[go: up one dir, main page]

WO2014090053A1 - 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途 - Google Patents

拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途 Download PDF

Info

Publication number
WO2014090053A1
WO2014090053A1 PCT/CN2013/086542 CN2013086542W WO2014090053A1 WO 2014090053 A1 WO2014090053 A1 WO 2014090053A1 CN 2013086542 W CN2013086542 W CN 2013086542W WO 2014090053 A1 WO2014090053 A1 WO 2014090053A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
variable region
chain variable
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2013/086542
Other languages
English (en)
French (fr)
Inventor
罗师平
胡红群
陈蕞
蔡明文
孙亚男
刘海云
周建鹰
宋晓琦
平小芽
陈思宇
石东红
徐一清
周群敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SUZHOU STAINWEI BIOTECH Inc
Original Assignee
SUZHOU STAINWEI BIOTECH Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SUZHOU STAINWEI BIOTECH Inc filed Critical SUZHOU STAINWEI BIOTECH Inc
Priority to EP13863651.9A priority Critical patent/EP2933266B1/en
Priority to ES13863651T priority patent/ES2719084T3/es
Priority to US14/651,785 priority patent/US9580498B2/en
Publication of WO2014090053A1 publication Critical patent/WO2014090053A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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

Definitions

  • the invention belongs to the field of biotechnology-monoclonal antibodies.
  • the present invention relates to a monoclonal antibody which antagonizes the inhibition of binding of vascular endothelial growth factor (VEGF) to its receptor (i.e., VEGF-R) and uses thereof.
  • VEGF vascular endothelial growth factor
  • Angiogenesis or hyperplasia Biologically refers to the process in which existing blood vessels (such as capillaries and tiny movements, veins) in the body produce new blood vessels by sprouting or splitting.
  • Angiogenesis is beneficial and necessary in maintaining many of the normal physiological processes of the body, such as tissue embryo development, healing and repair of traumatic wounds.
  • excessive angiogenesis or hyperplasia is also closely related to many pathological changes (such as tumor proliferation, inflammation, etc.).
  • the key to the continuous proliferation and growth of blood vessels in the body is the ability of the lining of the vascular endothelial cells to continually divide and proliferate and migrate into the existing vessel wall.
  • VEGF vascular endothelial growth factor
  • malignant tumor tissues such as colorectal cancer, gastric cancer, ovarian cancer, breast cancer, liver cancer, multiple myeloma, etc.
  • its expression level is highly positively correlated with tumor growth and metastasis recurrence ( Dvorak HF et al.: J Exp Med 1991; 174: 1275-8; Brown LF et al. Cancer Res 1993; 53: 4727-35; Weidner N, Semple JP, Welch WR and Folkman J: N Engl J Med 1991; 324: 1-8.4-5).
  • VEGF-R VEGF receptor
  • Mode 1 is to develop inhibitors of tyrosine protein kinase activity in the cell membrane antagonizing VEGF receptor (VEGF-R).
  • VEGF-R cell membrane antagonizing VEGF receptor
  • These antagonistic inhibitors are generally small molecule chemical drugs, and the representative drugs are 2006.
  • Pfizer Pfizer
  • Sutent Sunitinib
  • Bayer Bayer
  • Onyx Pharmaceuticals develops Nexavar for marketing.
  • the antibody also has the following disadvantages:
  • mouse monoclonal antibody A4.6.1 and its humanized antibody Avastin can only bind to a part of the VEGF epitope and cannot bind or cover the VEGF epitope. Numerous other sites.
  • Another technical problem to be solved by the present invention is to provide a DNA molecule or gene encoding the above antibody.
  • Another technical problem to be solved by the present invention is to provide a pharmaceutical composition containing the above antibody.
  • Still another technical problem to be solved by the present invention is to provide a method of preparing the above antibody.
  • the present invention provides a mouse monoclonal antibody which antagonizes the inhibition of binding of vascular endothelial growth factor (VEGF) to its receptor (VEGF-R), which is characterized by a variable light chain thereof.
  • Zone with SEQ The amino acid sequence of ID NO.: 1 has a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 2.
  • the murine monoclonal antibody is derived from the code name PV19-5 Mouse hybridoma cell line deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on March 12, 2012 (Accession No. CGMCC No) 5889. Deposit location: China, Beijing).
  • the present invention also provides a DNA molecule encoding the above-described murine monoclonal antibody, wherein the light chain variable region has the SEQ ID
  • the heavy chain variable region has the nucleotide sequence of SEQ ID NO.: 4.
  • Another aspect of the invention provides a humanized monoclonal antibody derived from the above-described murine monoclonal antibody.
  • Humanization compared to murine monoclonal antibodies
  • monoclonal antibodies have the advantages of long half-life in humans (up to 20 days) and low immunogenicity, and are convenient for long-term or repeated use in vivo.
  • the above-mentioned murine monoclonal antibody Both the heavy chain variable region and the light chain variable region genes have been humanized, including amino acid substitutions in the framework region and adjacent sites of the antigen binding region/complementarity determining region.
  • Humanized monoclonal The antibody is characterized in that its light chain variable region has the amino acid sequence shown in SEQ ID NO.: 5, and the heavy chain variable region has the SEQ ID NO.: The amino acid sequence shown in 6. Furthermore, the invention also provides DNA molecules encoding the variable regions of the humanized monoclonal antibodies. Its light chain variable region has SEQ ID The nucleotide sequence shown by NO.:7, the heavy chain variable region has the nucleotide sequence of SEQ ID NO.: 8.
  • a third aspect of the invention provides the above humanized monoclonal A derivative of an antibody, characterized in that the light chain antigen-complementarity determining region (CDR-L) of the derivative comprises SEQ ID NO.: 9, the amino acid sequence of SEQ ID NO.: 10 and SEQ ID NO.: 11, the heavy chain antigen complementarity determining region (CDR-H) comprises SEQ ID NO.: 12, SEQ ID NO.: 13 and the amino acid sequence of SEQ ID NO.: 14.
  • the light chain antigen-complementarity determining region (CDR-L) of the derivative comprises SEQ ID NO.: 9, the amino acid sequence of SEQ ID NO.: 10 and SEQ ID NO.: 11, the heavy chain antigen complementarity determining region (CDR-H) comprises SEQ ID NO.: 12, SEQ ID NO.: 13 and the amino acid sequence of SEQ ID NO.: 14.
  • an expression vector comprising the DNA encoding the above humanized monoclonal antibody A molecular sequence and an expression control sequence operably linked to the sequence.
  • the fifth aspect of the invention provides A recombinant host cell which is transformed from the above expression vector.
  • the recombinant host cell or a progeny cell thereof expresses the above-described humanized monoclonal antibody.
  • mAb monoclonal antibody
  • monoclonal antibodies differ from conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody being directed against a single determinant on the antigen.
  • monoclonal antibodies are also advantageous in that they are obtained by hybridoma or recombinant engineered cell culture without intermixing with other immunoglobulins.
  • the modifier 'monoclonal' indicates the identity of the antibody and is obtained from a homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the term 'humanized monoclonal antibody' as used herein In addition to retaining the complementarity determining region (CDR) of the amino acid sequence of the murine monoclonal antibody, all or most of the sequences (including the framework region sequence in the variable region) replace the amino acid sequence of the adult immunoglobulin to achieve the pass gene Engineering means to minimize the immunogenicity of murine monoclonal antibodies.
  • CDR complementarity determining region
  • 'antibody' and 'immunoglobulin' as used herein are isomeric polysaccharide proteins of the same structural feature of about 150,000 daltons, which consist of two identical light chains (L) and two identical heavy chains ( H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH). This is followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies to their particular antigen.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in the light and heavy chain variable regions into three segments in the complementarity determining region (CDR) or hypervariable region.
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the antibody heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker loop, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are tightly bound together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH). Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • Antibody constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity (ADCC) or complement-mediated toxicity (CDC) involved in antibodies.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-mediated toxicity
  • the antibody of the present invention can usually be produced by the following method.
  • a gene containing an antibody encoding the present invention is inserted into an expression vector containing a suitable expression control sequence.
  • expression control sequence' as used herein generally refers to a sequence involved in controlling the expression of a gene.
  • Expression control sequences include promoter and termination signals operably linked to a gene of interest.
  • Gene encoding the antibody of the present invention (DNA The sequences can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the protein sequences disclosed in accordance with the present invention or by PCR. Thereafter, synthetic or PCR amplification can be carried out by various methods well known in the art.
  • the DNA fragment is inserted into a suitable expression vector.
  • the expression vector used in the present invention may be a commercially available expression vector known to those skilled in the art, such as pCDNA3.1 of Invitrogen Corporation. Expression vector.
  • Suitable host cells for receiving expression vector transformation typically include prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
  • preferred host cells are mammalian cells, especially the Chinese hamster ovary ( CHO) cells.
  • the host cell transformed with the expression vector is cultured under suitable conditions (for example, adherent culture in a cell culture flask or a bioreactor in a serum-free medium), and the culture supernatant is harvested, and then the protein-A is included.
  • suitable conditions for example, adherent culture in a cell culture flask or a bioreactor in a serum-free medium
  • the culture supernatant is harvested, and then the protein-A is included.
  • the antibodies of the invention are obtained by affinity chromatography, ion exchange chromatography, filtration, etc., by conventional separation procedures or means well known to those skilled in the art.
  • the purified antibody of the present invention can be dissolved in a suitable solvent such as physiological saline or PBS liquid, and the desired final solubility can be prepared. Between 0.1 and 100 mg/ml.
  • the sixth aspect of the invention provides A pharmaceutical composition which comprises a pharmaceutically effective amount of a humanized monoclonal antibody, or a derivative thereof, as described in the present invention, and a pharmaceutically acceptable carrier.
  • 'pharmaceutically acceptable means that when the antibody and composition are suitably administered to an animal or a human, they do not produce an allergy or other adverse reaction.
  • a 'pharmaceutically acceptable carrier' should be compatible with the antibody protein of the present invention, i.e., can be blended therewith without substantially reducing the effectiveness of the pharmaceutical composition.
  • a pharmaceutically acceptable carrier or a component thereof examples include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil , corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; stabilizer; antioxidant; pyrogen-free sterile water for injection; physiological saline solution; phosphate buffer.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil , corn oil and cocoa butter
  • polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol
  • alginic acid such as Twe
  • the pharmaceutical composition of the present invention can be formulated into various dosage forms such as lyophilized powder, injection, eye drop, etc. as needed.
  • the dosage that is beneficial to the patient can be determined by the physician based on factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the seventh aspect of the present invention provides the use of the above pharmaceutical composition for the preparation of a pharmaceutical preparation for treating a disease associated with vascular proliferation.
  • the use of the humanized antibody to inhibit the growth of a variety of transplanted tumors such as human colon cancer, breast cancer, and rhabdomyosarcoma in vivo is described.
  • the present invention provides a method of preparing the above humanized monoclonal antibody, the method comprising the steps of:
  • an expression vector comprising DNA encoding the humanized monoclonal antibody a sequence and an expression control sequence operably linked to the sequence;
  • step b) transforming the host cell with the expression vector of step a);
  • the present invention selects a recombinant human VEGF165 protein expressed by yeast as an immune antigen, and repeatedly The mice were immunized subcutaneously to obtain a high-valence anti-VEGF polyclonal antibody; then the mice were picked and the spleen cells were taken, and the cells were fused with mouse myeloma cells in vitro, followed by drug screening and subcloning. A plurality of hybridoma monoclonal cells stably secreting anti-human VEGF antibodies were established.
  • the invention obtains the heavy chain and light chain variable region genes encoding the mouse antibody by means of protein isolation and purification and genetic engineering, and on the basis of the humanization transformation of the antibody.
  • the humanized transformed antibody gene was inserted into the expression vector (pCDNA3.1) and transferred into Chinese hamster ovary (CHO) cells to obtain recombinant engineering cells, which were isolated and purified from recombinant engineering cell culture medium to obtain antagonistic tumor growth in vivo. Humanized antibody protein.
  • the humanized antibody can be used as a medicine
  • the composition of the composition is prepared or prepared into a suitable pharmaceutical preparation, used alone or in combination with other therapeutic means such as chemotherapeutic drugs, and is used for broad-spectrum treatment of various solid tumors such as colon cancer, breast cancer, and rhabdomyosarcoma.
  • the invention also discloses a humanized preparation process of the antibody and an amino acid sequence of the humanized antibody heavy chain variable region and the light chain variable region.
  • the invention obtains the heavy chain and light chain variable region genes encoding the mouse antibody by means of protein isolation and purification and genetic engineering, and on the basis of the humanization transformation of the antibody.
  • the humanized transformed antibody gene was inserted into the expression vector (pCDNA3.1) and transferred into Chinese hamster ovary (CHO) cells to obtain recombinant engineering cells, which were isolated and purified from recombinant engineering cell culture medium to obtain antagonistic tumor growth in vivo. Humanized antibody protein.
  • the humanized antibody can be used as a medicine
  • the composition of the composition is prepared or prepared into a suitable pharmaceutical preparation, used alone or in combination with other therapeutic means such as chemotherapeutic drugs, and is used for broad-spectrum treatment of various solid tumors such as colon cancer, breast cancer, and rhabdomyosarcoma.
  • FIG. 1 embodiment to recombinant human VEGF165 protein coated plates, a schematic of mouse hybridoma by ELISA (PV19) results culture supernatants binding to recombinant human VEGF protein of the present invention.
  • M23 is a hybridoma cell supernatant which is known to secrete anti-human VEGF monoclonal antibody, and is a positive control.
  • P16 is a non-related hybridoma cell culture supernatant, which is a negative control; SP2/0 represents an unfused myeloma cell culture supernatant.
  • Fig. 2 is a diagram showing the murine PV19 monoclonal antibody protein purified from the culture supernatant of PV19 tumor cells by affinity chromatography column in SDS-PAGE analysis according to Example 2 of the present invention.
  • lane 1 is a DTT-reduced PV19 antibody protein
  • lane 2 is an unreduced PV19 antibody protein
  • Marker is a protein molecular weight marker.
  • FIG. 3 is a representative schematic diagram showing the results of competitive ELISA in Example 3 of the present invention.
  • mPV19&Biotin-VEGF was a PV19 murine antibody group
  • W10&Biotin-VEGF was a non-related antibody group as a negative control.
  • FIG. 4 is a diagram showing the humanized hPV19 monoclonal antibody protein purified from the culture supernatant by affinity chromatography column in SDS-PAGE analysis according to Example 8 of the present invention.
  • lane 1 is the DTT-reduced hPV19 antibody protein
  • lane 2 is the unreduced hPV19 antibody protein
  • Marker is the protein molecular weight marker.
  • Figure 5 is a comparison of the binding of humanized antibody (hPV19), chimeric antibody (chPV19) and murine antibody (mPV19) to human VEGF165 protein purified by expression supernatant in a direct ELISA method according to Example 8 of the present invention .
  • chP16 is a non-related chimeric antibody and serves as a negative control.
  • Figure 6 is a comparison of humanized antibody (hPV19), chimeric antibody (chPV19) and murine antibody (mPV19) in vitro to block biotinylated human VEGF165 protein (bio-VEGF) by competitive ELISA in Example 8.
  • biotinylated human VEGF165 protein bio-VEGF
  • VEGFR1 biotinylated human VEGF165 protein
  • W10 is a non-related chimeric antibody as a negative control.
  • Figure 7 is a graph showing the results of comparative analysis of the binding activity of humanized hPV19 antibody and Avastin antibody to wild type (VEGF165) and point mutant human VEGF protein (VEGF-G88/A) by ELISA in Example 9.
  • FIGS. 8A and 8B are schematic diagrams showing the results of inhibition of human Ls-174-T colon cancer growth by humanized hPV19 monoclonal antibody in nude mice in Example 10.
  • Figure 8A is a graph showing the relative tumor volume growth trend of each group during the experiment;
  • Figure 8B is the average tumor tumor weight reduction rate (% tumor suppression rate) of each treatment group compared with the negative control group at the end of the experiment.
  • FIGS. 9A and 9B are schematic diagrams showing the results of an experiment for inhibiting the growth of human MD231-MBA breast cancer in nude mice by humanized hPV19 monoclonal antibody in Example 11 of the present invention.
  • Figure 9A is a graph showing the relative tumor volume growth trend of each group during the experiment;
  • Figure 9B is the average tumor tumor weight reduction rate (% tumor suppression rate) of each treatment group compared with the negative control group at the end of the experiment.
  • FIG. 10A and 10B are diagrams showing the results of inhibiting human A673 rhabdomyosarcoma in nude mice by humanized hPV19 monoclonal antibody in Example 12 of the present invention.
  • FIG. 10A is a graph showing the relative tumor volume growth trend of each group during the experiment;
  • FIG. 10B is the average tumor tumor weight decrease rate (% tumor suppression rate) of each treatment group compared with the negative control group at the end of the experiment.
  • Figure 11A and Figure 11B are diagrams showing the results of inhibition of human HCT8 colon cancer in nude mice by humanized hPV19 monoclonal antibody in Example 13 of the present invention.
  • Figure 11A is a graph showing the relative tumor volume growth trend of each group during the experiment;
  • Figure 11B is the average tumor tumor weight reduction rate (% tumor inhibition rate) of each treatment group compared with the negative control group at the end of the experiment.
  • the mouse hybridoma cell line code-named PV19-5 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on March 12, 2012 (the deposit number is CGMCC No.). 5889; preservation location: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences).
  • Example 1 Establishment and screening of mouse hybridoma cell lines secreting anti-vascular endothelial growth factor antibody
  • a complete open reading frame sequence encoding human VEGF165 was amplified from human lung tissue cell cDNA library by PCR.
  • the gene fragment of reading frame, ORF) was sequenced and identified correctly. After restriction endonuclease treatment, it was cloned into yeast expression vector pPic9K.
  • the vector Invitrogen, the recombinant expression plasmid pPic9K-VEGF165 was obtained, and after transforming Pichia pastoris, the yeast was efficiently expressed.
  • the yeast is fermented and induced to express, and after isolation and purification, recombinant human VEGF165 protein having a purity of 95% or more is obtained.
  • the purified recombinant human VEGF165 protein was mixed with Freund's complete adjuvant, and Balb/c mice were injected subcutaneously (100). Ll/only, a total of 10 ⁇ g VEGF165 protein). After the first immunization for 2-3 weeks, the mice were given a subcutaneous injection of a mixture of VEGF165 protein and incomplete adjuvant to boost the 2-3. After that, a small amount of mouse serum was taken, and the titer of the anti-VEGF protein antibody in the serum of the mouse was detected by ELISA using a 96-plate coated with human VEGF165 protein, and the spleen cells of the mouse with high titer were used for the next step. Cell fusion.
  • mice spleen cell suspension was prepared aseptically, and mouse Sp2/0 myeloma cells (purchased from the Shanghai Institute of Bioscience, Chinese Academy of Sciences), in a ratio of 5:1 at 50% PEG- Fusion under the influence of 1000 (Sigma product).
  • the fusion was carried out according to a conventional method (Kohler G. and Milstein C: Nature 1975; 256: 495-497), and the amount of PEG was 1 ml, which was slowly added over 1 minute.
  • reaction was stopped in serum-free RPMI-1640 medium, centrifuged at 1000 rpm for 10 min, the supernatant was removed, and the cells under centrifugation were further subjected to 10% HAT (H is hypoxanthine, A-amino oxime). , T thymidine, Sigma product RPMI 1640-10% FCS medium to adjust the cell concentration to 1 ⁇ 10 6 /ml, add 96-well flat-bottomed cell culture plate (200 ⁇ l per well), at 37 ° C, 5% Incubate for 2-3 weeks in a CO2 incubator.
  • HAT hypoxanthine, A-amino oxime
  • Step 4 Enzyme-linked immunosorbent assay (ELISA) for screening antibody-positive mouse hybridoma cells
  • Recombinant human VEGF165 protein (2 ⁇ g/ml, pH 9.6, 0.1 M NaHCO3 The solution was coated with the ELISA plate and coated at 37 ° C for 2 hours or 4 ° C overnight; 2% bovine serum albumin (BSA) was blocked overnight at 4 ° C.
  • BSA bovine serum albumin
  • the positive cells obtained by the above screening were RPMI-1640-10%. Dilute the FCS medium to 1-10 cells per well, place it on a 96-well cell culture plate, and incubate at 37 ° C, 5% CO2 incubator 2-3 Week. The clones were grown, and the supernatant was taken for further detection by ELISA to identify the secretion of anti-VEGF antibody. After detection and identification, a plurality of antibody-secreting positive cell lines were obtained. Among them, after further subcloning and identification, a hybridoma cell line stably secreting anti-VEGF monoclonal antibody with the code P19-5 (referred to as PV19) was obtained.
  • Figure 1 shows the ELISA
  • the supernatant of the PV19 hybridoma cells was ligated to the recombinant human VEGF165 protein, and the supernatant of the hybridoma cells was confirmed to contain an antibody against the human VEGF165 protein. This antibody was identified as an IgG class.
  • the hybridoma cell line was further expanded and cultured for a long time and deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on March 12, 2012 (the deposit number is CGMCC). No 5889. Deposit location: China, Beijing).
  • mouse anti-human VEGF monoclonal antibody (PV19) was isolated and purified by affinity chromatography.
  • the purification steps are as follows:
  • the PV19 hybridoma cells were expanded, inoculated into 200 ml of serum-free 1640 medium, and cultured at 37 ° C for 5 days, and then the culture supernatant was collected, filtered through a 0.45 ⁇ m filter, and loaded to contain Protein.
  • G-Sepharose Fast Flow purchased from GE GE affinity chromatography column; the column was first rinsed with PBS solution to remove the heteroprotein, then low pH (2.7-3.0) The adsorbed PV19 antibody protein was eluted with glycine (0.1 M).
  • FIG. 1 is a DTT-reduced PV19 antibody and lane 2 is an unreduced PV19 antibody.
  • the DTT-reduced PV19 antibody was isolated into two major bands, wherein the upper band was the PV19 antibody heavy chain and the lower band was the PV19 antibody light. chain.
  • Example 3 Bioactivity assay of murine PV19 monoclonal antibody competitive ELISA assay to identify murine PV19 monoclonal antibody blocking the binding of VEGF to its receptor in vitro
  • One of the methods for determining the biological activity of the murine PV19 monoclonal antibody is the competitive ELISA assay to identify antibodies that block the binding of VEGF to its receptor in vitro.
  • the basic principle and process of the competitive ELISA method is to first mix the biotin-labeled human VEGF165 protein with different solubility monoclonal antibodies, and then transfer the mixture to a pre-coated recombinant soluble VEGF receptor protein (such as soluble VEGFR1). Protein-based 96-well plates, after incubation and elution, add enzyme-labeled Avidin (such as horseradish peroxidase-labeled Avidin). After incubation and elution, the substrate was added and the OD value was determined.
  • enzyme-labeled Avidin such as horseradish peroxidase-labeled Avidin
  • FIG 3 is a representative schematic representation of competitive ELISA results.
  • the OD value is inversely related to the amount of antibody protein: the higher the amount of PV19 antibody added, the lower the OD value.
  • the non-related antibody W10 & Biotin-VEGF
  • the OD value of each well was not affected by the amount of antibody protein added. This result indicates that the PV19 antibody blocks the binding of VEGF to its receptor (VEGFR1) in vitro.
  • Purified murine PV19 antibody was taken and the heavy and light chains were separated by SDS-PAGE under DTT reduction conditions. Then the electrophoresis band was transferred to PVDF membrane, and the color was amplified by Coomassie Brilliant Blue R250. The strand and light chain protein bands were subjected to N-terminal amino acid sequencing by Edman degradation to obtain the N-terminal amino acid sequence of the antibody light chain. Due to the N-terminal blockade of the antibody heavy chain, conventional amino acid N-terminal sequencing did not yield results.
  • Step 1 Using a kit (product of Jiangsu Haimen Biyuntian Co., Ltd.) to extract total RNA from mouse PV19 hybridoma cells;
  • Step 2 A cDNA template was obtained on an eppendorf tube by reverse transcription PCR (RT-PCR).
  • the sequence for the reverse transcription PCR primer (mIg-kappa) is: TGTCGTTCACTGCCATCAAT (SEQ ID NO.: 15);
  • the RT-PCR reaction system is as follows:
  • the reaction was carried out at a temperature of 42 ° C for 1 hour, then the temperature was raised to 75 ° C, and the cDNA obtained after 15 minutes of inactivation was obtained. Store at -20 ° C and store for later use.
  • Step 3 According to the N-terminal amino acid sequence information of the PV19 antibody light chain, the following pair of primers are designed:
  • Reverse primer AAT TGG ATC CAG TTG GTG CAG CAT CAG C (as shown in SEQ ID NO.: 17)
  • the reaction system for PCR amplification of light chain DNA is as follows:
  • the above primers and the cDNA template obtained from the mouse PV19 hybridoma cells were amplified by PCR (PCR reaction system and parameters similar to the above light chain PCR) to obtain the heavy chain variable region DNA.
  • Amplified DNA product at 1% agarose Electrophoresis analysis in gel. After the end of the electrophoresis, the isolated DNA band is excised and sequenced to obtain the nucleotide sequence of the antibody heavy chain variable region DNA.
  • the nucleotide sequence of the antibody heavy chain variable region DNA was determined as shown in SEQ ID. NO.: 4, the amino acid sequence of the antibody heavy chain variable region deduced from the DNA nucleotide sequence is shown in SEQ ID NO.: 2.
  • Complementary region of the heavy chain antigen The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.: 12, SEQ ID NO.: 13 and SEQ ID NO.: 14, respectively.
  • the PV19 antibody light chain variable region gene and heavy chain variable region gene obtained by clonal expansion in Example 4 were fused to human-kappa light chain constant region (C-domain) and human IgG1-heavy chain constant region gene fragment, respectively. , obtaining a human-mouse chimeric light chain gene ( chPV19L) and human-mouse chimeric heavy chain gene (chPV19H).
  • the light chain chimeric gene and the heavy chain chimeric gene were subsequently cloned into pcDNA3.1, respectively.
  • the expression plasmid was transferred to E. coli for amplification, and a large number of expression plasmids containing the human-mouse chimeric antibody gene were isolated.
  • Will contain a chimeric heavy chain gene (cPV19H), a chimeric light chain gene (cPV19L), a humanized heavy chain gene ( hPV19H) and humanized light chain gene (hPV19L) were cloned into pcDNA3.1 expression vector, and transferred into E. coli, and then amplified and isolated to obtain each expression plasmid. Thereafter, each expression plasmid was combined into two, and then transiently transfected into human CHO cells. 48 hours after transfection, the cell culture supernatant in the well was aspirated, and the VEGF165 protein was used as the coating antigen, and the HRP enzyme was labeled.
  • Goat-anti-human-IgG is a secondary antibody (purchased from Shanghai Xitang Biotechnology Co., Ltd.), and OPD is a chromogenic substrate.
  • OPD is a chromogenic substrate.
  • the activity of binding of the antibody in the supernatant of the transfected cells to the human VEGF165 antigen was detected by direct ELISA.
  • the protocol used in the present invention is to insert the gene containing the humanized hPV19 antibody heavy and light chains simultaneously.
  • a cloning plasmid containing the hamster dihydrofolate reductase (DHFR) gene (pCDNA3.1-DHFR) was obtained, and a recombinant plasmid (pQY-hPV19D-DHFR) containing both the heavy chain and light chain genes of the humanized hPV19 antibody was obtained.
  • the pCDNA3.1-DHFR plasmid containing the hamster dihydrofolate reductase (DHFR) gene was selected as a cloning vector, and the humanized hPV19 antibody light chain gene (hPV19L) and the heavy chain gene (hPV19) were simultaneously Inserted into the cloning vector, an expression plasmid named pQY-hPV19-DHFR was obtained.
  • the promoter for driving expression of the DHFR gene in mammalian cells is the SV40 early promoter (pSV40), and the human elongation factor 1- ⁇ subunit is used to drive expression of the hPV19 antibody light chain gene (hPV19L).
  • the promoter (pEF1-a) which is used to drive expression of the hPV19 antibody heavy chain gene (hPV19H), is the cytomegalovirus early promoter (pCMV).
  • the antibody reduced by DTT was divided into two main bands, wherein the upper band was the hPV19 antibody heavy chain, the lower band was the hPV19 antibody light chain, and the unreduced hPV19 antibody was located at 150. Near the kD, it is consistent with the theoretically expected molecular weight of the intact IgG1 antibody.
  • Figure 6 shows the comparison of humanized antibody (hPV19), chimeric antibody (chPV19) and murine antibody (mPV19) in vitro to block human VEGF165 protein (biotin-VEGF) and its receptor (VEGFR1) by competitive ELISA.
  • the specific activity of the binding, the specific method of competitive ELISA detection is the same as in Example 6, wherein W10 is a non-related chimeric antibody and is a negative control.
  • the results showed that the humanized antibody (hPV19) blocked the binding of VEGF to its receptor, and its blocking activity was very close to its predecessor mouse antibody (mPV19).
  • A4.6.1 Or the key epitope of Avastin antibody recognizing human VEGF protein is located near the 88th glycine (Gly, G88). If this glycine is mutated to alanine (G88/A point mutation) then A4.6.1 Or the binding strength of the Avastin antibody to the mutant VEGF protein is significantly decreased.
  • human VEGF165 (G88/A) containing G88/A point mutation was constructed in vitro, and then humanized hPV19 antibody was detected by ELISA. And the binding activity of the Avastin antibody to the wild-type and mutant VEGF proteins.
  • the specific implementation steps and results are described as follows:
  • Step 1 Expression and purification of G88/A mutant VEGF protein
  • Overlap-extension PCR (gene splicing by overlap) Extension PCR, referred to as SOE PCR)
  • SOE PCR Overlap-extension PCR
  • the use of primers with complementary ends allows the PCR products to form overlapping strands, thereby overlapping the amplified fragments from different sources in the subsequent amplification reaction by overlapping the overlapping strands.
  • This technique can be effective in vitro.
  • the gene is recombined and does not require endonuclease digestion and ligase treatment, and this technique can be used to quickly obtain other products that are difficult to obtain by restriction endonuclease digestion.
  • the key to the success of overlap-extension PCR technology is the design of overlapping complementary primers.
  • the overlap-extension PCR has wide and unique features in site-directed mutagenesis, fusion gene construction, long-segment gene synthesis, gene knockout, and amplification of target genes.
  • Application using a set of primers (forward primer: VEGFG88F: CCT) CAC CAA GCC CAG CAC ATA , as shown in SEQ ID NO.: 20, reverse primer: VEGFG88R: CTA TGT GCT GGG CTT GGT GAG as shown in SEQ ID NO.:21.
  • a point mutation was performed to obtain a cDNA encoding the G88/A mutant human VEGF165.
  • Step 2 Comparison of the binding ability of hPV19 antibody and Avastin to wild-type human VEGF165
  • Wild-type human VEGF165 or mutant VEGF protein (VEGF-G88/A) protein was coated on a 96-well-ELISA plate.
  • Example 10 Humanized hPV19 antibody inhibits the growth of human colon cancer cell line Ls-174-T in nude mice
  • Logarithmic growth phase human Ls-174-T colon cancer cells (purchased from the Shanghai Institute of Biosciences Cell Culture Collection, Chinese Academy of Sciences), inoculated 1 ⁇ 10 7 / only in the right axilla of 2 nude mice or existing tumor blocks Prepared into 1.5mm3 or so, inoculated into the right armpit of the animal to prepare tumor-bearing mice; until the tumor grows to about 400 ⁇ 600mm 3 , select tumor-bearing animals with good tumor growth and good health, take the tumor under sterile conditions, prepare It was about 1.5 mm3 and was inoculated under the skin of the right armpit of the animal for formal testing.
  • Step 2 Humanized PV19 antibody treatment of Ls-174-T transplanted colon cancer
  • mice inoculated with Ls-174-T colon cancer cells were divided into the following 5 groups (8 in each group), and administered twice a week by intraperitoneal injection for 4 weeks, and the 29th day (D29) was ended. .
  • High-dose group of humanized hPV19 antibody (high-dose group for test, 10 mg/kg body weight);
  • Avastin control group (5 mg/kg body weight);
  • the general clinical symptoms of the animals were observed twice a day, and the body weight and tumor diameter were measured twice a week. After the euthanasia, the tumor was removed and the tumor weight was weighed. The therapeutic effect was evaluated by the relative tumor proliferation rate T/C%, T/C% ⁇ 40% and P ⁇ 0.05 was effective, and the tumor weight inhibition rate was greater than 60% as the effective reference index.
  • Tumor long diameter and short diameter were measured twice a week after the first dose, and tumor volume V and its relative tumor volume RTV were calculated.
  • the average tumor weight of the saline negative control group was 2.957g.
  • the average tumor weight of the Avastin group was 0.767 g, which was significantly lower than that of the negative control group ( P ⁇ 0.01).
  • Example 11 Humanized hPV19 monoclonal antibody inhibits the growth of transplanted human MDA-MB-231 breast cancer in nude mice
  • the method of establishment is basically the same as that of Embodiment 10.
  • Tumor growth was observed after inoculation of human MDA-MB-231 breast cancer cells in nude mice.
  • the tumor volume was about 50-100 mm3, the tumor volume was screened. The tumor volume was too large and no tumorigenicity was selected. Different doses of the drug were administered in random groups after screening.
  • the drug administration group is as follows:
  • High-dose group of humanized hPV19 antibody (high-dose group for test, 10 mg/kg body weight);
  • the general clinical symptoms of the animals were observed twice a day, and the body weight and tumor diameter were measured twice a week. After the euthanasia, the tumor was removed and the tumor weight was weighed. The therapeutic effect was evaluated by the relative tumor proliferation rate T/C%, T/C% ⁇ 40% and P ⁇ 0.05 was effective, and the tumor weight inhibition rate was greater than 60% as the effective reference index.
  • the long and short diameters of the tumor were measured twice a week after the first dose, and the tumor volume V and its relative tumor volume were calculated.
  • RTV(Vt/V0) The relative volumetric growth trend of MDA-MA-231 in each group is shown in Figure 9A. The results show that humanized hPV19 antibody can significantly inhibit tumor growth compared with saline-negative control group.
  • the negative tumor control group had an average tumor weight of 0.890 g.
  • the average tumor weight of the Avastin group was 0.543 g, which was not significantly different from the negative control group (P>0.05). ).
  • Humanized hPV19 antibody The average tumor weight of low, medium and high dose groups was 0.576g, 0.298g, 0.357g There was no significant difference between the two groups (P>0.05).
  • the tumor weight inhibition rate (Fig. 9B) was calculated, and the tumor weight inhibition rate of Avastin group was 38.99%.
  • the tumor weight inhibition rates of the low, medium and high dose groups of humanized hPV19 antibody were 35.20%, 66.56% and 59.92%, respectively. This result indicates that humanized hPV19 in the middle and high dose groups Antibodies can inhibit tumor growth to varying degrees, and the degree of inhibition is higher than that of Avastin in the same dose group.
  • Example 12 Humanized hPV19 antibody inhibits growth of transplanted human A673 rhabdomyosarcoma in nude mice
  • Step 1 Establishment of a nude mouse xenograft model of human A673 rhabdomyosarcoma model
  • the method of establishment is basically the same as that of Embodiment 10.
  • the tumor growth was observed.
  • the tumor volume was about 50-100 mm3
  • the tumor volume was screened.
  • the tumor volume was too large and no tumorigenicity was selected.
  • Different doses of the drug were administered in random groups after screening.
  • Step 2 Humanized PV19 antibody treatment of human A673 rhabdomyomas
  • mice inoculated with human A673 rhabdomyosarcoma were divided into the following 6 groups (6 in each group), administered twice a week by intraperitoneal injection for 3 consecutive weeks, and the 22nd day (D22) test was completed.
  • the drug administration group is as follows:
  • High-dose group of humanized hPV19 antibody (high-dose group for test, 10 mg/kg body weight);
  • Avastin control group (5 mg/kg body weight);
  • the general clinical symptoms of the animals were observed twice a day, and the body weight and tumor diameter were measured twice a week. After the euthanasia, the tumor was removed and the tumor weight was weighed. The therapeutic effect was evaluated by the relative tumor proliferation rate T/C%, T/C% ⁇ 40% and P ⁇ 0.05 was effective, and the tumor weight inhibition rate was greater than 60% as the effective reference index.
  • the negative tumor control group had an average tumor weight of 8.726 g.
  • the average tumor weight of the Avastin group was 3.323 g, which was significantly lower than that of the negative control group (P ⁇ 0.05).
  • Humanized hPV19 antibody The average tumor weight of low, medium, high and ultra low dose groups was 3.811g, 3.046g, 2.499g, 7.056g. Compared with the negative control group, the mean tumor weight of the low-, medium-, and high-dose groups of humanized hPV19 antibody was significantly lower (P ⁇ 0.05).
  • the tumor weight inhibition rate (Fig. 10B) was calculated, and the tumor weight inhibition rate of the Avastin group was observed. 61.92%, humanized hPV19 antibody low, medium, high, ultra low dose group tumor weight inhibition rate were 56.33%, 65.09%, 71.36%, 19.13% . The results showed that the humanized hPV19 antibody in the medium and high dose groups can significantly inhibit tumor growth, and its inhibition is no less than Avastin.
  • Example 13 Humanized hPV19 antibody inhibits growth of transplanted human HCT-8 colon cancer in nude mice
  • Step 1 Establishment of a nude mouse xenograft model of human HCT-8 colon cancer model
  • the method of establishment is basically the same as that of Embodiment 10.
  • Tumor growth was observed after inoculation of human HCT-8 colon cancer in nude mice.
  • the tumor volume was about 50-100 mm3, the tumor volume was screened. The tumor volume was too large and no tumorigenicity was selected. Different doses of the drug were administered in random groups after screening.
  • Step 2 Humanized PV19 antibody treatment of human HCT-8 colon cancer
  • mice inoculated with human HCT-8 colon cancer were divided into the following 7 groups (8 in each group), administered twice a week by intraperitoneal injection for 4 consecutive weeks, and the 30th day (D30) test was completed.
  • the administration group was basically the same as in Example 9, but the cisplatin chemotherapeutic control group was added, and the cisplatin and hPV19 antibody were used in combination.
  • the drug administration group is as follows:
  • High-dose group of humanized hPV19 antibody (high-dose group for test, 10 mg/kg body weight);
  • the general clinical symptoms of the animals were observed twice a day, and the body weight and tumor diameter were measured twice a week. After the euthanasia, the tumor was removed and the tumor weight was weighed. The therapeutic effect was evaluated by the relative tumor proliferation rate T/C%, T/C% ⁇ 40% and P ⁇ 0.05 was effective, and the tumor weight inhibition rate was greater than 60% as the effective reference index.
  • the long and short diameters of the tumor were measured twice a week after the first dose, and the tumor volume V and its relative tumor volume were calculated. RTV(Vt/V0) .
  • the average tumor weight of the negative control group was 1.608 g.
  • the average tumor weight of cisplatin chemotherapy alone group was 1.201 g, which was not significantly different from the negative control group.
  • the average tumor weight of hPV19 monoclonal antibody in the low, medium, high and ultra low dose groups was 0.405g, 0.278g, 0.311g, 0.604g, significantly lower than the negative control group (P ⁇ 0.01 or P ⁇ 0.05); hPV19 monoclonal antibody and cisplatin chemotherapy combination group mean tumor weight 0.193 g was significantly lower than the negative control group (P ⁇ 0.01).
  • the tumor weight inhibition rate (see Figure 11B), the tumor weight inhibition rate of cisplatin chemotherapy alone group was 25.29%.
  • the tumor weight inhibition rates of the low, medium, high, ultra low dose and combination groups were 74.81%, 82.73%, 80.65%, 62.46% and 87.98%, respectively.
  • This result indicates that humanized hPV19 antibody in each dose group can significantly inhibit tumor growth; and in ultra-low dose (0.5 mg/kg) ) still valid.
  • the therapeutic effect was significantly better than the 5 mg/kg cisplatin group alone and the 5 mg/kg hPV19 monoclonal antibody group alone.
  • the invention obtains the heavy chain and light chain variable region genes encoding the mouse antibody by means of protein isolation and purification and genetic engineering, and on the basis of the humanization transformation of the antibody.
  • the humanized transformed antibody gene was inserted into the expression vector (pCDNA3.1) and transferred into Chinese hamster ovary (CHO) cells to obtain recombinant engineering cells, which were isolated and purified from recombinant engineering cell culture medium to obtain antagonistic tumor growth in vivo. Humanized antibody protein.
  • the humanized antibody can be used as a medicine
  • the composition of the composition is prepared or prepared into a suitable pharmaceutical preparation, used alone or in combination with other therapeutic means such as chemotherapeutic drugs, and is used for broad-spectrum treatment of various solid tumors such as colon cancer, breast cancer, and rhabdomyosarcoma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Endocrinology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种拮抗抑制血管内皮细胞生长因子（VEGF）与其受体（VEGF-R）结合的小鼠单克隆抗体及其重链可变区与轻链可变区氨基酸序列。本发明还公开了该抗体的人源化制备过程及该人源化抗体重链可变区与轻链可变区氨基酸序列。该人源化抗体或其衍生体可作为药物组合物成分或制备成合适药物制剂，单独给药或与化疗药物等其他治疗手段合并使用，用于广谱治疗结肠癌、乳腺癌、横纹肌肉瘤等各种实体瘤。

Description

拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途 技术领域
本发明属于生物技术-单克隆抗体领域。本发明涉及一种可拮抗抑制血管内皮细胞生长因子（VEGF）与其受体（即VEGF-R）结合的单克隆抗体及其用途。
背景技术
血管新生或增生 (angiogenesis) 在生物学上是指体内的已存在的血管(如毛细血管和微小动、静脉)通过出芽或分裂的方式而产生新的血管的过程。血管新生在维持机体的许多正常的生理过程如组织胚胎发育、外伤伤口的癒合与修复等是有益的和必需的。但过度的血管新生或增生也与许多病理变化（如肿瘤的增生扩散、炎症等）息息相关。体内的血管能够不断地增生与增长的关键是其内衬的血管内皮细胞具有不断分裂增生及定向迁移置入已有的血管管壁的能力。血管内皮细胞生长因子 (vascular endothelial growth factor,VEGF) 则是已知的最重要及最强烈的促血管内皮细胞分裂增生及血管增长的因子。VEGF在血管增生中的重要性已在VEGF基因剔除小鼠的研究中被充分证实：因为只要当VEGF基因中的一份被敲除后,小鼠胚胎在仅发育至11 至 12天时就会因血管增生受阻及异常而死亡 (Carmeliet P 等Nature 1996, 380: 435; Ferrara N 等 Nature 1996,380: 439)。
VEGF 在多种恶性肿瘤组织中（如结直肠癌、胃癌、卵巢癌、乳腺癌、肝癌、多发性骨髓瘤等）呈过度表达，且其表达水平高低与肿瘤生长与转移复发程度呈高度正相关 (Dvorak HF 等：J Exp Med 1991;174:1275-8；Brown LF 等Cancer Res 1993;53 :4727-35； Weidner N, Semple JP, Welch WR及Folkman J: N Engl J Med 1991； 324:1-8.4-5)。近年来国内外一系列动物试验及临床实践已证明：通过基因调控或给予外来药物来阻断体内VEGF及其受体（VEGF-R）介导的促血管增生，则可导致肿瘤组织因缺血而坏死，从而达到抑制肿瘤生长和转移及延长患者生存期的良好疗效。因此,以VEGF及其受体为靶点的抗肿瘤血管增生药物已成为当今国内外药物研发竞争热点。
在以VEGF及其受体为靶点的抗肿瘤血管增生药物的开发上，目前主要有两大模式：
模式一是研制拮抗VEGF受体（VEGF-R）中位于细胞膜内的酪氨酸蛋白激酶活性的抑制剂，这类拮抗抑制剂一般都属于小分子化学类药，其中的代表性药物为2006年由美国 Pfizer(辉瑞)开发上市的Sutent(舒尼替尼);及由德国Bayer(拜耳)与 美国Onyx Pharmaceuticals开发上市的Nexavar。
模式二是研制可直接阻断VEGF与其受体（VEGF-R）结合的抗体或抗体Fc融合蛋白等大分子蛋白类生物制剂。 该类代表性药物为由Roche/Genentech公司研发生产并于2004年2月获美国FDA批准上市的人源化抗VEGF单克隆抗体药Bevacizumab（贝伐单抗）,商品名Avastin （阿瓦斯丁)。 Avastin是目前为止全球市场上唯一的一个抗VEGF单克隆抗体药物。 Avastin通过与体内VEGF的高度特异结合, 从而阻止VEGF与其受体的结合，进而达到阻断血管增生与抑制肿瘤增生的疗效（Presta LG等 Cancer Res,1997,57: 4593; Hurwitz H 等 N Engl J Med,2004;350: 2335）。Avastin目前已获美国FDA批准用于治疗转移性结肠癌(metastatic colorectal cancer, mCRC) 、晚期肺癌(advanced non-squamous non-small cell lung cancer,NSCLC) 、脑胶质瘤(glioblastoma) 、 转移性肾癌(metastatic kidney cancer,mRCC)等多种实体瘤。Avastin 2010年2月获中国SFDA 批准用于治疗结肠癌。
Avastin 其前身可追溯到代号为A4.6.1的小鼠单克隆抗体。该小鼠单克隆抗体的来源及分泌它的杂交瘤细胞系与用途在专利号为US6,582,959的美国专利（发明人：Kim，Kyung Jin ，专利授权日期：June 24, 2003，专利名称：Antibodies to vascular endothelial cell growth factor）；及专利号为US7,227,004的美国专利（发明人：Kim，Kyung Jin 专利授权日期： June 5, 2007，专利名称：Antibodies to vascular endothelial cell growth factor）中都有所描述。该鼠源抗体及其人源化抗体rhuMab-VEGF（即Avastin）的序列及其制备方法在专利号为US6,054,297的美国专利（发明人：Carter; Paul J.及 Presta; Leonard G，专利申请日期： May 9， 1995，专利授权日期：April 25, 2000，专利名称：Humanized antibodies and methods for making them）公开并在国际期刊杂志上发表（Presta LG等 Cancer Res，1997，57: 4593）。
但该抗体还存在如下不足：
1）与大多数的单克隆抗体一样，小鼠单克隆抗体A4.6.1及其人源化抗体Avastin也仅只是能结合VEGF抗原表位中的部分位点，无法结合或涵盖VEGF抗原表位中众多的其他位点。
2）先前在动物实验中及近年来的临床研究中发现仅单独给予小鼠单克隆抗体A4.6.1或Avastin抗体，无法达到完全中和抑制体内VEGF及其介导的促血管新生。
因此，研制具有新的VEGF结合位点的，且可拮抗抑制VEGF与其受体（VEGF-R）结合的全新单克隆抗体或药物制剂显得很有意义与必要。
 技术问题
本发明要解决的技术问题是提供一种拮抗抑制血管内皮细胞生长因子（VEGF）与其受体（VEGF-R）结合的抗体或其衍生体。该抗体包括人源化抗体或其衍生体如抗体Fab片段 、 单链抗体等。
本发明的另一个要解决的技术问题是提供编码上述抗体的DNA分子或基因。
本发明的另一个要解决的技术问题是提供含有上述抗体的药物组合物。
本发明的还一个要解决的技术问题是提供制备上述抗体的方法。
 技术解决方案
为解决上述技术问题，本发明一方面提供了一种拮抗抑制血管内皮细胞生长因子（VEGF）与其受体（VEGF-R）结合的鼠源单克隆抗体，该抗体的特征是其轻链可变区具有SEQ ID NO.:1所示的氨基酸序列，重链可变区具有SEQ ID NO.:2所示的氨基酸序列。 该鼠源单克隆抗体来源于代号为 PV19-5 的小鼠杂交瘤细胞株,该杂交瘤细胞株已于 2012 年 03 月 12 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（保藏编号为 CGMCC No 5889. 保藏地点：中国，北京）。此外，本发明还提供编码上述鼠源单克隆抗体的DNA分子,其轻链可变区具有SEQ ID NO.：3所示的核苷酸序列，重链可变区具有SEQ ID NO.：4的核苷酸序列。
本发明另一方面提供了来源于上述鼠源单克隆抗体的人源化 单克隆 抗体。与鼠源单克隆抗体相比，人源化 单克隆 抗体作为治疗药物具有在人体中半衰期长（可长达20天）及免疫原性低等优点，便于长期或反复多次体内使用。本发明中将上述 鼠源单克隆抗体 的重链可变区和轻链可变区基因都进行了人源化改造，其中包括框架区和抗原结合区域/互补决定区临近位点的氨基酸置换。该人源化 单克隆 抗体的特征是其轻链可变区具有SEQ ID NO.:5所示的氨基酸序列，重链可变区具有SEQ ID NO.:6所示的氨基酸序列。此外，本发明还提供编码该人源化单克隆抗体可变区的DNA分子。其轻链可变区具有SEQ ID NO.：7所示的核苷酸序列，重链可变区具有SEQ ID NO.：8的核苷酸序列。
本发明的第三方面是提供上述人源化 单克隆 抗体的衍生体，其特征是该衍生体的轻链抗原互补决定区（complementarity-determining regions, CDR-L ）包含SEQ ID NO.：9，SEQ ID NO.：10及SEQ ID NO.：11的氨基酸序列，其重链抗原互补决定区(CDR-H)包含SEQ ID NO.：12，SEQ ID NO.：13及SEQ ID NO.：14的氨基酸序列。
本发明第四方面是提供了一种表达载体， 它含有编码上述人源化单克隆抗体的 DNA 分子序列以及与该序列操作性相连的表达调控序列。
本发明第五方面提 供了一种重组宿主细胞，它由上述表达载体转化而成。该重组宿主细胞或其子代细胞表达上述人源化单克隆抗体 。
本文所采用的术语'单克隆抗体（单抗）'指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂（通常是具有针对不同决定簇的不同抗体）不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语'单克隆'表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本文所采用的术语'人源化单克隆抗体' 系将鼠源单克隆抗体的氨基酸序列除保留互补决定区（CDR）外，其它序列（包括可变区中的框架区序列）全部或大部分替换成人免疫球蛋白的氨基酸序列，以达到通过基因工程手段最大限度地降低鼠源性单克隆抗体的免疫原性。
本文所用的术语'抗体'和'免疫球蛋白'是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链（L）和两个相同的重链（H）组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区（VH）。其后是多个恒定区。每条轻链的一端有可变区（VL），另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
本文所用的术语'可变'表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中成为互补决定区（CDR）或超变区中的三个片段中。可变区中较保守的部分称为构架区（Framework regions， FR）。抗体重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位（参见Kabat等,NIH Publ. No.91-3242,卷1,647-669页（1991））。抗体恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性（ADCC）或补体介导毒性（CDC）。
本发明的抗体通常可以通过以下方法来制备。
首先，将含有编码本发明的抗体的基因插入到含有合适的表达调控序列的表达载体中。
本文所用的术语'表达调控序列'通常指参与控制基因表达的序列。表达调控序列包括与目标基因操作性相连的启动子和终止信号。编码本发明抗体的基因（ 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蛋白质序列人工合成或用 PCR 法扩增得到。其后可用本领域熟知的各种方法将合成或 PCR 扩增得到的 DNA 片段插入到合适的表达载体中。本发明中所用的表达载体可以是本领域技术人员已知的市售表达载体，如 Invitrogen 公司的 pCDNA3.1 表达载体。
用于接纳表达载体转化的合适宿主细胞一般包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。在本发明中，较佳的宿主细胞是哺乳动物细胞，尤其是中华仓鼠卵巢（ CHO ）细胞。
表达载体转化的宿主细胞在合适的条件下（如以无血清培养基在细胞培养瓶或生物反应器中贴壁或悬浮培养）培养后，收获培养上清液，然后用包括 protein-A 亲和层析、离子交换层析、过滤等本领域技术人员熟知的常规分离步骤或手段纯化得到本发明的抗体。
纯化得到的本发明抗体可以溶于适当的溶剂如生理盐水或 PBS 液体中，理想的最终溶度可以制备成 0.1 至 100 mg/ml 之间。
本发明第六方面提 供了一种药物组合物，它含有药学上有效量的如本发明中描述的人源化单克隆抗体或其衍生体以及药学上可接受的载体。
本文所用的术语'药学上可接受的'是指当该抗体和组合物适当地给予动物或人时，它们不会产生过敏或其它不良反应。本文所用的'药学上可接受的载体'应当与本发明的抗体蛋白相容，即能与其共混而不会大幅度降低药物组合物的效果。可作为药学上可接受的载体或其组分的一些物质的具体例子包括糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如 Tween ；稳定剂；抗氧化剂；无热原无菌注射用水；生理盐水溶液；磷酸盐缓冲剂等。
本发明的药物组合物可根据需要制成各种剂型如冻干粉剂、注射剂、滴眼剂等给药 ，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用 。
本发明第七方面提供了上述 药物组合物在制备治疗与血管增生相关的疾病的药物制剂中的应用。 在本发明的具体实施实例中，描述了该人源化抗体 在体内抑制人结肠癌、乳腺癌、横纹肌肉瘤等多种移植肿瘤生长 的应用。
本发明第八方面是提供制备 上述人源化单克隆抗体的方法，该方法包括如下步骤：
a) 提供一表达载体，该表达载体含有编码所述人源化单克隆抗体的 DNA 序列以及与该序列操作性相连的表达调控序列；
b) 用步骤 a ）所述的表达载体转化宿主细胞；
c) 在适合所述人源化单克隆抗体表达的条件下培养步骤 b) 所得的宿主细胞：和
d) 从该宿主细胞培养液中分离纯化获得所述人源化单克隆抗体。
为获得可拮抗抑制人VEGF蛋白与其受体（VEGF-R）结合的单克隆抗体及分泌它的杂交瘤细胞系，本发明选取由酵母表达的重组人VEGF165蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌高效价的抗VEGF多克隆抗体；再从中挑取小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人VEGF抗体的杂交瘤单克隆细胞。 其中一代号为PV19小鼠杂交瘤细胞株，经ELISA、免疫印迹、免疫组化等多种方法鉴定，证实其所分泌的单克隆抗体能够特异识别并高亲合力结合人VEGF（包括VEGF121、165 及VEGF189 等主要亚型）。体外测试表明该鼠源单克隆抗体可高效抑制VEGF与其受体（VEGF-R）的结合。在人类移植肿瘤裸鼠模型上的测试结果还表明该鼠源单克隆抗体能有效抑制乳腺癌、横纹肌肉瘤等多种肿瘤在体内的生长。
本发明通过蛋白分离纯化及基因工程等手段获得了编码该鼠源抗体的重链及轻链可变区基因，并在此基础上完成了该抗体的人源化改造。人源化改造后的抗体基因插入表达载体（pCDNA3.1）后转入中华仓鼠卵巢（CHO）细胞，获得重组工程细胞，并从重组工程细胞培养液中分离纯化得到具拮抗肿瘤体内增长生物活性的人源化抗体蛋白。该人源化抗体可作为药 物组合物成分或制备成合适药物制剂，单独给药或与化疗药物等其他治疗手段合并使用，用于广谱治疗结肠癌、乳腺癌、横纹肌肉瘤等各种实体瘤。
本发明还公开了该抗体的人源化制备过程及该人源化抗体重链可变区与轻链可变区氨基酸序列。
 有益效果
本发明通过蛋白分离纯化及基因工程等手段获得了编码该鼠源抗体的重链及轻链可变区基因，并在此基础上完成了该抗体的人源化改造。人源化改造后的抗体基因插入表达载体（pCDNA3.1）后转入中华仓鼠卵巢（CHO）细胞，获得重组工程细胞，并从重组工程细胞培养液中分离纯化得到具拮抗肿瘤体内增长生物活性的人源化抗体蛋白。该人源化抗体可作为药 物组合物成分或制备成合适药物制剂，单独给药或与化疗药物等其他治疗手段合并使用，用于广谱治疗结肠癌、乳腺癌、横纹肌肉瘤等各种实体瘤。
附图说明
图1 为本发明实施例1中以重组人VEGF165蛋白包板，以ELISA法检测小鼠杂交瘤细胞（PV19）培养上清液与重组人VEGF蛋白结合的实验结果示意图。其中，M23为已知分泌抗人VEGF单抗的杂交瘤细胞上清液，为阳性对照。P16为非相关杂交瘤细胞培养上清液，为阴性对照；SP2/0代表未融合的骨髓瘤细胞培养上清液。
图2 为本发明实施例2中以SDS-PAGE 分析鉴定从PV19瘤细胞的培养上清液中经亲合层析柱纯化获得的鼠源PV19单克隆抗体蛋白。图2中，泳道1为DTT还原的PV19抗体蛋白；泳道2为未还原的PV19抗体蛋白；Marker为蛋白质分子量标志。
图3为本发明实施例3中竞争性ELISA结果的代表性示意图。其中的mPV19&Biotin-VEGF 为PV19鼠源抗体组，W10&Biotin-VEGF为非相关的抗体组，作为阴性对照。
图4 为本发明实施例8中以SDS-PAGE 分析鉴定从培养上清液中经亲合层析柱纯化获得的人源化hPV19单克隆抗体蛋白。图2中，泳道1为DTT还原的hPV19抗体蛋白；泳道2为未还原的hPV19抗体蛋白；Marker为蛋白质分子量标志。
图5 为本发明实施例8中以直接ELISA法比较由表达上清中纯化获得的人源化抗体（hPV19）、嵌合抗体（chPV19）及鼠源抗体（mPV19）与人VEGF165蛋白结合的相对活性结果示意图。其中的chP16为非相关嵌合抗体，作为阴性对照。
图6 为本发明实施例8中以竞争ELISA法比较人源化抗体（hPV19）、嵌合抗体（chPV19）及鼠源抗体（mPV19）体外阻断生物素标记的人VEGF165蛋白（bio-VEGF）与其受体（VEGFR1）结合的相对活性的结果示意图。其中的W10为非相关嵌合抗体，作为阴性对照。
图7 为本发明实施例9中以ELISA法比较分析人源化hPV19抗体及Avastin抗体与野生型（VEGF165）及点突变型人VEGF蛋白(VEGF-G88/A)的结合活性的结果示意图。
图8A及图8B为本发明实施例10中人源化hPV19单克隆抗体在裸鼠体内抑制人Ls-174-T结肠癌生长的实验结果示意图。 其中图8A为实验期间各组相对肿瘤体积增长趋势图；图8B为实验结束时与阴性对照组相比，各治疗组平均肿瘤瘤重下降率（瘤重抑制率%）。
图9A及图9B为本发明实施例11中人源化hPV19单克隆抗体在裸鼠体内抑制人MD231-MBA乳腺癌生长的实验结果示意图。其中图9A为实验期间各组相对肿瘤体积增长趋势图；图9B为实验结束时与阴性对照组相比，各治疗组平均肿瘤瘤重下降率（瘤重抑制率%）。
图10A及图10B 为本发明实施例12中人源化hPV19单克隆抗体在裸鼠体内抑制人人A673横纹肌瘤结果示意图。其中图10A为实验期间各组相对肿瘤体积增长趋势图；图10B为实验结束时与阴性对照组相比，各治疗组平均肿瘤瘤重下降率（瘤重抑制率%）。
图 11A 及图 11B 为本发明实施例 13 中人源化 hPV19 单克隆抗体在裸鼠体内抑制人 HCT8 结肠癌结果示意图。其中图 11A 为实验期间各组相对肿瘤体积增长趋势图；图 11B 为实验结束时与阴性对照组相比， 各治疗组平均肿瘤瘤重下降率（瘤重抑制率%）。
代号为PV19-5的小鼠杂交瘤细胞株已于2012年03月12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（保藏编号为CGMCC No. 5889；保藏地点：中国北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所）。
 本发明的最佳实施方式
本发明的实施方式
下面将结合实施实例来进一步描述本发明，这些实施例只是为了起说明作用，而不是用来限制本发明。
实施例1: 分泌抗血管内皮细胞生长因子抗体 的小鼠杂交瘤细胞系的建立与筛选鉴定
步骤1.重组人VEGF165蛋白（免疫抗原）的 制备
利用PCR技术从人肺组织细胞cDNA文库中扩增得到编码人VEGF165完整开放读码框架序列(open reading frame, ORF）的基因片断，经序列测定鉴定正确，用限制性内切酶处理后，将其克隆到酵母表达载体pPic9K (Invitrogen公司)载体中，获得重组表达质粒pPic9K-VEGF165，用其转化毕赤酵母菌后，筛选出高效表达酵母菌。酵母菌经发酵、诱导表达，分离纯化后，获得纯度95％以上的重组人VEGF165蛋白。
步骤2、动物免疫
将上述纯化的重组人VEGF165蛋白与弗氏完全佐剂混合，于皮下多点注射Balb/c小鼠（100 μl/只，共10μg VEGF165蛋白）。首次免疫 2-3周后，小鼠再给予皮下多点注射含VEGF165蛋白与不完全佐剂的混合物，加强免疫2-3 次后，取少量小鼠血清，用包被人VEGF165蛋白的96-板以ELISA法检测小鼠血清中抗VEGF蛋白抗体的效价，取效价高者小鼠的脾细胞用于下一步的细胞融合。
步骤3、细胞融合
在末次免疫后3天，无菌制备小鼠脾细胞悬液，与小鼠Sp2/0骨髓瘤细胞 (购自中国科学院上海生命科学院细胞保藏中心），以5：1的比例在50%PEG-1000（Sigma 公司产品）作用下融合。融合按常规法（Kohler G. and Milstein C: Nature 1975;256:495-497）,PEG用量1ml，1分钟内缓慢加完。反应90秒后，以无血清的RPMI-1640培养基终止反应，1000rpm离心10 min,去除上清液，再将离心沉淀下的细胞以含10% HAT(H为次黄嘌呤、A氨基碟呤、T胸腺嘧啶核苷，为Sigma公司产品) 的RPMI 1640-10% FCS培养基将细胞浓度调节至1Х106/ml，加入96孔平底细胞培养板（每孔200μl），于37℃，5%CO2培养箱中培养2-3 周。
步骤4、酶联免疫吸附试验（ELISA）筛选抗体分泌阳性的小鼠杂交瘤细胞
以重组人VEGF165蛋白（ 2 μg/ml， pH 9.6， 0.1 M NaHCO3 液）包被酶标板，37℃包被2小时或4℃过夜；2%牛血清白蛋白（BSA）4℃封闭过夜。经PBS-0.1% Tween20 液洗涤后加入待检杂交瘤细胞培养上清（以未融合的SP2/0骨髓瘤细培养上清为阴性对照）37℃孵育2小时； 经PBS-0.1% Tween20液洗涤后，加入辣根过氧化物酶（HRP）-标记的羊抗小鼠IgG（Sigma 公司产品），37℃孵育1小时；再经PBS-0.1% Tween20 液充分洗涤后，加入邻苯二胺（OPD）-0.1%H2O2底物液显色10-15min,以0.1MHCl终止反应。 在MK3-Multiskan酶标仪 (Thermo Scientific公司产品)中读取492nm 处OD值。测得的OD 492值比阴性对照高5-10倍的杂交瘤细胞再克隆化，并进行扩增冻存。
步骤5、阳性杂交瘤细胞的亚克隆-有限稀释法
将上述初筛得到的阳性细胞以RPMI-1640-10% FCS培养基稀释至每孔1-10个细胞，铺于96-孔细胞培养板，于37℃，5% CO2培养箱中培养2-3 周。待克隆长成，取上清液以ELISA再次检测鉴定抗VEGF抗体的分泌。经检测鉴定，获得多个抗体分泌阳性细胞株。其中，经再次亚克隆鉴定，获得了一株代号为P19-5（简称PV19)的稳定分泌抗VEGF单克隆抗体的杂交瘤细胞株。图1为以ELISA 法检测鉴定PV19杂交瘤细胞上清液与重组人VEGF165蛋白结合，结果证明该杂交瘤细胞上清液含高效价抗人VEGF165蛋白的抗体。该抗体经鉴定为IgG类。该杂交瘤细胞株再经大量扩增，长期传代培养并于2012年03月12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（保藏编号为CGMCC No 5889. 保藏地点：中国，北京）。
实施例2. 鼠源 抗人VEGF单克隆抗体（PV19）的体外制备及纯化
本实施例中，鼠抗人VEGF单克隆抗体（PV19）的分离纯化采用亲合层析法。
其纯化步骤如下：
将PV19杂交瘤细胞扩增后，接种于200ml无血清的1640培养基中，37℃培养5天，随后收集培养上清，经过0.45 μm滤膜过滤后上样至含Protein G-Sepharose Fast Flow（购自通用电气GE公司）亲合层析柱；层析柱先以PBS液漂洗去除杂蛋白后, 再以低pH(2.7-3.0) 甘氨酸(0.1M) 液洗脱被吸附的PV19抗体蛋白。 洗脱液以1 mol/L Tris(pH 8.5-9.0)调节pH至7.0，再对10 倍体积的PBS液透析12～16后（期间换液2-3次），透析后的样品再经0.45 μm滤膜过滤后即获得纯化的PV19抗体。
将纯化的PV19抗体按常规方法在DTT还原及未还原条件下进行经聚丙烯酰胺凝胶电泳分析（SDS-PAGE）（分离胶为10%，浓缩胶5%）。图2为该电泳分析结果图谱，其中泳道1为DTT还原的PV19抗体，泳道2为未还原的PV19抗体。如图2所示：与未还原的PV19抗体样品相比后，DTT还原的PV19抗体分离为2条主带，其中处于上面的条带为PV19抗体重链，处于下面的条带为PV19抗体轻链。
实施例3 鼠源PV19单克隆抗体生物活性测定：竞争性ELISA法检测鉴定鼠源PV19单克隆抗体体外阻断VEGF与其受体的结合
鼠源PV19单克隆抗体的生物活性测定方法之一是竞争性ELISA法检测鉴定抗体体外阻断VEGF与其受体的结合。该竞争性ELISA法的基本原理与过程是先将生物素标记的人VEGF165蛋白与不同溶度的单克隆抗体混合，之后再将混合物转入预先包被有重组可溶性VEGF受体蛋白（如可溶性VEGFR1蛋白）的96-孔板，经孵育及洗脱后，加入酶标记的Avidin（如辣根过氧化物酶标记的Avidin）。 再经孵育及洗脱后，加入底物显示并测定OD值。
检测具体步骤如下：
1 ）用重组可溶性人VEGFR1蛋白（美国R&D公司产品）包被96-孔板（2 μg/ml,50 μl/孔)，4℃过夜；
2 ）经PBS液漂洗及2% BSA (稀释在PBS-0.1% tween20液中) 室温封闭后,分别加入固定溶度的生物素标记的VEGF165单克隆抗体（Bio-VEGF，1：1000）与不同溶度的PV19抗体，或非相关的抗体（W10）， 37℃孵育2 h；
3 ）经PBS-T洗脱后，加入辣根过氧化物酶标记的Avidin（1：5000），37℃孵育1 h；
4 ）经PBS-T洗脱后，加入显色液(邻苯二胺)-3% 双氧水，室温10min至显色；
5 ）加入 HCL终止反应，以酶联免疫仪测定492 nm波长处各孔的吸光值。
图3为竞争性ELISA结果的代表性示意图。如图3所示:在PV19抗体（mPV19&Biotin-VEGF）组中，其 OD值与抗体蛋白量成反比关系：即加入的PV19抗体的量越高，其OD值越低。而非相关的抗体（W10&Biotin-VEGF）组，各孔OD值不受加入的抗体蛋白量影响。 此结果表明PV19抗体体外可阻断VEGF与其受体（VEGFR1）的结合。
实施例4.编码鼠源PV19抗体可变区基因的克隆
取纯化的鼠源PV19抗体，在DTT还原条件下经SDS-PAGE分离重链和轻链，然后将电泳条带转印至PVDF膜，用考马斯亮蓝R250显色后，分别剪取PV19抗体重链和轻链蛋白条带，用Edman降解法进行N-端氨基酸测序，获得该抗体轻链的N-端氨基酸序列。抗体重链由于N-端封闭，常规氨基酸N-端测序未获结果。
1 ）．PV19抗体轻链可变区基因的克隆
步骤1、 采用试剂盒（江苏海门碧云天公司产品）从小鼠PV19杂交瘤细胞中提取出总RNA；
步骤2、 采用逆转录PCR(RT-PCR)方法在 eppendorf 管 获得cDNA模板。 其中用于逆转录PCR引物（mIg-kappa）序列为：TGTCGTTCACTGCCATCAAT（ SEQ ID NO.:15 ） ；
其中 RT-PCR 反应体系如下：
引物 2 µ l
RNA 模板 30 µ l
72℃ 孵育10分钟，然后冰上放置2分钟 。
随后加入：
5 ×RT-PCR反应 缓冲液 10 µ l
dNTPs 5 µ l
PrimeScript 逆转录酶 1.5 µ l
蒸馏水 1.5 µ l
总体积 50µl
于42℃温度下反应1小时，随后温度升至75℃，15分钟灭活后将获得的cDNA 置于-20℃，保存备用。
步骤3、 根据PV19抗体轻链的N-端氨基酸序列信息，设计如下一对引物：
正向引物（5'primer）: GAC ATT GTG ATG TCA CAG TCT CCA T（如 SEQ ID NO.:16 所示 ）
反向引物（3'primer）：AAT TGG ATC CAG TTG GTG CAG CAT CAG C （如 SEQ ID NO.:17 所示）
以上述引物及步骤2 中获得的cDNA为模板进行PCR扩增获得PV19抗体轻链可变区编码基因。
其中用于PCR扩增轻链DNA的反应体系如下：
正向引物 5 µ l
反向引物 5 µ l
2.5mmol/L dNTPs 5 µ l
10 ×反应 缓冲液 5 µ l
cDNA 模板 2 µ l
pfu DNA 聚合酶 0.5 µ l
蒸馏水 27.5 µ l
总体积 50 µ l
该PCR反应循环参数设置如下：
94 ℃ 5 分钟
94 ℃ 30 秒 循环30次
56 ℃ 30 秒
72 ℃ 1 分钟
72 ℃ 10 分钟
步骤4、 PCR 结束后将反应产物在1% agarose 胶中电泳。电泳结束后，将分离的DNA条带切下并进行测序获得抗体轻链可变区DNA的核苷酸序列。
测得的该抗体轻链可变区DNA的核苷酸序列见SEQ ID NO.:3，由该DNA的核苷酸序列推测得到的抗体轻链可变区氨基酸序列见SEQ ID NO.:1。该轻链抗原互补决定区（complementarity-determining regions， CDR）的 CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:9、SEQ ID NO.:10 和SEQ ID NO.:11。
2 ）．PV19抗体重链可变区基因的克隆
重链由于缺乏N-端氨基酸序列信息，故采用简并引物PCR法，克隆获得重链可变区编码基因。其中用于克隆扩增该抗体重链可变区基因的一对引物为：
正向引物（5'primer）：TCA G GCC ATT ACG GCC MMY CWM ACC AT （如 SEQ ID NO.:18 所示） ， 其中M=A或C， Y=C或T代表: W=A或T。
反向引物（3'primer）：AAT TGG ATC CTG GGG GTG TCG TTT TGG C （如 SEQ ID NO.:19 所示）
用上述引物及从小鼠PV19杂交瘤细胞中获得的cDNA模板采用PCR（PCR反应体系与参数类同上述轻链PCR）扩增获得重链可变区DNA. PCR 扩增得到的DNA产物在1% agarose 胶中电泳分析。电泳结束后，将分离的DNA条带切下并进行测序获得抗体重链可变区DNA的核苷酸序列。测得的该抗体重链可变区DNA的核苷酸序列见SEQ ID NO.:4，由该DNA核苷酸序列推测得到的抗体重链可变区氨基酸序列见SEQ ID NO.:2。该重链抗原互补决定区的 CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:12、SEQ ID NO.:13 和SEQ ID NO.:14。
实施例5. 人-鼠嵌合抗体（chPV19）的制备
将实施例4中克隆扩增获得的PV19抗体轻链可变区基因和重链可变区基因分别与人-kappa轻链恒定区（C-domain）和人IgG1-重链恒定区基因片段融合，获得人-鼠嵌合轻链基因（ chPV19L ）及 人-鼠嵌合重链基因（ chPV19H ）。 其后将轻链嵌合基因与重链嵌合基因分别克隆至pcDNA3.1 表达质粒，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体基因的表达质粒。
含人-鼠嵌合抗体基因的表达质粒再与脂质体（RocheFugen6）混合后共转染入CHO细胞。细胞转染后2-3天，取培养上清液，用包被人VEGF165蛋白的96-孔板，用 HRP 酶标记的 Goat-anti-human-IgG 为二抗（购自上海西塘生物公司 ) ，为检测二抗，以ELISA法检测上清中的嵌合抗体（chPV19）与人VEGF165蛋白结合。 ELISA法检测结果表明表达质粒转染的CHO细胞上清中含有可与人VEGF的蛋白结合的嵌合抗体。
实施例6.人源化抗体（hPV19）的初步制备与鉴定
在ELISA法检测初步证明人-鼠嵌合抗体（chPV19）保持有与人VEGF蛋白高亲合力结合活性的基础上，采用PCR等系列基因工程克隆手段将该嵌合抗体轻链和重链中的抗原互补决定区(CDR)基因片段分别移植到对应人kappa-轻链和IgG1-重链可变区骨架（framework regions，FR）上，获得人源化的hPV19抗体。
1 ）抗体轻链人源化
通过氨基酸序列分析，确定人免疫球蛋白Kappa轻链第四V区胚系基因的表达产物(IgKV4-1,Gene ID: 28908)与PV19轻链具有最高同源性。据此,将PV19轻链可变区基因与人免疫球蛋白Kappa轻链的恒定区(GenBank: BC095489.1)编码序列拼接,获得嵌合型轻链全长基因(cPV19-L)。然后,将cPV19-L轻链骨架区(FR)用人IgKV4-1的同源序列替换,经基因重组克隆最后成功获得人源化的轻链编码基因(hPV19-L)。其中人源化hPV19抗体轻链可变区的氨基酸序列见SEQ ID NO.:5。
2 ）抗体重链人源化
通过氨基酸序列分析，确定人免疫球蛋白重链第七V区胚系基因的表达产物(IgHV7-81，NCBI Gene: 28378)与PV19抗体重链具有最高同源性。据此，将PV19抗体重链可变区基因与人免疫球蛋白IgG-Gamma1链恒定区(GenBank: BC073782.1)编码序列拼接，获得嵌合型重链全长基因(cPV19-H)。然后，将cPV19-H重链骨架区(FR)用人IgHV7-81的同源序列替换，获得人源化的重链编码基因(hPV19-H)。其中人源化hPV19抗体重链可变区的氨基酸序列见SEQ ID NO.:6。
将含嵌合型重链基因（ cPV19H ） 、嵌合型轻链基因（ cPV19L ） 、人源化重链基因（ hPV19H ） 、人源化轻链基因（ hPV19L ）分别克隆到 pcDNA3.1 表达载体，转入大肠杆菌后扩增分离获得各表达质粒。 其后将各表达质粒两两组合后，瞬时转染人CHO细胞。转染48小时后，吸取孔内细胞培养上清，以VEGF165蛋白为包被抗原， HRP 酶标记的 Goat-anti-human-IgG 为检测二抗（购自上海西塘生物公司 ) ， OPD 为显色底物，以 直接ELISA法检测转染细胞上清中抗体与人VEGF165抗原结合的活性。
下表1为ELISA代表性检测结果。如表1中结果所示：与嵌合型抗体一样，人源化hPV19抗体保持与人VEGF165蛋白结合的活性。
表1 ELISA分析瞬时转染细胞培养上清结合人VEGF165蛋白活性
上清稀释
倍数		 OD 显色值
cPV19H+cPV19L		 OD 显色值
cPV19H+hPV19L		 OD 显色值
hPV19H+cPV19L		 OD 显色值
hPV19H+hPV19L
2 2.933 2.904 2.688 2.632
4 2.762 2.718 1.976 2.291
8 2.451 2.438 1.33 1.745
16 1.745 1.769 0.888 1.207
32 0.967 1.14 0.591 0.874
64 0.762 0.776 0.408 0.598
128 0.632 0.585 0.337 0.476
256 0.524 0.465 0.312 0.517
实施例7稳定高效表达人源化hPV19抗体的重组工程细胞的筛选与建立
为了获得具产业化意义的、可稳定高效分泌表达hPV19抗体的重组工程细胞株，在本发明实施例中采用的方案是：先将含人源化hPV19抗体重链及轻链的基因同时插入到已含仓鼠二氢叶酸还原酶（DHFR）基因的克隆质粒中（pCDNA3.1-DHFR），获得同时含人源化hPV19抗体重链及轻链基因的重组质粒(pQY-hPV19D-DHFR)。再将该重组质粒与脂质体混合后共转染人DHFR缺陷型中华仓鼠卵巢细胞细胞（CHO-DHFR-/-），通过逐渐增加细胞培养基中的氨甲喋呤（MTX）的溶度而提高转染细胞中DHFR基因（及抗体基因）的拷贝数及抗体蛋白的表达，从而筛选获得稳定高效表达人源化hPV19抗体的重组工程细胞株。
有关该重组质粒的构建及重组工程细胞株的筛选与建立过程如下：
1 ）含人源化hPV19抗体基因及DHFR基因的表达质粒(phPV19D-DHFR)的构建
在本实施例中，选取含仓鼠二氢叶酸还原酶（DHFR）基因的pCDNA3.1-DHFR质粒作为克隆载体，将含人源化hPV19抗体轻链基因（hPV19L）和重链基因（hPV19）同时插入到该克隆载体，获得名为pQY-hPV19-DHFR的表达质粒。该表达质粒中，用于驱动DHFR基因在哺乳动物细胞中表达的启动子是SV40早期启动子(pSV40)，用于驱动hPV19抗体轻链基因(hPV19L)表达的是人延伸因子1-α亚基启动子(pEF1-a)，用于驱动hPV19抗体重链基因(hPV19H)表达的是巨细胞病毒早期启动子(pCMV)。
2 ）重组工程细胞的初步筛选
采用罗氏（Roche）公司生产的FUGENE-HD脂质体作为转染试剂，将上述含人源化hPV19抗体基因的表达质粒phPV19D-DHFR(HEX)与FUGENE-HD脂质体按一定比例混合后，转染至DHFR缺陷型中华仓鼠卵巢细胞（CHO-DHFR-）。 转染48小时后，吸取孔内细胞培养上清，在包被人VEGF165蛋白的96-孔板中以实施例6描述的直接ELISA法检测转染细胞上清中hPV19抗体的表达。 ELISA法检测结果表明转染细胞上清中含有可与人VEGF165抗原结合的hPV19抗体。其后转染的细胞用胰蛋白酶液处理后再重悬于不含HT的IMDM培养基，以有限稀释法（limited dilution）接种培养于多个96孔细胞培养板中，培养5-7天后再挑取细胞上清以ELISA方法初步筛选检测细胞克隆。ELISA方法共筛选检测了400个样品孔上清，结果显示有380个样品为阳性克隆（上清阳性的克隆比例为95%），表达量介于1-10μg/ml 之间。
3 ）重组工程细胞在含氨甲喋呤的培养基中加压筛选
上述初筛获得的几个表达相对较高的阳性克隆再接种培养在24-孔细胞培养板中，通过逐渐增加细胞培养基中氨甲喋呤（MTX）的溶度而提高抗体基因与抗体蛋白的表达。
首轮（初始）MTX筛选浓度为 5nM ，培养3-4天后取细胞上清液，以ELISA法检测细胞上清中hPV19抗体蛋白浓度，从中挑选出表达量相对较高的细胞再铺至新的24孔细胞培养板中，进人下一轮（第２轮）MTX筛选（该轮MTX 的筛选浓度增至为10nM）；以此类推，直至到培养基中MTX浓度增至320nM以上轮筛选结束。经7-8轮筛选最终获得了多个上清中抗体蛋白表达量高达150-200μg/ml的细胞株（与MTX筛选前相比，该轮筛选结束后抗体蛋白表达量增加20倍以上）。其中多株细胞经过多轮无血清培养基中悬浮培养与驯化后，在未经培养基配方与工艺优化条件下，其上清中hPV19抗体的表达浓度介于150-200μg/ml，这与驯化前的细胞株表达量相同。
实施例8.人源化抗体（hPV19）的大规模制备与鉴定
将无血清培养基悬浮培养驯化后的细胞株在培养瓶或生物反应器中规模培养。培养结束后收集细胞上清，上样至protein-A亲合层析柱纯化，再经过离子交换、病毒灭活及过滤等步骤，获得纯度达99%以上的人源化hPV19抗体。图4为从培养液中纯化获得的人源化抗体（hPV19）在DTT还原及未还原条件下的SDS-PAGE电泳一代表性图谱，其中泳道1为DTT还原的抗体，泳道2为未还原的抗体。结果显示DTT还原的抗体分为2条主带，其中处于上面的条带为hPV19抗体重链，处于下面的条带为hPV19抗体轻链；未还原的hPV19抗体则位于150 kD 附近，与理论预期的完整IgG1抗体的分子量一致。
图5为以直接ELISA法比较纯化获得的人源化抗体（hPV19）、嵌合抗体（chPV19）及鼠源抗体（mPV19）与人VEGF165蛋白结合的相对活性的代表性结果图谱，ELISA检测具体方法同实施例5，其中的chP16为非相关嵌合抗体，为阴性对照。检测结果显示：纯化的人源化抗体（hPV19）保持与人VEGF165蛋白高度结合的活性，且其结合活力与其前身鼠源抗体（mPV19）非常接近。
图6则为以竟争性ELISA法检测比较人源化抗体（hPV19）、嵌合抗体（chPV19）及鼠源抗体（mPV19）体外阻断人VEGF165蛋白（biotin-VEGF）与其受体（VEGFR1）结合的相对活性，竟争性ELISA检测具体方法同实施例6，其中的W10为非相关嵌合抗体，为阴性对照。 检测结果显示：该人源化抗体（hPV19）可以阻断VEGF与其受体的结合，且其阻断活性与其前身鼠源抗体（mPV19）非常接近。
实施例9. 人源化hPV19抗体及Avastin抗体在VEGF抗原的识别位点比较
根据公开文献报道，A4.6.1 或Avastin抗体识别人VEGF蛋白的关键抗原表位在于第88位的甘氨酸（Gly,G88）附近，若将此甘氨酸突变为丙氨酸（G88/A点突变）则A4.6.1 或Avastin抗体与该突变型VEGF蛋白的结合强度则明显下降。为了比较hPV19抗体与Avastin在识别人VEGF抗原的位点方面是否存在差异，在此先体外构建含G88/A点突变的人VEGF165（G88/A），再以ELISA法分析检测人源化hPV19抗体及Avastin抗体与野生型及突变型VEGF蛋白的结合活性。其中具体实施步骤及结果描述如下：
实施步骤1、G88/A突变型VEGF蛋白的表达、纯化
采用重叠－延伸PCR法（重叠-延伸PCR技术(gene splicing by overlap extension PCR,简称SOE PCR)由于采用具有互补末端的引物,使PCR产物形成了重叠链，从而在随后的扩增反应中通过重叠链的延伸，将不同来源的扩增片段重叠拼接起来，此技术能够在体外进行有效的基因重组，而且不需要内切酶消化和连接酶处理,可利用这一技术很快获得其它依靠限制性内切酶消化的方法难以得到的产物。重叠延伸PCR技术成功的关键是重叠互补引物的设计，重叠延伸PCR在基因的定点突变、融合基因的构建、长片段基因的合成、基因敲除以及目的基因的扩增等方面有其广泛而独特的应用），利用一组引物（正向引物：VEGFG88F：CCT CAC CAA GCC CAG CAC ATA ，如SEQ ID NO.:20所示，反向引物：VEGFG88R：CTA TGT GCT GGG CTT GGT GAG ，如 SEQ ID NO.:21 所示 ）进行点突变，获得编码G88/A突变型人VEGF165的cDNA。其后将突变编码序列插入pPic9K表达载体中，然后将质粒导入毕赤酵母GS115菌株，诱导表达、纯化后获得突变型VEGF蛋白(VEGF G88/A)。
实施步骤2、 hPV19 抗体及Avastin与对野生型人VEGF165结合力比较
实验方法：
1）将野生型人VEGF165或突变型VEGF蛋白(VEGF-G88/A)蛋白包被在96孔-ELISA板。
2 ）hPV19 抗体及 Avastin 各对倍梯度稀释后，加至96孔板中，每孔100μl。37℃放置1小时。
3 ）洗板后，加入1:2000稀释的HRP-Goat anti-human Fc 抗体，每孔100μl。
4 ）洗板后，加入底物OPD显色。
ELISA 检测结果如图7。如图7所示：hPV19抗体及Avastin与野生型人VEGF165的结合强度几乎相近。 hPV19抗体及Avastin与突变型VEGF蛋白(VEGF G88/A) 蛋白的结合强度则明显不同。与野生型人VEGF165的结合相比，Avastin与突变型VEGF蛋白(VEGF G88/A)蛋白的结合强度下降约25-50倍,而hPV19与突变型VEGF蛋白(VEGF G88/A)蛋白的结合强度仅下降约3-6倍。此结果表明hPV19抗体结合人VEGF蛋白的位点与Avastin的结合位点并不完全相同。
实施例10. 人源化hPV19抗体在裸鼠体内抑制人结肠癌细胞Ls-174-T移植瘤的生长
步骤1：人Ls-174-T移植结肠癌裸鼠移植瘤模型的建立
将对数生长期人Ls-174-T结肠癌细胞（购自中国科学院上海生命科学院细胞保藏中心），按1×107个/只接种于2只裸鼠右侧腋窝皮下或已有瘤块制备成1.5mm3左右，接种于动物右侧腋窝皮下，制备荷瘤种鼠；待肿瘤生长至400 ~ 600mm 3 左右，选择肿瘤生长及健康情况良好的荷瘤动物，无菌条件下取瘤，制备成1.5mm3左右，接种于动物右侧腋窝皮下，用于正式试验。
裸鼠接种后观察肿瘤生长情况，待肿瘤体积为50-100mm3左右时，按瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选。筛选后随机分组给予不同剂量药物。
步骤2：人源化PV19抗体治疗Ls-174-T移植结肠癌
将上述接种了Ls-174-T结肠癌细胞的裸鼠分成以下5组（每组8只）给药，每周通过腹腔注射给药2次，连续4周，第29天（D29）试验结束。
给药分组情况为：
1) 人源化hPV19抗体高剂量组（供试品高剂量组，10mg/kg体重）；
2) 人源化hPV19抗体中剂量组（供试品中剂量组，5mg/kg体重）；
3) 人源化hPV19抗体低剂量组（供试品低剂量组，2.5mg/kg体重)；
4 ）Avastin对照组（5mg/kg 体重）；
5) 生理盐水阴性对照组。
给药期间每天2次观察动物一般临床症状、每周进行2次体重和瘤径测量，安乐死后剥取肿瘤，称量瘤重。以相对肿瘤增殖率T/C%来评价疗效，T/C%≤40%且P<0.05为有效，同时以瘤重抑制率大于60%为有效性参考指标。
试验结果：
1 ）对肿瘤生长情况的影响
首次给药后每周 2 次测量肿瘤长径和短径，计算肿瘤体积 V 及其相对肿瘤体积 RTV 。计算公式为 RTV=Vt/V0, 其中 Vt 为各时间点（天）测量得到的肿瘤体积， Vo 为初始（给药前 ) 肿瘤体积。
各组 Ls-174-T 肿瘤相对体积增长趋势见图 8A ，结果表明与生理盐水阴性对照组相比，人源化 hPV19 抗体可明显抑制肿瘤的生长。
2 ）对肿瘤重量的影响
D29 试验结束，动物安乐死，剥离肿瘤并称重，计算各组间平均瘤重差异：
生理盐水 阴性对照组平均瘤重 2.957g 。
阿瓦斯汀 Avastin 组平均瘤重 0.767g ，显著低于阴性对照组（ P ≤0.01 ）。
供试品（ 人源化hPV19抗体） 低、中、高剂量组平均瘤重 0.883g 、 0.631g 、 0.467g ，显著低于阴性对照组（ P ≤0.01 或 P ≤0.001 ）。
根据各组平均瘤重计算瘤重抑制率（图 8B ），可见 Avastin 组的瘤重抑制率为 74.05% ， 人源化hPV19抗体 低、中、高剂量组的瘤重抑制率分别为 70.15% 、 78.66% 、 84.21% 。此结果表明各剂量组的人源化 hPV19 抗体均可明显抑制肿瘤生长，且其抑制程度不亚于 Avastin 。
实施例11. 人源化hPV19单克隆抗体在裸鼠体内抑制移植人MDA-MB-231乳腺癌的生长
步骤1：人MDA-MB-231乳腺癌模型的裸鼠移植瘤模型的建立
建立方法基本同实施例10。
裸鼠接种人MDA-MB-231乳腺癌细胞后观察肿瘤生长情况，待肿瘤体积为50-100mm3左右时，按瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选。筛选后随机分组给予不同剂量药物。
步骤2：人源化PV19抗体治疗人MDA-MB-231乳腺癌
将上述接种了人MDA-MB-231乳腺癌的裸鼠分成以下5组（每组7只）给药，每周通过腹腔注射给药2次，连续5周， 第36天（D36）试验结束。
给药分组情况为：
1) 人源化hPV19抗体高剂量组（供试品高剂量组，10mg/kg体重）；
2) 人源化hPV19抗体中剂量组（供试品中剂量组，5mg/kg体重）；
3) 人源化hPV19抗体低剂量组（供试品低剂量组，2.5mg/kg体重)；
4 ）Avastin对照组（5mg/kg 体重）；
5) 生理盐水阴性对照组。
给药期间每天2次观察动物一般临床症状、每周进行2次体重和瘤径测量，安乐死后剥取肿瘤，称量瘤重。以相对肿瘤增殖率T/C%来评价疗效，T/C%≤40%且P<0.05为有效，同时以瘤重抑制率大于60%为有效性参考指标。
试验结果：
1 ）对肿瘤生长情况的影响
首次给药后每周 2 次测量肿瘤长径和短径，计算肿瘤体积 V 及其相对肿瘤体积 RTV(Vt/V0) 。各组 MDA-MA-231 肿瘤相对体积增长趋势见图 9A ，结果表明与生理盐水阴性对照组相比，人源化 hPV19 抗体可明显抑制肿瘤的生长。
2 ）对肿瘤重量的影响
D36 试验结束，动物安乐死，剥离肿瘤并称重，计算各组间平均瘤重差异：
阴性对照组平均瘤重 0.890g 。
Avastin 组平均瘤重 0.543g ，与阴性对照组比较差异不显著（ P>0.05 ）。
人源化hPV19抗体 低、中、高剂量组平均瘤重 0.576g 、 0.298g 、 0.357g ，与阴性对照组比较差异均不显著（ P>0.05 ）。
根据各组平均瘤重计算瘤重抑制率（图 9B ），可见 Avastin 组的瘤重抑制率为 38.99% ，人源化 hPV19 抗体低、中、高剂量组的瘤重抑制率分别为 35.20% 、 66.56% 、 59.92% 。此结果表明中、高剂量组的人源化 hPV19 抗体均可不同程度抑制肿瘤生长，且其抑制程度高于同剂量组的 Avastin 。
实施例12. 人源化hPV19抗体在裸鼠体内抑制移植人A673横纹肌瘤的生长
步骤1：人A673横纹肌瘤模型的裸鼠移植瘤模型的建立
建立方法基本同实施例10。
裸鼠接种人A673横纹肌瘤细胞后观察肿瘤生长情况，待肿瘤体积为50-100mm3左右时，按瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选。筛选后随机分组给予不同剂量药物。
步骤2：人源化PV19抗体治疗人A673横纹肌瘤
将上述接种了人A673横纹肌瘤的裸鼠分成以下6组（每组6只）给药，每周通过腹腔注射给药2次，连续3周，第22天（D22）试验结束。
给药分组情况为：
1) 人源化hPV19抗体高剂量组（供试品高剂量组，10mg/kg体重）；
2) 人源化hPV19抗体中剂量组（供试品中剂量组，5mg/kg体重）；
3) 人源化hPV19抗体低剂量组（供试品低剂量组，2.5mg/kg体重)；
4) 人源化hPV19抗体超低剂量组（供试品超低剂量组，0.5mg/kg体重)；
5 ）Avastin对照组（5mg/kg 体重）；
6) 生理盐水阴性对照组。
给药期间每天2次观察动物一般临床症状、每周进行2次体重和瘤径测量，安乐死后剥取肿瘤，称量瘤重。以相对肿瘤增殖率T/C%来评价疗效，T/C%≤40%且P<0.05为有效，同时以瘤重抑制率大于60%为有效性参考指标。
试验结果：
1 ）对肿瘤生长情况的影响
首次给药后每周 2 次测量肿瘤长径和短径，计算肿瘤体积 V 及其相对肿瘤体积 RTV(Vt/V0) 。各组 A673 肿瘤相对体积增长趋势见图 10A ，结果表明与生理盐水阴性对照组相比，人源化 hPV19 抗体可明显抑制肿瘤的生长。
2 ）对肿瘤重量的影响
D22 试验结束，动物安乐死，剥离肿瘤并称重，计算各组间平均瘤重差异：
阴性对照组平均瘤重 8.726g 。
Avastin 组平均瘤重 3.323g ，显著低于阴性对照组（ P ≤ 0.05 ）。
人源化hPV19抗体 低、中、高、超低剂量组平均瘤重 3.811g 、 3.046g 、 2.499g 、 7.056g 。与阴性对照组比较， 人源化hPV19抗体 低、中、高剂量组平均瘤重显著降低（ P ≤ 0.05 ）。
根据各组平均瘤重计算瘤重抑制率（图 10B ），可见 Avastin 组的瘤重抑制率为 61.92% ， 人源化hPV19抗体 低、中、高、超低剂量组的瘤重抑制率分别为 56.33% 、 65.09% 、 71.36% 、 19.13% 。结果表明中、高剂量组的人源化 hPV19 抗体均可明显抑制肿瘤生长，且其抑制程度不亚于 Avastin 。
实施例13. 人源化hPV19抗体在裸鼠体内抑制移植人HCT-8结肠癌的生长
步骤1：人HCT-8结肠癌模型的裸鼠移植瘤模型的建立
建立方法基本同实施例10。
裸鼠接种人HCT-8结肠癌后观察肿瘤生长情况，待肿瘤体积为50-100mm3左右时，按瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选。筛选后随机分组给予不同剂量药物。
步骤2：人源化PV19抗体治疗人HCT-8结肠癌
将上述接种了人HCT-8结肠癌的裸鼠分成以下7组（每组8只）给药，每周通过腹腔注射给药2次，连续4周， 第30天（D30）试验结束。
给药分组情况基本同实施例9，但增加顺铂化疗药对照组，及顺铂与hPV19抗体合并使用的联合给药组。
给药分组情况具体如下：
1) 人源化hPV19抗体高剂量组（供试品高剂量组，10mg/kg体重）；
2) 人源化hPV19抗体中剂量组（供试品中剂量组，5mg/kg体重）；
3) 人源化hPV19抗体低剂量组（供试品低剂量组，2.5mg/kg体重)；
4) 人源化hPV19抗体超低剂量组（供试品超低剂量组，0.5mg/kg体重)；
5 ）顺铂化疗对照组（5mg/kg 体重）；
6 ）联合给药组（供试品hPV19抗体5mg/kg +顺铂5mg/kg 体重）；
7) 生理盐水阴性对照组。
给药期间每天2次观察动物一般临床症状、每周进行2次体重和瘤径测量，安乐死后剥取肿瘤，称量瘤重。以相对肿瘤增殖率T/C%来评价疗效，T/C%≤40%且P<0.05为有效，同时以瘤重抑制率大于60%为有效性参考指标。
试验结果：
1 ）对肿瘤生长情况的影响
首次给药后每周 2 次测量肿瘤长径和短径，计算肿瘤体积 V 及其相对肿瘤体积 RTV(Vt/V0) 。
各组 HCT-8 肿瘤相对体积增长趋势见图 11A ，结果表明与生理盐水阴性对照组相比，各剂量组的人源化 hPV19 抗体均可明显抑制肿瘤的生长。
2 ）对肿瘤重量的影响
D30 试验结束，动物安乐死，剥离肿瘤并称重，计算各组间平均瘤重差异：
阴性对照组组平均瘤重 1.608g 。
顺铂化疗单用组平均瘤重 1.201g ，与阴性对照组比较差异不显著。
hPV19 单抗供试品低、中、高、超低剂量组平均瘤重 0.405g 、 0.278g 、 0.311g 、 0.604g ，显著低于阴性对照组（ P ≤ 0.01 或 P ≤ 0.05 ）； hPV19 单抗与顺铂化疗联合用药组平均瘤重 0.193g ，显著低于阴性对照组（ P ≤ 0.01 ）。
根据各组平均瘤重计算瘤重抑制率（见图 11B ），可见顺铂化疗单用组的瘤重抑制率为 25.29% ，供试品低、中、高、超低剂量组及联合用药组的瘤重抑制率分别为 74.81% 、 82.73% 、 80.65% 、 62.46% 和 87.98% 。 此结果表明各剂量组的人源化 hPV19 抗体均可明显抑制肿瘤生长；且在超低剂量（ 0. 5mg/kg ）依然有效。此外，在与顺铂化疗药合并使用治疗，其治疗效果明显优于单用5mg/kg顺铂组和单用5mg/kg hPV19单克隆抗体组。
 工业实用性
本发明通过蛋白分离纯化及基因工程等手段获得了编码该鼠源抗体的重链及轻链可变区基因，并在此基础上完成了该抗体的人源化改造。人源化改造后的抗体基因插入表达载体（pCDNA3.1）后转入中华仓鼠卵巢（CHO）细胞，获得重组工程细胞，并从重组工程细胞培养液中分离纯化得到具拮抗肿瘤体内增长生物活性的人源化抗体蛋白。该人源化抗体可作为药 物组合物成分或制备成合适药物制剂，单独给药或与化疗药物等其他治疗手段合并使用，用于广谱治疗结肠癌、乳腺癌、横纹肌肉瘤等各种实体瘤。
序列表自由内容
序列表
<110> 苏州思坦维生物技术有限责任公司
<120> 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途
<130>DAPCT-1797
<160> 21
<170> PatentIn version 3.4
<210> 1
<211> 108
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 1
DIVMSQSPSS LAVSAGEKVT LSCKSSQSLL NSRTRKNFLA WYQQKPGQSP KLLIYWASTR 60
ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL YTFGGGPT 108
<210> 2
<211> 116
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 2
QPGGAFWMCV SGGKVTISCK ASGYSFTNSG INWVKQAPGK GLKWMGWINT YTGEPTYADD 60
FKGRFAFSLE TSASSAYLQI NNLKNEDTAT YFCARFGDGY YWFFDVWGAG TTVTVS 116
<210> 3
<211> 324
<212> DNA
<213> 小鼠 (Mus musculus)
<400> 3
gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60
ttgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaaaaa cttcttggct 120
tggtaccagc agaaaccagg gcagtctcct aaactactga tctactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 300
tacacgttcg gcgggggacc aacc 324
<210> 4
<211> 348
<212> DNA
<213> 小鼠 (Mus musculus)
<400> 4
cagcctgggg gtgccttttg gatgtgtgtt tctggcggga aagtcactat ctcctgcaag 60
gcttctggat attccttcac aaactctgga attaactggg tgaagcaggc tccaggaaag 120
ggtttaaagt ggatgggctg gataaacacc tacactggag agccaacata tgctgatgac 180
ttcaagggac ggtttgcctt ctctttggaa acctctgcca gctctgccta tttgcagatc 240
aacaacctca aaaatgagga cacggctaca tatttctgtg caagattcgg agatggttac 300
tactggttct tcgatgtctg gggcgcaggg accacggtca ccgtctcc 348
<210> 5
<211> 113
<212> PRT
<213> 智人(Homo sapiens)
<400> 5
DIVMTQSPDS LAVSLGERAT INCKSSQSLL NSRTRKNFLA WYQQKPGQSP KLLIYWASTR 60
ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL YTFGGGTNLE IKR 113
<210> 6
<211> 116
<212> PRT
<213> 智人(Homo sapiens)
<400> 6
DVQLVQSGVE VKNPGASVKV SCRASGYSFT NSGINWVKQA PGKGLKWMGW INTYTGEPTY 60
ADDFKGRFAF SLETSASSAY LQINNLKNED TATYFCARFG DGYYWFFDVW GAGTTV 116
<210> 7
<211> 339
<212> DNA
<213> 智人(Homo sapiens)
<400> 7
gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60
atcaactgca agtccagcca gagtctgctc aacagtagaa cccgaaaaaa cttcttggct 120
tggtaccagc agaaaccagg gcagtctcct aaactactga tctactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 300
tacacgttcg gcggggggac caacctggaa ataaaacgt 339
<210> 8
<211> 357
<212> DNA
<213> 智人(Homo sapiens)
<400> 8
gacgtccagc tggtgcagtc tggagttgag gtgaagaacc ctggggcctc agtaaaggtc 60
tcctgcaggg cttctggtta ctccttcaca aactctggaa ttaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacatat 180
gctgatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag ctctgcctat 240
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagattcgga 300
gatggttact actggttctt cgatgtctgg ggcgcaggga ccacggtcac cgtctcc 357
<210> 9
<211> 17
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 9
KSSQSLLNSR TRKNFLA 17
<210> 10
<211> 7
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 10
WASTRES 7
<210> 11
<211> 11
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 11
KQSYNLYTFG G 11
<210> 12
<211> 10
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 12
GYSFTNSGIN 10
<210> 13
<211> 14
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 13
INTYTGEPTY ADDF 14
<210> 14
<211> 10
<212> PRT
<213> 小鼠 (Mus musculus)
<400> 14
FGDGYYWFFD 10
<210> 15
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 15
tgtcgttcac tgccatcaat 20
<210> 16
<211> 25
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 16
gacattgtga tgtcacagtc tccat 25
<210> 17
<211> 28
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 17
aattggatcc agttggtgca gcatcagc 28
<210> 18
<211> 27
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 18
tcaggccatt acggccmmyc wmaccat 27
<210> 19
<211> 28
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 19
aattggatcc tgggggtgtc gttttggc 28
<210> 20
<211> 21
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 20
cctcaccaag cccagcacat a 21
<210> 21
<211> 21
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<223> 引物
<400> 21
ctatgtgctg ggcttggtga g 21

Claims (1)

  1. 1. 一种拮抗抑制血管内皮生长因子与其受体结合的鼠源单克隆抗体，其特征在于，所述抗体的轻链可变区具有 SEQ ID NO：1 所示的氨基酸序列,重链可变区具有 SEQ ID NO：2 的氨基酸序列。
    2 、一种编码权利要求 1 所述抗体的 DNA 分子或基因，其特征在于，其轻链可变区具有 SEQ ID NO ： 3 所示的核苷酸序列，重链可变区具有 SEQ ID NO ： 4 的核苷酸序列。
    3 、一种拮抗抑制血管内皮生长因子与其受体结合的人源化单克隆抗体，其特征在于， 将权利要求1所述的重链可变区和轻链可变区基因进行人源化改造，包括框架区和抗原结合区域/互补决定区的氨基酸置换； 所述人源化单克隆抗体的轻链可变区具有 SEQ ID NO:5 所示的氨基酸序列，重链可变区具有 SEQ ID NO:6 所示的氨基酸序列。
    4 、一种编码权利要求 3 所述人源化单克隆抗体的 DNA 分子或基因，其特征在于，其轻链可变区具有 SEQ ID NO ： 7 所示的核苷酸序列，重链可变区具有 SEQ ID NO ： 8 的核苷酸序列。
    5 、一种如权利要求 3 所述的人源化单克隆抗体的衍生体，其特征在于，所述衍生体的轻链抗原互补决定区具有 SEQ ID NO ： 9 ， SEQ ID NO ： 10 及 SEQ ID NO ： 11 的氨基酸序列；其重链抗原互补决定区具有 SEQ ID NO ： 12 ， SEQ ID NO ： 13 及 SEQ ID NO ： 14 的氨基酸序列。
    6 、一种表达载体，其特征在于，它含有权利要求 4 所述的 DNA 分子序列以及与该序列操作性相连的表达调控序列。
    7 、一种重组宿主细胞，其特征在于，它由权利要求 6 所述的表达载体转化而成。
    8 、根据权利要求 7 所述的重组宿主细胞或其子代细胞,所述重组宿主细胞或其子代细胞表达权利要求 3 的人源化单克隆抗体。
    9 、一种药物组合物，其特征在于，它含有药学上有效量的权利要求 3 所述的人源化单克隆抗体或权利要求 5 所述的人源化单克隆抗体的衍生体以及药学上可接受的载体。
    10 、权利要求 9 所述的药物组合物在制备治疗与血管增生相关的疾病的药物制剂中的应用。
    11 、如权利要求 10 所述的应用，其特征在于，所述疾病包括结肠癌、乳腺癌、横纹肌肉瘤。
    12 、一种制备权利要求 3 所述的人源化单克隆抗体的方法，其特征在于，该方法包括如下步骤：
    a) 提供一表达载体，该表达载体含有权利要求 4 所述的 DNA 分子序列以及与该序列操作性相连的表达调控序列；
    b) 用步骤 a ）所述的表达载体转化宿主细胞；
    c) 在适合所述人源化单克隆抗体表达的条件下培养步骤 b) 所得的宿主细胞：和
    d) 采用亲合层析从宿主细胞培养液中分离纯化获得所述人源化单克隆抗体。
PCT/CN2013/086542 2012-12-14 2013-11-05 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途 Ceased WO2014090053A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP13863651.9A EP2933266B1 (en) 2012-12-14 2013-11-05 Monoclonal antibody for antagonizing and inhibiting binding of vascular endothelial cell growth factor and its receptor, and coding sequence and use thereof
ES13863651T ES2719084T3 (es) 2012-12-14 2013-11-05 Anticuerpo monoclonal para antagonizar e inhibir la unión de factor de crecimiento celular endotelial vascular y su receptor, y secuencia de codificación y uso de este
US14/651,785 US9580498B2 (en) 2012-12-14 2013-11-05 Monoclonal antibody for antagonizing and inhibiting binding of vascular endothelial growth factor to its receptor, and coding sequence

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210543652.0A CN103864932B (zh) 2012-12-14 2012-12-14 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途
CN201210543652.0 2012-12-14

Publications (1)

Publication Number Publication Date
WO2014090053A1 true WO2014090053A1 (zh) 2014-06-19

Family

ID=50903988

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/086542 Ceased WO2014090053A1 (zh) 2012-12-14 2013-11-05 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途

Country Status (6)

Country Link
US (1) US9580498B2 (zh)
EP (1) EP2933266B1 (zh)
CN (1) CN103864932B (zh)
ES (1) ES2719084T3 (zh)
HU (1) HUE042434T2 (zh)
WO (1) WO2014090053A1 (zh)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10713726B1 (en) 2013-01-13 2020-07-14 United Services Automobile Association (Usaa) Determining insurance policy modifications using informatic sensor data
US9947051B1 (en) 2013-08-16 2018-04-17 United Services Automobile Association Identifying and recommending insurance policy products/services using informatic sensor data
US11416941B1 (en) 2014-01-10 2022-08-16 United Services Automobile Association (Usaa) Electronic sensor management
US12100050B1 (en) 2014-01-10 2024-09-24 United Services Automobile Association (Usaa) Electronic sensor management
US11087404B1 (en) 2014-01-10 2021-08-10 United Services Automobile Association (Usaa) Electronic sensor management
US10552911B1 (en) 2014-01-10 2020-02-04 United Services Automobile Association (Usaa) Determining status of building modifications using informatics sensor data
US11847666B1 (en) 2014-02-24 2023-12-19 United Services Automobile Association (Usaa) Determining status of building modifications using informatics sensor data
US10614525B1 (en) 2014-03-05 2020-04-07 United Services Automobile Association (Usaa) Utilizing credit and informatic data for insurance underwriting purposes
WO2018195912A1 (zh) * 2017-04-28 2018-11-01 苏州思坦维生物技术股份有限公司 一种眼用药物组合物及其用途
CN108997497B (zh) 2018-03-30 2022-02-25 华兰基因工程有限公司 特异结合人质膜膜泡关联蛋白pv-1的单克隆抗体及其制备方法与应用
IL321888A (en) 2018-04-15 2025-09-01 Immvira Co Ltd Antibodies that bind PD-1 and their uses
CN109053892B (zh) 2018-09-19 2021-03-26 苏州思坦维生物技术股份有限公司 特异结合人及猴cd38抗原的单克隆抗体及其制备方法与应用
WO2025193962A1 (en) * 2024-03-15 2025-09-18 OncoC4, Inc. Anti-vegf and pd-1 bispecific antibodies and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6582959B2 (en) 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
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
CN101935349A (zh) * 2010-01-12 2011-01-05 江苏泰康生物医药有限公司 一种人源化抗人血管内皮生长因子单克隆抗体及其制备与应用
CN102276722A (zh) * 2011-01-14 2011-12-14 中国科学院北京基因组研究所 新型血管内皮生长因子人源化单克隆抗体

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100260668A1 (en) * 2008-04-29 2010-10-14 Abbott Laboratories Dual Variable Domain Immunoglobulins and Uses Thereof
US8834883B2 (en) * 2010-06-14 2014-09-16 Vaccinex, Inc. Anti-VEGF antibodies and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582959B2 (en) 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US7227004B2 (en) 1991-03-29 2007-06-05 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
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
CN101935349A (zh) * 2010-01-12 2011-01-05 江苏泰康生物医药有限公司 一种人源化抗人血管内皮生长因子单克隆抗体及其制备与应用
CN102276722A (zh) * 2011-01-14 2011-12-14 中国科学院北京基因组研究所 新型血管内皮生长因子人源化单克隆抗体

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BROWN LF, CANCER RES, vol. 53, 1993, pages 4727 - 35
CARMELIET P, NATURE, vol. 380, 1996, pages 435
DVORAK HF, J EXP MED, vol. 174, 1991, pages 1275 - 8
FERRARA N, NATURE, vol. 380, 1996, pages 439
HURWITZ H, N ENGL J MED, vol. 350, 2004, pages 2335
KABAT, NIH PUBL. NO. 91-3242, vol. 1, 1991, pages 647 - 669
KOHLER G.; MILSTEIN C, NATURE, vol. 256, 1975, pages 495 - 497
PRESTA LG, CANCER RES, vol. 57, 1997, pages 4593
WEIDNERN; SEMPLE JP; WELCH WR; FOLKMAN J, N ENGL J MED, vol. 324, 1991, pages 1 - 8.4,5

Also Published As

Publication number Publication date
HUE042434T2 (hu) 2019-07-29
EP2933266A1 (en) 2015-10-21
CN103864932A (zh) 2014-06-18
ES2719084T3 (es) 2019-07-08
EP2933266A4 (en) 2016-08-03
CN103864932B (zh) 2015-11-25
US20160039921A1 (en) 2016-02-11
EP2933266B1 (en) 2019-01-09
US9580498B2 (en) 2017-02-28

Similar Documents

Publication Publication Date Title
WO2014090053A1 (zh) 拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途
WO2015058573A1 (zh) 拮抗抑制程序性死亡受体pd-1与其配体结合的单克隆抗体及其编码序列与用途
WO2019098682A1 (ko) 항-her2 항체 또는 그의 항원 결합 단편, 및 이를 포함하는 키메라 항원 수용체
WO2016137108A1 (en) Novel antibody binding to tfpi and composition comprising the same
WO2017052241A1 (en) Novel anti-mesothelin antibody and composition comprising the same
WO2019225787A1 (ko) 항-b7-h3 항체 및 그 용도
WO2018194381A1 (ko) Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
WO2020080908A1 (ko) 항-l1cam 항체 또는 그의 항원결합 단편, 및 이를 포함하는 키메라 항원 수용체
WO2022177394A1 (ko) Pd-l1 및 cd47에 대한 이중특이적 단일 도메인 항체 및 이의 용도
WO2022039490A1 (en) Anti-b7-h4/anti-4-1bb bispecific antibodies and use thereof
WO2019112347A2 (ko) 악성 b 세포를 특이적으로 인지하는 항체 또는 그의 항원 결합 단편, 이를 포함하는 키메라 항원 수용체 및 이의 용도
WO2022035201A1 (ko) Il-12 및 항-fap 항체를 포함하는 융합단백질 및 이의 용도
WO2018026248A1 (ko) 프로그램화된 세포 사멸 단백질(pd-1)에 대한 신규 항체 및 이의 용도
WO2022169269A1 (ko) 항 ctla-4 항체 및 이의 용도
WO2023068818A1 (ko) 두개의 fc 도메인을 포함하는 항원 결합 단백질 및 이의 용도
WO2021020846A1 (en) Anti-her2/anti-4-1bb bispecific antibody and use thereof
WO2020251316A1 (ko) α-SYN/IGF1R에 대한 이중 특이 항체 및 그 용도
WO2020101365A1 (ko) 안정성이 향상된 항 c-met 항체 또는 그의 항원 결합 단편
WO2021020845A1 (en) Anti-egfr/anti-4-1bb bispecific antibody and use thereof
CN1326880C (zh) G－csf受体兴奋剂抗体及其筛选方法
WO2018026249A1 (ko) 프로그램화된 세포 사멸 단백질 리간드-1 (pd-l1)에 대한 항체 및 이의 용도
WO2022085905A1 (ko) SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 및 이의 용도
WO2023224412A1 (ko) Pd-l1 및 cd47에 대한 이중특이적 인간화 단일 도메인 항체 및 이의 용도
WO2023239228A1 (ko) 항-tigit 항체, 인터류킨-15, 및 인터류킨-15 수용체 알파 스시 도메인을 포함하는 융합 단백질 및 이의 용도
WO2022177392A1 (ko) Cd47에 대한 단일 도메인 항체 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13863651

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 14651785

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2013863651

Country of ref document: EP