WO2011023130A1 - 一种抗vegf的单克隆抗体及含有该抗体的药物组合物 - Google Patents

一种抗vegf的单克隆抗体及含有该抗体的药物组合物 Download PDF

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WO2011023130A1
WO2011023130A1 PCT/CN2010/076420 CN2010076420W WO2011023130A1 WO 2011023130 A1 WO2011023130 A1 WO 2011023130A1 CN 2010076420 W CN2010076420 W CN 2010076420W WO 2011023130 A1 WO2011023130 A1 WO 2011023130A1
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seq
antibody
amino acid
acid sequence
monoclonal antibody
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PCT/CN2010/076420
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English (en)
French (fr)
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李森伟
柯耀煌
张永克
朱伟民
余国良
马梵辛
樊馨
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江苏先声药物研究有限公司
宜康公司
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Priority to EP10811290.5A priority Critical patent/EP2471814B1/en
Priority to CN201080018409.6A priority patent/CN102448987B/zh
Priority to US13/393,185 priority patent/US8986692B2/en
Priority to JP2012525877A priority patent/JP5738294B2/ja
Priority to ES10811290.5T priority patent/ES2657226T3/es
Publication of WO2011023130A1 publication Critical patent/WO2011023130A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of genetic engineering antibody technology, in particular to vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • human VEGF protein was successfully purified and identified by American scientists in 1989, and its gene sequence was cloned and determined.
  • Vascular endothelial growth factor has an effect of promoting angiogenesis. All members of the VEGF family can activate cellular responses by binding to corresponding receptors on the cell surface (VEGFRs), dimerizing and activating by phosphorylation.
  • the vascular endothelial growth factor receptor contains seven immunoglobulin-like extracellular domains, a transmembrane domain and an intracellular domain containing a tyrosine kinase domain.
  • Vascular endothelial growth factor A binds to vascular endothelial growth factor receptor 1 (receptor Flt-1) and vascular endothelial growth factor receptor-2 (KDR/Flk-1). Vascular endothelial growth factor receptor-2 almost mediates all known cellular responses to VEGF.
  • Vascular endothelial growth factor, its biological activity and its receptors have been elaborated and studied by Matsumoto et al. and Marti et al. (see Angiogenesis in ischemic disease. Thromb Haemost. 1999 Suppl 1 : 44-52; VEGF receptor signal transduction Sci STKE. 2001 : RE21).
  • VEGF is a highly conserved homodimeric glycoprotein composed of two disulfide bonds of two single strands each having a molecular weight of 24 kDa. Due to different splicing patterns of mRNA, at least 5 proteins such as VEGF 121, VEGF 145, VEGF 165, VEGF 185 and VEGF206 were produced, respectively. Form, wherein VEGF121, VEGF 145, VEGF 165 are secreted soluble proteins, which can directly act on vascular endothelial cells, promote proliferation and migration of vascular endothelial cells, and increase vascular permeability.
  • VEGF-associated diseases are usually characterized by excessive vascular endothelial cell proliferation, increased vascular permeability, tissue edema and inflammation such as cerebral edema caused by injury, stroke or tumor; edema caused by inflammatory diseases such as psoriasis or joints Inflammation, including rheumatoid arthritis; asthma; general edema associated with burns; ascites and pleural effusion caused by tumors, inflammation or trauma; chronic bronchitis; capillary leak syndrome; septicemia; Kidney disease; target diseases such as age-related macular degeneration and diabetic retinopathy; tumors, including breast cancer, lung cancer, colorectal cancer, glioma, and kidney cancer.
  • antibody and “immunoglobulin” are used interchangeably herein. These terms are all terms well known to those skilled in the art and specifically refer to proteins consisting of one or more polypeptides that specifically bind antigen.
  • One form of antibody constitutes the basic building block of an antibody. This form It is a tetramer composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together to bind the antigen, while the constant region is responsible for the effector function of the antibody.
  • polypeptides include immunoglobulin ⁇ and ⁇ light chains, and ⁇ , ⁇ (IgGi, IgG 2 , IgG 3, IgG 4), ⁇ , ⁇ and ⁇ heavy chains, or other type of equivalents thereof.
  • Full-length immunoglobulin "light chains” (about 25kDa or about 214 amino acids) comprising a a 2 NH - variable region of about 110 amino acids formed by the end, and a COOH- ⁇ or ⁇ constant region end.
  • the full length immunoglobulin "heavy chain” (approximately 50 kDa or approximately 446 amino acids) also contains a variable region (approximately 116 amino acids) and one of the heavy chain constant regions, such as gamma (approximately 330 amino acids).
  • antibody and “immunoglobulin” include antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, human sources An antibody, a single-chain antibody, and a fusion protein comprising an antigen-binding portion of the antibody and a non-antibody protein.
  • the antibody can be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like, and detected.
  • the antibody may also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.
  • the term also includes Fab, Fv, F(ab,) 2 and/or other antibody fragments and monoclonal antibodies that specifically bind to an antigen.
  • Antibodies can also exist in a variety of forms, including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies (eg, literature, Lanzavecchia et al, Eur. J. Immunol, 1987; 17, 105) and single-stranded Forms (e.g., Huston et al, Proc. NatL Acad. Sci. USA, 1988; 85, 5879 and Bird et al, Science, 1988; 242, 423, incorporated herein by reference).
  • the heavy or light chain variable region of an immunoglobulin consists of three hypervariable regions (also referred to as "complementarity determining regions" or CDRs) that are separated by a framework region (FR).
  • a chimeric antibody is an antibody whose heavy and light chain genes have been constructed, in particular, genetically engineered antibody variable and constant region genes belonging to different species.
  • variable region fragment of a murine monoclonal antibody gene can be ligated into human antibody constant region fragments such as gamma ⁇ and ⁇ 3.
  • a therapeutic chimeric antibody is a chimeric protein which is produced by a rabbit antibody variable region fragment or antigen binding region fragment and a human antibody constant region or effector region (as prepared by cells of ATCC Deposit No. CRL 9688).
  • Anti-Tac chimeric antibodies of course, other mammalian species can also be used as a source of chimeric antibodies.
  • humanized antibody has the same meaning as “humanized immunoglobulin”, and generally a humanized antibody will reduce the immune response produced in a human host compared to a non-humanized form of the same antibody.
  • humanized antibodies designed and produced in accordance with the present invention may be substituted for certain conserved amino acids which have substantially no effect on antigen binding or other functions of the antibody.
  • amino acids inside each combination can be substituted for each other.
  • Amino acids that are not present in the same group are "substantially different" amino acids.
  • the affinity between an antibody and its target is characterized by K D (dissociation constant), which is less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or approximately 10_ 12 M or more.
  • variable region of an antibody heavy or light chain is the N-terminal mature region of the chain. All regions, CDRs, and residue numbers are defined by sequence alignment based on existing structural knowledge. Identification and numbering of framework regions and CDR residues are described by Chothia and Others (Chothia, Structural determinants in the sequences of immunoglobulin variable domain. J Mol Biol. 1998; 278, 457).
  • VH is the variable region of an antibody heavy chain.
  • VL is the variable region of the antibody light chain and may have ⁇ and ⁇ isotypes.
  • the K-1 antibody has a kappa-1 isotype and the ⁇ -2 antibody has a kappa-2 isotype, and ⁇ is a variable lambda light chain.
  • polypeptide and protein are used interchangeably herein and refer to a polymeric form of amino acid of any length, which may include both coding and non-coding amino acids, amino acids modified or derived by chemical or biochemical modification, and modifications.
  • a polypeptide of a peptide backbone includes fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence; heterologous and homologous leader a fusion protein with or without an N-terminal methionine residue; an immunolabeled protein; a fusion protein with a detectable fusion partner, for example, a fluorescent protein, beta-galactosidase, Fluorescein or the like as a fusion partner for fusion proteins and the like.
  • the polypeptide may be of any size, and the term “peptide” refers to a polypeptide having a length of from 8 to 50 residues (e.g., from 8 to 20 residues).
  • subject refers to any mammal, especially a human, that is diagnosed or treated.
  • Other subjects may include monkeys, cows, dogs, cats, guinea pigs, rabbits, rats, mice, and horses.
  • corresponding amino acid refers to an amino acid residue which is located at the same position (i.e., they correspond to each other) when two or more amino acid sequences are aligned.
  • Methods for antibody sequence alignment and numbering are detailed in Chothia, supra, Kabat, supra, and others.
  • One of ordinary skill in the art is known (see, e.g., Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), sometimes one, two or three gaps and/or insertions can be made in one or two amino acids of an antibody. 1, 2, 3 or 4 residues or up to about 15 residues (especially in the L3 and H3 CDRs;) to complete an alignment.
  • Substitutable position refers to a specific position of an antibody which can be substituted with a different amino acid without significantly reducing the binding activity of the antibody. Methods for identifying alternative positions and how they can be substituted are described in more detail below.
  • the replaceable position can also be referred to as the "variable tolerance position.”
  • a “parent” antibody refers to a target antibody that is substituted as an amino acid.
  • a “donor” antibody will “gift” an amino acid to a parent antibody to generate an altered antibody.
  • “Relevant antibody” refers to an antibody that has a similar sequence and is produced by a cell having a common B cell ancestor. Such B cell progenitors contain a genome having a rearranged light chain VJC region and a rearranged heavy chain VDJC region, and produce antibodies that have not undergone affinity maturation. "Pure” or “primitive” B cells present in spleen tissue are the common ancestor of B cells. The binding of related antibodies to the same epitope is generally very similar in sequence, in particular their L3 and H3 CDRs.
  • the H3 and L3 CDRs of the relevant antibodies all have the same length and nearly identical sequence (different from 0-4 amino acid residues).
  • Related antibodies are associated by antibodies raised by the common antibody ancestor, the original B cell ancestor. Summary of the invention It is an object of the present invention to provide a monoclonal antibody having a higher affinity for VEGF.
  • the VEGF monoclonal antibody of the present invention has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 1, SEQ ID N0.2 and SEQ ID N0.3, and/or its light chain variable region comprises SEQ Amino acid sequence of ID N0.4, SEQ ID NO. 5 and SEQ ID N0.6.
  • the "antibody” of the present invention should be construed as encompassing any specific binding factor that has a binding domain of the desired specificity.
  • the term encompasses antibody fragments, derivatives, humanized antibodies, and functional equivalents and homologs of the antibodies, as well as any polypeptides containing the antigen-binding domain, whether natural or synthetic. of.
  • antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD and IgA) and subtypes thereof; or fragments comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; Double-chain antibodies (diabodies).
  • a chimeric molecule or equivalent comprising an antigen binding domain fused to another polypeptide is also included.
  • the cloning and expression of chimeric antibodies are described in EP. A-0120694 and EP. A. 0125023.
  • the monoclonal antibody of the present invention may be, for example, a monovalent or single-chain antibody, a double-chain antibody, a chimeric antibody, a humanized antibody, and derivatives, functional equivalents and homologs of the above antibodies, including Antibody fragments and any polypeptide comprising an antigen binding domain.
  • Antibodies can be modified in a number of ways, and DNA recombination techniques can be used to generate additional antibodies or chimeric molecules that retain the original antibody specificity. Such techniques may involve introducing DNA encoding the immunoglobulin variable regions or complementarity determining regions (CDRs) of the antibody into the constant or constant region plus truss regions of different immunoglobulins. See, EP. A. 184187, GB 2188638A or EP. A. 239400. Genetic mutations or other alterations can also be made to hybridoma cells or other cells that produce antibodies, which may or may not alter the binding specificity of the antibody produced.
  • CDRs complementarity determining regions
  • the monoclonal antibodies of the present invention are in addition to the highly variable regions CDR1, CDR2 and CDR3 and the ligated sequences in the heavy and light chains, and the others are framework regions.
  • the framework region can be replaced by other sequences under the condition that the desired three-dimensional structure is not affected.
  • the molecular basis of antibody specificity is mainly derived from its highly variable regions CDR1, CDR2 and CDR3, which are the key to antigen binding. Part.
  • the sequence of the CDRs should be retained as much as possible. However, some amino acid changes may be required to optimize the binding properties, and those skilled in the art can use standard practices to achieve this.
  • a monoclonal antibody comprises a variable region comprising: a heavy chain variable region comprising SEQ ID N0.7, comprising CDR1 (SN DVMCW; SEQ ID NO. 1), CDR2 (GCIMTTDVVTEYANWAKS; SEQ ID 2 2) and CDR3 (RDSVGSPLMSFDLW; SEQ ID NO. 3); and a light chain variable region comprising SEQ ID NO. 8, CDR1 (QASQSIYN ELS; SEQ ID NO. 4) , CDR2 (RASTLAS; SEQ ID NO. 5), and CDR3 (GGYKSYSNDGNG; SEQ ID 6 ⁇ 6).
  • Variants of the variable region CDRs are substantially identical to the above CDR regions except for up to 6 amino acid substitutions (eg, 1, 2, 3, 4 or 5 amino acid substitutions), in the CDRs of the monoclonal antibody
  • the region has binding activity to VEGF.
  • the antibody may comprise: a) a heavy chain variable region having an amino acid sequence differing from SEQ ID N0.7 by up to 6 amino acid sequence substitutions, eg, 1, 2, 3, 4, 5 or 6 substitutions; and b) a light chain variable region having an amino acid sequence differing from SEQ ID N0.8 by up to 6 amino acid sequence substitutions, for example 1, 2, 3, 4, 5, Or 6 amino acid substitutions.
  • the antibody of interest may comprise any one or combination of these substitutions.
  • an antibody having any of these substitution positions and an antibody having all of the substitution positions also have VEGF-binding activity.
  • Amino acid substitutions may be present in both the framework and CDR regions, or in the framework or CDR regions alone.
  • the amino acid sequence of the framework region of the heavy chain variable region may differ from SEQ ID N0.7 by a maximum of 6 amino acid sequence substitutions, such as 1, 2, 3, 4, 5, Or the amino acid sequence of the framework regions of the 6 substitutions, and the light chain variable region may have a maximum of 6 amino acid sequence substitutions compared to SEQ ID N0.8, such as 1, 2, 3, 4, 5, or 6 Replace.
  • amino acid substitutions may be distributed across multiple CDR regions.
  • the amino acid sequence of a plurality of CDR regions of the heavy chain variable region may differ from SEQ ID N0.7 by up to 6 amino acid sequence substitutions, such as 1, 2, 3, 4, 5, or 6 substitutions.
  • the amino acid sequences of the plurality of CDR regions of the variable region of the light chain and the light chain may differ from SEQ ID NO. 8 by up to 6 amino acid sequence substitutions, such as 1, 2, 3, 4, 5, or 6 substitutions.
  • the antibody may comprise a) a heavy chain variable region, the amino acid sequence of which is identical to SEQ ID NO. 7 and b) a light chain variable region, the amino acid sequence and SEQ ID ⁇ 8 is the same.
  • the antibody may comprise a) a heavy chain variable region, the amino acid sequence of which is at least 95% identical to SEQ ID NO. 7, and b) a light chain variable region, the amino acid sequence thereof At least 95% identity to SEQ ID NO.
  • the antibody of interest may comprise a) a heavy chain variable region having an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 7 and b) a light chain variable region having an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.
  • the antibody may comprise a) a heavy chain having an amino acid sequence SEQ ID NO. 9 and b) a light chain having an amino acid sequence identical to SEQ ID NO.
  • the antibody may comprise a) a heavy chain having an amino acid sequence at least 95% identical to SEQ ID N0.9 and b) a light chain having an amino acid sequence of SEQ ID NO. At least 95% consistency.
  • the antibody of interest may comprise a) a heavy chain having an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 9 and b)
  • the light chain has an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.
  • the antibody of interest may have additional amino acids at both ends of the heavy or light chain.
  • the antibody of interest may comprise at least 1, 2, 3, 4, 5 or 6 or more additional amino acids at the C or N terminus of the heavy and/or light chain, respectively.
  • the antibody of interest may be shorter than the exemplary amino acids described herein, with the primary difference being that the ends of the heavy and light chains are 1, 2, 3, 4, 5 or 6 less than the exemplary amino acids, respectively. Amino acids.
  • the target antibody may be humanized.
  • a humanized antibody produces a modified antibody by amino acid substitution in the framework region of the parent antibody, and the humanized antibody has less immunogenicity than the parent antibody.
  • Antibodies can be humanized by a number of techniques well known in the art, including, for example, CDR grafting (ERA-239, 400; PCT Publication WO 91/09967; US Pat. No. 5,225,539; 5,530,101; and 5,585,089), and strand replacement ( US Pat. No. 5,565,332).
  • framework substitution is to confirm the importance of framework residues for antigen binding by mimicking the interaction of CDRs and framework residues and to identify unusual framework residues for specific sites by sequence alignment.
  • the amino acid sequence of the variable region of the parent antibody is typically compared to the amino acid sequence in the human antibody database, and such a humanized antibody having a similar amino acid sequence to the parent antibody is selected.
  • the sequences of the parent antibody and the humanized antibody are compared (eg, sequence alignment), and the amino acid at one or more of the variable tolerance positions in the parent antibody is replaced with an amino acid at a corresponding position in the human antibody.
  • the substitution method of the variation tolerance position discussed above has been readily combined with any known humanization method and is also readily applied to the production of humanized antibodies comprising CDR regions in which the CDR regions are It is modified based on the CDR regions of the parent antibody.
  • the invention also provides a humanized VEGF neutralizing antibody comprising a plurality of CDR regions that are altered from a parent antibody.
  • the dissociation constant Kd of AVASTIN and VEGF is lower (the monoclonal antibody Kd of the present invention is 0.485 nM, and the AVASTIN is 47.9 nM), and the monoclonal antibody of the present invention has higher affinity with VEGF, indicating that the present invention has stronger VEGF. Inhibition. It has been shown in mouse model tests that the anti-tumor rate of the antibody of the present invention is significantly higher than that of AVASTIN (see Example 5), so theoretically, the potential clinical efficacy of the present invention is higher than that of AVASTIN.
  • the monoclonal antibody of the present invention is produced by a cell line having the accession number CGMCC No. 3233, the heavy chain amino acid sequence thereof is set forth in SEQ ID N0.9, and the light chain is set forth in SEQ ID NO.
  • Its dissociation constant with VEGF is 0.485 nM, which is 1/100 of AVASTIN, indicating that EPI0030 has stronger binding ability to VEGF than AVASTIN.
  • the invention also provides a cell line deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 3233. It produces a monoclonal antibody having the heavy chain amino acid sequence as set forth in SEQ ID N0.9 and the light chain as set forth in SEQ ID NO.
  • EPI0030 antibody cell test and animal in vivo test prove that VEGF-induced endothelial cell proliferation and migration can be inhibited in vitro, and tumor growth can be inhibited in animals, and can be used for treating VEGF-related Sexual disease.
  • the invention also provides the use of the monoclonal antibody described in the manufacture of a medicament for the treatment of a VEGF-related disease.
  • VEGF-related diseases include tumors, age-related macular degeneration, neurodegenerative diseases, obesity, and diabetes.
  • the target antibody can be used in scientific research related to VEGF, such as developmental biology, cell biology, metabolism, structural biology, functional genomics, and other fields of scientific research, or tumor, age-related macular degeneration (AMD), Medical and pharmaceutical applications such as neurodegenerative diseases, obesity, and diabetes.
  • the present invention also provides a pharmaceutical composition comprising an effective amount of the above monoclonal antibody and a pharmaceutically acceptable carrier.
  • the invention also provides a reagent, kit or chip comprising the monoclonal antibody described above.
  • the present invention also provides a method of inhibiting VEGF activity using an antibody of interest, and using the antibody of interest for the treatment of a VEGF-related disease or using a kit containing the antibody for VEGF-related diagnosis and detection.
  • the antibody molecule of the invention can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (eg, ion exchange chromatography, affinity chromatography, particularly by protein). A affinity chromatography for specific antigens and other column chromatography), centrifugation, utilization of solubility differences, or by any other standard technique for purifying proteins.
  • the antibody is secreted from the cell into the culture medium, and the antibody is obtained by collecting the medium and purifying it.
  • Antibodies can be modified in a number of ways, and DNA recombination techniques can be used to generate additional antibodies or chimeric molecules that retain the original antibody specificity. Such techniques can include introducing DNA encoding immunoglobulin variable regions or complementarity determining regions (CDRs) of an antibody into the constant region or constant region plus framework regions of different immunoglobulins. See, EP. A-184187, GB 2188638A or EP. A-239400. Genetic mutations or other alterations can also be made to hybridoma cells or other cells that produce antibodies, which may or may not alter the binding specificity of the antibody produced.
  • CDRs complementarity determining regions
  • Monoclonal antibodies for use in the present invention can also be produced by the hybridoma method, as the DNA sequences encoding the humanized antibodies of the present invention can be obtained by conventional means well known to those skilled in the art, such as
  • the amino acid sequences disclosed in the present invention are artificially synthesized or amplified by PCR, and thus the recombinant DNA method can be used, and the sequence can be ligated into a suitable expression vector by various methods well known in the art.
  • the transformed host cells are cultured under conditions suitable for expression of the antibody of the present invention, and then the monoclonal antibodies of the present invention are purified by those skilled in the art using well-known conventional separation and purification means.
  • the invention also provides reagents, kits or chips for use in practicing the antibodies of the invention.
  • the reagent, kit or chip comprises at least one or more of the following: an antibody produced according to the above method, a nucleotide encoding the antibody, or a eukaryotic cell, a prokaryotic cell and a virus comprising the antibody.
  • the antibody can be humanized.
  • reagent, kit or chip include: restriction enzymes, primers and plasmids, buffers, etc., for performing assays for detecting antibody activity.
  • the nucleic acid of the reagent, kit or chip may also have restriction enzyme sites, multiple cloning sites, primer sites, and the like to facilitate their attachment to non-rabbit antibody nucleic acids.
  • the components of the reagent, kit or chip may be stored separately in separate containers, or some compatible components may be pre-assembled into a single container as needed.
  • a pharmaceutically acceptable carrier can be added to prepare the antibody of interest.
  • pharmaceutically acceptable carrier refers to one or more organic or inorganic ingredients which may be natural or synthetic and which, when combined with an antibody, may facilitate its use.
  • Acceptable carriers include sterile physiological saline or other pharmaceutically acceptable and water or nonaqueous isotonic solutions and sterile suspensions which are well known in the art.
  • Effective dose refers to a dose that is capable of ameliorating or delaying the progression of a pathological, degenerative or damaged condition.
  • Effective doses are defined on an individual basis and will be based on this, specifically considering treatment symptoms and finding results.
  • the effective dose can be determined by one of ordinary skill in the art, and the factors used will not exceed conventional experimentation.
  • the cell line with the accession number CGMCC No.3233 was deposited on August 20, 2009 at the General Microbiology Center of the China Microbial Culture Collection Management Committee. The address is Datun Road, Chaoyang District, Beijing, and is classified as Chinese hamster ovary cells. DRAWINGS
  • Figure 1 shows that recombinant antibodies expressed by HZD-V1, HZD-V2, HZD-V5, and HZD-V6 clones competitively inhibit the binding of VEGF to KDR;
  • Figure 2 shows the purity of the HZD-V6 clone expressing EPI0030 antibody by SDS-PAGE; Lane 1 is the reduction electricity: ⁇ ;
  • Lane 2 is a molecular weight standard of 170kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD and lOkD from top to bottom;
  • Lane 3 is a non-reductive electrophoresis
  • FIG. 3 is a SEC-HPLC analysis chart HZD-V6 Cloning and Expression of purity EPI0030 antibody; Measurement of binding activity antibody to human VEGF
  • Figure 4 shows VEGF direct plated bonding method;
  • FIG. 5 illustrates EPI0030, AVASTIN inhibit VEGF to KDR binding IC 50 Determination
  • the IC 50 of EPI0030 was 166.3 ng/ml, and the IC 50 of AVASTIN was 253.7 ng/ml.
  • Figure 6 shows the effect of 5 mg/kg EPI0030 and AVASTIN on HCT-116 tumor growth
  • Figure 7 shows the effect of 5 mg/kg EPI0030 and AVASTIN on NCI-H460 tumor growth
  • Figure 8 shows 1.5 mg/kg EPI0030 pair The effect of NCI-H460 tumor growth.
  • Example 1 Preparation of hybridoma cells expressing human anti-human VEGF165 rabbit monoclonal antibody and gene cloning Rabbit monoclonal antibodies were prepared by hybridoma cell technology. See US Patent: 7,429,487 for the experimental protocol, especially Example 1-4.
  • the IgG Fc-hVEGF-A (Human VEGF 165) fusion protein was first prepared by recombinant techniques, wherein the IgG Fc sequence was a rabbit source.
  • the DNA sequence of IgG Fc-hVEGF-A was cloned into the pTT5 plasmid, and the plasmid was transiently transfected into the 293 293-6 cell line, serum-free cells were cultured, and the culture supernatant was collected, and the transiently expressed IgG Fc- was purified by Protein A column. hVEGF-A fusion protein.
  • IgG Fc-hVEGF-A (as an antigen component) was mixed with complete Freund's adjuvant for subcutaneous multiple injection, New Zealand rabbits were first immunized, and purified protein was used once every three weeks. Rabbits were boosted by subcutaneous injection with incomplete Freund's adjuvant, and rabbits were immunized intravenously with antigen plus PBS 4 days before spleen. According to the method of U.S. Patent No.
  • the rabbit spleen cells are fused with the same immortalized HRGTP-B lymphocyte 240E-W2 cells of the same size as the spleen cells, and cultured in a 96-well plate in HAT medium, followed by Hybridoma cells were screened and the resulting cell clones were screened for new IgG Fc-hVEGF-A binding.
  • the identification screening process was divided into two positive clone screening steps: 1 immobilized IgG Fc-hVEGF-A antigen on 96-well enzyme-linked immunosorbent plate, added cloned expression supernatant for 1 h, washed with PBS 3 times, using enzyme The labeled antibody was identified to have an IgG Fc-hVEGF-A binding active cell clone supernatant, thereby obtaining a positive clone that can directly bind to IgG Fc-hVEGF-A. 2 The positive clones in step 1 were then transferred to 24-well plate culture to obtain more expression products.
  • the extracellular domain of IgG Fc-VEGFR2 ( KDR/Flk-1 ) was immobilized on a 96-well ELISA plate, and IgG Fc-hVEGF-A and cloned expression products were added for incubation for 1 h, and then washed 3 times with PBS.
  • the enzyme-labeled antibody detects the content of IgG Fc-hVEGF-A to identify the inhibitory effect of the clone on VEGF-VEGFR2 binding activity, thereby identifying a positive clone capable of blocking VEGF-VEGFR2 binding.
  • the positive clones of the selected positive clones were lysed, and mRNA was extracted and reverse-transcribed to obtain cDNA.
  • the light chain and heavy chain variable region nucleic acid sequences of rabbit IgG antibody were amplified by PCR, and the heavy chain variable region and the light chain variable region were analyzed, and the encoded heavy chain was variable.
  • the region comprises the parental sequence of SEQ ID NO. 1 ( Ser Asn Asn Asp Val Met Cys Trp ), the parental sequence of SEQ ID N0.2 (Gly Cys lie Met Thr Thr Asp Val Val Thr Glu Tyr Ala Asn Trp Ala Lys Ser ) and The parental sequence of SEQ ID NO.
  • the light chain variable region comprising the parent sequence of SEQ ID ⁇ 4 (Gin Ala Ser Gin Ser Val Tyr Gly Asn Asn Glu Leu Ser ), the parental sequence of SEQ ID N0.5 (Arg Ala Ser Thr Leu Ala Ser ) and the parental sequence of SEQ ID N0.6 (Gly Gly Tyr Lys Ser Tyr Ser Asn Asp Gly Asn Gly ).
  • the light chain nucleic acid sequence was cloned into the pTT5 plasmid.
  • the heavy chain variable region nucleic acid sequence was cloned into the ⁇ 5 plasmid having the heavy chain constant region.
  • the human sequences VKI-2-l-(U)-A20-JK4 and VH3-1-3-3-21-JH4 were used as reference sequences using the technique described in U.S. Patent No. 7,462,697. After the human rabbit anti-VEGF monoclonal antibody sequence was humanized, four versions of VK and VH were obtained.
  • the light chain variable region includes VK-HZD1 (such as SEQ ID NO. 12), VK-HZD2 (such as SEQ ID NO. 14), VK-HZD5 (such as SEQ ID NO. 16), and VK-HZD6 (such as SEQ ID NO. .8);
  • the heavy chain variable region includes VH-HZD1 (such as SEQ ID NO.
  • VH-HZD2 (such as SEQ ID NO. 13), VH-HZD5 (such as SEQ ID NO. 15), and VH. - HZD6 (eg SEQ ID NO. 7)
  • VK-HZD2 has 2 different residues in the CDR1 region compared to VK-HZD1. After adding 2 additional amino acid residues at the N-terminus of VK-HZD1 and VK-HZD2 That is, VK-HZD5 and VK-HZD6, respectively.
  • VH-HZD1 is different from the 71 residue of VH-HZD2, 71 of VH-HZD1 is K, and 71 of VH-HZD2 is R.
  • VK(H)-HZD1 and The VK(H)-HZD 2 sequence contains a rabbit-derived signal peptide, while the sequences of VK(H)-HZD5 and VK(H)-HZD6 contain a human signal peptide.
  • the four versions of the VK and VH DNA sequences were artificially synthesized and cloned into the pTT5 plasmid of the human CK sequence and the human CH sequence, respectively, and the antibody was expressed by the human signal peptide.
  • the above two plasmids were co-transfected into 293 ⁇ -6 ⁇ cells, and the humanized anti-VEGF antibody was transiently expressed.
  • a suitable affinity HZD-V6 clone was selected as the final clone, and its expression was
  • the humanized anti-VEGF antibody is referred to as EPI0030, and its amino acid sequence is the heavy chain shown in SEQ ID N0.9 and the light chain shown in SEQ ID NO.
  • VEGF antibodies inhibit the VEGF signaling pathway by binding to VEGF to block its binding to the receptor KDR.
  • HZD-V1, HZD-V2, HZD-V5, HZD-V6 1:3 series dilution of recombinantly expressed different antibodies (HZD-V1, HZD-V2, HZD-V5, HZD-V6) and after AVASTIN (9 ( ⁇ g/ml-45ng/ml) and IgG Fc-hVEGF ( ⁇ g /ml), the mixed antibody-VEGF complex was added to the well of the IgG Fc-VEGFR2 plate, and the mouse anti-human VEGF antibody was added and detected by goat anti-mouse IgG antibody-AP. The results showed that the expression was expressed.
  • Recombinant antibodies compete with AVASTIN for the competitive inhibition of VEGF binding to KDR.
  • the results showed that antibodies expressed by HZD-V1, HZD-V2, HZD-V5, HZD-V6 clones can block the binding of VEGF to KDR (see figure 1 ).
  • the IC 5 of EPI0030 and AVASTIN was determined by referring to the above experimental method. The value is the half-inhibitory concentration, and the results are shown in Fig. 5, which shows that EPI0030 and AVASTIN have similar competitive inhibition activities.
  • the dissociation constant Kd of EPI0030 and VEGF was determined using BIAcore-3000. Human VEGF was immobilized on a CM5 chip, and 2-fold serial dilutions of EPI0030 and AVASTIN were injected into HBS-EP buffer at a flow rate of 30 ul/min. Kd is k. Ff /k. n , Table 1 shows the dissociation constants of EPI0030 and AVASTIN and human VEGF.
  • the dissociation constant of EPI0030 and VEGF is 0.485nM, which is 1/100 of AVASTIN, indicating that the binding ability of EPI0030 to VEGF is stronger than that of AVASTIN o.
  • Example 4 Identification and in vitro activity assay of the monoclonal antibody EPI0030 of the present invention
  • HEK 293-6E cells The EPI0030 antibody transiently expressed in the HZD-V6 clone was purified and tested for quality.
  • the specific identification items are as follows:
  • the purity was analyzed by SDS-PAGE (reduction and non-reduction) and SEC-HPLC. The results are shown in Fig. 2 and Fig. 3, which shows that the purity of the EPI0030 antibody is greater than 98%, and the multimer content is less than 5%.
  • the main peak (retention time 10.572 minutes) covers an area of total area (including peaks from left to right 1, 2, 3) greater than 95%, and peaks 1 and 2 are multimers.
  • Example 5 Detection of in vivo activity of monoclonal antibody EPI0030 of the present invention
  • mice 5 l0 7 /ml, the cell suspension was placed on ice and inoculated into the ventral side of 6-8 week old rats. Each mouse was inoculated with 0.1 ml, ie 5 ⁇ 10 6 cells/cell. The tumor diameter of the mice was measured every two days with a vernier caliper. The tumor length of each mouse was 100 mm 3 -300 mm 3 , and the tumor volume was SD ⁇ 1/3. The mice were randomly divided into 5 groups, each group. 6 only.
  • the human colon cancer HCT-116 model group was model control group (1 group), 5 mg/kg AVASTIN (group 1) and 5 mg/kg EPI-0030 (group 1) group, and AVASTIN and
  • EPI-0030 was diluted to 0.5 mg/ml with physiological saline.
  • Human non-small cell lung cancer NCI-H460 model components are model control group (1 group), 5 mg/kg AVASTIN (group 1) and 1.5 mg/kg, 5 mg/kg
  • the EPI-0030 (Group 2) group diluted AVASTIN and EPI-0030 with physiological saline to 0.5 mg/ml and 0.15 mg/ml.
  • the drug-administered group was given a single intraperitoneal injection of 0.2 ml per week for 1, 3, and 5 days.
  • AVASTIN and EPI-0030, the model control group was administered 0.2 ml of physiological saline at the same time and manner for 3 weeks (21 days, 9 times in total).
  • human colon cancer HCT-116 and human non-small cell lung cancer NCI-H460 mouse xenograft tumor model were selected to verify the inhibitory effect of EPI-0030 on human colon cancer and human non-small cell lung cancer, proving EPI-0030 It has a significant inhibitory effect on human colon cancer and human non-small cell lung cancer xenograft tumors.
  • EPI-0030 is more potent than AVASTIN in the model.

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Description

一种抗 VEGF的单克隆抗体及含有该抗体的药物组合物
本申请要求于 2009 年 8 月 28 日提交中国专利局、 申请号为 200910171550.9、 发明名称为"一种抗 VEGF的单克隆抗体及含有该抗体 的药物组合物 "的中国专利申请的优先权， 其全部内容通过引用结合在本 申请中。 技术领域
本发明涉及基因工程抗体技术领域， 具体涉及与血管内皮生长因子
VEGF 特异性结合的基因工程抗体以及含有该抗体的药物组合物和试剂 合
。
背景技术
血管内皮生长因子， vascular endothelial growth factor, 筒称 VEGF , 是血管内皮细胞特异性的肝素结合生长因子 （ heparin-binding growth factor ), 可在体内诱导血管新生。 人的 VEGF蛋白于 1989年由美国的科 学家成功纯化与鉴定， 并克隆与测定了其基因序列。
血管内皮生长因子具有促进血管生成的作用。 所有 VEGF 家族的成 员都能通过结合细胞表面的相应受体（ VEGFRs)来激活细胞反应， 通过磷 酸作用从而二聚化并活化。 血管内皮生长因子受体含有 7个免疫球蛋白 样的胞外结构域， 一个跨膜结构域和一个含酪氨酸激酶域的胞内结构域。 血管内皮生长因子 A能与血管内皮生长因子受体 1 (受体 Flt - 1 )和血管 内皮生长因子受体- 2 ( KDR/Flk-1 )结合。血管内皮生长因子受体- 2几乎 介导了 VEGF所有已知的细胞反应。 血管内皮生长因子， 它的生物活性 和它的受体已经被 Matsumoto 等人和 Marti 等人详细阐述和研究（参见 Angiogenesis in ischemic disease. Thromb Haemost. 1999 Suppl 1 :44-52; VEGF receptor signal transduction Sci STKE. 2001 :RE21)。
VEGF 是高度保守的同源二聚体糖蛋白， 由两条分子量各为 24kDa 的单链以二硫键组成二聚体。 由于 mRNA不同的剪切方式， 分别产生出 VEGF 121 , VEGF 145 , VEGF 165 , VEGF 185 , VEGF206等至少 5种蛋白 形式， 其中 VEGF121、 VEGF 145, VEGF 165是分泌型可溶性蛋白， 能直 接作用于血管内皮细胞，促进血管内皮细胞增殖和迁移，增加血管通透性。
VEGF相关疾病通常的特征为： 过度的血管内皮细胞的增殖、 血管通透 性增加、组织水肿和炎症如由损伤、 中风或肿瘤引起的脑水肿； 炎症疾病 引起的水肿， 如银屑病或关节炎， 包括类风湿性关节炎； 哮喘； 烧伤相关 的普遍性水肿； 肿瘤、 炎症或外伤引起的腹水和胸腔积液； 慢性气管炎 ； 毛细血管漏综合症； 败血病； 蛋白质渗漏相关的肾脏疾病； 目艮部疾病， 如老年性黄斑变性和糖尿病性视网膜病变； 肿瘤， 包括乳腺癌、 肺癌、 结 直肠癌、 脑胶质瘤和肾癌等。
抗体与其靶点的结合是特异性的， 能够起到介导免疫效应机制的作 用，并且在血清中具有较长的半衰期。这些特性使抗体具有很强的治疗应 用。
目前， FDA 和欧洲已批准了重组人源化鼠抗 VEGF 单克隆抗体 AVASTIN用于治疗结直肠癌、 非小细胞肺癌、 乳腺癌、 脑胶质瘤、 肾癌 和老年性黄斑变性 （AMD ), 2008 年的销售额达到 48 亿美元。 但是 AVASTIN抗体对 VEGF亲和力不高， 另外由于独家生产， 病人需要花费 高额的费用， 目前一个病人用药一年的费用约在 5万至 10万美元。所以， 研发新的抗 VEGF单克隆抗体， 从而减少病人负担， 降低治疗费用是一 个亟待解决的问题。 定义
在进一步阐述本发明之前， 我们有必要认识到，本发明并不局限于描 述的特定的实施方案， 也就是说， 在具体形式上可能存在着变化。还有一 点需要提醒的是， 由于本发明的范围受附加的权利要求书的限制， 因此， 本文使用的术语只是为了描述特定实施方案的目的，而不是为了限制本发 明的目的。
术语"抗体"和"免疫球蛋白"在本文中可以互换使用。这些术语均为本 领域技术人员所熟知的术语，具体是指由能特异结合抗原的一种或多种多 肽构成的蛋白质。抗体的一种形式构成了抗体的基本结构单元。这种形式 是四聚物，它由两对完全相同的抗体链构成，每一对都有一个轻链和一个 重链。在每对抗体链中，轻链和重链的可变区联合在一起共同负责结合抗 原， 而恒定区则负责抗体的效应器功能。
目前已知的免疫球蛋白多肽包括 κ和 λ轻链， 以及 α , γ (IgGi, IgG2, IgG3, IgG4), δ, ε和 μ重链或它们的其它类型等价物。全长的免疫球蛋白"轻 链"（大约 25kDa或大约 214个氨基酸）包含一个由 NH2-末端上大约 110 个氨基酸形成的可变区， 以及一个 COOH-末端上的 κ或 λ恒定区。 全长 的免疫球蛋白"重链"（大约 50kDa或大约 446个氨基酸）， 同样包含一个 可变区 （大约 116个氨基酸）， 以及重链恒定区之一， 例如 γ (大约 330 个氨基酸）。
术语"抗体"和"免疫球蛋白"包括任何同型体的抗体或免疫球蛋白，或 保持与抗原特异结合的抗体片段， 包括但不限于 Fab, Fv, scFv和 Fd片 段、嵌合抗体、人源化抗体、单链抗体以及包含抗体的抗原结合部分和非 抗体蛋白质的融合蛋白质。 抗体可以被标记和检测， 例如， 可以通过放射 性同位素、 能产生可检测物的酶、 荧光蛋白质、 生物素等等进行标记并被 检测。抗体还可以结合于固相载体， 包括但不限于聚苯乙烯平板或珠粒等 等。 该术语还包括 Fab，、 Fv、 F(ab，)2和 /或其它能与抗原特异性结合的抗 体片段和单克隆抗体。
抗体还可以以多种形式存在， 例如包括 Fv、 Fab和 (Fab')2, 以及双功 能杂合抗体（例如文献， Lanzavecchia等， Eur. J. Immunol , 1987; 17, 105) 以及以单链形式 （例如, Huston等， Proc. NatL Acad. Sci. U.S.A. , 1988; 85, 5879和 Bird等， Science, 1988; 242, 423, 在此引用作为参考）存在。 免疫球蛋白的重链或轻链可变区由三个超变区 （也称为"互补决定区"或 CDR )组成， 这些超变区被 架区 （FR ) 间隔。 架区和互补决定区的 范围已被精确定义 (参见" Sequences of Proteins of Immunological Interest," E. Kabat等, U.S. Department of Health and Human Services, 1991)。 此处所 讨论的所有抗体氨基酸序列的排序都参照 Kabat系统。同一物种不同的轻 链和重链框架区序列相对保守。抗体的框架区用于定位和校准 CDR。 CDR 主要负责结合抗原的表位。 嵌合抗体是其重链和轻链基因经过构建的抗体，特别是利用基因工程 改造的属于不同物种的抗体可变区和恒定区基因。例如，可以将鼠单克隆 抗体基因的可变区片段连接到人抗体恒定区片段如 γΐ和 γ3。 例如治疗用 嵌合抗体是一种嵌合蛋白质，它由来源于兔抗体可变区片段或抗原结合区 片段和人抗体恒定区或效应区结合（如由 A.T.C.C.保藏登记号 CRL 9688 的细胞制备的抗 Tac嵌合抗体 ), 当然， 嵌合抗体的基因来源也可以使用 其它哺乳动物物种。
术语"人源化抗体"与"人源化免疫球蛋白"含义相同，通常人源化抗体 与同一种抗体的非人源化形式相比， 会降低在人宿主中产生的免疫反应。
可以理解本发明设计和生产的人源化抗体可能会替代某些保守性氨 基酸，这些氨基酸对抗原结合或抗体其他功能基本上没有影响。换而言之， gly和 ala; val、 ile和 leu; asp和 glu; asn和 gin; ser和 thr; lys和 arg; phe和 tyr, 以上各组合内部的氨基酸可相互取代。 未存在于同一组中的 氨基酸属于"基本上不同的"氨基酸。
在某些实施方案中， 抗体与其靶点之间的亲和力用 KD (解离常数） 来表征， 它低于 10-6 M、 10-7 M、 10-8 M、 10-9 M、 10-10 M、 10-11 M或者 约 10_12 M或更氐。
抗体重链或轻链的"可变区 "是该链的 N端成熟区域。所有区域、 CDR 和残基编号均以序列比对、按已有的结构知识为基础进行定义。框架区和 CDR 残基的鉴定和编号按 Chothia 和他人所述（Chothia, Structural determinants in the sequences of immunoglobulin variable domain. J Mol Biol. 1998; 278,457)。
VH是抗体重链的可变区。 VL是抗体轻链的可变区， 它可能具有 κ 和 λ同种型。 K-1抗体具有 κ-1 同种型而 Κ-2抗体具有 κ-2同种型， νλ 是可变的 λ轻链。
术语"多肽"和"蛋白质 "在本文中可以互换使用，它们都是指任何长度 的聚合形式的氨基酸，可以包括编码和非编码的氨基酸、通过化学或生物 化学修饰或衍生的氨基酸以及具有修饰肽骨架的多肽。该术语包括融合蛋 白， 包括但不限于具有异源氨基酸序列的融合蛋白；具有异源和同源前导 序列， 带有或不带有 N-末端曱硫氨酸残基的融合蛋白； 带有免疫标签的 蛋白； 带有可检测融合伴侣的融合蛋白， 例如包括荧光蛋白质、 β-半乳糖 苷酶、荧光素等等作为融合伴侣的融合蛋白等等。多肽可以具有任何大小， 术语"肽"是指长度为 8-50个残基（如 8-20个残基 ) 的多肽。
术语"受试者"、 "宿主"、 "患者 "以及 "个体 "在本文中可以交替使用， 具体是指接受诊断或治疗的任何哺乳动物，尤其是指人类。其它对象可能 包括猴、 牛、 狗、 猫、 豚鼠、 兔、 大鼠、 小鼠和马等。
"相应的氨基酸"，是指当两个或多个氨基酸序列比对时，位于相同位 置（也就是它们彼此对应）的氨基酸残基。抗体序列比对和编号的方法在 Chothia, 见上， Kabat, 见上和其他中得到详尽阐述。 本领域普通技术人 员已知 (参见如 Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), 有时可以在抗体的一个或两个氨基酸中制造一 个、 两个或三个缺口和 /或插入 1、 2、 3或 4个残基或者至多约 15个残基 (特别是在 L3和 H3 CDR中；)， 从而完成一次比对。
"可取代位置"，指的是抗体的一个特殊位置， 其可以被不同的氨基酸 取代而不会使抗体的结合活性显著降低。鉴定可取代位置的方法和它们可 以被如何取代在下面将进行更加详细的描述。 可取代位置也可以称为"变 异耐受位置"。
"亲本 "抗体是指作为氨基酸取代的靶抗体。 在某些实施方案中， "供 体"抗体会将氨基酸"赠捐"给亲本抗体以生成改变的抗体。 "相关抗体"是 指具有相似序列并且由具有共同 B细胞祖先的细胞产生的抗体。 这种 B 细胞祖先含有具有重排轻链 VJC区和重排重链 VDJC区基因组， 并且产 生还未经历亲和力成熟的抗体。存在于脾脏组织中的 "纯真 "或"原始" B细 胞是 B细胞的共同祖先。 相关抗体与相同的抗原表位结合通常在序列上 极为相似， 特别是它们的 L3和 H3 CDR。 相关抗体的 H3和 L3 CDR都 具有相同的长度和近乎一致的序列 （有 0-4个氨基酸残基不同）。 相关抗 体通过共同抗体祖先， 即原初 B细胞祖先产生的抗体相关联。 发明内容 本发明的目的是提供一种对 VEGF 亲和力更高的单克隆抗体。 本发 明所述 VEGF单克隆抗体，其重链可变区含有 SEQ ID NO.l、 SEQ ID N0.2 和 SEQ ID N0.3 所示的氨基酸序列， 和 /或其轻链可变区含有 SEQ ID N0.4、 SEQ ID NO.5和 SEQ ID N0.6所示的氨基酸序列。
本发明所述"抗体"应该解释为涵盖具有所需特异性的结合结构域的 任意特异性结合因子。 因而， 这个术语涵盖了与之同源的抗体片段、衍生 物、人源化抗体以及抗体的功能等同物和同源物，也包括含有抗原结合结 构域的任何多肽，无论是天然的还是合成产生的。抗体的实例是免疫球蛋 白亚型（如 IgG, IgE, IgM, IgD和 IgA)及其亚型亚类； 也可以是包含抗 原结合结构域的片段如 Fab、 scFv、 Fv、 dAb、 Fd; 和双链抗体 (diabodies)。 融合至另一多肽的、包含抗原结合结构域的嵌合体分子或者等同物也包括 在其中。 嵌合抗体的克隆与表达在 EP. A-0120694和 EP. A. 0125023 中描述。
本发明所述单克隆抗体可以是， 例如， 单价的或是单链抗体、 双链 抗体、 嵌合抗体、 人源化抗体、 以及上述抗体的衍生物、 功能等同物和同 源物， 也包括抗体片段和含有抗原结合结构域的任何多肽。
抗体可以通过许多方式修饰， 可用 DNA重组技术来产生保留原来抗 体特异性的其它抗体或嵌合分子。这种技术可以包括将编码抗体的免疫球 蛋白可变区或互补性决定区 (CDRs)的 DNA 引入不同免疫球蛋白的恒定 区或恒定区加才匡架区。 参见， EP . A . 184187 , GB 2188638A 或. EP. A. 239400。 还可以对杂交瘤细胞或产生抗体的其它细胞进行遗 传突变或其它改变， 这可以改变或者不改变所产生抗体的结合特异性。
本发明所述单克隆抗体除了重链和轻链中的高度可变区 CDR1、 CDR2和 CDR3和连接序列夕卜， 其它为框架区。框架区可在结合所需的三 维结构不受影响的条件下被其他序列置换，抗体特异性的分子基础主要来 自于它的高度可变区 CDR1、 CDR2和 CDR3 , 这些区域是与抗原结合的 关键部位。 为维持优选的结合特性， CDR的序列应尽可能保留， 然而， 可能需要一些氨基酸改变使结合特性最优化，本领域的技术人员可以用标 准做法来达到此目的。 在某些优选例中，一个单克隆抗体包含如下内容的可变区： 一个包含 了如 SEQ ID N0.7的重链可变区， 其含有 CDR1 (SN DVMCW; SEQ ID NO.l), CDR2 (GCIMTTDVVTEYANWAKS; SEQ ID ΝΟ·2)和 CDR3 (RDSVGSPLMSFDLW; SEQ ID NO.3); 和一个包含了如 SEQ ID NO.8的 轻链可变区， 其 CDR1 (QASQSIYN ELS; SEQ ID NO.4), CDR2 (RASTLAS; SEQ ID NO.5), 和 CDR3 (GGYKSYSNDGNG; SEQ ID ΝΟ·6)。
可变区 CDR的变体与上述 CDR区除了有至多 6个氨基酸取代的不 同外基本上是一致的 （例如， 1、 2、 3、 4或 5个氨基酸取代）， 在该单克 隆抗体的 CDR区具有与 VEGF结合活性。
在其它实施方案中， 抗体可包含： a) —个重链可变区， 其氨基酸序 列与 SEQ ID N0.7相比有至多 6个氨基酸序列取代的不同，例如 1、 2、 3、 4、 5、或 6个取代；和 b) —个轻链可变区，其氨基酸序列与 SEQ ID N0.8 相比有至多 6个氨基酸序列取代的不同， 例如 1、 2、 3、 4、 5、 或 6个氨 基酸取代。 目标抗体可能包含这些取代中的任何一个或其组合。
拥有这些取代位置中任一个的抗体和拥有所有取代位置的抗体同样 具有结合 VEGF的活性。 氨基酸取代可同时存在于框架区和 CDR区， 或 单独出现在框架区或 CDR区。 因此， 在某些优选例中， 重链可变区的框 架区的氨基酸序列与 SEQ ID N0.7相比可能有最多 6个氨基酸序列取代 的不同， 例如 1、 2、 3、 4、 5、 或 6个取代， 和轻链可变区的框架区的氨 基酸序列与 SEQ ID N0.8相比可能有最多 6个氨基酸序列取代的不同， 例如 1、 2、 3、 4、 5、 或 6个取代。
在一些抗体中， 氨基酸取代可能分布在多个 CDR区。 因此， 重链可 变区的多个 CDR区的氨基酸序列与 SEQ ID N0.7相比可能有至多 6个氨 基酸序列取代的不同， 例如 1、 2、 3、 4、 5、 或 6个取代， 和轻链可变区 的多个 CDR区的氨基酸序列与 SEQ ID NO.8相比可能有至多 6个氨基酸 序列取代的不同， 例如 1、 2、 3、 4、 5、 或 6个取代。
在特殊的优选例中， 抗体可能包含 a) —个重链可变区， 其氨基酸序 列与 SEQ ID NO. 7一致和 b) —个轻链可变区， 其氨基酸序列与 SEQ ID ΝΟ· 8一致。
在特殊的优选例中， 抗体可能包含 a)—个重链可变区， 其氨基酸序 列与 SEQ ID NO. 7至少有 95%的一致性和 b)—个轻链可变区， 其氨基酸 序列与 SEQ ID NO. 8至少有 95%的一致性。 因此， 目标抗体可能包含 a) 一个重链可变区， 其氨基酸序列与 SEQ ID NO. 7至少有约 95%、 96%、 97%、 98%、 99%或 100%的一致性和 b) —个轻链可变区， 其氨基酸序列 与 SEQ ID NO. 8至少有约 95%、 96%、 97%、 98%、 99%或 100%的一致 性。
在特殊的优选例中，抗体可能包含 a) —个重链，其氨基酸序列与 SEQ ID NO. 9—致和 b) —个轻链， 其氨基酸序列与 SEQ ID NO. 10一致。
在特殊的优选例中，抗体可能包含 a)—个重链，其氨基酸序列与 SEQ ID N0.9至少有 95%的一致性和 b)—个轻链，其氨基酸序列与 SEQ ID NO. 10至少有 95%的一致性。 因此， 目标抗体可能包含 a)—个重链， 其氨基 酸序列与 SEQ ID NO. 9至少有约 95%、 96%、 97%、 98%、 99%或 100% 的一致性和 b) —个轻链，其氨基酸序列与 SEQ ID N0. 10至少有约 95%、 96%、 97%、 98%、 99%或 100%的一致性。 除了上面所描述的氨基酸取代， 目标抗体可能在重链或轻链的两端有附加的氨基酸。例如， 目标抗体在重 链和 /或轻链的 C或 N末端分别可能包含至少 1、 2、 3、 4、 5或 6或更多 的附加氨基酸。在某些实施例中， 目标抗体可能比这里所描述的示范性氨 基酸短， 其主要区别为在重链和轻链的两端分别比示范性氨基酸少 1、 2、 3、 4、 5或 6个氨基酸。
目标抗体可能是人源化的。一般而言，人源化抗体是通过在亲本抗体 的框架区进行氨基酸取代而产生修饰的抗体，并且人源化的抗体与亲本抗 体相比具有较小的免疫源性。抗体可通过本领域熟知的很多技术进行人源 化， 包括， 例如， CDR移植（ERA-239,400; PCT公开文本 WO 91/09967; U.S. Pat. No. 5,225,539; 5,530,101; 和 5,585,089), 和链替换 (U.S. Pat. No. 5,565,332)。 在某些优选例中， 框架替换是通过模拟 CDR和框架残基的相 互作用来确认框架残基对于抗原结合的重要性和通过序列比对确认特殊 位点的非寻常框架残基。 （见，例如， U.S. Pat. No. 5,585,089; Riechmann et al.， Nature, 1988; 332, 323)。 抗体的人源化方法的具体细节可参照美国 专利申请 10/984,473 , 即在 2004年 11月 8日提交， 标题为 "Methods for antibody engineering" , 该申请已经全部弓 |入本文作为参考。 一般而言， 这种人源化的方法包括通过比对能够结合相同抗原的抗体的序列来确定 合适的位点，并且用相似氨基酸位于相同位点的不同氨基酸来取代该位点 的氨基酸。在这些方法中，将亲本抗体的氨基酸序列与其它相关的抗体进 行比较（例如， 序列比对）， 从而识别变异耐受位置。 亲本抗体可变区的 氨基酸序列通常与人类抗体数据库中的氨基酸序列进行比较，并且选出这 种与亲本抗体具有相似的氨基酸序列的人源化抗体。将亲本抗体和人源化 抗体的序列进行比较（例如， 序列比对）， 亲本抗体中一个或多个变异耐 受位置上的氨基酸被人源抗体中相应位置上的氨基酸所取代。
上面所讨论的变异耐受位置的取代方法已很容易地与任何已知的人 源化方法结合， 并且也很容易地应用于生产包含 CDR区的人源化抗体， 该抗体的 CDR区是在忠实于亲本抗体的 CDR区的基础上进行修改的。 因 此， 本发明还提供了包含从亲本抗体改变版本的多个 CDR区的人源化 VEGF中和抗体。
本发明所论述的人源化兔源抗 VEGF 单克隆抗体比现有技术产物
AVASTIN与 VEGF 的解离常数 Kd要低（本发明的单克隆抗体 Kd为 0.485 nM, AVASTIN为 47.9 nM ), 本发明的单克隆抗体与 VEGF的亲和力更高， 表明本发明对 VEGF有更强的抑制作用。 在小鼠模型试验上显示本发明所 述抗体抑瘤率明显高于 AVASTIN (见实施例 5 )，所以， 理论上本发明的潜 在临床疗效要高于 AVASTIN。
在一个实施例中， 本发明所述单克隆抗体由保藏编号为 CGMCC No.3233的细胞株产生， 其重链氨基酸序列如 SEQ ID N0.9所示， 轻链如 SEQ ID NO.10所示的， 其与 VEGF解离常数为 0.485nM, 是 AVASTIN 的 1/100, 说明 EPI0030与 VEGF的结合能力强于 AVASTIN。
本发明还提供一种细胞株，保藏在中国微生物菌种保藏管理委员会普 通微生物中心， 保藏编号为 CGMCC No.3233。 其产生一种单克隆抗体， 所述抗体重链氨基酸序列如 SEQ ID N0.9所示，轻链如 SEQ ID NO.10所 示， 在本发明的实施例中， 命名为 EPI0030抗体， 细胞试验及动物体内试 验证明， 可在体外抑制 VEGF诱导的内皮细胞增殖和迁移， 并且可在动 物体内抑制肿瘤生长， 可用于治疗 VEGF相关性疾病。
本发明还提供所述的单克隆抗体在制备用于治疗 VEGF相关性疾病 药物中的用途。 所述 VEGF相关性疾病包括肿瘤、 老年性黄斑变性、 神 经退行性疾病、 肥胖、 糖尿病。 该目标抗体可以用于与 VEGF相关的科 学研究， 如发育生物学、 细胞生物学、 代谢、 结构生物学、 功能基因组学 等多个领域的科学研究、 或肿瘤、 老年性黄斑变性（AMD )、 神经退行性 疾病、 肥胖、 糖尿病等医学和药学的应用研究。
本发明还提供一种药物组合物，其特征在于，含有有效量的上述的单 克隆抗体及药学上可接受的载体。
本发明还提供一种试剂、 试剂盒或芯片， 包含上述的单克隆抗体。 本发明还提供了使用目标抗体来抑制 VEGF活性的方法， 以及使用 目标抗体用于治疗 VEGF 相关性疾病或使用含有该抗体的试剂盒进行 VEGF相关诊断与检测。
一旦制得本发明的抗体分子，就可以通过本领域已知的纯化免疫球蛋白 分子的任何方法对其进行纯化，例如，通过色谱法（例如， 离子交换色谱， 亲和色谱， 特别是通过蛋白 A对特异性抗原的亲和色谱和其它柱色谱）、 离心、 利用溶解度差异， 或通过任何其它纯化蛋白质的标准技术。 在许多 实施方案中，抗体从细胞分泌到培养基中，通过收集培养基并进行纯化得 到抗体。
抗体可以通过许多方式修饰， 可用 DNA重组技术来产生保留原来抗 体特异性的其它抗体或嵌合分子。这种技术可以包括将编码抗体的免疫球 蛋白可变区或互补决定区 (CDRs)的 DNA 引入不同免疫球蛋白的恒定区 或恒定区加框架区。参见， EP. A-184187, GB 2188638A或 EP. A-239400。 还可以对杂交瘤细胞或产生抗体的其它细胞进行遗传突变或其它改变，这 可以改变或者不改变所产生抗体的结合特异性。
用于本发明的单克隆抗体也可用杂交瘤方法制得， 因为编码本发明 人源化抗体的 DNA序列可用本领域技术人员熟知的常规手段， 如根据本 发明公开的氨基酸序列人工合成或用 PCR法扩增得到， 因而也可用重组 DNA方法，可用本领域熟知的各种方法将该序列连入合适的表达载体中。 最后， 在适合本发明抗体表达的条件下， 培养转化所得的宿主细胞， 然后 本领域技术人员应用熟知的常规分离纯化手段纯化得到本发明的单克隆 抗体。
如上所述， 本发明还提供了用于实施本发明抗体的试剂、 试剂盒或 芯片。 试剂、 试剂盒或芯片至少包括如下一种或多种： 根据以上方法制成 的抗体， 编码该抗体的核苷酸， 或包含该抗体的真核细胞、原核细胞和病 毒。 可以对抗体进行人源化。
试剂、 试剂盒或芯片的其它任选组分包括： 限制性内切酶、 引物和 质粒、 緩沖液等， 用于进行检测抗体活性的实验。 该试剂、 试剂盒或芯片 的核酸还可以具有限制性酶切位点、 多克隆位点、 引物位点等等， 以利于 它们与非兔抗体核酸的连接。该试剂、试剂盒或芯片的各组分可以单独存 在于分开的容器中，也可以根据需要，把某些相容的组分预先组装到单一 的容器中。
制备目标抗体时可加入药学上可接受的载体。 术语"药学上可接受载 体" 是指一种或多种有机或无机成分， 它可以是天然的或合成的， 与抗体 组合后可促进其应用。可接受的载体包括无菌的生理盐水或是其它药学上 可获得的且为本领域所熟知的水或非水的等渗溶液和灭菌混悬剂。 "有效 剂量"是指能够改善或延緩病态的、 退变的或损坏的状况的进程的剂量。
有效剂量定义在个人基础之上，并且将以此为基础，具体来说即考虑 治疗症状和寻找结果。有效剂量可通过本领域的一种普通技术来决定，并 且使用的这些因素将不会超过常规实验。 生物材 ·藏说明
保藏编号为 CGMCC No.3233的细胞株于 2009年 8月 20日保藏在中 国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区大屯 路， 分类命名为中国仓鼠卵巢细胞。 附图说明
图 1示 HZD-V1、 HZD-V2、 HZD-V5、 HZD-V6克隆表达的重组抗体竟争 性抑制 VEGF与 KDR结合；
图 2示 SDS-PAGE测定 HZD-V6克隆表达 EPI0030抗体的纯度； 泳道 1为还原电:^；
泳道 2为分子量标准一从上至下依次为 170kD、 130 kD、 100kD、 70kD、 55kD、 40kD、 35kD、 25kD、 15kD和 lOkD;
泳道 3为非还原电泳；
图 3为 HZD-V6克隆表达 EPI0030抗体的纯度的 SEC-HPLC分析图； 图 4示 VEGF直接铺板结合法测定抗体与人 VEGF的结合活力； 图 5示 EPI0030、 AVASTIN抑制 VEGF与 KDR结合的 IC50测定；
EPI0030的 IC50=166.3ng/ml, AVASTIN的 IC50=253.7ng/ml。
图 6示 5 mg/kg的 EPI0030和 AVASTIN对 HCT-116肿瘤生长的影响； 图 7示 5 mg/kg的 EPI0030和 AVASTIN对 NCI-H460肿瘤生长的影响； 图 8示 1.5 mg/kg的 EPI0030对 NCI-H460肿瘤生长的影响。 具体实施方式
实施例 1 : 制备表达抗人 VEGF165兔单抗的杂交瘤细胞及其基因克隆 通过杂交瘤细胞技术制备兔单克隆抗体。 有关实验方案参见 US Patent: 7,429,487, 特别是 Example 1-4。
首先通过重组技术制备 IgG Fc-hVEGF-A (Human VEGF 165)融合蛋 白，其中 IgG Fc序列为兔源。将 IgG Fc-hVEGF-A的 DNA序列克隆入 pTT5 质粒， 瞬时转染该质粒进入 ΗΕΚ 293-6Ε细胞株， 无血清培养细胞， 收集 培养上清液， 用 Protein A柱纯化瞬时表达的 IgG Fc-hVEGF-A融合蛋白。
用纯化的 IgG Fc-hVEGF-A (作为抗原组分）与完全弗氏佐剂混合进 行皮下多点注射， 对新西兰白兔（ New Zealand rabbits )进行首次免疫， 此后每三周 1次用纯化蛋白和不完全弗氏佐剂混合后皮下注射对兔进行加 强免疫， 在取脾脏前 4天用抗原加 PBS对兔静脉注射进行最终免疫。 根据美国专利 US7429487的方法，将兔脾细胞与免疫脾细胞来源相同 的永生化 HRGTP-的 B淋巴细胞 240E-W2细胞按 2: 1比例融合， 在 96孔板 中以 HAT培养基培养， 然后进行杂交瘤细胞筛选，所得细胞克隆进入新的 IgG Fc-hVEGF-A结合筛选。
鉴定筛选过程分为 2个阳性克隆筛选步骤：①将 IgG Fc-hVEGF-A抗原 固定化于 96孔酶联免疫吸附板上， 加入克隆表达上清孵育 lh后， 用 PBS 洗涤 3次，使用酶标记的抗体鉴定具有 IgG Fc-hVEGF-A结合活性细胞克隆 上清， 从而获得可与 IgG Fc-hVEGF-A直接结合的阳性克隆。 ②随后将步 骤①中的阳性克隆转移入 24孔板培养， 以获得更多的表达产物。 将 IgG Fc-VEGFR2 ( KDR/Flk-1 )胞外区固定化于 96孔酶联免疫吸附板上， 加入 IgG Fc-hVEGF-A和克隆表达产物共同孵育 lh, 再用 PBS洗涤 3次， 使用酶 标记的抗体检测 IgG Fc-hVEGF-A的含量， 以鉴定克隆对 VEGF-VEGFR2 结合活性的抑制作用，从而鉴别出能够阻断 VEGF-VEGFR2结合的阳性克 隆。
将所筛选的阳性克隆的杂交瘤细胞裂解， 提取 mRNA后逆转录得 cDNA。 以此 cDNA为模板， 采用 PCR方法分别扩增出兔 IgG抗体的轻链和 重链可变区核酸序列， 对重链可变区、 轻链可变区进行分析， 其编码的重 链可变区含有 SEQ ID NO.1的亲本序列 （ Ser Asn Asn Asp Val Met Cys Trp ), SEQ ID N0.2的亲本序列 （ Gly Cys lie Met Thr Thr Asp Val Val Thr Glu Tyr Ala Asn Trp Ala Lys Ser )和 SEQ ID NO.3的亲本序列 （ Arg Asp Ser Val Gly Ser Pro Leu Met Ser Phe Asp Leu Trp )、 轻链可变区含有 SEQ ID ΝΟ·4的亲本序列 （ Gin Ala Ser Gin Ser Val Tyr Gly Asn Asn Glu Leu Ser )、 SEQ ID N0.5的亲本序列（ Arg Ala Ser Thr Leu Ala Ser )和 SEQ ID N0.6的 亲本序列（Gly Gly Tyr Lys Ser Tyr Ser Asn Asp Gly Asn Gly )。 轻链核酸序 列被克隆入 pTT5质粒。 重链可变区核酸序列被克隆入已有重链恒定区的 ρΤΤ5质粒。 共转染轻、 重链质粒至 ΗΕΚ 293-6Ε细胞株， 培养 5天后， 用 Protein Α纯化上清， 最终获得重组表达的兔抗人 VEGF 165单克隆抗体。采 用上述阳性克隆筛选方法， 对表达的重组抗体进行亲和力确认。 实施例 2: 本发明所述人源化兔抗 VEGF单抗的制备
人源化技术参见美国专利 US 7,462,697, 特别是优化实施方式的详细 描述部分（ DETAILED DESCRIPTION OF PREFERRED
EMBODIMENTS )。
采用美国专利 US7,462,697所描述的技术， 用人序列 VKI-2-l- ( U ) -A20— JK4和 VH3-1-3-3-21— JH4作为参比序列。 表达的兔抗 VEGF单抗序 列经人源化后，各得到 4个版本的 VK和 VH。轻链可变区包括 VK-HZD1 (如 SEQ ID NO.12 ), VK-HZD2 (如 SEQ ID NO.14 ), VK-HZD5 (如 SEQ ID NO.16 )和 VK-HZD6 (如 SEQ ID NO.8 ); 重链可变区包括与 VH-HZD1 (如 SEQ ID NO.ll )所示、 VH- HZD2 (如 SEQ ID NO.13 )、 VH- HZD5 (如 SEQ ID NO.15 )和 VH- HZD6 (如 SEQ ID NO.7 )„与 VK-HZD1比较， VK-HZD2 在 CDR1区具有 2个不同的残基。 在 VK-HZD1和 VK-HZD2 N端添加 2个额 外的氨基酸残基后即分别成为 VK-HZD5和 VK-HZD6。 VH-HZD1与 VH-HZD2的 71位残基不同， VH-HZD1的 71位是 K, VH-HZD2的 71位是 R。 VK(H)-HZD1和 VK(H)-HZD 2序列中含有兔源信号肽， 而 VK(H)-HZD5和 VK(H)-HZD6的序列中含有人源信号肽。
将 4个版本的 VK和 VH的 DNA序列通过人工合成后分别克隆进已 有人 CK序列和人 CH序列的 pTT5质粒中， 通过人信号肽表达抗体。 将 上述两个质粒共转染 ΗΕΚ 293-6Ε细胞， 瞬时表达人源化抗 VEGF抗体， 使用实施例 1 中的筛选方法， 选定亲和力适宜的 HZD-V6克隆作为最终 使用的克隆， 其表达的人源化抗 VEGF抗体称为 EPI0030, 其氨基酸序列 如 SEQ ID N0.9所示的重链和 SEQ ID NO.10所示的轻链。
为提高产量， 获得工业用生产细胞株，将氨基酸序列如 SEQ ID N0.9 所示的重链和 SEQ ID NO.10所示的轻链表达质粒共转染入中国仓鼠卵巢 细胞株 ( CHO ) , 于 2009年 8月 20日保藏在中国微生物菌种保藏管理委 员会普通微生物中心， 地址为北京市朝阳区大屯路，保藏编号为 CGMCC Νο·3233。 实施例 3： 本发明所述单克隆抗体 ΕΡΙ0030与 VEGF结合的检测 VEGF抗体通过结合 VEGF从而阻断其与受体 KDR的结合， 来抑制 VEGF信号通路。 1 :3系列稀释重组表达的不同抗体（ HZD-V1、 HZD-V2、 HZD-V5、 HZD-V6 )和 AVASTIN后 （ 9(^g/ml-45ng/ml )与 IgG Fc-hVEGF ( ^g/ml )混合， 加入混合好的抗体 -VEGF复合物至铺 IgG Fc-VEGFR2板 的孔中， 加入鼠抗人 VEGF抗体后用羊抗鼠 IgG抗体 -AP显色进行检测。 结 果显示所表达的重组抗体与 AVASTIN相似的竟争性抑制 VEGF与 KDR结 合的活性。 测定结果显示 HZD-V1、 HZD-V2、 HZD-V5、 HZD-V6克隆表 达的抗体均可以阻断 VEGF与 KDR的结合（见图 1 )。
参照上述实验方法， 测定 EPI0030和 AVASTIN的 IC5。值即半数 抑制浓度， 结果如图 5所示， 显示 EPI0030和 AVASTIN有相似的竟争抑 制活力。
采用 BIAcore-3000测定 EPI0030与 VEGF的解离常数 Kd。人 VEGF 被固定在 CM5芯片上， 2倍系列稀释的 EPI0030和 AVASTIN被注射进流 速为 30ul/min的 HBS-EP緩沖液中。 Kd 为 k。ff/k。n，表 1 中显示 EPI0030 和 AVASTIN与人 VEGF 的解离常数， EPI0030与 VEGF解离常数为 0.485nM,是 AVASTIN的 1/100,说明 EPI0030与 VEGF的结合能力强于 AVASTIN o
表 1.EPI0030和 AVASTIN与人 VEGF的解离常数
Figure imgf000017_0001
实施例 4: 本发明所述单克隆抗体 EPI0030的鉴定和体外活性的测定
HEK 293-6E细胞 HZD-V6克隆瞬时表达的 EPI0030抗体经过纯化后， 进行了有关质量的检定， 具体鉴定项目如下：
A: 纯度
采用 SDS-PAGE (还原和非还原）及 SEC-HPLC来分析纯度， 结果 见图 2、 图 3 , 显示 EPI0030抗体的纯度大于 98%, 多聚体含量小于 5%, 主峰（保留时间 10.572分钟）面积占总面积（包括从左至右的峰 1、 2、 3 ) 大于 95%, 峰 1、 2为多聚体。
B: 体外结合活性
IgG Fc-hVEGF铺板， 1%BSA封闭， 1:3 系列稀释 EPI0030抗体和 AVASTIN 8个梯度（ l g/ml-0.46ng/ml ), 加入至已铺好 IgG Fc-hVEGF的 孔中， 加驴抗人 IgG抗体 -AP检测。 结果显示 EPI0030抗体和 AVASTIN 有相似的结合活力。
采用 ELISA方法， 包括 IgG Fc-hVEGF直接铺板法（见实施例 1 )和 KDR竟争结合法（见实施例 3 ), 结果显示 EPI0030抗体与 VEGF的结合 以及抑制 VEGF与 KDR的结合呈剂量相关性， 结果见图 4和图 5。 实施例 5: 本发明所述单克隆抗体 EPI0030的体内活性检测
在实验前两周从液氮罐中取出一支冻存的人结肠癌 HCT- 116细胞和 人非小细胞肺癌 NCI-H460细胞（约 Ι χΙΟ7细胞）迅速放至 37°C水浴中融化， 随后分别用预热至 37°C添加了 10%胎牛血清（购自 GIBCO )的 McCoy's 5A 培养基和 DMEM培养基将细胞接种至 75CM2的细胞培养瓶中，待细胞生长 至 80%融合率时按 1 : 5进行传代， 在体外连续传代三次以上。 当细胞总量 达到接种所需，将细胞用胰酶消化、 离心，然后用 PBS洗涤细胞去除血清， 最后分别用无血清、 无抗生素 McCoy's 5A培养基和 DMEM培养基调整处 理好的人结肠癌 HCT- 116和人非 d、细胞肺癌 NCI-H460细胞密度至
5 l07/ml, 细胞悬液放置于冰上， 接种于 6-8周龄棵鼠腹侧部， 每只小鼠 接种 0.1 ml, 即 5x l 06细胞 /只。 以游标卡尺每 2天测量棵小鼠的肿瘤直径， 待每只小鼠的肿瘤均长至 100 mm3-300 mm3,按瘤体积 SD <1/3入组后随机 分为 5组， 每组 6只。 人结肠癌 HCT-116模型组分别为模型对照组（1组）、 5mg/kg AVASTIN(l组)和 5mg/kg EPI-0030(1组)组， 将 AVASTIN和
EPI-0030用生理盐水稀释至 0.5mg/ml。人非小细胞肺癌 NCI-H460模型组分 别为模型对照组（1组）、 5mg/kg AVASTIN(l组)和 1.5mg/kg、 5mg/kg
EPI-0030(2组)组将 AVASTIN和 EPI-0030用生理盐水稀释至 0.5mg/ml和 0.15mg/ml。 给药组分别每周 1 , 3 , 5d分别给予一次性腹腔注射 0.2ml的 AVASTIN和 EPI-0030, 模型对照组以同样的时间和方式给予生理盐水 0.2ml, 连续给药 3周 （21天， 共 9次）。
测量棵鼠的肿瘤瘤结节最大直径 a和最小径 b, 按公式 V = 0.5xaxb2计 算肿瘤体积， 以相对肿瘤增殖率 T/C %作为疗效评价指标。
结果显示， 人结肠癌 HCT-116模型中， 5mg/kg剂量组中， EPI-0030 与 AVASTIN的抑瘤率分别为 65.7%和 15.7% (见图 6 )。 人非小细胞肺癌 NCI-H460模型中， EPI-0030与 AVASTIN的抑瘤率分别为 95.5%和 66.7% (见图 7 ); 1.5mg/kg剂量组中 EPI-0030的抑瘤率为 57.6% (见图 8 )。 结 果说明 EPI-0030有显著的抑制肿瘤活性的作用。
参考 AVASTIN临床适应症，选择人结肠癌 HCT-116和人非小细胞肺 癌 NCI-H460棵鼠异种移植瘤模型验证 EPI-0030对人结肠癌和人非小细 胞肺癌的抑制作用， 证明 EPI-0030具有显著抑制人结肠癌和人非小细胞 肺癌异种移植瘤的作用。 EPI-0030在模型中抑瘤活性强于 AVASTIN。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的 普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进 和润饰， 这些改进和润饰也应视为本发明的保护范围。

Claims

权 利 要 求
1. 一种单克隆抗体，其特征在于，其重链可变区含有 SEQ ID NO.1 , SEQ ID N0.2和 SEQ ID N0.3所示的氨基酸序列，和 /或其轻链可变区含有 SEQ ID N0.4、 SEQ ID N0.5和 SEQ ID NO.6所示的氨基酸序列。
2. 根据权利要求 1所述的单克隆抗体，其特征在于，所述单克隆抗体包 括单链抗体、双链抗体、嵌合抗体、人源化抗体、以及上述抗体的衍生物、 功能等同物和同源物， 也包括抗体片段和含有抗原结合结构域的任何多 肽。
3. 根据权利要求 1或 2所述的单克隆抗体，其特征在于，其重链可变区 氨基酸序列如 SEQ ID N0.7所示； 或其重链可变区由 SEQ ID N0.7所示 的氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与 SEQ ID NO. 7的氨基酸序列至少有 95%的一致性，且所述单克隆抗体具有特异性 结合 VEGF的活性。
4. 根据权利要求 1或 2所述的单克隆抗体，其特征在于，其轻链可变区 如 SEQ ID N0.8所示的氨基酸序列； 或其轻链可变区由 SEQ ID N0.8所 示的氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与 SEQ ID NO. 8的氨基酸序列至少有 95%的一致性， 且所述单克隆抗体具有特 异性结合 VEGF的活性。
5. 根据权利要求 1或 2所述的单克隆抗体，其特征在于，其重链如 SEQ ID N0.9所示的氨基酸序列；或其重链由 SEQ ID N0.9所示的氨基酸序列 经过取代、 缺失、 或添加一个或几个氨基酸衍生的与 SEQ ID NO. 9的氨 基酸序列至少有 95%的一致性，且所述单克隆抗体具有特异性结合 VEGF 的活性。
6. 根据权利要求 1或 2所述的单克隆抗体，其特征在于，其轻链氨基酸 序列如 SEQ ID NO.10所示； 或其轻链由 SEQ ID NO.10所示的氨基酸序 列经过取代、 缺失、 或添加一个或几个氨基酸衍生的与 SEQ ID NO. 10 的氨基酸序列至少有 95%的一致性， 且所述单克隆抗体具有特异性结合 VEGF的活性。
7. 根据权利要求 1-6任一项所述的单克隆抗体，其特征在于由保藏编号 为 CGMCC No.3233的细胞株产生。
8. 一种细胞株，其特征在于，保藏在中国微生物菌种保藏管理委员会普 通 生物中心， 保藏编号为 CGMCC No.3233。
9. 权利要求 1至 7任一项所述的单克隆抗体在制备用于治疗 VEGF相 关性疾病药物中的用途。
10. 根据权利要求 9所述的用途， 其特征在于， 所述 VEGF相关性疾病 包括肿瘤、 老年性黄斑变性、 神经退行性疾病、 肥胖、 糖尿病。
11. 一种药物组合物， 其特征在于，含有有效量的权利要求 1-7任一项所 述的单克隆抗体及药学上可接受的载体。
12. 一种试剂、试剂盒或芯片， 包含权利要求 1至 7任一项所述的单克隆 抗体。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2259795A1 (en) * 2008-03-26 2010-12-15 Epitomics, Inc. Anti-vegf antibody
US8986692B2 (en) 2009-08-28 2015-03-24 Jiangsu Simcere Pharmaceutical R & D Co., Ltd. Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody
US10919947B2 (en) 2015-09-01 2021-02-16 Il Dong Pharmaceutical Co., Ltd. Pharmaceutical composition containing, as active ingredient, fusion protein in which tumor-penetrating peptide and anti-angiogenesis agent are fused, for preventing and treating cancer or angiogenesis-related diseases
EP3973977A1 (en) 2015-03-31 2022-03-30 Ildong Pharm Co., Ltd. Pharmaceutical composition for preventing and treating eye diseases, containing, as active ingredient, fusion protein in which tissue-penetrating peptide and anti-vascular endothelial growth factor preparation are fused

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875676B (zh) * 2011-07-13 2014-11-05 无锡天演生物技术有限公司 全人源抗人vegf单抗分子及其应用
CN103417965B (zh) * 2012-05-17 2018-04-13 江苏先声药业有限公司 一种含有抗vegf抗体的药物组合物
CN103417964A (zh) * 2012-05-17 2013-12-04 江苏先声药物研究有限公司 一种抗vegf抗体的用途
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
AU2015376558B9 (en) * 2015-01-06 2021-07-01 Zhuhai Essex Bio-Pharmaceutical Co., Ltd. Anti-VEGF antibody
CA2998343A1 (en) * 2015-09-14 2017-03-23 Galaxy Biotech Llc Highly potent monoclonal antibodies to angiogenic factors
AU2016381964B2 (en) 2015-12-30 2024-02-15 Kodiak Sciences Inc. Antibodies and conjugates thereof
US11008387B2 (en) 2016-01-06 2021-05-18 Order-Made Medical Research Inc. Antibody inhibiting binding of VEGF to NRP1
KR102293755B1 (ko) 2016-01-06 2021-08-24 오더-메이드 메디컬 리서치 인코포레이티드 고친화성 항vegf 항체
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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (en) 1983-03-25 1984-10-03 Celltech Limited Processes for the production of multichain polypeptides or proteins
EP0125023A1 (en) 1983-04-08 1984-11-14 Genentech, Inc. Recombinant immunoglobulin preparations, methods for their preparation, DNA sequences, expression vectors and recombinant host cells therefor
EP0184187A2 (en) 1984-12-04 1986-06-11 Teijin Limited Mouse-human chimaeric immunoglobulin heavy chain, and chimaeric DNA encoding it
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
CN1445242A (zh) * 2002-03-20 2003-10-01 上海中信国健药业有限公司 人源化抗血管内皮生长因子单克隆抗体及其制法和药物组合物
CN101148474A (zh) * 2006-09-21 2008-03-26 上海杰隆生物工程股份有限公司 人源人血管内皮生长因子单克隆抗体及其制备方法
US7429487B2 (en) 2005-07-05 2008-09-30 Epitomics, Inc. Fusion partner for production of monoclonal rabbit antibodies
US7462697B2 (en) 2004-11-08 2008-12-09 Epitomics, Inc. Methods for antibody engineering
CN101487005A (zh) * 2008-09-04 2009-07-22 林植华 人血管内皮生长因子单克隆抗体的基因及其应用

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1185559A2 (en) * 1999-04-28 2002-03-13 Board Of Regents, The University Of Texas System Compositions and methods for cancer treatment by selectively inhibiting vegf
US20040086979A1 (en) 2002-08-15 2004-05-06 Dongxiao Zhang Humanized rabbit antibodies
US20040067496A1 (en) 2002-10-07 2004-04-08 Robert Pytela System for production and screening of monoclonal antibodies
US8071322B2 (en) 2002-11-12 2011-12-06 Epitomics, Inc. Method for identifying differentially expressed proteins
US7402409B2 (en) 2003-01-23 2008-07-22 Epitomics, Inc. Cell fusion method
WO2005007671A2 (en) 2003-04-29 2005-01-27 Epitomics, Inc. Compositions and methods for treating sars
US20050048578A1 (en) 2003-06-26 2005-03-03 Epitomics, Inc. Methods of screening for monoclonal antibodies with desirable activity
JP2007501011A (ja) * 2003-08-01 2007-01-25 ジェネンテック・インコーポレーテッド 制限多様性配列を有する結合型ポリペプチド
CA2533830A1 (en) 2003-08-07 2005-02-24 Epitomics, Inc. Methods for humanizing rabbit monoclonal antibodies
US20050277127A1 (en) 2003-11-26 2005-12-15 Epitomics, Inc. High-throughput method of DNA immunogen preparation and immunization
US20050287118A1 (en) 2003-11-26 2005-12-29 Epitomics, Inc. Bacterial plasmid with immunological adjuvant function and uses thereof
US7431927B2 (en) 2005-03-24 2008-10-07 Epitomics, Inc. TNFα-neutralizing antibodies
JP2010507594A (ja) * 2006-10-20 2010-03-11 シェーリング コーポレイション 完全ヒト抗ｖｅｇｆ抗体および使用方法
MX2009012493A (es) * 2007-05-21 2010-01-20 Alder Biopharmaceuticals Inc Metodos de humanizacion de anticuerpo de conejo novedosos y anticuerpos de conejo humanizados.
TWI580694B (zh) * 2007-11-30 2017-05-01 建南德克公司 抗-ｖｅｇｆ抗體
JP5823858B2 (ja) 2008-03-26 2015-11-25 エピトミクス インコーポレーティッド 抗ｖｅｇｆ抗体
US9365644B2 (en) 2008-04-23 2016-06-14 Epitomics, Inc. Anti-TNFα antibody
US9079942B2 (en) 2009-02-09 2015-07-14 Epitomics, Inc. CDR-anchored amplification method
CN102002104A (zh) 2009-08-28 2011-04-06 江苏先声药物研究有限公司 一种抗vegf的单克隆抗体及含有该抗体的药物组合物
US8293483B2 (en) 2009-09-11 2012-10-23 Epitomics, Inc. Method for identifying lineage-related antibodies
US9085621B2 (en) 2010-09-10 2015-07-21 Apexigen, Inc. Anti-IL-1β antibodies
AU2012223449A1 (en) 2011-03-03 2013-05-02 Apexigen, Inc. Anti-IL-6 receptor antibodies and methods of use
CN106928362B (zh) 2011-04-29 2021-10-26 埃派斯进有限公司 抗-cd40抗体及其使用方法
US9057728B2 (en) 2011-07-12 2015-06-16 Epitomics, Inc. FACS-based method for obtaining an antibody sequence
EP2914627B1 (en) 2012-10-30 2021-04-07 Apexigen, Inc. Anti-cd40 antibodies and methods of use

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (en) 1983-03-25 1984-10-03 Celltech Limited Processes for the production of multichain polypeptides or proteins
EP0125023A1 (en) 1983-04-08 1984-11-14 Genentech, Inc. Recombinant immunoglobulin preparations, methods for their preparation, DNA sequences, expression vectors and recombinant host cells therefor
EP0184187A2 (en) 1984-12-04 1986-06-11 Teijin Limited Mouse-human chimaeric immunoglobulin heavy chain, and chimaeric DNA encoding it
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
GB2188638A (en) 1986-03-27 1987-10-07 Gregory Paul Winter Chimeric antibodies
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
CN1445242A (zh) * 2002-03-20 2003-10-01 上海中信国健药业有限公司 人源化抗血管内皮生长因子单克隆抗体及其制法和药物组合物
US7462697B2 (en) 2004-11-08 2008-12-09 Epitomics, Inc. Methods for antibody engineering
US7429487B2 (en) 2005-07-05 2008-09-30 Epitomics, Inc. Fusion partner for production of monoclonal rabbit antibodies
CN101148474A (zh) * 2006-09-21 2008-03-26 上海杰隆生物工程股份有限公司 人源人血管内皮生长因子单克隆抗体及其制备方法
CN101487005A (zh) * 2008-09-04 2009-07-22 林植华 人血管内皮生长因子单克隆抗体的基因及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423
CHOTHIA: "Structural determinants in the sequences of immunoglobulin variable domain", J MOL BIOL., vol. 278, 1998, pages 457
HUSTON ET AL., PROC. NATL ACAD. SCI. U.S.A., vol. 85, 1988, pages 5879
LANZAVECCHIA ET AL., EUR. J. IMMUNOL, vol. 17, 1987, pages 105
MATSUMOTO; MARTI ET AL.: "Angiogenesis in ischemic disease", THROMB HAEMOST., vol. 1, 1999, pages 44 - 52
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2259795A1 (en) * 2008-03-26 2010-12-15 Epitomics, Inc. Anti-vegf antibody
EP2259795A4 (en) * 2008-03-26 2012-06-06 Epitomics Inc ANTI-VEGF ANTIBODY
US8986692B2 (en) 2009-08-28 2015-03-24 Jiangsu Simcere Pharmaceutical R & D Co., Ltd. Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody
EP3973977A1 (en) 2015-03-31 2022-03-30 Ildong Pharm Co., Ltd. Pharmaceutical composition for preventing and treating eye diseases, containing, as active ingredient, fusion protein in which tissue-penetrating peptide and anti-vascular endothelial growth factor preparation are fused
US10919947B2 (en) 2015-09-01 2021-02-16 Il Dong Pharmaceutical Co., Ltd. Pharmaceutical composition containing, as active ingredient, fusion protein in which tumor-penetrating peptide and anti-angiogenesis agent are fused, for preventing and treating cancer or angiogenesis-related diseases

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