 26 and/or heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 28, and/or
a light chain variable region comprises light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 30, light chain CDR2 (CDR-L2) comprising or consisting of the amino acid sequence that consists of glycine (G)-threonine (T)-asparagine (N) (amino acid sequence Gly-Thr-Asn), and/or light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence represented by SEQ ID NO: 32.

[0088] Moreover, examples of the antibody fragment of the present invention include, but are not limited to, an antibody fragment wherein a heavy chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 34 or 38, and/or a light chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 36 or 40.

[0089] An antibody fragment (peptide) comprising CDR is composed of at least one region of VH or VL CDRs (CDR1-3). An antibody fragment comprising a plurality of CDRs can be bound directly or via an appropriate peptide linker. An antibody fragment comprising CDR can be produced by constructing DNA encoding antibody VH and VL CDRs, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into prokaryotes or eukaryotes for expression. Furthermore, a peptide comprising CDR can also be produced by a chemical synthesis method such as a Fmoc method (fluorenylmethyloxycarbonyl method) and a tBoc method (t-butyloxycarbonyl method).

[0090] Examples of DNA encoding VH CDR1-3 include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 13, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 15 and DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 17, respectively. Examples of DNA encoding VL CDR1-3 include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 19, DNA comprising or consisting of the nucleotide sequence that consists of thymine (T)-guanine (G)-guanine (G)-guanine (G)-cytosine (C)-adenine (A)-thymine (T)-cytosine (C)-cytosine (C) (also referred to as “nucleotide sequence TGGGCATCC”), and DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 21, respectively.

[0091] In another aspect, examples of DNA encoding VH CDR1-3 include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 23, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 25 and DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 27, respectively. Examples of DNA encoding VL CDR1-3 include, but are not limited to, DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 29, DNA comprising or consisting of the nucleotide sequence that consists of guanine (G)-guanine (G)-thymine (T)-adenine (A)-cytosine (C)-cytosine (C)-adenine (A)-adenine (A)-cytosine (C) (may also be referred to as “nucleotide sequence GGTACCAAC”), and DNA comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 31, respectively.

(6) Binding Affinity

[0092] Binding affinity can be determined by the binding constant (KA) and dissociation constant (KD). Affinity equilibrium constant (K) is expressed by the ratio of the KA/KD. Its binding affinity can be detected as follows.

[0093] Binding constant (KA) and dissociation constant (KD) can be measured using surface plasmon resonance (SPR), and known devices and methods for detecting binding rates in real time and monitoring can be employed (e.g. Biacore®-3000 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.).

[0094] The antibody of the present invention can inhibit its target with an IC50 of 1×10⁻⁷ mol/L or less (for example, 1×10⁻⁷ mol/L or less, 1×10⁻⁸ mol/L or less, 1×10⁻⁹ mol/L or less, preferably 1×10⁻¹⁰ mol/L or less, more preferably 1×10⁻¹¹ mol/L or less).

[0095] The present invention provides an antibody that binds to a site to which the antibody against VEGF of the present invention binds. In detail, examples of the antibody against VEGF of the present invention include an antibody that binds to a site, to which a monoclonal antibody against VEGF that binds to VEGF with a dissociation constant of 1×10⁻¹¹ mol/L or less.

[0096] A site to which the anti-VEGF antibody of the present invention binds is not limited, as long as it is at least a partial region of VEGF that is an antigen. Examples of a site to which the anti-VEGF antibody of the present invention binds include, at least one region selected from the group consisting of exon 1, 2, 3, 4, 5, 6a, 6b, 7a, 7b, and 8a, and exon 1 to 5 regions are preferred. Persons skilled in the art can specify exon 1 to 5 regions of various VEGFs based on known information such as Genbank. For example, the amino acid sequences of mouse, rat, canine, human VEGF exon 1 to 5 are represented by SEQ ID NO: 51, 52, 53 and 54, respectively.

[0097] Persons skilled in the art can specify sites (e.g., regions and epitopes) or polypeptides containing them, to which the anti-VEGF antibody of the present invention binds, based on the Description and known techniques such as epitope mapping or X-ray structural analysis.

[0098] Furthermore, an antibody that binds to a site, to which the above monoclonal antibodies bind, may be a polyclonal antibody or a monoclonal antibody. When such an antibody is a monoclonal antibody, the antibody may be a recombinant antibody, such as a chimeric antibody, a humanized antibody, or a caninized antibody.

[0099] An antibody that binds to a site, to which the antibody against VEGF of the present invention binds, competes with the antibody against VEGF of the present invention for binding with VEGF. Persons skilled in the art can understand that an antibody competing with the antibody against VEGF of the present invention for binding with VEGF has specificity and/or activity equivalent to that of the antibody of the present invention (Specifically, a monoclonal antibody against VEGF, which binds to VEGF with a dissociation constant of 1×10⁻¹¹ mol/L or less).

[0100] A competition test using an antibody is a technique established in the technical field of antibodies as a technique for examining if a set of antibodies bind to the same (or redundant) site (see e.g., Ju-Won Kwak et al., Journal of Immunological Methods 191 (1996) 49-54). In the competition test, when the binding of the antibody against VEGF of the present invention is competitively inhibited by an anti-VEGF antibody to be tested, the test subject anti-VEGF antibody can be identified to be an antibody that binds to a site, to which the antibody against VEGF of the present invention binds. Furthermore, the test method does not require information about the structure of the relevant site for examining if a set of antibodies bind to the same (or redundant) site.

[0101] That is, persons skilled in the art can obtain an antibody binding to a site, to which the antibody against VEGF of the present invention binds, by conducting a competition test using the antibody against VEGF of the present invention without excessive experimentation.

4. Pharmaceutical Composition

[0102] The pharmaceutical composition of the present invention comprises the antibody or a fragment thereof described in the above “3. Antibody against VEGF” as an active ingredient, and is used for a target disease. The pharmaceutical composition of the present invention is used for prevention or treatment of target diseases and is effective for the prevention or the treatment.

[0103] Diseases targeted by the pharmaceutical composition of the present invention are not limited, as long as they are VEGF-associated diseases (including symptoms (conditions)), and examples of such diseases include solid tumors, cancers including particularly problematic metastatic tumors, and VEGF-mediated eye diseases, and other immune diseases.

[0104] Tumor vascular structures are known to supply blood, and also function as a vascular niche environment supporting cancer stem cells. The vascular niche environment refers to an environment involving maintenance, proliferation and differentiation of hematopoietic stem cells that are sources of blood and blood vessels, and is considered to be important for cancer stem cell proliferation.

[0105] Unlike normal cells, cancer cells are characterized by high proliferative ability, unlimited number of cell divisions, and ability to cause invasion and metastasis to peripheral tissues. In recent years, it has become considered that not all cancer cells in a cancer tissue have such properties, and limited partial cells have such properties. That is, these partial cancer cells are cells having properties observed in common among stem cells such as embryonic stem cells and somatic stem cells, including self-replicating ability, by which the cells can create cells completely identical thereto, and pluripotency, by which the cells are capable of differentiating into many cell types. The partial cancer cells are considered to be cancer stem cells that function as a source for creating the majority of peripheral cancer cells via differentiation while maintaining cells identical to themselves in the cancer tissue through self-replication.

[0106] Cancer stem cells are considered to be a major cause of cancer recurrence and cancer metastasis, and thus importance of targeting cancer stem cells in cancer treatment has been indicated. If significant inhibition of cancer stem cell proliferation within tumor tissue becomes possible, a novel treatment capable of killing entire cancer cells effectively can be developed.

[0107] The term “cancer sternness” in the present invention refers to a property of a cell population exerting the properties of cancer stem cells. Specifically, cancer sternness refers to properties of having self-replication and pluripotency peculiar to stem cells, and a property of cells functioning as a source of cancer.

[0108] In the present invention, examples of cancer include brain tumor, cervical cancer, esophageal cancer, cancer on the tongue, lung cancer, breast cancer, pancreatic cancer, gastric cancer, cancer of small bowel, duodenal cancer, colon cancer, bladder cancer, renal cancer, liver cancer, prostate cancer, uterine cancer, uterine cervix cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, melanoma, leukemia, lymphoma, and multiple myeloma.

[0109] In the present invention, VEGF-mediated eye disease is VEGF-associated disease that is caused by pathological angiogenesis or vascular hyperpermeability in the eye. That is, the VEGF-mediated eye disease can be treated or prevented by inhibiting binding of VEGF to a VEGF receptor using the anti-VEGF antibody. Examples of such disease include age-related macular degeneration (including special types such as polypoidal choroidal vasculopathy and retinal angiomatous proliferation), diabetic retinopathy, diabetic macular edema, neovascular glaucoma, retinal vein occlusion, retinopathy of prematurity, choroidal neovascularization associated with pathological myopia, pterygium, rubeosis, pannus, Stevens-Johnson syndrome, and immunological rejection of transplanted tissue (e.g., corneal tissue) of the eye.

[0110] In the present invention, the term “pathological angiogenesis” refers to angiogenesis that takes place, in morbidity or a state inducing morbidity, when new blood vessels grow excessively, insufficiently, or inappropriately (for example, the position or timing is undesirable from a medical viewpoint, or in development) compared with normal physiological angiogenesis (vasculogenesis).

[0111] In the present invention, the term “treatment”, “treat”, or “treating” refers to contacting (for example, administrating) the antibody or a fragment thereof or the pharmaceutical composition of the present invention (hereafter, may also be referred to as “the pharmaceutical composition of the present invention or the like”) with a subject after the onset of the disease, thereby alleviating the symptoms of the disease, compared to when the subject is not contacted with the pharmaceutical composition of the present invention or the like. The treatment as used herein does not always refer to completely suppressing the symptoms of a disease. The term “onset of a disease” refers to the appearance of the symptoms of the disease in the body.

[0112] In the present invention, the term “prevention”, “prevent”, or “preventing” refers to contacting (for example, administrating) the pharmaceutical composition of the present invention or the like with a subject before the onset of a disease, thereby alleviating symptoms of the disease after the onset of the disease, compared to when the subject is not contacted with the pharmaceutical composition of the present invention or the like. The term “prevention” does not always refer to completely suppressing the onset of the disease.

[0113] The pharmaceutical composition of the present invention can comprise, in addition to the antibody against VEGF of the present invention, a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to any carrier (e.g., liposome, lipid microcapsule, and micelle), diluent, excipient, wetting agent, buffering agent, suspending agent, lubricant, adjuvant, emulsifier, disintegrator, absorbent, preservative, surfactant, colorant, flavoring, or sweetener appropriate for the pharmaceutical composition for immune diseases.

[0114] The pharmaceutical composition of the present invention or the like can be formulated into preparations for injection, freeze-dried products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, cataplasm, ointments, cream pharmaceuticals, eye drops, and the like. Liquid preparations such as preparations for injection may be in the form of powder to be prepared before use (for example, freeze-dried powder), which is dissolved in saline or the like before use.

[0115] The pharmaceutical composition of the present invention or the like can be administered topically or systemically through any means known by persons skilled in the art. The route of administration of the pharmaceutical composition of the present invention can be oral administration and parenteral administration. In the case of parenteral administration, intratissue administration (e.g., subcutaneous administration, intraperitoneal administration, intramuscular administration, and intravenous administration), intradermal administration, local administration (e.g., transdermal administration) or transrectal administration can be performed. The pharmaceutical composition of the present invention can be administered in dosage forms appropriate for these routes of administration.

[0116] The dosage of the pharmaceutical composition of the present invention or the like can be varied depending on factors such as a subject's age, body weight, health status, gender, and symptoms, and the animal species of a subject, the route of administration, the frequency of administration, and a dosage form, and specific procedures for administration can be determined by persons skilled in the art. The dosage of the antibody of the present invention for treatment of cancer ranges from, for example, 0.1 mg to 100 mg/day, preferably 1 mg to 15 mg/day, and more preferably 2 mg to 12 mg/day per kg body weight of a subject, but the examples are not limited thereto. Regarding the frequency of administration, the pharmaceutical composition can be administered once to 5 times a day. The dosage of the antibody of the present invention for treatment of VEGF-mediated eye disease ranges from, for example, 0.01 mg to 100 mg/eye, and more preferably, 0.1 mg to 10 mg/eye. Regarding the frequency of administration, the antibody can be administered once a day to once every two months, but the examples are not limited thereto.

[0117] The timing of administration can be appropriately determined depending on symptoms, and several dosages can be administered simultaneously or separately at intervals. Furthermore, the pharmaceutical composition of the present invention may be administered to a subject before the onset of a disease or after the onset of the disease.

[0118] The pharmaceutical composition of the present invention can be administered to a mammal as a subject. Examples of mammals include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cattle, horses, monkeys, and humans.

5. Method for Treating or Preventing a Cancer or a VEGF-Mediated Eye Disease

[0119] According to the present invention, a cancer or a VEGF-mediated eye disease can be treated or prevented by administrating to a subject the antibody against VEGF or a fragment thereof or the pharmaceutical composition comprising the same. That is, the present invention provides a method for treating or preventing a cancer or a VEGF-mediated eye disease, comprising a step of administering to a subject a therapeutically effective amount of the antibody of the present invention or a fragment thereof or the pharmaceutical composition comprising the same. The therapeutically effective amount of the antibody of the present invention or a fragment thereof or the pharmaceutical composition comprising the same is varied depending on factors such as a subject's age, body weight, health status, gender, and symptoms, the route of administration, the frequency of administration, and a dosage form. Persons skilled in the art can easily determine the therapeutically effective amount required for treatment or prevention of a cancer or a VEGF-mediated eye disease. In the present invention, “subject” includes subjects in need of treatment or prevention of a cancer or a VEGF-mediated eye disease. In addition, mammals to be subjected to treatment or prevention as “subject” are as described above.

[0120] In the method for treating or preventing a cancer or a VEGF-mediated eye disease of the present invention, “cancer”, “VEGF-mediated eye disease”, “treatment” and “prevention” are as described above. Moreover, the dosage form, the route of administration, the dosage, the timing of administration and the like of the antibody of the present invention or a fragment thereof or the pharmaceutical composition comprising the same are also as described above.

6. Angiogenesis Inhibitor

[0121] The antibody of the present invention inhibits binding of VEGF to a VEGF receptor, and thus can inhibit angiogenesis resulting from the binding. Thus, the present invention provides an angiogenesis inhibitor comprising the antibody or a fragment thereof described in the above “3. Antibody against VEGF” as an active ingredient.

[0122] The angiogenesis inhibitor of the present invention can be used as a reagent or used for treatment of a mammal, and its dosage form, additives, route of administration, administration target, dosage and the like can be appropriately selected according to the description of the above “4. Pharmaceutical composition”. However, the angiogenesis inhibitor of the present invention may be an inhibitor comprising the antibody of the present invention or a fragment thereof alone.

7. Use of Antibody

[0123] The antibody of the present invention or a fragment thereof can be used in a method for treating or preventing a cancer or a VEGF-mediated eye disease, or the manufacture of a medicament for treatment or prevention of a cancer or a VEGF-mediated eye disease. Specifically, the present invention provides the anti-VEGF antibody of the present invention or a fragment thereof for use in the method for treating or preventing a cancer or a VEGF-mediated eye disease. Moreover, the present invention provides the anti-VEGF antibody of the present invention or a fragment thereof for use in the manufacture of a medicament for treatment or prevention of a cancer or a VEGF-mediated eye disease. Furthermore, the present invention provides the anti-VEGF antibody or a fragment thereof for use in the manufacture of an angiogenesis inhibitor.

[0124] In these aspects, “cancer”, “VEGF-mediated eye disease”, “treatment” and “prevention” are as described above.

8. Combination Therapy

[0125] The pharmaceutical composition of the present invention can be used for administration in combination with at least one of other anticancer agents. Examples of anticancer agents to be used in the present invention include sorafenib (Nexavar®), sunitinib (Sutent®), bevacizumab (Avastin®), cisplatin (cDDP), carboplatin (Paraplatin®), paclitaxel (Taxol®), docetaxel (Taxotere®), gemcitabine hydrochloride (Gemzar®), gefitinib (Iressa®), erlotinib (Tarceva®), irinotecan hydrochloride (CPT-11), and 5-fluorouracil (5-FU).

[0126] It can be expected that combined administration of the pharmaceutical composition of the present invention and at least one of anticancer agents exert more advantageous effects than an independent use thereof. Such advantageous effects include an effect on alleviating adverse effects more than conventional therapies while maintaining the therapeutic effects.

[0127] The term “used in combination” in the present invention refers to simultaneous administration of or separate administration of the pharmaceutical composition of the present invention and at least one of the above anticancer agents. The term “simultaneously” means that administration is performed at the same timing in a single administration schedule, but the times; that is, hours and minutes of administration are not required to be completely the same. The term “separately” means that administration is performed at different timings in a single administration schedule.

[0128] The dosage form, the route of administration, and the administration target of the pharmaceutical composition or the like and an anticancer agent to be used for combination therapy in the present invention are not particularly limited, and can be appropriately selected according to the description of the above “4. Pharmaceutical composition”. Moreover, the dosage forms or dosages of agents to be used in combination may differ from each other, and can be appropriately adjusted depending on a combination employed.

[0129] When the pharmaceutical composition of the present invention is used in combination with another anticancer agent, the dosage can be decreased as appropriate. Therefore, when the pharmaceutical composition of the present invention is combined with another anticancer agent, the following combinations of

(i) an effective dose of the pharmaceutical composition of the present invention and an effective dose of another anticancer agent,

(ii) an effective dose of the pharmaceutical composition of the present invention and a subeffective dose of another anticancer agent,

(iii) a subeffective dose of the pharmaceutical composition of the present invention and an effective dose of another anticancer agent, and

(iv) a subeffective dose of the pharmaceutical composition of the present invention and a subeffective dose of another anticancer agent can be employed.

[0130] Even in an aspect in which one of or both the pharmaceutical composition and an anticancer agent are used in subeffective doses, the two can be administered in combination in such an aspect, when the combination can exert pharmacological effects.

9. Reagent, Kit

[0131] The monoclonal antibody of the present invention or a fragment thereof can be included in a reagent for detection of VEGF or a kit. That is, the present invention provides a reagent and a kit comprising the monoclonal antibody of the present invention or a fragment thereof. The reagent and the kit of the present invention can be used as a reagent for detecting VEGF or a kit, for example.

[0132] In the reagent and the kit of the present invention, the monoclonal antibody of the present invention or a fragment thereof may be treated by a method such as freezing for easy handling, and then directly or mixed with a known pharmaceutically acceptable carrier, such as an excipient, an extending agent, a binder, and a lubricant, and a known additive (including a buffering agent, a tonicity agent, a chelating agent, a colorant, a preservative, an aroma chemical, a flavoring agent, a sweetening agent and the like), for example.

[0133] The kit of the present invention can comprise, in addition to the monoclonal antibody of the present invention or a fragment thereof, a buffer, an enzyme solution, a secondary antibody, a solution for dilution, instructions, and the like.

[0134] Hereafter, the present invention is described in detail by the Examples, but the present invention is not limited to the Examples.

Preparation of Monoclonal Antibody

(1) Preparation of Antigen

[0135] Human recombinant VEGF₁₆₅ (hereafter, may also be referred to as “VEGF”, “rhVEGF₁₆₅” or “rhVEGF”) expressed by CHO cells (PROSPEC) was used as an antigen. For the purpose of suppressing the effect of VEGF on mice, an antigen bound to KLH (Keyhole Limpet Hemocyanin) was prepared. In this case, VEGF and KLH were mixed so that the molar ratio of VEGF to KLH was 4:1 in PBS (−) (0.01 M sodium-phosphate buffer, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), and then 1% glutaraldehyde was added, and then the mixture was allowed to react at room temperature for 1 hour, thereby cross-linking KLH and rhVEGF₁₆₅. The thus prepared protein may also be referred to as “KLH-VEGF” in the followings.

(2) Immunization

[0136] rhVEGF or KLH-VEGF and Freund's complete adjuvant were mixed in equal amounts, and then the mixture was administered intraperitoneally to BALB/c mice in an amount of 100 μl (about 40 μg/mouse). Subsequently, about 40 μg of KLH-VEGF or rhVEGF was administered with an incomplete Freund's adjuvant intraperitoneally to mice once every 3 weeks for boosters (additional immunization). Boosters were performed not more than 12 times. Final immune stimulation was performed by administering saline containing about 40 μg of KLH-VEGF or rhVEGF into the tail vein of each mouse on day 3 before splenectomy.

(3) Cell Fusion

[0137] Isolated splenocytes and a mouse myeloma cell line, P3-x63-Ag8U. 1 (DS PHARMA BIOMEDICAL), were mixed at 5:1 in terms of the number of cells, and then cell fusion was performed using 50% polyethylene glycol 3350 (Sigma). Cells were suspended in HAT medium (RPMI1640 medium supplemented with 1/2×HT (MP), 1/2×HAT (MP), 10% FBS, 50 ng/L mouse IL-6, and 500 mg/mL D-Glucose), dispensed in a 96-well microculture plate and then cultured.

(4) ELISA

[0138] Culture supernatants of wells in which hybridomas had grown were collected, and then hybridomas producing monoclonal antibodies reactive to rhVEGF₁₆₅ were selected. rhVEGF₁₆₅ was diluted with PBS (−), dispensed into a 96-well ELISA plate (Nunc) at 1 μg/well, allowed to stand overnight at 4° C., and thus bound onto the plate surface. Next, after 3 times of washing with 350 μL of 0.05% Tween 20-containing PBS (−) (hereafter, denoted as “PBS-T”), 300 μL of 1% skim milk-containing PBS-T was dispensed into each well, followed by blocking at room temperature for 1 hour. After washing with PBS-T, the culture supernatant of the hybridomas was dispensed into an ELISA plate on which rhVEGF₁₆₅ had been immobilized, and then allowed to react for 1 hour at room temperature. After washing with PBS-T, 100 μL of peroxidase (hereafter, denoted as “POD”)-labeled anti-mouse immunoglobulin antibody (BETHYL) diluted 10000-fold was dispensed into each well, and then allowed to react for 1 hour at room temperature. After similar washing was performed, a POD substrate that had been prepared to a concentration of 1 mg/mL POD was added, and then the resultant was allowed to develop color at room temperature for 5 minutes. The reaction was stopped with 1.5 N sulfuric acid, and then absorbance at 490 nm was measured using a plate reader (Molecular Devices).

(5) Establishment of Anti-VEGF Monoclonal Antibody

[0139] Based on the absorbance of a plate to which no rhVEGF₁₆₅ had been immobilized, wells each found to have an absorbance three or more times the baseline value were determined to be positive, and then cloning was performed by a limiting dilution method. Cell supernatants of wells containing single colonies were examined for antibody activity by the method of the above (4), isolated, and then cultured, thereby establishing a hybridoma cell line producing a monoclonal antibody reactive to rhVEGF₁₆₅. From the thus established hybridomas, two positive clones were selected, antibodies produced from these hybridomas were designated as KLHa505 and KLHb1501, respectively.

Evaluation of the Capability of Monoclonal Antibodies to Inhibit Binding of VEGF to its Receptor

[0140] An anti-VEGF antibody binds to VEGF to block the binding of VEGF to its receptors, VEGFR-1 and/or VEGFR-2, to be able to inhibit signal transduction through mediation of VEGF.

[0141] KLHa505 and KLHb1501 were separated and purified from the culture supernatants of the two positive clones using Protein G.

[0142] Next, IgG Fc-VEGFR-1 or IgG Fc-VEGFR2 was immobilized on a 96-well ELISA plate. After blocking with 2% bovine serum albumin, a purified antibody mixed with rhVEGF was added to the plate, followed by reaction at room temperature for 1 hour. A solution was prepared by mixing with rhVEGF, and then washed 3 times with 0.05% TWEEN® 20-containing TBS (TBS: 50 mM Tris-HCl (pH7.4), 500 mM NaCl; hereafter, referred to as “TBS-T”). Thereafter, through color development using rabbit anti-human VEGF polyclonal antibody-HRP, the rhVEGF content was determined.

[0143] As a result, it was demonstrated that the KLHa505 antibody competitively inhibits binding of VEGF to VEGFR-1 and VEGFR-2, and the KLHb1501 antibody competitively inhibits binding of VEGF to VEGFR-2 (FIG. 1).

[0144] That is, it was demonstrated in this Example that the antibodies of the present invention, KLHa505 and KLHb1501, can block VEGF-associated signal transduction.

Measurement of Dissociation Constant of KLHa505 and KLHb1501 for Binding to rhVEGF

[0145] With the use of BIAcore-3000, Kd of KLHa505 and Kd of KLHb1501 for binding to VEGF were determined. rhVEGF was immobilized on a CMS chip, serial dilution was performed twice with HBS-EP buffer, and then each antibody was injected at a flow rate of 30 μL/min. Kd is K_off/K_on.

[0146] As a result, it was demonstrated that the dissociation constant for binding of KLHa505 to rhVEGF was 4.23 pmol/L, and the dissociation constant for binding of KLHb1501 to rhVEGF was 0.60 pmol/L.

[0147] When compared with the dissociation constant for an existing antibody (for example, the dissociation constant for Avastin (bevacizumab) is 4.9 nmol/L), the dissociation constants for the antibodies of the present invention were each 1/1000 or less that for the existing antibody. Hence, the binding activity of the antibodies that bind to VEGF is significantly high.

[0148] That is, in this Example, the antibodies (high-affinity anti-VEGF antibodies) having extremely high affinity for VEGF were obtained.

Examination of the Inhibiting Effect of the Monoclonal Antibodies, KLHa505 and KLHb1501 on HUVEC Proliferation

[0149] It was examined if KLHa505 and KLH1501 inhibited VEGF activity exerted to normal human umbilical vein-derived endothelial cells (hereafter, referred to as “HUVEC”). HUVEC was used for the test to such an extent that the passage number did not exceed 8. With 1% fetal calf serum-containing Medium 200, 10000 cells each were seeded on a 96-well cell culture plate, rhVEGF was added at a final concentration of 50 ng/mL and KLHa505 or KLHb1501 was added at a final concentration of 2500 ng/mL each, cells were cultured for 24 hours, and then the numbers of cells were determined.

[0150] As a result, compared to positive controls (VEGF and isotype control antibody (“Control IgG” in FIG. 2)), cells with KLHa505 or KLHb1501 were found to exert a significant decrease in the number of cells (FIG. 2).

[0151] It was thus demonstrated in this Example that the high-affinity antibodies of the present invention can inhibit blood vessel-derived cells' cell proliferation activity due to VEGF, and can suppress angiogenesis.

Examination of the In Vivo Inhibiting Effect of the Monoclonal Antibodies, KLHa505 and KLHb1501, on Cell Proliferation

[0152] One lineage of human colon adenocarcinoma LS174T cells (about 1×10⁷ cells) cryopreserved in liquid nitrogen were thawed in a warm bath at 37° C. two weeks before the experiment. Subsequently, to a 100-mm cell dish added with McCoy's 5A medium containing 10% fetal calf serum warmed in advance at 37° C., the thawed cell suspension was added, and then cells were grown to 80% confluency. Thereafter, cells were subcultured at 1:5, followed by 3 times of continuous subculturing. When all cells reached the number of cells required for inoculation, cells were removed from the dish using a 0.25% trypsin-EDTA solution, and then washed with PBS. Saline containing 50% MatriGel® was added to the washed cells to a concentration of 2×10⁷/mL, and then the resultant was inoculated subcutaneously to the abdomen of each Balb/C nude mouse at 100 μL/mouse, specifically at 2×10⁶ cells/mouse.

[0153] On the next day of cell inoculation, administration of KLHa505 and KLHb1501 was initiated. Regarding the dosage of KLHa505, administration was performed for 5 mouse per group, and thus (i) model control group (saline alone), (ii) 5 mg/kg KLHa505 group, (iii) 2.5 mg/kg KLHa505 group and (iv) 0.5 mg/kg KLHa505 group were prepared. Similar administration groups were prepared also for KLHb1501.

[0154] Regarding the administration method, KLHa505 or KLHb1501 dissolved in saline in an amount of 200 μL per mouse was administered once on day 2 and on day 5 in each week through intraperitoneal injection. To a model control group, 200 μL of saline was simultaneously administered by the same method.

[0155] Regarding the administration period, administration was continuously performed for 18 days for KLHa505 and 22 days for KLHb1501. When the antibodies were administered, the maximum diameter “a” and the minimum diameter “b” of a tumor nodule of each nude mouse were measured, and the tumor volume was calculated according to the formula V=0.5×a×b².

[0156] As a result, KLHa505 and KLHb1501 were demonstrated to have capability of significantly inhibiting in vivo tumor growth (FIG. 3 and FIG. 4).

[0157] That is, this Example demonstrated that the high-affinity antibodies of the present invention are effective for treatment or prevention of a cancer.

Determination of the Nucleotide Sequences and Amino Acid Sequences of Monoclonal Antibody Variable Regions

[0158] mRNAs were extracted from hybridomas that produce KLHa505 and KLHb1501, cDNAs were prepared using reverse transcriptase. With the use of these cDNAs as templates, the nucleotide sequences of mouse IgG antibody heavy chain and light chain variable regions were amplified by PCR, and then the nucleotide sequences were analyzed, thereby the amino acid sequences of complementarity determining regions contained in the light chain and heavy chain variable regions were determined.

[0159] Furthermore, the amino acid sequences of CDR1, CDR2 and CDR3 of KLHa505 and KLHb1501 variable region were determined using IMGT/V-QUEST software, version 3.3.0, on the immunoglobulin database “the international ImMunoGeneTics information System®” (IMGT/GENE-DB).

[0160] The results are each shown in the following Table 1 and Table 2.

[0161]

TABLE 1
KLHa505
Heavy chain (V-D-J region)
Nucleotide	cagatccagttggtgcagtctggacctgagctgaagaagc
sequence	ctggagagacagtcaagatctcctgcaaggcttctgggta
	taccttcacaaactatggaatgaactgggtgaagcaggct
	ccaggaaagggtttaaagtggatgggctggataaacacct
	acactggagagccaacatatcttgatgacttcaagggacg
	gtttgccttctctttggaaacctctgccagcactgcctac
	ttgcagatcaacaacctcaaaaatgaggacacgtctacat
	atttctgtgcaagattttcctacggtattacctggttctt
	cgatgtctggggcgcagggaccacggtcaccgtctcctca
	(SEQ ID NO: 33)
Peptide	QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQA
sequence	PGKGLKWMGWINTYTGEPTYLDDFKGRFAFSLETSASTAY
	LQINNLKNEDTSTYFCARFSYGITWFFDVWGAGTTVTVSS
	(SEQ ID NO: 34)
Light chain (V-J region)
Nucleotide	gacattgtgatgtcacagtctccatcctccctggctgtgt
sequence	cagcaggagagaaggtcactatgagctgcaaatccagtca
	gagtctgctcaacagtagaacccgaaagaactacttggct
	tggtaccagcagaaaccagggcagtctcctaaactgctga
	tctactgggcatccactagggaatctggggtccctgatcg
	cttcacaggcagtggatctgggacagatttcactctcacc
	atcagcagtgtgcaggctgaagacctggcagtttattact
	gcaagcaatcttataatctgtacacgttcggaggggggac
	caagctggaaataaaac (SEQ ID NO: 35)
Peptide	DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLA
sequence	WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT
	ISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK (SEQ ID
	NO: 36)
Sequence
Heavy chain
VH-CDR1	GYTFTNYG (SEQ ID NO: 14)
VH-CDR2	INTYTGEP (SEQ ID NO: 16)
VH-CDR3	ARFSYGITWFFDV (SEQ ID NO: 18)
Light chain
VL-CDR1	QSLLNSRTRKNY (SEQ ID NO: 20)
VL-CDR2	WAS
VL-CDR3	KQSYNLYT (SEQ ID NO: 22)

[0162]

TABLE 2
KLHb1501
Heavy chain (V-D-J region)
Nucleotide	gatgtgcagcttcaggagtcgggacctggcctggtgaaac
sequence	cttctcagtctctgtccctcacctgcactgtcactggcta
	ctcaatcaccagtgattatgcctggacctggatccggcag
	tttccaggagacaaactggagtggatgggctacataagct
	acagtggtagcactcgctacaacccatctctcaaaagtcg
	aatctctatcactcgagacacatccaagaaccagttcttc
	ctgcacttgaattctgtgactactgaggacacggccacat
	atttctgtgcaagagggggggattactacggtagtcgccc
	ctggtttgcttactggggccaagggactctggtcactgtc
	tctgcag (SEQ ID NO: 37)
Peptide	DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWTWIR
sequence	QFPGDKLEWMGYISYSGSTRYNPSLKSRISITRDTSKNQ
	FFLHLNSVTTEDTATYFCARGGDYYGSRPWFAYWGQGTL
	VTVSA (SEQ ID NO: 38)
Light chain (V-J region)
Nucleotide	caggctgttgtgactcaggaatctgcactcaccacatcac
sequence	ctggtgaaacagtcacactcacttgtcgctcaagtactgg
	ggctgttacaattagtaactacgccaactgggtccaagaa
	aaaccagatcatttattcactggtctaataggtggtacca
	acaaccgagctccagatgttcctgccagattctcaggctc
	cctgattggagacaaggctgccctcaccatcacaggggca
	cagactgaggatgaggcaatatatttctgtgctctctggt
	acagcaaccatttggtgttcggtgaaggaaccaaactgac
	tgtcctag (SEQ ID NO: 39)
Peptide	QAVVTQESALTTSPGETVTLTCRSSTGAVTISNYANWVQE
sequence	KPDHLFTGLIGGTNNRAPDVPARFSGSLIGDKAALTITGA
	QTEDEAIYFCALWYSNHLVFGEGTKLTVL (SEQ ID
	NO: 40)
Sequence
Heavy chain
VH-CDR1	GYSITSDYA (SEQ ID NO: 24)
VH-CDR2	ISYSGST (SEQ ID NO: 26)
VH-CDR3	ARGGDYYGSRPWFAY (SEQ ID NO: 28)
Light chain
VL-CDR1	TGAVTISNY (SEQ ID NO: 30)
VL-CDR2	GTN
VL-CDR3	ALWYSNHLV (SEQ ID NO: 32)

Preparation of Human-IgG1 Chimeric KLHa505 Antibody (KLHa505HC Antibody)

[0163] From the hybridoma that produces the KLHa505 antibody, the KLHa505 antibody heavy chain variable region gene (nucleotide sequence: SEQ ID NO: 33, amino acid sequence: SEQ ID NO: 34) and light chain variable region gene (nucleotide sequence: SEQ ID NO: 35, amino acid sequence: SEQ ID NO: 36) were cloned. Next, these genes were each ligated in-frame to the nucleotide sequence of the human IgG1 heavy chain constant region gene or light chain (κ chain) constant region gene. PCR was carried out using a primer having the 5′-terminal nucleotide sequence of the heavy chain variable region, Kozak sequence, and a restriction enzyme Mlu I sequence and an antisense primer having a complementary sequence of the 3′-terminal nucleotide sequence and a restriction enzyme Nhe I sequence. Next, PCR was carried out using a primer having the 5′-terminal nucleotide sequence of the light chain variable region gene and a restriction enzyme BamH I sequence, and an antisense primer having a complementary sequence of the 3′-terminal nucleotide sequence and a restriction enzyme BsiW I sequence. The thus obtained amplification product was treated with restriction enzymes Mlu I and Nhe I or BamH I and BsiW I, and then the resultant was incorporated into the Mlu I-Nhe I site of the human IgG1 heavy chain constant region expression plasmid (pEF6/G1) or the BamH I-BsiW I site of the human Ig light chain (κ chain) constant region expression plasmid (pEF1/G1k). In pEF6/G1, the human IgG1 heavy chain constant region gene (nucleotide sequence: SEQ ID NO: 41, amino acid sequence: SEQ ID NO: 42) was cloned into the plasmid pEF6/myc-His (Invitrogen). In pEF1/G1-k, the human Ig light chain (κ chain) constant region gene (nucleotide sequence: SEQ ID NO: 43, amino acid sequence: SEQ ID NO: 44) was cloned into the plasmid pEF1/myc-His (Invitrogen). The mouse heavy chain variable region and the human heavy chain constant region were linked by a restriction enzyme Nhe I, and the mouse light chain variable region and the human light chain constant region were linked by a restriction enzyme BsiW I sequence.

[0164] The plasmids pEF6/G1-KLHa505 and pEF1/G1-K-KLHa505 were introduced into 293F cells using a FreeStyle™ 293 Expression System (Thermo Fisher Scientific K.K.), and thus the human IgG1 chimeric KLHa505 antibody (hereafter, may also be referred to as “KLHa505HC antibody”) was transiently expressed. Next, the KLHa505HC antibody was purified from a culture supernatant of 293F cells, into which the chimeric gene had been introduced, using Protein G Sepharose 4 Fast Flow (GE Healthcare Japan). A solvent was substituted with D-PBS (−) using an Amicon Ultra-15 centrifugal filter unit (Merck millipore). The concentration of the purified KLHa505HC antibody was determined using Nano drop 1000 (Thermo Fisher Scientific K.K.). Binding of the KLHa505HC antibody to VEGF was evaluated by the method described in the above “Purification of monoclonal antibody (4) ELISA”. As a result, specific binding of the KLHa505HC antibody to VEGF was confirmed.

[0165] That is, in this Example, the chimeric antibody of the high-affinity anti-VEGF antibody of the present invention was obtained.

Preparation of Human-IgG1 Chimeric KLHb1501 Antibody (KLHb1501HC Antibody)

[0166] From hybridomas that produce the KLHb1501 antibody, the KLHb1501 antibody heavy chain variable region gene (nucleotide sequence: SEQ ID NO: 37, amino acid sequence: SEQ ID NO: 38) and light chain variable region gene (nucleotide sequence: SEQ ID NO: 39, amino acid sequence: SEQ ID NO: 40) were cloned. Next, these genes were each ligated in-frame to the nucleotide sequence of a human IgG1 heavy chain constant region gene or light chain (λ chain) constant region gene. PCR was carried out using a primer having the 5′ terminal nucleotide sequence of the heavy chain variable region, Kozak sequence, and a restriction enzyme Xho I sequence, and an antisense primer having the complementary sequence of the 3′ terminal nucleotide sequence and a restriction enzyme Nhe I sequence. Next, PCR was carried out using a primer having the 5′ terminal nucleotide sequence of the light chain variable region gene and a restriction enzyme EcoR I sequence, and an antisense primer having the complementary sequence of the 3′ terminal nucleotide sequence and a restriction enzyme Avr II sequence. The thus obtained amplification products were treated with restriction enzymes Xho I and Nhe I, or EcoR I and Avr II, and then incorporated into the Xho I-Nhe I site of a human IgG1 heavy chain constant region expression plasmid (pFUSE-CHIg-hG1; InvivoGen), or the EcoR I-Avr II site of a human Ig light chain (λ chain) constant region expression plasmid (pFUSE2ss-CLIg-hl2: InvivoGen). In pFUSE-CHIg-hG1, the human IgG1 heavy chain constant region gene (nucleotide sequence: SEQ ID NO: 41, amino acid sequence: SEQ ID NO: 42) was cloned. In pFUSE2ss-CLIg-hl2, the human Ig light chain (λ chain) constant region gene (nucleotide sequence: SEQ ID NO: 43, amino acid sequence: SEQ ID NO: 44) was cloned. The mouse heavy chain variable region and the human heavy chain constant region were linked by the restriction enzyme Nhe I, and the mouse light chain variable region and the human light chain constant region were linked by the restriction enzyme Avr II sequence.

In Vivo Inhibiting Effect of Human IgG1 Chimeric KLHb1501 Antibody (KLHb1501HC Antibody) on Tumor Growth

[0167] Human colon cancer cell line LS174T cells were subcutaneously injected at a rate of 2×10⁶ cells per mouse into the right flanks of the 20 immunodeficient mice. On the next day of injection, 0.9% NaCl (control group A) or KLHb1501HC antibody (control group B) was intraperitoneally administered. The KLHb1501HC antibody was administered to the mice of control group B twice a week at 0.5 mg/Kg, 2.5 mg/Kg, or 5 mg/Kg. Furthermore, similarly to the antibody, 0.9% NaCl was administered twice a week to the mice of control group A. On days 1, 5, 8, 12, 15, 19 and 22 after inoculation of cells, tumor sizes were measured, and then tumor volumes were calculated using the formula: V (mm³)=(d: minor axis)²×(D: major axis)/2. The results are shown in FIG. 5. In FIG. 5, group A (0.9% NaCl control) is indicated with (⋄), group B (mice injected with the anti-VEGF antibody) is indicated with (□: 0.5 mg/Kg), (Δ: 2.5 mg/Kg), or (◯: 5.0 mg/Kg). As shown in FIG. 5, significant decreases in tumor volume were observed in mice injected with the KLHb1501HC antibody. These results demonstrated that the KLHb1501HC antibody; that is an antibody of the present invention, inhibited (suppressed) the tumor growth even in vivo. This demonstrated that a pharmaceutical composition comprising the human IgG1 chimeric anti-VEGF antibody of the present invention is significantly effective for treatment or prevention of cancer.

Preparation of Canine-IgGB Chimeric KLHa505 Antibody (KLHa505CC Antibody)

[0168] From the hybridoma that produces the KLHa505 antibody, the KLHa505 antibody heavy chain variable region gene (nucleotide sequence: SEQ ID NO: 33, amino acid sequence: SEQ ID NO: 34) and light chain variable region gene (nucleotide sequence: SEQ ID NO: 35, amino acid sequence: SEQ ID NO: 36) were cloned. Next, these genes were each ligated in-frame to the nucleotide sequence of the canine IgGB heavy chain constant region gene or light chain (κ chain) constant region gene. PCR was carried out using a primer having the 5′-terminal nucleotide sequence of the heavy chain variable region, Kozak sequence, and a restriction enzyme EcoR V sequence, and an antisense primer having a complementary sequence of the 3′-terminal nucleotide sequence and a restriction enzyme Nhe I sequence. Next, PCR was carried out using a primer having the 5′-terminal nucleotide sequence of the light chain variable region gene and a restriction enzyme EcoR I sequence, and an antisense primer having a complementary sequence of the 3′-terminal nucleotide sequence and a restriction enzyme Xho I sequence. The thus obtained amplification product was treated with restriction enzymes Eco V and Nhe I, or EcoR I and Xho I, and then the resultant was incorporated into the EcoR V-Nhe I site of a canine IgGB heavy chain constant region expression plasmid (pFUSE2ss-CHIg-dGB), or the EcoR I-Xho I site of a canine Ig light chain constant region expression plasmid (pFUSE2ss-CHIg-dK). In pFUSE2ss-CHIg-dGB, the canine IgGB heavy chain constant region gene (nucleotide sequence: SEQ ID NO: 45, amino acid sequence: SEQ ID NO: 46) was cloned into plasmid pFUSE2ss-CLIg-hl2 (InvivoGen). In pFUSE2ss-CHIg-dK, the canine Ig light chain (κ chain) constant region gene (nucleotide sequence: SEQ ID NO: 47, amino acid sequence: SEQ ID NO: 48) was cloned into plasmid pFUSE2ss-CLIg-hl2. The mouse heavy chain variable region and the canine heavy chain constant region were linked by the restriction enzyme Nhe I sequence, and the mouse light chain variable region and the canine light chain constant region were linked by the restriction enzyme Xho I sequence.

Preparation of Canine-IgGB Chimeric KLHb1501 Antibody (KLHb1501CC Antibody)

[0169] From hybridomas that produce the KLHb1501 antibody, the KLHb1501 antibody heavy chain variable region gene (nucleotide sequence: SEQ ID NO: 37, amino acid sequence: SEQ ID NO: 38) and light chain variable region gene (nucleotide sequence: SEQ ID NO: 39, amino acid sequence: SEQ ID NO: 40) were cloned. Next, these genes were each ligated in-frame to the nucleotide sequence of a canine IgGB heavy chain constant region gene or light chain (λ chain) constant region gene. PCR was carried out using a primer having the 5′ terminal nucleotide sequence of the heavy chain variable region, Kozak sequence, and a restriction enzyme EcoR V sequence, and an antisense primer having the complementary sequence of the 3′ terminal nucleotide sequence and a restriction enzyme Nhe I sequence. Next, PCR was carried out using a primer having the 5′ terminal nucleotide sequence of the light chain variable region gene and a restriction enzyme EcoR I sequence and an antisense primer having the complementary sequence of the 3′ terminal nucleotide sequence and a restriction enzyme Sca I sequence. The thus obtained amplification products were treated with restriction enzymes Eco V and Nhe I, or EcoR I and Sca I, and then incorporated into the EcoR V-Nhe I site of a canine IgGB heavy chain constant region expression plasmid (pFUSE2ss-CHIg-dGB) or the EcoR I-Sca I site of a canine Ig light chain constant region expression plasmid (pFUSE2ss-CHIg-dK). In pFUSE2ss-CHIg-dGB, the canine IgGB heavy chain constant region gene (nucleotide sequence: SEQ ID NO: 45, amino acid sequence: SEQ ID NO: 46) was cloned to plasmid pFUSE2ss-CLIg-hl2 (InvivoGen). In pFUSE2ss-CHIg-dK, the canine Ig light chain (λ chain) constant region gene (nucleotide sequence: SEQ ID NO: 49, amino acid sequence: SEQ ID NO: 50) was cloned into plasmid pFUSE2ss-CLIg-hl2. The mouse heavy chain variable region and the canine heavy chain constant region were linked by the restriction enzyme Nhe I, and the mouse light chain variable region and the canine light chain constant region were linked by the restriction enzyme Sca I sequence.

Cross-Reactivity of Canine-IgGB Chimeric KLHb1501 Antibody (KLHb1501CC Antibody) to Human VEGF165 and Canine VEGFA

[0170] Whether or not the thus prepared canine-IgGB chimeric antibody had capability of binding to human VEGF165 and canine VEGFA (VEGF164) was confirmed by ELISA. Human VEGF165 or canine VEGFA were diluted with TBS, dispensed to a 96-well ELISA plate (Nunc) at 20 ng per well, and then incubated at 37° C. for 1 hour for binding to the plate surface. Next, after 3 times of washing with 300 μL of 0.02% Tween 20-containing PBS (−) (hereafter, denoted as “PBS-T”), 300 μL of 3% skim milk-containing PBS (−) was dispensed into each well, followed by blocking at 37° C. for 1 hour. After washing with PBS-T, the canine-IgGB chimeric KLHb1501 antibody (KLHb1501CC antibody) serially diluted from the concentration of 1 μg/ml was dispensed to an ELISA plate to which VEGF had been immobilized, and then allowed to react at 37° C. for 1 hour. After washing with PBS-T, a peroxidase-labeled anti-canine immunoglobulin antibody (BETHYL) prepared to have a concentration of 100 ng/ml was dispensed at 100 μL per well, and then allowed to react at 37° C. for 1 hour. After washing similarly, TMB Single Solution was added for color development at room temperature. After the reaction was stopped with 1 N sulfuric acid, absorbance at 450 nm was measured using a plate reader (Molecular Devices). The results are shown in FIG. 6. It was demonstrated that the KLHb1501CC antibody binds to canine VEGFA with the same degree of affinity as that to human VEGF165.

[0171] These results demonstrated that a pharmaceutical composition comprising the canine IgGB chimeric anti-VEGF antibody of the present invention can be used for treatment or prevention of diseases of dogs.

In Vivo Inhibiting Effect of Canine IgGB Chimeric KLHb1501 Antibody (KLHb1501CC Antibody) on Tumor Growth in Human Cancer Cell Line

[0172] Human colon cancer cell line LS174T cells were subcutaneously injected at a rate of 2×10⁶ cells per mouse to the right flanks of 20 immunodeficient mice. On the next day of injection, 0.9% NaCl (control group A) or the KLHb1501CC antibody (control group B) was administered intraperitoneally. The KLHb1501CC antibody was administered twice a week to the mice of control group B at 0.5 mg/Kg, 2.5 mg/Kg, or 5 mg/Kg. Furthermore, similarly to the antibody, 0.9% NaCl was administered twice a week to the mice of control group A. On days 5, 8, 12, 15, 19 and 22 after inoculation of cells, tumor sizes were measured and tumor volumes were calculated using the formula: V (mm³)=(d: minor axis)²×(D: major axis)/2. The results are shown in FIG. 7. In FIG. 7, group A (0.9% NaCl control) is indicated with (▪), group B (mice injected with the anti-VEGF antibody) is indicated with (●: 0.5 mg/Kg), (▴: 2.5 mg/Kg), or (A: 5.0 mg/Kg). As shown in FIG. 7, significant decreases in tumor volume were observed in mice injected with the KLHb1501CC antibody.

[0173] These results demonstrated that the KLHb1501CC antibody of the present invention also inhibits (suppresses) in vivo the growth of human tumors. These results also demonstrated that a pharmaceutical composition comprising the canine IgGB chimeric anti-VEGF antibody of the present invention is significantly effective for treatment or prevention of cancer.

In Vivo Inhibiting Effect of Canine IgGB Chimeric KLHb1501 Antibody (KLHb1501CC Antibody) on Tumor Growth in Canine Cancer Cell Line

[0174] Canine bone sarcoma cell line D-17 cells were subcutaneously injected at a rate of 1×10⁷ cells per mouse to the right flanks of 20 immunodeficient mice. After the mean tumor volume reached 150 mm³, 0.9% NaCl (control group A) or the KLHb1501CC antibody (control group B) was intraperitoneally administered. The KLHb1501CC antibody was administered twice a week to the mice of control group B at 0.5 mg/Kg, 2.5 mg/Kg, or 5 mg/Kg. Similarly to the antibody, 0.9% NaCl was administered twice a week to the mice of control group A. On days 50, 54, 57, 61, 64, 67, 70, 74, 77 and 81 after inoculation of cells, tumor sizes were measured, and the tumor volumes were calculated using the formula: V (mm³)=(d: minor axis)²×(D: major axis)/2. The results are shown in FIG. 8. In FIG. 8, group A (0.9% NaCl control) is indicated with (▪), and group B (mice injected with the anti-VEGF antibody) is indicated with (●: 0.5 mg/Kg), (▴: 2.5 mg/Kg), or (Δ: 5.0 mg/Kg). As shown in FIG. 8, significant decreases in tumor volume were observed in mice injected with the KLHb1501CC antibody.

[0175] These results demonstrated that the KLHb1501CC antibody of the present invention inhibits (suppresses) the growth of tumors of dogs even in vivo. These results also demonstrated that a pharmaceutical composition comprising the canine IgGB chimeric anti-VEGF antibody of the present invention is significantly effective for treatment or prevention of cancer of dogs.

Examination of the Antigen Binding Site of the Antibody of the Present Invention

[0176] To examine the binding site of the antibody of the present invention in VEGF, a binding experiment based on the ELISA method was conducted as follows.

[0177] In this Example, as anti-VEGF antibodies, three anti-VEGF antibodies including KLHa505HC and KLHb1501HC obtained in Examples 7 and 8 were used.

[0178] Furthermore, as VEGF, three types of VEGF, (i) VEGF165 containing exon 8a that is the C-terminal region of VEGF, (ii) VEGF121 lacking heparin binding region, and (iii) VEGF165b having an amino acid sequence (exon 8b) differing from the C-terminal region of VEGF165 were used.

[0179] The binding experiment was specifically conducted as follows.

[0180] Human VEGF165 (consisting of exon 1, 2, 3, 4, 5, 7 and 8a), human VEGF165b (consisting of exon 1, 2, 3, 4, 5, 7 and 8b) or human VEGF121 (consisting of exon 1, 2, 3, 4, 5 and 8a) was diluted with TBS, dispensed to a 96-well ELISA plate (Nunc) at 20 ng per well, and then incubated at 37° C. for 1 hour, for binding onto the plate surface. Next, after 3 times of washing with 300 μL of 0.02% Tween 20-containing PBS (−) (hereafter, denoted as “PBS-T”), 300 μL of 3% skim milk-containing PBS (−) was dispensed to each well, followed by blocking at 37° C. for 1 hour. After washing with PBS-T, the KLHa505HC antibody or KLHb1501HC antibody serially diluted from the concentration of 1 μg/ml was dispensed to an ELISA plate to which VEGF had been immobilized, and then allowed to react at 37° C. for 1 hour. After washing with PBS-T, a peroxidase-labeled anti-human immunoglobulin antibody (BETHYL) prepared to have a concentration of 100 ng/ml was dispensed at 100 μL per well, and then allowed to react at 37° C. for 1 hour. After washing similarly, TMB Single Solution was added for color development at room temperature. After the reaction was stopped with 1 N sulfuric acid, absorbance at 450 nm was measured using a plate reader (Molecular Devices).

[0181] As a result, all of the three anti-VEGF antibodies including KLHa505HC and KLHb1501HC that is the anti-VEGF antibody of the present invention were found to bind to all of VEGF165, VEGF121 and VEGF165b above (FIG. 9, 10).

[0182] The result demonstrates that the antibody of the present invention binds to exon 1 to 5 regions of VEGF.

[0183] According to the present invention, novel antibodies capable of binding to VEGF with significantly high affinity compared to the prior art can be provided.

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