WO2022012311A1 - 一种结合密蛋白的用于治疗癌症的人源化抗体 - Google Patents

一种结合密蛋白的用于治疗癌症的人源化抗体 Download PDF

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WO2022012311A1
WO2022012311A1 PCT/CN2021/102664 CN2021102664W WO2022012311A1 WO 2022012311 A1 WO2022012311 A1 WO 2022012311A1 CN 2021102664 W CN2021102664 W CN 2021102664W WO 2022012311 A1 WO2022012311 A1 WO 2022012311A1
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seq
amino acid
lcdr2
acid sequence
lcdr1
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French (fr)
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许铮
李响
刘影
熊国裕
李峰
史继峰
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北京凯因科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention relates to a humanized antibody for treating cancer that binds to claudin 18.2, and belongs to the technical field of antibody drugs.
  • Gastric cancer is one of the most common cancers worldwide and the second most common tumor in China. According to the 12th International Gastric Cancer Conference held in Beijing in April 2017, there are about 680,000 newly discovered cases of gastric cancer in China each year, accounting for about half of the global total. rate is over 13%.
  • the mortality rate of gastric cancer in China is 4 to 8 times that of developed countries in Europe and the United States, and about 1 Chinese person dies of gastric cancer every 2 to 3 minutes. Compared with other countries, the situation of gastric cancer in China is more severe.
  • Intercellular tight junctions are a transmembrane protein complex, and the stability of tight junctions requires the coordinated activities of several different proteins to maintain, and Claudin protein is the main protein that ensures the permeability of tight junctions with specificity. Twenty-seven members of the Claudin family have been found in mammals so far.
  • the Claudin protein family has a molecular weight of 20-27KD, and its structure includes four transmembrane regions, two extracellular loops and one intracellular loop, and its N-terminus and C-terminus are in the cytoplasm. Two extracellular loops make it an ideal antibody target.
  • Claudin protein is a skeletal protein that constitutes a tight junction structure. It is located on the top side of the adjacent intercellular space. Its distribution is tissue-organ-specific. Its functions are mainly intercellular adhesion, maintenance of cell polarity, regulation of paracellular permeability and participation in cell proliferation. , Differentiation regulation.
  • Claudin18 protein The molecular weight of Claudin18 protein is about 26KD, and Claudin protein can be transformed into Claudin isoforms with different properties through selective splicing: Claudin18.1 and Claudin18.2. Although there are only eight amino acid differences between the first ectodomains of Claudin18.1 and Claudin18.2, the expression distribution is different. Claudin18.1 is selectively expressed in the epithelium of normal lung and stomach, while Claudin18.2 is only expressed in the epithelium of normal lung and stomach.
  • Claudin18.2 is significantly upregulated in a variety of malignancies, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors , 30% of esophageal cancer and 25% of non-small cell lung cancer. In tumors, the tight junctions between cells are destroyed and Claudin18.2 cannot perform its normal function. Therefore, Claudin18.2 is a suitable tumor therapeutic target.
  • the first aspect of the present invention provides a humanized antibody that binds to Claudin18.2, the antibody includes a heavy chain hypervariable region (HCDR, or a heavy chain complementarity determining region) and a light chain hypervariable region (LCDR, or called light chain complementarity determining regions), the heavy chain hypervariable regions include:
  • HCDR1 whose amino acid sequence is GYX 1 FTNYG
  • HCDR2 whose amino acid sequence is INTNTGEP
  • HCDR3 whose amino acid sequence is ARLGFGNAMDY
  • the light chain hypervariable region includes:
  • LCDR1 whose amino acid sequence is QX 2 X 3 LNX 4 X 5 NX 6 KNY, LCDR2 whose amino acid sequence is WAX 7 , and LCDR3 whose amino acid sequence is QX 8 DYX 9 YPLT,
  • amino acid X 1 in the HCDR1 is selected from S or T;
  • LCDR1 the amino acid X 2 is selected from T or S, X 3 is selected from L or V, X4 is selected from T or S, X5 is selected from a G or S, X 6 is selected from Q or N;
  • the LCDR2 amino acid X 7 is selected from T or S;
  • amino acid X 8 is selected from N or Q, and X 9 is selected from T or S.
  • the antibody comprises HCDR and LCDR as described below,
  • the amino acid sequence is CDR1 (HCDR1) of GYSFTNYG (SEQ ID NO: 1) or GYTFTNYG (SEQ ID NO: 2);
  • the amino acid sequence is CDR2 (HCDR2) of INTNTGEP (SEQ ID NO: 3);
  • CDR3 (HCDR3) whose amino acid sequence is ARLGFGNAMDY (SEQ ID NO: 4);
  • amino acid sequences are QTLLNTGNQKNY(SEQ ID NO:5), QSVLNSGNQKNY(SEQ ID NO:6), QSLLNSSNNKNY(SEQ ID NO:7), QSVLNSSNNKNY(SEQ ID NO:8), QTLLNSGNQKNY(SEQ ID NO:9), QTLLNSGNNKNY( SEQ ID NO: 10), QTLLNSSNQKNY (SEQ ID NO: 11), QTLLNSSNNKNY (SEQ ID NO: 12), QSLLNSSNQKNY (SEQ ID NO: 13), QSLLNSGNNKNY (SEQ ID NO: 14), QSLLNSGNQKNY (SEQ ID NO: 15) ), CDR1 (LCDR1) of QSVLNSSNQKNY (SEQ ID NO: 16), QSVLNSGNNKNY (SEQ ID NO: 17) or QTVLNSGNNKNY (SEQ ID NO: 18);
  • the amino acid sequence is CDR2 (LCDR2) of WAT (SEQ ID NO: 19) or WAS (SEQ ID NO: 20);
  • the amino acid sequence is CDR3 (LCDR3) of QNDYTYPLT (SEQ ID NO:21), QQDYTYPLT (SEQ ID NO:22), QQDYSYPLT (SEQ ID NO:23) or QNDYSYPLT (SEQ ID NO:24).
  • the humanized antibody comprises the following combination of a heavy chain hypervariable region and a light chain hypervariable region:
  • HCDR1 of SEQ ID NO:17 HCDR2 of SEQ ID NO:32, HCDR3 of SEQ ID NO:54, and LCDR1 of SEQ ID NO:61, LCDR2 of SEQ ID NO:63, LCDR2 of SEQ ID NO:66 LCDR3.
  • an antibody having the following amino acid sequence having the following heavy chain variable region (VH) and light chain variable region (VL) sequences:
  • VH amino acid sequence shown QVQLVQSGX a1 ELKKPGASVKISCKASZ H1 MNWVRQAPGQGLKWMGZ H2 TYAEEFKGRFVFS LDTSVSTAYLQISSLKAEDTAVYX a2 CZ H3 WGQGTLVTVSS; and VL amino acid sequence is shown DIVMTQSPDSLAVSLGERATMNCKSSZ L1 LTWYQQKPGQPPKLLIYZ L2 T RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCZ L3 FGAGTKLEIK of
  • X a1 is P or S
  • X a2 is F or Y
  • Z H1 , Z H2 and Z H3 are respectively the three CDR regions of the heavy chain
  • Z L1 , Z L2 and Z L3 are the three CDR regions of the light chain
  • the amino acid sequence of Z H1 is GYX 1 FTNYG
  • the amino acid sequence of Z H2 is INTNTGEP
  • the amino acid sequence of Z H3 is ARLGFGNAMDY
  • the amino acid sequence of Z L1 is QX 2 X 3 LNX 4 X 5 NX 6 KNY
  • the amino acid sequence of Z L2 is is WAX 7
  • the amino acid sequence of Z L3 is QX 8 DYX 9 YPLT;
  • X 1 is selected from S or T;
  • the humanized antibody has the following VH and VL sequences,
  • amino acid sequence is the VH set forth in SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51, and
  • amino acid sequence is the VL set forth in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52.
  • Exemplary humanized antibodies have VH and VL sequences as described in any of the following groups:
  • the weight constant region is selected from human IgG series, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1;
  • the constant region of the light chain is selected from kappa or lambda chain, preferably kappa chain.
  • the antibody name and corresponding sequence of the present invention are as follows:
  • VH complementarity determining regions CDR1: GYTFTNYG, CDR2: INTNTGEP; CDR3: ARLGFGNAMDY
  • VH framework region FR1: QIQLVQSGPELKKPGETVKISCKAS; FR2: MNWVKQAPGKGLKWMGW; FR3: TYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFC; FR4: WGQGTSVTVSS
  • VL hypervariable regions/complementarity determining regions CDR1: QSLLNSGNQKNY; CDR2: WAS; CDR3: QNDYSYPLT.
  • VL framework regions FR1: DIVMTQSPSSLTVTAGEKVTMSCKSS; FR2: LTWYQQKPGQPPKLLIY; FR3: TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC; FR4: FGAGTKLELK.
  • the constant region of the HVR is selected from human IgG series, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1;
  • the constant region of the LVR is selected from the ⁇ or ⁇ chain, preferably kappa chain.
  • the third aspect of the present invention provides and a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, antioxidants (eg, ascorbic acid and sodium bisulfate), preservatives (eg, benzyl alcohol, methylparaben, ethylparaben, or n-propyl), emulsifiers , suspending agents, dispersing agents, solvents, fillers, bulking agents, buffers, carriers, diluents and/or adjuvants.
  • a suitable carrier may be physiological saline solution or citrate buffered saline, possibly supplemented with other materials commonly found in parenterally administered pharmaceutical compositions.
  • ZTE buffered saline or saline mixed with serum albumin are other exemplary carriers.
  • Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.
  • Buffer components also include water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.
  • compositions and can be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or freeze-dried powders. These compositions can be stored as ready-to-use forms, freeze-dried forms that require reconstitution before use, liquid forms that require dilution before use, or other useful forms.
  • the present invention also relates to an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; an expression vector containing the nucleic acid molecule of the present invention, and a host cell comprising the expression vector of the present invention , preferably eukaryotic cells.
  • the fourth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof that specifically binds to Claudin18.2, the method comprising expressing the antibody or its antigen-binding fragment of the present invention under conditions favorable for expression of the antibody or its antigen-binding fragment.
  • the nucleic acid molecule of the present invention is recovered, and the expressed antibody or antigen-binding fragment thereof is recovered.
  • the medium used to culture the cells can be any conventional medium used to culture the host cells, such as minimal medium or complex medium with appropriate supplements. Suitable media can be obtained commercially, or prepared according to published methods.
  • the polypeptide produced by the host cell can then be recovered from the culture medium by conventional methods, such as precipitating the protein fraction in the supernatant or filtrate with a salt such as ammonium sulfate, using various chromatographic methods depending on the type of peptide of interest, for example Exchange chromatography, gel filtration chromatography, affinity chromatography, etc. are used for further purification.
  • the above-described coding DNA sequences can be inserted into any suitable vector.
  • the choice of vector is often dependent on the host cell into which the vector is to be introduced, and thus, the vector may be an autonomously replicating vector, ie a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid.
  • the vector may be of a type that, when introduced into a host cell, will integrate into the host cell genome and replicate together with the chromosome into which it is integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to other segments required for transcription of the DNA, such as a promoter.
  • promoters suitable for directing the transcription of DNA encoding the peptides of the invention in a variety of host cells are well known in the art, see, eg, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989.
  • the vector may also contain a selectable marker, such as a gene whose gene product will complement a defect in the host cell, or confer resistance to drugs such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin , streptomycin or methotrexate resistance.
  • a selectable marker such as a gene whose gene product will complement a defect in the host cell, or confer resistance to drugs such as ampicillin, doxorubicin, tetracycline, chloramphenicol, neomycin , streptomycin or methotrexate resistance.
  • a secretory signal sequence (also referred to as a leader sequence) can be provided in the recombinant vector.
  • the secretion signal sequence is linked in correct reading frame to the DNA sequence encoding the peptide.
  • the secretion signal sequence is usually located 5' to the DNA sequence encoding the peptide.
  • the secretion signal sequence may be the secretion signal sequence normally linked to the peptide, or may be derived from a gene encoding another secreted protein.
  • Expression vectors for use in eukaryotic host cells will also contain sequences required to terminate transcription and stabilize mRNA.
  • eukaryotic host cells fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms
  • sequences required to terminate transcription and stabilize mRNA usually available from the 5' and (occasionally) 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the humanized antibody that binds Claudin18.2.
  • the host cell into which the DNA sequence or recombinant vector is to be introduced can be any cell capable of producing the peptides of the invention, including bacterial, viral, yeast, fungal and higher eukaryotic cells.
  • suitable hosts well known and used by those skilled in the art include, but are not limited to, viruses.
  • Antibodies or antigen-binding fragments of the invention can be recovered in various forms from culture medium or host cell lysates, and if membrane-bound, can be recovered using a suitable detergent solution (eg Triton-X 100) or by enzymatic cleavage. It is released from the membrane and the cells used to express the Claudin18.2 binding antibodies of the invention can be disrupted by various physical or chemical methods such as freeze-thaw cycling, sonication, mechanical disruption or cell lysis reagents.
  • a suitable detergent solution eg Triton-X 100
  • the following methods are examples of suitable purification methods: separation by fractionation on ion exchange columns; ethanol precipitation; reverse phase HPLC; Chromatography on cation exchange resins such as DEAE; chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using eg Sephadex G-75; Protein A Sepharose columns to remove contaminants such as IgG.
  • the present invention relates to the use of antibodies or antigen-binding fragments thereof in the preparation of medicaments for preventing or treating cancer-related diseases, especially gastric cancer.
  • the fifth aspect of the present invention provides a method for preventing or treating cancer, comprising administering the antibody that binds to Claudin18.2 of the present invention; wherein the cancer may include, for example, gastrointestinal cancer, pancreas cancer, esophageal cancer or non-small cell lung cancer; the gastrointestinal cancer is preferably advanced gastric cancer.
  • a sixth aspect of the present invention provides a product or kit comprising a container and a package insert, wherein the container contains the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition of the present invention , the package insert contains the instructions for use of the drug.
  • the article of manufacture or kit further comprises one or more containers containing one or more other drugs for preventing or treating cancer.
  • Suitable containers include, for example, ampoules, vials, syringes, and the like.
  • the container can be formed from a variety of substances (such as glass or plastic), the container holds or contains a small composition for treatment and can have a sterile access port (for example, the container can be an intravenous solution pack or have a hypodermic needle pierceable bottle with a cork).
  • At least one active agent in the composition is an antibody or antigen-binding fragment of the invention.
  • the label or package insert indicates that the composition is used to treat a metabolic-related disease, disorder, or symptom in an individual suffering from a metabolic-related disease, disorder, or symptom under specific guidance regarding the time interval between the dosages of the antibody and any other drug provided. .
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable dilution buffer, plus bacteriostatic water for injection, phosphate buffered saline, Ringer's solution and dextrose solution.
  • the article of manufacture may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • Use "package inserts" typically include instructions in commercial packaging of therapeutic products that contain warnings about instructions, usage, dosage, administration, contraindications, other therapeutic products in combination with the packaged product and/or warnings about the use of these therapeutic products etc. information.
  • the article of manufacture may also include other components, each component of the article of manufacture may be packaged in a single container and all multiple containers may be placed in a single package.
  • an “antibody” as described herein is an immunoglobulin molecule consisting of four peptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, each heavy chain comprising a heavy chain variable region ( HVR or VH) and a heavy chain constant region, the heavy chain constant region includes three domains of CH1, CH2 and CH3, each light chain includes a light chain variable region (LVR or VL) and a light chain constant region, the light chain constant region Comprising a single structural domain (CL1), the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs), each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the heavy chain constant region may be selected from IgGl, IgG2, IgG
  • variable region is used herein to describe the regions of certain parts of an antibody that differ between antibody sequences and are involved in the binding and specificity of a particular antibody for its particular antigen.
  • variability is usually not evenly distributed throughout the variable region of an antibody. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions.
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each comprise four FRs, mostly adopting a ⁇ -sheet conformation, connected by three CDRs that form loops linking and in some cases forming part of the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity.
  • CDRs complementarity determining regions
  • hypervariable regions or complementarity determining regions (CDRs) present in the variable regions of antibody light or heavy chains
  • CDRs complementarity determining regions
  • hypervariable regions as defined by Kabat et al. ("Sequences of Proteins of Immunological Interest,” Kabat E., et al., US Sept. of Health and Human Services, 1983) or hypervariable loops in the three-dimensional structure of antibodies (Chothia and Lesk, J Mol. Biol. 196901 -917 (1987)).
  • the CDRs in each chain are held in close proximity by the framework regions, and together with CDRs from other chains facilitate the formation of the antigen binding site.
  • frame region and "FR” refer to one or more of the framework regions within the variable regions of antibody light and heavy chains (see, Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)) . These terms include those amino acid sequences in antibody light and heavy chains that are located between the amino terminus and the first CDR, those between the CDRs, and those between the third CDR and the start of the constant region.
  • CDR and FR residues can be defined according to standard sequence definitions (Kabat et al., Sequences of Proteins of Immunological Interes, National Institutes of Health, Bethesda Md. (1987)) and structural definitions (Chothia and Lesk, J. Mot. Biol. 196:901-217 (1987)). Sure.
  • Kabat utilizes a method of assigning a residue number to each amino acid in the listed sequence, And this method of assigning residue numbering has become standard in the art.
  • Kabat numbering is explicitly indicated, this specification follows the Kabat numbering scheme.
  • sequential amino acid sequence numbering is used (i.e., the amino acids in the sequence use Sequential integers (1, 2, 3, etc.) are numbered from left to right in the amino-terminal to carboxy-terminal direction.
  • the "affinity" of an antibody for an antigen or epitope is a term well understood in the art and refers to the degree or strength of binding of an antibody to an epitope. Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (KD or Kd, which can be defined as the ratio of the off-rate to on-rate of the antibody, i.e., K off / K on ), apparent equilibrium dissociation constant (KD' or Kd'), EC50 (amount required to achieve 50% binding to antigen) and IC50 (amount required to achieve 50% inhibition in a competition assay); human
  • KD or Kd equilibrium dissociation constant
  • KD' or Kd' apparent equilibrium dissociation constant
  • EC50 amount required to achieve 50% binding to antigen
  • IC50 amount required to achieve 50% inhibition in a competition assay
  • human The relative affinities of IgG antibodies can also be determined by comparison to, for example, related murine
  • affinity is the average affinity of a particular population of antibodies that bind an antigen or epitope.
  • the affinity of the antibody can be measured using enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS) assay.
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • the ELISA method is used to determine the binding affinity of the antibody of the present invention to Claudin18.2. described.
  • Antibodies of the invention can be produced by any available method, such as recombinant expression techniques. Nucleic acids encoding light and heavy chain variable regions operably linked to constant regions can be inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain can be operably linked to control sequences of the expression vector that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. In one embodiment, the expression control sequence is a prokaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. Once the vector is introduced into the appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the antibody.
  • promoters eg, naturally associated or heterologous promoters
  • signal sequences
  • Expression vectors can be replicable in any host organism, either as episomes or as an integral part of the host chromosomal DNA.
  • the expression vector contains a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence.
  • Expression vectors can be used to express the antibodies of the invention from any host cell, including prokaryotic host cells (eg, E. coli), yeast host cells, mammalian host cells, plant host cells, and insect host cells.
  • E. coli is used to produce the antibodies of the invention.
  • Other prokaryotic hosts suitable for this application include bacilli, such as Bacillus, and other Enterobacteriaceae, such as Salmonella, Saierella, and various Pseudomonas species.
  • expression vectors can also be prepared, which typically contain expression control sequences (eg, origins of replication) that are compatible with the host cell.
  • expression control sequences eg, origins of replication
  • promoters such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from lambda phage. Promoters typically control expression, optionally together with operator sequences, and have ribosome binding site sequences, etc., for initiating and completing transcription and translation.
  • yeast can also be used to express the antibodies of the invention.
  • Saccharomyces spp. can be used as yeast hosts with suitable promoters containing expression control sequences (eg, promoters), origins of replication, termination sequences, and other sequences as desired.
  • Promoters used in yeast expression techniques include 3-phosphoglycerate kinase and other glycolytic enzyme promoters.
  • Inducible yeast promoters include, but are not limited to, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • mammalian tissue cell cultures can be used to express and produce the antibodies of the invention.
  • Any mammalian tissue cell can be used for such methods, and many suitable host cell lines capable of secreting heterologous proteins (eg, intact immunoglobulins) have been developed in the art, including mammalian BHK or CHO cell lines, various Cos Cell lines, HeLa cell lines, preferably myeloma cell lines or transformed B cells or hybridomas.
  • the cells are non-human.
  • Expression vectors for mammalian cells can include expression control sequences, such as origins of replication, promoters, and enhancers, as well as necessary processing signal sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequence.
  • the expression control sequence is a promoter derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like.
  • Vectors containing polynucleotide sequences of interest can be transferred to host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, bioballistics or viral transfection can be used in other cellular hosts. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, transgenes can be microinjected into fertilized eggs, or they can be introduced into the genome of embryonic stem cells whose nuclei are transferred into enucleated oocytes.
  • transgenes can be microinjected into fertilized eggs, or they can be introduced into the genome of embryonic stem cells whose nuclei are transferred into enucleated oocytes.
  • the vectors can be co-transfected to express and assemble the complete immunoglobulin.
  • the intact antibody, its dimers, individual light and heavy chains, or other immunoglobulin forms of the antibody can be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity column, column chromatography, HPLC purification , gel electrophoresis, etc.
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity can be prepared for pharmaceutical use.
  • substantially pure humanized antibodies of at least about 98 to 99% or greater homogeneity can be produced for use in pharmaceutical formulation and methods.
  • the present invention provides a method of expressing an antibody of the present invention, comprising: (a) transforming a host cell with a nucleic acid molecule encoding the antibody described herein, and (b) culturing the transformed host cell under conditions permitting expression of the antibody.
  • a method of expressing an antibody of the present invention comprising: (a) transforming a host cell with a nucleic acid molecule encoding the antibody described herein, and (b) culturing the transformed host cell under conditions permitting expression of the antibody.
  • Known techniques for including a selectable marker on a vector can be used so that the labeled host cells expressing the antibody can be readily selected.
  • an "antigen-binding fragment" of an antibody or referred to as an “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, Claudin 18.2). It has been demonstrated that the antigen-binding function of antibodies can be accomplished by certain fragments of full-length antibodies.
  • antigen-binding fragments include, but are not limited to: (i) Fab fragments, ie, monovalent fragments consisting of the VL, VH, CL1 and CH1 domains; (ii) F(ab')2 fragments, ie, consisting of hinge A bivalent fragment composed of two F(ab') fragments linked by disulfide bonds in the region; (iii) an Fd fragment composed of VH and CH1 domains; (iv) a one-armed VL and VH domain composed of Fv fragments; (v) dAb fragments consisting of VH domains; and (vi) CDRs.
  • the two domains of the Fv fragment, VL and VH are encoded by different genes, they can be joined together by recombinant methods by a synthetic linker into a single linked chain in which the VL and VH domains are paired Monovalent molecules (called single-chain Fv, scFv) are formed.
  • single chain Fv Single-chain Fv
  • scFv Monovalent molecules
  • the antibodies of the invention can be prepared by a method comprising culturing a host cell containing a DNA sequence encoding the antibody and capable of expressing the antibody under conditions that permit expression of the antibody, and recovering the antibody produced from the culture .
  • the medium used to culture the cells can be any conventional medium used to culture the host cells, such as minimal medium or compatible medium with appropriate supplements. Suitable media can be obtained commercially, or prepared according to published methods.
  • the polypeptides produced by the cells can then be recovered from the culture medium by conventional methods including isolating the host cells from the culture medium by centrifugation or filtration, and precipitating the protein components in the supernatant or filtrate with salts such as ammonium sulfate, according to Various chromatographic methods such as exchange chromatography, gel filtration chromatography, affinity chromatography, etc. are used for purification according to the type of the target peptide.
  • the present invention provides a pharmaceutical composition comprising the Claudin18.2-binding antibody or antigen-binding fragment thereof of the present invention.
  • the pharmaceutical compositions of the present invention will be administered with appropriate carriers, excipients and other formulations. These formulations are formulated to improve delivery and tolerability, among others. A large number of suitable formulations can be found in the pharmacopoeia well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA).
  • the administered dose is adjusted according to the age and body size of the subject, target disease, symptoms, administration route, and the like.
  • the antibody of the present invention can be administered intravenously, usually at a single dose of about 0.01 to about 20 mg/kg per kilogram of body weight, more preferably is about 0.1 to about 15, about 1 to about 10, or about 3 to about 10 mg/kg body weight.
  • the frequency and duration of treatment can be adjusted.
  • compositions of the present invention such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis effect (see eg Wu et al. (1987), J. Biol. Chem. 262:4429-4432).
  • Methods of drug administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, dural, and oral routes.
  • the pharmaceutical composition may be administered by any convenient route, such as by infusion or intravenous bolus, absorption from epithelia and mucosal layers (eg, oral mucosa, rectal and small intestinal mucosa), and may be administered with other biologically active agents.
  • the mode of administration can be systemic or local.
  • the pharmaceutical composition can also be delivered by vesicles, especially liposome vesicles (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327).
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump can be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials may be employed (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974). For a discussion of other controlled release systems see Langer (1974). 1990) Science 249: 1527-1533.
  • injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, instillation, and the like. These injectable formulations can be prepared by published methods. For example, preparations for injection can be prepared by dissolving, suspending or emulsifying the antibody or salt thereof in sterile aqueous or oily vehicles conventionally used for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like.
  • solubilizers such as alcohols (eg ethanol), polyols (eg propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80, HCO-50 (polyoxygenated hydrogenated castor oil) Ethylene (50 mol) adduct) and the like are used in combination.
  • the oil medium used is, for example, sesame oil, soybean oil and the like. They can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, and the like.
  • the injections thus prepared are preferably enclosed in suitable ampoules.
  • the antibodies and antibody fragments of the present invention can be improved for therapy by disrupting the tight junctions between cells so that Claudin 18.2 cannot perform its normal function.
  • Combination therapy is encompassed by the present invention, wherein an antibody or antibody fragment that binds Claudin 18.2 is administered in combination with one or more therapeutic agents (or a second therapeutic agent).
  • Co-administration and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which the anti-Claudin 18.2 antibody or antibody fragment is administered at least once in a course of treatment involving the administration of at least one other therapeutic agent to the patient.
  • the second therapeutic agent may be another gastric cancer (eg, gastric cancer) drug.
  • the present invention also includes the use of any anti-Claudin18.2 antibody or antigen-binding fragment described herein in the preparation of a medicament for the treatment of diseases or symptoms, wherein the diseases or symptoms are caused by breaking the tight junctions between cells, so that Claudin18.2 cannot function its normal function.
  • a pharmaceutical composition which comprises the antibody or antigen-binding fragment thereof of the present invention that binds to Claudin18.2, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable excipients refer to nontoxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients.
  • diluents such as microcrystalline cellulose, mannitol, etc.
  • fillers such as starch, sucrose, etc.
  • binders such as starch, cellulose derivatives, alginates, gelatin, and/or polyethylene Pyrrolidone
  • disintegrants such as calcium carbonate and/or sodium bicarbonate
  • absorption enhancers such as quaternary ammonium compounds
  • surfactants such as cetyl alcohol
  • carriers, solvents such as water, physiological saline, kaolin, bentonite, etc.
  • Lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc.
  • the pharmaceutical composition of the present invention is preferably an injection.
  • the antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is present at a concentration of 1 mg/ml to 1000 mg/ml, preferably at a concentration of 10 mg/ml to 1000 mg/ml, more preferably at a concentration of 1 mg/ml to 1000 mg/ml It is present at a concentration of 50 mg/ml to 500 mg/ml, more preferably at a concentration of 100 mg/ml to 300 mg/ml.
  • the pharmaceutical composition of the present invention preferably has a pH of 3.0 to 9.0. Among them, buffer systems, preservatives, surface tension agents, chelating agents, stabilizers and surfactants may be further included.
  • the pharmaceutical composition of the present invention is an aqueous formulation. Such formulations are usually solutions or suspensions.
  • the pharmaceutical composition is a stable aqueous solution.
  • the pharmaceutical composition is a lyophilized formulation, and the physician or patient adds a solvent and/or a diluent to dissolve the lyophilized formulation before use.
  • Figure 1 is a graph showing the binding activity of anti-Claudin18.2 antibody screening-cell ELISA.
  • Figure 2 is a graph showing anti-Claudin18.2 antibody screening-proliferation inhibitory activity.
  • Figure 3 is a graph of anti-Claudin18.2 antibody screening-antibody-dependent cytotoxicity.
  • Figure 4 is a graph of anti-Claudin18.2 antibody screening-complement-dependent cytotoxicity.
  • Figure 5 is a graph showing the effect of the candidate antibodies on the relative tumor volume (RTV) of the KATO cell nude mouse xenograft model.
  • Figure 6 (6-1, 6-2) is a graph showing the effect of candidate antibodies on the relative tumor volume (RTV) of a NUGC cell nude mouse xenograft model.
  • the pcDNA3.1 vector encoding human Claudin18.1 and Claudin18.2 antigens was transfected into HEK293 cells (purchased from ATCC), and 200 ⁇ g/mL Geneticin was used as the screening pressure to obtain stable expression of Claudin18.1 and Claudin18.1 and HEK293 cells with Claudin18.2 antigen.
  • the Claudin18.2 antibody IMAB362 of Ganymed self-made, transiently expressed in CHO-S cells, and further purified by chromatography, refer to Examples 2 and 3, BY0-0 in the present invention) was used as the positive antibody, and the stability was screened by FACS method.
  • HEK293 cells expressing human Claudin18.1 and Claudin18.2 antigens purchased from Invitrogen
  • the HindIII restriction site ( AAGCTT ), KoZAK sequence ( GCCGCCACC ), ATG, signal peptide gene GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and the heavy chain coding gene of the antibody (including the heavy chain variable region coding gene SEQ ID NO: 59 and the constant region IgG1 coding gene SEQ ID NO: 59) ID NO: 63), the termination code TAA and the EcoRI encoding gene GAATTC were fused in series in sequence, and the gene fragment was obtained by chemical synthesis.
  • the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.4(+) (purchased from Invitrogen) through the EcoRI and HindIII sites and verified by sequencing to obtain the expression plasmid pCDNA3.4(+)-BY6-4 of the antibody heavy chain .
  • the HindIII restriction site ( AAGCTT ), KoZAK sequence ( GCCGCCACC ), ATG, signal peptide gene GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT and the light chain encoding gene of the antibody (including the light chain variable region encoding gene SEQ ID NO: 60 and the constant region kappa encoding gene SEQ ID NO: 60) ID NO: 64), the termination code TAA and the EcoRI encoding gene GAATTC were fused in series in sequence, and the gene fragment was obtained by chemical synthesis.
  • the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.4(+) through the EcoRI and HindIII sites and sequenced for verification to obtain the expression plasmid pCDNA3.4(+)-BY6-4 of the antibody light chain.
  • the heavy chain constant region of each candidate antibody is IgG1, and the light chain constant region is ⁇ .
  • each instantaneous go CHO-S cells (Invitrogen Corp.), expressing the antibody according to Manual CHO-S cells (Freedom TM CHO-S TM KitUSER GUIDE), the plasmid one day cell density was adjusted to 1x10 6 cells / ml before transfection.
  • EXPICHO EXPRESSION MEDIUM cell culture medium purchased from Invitrogen
  • EXPICHO EXPRESSION MEDIUM cell culture medium purchased from Invitrogen
  • EXPICHO EXPRESSION MEDIUM cell culture medium purchased from Invitrogen
  • collect cell fluid remove cells by centrifugation, 0.2
  • protein A affinity chromatography was used for purification, and the pH of the collected samples was adjusted to 5.5 and stored at 2-8 °C.
  • the purified antibody was detected by SDS PAGE and SEC, and the purity was above 95%.
  • the HEK293 cells stably transfected with Claudin18.2 and Claudin18.1 were coated by ELISA to screen the antibody by ELISA, and the double antibody sandwich method was used for detection (at 0.1mg/ 96-well cell culture plates were coated with mL of polylysine. After 5 min, the coating solution was removed, and 5 ⁇ 10 4 /well HEK293-Claudin18.2 or HEK293-Claudin18.1 cells were added.
  • the cells were incubated at 37°C, 5% After culturing in a CO 2 incubator for 24 h to adhere, remove the supernatant, add PBST to each well to wash the cells 3 times, add 100 ⁇ l/well of gradient dilution antibody solution, 37 °C for 2 hours, wash the plate 3 times with washing solution; Use washing solution to prepare 2% bovine serum albumin solution) 1/5000 dilution of HRP-labeled secondary antibody (number ab6858, the product comes from Abcam company), add 100 ⁇ l/well to the ELISA plate, 37 °C for 1 hour; use washing solution Wash the plate 3 times; add 100 ⁇ l/well of TMB chromogenic solution, leave the reaction at room temperature for 5-10 minutes, add 50 ⁇ l/well of stop solution to stop the reaction, and measure the absorbance at a wavelength of 450 nm), measure each candidate
  • the relative binding activity of each candidate antibody was calculated based on the EC50 value of the antibody and Claudin18.2, and
  • the cell ELISA binding activity results showed that the specificity of the candidate antibody was good, and it did not bind to Claudin18.1, but had significant differences in binding activity to Claudin18.2, among which BY6-1, BY6-2, BY6-3, BY6-4 , BY6-6, BY6-7, BY6-8, BY6-9, BY6-13 and BY6-19 had better binding activity to Claudin18.2, while BY5-2, BY5-3, BY5-4, BY5-6 , BY5-7, BY5-8, BY6-10, BY6-12, BY6-14, BY6-17, BY6-18 had poor binding activity to Claudin18.2.
  • Anti-Claudin18.2 antibodies mediate four independent, highly potent mechanisms of action to induce tumor cell killing and apoptosis: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of Target crosslinking-induced apoptosis and direct inhibition of proliferation.
  • the second round of antibody screening is to screen 7 antibodies with higher affinity (BY6-1, BY6-2, BY6-3, BY6-4, BY6-6, BY6-7, and BY6-9) for further functional (ADCC, CDC, proliferation inhibition) identification on cell models to provide a basis for the determination of clinical drug candidates.
  • KATO III cells purchased from ATCC
  • CCK8 method The proliferation inhibition effect of anti-Claudin18.2 antibody on KATO III cells was studied by CCK8 method: KATO III cells grown in log phase were taken, and the cell concentration was adjusted to 2 with IMDM (purchased from Invitrogen) + 10% FBS medium. ⁇ 10 5 /ml, 37°C, 5% CO2 incubator for use.
  • sample and reference substance (Ch163, or BY0-0) were pre-diluted with IMDM+5% FBS medium to a final concentration of 400 ⁇ g/ml, and then diluted twice, with a total of 14 gradients: 400ug/ml, 200 ⁇ g/ml, 100 ⁇ g/ml, 50 ⁇ g/ml, 25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 ⁇ g/ml, 3.125 ⁇ g/ml, 1.56 ⁇ g/ml, 0.78 ⁇ g/ml, 0.39 ⁇ g/ml, 0.19 ⁇ g/ml, 0.097 ⁇ g/ ml, 0 ⁇ g/ml.
  • the results of the growth inhibitory activity showed that the antibodies of the candidate antibodies BY6-2, BY6-3, and BY6-4 had obvious inhibitory effects on cell proliferation in vitro.
  • ADCC Antibody Dependent Cytotoxicity
  • HEK18.2 cells Using stably transfected HEK18.2 cells as target cells, and PBMC cells isolated from fresh human peripheral blood as effector cells, the ADCC activity of the antibody to be tested was detected. Trypsin-digested NUGC cells (purchased from JCRB) and HEK18.2 cells were used as target cells to detect 7 kinds of antibodies, 5000 cells per well.
  • PBMCs were extracted from 30 ml of blood from 2 healthy volunteers, and cultured overnight with RPMI-1640 (purchased from Invitrogen) + 10% FBS, and 2.5 ⁇ 10 5 effector cells were used per well.
  • the initial concentration of the test drug was 10 ⁇ g/ml (diluted to 40 ⁇ g/ml before use), and it was diluted to 8 samples by a 5-fold dilution method.
  • RPMI-1640+2% FBS (inactivated serum) was used as the experimental medium for ADCC.
  • ESR ESR well absorbance-CMB well absorbance
  • TSR TSR well absorbance - CMB well absorbance
  • TMR TMR well absorbance - VCC well absorbance
  • Cytotoxicity (%) (ER-ESR-TSR)/(TMR-TSR) ⁇ 100%)
  • the amount of dead cells and damaged cells was detected by Cytotoxicity LDH Assay Kit-WST kit (purchased from Nippon Dojin Chemical). The kit measures cell damage by measuring the lactate dehydrogenase (LDH) activity released by cells into the culture medium. The test results are shown in Figure 3.
  • the ADCC activity results showed that the antibodies of the candidate antibodies BY6-1, BY6-2, BY6-3 and BY6-4 had obvious inhibitory effect on ADCC activity in vitro.
  • the CDC effect of anti-Claudin18.2 antibody, human serum and HEK8.2 cells was detected by CCK8 method.
  • Serum for complement lysis assay was obtained from healthy volunteers, centrifuged at 600g for 20min, and the serum was harvested and stored at -20°C.
  • HEK18.2 cells were fully cultured with RPMI-1640 at 37°C, 5% CO 2 to logarithmic growth phase.
  • HEK18.2 cells were digested and washed twice with serum-free RPMI-1640 medium. Cell counts, and formulated into a cell suspension containing 5 ⁇ 10 5 / ml with serum-free RPMI-1640 medium.
  • the screening antibody was routinely diluted 1000 ⁇ g/mL (with RPMI-1640 medium), and the 5-fold gradient was continuously diluted (1000, 300, 100, 30, 10 ⁇ g/mL); the positive control antibody Rituxan (purchased from Roche) was routinely diluted 500 ⁇ g/mL ( With RPMI-1640 medium), 5-fold serial dilution (500, 100, 20, 4 ⁇ g/mL).
  • the volume of HEK18.2 cells suspended in RPMI-1640 medium was 85 ⁇ L, the number of cells was 5.0 ⁇ 10 4 /well, 5 ⁇ L of human serum (complement) (1:30 dilution); 10 ⁇ L of antibodies with different concentrations (For positive control, add RPMI-1640 medium for antibody dilution, and set up cell-free medium control wells); the final concentration of screening antibody is (100, 30, 10, 3, 1 ⁇ g/mL), and the final concentration of Rituxan mAb is The concentrations were (50, 10, 2, 0.4 ⁇ g/mL).
  • the above-mentioned 96-well cell culture plate was placed in 5% CO 2 , incubated at 37°C for 4 hours, and 10 microliters of CCK8 was added. After 4 hours, detect the absorbance: put it into a microplate reader and read: the wavelength is 450nm;
  • KATO human gastric cancer
  • NUGC cell lines purchased from: JCRB
  • Claudin18.2 a subcutaneously transplanted tumor model in nude mice (purchased from: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) block inoculation.
  • BY0-0, BY6-1, BY6-2, BY6-4 were administered at a dose of 10 mg/kg/beast, and the negative control group was given PBS, twice a week on Monday and Thursday for 4 weeks. for 7 consecutive times, counted as the first day of dosing D0, twice weekly tumor growth in mice detecting (measuring mouse weight, caliper measurements of tumor length volume was calculated (tumor volume of the control group to more than 1000mm 3 end of the experiment)) .
  • Tumor tumor weight inhibition rate (%) (tumor tumor weight in control group - tumor tumor weight in administration group)/tumor tumor weight in control group ⁇ 100%
  • test results are shown in Tables 3-1, 3-2, Figures 5-1, 5-2 and 6-1/6-2.
  • mice xenograft models of two human gastric cancer cell lines showed that the inhibitory effect of the candidate antibodies on the growth of human gastric cancer KATO and NUGC cells in nude mice xenografts was positively correlated with the dose. It has obvious inhibitory effect on the growth of human gastric cancer KATO and NUGC cell xenograft tumor in nude mice within a certain dose and administration time.
  • BY6-1, BY6-2 and BY6-4, BY6-4 had the best effect in the nude mice xenografts of the two gastric cancer cells.
  • the zero-point detection results showed that the monomer purity of CHO-S expression and one-step affinity purity was higher than 98% in SEC detection, and the main peak content in IEX detection was about 70%. There was no significant difference between the 8 antibodies, and the detection after carboxypeptidase digestion , the modification ratio of lysine is different. Except for BY6-15, the lysine modification ratio of 7 antibodies is 3%-5%, and the lysine modification ratio of BY6-15 is about 10%.
  • the SDS-PAGE results show the molecular weight of the antibodies. Normal, the light and heavy chains are intact, there is no significant difference between the 8 antibodies, and the relative binding activity of Claudin18.2 is relatively different. BY6-1, BY6-2, BY6-3 and BY6-4 have higher activities than BY0-0 The activities of BY6-6, BY6-15 and BY6-19 were lower than that of BY0-0, but were consistent with the relative activities in the antibody screening stage.
  • the purity of the main peak of BY6-3 decreased significantly by IEX, about 20%, and the purity of the main peak of BY6-1/2 decreased by 10%.
  • the relative binding activity of BY6-6/16 and BY6-19 was low, and there was no significant difference in other.

Abstract

一种结合密蛋白18.2的用于治疗癌症的人源化抗体，所述抗体与Claudin18.1均不结合，但是与Claudin18.2选择性结合，破坏细胞间的紧密连接，使Claudin18.2无法发挥其正常功能，可用于预防或***，尤其是晚期胃癌。

Description

一种结合密蛋白的用于治疗癌症的人源化抗体 技术领域
本发明涉及一种结合密蛋白18.2的用于治疗癌症的人源化抗体，属于抗体药物技术领域。
背景技术
胃癌在全世界范围内是发病率最高的癌症之一，是中国的第二大常见肿瘤。据2017年4月在北京召开的第12届国际胃癌大会上披露，中国胃癌每年新发现病例约为68万例，占全球发病总数的一半左右，同比2012年公布的44.65万例，年平均增长率超过了13％。中国胃癌死亡率是欧美发达国家的4～8倍，约每2～3分钟就有1名中国人死于胃癌。相比其他国家，中国的胃癌形势更加严峻。
中国大部分胃癌患者确诊时，病情已经进入中晚期，手术后效果极不理想，且预后极差，是临床上极为棘手的恶性肿瘤。
细胞间紧密连接(tight junction，TJs)是一种跨膜蛋白复合体，紧密连接的稳定需要几种不同蛋白的协调活动来维持，而Claudin蛋白是保证紧密连接渗透性具有特异性的主要蛋白。迄今在哺乳动物中已发现27个Claudin家族成员。Claudin蛋白家族分子量为20～27KD，结构中包括4个跨膜区域、两个细胞外环和一个细胞内环，其N端和C末端在胞浆内。两个细胞外环使其成为理想的抗体靶点。Claudin蛋白是构成紧密连接结构的骨架蛋白，位于相临细胞间隙顶侧，其分布具有组织器官特异性，功能主要为细胞间粘附、维持细胞极性、调节细胞旁通透性及参与细胞增殖、分化调节。
Claudin18蛋白分子量约为26KD，可以通过选择性剪切使Claudin蛋白变成具有不同特性的Claudin亚型：Claudin18.1和Claudin18.2。Claudin18.1和Claudin18.2的第一胞外结构域之间虽然只有八个氨基酸的差异，但表达分布却不同，Claudin18.1在正常肺和胃的上皮中选择性表达，Claudin18.2只在短暂分化的胃上皮细胞上表达，在任何其他正常人器官中完全检测不到，但是Claudin18.2在多种恶性肿瘤中有显著上调，包括80％的胃肠道腺瘤、60％的胰腺肿瘤、30％食道癌以及25％非小细胞肺癌。在肿瘤中，细胞间的紧密连接遭到破坏，Claudin18.2无法发挥其正常功能。因此，Claudin18.2是一个合适的肿瘤治疗靶标。
发明内容
本发明的第一方面，提供一种结合Claudin18.2人源化抗体，所述抗体包括重链高变区(HCDR，或称重链互补决定区)和轻链高变区(LCDR，或称轻链互补决定区)，所述重链高变区包括：
氨基酸序列为GYX 1FTNYG的HCDR1，氨基酸序列为INTNTGEP的HCDR2，以及氨基酸序列为ARLGFGNAMDY的HCDR3；
所述轻链高变区包括：
氨基酸序列为QX 2X 3LNX 4X 5NX 6KNY的LCDR1，氨基酸序列为WAX 7的LCDR2，以及氨基酸序列为QX 8DYX 9YPLT的LCDR3，
其中所述HCDR1中氨基酸X 1选自S或T；
所述LCDR1中氨基酸X 2选自T或S，X 3选自L或V，X4选自T或S，所述X5选自G或S，X 6选自Q或N；
所述LCDR2中氨基酸X 7选自T或S；
所述LCDR3中氨基酸X 8选自N或Q，X 9选自T或S。
一个优选的实施方案中，所述抗体包含如下所述的HCDR及LCDR，
氨基酸序列为GYSFTNYG(SEQ ID NO：1)或GYTFTNYG(SEQ ID NO：2)的CDR1(HCDR1)；
氨基酸序列为INTNTGEP(SEQ ID NO：3)的CDR2(HCDR2)；
氨基酸序列为ARLGFGNAMDY(SEQ ID NO：4)的CDR3(HCDR3)；以及
氨基酸序列为QTLLNTGNQKNY(SEQ ID NO:5)、QSVLNSGNQKNY(SEQ ID NO:6)、QSLLNSSNNKNY(SEQ ID NO:7)、QSVLNSSNNKNY(SEQ ID NO:8)、QTLLNSGNQKNY(SEQ ID NO:9)、QTLLNSGNNKNY(SEQ ID NO:10)、QTLLNSSNQKNY(SEQ ID NO:11)、QTLLNSSNNKNY(SEQ ID NO:12)、QSLLNSSNQKNY(SEQ ID NO:13)、QSLLNSGNNKNY(SEQ ID NO:14)、QSLLNSGNQKNY(SEQ ID NO:15)、QSVLNSSNQKNY(SEQ ID NO:16)、QSVLNSGNNKNY(SEQ ID NO:17)或QTVLNSGNNKNY(SEQ ID NO:18)的CDR1(LCDR1)；
氨基酸序列为WAT(SEQ ID NO：19)或WAS(SEQ ID NO：20)的CDR2(LCDR2)；
氨基酸序列为QNDYTYPLT(SEQ ID NO:21)、QQDYTYPLT(SEQ ID NO:22)、QQDYSYPLT(SEQ ID NO:23)或QNDYSYPLT(SEQ ID NO:24)的CDR3(LCDR3)。
本发明一个更为优选的实施方案中，所述的人源化抗体，其包含如下组合的重链高变区和轻链高变区：
(1)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:5的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；或
(2)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:6的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:22的LCDR3；或
(3)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；或
(4)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:8的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；或
(5)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:9的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(6)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:5的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(7)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:9的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；或
(8)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:10的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(9)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:11的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(10)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:12的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(11)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID  NO:6的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(12)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:13的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(13)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:14的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(14)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:15的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；或
(15)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(16)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:13的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；或
(17)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:16的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(18)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:14的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；或
(19)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:17的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(20)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:8的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；或
(21)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；或
(22)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:18的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
(23)SEQ ID NO:17的HCDR1、SEQ ID NO:32的HCDR2、SEQ ID NO:54的HCDR3，和SEQ ID NO:61的LCDR1、SEQ ID NO:63的LCDR2、SEQ ID NO:66的LCDR3。
本发明的第二方面，提供了具有如下所述氨基酸序列的抗体，所述抗体具有如下的重链可变区(VH)和轻链可变区(VL)序列：
氨基酸序列为QVQLVQSGX a1ELKKPGASVKISCKASZ H1MNWVRQAPGQGLKWMGZ H2TYAEEFKGRFVFS LDTSVSTAYLQISSLKAEDTAVYX a2CZ H3WGQGTLVTVSS所示的VH；以及氨基酸序列为DIVMTQSPDSLAVSLGERATMNCKSSZ L1LTWYQQKPGQPPKLLIYZ L2T RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCZ L3FGAGTKLEIK所示的VL，
其中X a1为P或S，X a2为F或Y，Z H1、Z H2及Z H3分别为重链的三个CDR区，Z L1、Z L2及Z L3为轻链的三个CDR区，
其中Z H1的氨基酸序列为GYX 1FTNYG，Z H2的氨基酸序列为INTNTGEP，Z H3的氨基酸序列为ARLGFGNAMDY；Z L1的氨基酸序列为QX 2X 3LNX 4X 5NX 6KNY，Z L2的氨基酸序列为WAX 7，Z L3的氨基酸序列为QX 8DYX 9YPLT；
X 1选自S或T；
X 2选自T或S；X 3选自L或V；X 4选自T或S；X 5选自G或S；X 6选自Q或N；X 7 选自T或S；X 8选自N或Q；X 9选自T或S。
本发明一个更为具体的实施方案中，所述的人源化抗体，具有如下所述的VH和VL序列，
氨基酸序列为SEQ ID NO：25、27、29、31、33、35、37、39、41、43、45、47、49或51所示的VH，以及
氨基酸序列为SEQ ID NO：26、28、30、32、34、36、38、40、42、44、46、48、50或52所示的VL。
列举性的人源化抗体，具有如下任意一组所述的VH和VL序列：
SEQ ID NO：25所示氨基酸序列的VH，以及SEQ ID NO：26所示氨基酸序列的VL(BY5-1)；SEQ ID NO：27所示氨基酸序列的VH，以及SEQ ID NO：28所示氨基酸序列的VL(BY5-5)；SEQ ID NO：29所示氨基酸序列的VH，以及SEQ ID NO：30所示氨基酸序列的VL(BY6-1)；SEQ ID NO：31所示氨基酸序列的VH，以及SEQ ID NO：32所示氨基酸序列的VL(BY6-2)；SEQ ID NO：33所示氨基酸序列的VH，以及SEQ ID NO：34所示氨基酸序列的VL(BY6-3)；SEQ ID NO：35所示氨基酸序列的VH，以及SEQ ID NO：36所示氨基酸序列的VL(BY6-4)；SEQ ID NO：37所示氨基酸序列的VH，以及SEQ ID NO：38所示氨基酸序列的VL(BY6-6)；SEQ ID NO：39所示氨基酸序列的VH，以及SEQ ID NO：40所示氨基酸序列的VL(BY6-7)；SEQ ID NO：41所示氨基酸序列的VH，以及SEQ ID NO：42所示氨基酸序列的VL(BY6-8)；SEQ ID NO：43所示氨基酸序列的VH，以及SEQ ID NO：44所示氨基酸序列的VL(BY6-9)；SEQ ID NO：45所示氨基酸序列的VH，以及SEQ ID NO：46所示氨基酸序列的VL(BY6-13)；SEQ ID NO：47所示氨基酸序列的VH，以及SEQ ID NO：48所示氨基酸序列的VL(BY6-15)；SEQ ID NO：49所示氨基酸序列的VH，以及SEQ ID NO：50所示氨基酸序列的VL(BY6-19)。
所述各候选抗体中，重量恒定区选自人IgG系列，如IgG1、IgG2、IgG3或IgG4，优选IgG1；所述轻链的恒定区选自κ或λ链，优选κ链。
一个具体的实施方案中，本发明所述抗体名称、对应序列如下表：
VH序列：
QVQLVQSGX a1ELKKPGASVKISCKASGYX 1FTNYGMNWVRQAPGQGLKWMGWINTNTGEPTYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYX a2CARLGFGNAMDYWGQGTLVTVSS；
VL序列：
DIVMTQSPDSLAVSLGERATMNCKSSQX 2X 3LNX 4X 5NX 6KNYLTWYQQKPGQPPKLLIYWAX 7TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQX 8DYX 9YPLTFGAGTKLEIK所示的VL，
表1结合Claudin18.2的人源化抗体名称及对应的VH、VL序列
Figure PCTCN2021102664-appb-000001
Figure PCTCN2021102664-appb-000002
BY0-0的VH及VL氨基酸序列(SEQ ID NO：51及52)
VH互补决定区：CDR1:GYTFTNYG，CDR2:INTNTGEP；CDR3:ARLGFGNAMDY
VH框架区：FR1：QIQLVQSGPELKKPGETVKISCKAS；FR2：MNWVKQAPGKGLKWMGW；FR3：TYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFC；FR4：WGQGTSVTVSS
VL高变区/互补决定区：CDR1：QSLLNSGNQKNY；CDR2：WAS；CDR3：QNDYSYPLT。
VL框架区：FR1：DIVMTQSPSSLTVTAGEKVTMSCKSS；FR2：LTWYQQKPGQPPKLLIY；FR3：TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC；FR4：FGAGTKLELK。
本发明所述的抗体或抗原结合片段，其中所述HVR的恒定区选自人IgG系列，如IgG1、IgG2、IgG3或IgG4，优选IgG1；所述LVR的恒定区选自κ或λ链，优选κ链。
本发明的第三方面，提供了及一种药物组合物，其包含本发明所述的抗体或其抗原结合 片段和可药用载体。
适合的可药用载体，包括但不限于：抗氧化剂(例如抗坏血酸和硫酸氢钠)、防腐剂(例如苯甲醇、对羟基苯甲酸甲酯、羟基苯酸乙酯或正丙酯)、乳化剂、助悬剂、分散剂、溶剂、填充剂、膨胀剂、缓冲剂、载体、稀释剂和/或佐剂。例如，适合的载体可以是生理盐水溶液或柠檬酸盐缓冲盐水，其可能补充了在肠外施用药物组合物中常见的其它材料。中兴缓冲盐水或与血清白蛋白混合的盐水是其它的示例性载体。本领域那些技术人员将容易地分辨可以在本发明中使用的药物组合物和剂量形式中使用的多种缓冲剂。典型的缓冲剂包括但不限于，药物可用的弱酸、弱碱或其混合物。缓冲液组分还包括水溶性材料，如磷酸、酒石酸、乳酸、丁二酸、柠檬酸、乙酸、抗坏血栓、门冬氨酸、谷氨酸及其盐。
药物组合物和可以作为溶液、混悬液、凝胶、乳浊液、固体或者脱水或冷冻干燥的粉末保存在无菌小瓶中。这些组合物可以作为即可使用的形式、使用前需要复原的冷冻干燥形式、使用前需要稀释的液体形式或其它可用形式存储。
本发明还涉及一种分离的核酸分子，其编码本发明所述的抗体或其抗原结合片段；一种表达载体，含有本发明所述核酸分子，以及包含本发明所述的表达载体的宿主细胞，优选真核细胞。
本发明第四方面，提供了一种制备与Claudin18.2特异性结合的抗体或其抗原结合片段的方法，该方法包括在有利于本发明所述的抗体或其抗原结合片段表达的条件下表达本发明所述的核酸分子，并回收表达的抗体或其抗原结合片段。
用来培养细胞的培养基可以是用于培养该宿主细胞的任何常规培养基，如基本培养基或含有适宜添加物的复合培养基。可以通过市售得到适宜的培养基，或根据已公开的制法制备适宜的培养基。然后可以通过常规方法从培养基中回收由所述宿主细胞产生的多肽，例如用盐如硫酸铵沉淀上清液或滤液中的蛋白质成分，根据目的肽的种类而选用各种层析方法如例子交换层析、凝胶过滤层析、亲和层析等进行进一步纯化。
可以将上述编码DNA序列***任何适当的载体中。通常，载体的选择常常取决于该载体将要被引入的宿主细胞，因此，载体可以是一种自主复制型载体，即作为染色体外实体存在的载体，其复制不依赖于染色体复制，如质粒。或者，载体可以是这样一种类型，当将其引入宿主细胞时，它将整合到宿主细胞基因组中，并与它所整合入的染色体一起复制。
载体优选是一种表达载体，其内编码所述肽的DNA序列与该DNA转录所需的其它区段(如启动子)有效相连。本领域熟知适合于在多种宿主细胞中指导编码本发明肽的DNA进行转录的启动子例子，参见例如Sambrook，J，Fritsch，EF和Maniatis，T，分子克隆：实验操作指南，Cold Spring Harbor Laboratory Press，纽约，1989中所述。
载体还可以含有选择标记，如可以是这样的一种基因，其基因产物将弥补宿主细胞内的一个缺陷，或者能赋予对药物如氨苄青霉素、阿霉素、四环素、氯霉素、新霉素、链霉素或氨甲喋呤等的抗性。
为将本发明表达的肽引入宿主细胞的分泌途径，可以在重组载体中提供分泌信号序列(也称之为前导序列)。分泌信号序列以正确读框与编码该肽的DNA序列连接。分泌信号序列通常位于编码该肽的DNA序列的5’侧。分泌信号序列可以是正常地与该肽连接的分泌信号序列，或可以源于编码另一种分泌蛋白质的基因。
用于分别连接编码本发明肽的DNA序列，启动子和可选择的终止子和/或分泌信号肽序列，并将其***到适宜的含有复制所必需的信息的载体中的方法，对本领域的技术人员是已 知的。
用于真核宿主细胞(酵母、真菌、昆虫、植物、动物、人或来自其他多细胞生物的成核细胞)中的表达载体还将包含终止转录和稳定mRNA所需的序列，这种个序列通常可从真核或病毒DNA或cDNA的5’和(偶尔的)3’末翻译区获得。这些区域包含作为编码结合Claudin18.2的人源化抗体的mRNA的未翻译部分中的多腺苷酸化片段转录的核苷酸区段。
将导入DNA序列或重组载体的宿主细胞可以是能够产生本发明肽的任何细胞，包括细菌、病毒、酵母、真菌和高等真核生物细胞。本领域技术人员熟知并使用的适宜的宿主的例子包括但不限病毒。
可以从培养基或宿主细胞裂解液回收多种形式的本发明抗体或抗原结合片段，如果是膜结合的，则可以使用合适的去垢剂溶液(例如Triton-X 100)或通过酶切来使其从膜释放，可以通过多种物理或化学方法如冻融循环、超声、机械破碎或细胞裂解试剂来破裂用于表达本发明所述结合Claudin18.2的抗体的细胞。
可能需要的是从重组细胞蛋白纯化本发明抗体或抗原结合片段，以下方法是合适纯化方法的示例：通过在离子交换柱上的分部分离；乙醇沉淀；反相HPLC；在二氧化硅或在阳离子交换树脂如DEAE上的层析；色谱聚焦；SDS-PAGE；硫酸铵沉淀；使用例如Sephadex G-75的凝胶过滤；除去污染物如IgG的蛋白A Sepharose柱。
另一方面，本发明涉及抗体或其抗原结合片段在制备药物中的用途，所述药物用于预防或治疗癌症相关的疾病，尤其是胃癌。
本发明的第五方面，提供了一种预防或治疗癌症药的方法，包括给药受试本发明所述的结合Claudin18.2的抗体；其中所述癌症例如可以是包括胃肠道癌、胰腺癌、食道癌或非小细胞肺癌；所述胃肠道癌优选为晚期胃癌。
本发明的第六方面，提供了一种制品或药盒，包含容器和包装插页，其中所述容器中装有本发明所述的抗体或其抗原结合片段，或者本发明所述的药物组合物，所述包装插页上载有药物的使用说明书。在一个优选实施方案中，该制品或药盒进一步包含一个或多个容器，该容器中装有一种或多种预防或治疗癌症的其它药物。
合适的容器包括例如安瓿、针剂药水瓶、注射器等。容器可由多种物质(如玻璃或塑料)形成，容器盛有或容纳对于治疗由小的组合物并且可以具有无菌接入端(例如该容器可以是静脉溶液包或具有可被皮下注射针头刺穿的塞子的瓶)。组合物中的至少一种活性药剂是本发明抗体或抗原结合片段。标签或药品说明书表明该组合物在关于抗体和提供的任何其它药物的用药量的间隔时间的具体指导下被用于治疗遭受代谢相关的疾病、病症或症状的个体的代谢相关疾病、病症或症状。该制品还可以包括第二容器、所述第二容器包含药用稀释缓冲液、加注射用抑菌水、磷酸盐缓冲液、Ringer氏溶液和葡萄糖溶液。该制品还可以包括从商业和使用者角度看是需要的其它物质，包括其它缓冲、稀释剂、过滤器、针头和注射器。使用“药品说明书”通常包括在治疗产品的商业包装中的说明书，其包含关于指示、用法、剂量、给药、禁忌、与所包装产品结合的其它治疗产品和/或关于使用这些治疗产品的警告等的信息。
制品还可以包括其它组分，制品的每种组分可以包装在单个容器内并且可以将所有多个容器放置在单个包装内。
发明详述
1.定义：
本文所述“抗体”由四条肽链即由二硫键互联的两条重链(H)和两条轻链(L)组成的免疫球蛋白分子，每条重链包含重链可变区(HVR或VH)和重链恒定区，重链恒定区包含CH1、CH2和CH3三个结构域，每条轻链包括轻链可变区(LVR或VL)和轻链恒定区，轻链恒定区包含一个结构域(CL1)，VH和VL区可进一步分成被称为互补决定区(CDR)的高变区，其中散布着较保守的被称为框架区(FR)的区域，每个VH和VL由三个CDR和四个FR组成，从氨基末端到羧基末端按下列次序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。本文中，重链恒定区可选自IgG1、IgG2、IgG3或IgG4，优选IgG4，而轻链恒定区选自κ或λ。
术语“可变区”在本文中用于描述抗体的某些部分在抗体序列间有差异且涉及特定抗体对其特定抗原的结合和特异性的区域。然而，变异性通常不是均匀分布于抗体的整个可变区的。它典型的集中于轻链和重链可变区中称作互补决定区(CDR)或高变区的三个区段。可变区中相对比较保守的部分称作框架区(FR)。天然重链和轻链的可变区各自包含四个FR，它们大多采取β-折叠片构象，通过形成环状连接且在有些情况中形成β-折叠片结构一部分的三个CDR连接。每条链中的CDR通过FR非常接近的保持在一起，并与另一条链的CDR一起促成抗体的抗原结合位点的形成。恒定区不直接参与抗体与抗原的结合，但展现出多种效应物功能，诸如抗体依赖性细胞毒性中抗体的参与。
术语“互补决定区”、“高变区”和“CDR”指抗体轻链或重链的可变区中存在的高变区或互补决定区(CDR)的一个或多个(参见，Kabat，E.A.等人，SequencesofProteinsofImmunologicalInterest，NationalInstitutesofHealth，Bethesda，Md.，(1987))。这些术语包括Kabat等人定义的高变区(“Sequences ofProteinsofImmunologicalInterest，”KabatE.，等人，USDept.ofHealth andHumanServices，1983)或抗体三维结构中的高变环(Chothia和Lesk，J Mol.Biol.196901-917(1987))。每条链中的CDR通过构架区保持紧密靠近，并且与来自其它链的CDR一起促进抗原结合位点的形成。
术语“构架区”和“FR”指抗体轻链和重链的可变区内构架区的一个或多个(参见，Kabat，E.A.等人，SequencesofProteinsofImmunologicalInterest，NationalInstitutesofHealth，Bethesda，Md.，(1987))。这些术语包括抗体轻链和重链中位于氨基末端和第一CDR之间的那些氨基酸序列，介于CDR之间的那些以及第三CDR和恒定区起点之间的那些氨基酸序列。
CDR和FR残基可以根据标准序列定义(Kabat等人，Sequencesof ProteinsofImmunologicalInteres,NationalInstitutesofHealth，BethesdaMd.(1987))和结构定义(Chothia和Lesk，J.Mot.Biol.196：901-217(1987))来确定。
在本文指出时，参考Kabat，E.A.，等人，SequencesofProteinsof ImmunologicalInterest(NationalInstitutesofHealth，Bethesda，Md.(1987)和(1991)的编号方案。Kabat利用为所列序列中每个氨基酸分配残基编号的方法，并且分配残基编号的该方法已经成为本领域的标准。当明确指出使用Kabat编号时，本说明书遵循Kabat编号方案。当未指出Kabat编号时，使用顺序氨基酸序列编号(即，序列中的氨基酸使用顺序整数(1、2、3等)以氨基末端到羧基末端的方向从左到右编号。
抗体对抗原或表位的“亲和力”是本领域很好理解的术语，并且表示抗体对表位结合的程度或强度。亲和力可以本领域已知的多种方式测量和/或表示，包括但不限于平衡解离常数(KD或Kd，它可以被定义为抗体的解离速率与结合速率的比率，即，K off/K on)、表观平衡解离常数(KD′或Kd′)、EC50(实现与抗原结合50％时所需要的量)和IC50(实现竞争测定中50％抑 制所需的量)；人源化抗体的相对亲和力也可以通过与例如相关的鼠或嵌合抗体比较来确定。对于本发明目的而言，亲和力是结合抗原或表位的具体抗体群体的平均亲和力。抗体的亲和力可以使用酶联免疫吸附测定(ELISA)或荧光激活的细胞分选(FACS)测定来测量，本文实施例采用了ELISA方法测定本发明抗体与Claudin18.2结合的亲和力，参见实施例所述。
2.本发明抗体的制备方法
本发明抗体可以通过任何可用的方法生产，例如重组表达技术。编码与恒定区可操作连接的轻链和重链可变区的核酸可以被***表达载体。轻链和重链可以在相同或不同表达载体中克隆。编码免疫球蛋白链的DNA区段可以被可操作连接至确保免疫球蛋白多肽表达的表达载体的控制序列。表达控制序列包括但不限于启动子(例如，天然结合的或异源的启动子)、信号序列、增强子元件和转录终止序列。在一个实施方案中，表达控制序列是能够转化或转染真核宿主细胞的载体中的原核启动子***。一旦载体被引入适当的宿主，宿主被保持在适合高水平表达核苷酸序列和收集并纯化抗体的条件下。
表达载体可以是在任何宿主生物体中可复制的，作为附加体或作为宿主染色体DNA的整合部分。在一个实施方案中，表达载体含有选择标记(例如，氨苄青霉素抗性、潮霉素抗性、四环素抗性或新霉素抗性)以允许检测经预期DNA序列转化的那些细胞。
表达载体可用于从任何宿主细胞表达本发明的抗体，包括原核宿主细胞(例如大肠杆菌)、酵母宿主细胞、哺乳动物宿主细胞、植物宿主细胞和昆虫宿主细胞。
在一个实施方案中，使用大肠杆菌生产本发明抗体。适合这种应用的其他原核宿主包括杆菌，例如芽孢杆菌和其他肠杆菌科，例如沙门氏菌属、赛氏杆菌属和各种假单胞杆菌种。在这些原核宿主中，还可以制备表达载体，该表达载体通常含有与宿主细胞相容的表达控制序列(例如复制起点)。此外，将存在任何数目的多种公知启动子，例如乳糖启动子***、色氨酸(trp)启动子***、β-内酰胺酶启动子***或来自λ噬菌体的启动子***。启动子通常任选地与操纵子序列一起控制表达，并且具有核糖体结合位点序列等，用于起始和完成转录和翻译。
其他微生物例如酵母也可用于表达本发明抗体。例如，酵母菌属可用作酵母宿主，具有含有表达控制序列(例如启动子)、复制起点、终止序列及所需其他序列的适合启动子。用于酵母表达技术的启动子包括3-磷酸甘油酸激酶和其他糖酵解酶启动子。可诱导的酵母启动子包括但不限于来自醇脱氢酶、异细胞色素C和负责麦芽糖和半乳糖利用的酶的启动子。
在另一个实施方案中，哺乳动物组织细胞培养物可用于表达和生产本发明抗体。任何哺乳动物组织细胞可以用于此类方法，并且本领域已经开发了能够分泌异源蛋白(例如完整免疫球蛋白)的许多适合的宿主细胞系，包括哺乳动物BHK或CHO细胞系、各种Cos细胞系、HeLa细胞系、优选骨髓瘤细胞系或转化的B细胞或杂交瘤。在一个实施方案中，细胞是非人的。哺乳动物细胞的表达载体可以包括表达控制序列，例如复制起点、启动子和增强子，以及必要的加工信号位点，例如核糖体结合位点、RNA剪接位点、聚腺苷酸化位点和转录终止子序列。在一个实施方案中，表达控制序列是源自免疫球蛋白基因、SV40、腺病毒、牛***瘤病毒、巨细胞病毒等的启动子。
含有感兴趣的多核苷酸序列(例如重链和轻链编码序列和表达控制序列)的载体可以通过公知方法转移至宿主细胞，所述方法根据细胞宿主类型而改变。例如，氯化钙转染常用于原核细胞，而磷酸钙处理、电穿孔、脂质转染、生物弹道术或病毒转染可以用于其它细胞宿主。用于转化哺乳动物细胞的其他方法包括使用凝聚胺(polybrene)、原生质体融合、脂质体、 电穿孔和显微注射。为了生产转基因动物，转基因可以被显微注射入受精卵，或者可以被引入胚胎干细胞基因组，此类细胞的核被转移入去核***。
当编码重链和轻链的核酸分子被克隆入单独表达载体时，该载体可以被共转染以获得表达并组装完整免疫球蛋白。一经表达，完整抗体、其二聚体、单个的轻链和重链、或抗体的其它免疫球蛋白形式可以根据本领域的标准方法纯化，包括硫酸铵沉淀、亲和柱、柱色谱、HPLC纯化、凝胶电泳等。至少约90至95％同质性的基本上纯的免疫球蛋白可以被制备用于药物用途。在另一个实施方案中，至少约98至99％或更高同质性的基本上纯的人源化抗体可以被生产用于药物配制和方法。
因此，本发明提供了表达本发明抗体的方法，包括：(a)用编码本文所述抗体的核酸分子转化宿主细胞，和(b)在允许表达该抗体的条件下培养转化的宿主细胞。可以使用在载体上包括选择标记的已知技术，使得表达所述抗体的所述标记的宿主细胞可以容易被选择。
所述抗体的“抗原结合片段”或称为抗体的“抗原结合部分”指抗体保留了与抗原(如Claudin18.2)特异性结合能力的一个或多个片段。业已证明，抗体的抗原结合功能可由全长抗体的某些片段来实现。抗体的“抗原结合片段”的实例包括但不限于：(i)Fab片段，即由VL、VH、CL1和CH1结构域组成的单价片段；(ii)F(ab’)2片段，即由铰链区的二硫键连接的两个F(ab’)片段组成的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂VL和VH结构域组成的Fv片段；(v)由VH结构域组成的dAb片段；以及(vi)CDR。此外，尽管Fv片段的两个结构域VL和VH是由不同的基因编码，但它们可通过重组方法，由一种合成的接头连接在一起而成为单独的相连的链，其中VL和VH区配对形成单价分子(称为单链Fv，scFv)。这样的单链抗体也涵盖在抗体的“抗原结合片段”范围内。
可以通过这样一种方法制备本发明所述抗体，该方法包括在允许抗体表达的条件下，培养含有编码该抗体的DNA序列并能表达该抗体的宿主细胞，然后从培养物中回收产生的抗体。
用来培养细胞的培养基可以是用于培养该宿主细胞的任何常规培养基，如基本培养基或含有适宜添加物的符合培养基。可以通过市售得到适宜的培养基，或根据已公开的制法制备适宜的培养基。然后可以通过常规方法从培养基中回收由该细胞产生的多肽，这些方法包括通过离心或过滤而从培养基中分离宿主细胞，用盐如硫酸铵沉淀上清液或滤液中的蛋白质成分，根据目的肽的种类而选用各种层析方法如例子交换层析、凝胶过滤层析、亲和层析等进行纯化。
3.治疗方法和药剂
本发明提供了包含本发明的结合Claudin18.2的抗体或其抗原结合片段的药物组合物。本发明的药物组合物将与适当的载体、赋形剂以及其它制剂一起施用。这些制剂纳入配方是为了改善递送和耐受性等。在所有药剂化学家熟知的药典：雷氏药学大全(Remington′s Pharmaceutical Sciences,Mack Publishing Company，Easton，PA)中可找到大量适当的配方。
给药剂量随受试者年龄和体型大小、目标疾病、症状、施用途径等不同而调整。当本发明的抗体用来治疗成人的各种与癌症相关的病症和疾病时，可采用静脉给药本发明抗体，通常单剂按每公斤体重计约0.01至约20mg/kg给药，更优选的是约0.1至约15、约1至约10，或约3至约10mg/kg体重。根据病症的严重性，可对治疗的频度和持续时间进行调整。
已知有各种药物递送***可用来施用本发明的药物组合物，例如包在脂质体、微颗粒、微胶囊中的胶囊化，能够表达变异病毒的重组细胞，受体介导的胞吞作用(参阅如Wu等人(1987)，J.Biol.Chem.262：4429-4432)。给予药物的方法包括但不限于皮内、肌肉内、腹膜内、静脉内、皮下、鼻内、硬膜，和口腔等途径。该药物组合物可采用任何方便的途径施用，如通过灌注或静脉团注、上皮和粘膜层(如口腔粘膜、直肠和小肠粘膜)吸收，并可与其它生物活性药剂一起施用。施用方式可为全身性或局部性施用。
该药物组合物也可通过液囊递送，尤其是通过脂质体液囊递送(参阅Langer(1990)Science249：1527-1533；Treat等人(1989)in Liposomes in the Therapy of Infectious Disease and Cancer，Lopez Berestein and Fidler(编著)，Liss，New York，第353-365页；Lopez-Berestein，同上，第317-327页)。
在某些情况下，该药物组合物可以受控释放***递送。在一个实施方案中，可使用泵(参阅Langer，如上；Sefton(1987)CRC Crit.Ref.Biomed.Eng.14：201)。在另一个实施方案中，可采用聚合物材料(参阅Medical Applications of Controlled Release，Langer and Wise(编著)，CRC Pres.，Boca Raton，Florida(1974)。关于其它受控释放***的讨论见Langer(1990)Science249：1527-1533。
注射制剂可包括用于静脉内、皮下、皮内和肌肉内注射、滴注等的剂型。这些注射制剂可用已公开的方法制备。例如，注射制剂可通过将抗体或其盐溶于、悬浮于或乳化于惯常用于注射的无菌水介质或油介质的方式来制备。用于注射的水性介质有，例如生理盐水、含有葡萄糖和其它辅助剂的等渗溶液等。它们可与适当的增溶剂如醇(如乙醇)、多元醇(如丙二醇、聚乙二醇)、非离子型表面活性剂[如聚山梨醇酯80、HCO-50(氢化蓖麻油的聚氧乙烯(50摩尔)加成物)等结合使用。所用的油介质有，如芝麻油、大豆油等。它们可与增溶剂如苯甲酸苄酯、苯甲醇等结合使用。这样制备的注射剂最好封装在适当的安瓿瓶中。
单一治疗和联合治疗本发明的抗体和抗体片段对于治疗可通过破坏细胞间的紧密连接，使Claudin18.2无法发挥其正常功能而得到改善。
本发明涵盖联合治疗，其中结合Claudin18.2的抗体或抗体片段与一种或多种治疗药剂(或称第二种治疗药剂)联合施用。共同给药和联合治疗并不限于同时给药，而是还包括在涉及给予患者至少一种其它治疗药剂的疗程中至少给予一次抗Claudin18.2抗体或抗体片段的治疗方案。第二种治疗药剂可能为另一种胃癌症(如胃癌)治疗药物。
本发明还包括本文所述的任何抗Claudin18.2抗体或抗原结合片段在制备治疗疾病或症状的药物中的应用，其中的疾病或症状是通过破坏细胞间的紧密连接，使得Claudin18.2无法发挥其正常功能而实现。
本发明的一方面，提供了一种药物组合物，其包括本发明与Claudin18.2结合的抗体或其抗原结合片段，以及药学上可接受的载体。
在本文中，如无矛盾或特别说明，药物、药物组合物和药物制剂(药剂)可以互换使用。在本文中，药学上可接受的辅料指无毒的填充剂、稳定剂、稀释剂、载体、溶剂或其他制剂辅料。例如，稀释剂、赋形剂，如微晶纤维素、甘露醇等；填充剂，如淀粉、蔗糖等；粘合剂，如淀粉、纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮；崩解剂，如碳酸钙和/或碳酸氢钠；吸收促进剂，如季铵化合物；表面活性剂，如十六烷醇；载体、溶剂，如水、生理盐水、高岭土、皂粘土等；润滑剂，如滑石粉、硬脂酸钙/镁、聚乙二醇等。另外，本发明的药物组合物优选为注射剂。
在本发明的一些实施方案中，本发明药物组合物中的抗体或其抗原结合片段以1mg/ml至1000mg/ml的浓度存在，优选以10mg/ml至1000mg/ml的浓度存在，更优选以50mg/ml至500mg/ml的浓度存在，更优选以100mg/ml至300mg/ml的浓度存在。
本发明的药物组合物优选具有3.0至9.0的pH。其中，可进一步包含缓冲***、防腐剂、表面张力剂、螯合剂、稳定剂和表面活性剂。在本发明的一个实施方案中，本发明的药物组合物是含水制剂。这种制剂通常是溶液或悬浮。本发明的具体实施方案中，该药物组合物是稳定的含水溶液。在本发明的另一个具体实施方案中中，该药物组合物是一种冻干制剂，在使用前医师或患者加入溶剂和/或稀释液溶解所述冻干制剂。
附图说明
图1为抗Claudin18.2抗体筛选-细胞ELISA结合活性图。
图2为抗Claudin18.2抗体筛选-增殖抑制活性图。
图3为抗Claudin18.2抗体筛选-抗体依赖性细胞毒性图。
图4为抗Claudin18.2抗体筛选-补体依赖性细胞毒性图。
图5(5-1,5-2)为候选抗体对KATO细胞裸鼠移植瘤模型相对肿瘤体积(RTV)的影响图。
图6(6-1,6-2)为候选抗体对NUGC细胞裸鼠移植瘤模型相对肿瘤体积(RTV)的影响图。
具体实施方式
实施例1表达Claudin18.1及Claudin18.2抗原的HEK293细胞的构建
将编码人Claudin18.1及Claudin18.2抗原的pcDNA3.1载体(购自Invitrogen公司)转染HEK293细胞(购自ATCC)，采用200μg/mL的遗传霉素作为筛选压力得到稳定表达Claudin18.1及Claudin18.2抗原的HEK293细胞。以Ganymed公司的Claudin18.2抗体IMAB362(自制，CHO-S细胞瞬转表达，并进一步层析纯化，参考实施例2及3，本发明中为BY0-0)为阳性抗体，通过FACS方法筛选稳定表达人Claudin18.1及Claudin18.2抗原的HEK293细胞。
实施例2候选抗体BY6-4表达载体的构建
将HindIII酶切位点( AAGCTT)、KoZAK序列( GCCGCCACC)、ATG、信号肽基因GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT和抗体的重链编码基因(包括重链可变区编码基因SEQ ID NO：59和恒定区IgG1编码基因SEQ ID NO：63)、终止码TAA和EcoRI编码基因GAATTC依次串联融合，并使用化学合成的方式获得基因片段。通过EcoRI和HindIII位点，将上述片段***真核表达质粒pCDNA 3.4(+)((购自Invitrogen公司))中并测序验证，得到抗体重链的表达质粒PCDNA3.4(+)-BY6-4。
将HindIII酶切位点( AAGCTT)、KoZAK序列( GCCGCCACC)、ATG、信号肽基因GAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT和抗体的轻链编码基因(包括轻链可变区编码基因SEQ ID NO：60和恒定区κ编码基因SEQ ID NO:64)、终止码TAA和EcoRI编码基因GAATTC依次串联融合，并使用化学合成的方式获得基因片段。通过EcoRI和HindIII位点，将上述片段***真核表达质粒pCDNA 3.4(+)中并测序验证，得到抗体轻链的表达质粒PCDNA3.4(+)-BY6-4。
将BY6-1的重链编码基因(SEQ ID NO：53)和轻链编码基因(SEQ ID NO：54)、BY6-2的重链编码基因(SEQ ID NO：55)和轻链编码基因(SEQ ID NO：56)、BY6-3的重链编码基因(SEQ ID NO：57)和轻链编码基因(SEQ ID NO：58)、BY0-0的重链编码基因(SEQ  ID NO：61)和轻链编码基因(SEQ ID NO：62)，替换BY6-4的重轻链编码基因，可得到相应的重轻链表达质粒。
在知道所述抗体氨基酸序列的情况下，获得这些抗体的编码基因是本领域所熟知的。依据上述同样方法，根据不同候选抗体氨基酸序列，可以得到一系列候选抗体的表达质粒：BY5-1、BY5-2、BY5-3、BY5-4、BY5-5、BY5-6、BY5-7、BY5-8、BY5-9、BY6-5、BY6-6、BY6-7、BY6-8、BY6-9、BY6-10、BY6-11、BY6-12、BY6-13、BY6-14、BY6-15、BY6-16、BY6-17、BY6-18、BY6-19的重链及轻链表达质粒
各候选抗体的重链恒定区均为IgG1，轻链恒定区为κ。
实施例3抗Claudin18.2抗体的表达、纯化
使用实施例2所述DNA构建体，分别瞬转至CHO-S细胞(购自Invitrogen公司)，表达目的抗体，按照CHO-S细胞操作手册(Freedom TM CHO-S TMKitUSER GUIDE)，在质粒转染前一天将细胞密度调整至1x10 6个/毫升。在质粒转染当天，与转染试剂混合后加入EXPICHO EXPRESSION MEDIUM细胞培养基(购自Invitrogen公司)中，37℃，8％CO2持续培养至第8天后收集细胞液，采用离心方式去除细胞，0.2μm过滤后，ProteinA亲和层析纯化，收集的样品的pH调至5.5，2～8℃保存。纯化后的抗体进行SDS PAGE、SEC检测，纯度在95％以上。
实施例4特异性与亲和力鉴定
以Claudin18.2 N端胞外结构域的合成肽部分为抗原，通过ELISA包被稳转Claudin18.2及Claudin18.1的HEK293细胞对抗体进行ELISA筛选，采用双抗体夹心法检测(以0.1mg/mL的多聚赖氨酸包被96孔细胞培养板，5min后，除去包被液，加入5×10 4/孔HEK293-Claudin18.2或HEK293-Claudin18.1细胞，细胞在37℃、5％CO 2培养箱中培养24h贴壁后，去上清，每孔加PBST洗涤细胞3次，以100μl/孔加入梯度稀释抗体溶液，37℃2小时，用洗涤液洗板3次；稀释液(使用洗涤液配置2％牛血清白蛋白溶液)1/5000稀释HRP标记二抗(编号ab6858，产品来源于Abcam公司)，以100μl/孔加至酶标板内，37℃1小时；用洗涤液洗板3次；以100μ1/孔加入TMB显色液，置室温避光反应5～10分钟，以50μl/孔加人终止液终止反应，并在波长4 5 0nm处测定吸光度)，测定各候选抗体与Claudin18.2的EC50值，并以BY0-0的EC50值为基准，计算各候选抗体的相对结合活性(结构见表2)。计算方法如下，相对结合活性＝参考品BY0-0的EC50/各候选抗体的EC50值。
表2各候选抗体的相对结合活性
样品 相对结合活性 样品 相对结合活性
BY0-0 100％ BY6-6 51.5％
BY5-1 56.7％ BY6-7 106.1％
BY5-2 0.38％ BY6-8 73.7％
BY5-3 11.7％ BY6-9 103.5％
BY5-4 0.64％ BY6-10 0.7％
BY5-5 42.0％ BY6-11 24.9％
BY5-6 2.1％ BY6-12 0.7％
BY5-7 0.2％ BY6-13 78.7％
BY5-8 0.82％ BY6-14 0.1％
BY5-9 26.4％ BY6-15 88.2％
BY6-1 144.1％ BY6-16 16.1％
BY6-2 183.7％ BY6-17 0.1％
BY6-3 198.3％ BY6-18 0.1％
BY6-4 248.4％ BY6-19 45.7％
BY6-5 60.9％    
细胞ELISA结合活性结果表明，候选抗体的特异性较好，与Claudin18.1均不结合，但是与Claudin18.2结合活性有显著差异，其中BY6-1、BY6-2、BY6-3、BY6-4、BY6-6、BY6-7、BY6-8、BY6-9、BY6-13及BY6-19与Claudin18.2的结合活性较优，而BY5-2、BY5-3、BY5-4、BY5-6、BY5-7、BY5-8、BY6-10、BY6-12、BY6-14、BY6-17、BY6-18与Claudin18.2结合活性则较差。
实施例5细胞体外功能活性
抗Claudin18.2抗体介导四种独立的高度有效的作用机制来诱导肿瘤细胞的杀伤和凋亡：抗体依赖性细胞毒性(ADCC)、补体依赖性细胞毒性(CDC)、诱导肿瘤细胞表面上的靶标交联诱导的凋亡和直接抑制增殖。第二轮抗体筛选是对第一轮全人源抗体库筛选出来的亲和力较高的7个抗体(BY6-1、BY6-2、BY6-3、BY6-4、BY6-6、BY6-7、及BY6-9)进行进一步的细胞模型上的功能性(ADCC、CDC、增殖抑制)鉴定，为临床候选药物的确定提供依据。
(1)增殖抑制活性
利用CCK8法研究抗Claudin18.2抗体对KATOIII细胞(购自ATCC)的增值抑制作用：取对数期生长的KATO III细胞，用IMDM(购自Invitrogen)+10％FBS培养基调整细胞浓度到2×10 5/ml，37℃，5％CO2培养箱中待用。将样品及参考品(Ch163，或称BY0-0)用IMDM+5％FBS培养基预稀释终浓度为400μg/ml，然后进行2倍稀释，共14个梯度：400ug/ml、200μg/ml、100μg/ml、50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.125μg/ml、1.56μg/ml、0.78μg/ml、0.39μg/ml、0.19μg/ml、0.097μg/ml、0μg/ml。在平底96孔板中每孔加入60ul细胞悬液，再依次加入梯度稀释好的抗CLDN18.2抗体60μl。轻轻震荡，混匀。细胞培养板置含5％CO 2的37℃的培养箱孵育72h。孵育结束后，加入CCK-8显色液，12μl/孔，置含5％CO 2的37℃的培养箱孵育2-4h。在450nm波长处读取各孔吸光度值。
数据处理使用GraphPad.Prism.v5.01软件，计算样品和参考品的IC50值。试验结果如图2所示。
增殖抑制活性结果表明，候选抗体BY6-2、BY6-3、BY6-4的抗体的体外细胞增殖抑制效果明显。
(2)抗体依赖性细胞毒性(ADCC)
以稳转HEK18.2细胞作为靶细胞，从新鲜的人外周血中分离出来的PBMC细胞作为效应细胞检测待检抗体的ADCC活性。分别采用胰酶消化的NUGC细胞(购自JCRB)和HEK18.2细胞作为靶细胞对7种抗体进行检测，每孔5000个细胞。
试验前一天分别提取2个健康自愿者各30ml血液中的PBMC，以RPMI-1640(购自Invitrogen) +10％FBS培养过夜，效应细胞每孔使用2.5×10 5个。受试药初始浓度为10μg/ml(使用前稀释到40μg/ml)，以5倍稀释法稀释至8个样品。采用RPMI-1640+2％FBS(灭活血清)为ADCC的实验培养基。
结果计算方法
ER＝ER孔吸光度-CMB孔吸光度
ESR＝ESR孔吸光度-CMB孔吸光度
TSR＝TSR孔吸光度-CMB孔吸光度
TMR＝TMR孔吸光度-VCC孔吸光度
细胞毒性(％)＝(ER-ESR-TSR)/(TMR-TSR)×100％)
死细胞和受损细胞的量通过Cytotoxicity LDH Assay Kit-WST试剂盒(购自日本同仁化学)进行检测。该试剂盒是通过测定细胞释放到培养基中的乳酸脱氢酶(LDH)活性进而测定细胞损害。试验结果如图3所示。
ADCC活性结果表明，候选抗体BY6-1、BY6-2、BY6-3、BY6-4的抗体的体外ADCC活性抑制效果明显。
(3)补体依赖性细胞毒性
利用CCK8方法检测抗Claudin18.2抗体、人血清与HEK8.2细胞作用后的CDC效应。补体裂解试验用血清取自于健康自愿者，600g离心20min，收获血清，并保存在-20℃。HEK18.2细胞用RPMI-1640完全培养基于37℃、5％CO 2条件下培养至对数生长期。消化HEK18.2细胞，使用无血清RPMI-1640培养基洗两遍。细胞计数，并用无血清RPMI-1640培养基配制成含5×10 5/ml的细胞悬液。筛选抗体常规稀释1000μg/mL(用RPMI-1640培养基)，5倍梯度继续稀释(1000、300、100、30、10μg/mL)；阳性对照抗体Rituxan(购自罗氏)常规稀释500μg/mL(用RPMI-1640培养基)，5倍梯度稀释(500、100、20、4μg/mL)。每孔100μl，其中；RPMI-1640培养液悬浮的HEK18.2细胞的体积85μL，其细胞数量为5.0×10 4/孔，人血清(补体)5μL(1:30稀释)；不同浓度的抗体10μL(阳性对照加稀释抗体用的RPMI-1640培养基，同时设立无细胞的培养基对照孔)；筛选抗体的终浓度为(100、30、10、3、1μg/mL)，Rituxan单抗的终浓度为(50、10、2、0.4μg/mL)。上述96孔细胞培养板置于5％CO 2，于37℃培养4小时，加入10微升CCK8。4小时后，检测吸光度：放入酶标仪内测读：波长为450nm；
计算处理样品实际吸光读数：处理样品孔吸光读数－无细胞培养基对照孔吸光读数。
细胞死亡率(％)计算：
[(未加抗体药物孔最大实际吸光值－处理样品实际吸光值)÷未加抗体药物孔最大实际吸光值]×100％
试验结果如4所示。
CDC活性结果表明，候选抗体BY6-1、BY6-3、BY6-4的抗体的体外细胞CDC活性效果明显。
实施例6体内抑瘤活性
使用表达Claudin18.2的人胃癌KATO(购自：ATCC)及NUGC细胞系(购自：JCRB)构建裸鼠(购自：北京维通利华实验动物技术有限公司)皮下移植瘤模型，采用瘤块接种法。
无菌条件下收集KATO及NUGC细胞，用灭菌生理盐水调整细胞密度至1×10 7vc/ml，取0.2ml接种于裸鼠腋窝皮下，待肿瘤生长至直径1000mm 3大小，无菌条件下取出，切成 1mm×1mm大小的瘤块，均匀接种于裸鼠腋窝皮下。待肿瘤体积长至100～300mm 3大小时，按肿瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选，随机分组每组10只，KATO细胞、NUGC细胞各5组，分别腹腔注射给药BY0-0、BY6-1、BY6-2、BY6-4，给药剂量为10mg/kg/只，阴性对照组给予PBS，在4周中，每周周一、周四给药两次，连续给药7次，首次给药当天计为D0，每周两次检测小鼠的肿瘤生长(测小鼠体重、游标卡尺测量肿瘤长短计算体积(对照组肿瘤体积超过1000mm 3即可结束实验))。
肿瘤瘤重抑制率(％)＝(对照组肿瘤瘤重-给药组肿瘤瘤重)/对照组肿瘤瘤重×100％
试验结果如表3-1、3-2，图5-1、5-2及6-1/6-2所示。
表3-1抗Claudin18.2抗体对KATO细胞裸鼠移植瘤生长的影响
Figure PCTCN2021102664-appb-000003
表3-2抗Claudin18.2抗体对NUGC细胞裸鼠移植瘤生长的影响
Figure PCTCN2021102664-appb-000004
两种人胃癌细胞系小鼠异种移植模型试验结果表明，候选抗体抑制人胃癌KATO及NUGC细胞裸鼠异体移植瘤生长的作用与剂量呈正相关。在一定的剂量和给药时间内对人胃癌KATO及NUGC细胞裸鼠异体移植瘤的生长有明显的抑制作用。在BY6-1、BY6-2、BY6-4三种候选抗体中，BY6-4在两种胃癌细胞的裸鼠异体移植瘤中效果最优。
实施例7稳定性实验
考察候选抗体在高温、强光、反复冻融、强酸、强碱条件等因素影响下的稳定性并按照检验重点考察项目进行检测。具体实验设计、检测项目、取样方式如表4-1、表4-2、表4-3所示；稳定性结果见表4-4。
表4-1候选抗体成药性分析实验设计
Figure PCTCN2021102664-appb-000005
*每个考察条件放置相同包装的buffer，用于空白对照。
表4-2候选抗体成药性分析检测方法
Figure PCTCN2021102664-appb-000006
表4-3候选抗体成药性分析取样表格
Figure PCTCN2021102664-appb-000007
表4-4候选抗体稳定性实验结果
Figure PCTCN2021102664-appb-000008
Figure PCTCN2021102664-appb-000009
零点检测结果显示，CHO-S表达及一步亲和纯度的抗体，SEC检测单体纯度均高于98％，IEX检测主峰含量70％左右，8种抗体无明显差异，羧肽酶酶切后检测，赖氨酸的修饰比例 不同，除BY6-15外，7种抗体的赖氨酸修饰比例3％-5％，BY6-15赖氨酸修饰比例10％左右，SDS-PAGE结果显示抗体分子量大小正常，轻重链完整，8种抗体无明显的差异，与Claudin18.2的相对结合活性差异比较大，BY6-1、BY6-2、BY6-3与BY6-4相对BY0-0的活性较高，BY6-6、BY6-15与BY6-19相对BY0-0的活性较低，但是与抗体筛选阶段的相对活性一致。
高温稳定性实验结果显示：1)SEC检测：8种抗体高温稳定性的SEC检测无显著性差异，20天50℃高温检测的SEC结果均等于或优于对照抗体BY0-0；2)IEX检测：除BY6-15在20天50℃高温主峰比例较低，酸峰比例较高外，其余7种抗体的20天50℃高温稳定性IEX检测无显著差异；3)SDS-PAGE：非还原SDS结果中，BY6-3纯度略低；4)相对结合活性：8种抗体的相对结合活性随着50℃高温孵育时间的延长均呈下降趋势，其中BY6-1和BY6-4的相对结合活性优于BY0-0，BY6-2和BY6-3的相对结合活性与BY0-0较一致，BY6-6、BY6-15和BY6-19相对结合活性则偏低。
强酸稳定性实验结果显示：1)各抗体的SEC、SDS及IEX的主峰比例均呈下降趋势，其中BY6-3/4的SEC、SDS与IEX的主峰下降比例相比其他抗体较为显著。2)BY6-3/4抗体的相对结合活性在稳定性研究期间呈下降趋势，但仍然是对照抗体相对活性的80％；BY6-19相对结合活性在起始点偏低(59.2％)，但是在稳定性研究中相对活性升高(130.4％)；其余各抗体的相对结合活性在稳定性研究期间无显著变化趋势。BY6-6及BY6-15的相对结合活性起始点和终点仍较低。
光照稳定性实验结果显示：1)SEC检测：在稳定性研究期间，各抗体SEC主峰纯度均呈下降趋势，但都不低于对照抗体BY0-0；2)IEX检测：在稳定性研究期间，各抗体的IEX主峰比例均下降，酸峰比例均上升，除BY6-3的主峰纯度略低外，其余各抗体的主峰和酸峰比例无明显差异；3)SDS-PAGE：除BY6-6的还原SDS-PAGE主带纯度偏低，其余各抗体无显著差异；4)相对结合活性：在稳定性研究期间，各抗体无显著变化趋势。
IEX检测BY6-3主峰纯度下降显著，约20％，BY6-1/2主峰纯度下降10％，BY6-6/16与BY6-19相对结合活性偏低，其他无显著差异。
反复冻融稳定性实验结果显示：1)SEC及IEX检测：在稳定性研究期间，各抗体无显著变化趋势；2)SDS-PAGE：BY6-6的轻重链含量偏低，其他抗体无明显差异；3)相对结合活性：BY6-1//2/3/4/6相对活性均显著降低，只有BY6-1相对活性高于对照80％，且BY6-15与BY6-19相对活性无变化。提示BY6-1//2/3/4/6尽量避免反复冻融。

Claims (11)

  1. 一种结合密蛋白18.2的人源化抗体，所述抗体包括重链高变区(HCDR)和轻链高变区(LCDR)，所述重链高变区包括：
    氨基酸序列为GYX 1FTNYG的HCDR1，氨基酸序列为INTNTGEP的HCDR2，以及氨基酸序列为ARLGFGNAMDY的HCDR3；所述轻链高变区包括：
    氨基酸序列为QX 2X 3LNX 4X 5NX 6KNY的LCDR1，氨基酸序列为WAX 7的LCDR2，以及氨基酸序列为QX 8DYX 9YPLT的LCDR3，
    其中所述HCDR1中氨基酸X 1选自S或T；
    所述LCDR1中氨基酸X 2选自T或S，X 3选自L或V，X 4选自T或S，所述X 5选自G或S，X 6选自Q或N；
    所述LCDR2中氨基酸X 7选自T或S；
    所述LCDR3中氨基酸X 8选自N或Q，X 9选自T或S。
  2. 根据权力要求1所述的人源化抗体，其中所述抗体包括重链高变区和轻链高变区，其中重链高变区包含如下CDR序列，
    氨基酸序列为GYSFTNYG(SEQ ID NO：1)、GYTFTNYG(SEQ ID NO：2)的CDR1(HCDR1)；氨基酸序列为INTNTGEP(SEQ ID NO：3)的CDR2(HCDR2)；
    氨基酸序列为ARLGFGNAMDY(SEQ ID NO：4)的CDR3(HCDR3)；
    所述轻链高变区包含如下CDR序列，
    氨基酸序列为QTLLNTGNQKNY(SEQ ID NO:5)、QSVLNSGNQKNY(SEQ ID NO:6)、QSLLNSSNNKNY(SEQ ID NO:7)、QSVLNSSNNKNY(SEQ ID NO:8)、QTLLNSGNQKNY(SEQ ID NO:9)、QTLLNSGNNKNY(SEQ ID NO:10)、QTLLNSSNQKNY(SEQ ID NO:11)、QTLLNSSNNKNY(SEQ ID NO:12)、QSLLNSSNQKNY(SEQ ID NO:13)、QSLLNSGNNKNY(SEQ ID NO:14)、QSLLNSGNQKNY(SEQ ID NO:15)、QSVLNSSNQKNY(SEQ ID NO:16)、QSVLNSGNNKNY(SEQ ID NO:17)、QTVLNSGNNKNY(SEQ ID NO:18)的CDR1(LCDR1)；
    氨基酸序列为WAT(SEQ ID NO：19)、WAS(SEQ ID NO：20)的CDR2(LCDR2)；氨基酸序列为QNDYTYPLT(SEQ ID NO:21)、QQDYTYPLT(SEQ ID NO:22)、QQDYSYPLT(SEQ ID NO:23)、QNDYSYPLT(SEQ ID NO:24)的CDR3(LCDR3)。
  3. 根据权力要求2所述的人源化抗体，其包含如下任意一组重链高变区和轻链高变区：
    (1)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:5的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；
    (2)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:6的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:22的LCDR3；
    (3)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；
    (4)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:8的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；(5)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:9的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；
    (6)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:5的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；
    (7)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:9的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:21的LCDR3；
    (8)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:10的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；
    (9)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:11的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；
    (10)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:12的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；
    (11)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:6的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (12)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:13的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (13)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:14的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (14)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:15的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；
    (15)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (16)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:13的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；
    (17)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:16的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (18)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:14的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；
    (19)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:17的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (20)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:8的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:24的LCDR3；
    (21)SEQ ID NO:2的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:7的LCDR1、SEQ ID NO:20的LCDR2、SEQ ID NO:23的LCDR3；
    (22)SEQ ID NO:1的HCDR1、SEQ ID NO:3的HCDR2、SEQ ID NO:4的HCDR3，和SEQ ID NO:18的LCDR1、SEQ ID NO:19的LCDR2、SEQ ID NO:24的LCDR3；或
    (23)SEQ ID NO:17的HCDR1、SEQ ID NO:32的HCDR2、SEQ ID NO:54的HCDR3，和SEQ ID NO:61的LCDR1、SEQ ID NO:63的LCDR2、SEQ ID NO:66的LCDR3。
  4. 根据权力要求1-3中任意一权利要求所述的人源化抗体，其中所述抗体具有如下的重链可变区(VH)和轻链可变区(VL)序列：
    氨基酸序列为QVQLVQSGX a1ELKKPGASVKISCKASZ H1MNWVRQAPGQGLKWMGWZ H2TYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYX a2CZ H3WGQGTLVTVSS所示的VH；以及
    氨基酸序列为DIVMTQSPDSLAVSLGERATMNCKSSZ L1LTWYQQKPGQPPKLLIYZ L2T RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCZ L3FGAGTKLEIK所示的VL，
    其中X a1为P或S，X a2为F或Y，Z H1、Z H2及Z H3分别为重链的三个CDR区，Z L1、Z L2及Z L3为轻链的三个CDR区，
    其中Z H1的氨基酸序列为GYX 1FTNYG，Z H2的氨基酸序列为INTNTGEP，Z H3的氨基酸序列为ARLGFGNAMDY；ZL1的氨基酸序列为QX 2X 3LNX 4X 5NX 6KNY，ZL2的氨基酸序列为
    WAX7，ZL3的氨基酸序列为QX 8DYX 9YPLT；
    X 1选自S或T；
    X 2选自T或S；X 3选自L或V；X 4选自T或S；X 5选自G或S；X 6选自Q或N；X 7选自T或S；X 8选自N或Q；X 9选自T或S。
  5. 根据权力要求4所述的人源化抗体，其中所述抗体具有如下所述的VH和VL序列，
    氨基酸序列为SEQ ID NO：25、27、29、31、33、35、37、39、41、43、45、47、49或51所示的VH，以及
    氨基酸序列为SEQ ID NO：26、28、30、32、34、36、38、40、42、44、46、48、50或52所示的VL。
  6. 根据权力要求5所述的人源化抗体，其中所述抗体具有如下任意一组所述的VH和VL序列，
    SEQ ID NO：25所示氨基酸序列的VH，以及SEQ ID NO：26所示氨基酸序列的VL；SEQ ID NO：27所示氨基酸序列的VH，以及SEQ ID NO：28所示氨基酸序列的VL；SEQ ID NO：29所示氨基酸序列的VH，以及SEQ ID NO：30所示氨基酸序列的VL；SEQ ID NO：31所示氨基酸序列的VH，以及SEQ ID NO：32所示氨基酸序列的VL；SEQ ID NO：33所示氨基酸序列的VH，以及SEQ ID NO：34所示氨基酸序列的VL；SEQ ID NO：35所示氨基酸序列的VH，以及SEQ ID NO：36所示氨基酸序列的VL；SEQ ID NO：37所示氨基酸序列的VH，以及SEQ ID NO：38所示氨基酸序列的VL；SEQ ID NO：39所示氨基酸序列的VH，以及SEQ ID NO：40所示氨基酸序列的VL；SEQ ID NO：41所示氨基酸序列的VH，以及SEQ ID NO：42所示氨基酸序列的VL；SEQ ID NO：43所示氨基酸序列的VH，以及SEQ ID NO：44所示氨基酸序列的VL；SEQ ID NO：45所示氨基酸序列的VH，以及SEQ ID NO：46所示氨基酸序列的VL；SEQ ID NO：47所示氨基酸序列的VH，以及SEQ ID NO：48所示氨基酸序列的VL；SEQ ID NO：49所示氨基酸序列的VH，以及SEQ ID NO：50所示氨基酸序列的VL。
  7. 根据权利要求1-6中任意权利要求所述的人源化抗体，其中所述重链的恒定区选自人IgG系列，如IgG1、IgG2、IgG3或IgG4，优选IgG1；所述轻链的恒定区选自κ或λ链。
  8. 一种分离的核酸分子，其编码权利要求1-7中任一权利要求所述的人源化抗体。
  9. 药物组合物，其包含权力要求1-7中任意一权利要求所诉人源化抗体，以及制药上可接受的赋形剂。
  10. 权利要求1-7中任一项所述的人源化抗体在制备具有治疗和/或预防癌症药物中的应用；其中所述癌症例如可以是包括胃肠道癌、胰腺癌、食道癌或非小细胞肺癌；所述胃肠道癌优选为晚期胃癌。
  11. 一种制品或药盒，包含容器和包装插页，其中所述容器中装有权力要求1-7中任意一权力要求所述的人源化抗体，或者权力要求9所述的药物组合物，所述包装插页上载有药物的使用说明书。
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