WO2024067759A1 - 一种能够结合cldn18.2的抗体或其抗原结合片段及其应用 - Google Patents

一种能够结合cldn18.2的抗体或其抗原结合片段及其应用 Download PDF

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WO2024067759A1
WO2024067759A1 PCT/CN2023/122392 CN2023122392W WO2024067759A1 WO 2024067759 A1 WO2024067759 A1 WO 2024067759A1 CN 2023122392 W CN2023122392 W CN 2023122392W WO 2024067759 A1 WO2024067759 A1 WO 2024067759A1
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antibody
cells
antigen
binding
amino acid
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French (fr)
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欧阳雪松
张红娟
王晓燕
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北京诺诚健华医药科技有限公司
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    • 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
    • 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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to the field of biomedicine, and specifically relates to an antibody or an antigen-binding fragment thereof capable of binding to CLDN18.2.
  • the present invention also relates to the application of the antibody.
  • Claudins are important members of the tight junction protein family and play an important role in the connection between cells and cells and between cells and the matrix.
  • Claudin18 is encoded by the gene CLDN18. It is a four-transmembrane protein with two extracellular regions. It is widely distributed in tissues such as the stomach, pancreas and lungs. Claudin18 is considered to be a diagnostic marker and therapeutic target (Krause, G., Winkler, L., Mueller, S.L., Haseloff, R.F., Piontek, J., & Blasig, I.E. (2008). Structure and function of claudins. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1778 (3), 631-645.).
  • the Claudin18 family includes two variants, Claudin18.1 (CLDN18.1) and Claudin18.2 (CLDN18.2).
  • CLDN18.1 is specifically expressed in alveolar epithelial cells (Yasui, W., Sentani, K., Sakamoto, N., Anami, K., Naito, Y., & Oue, N. (2011).
  • Molecular pathology of gastric cancer Research and practice. Pathology-Research and Practice, 207(10), 608-612.), and has only 21 amino acid differences from CLDN18.2 (Sahin et al., 2008).
  • CLDN18.2 is widely present in gastric tumor tissues; it has been recently discovered that it is also expressed in pancreatic cancer, esophageal cancer and lung cancer (Jovov et al., 2007; Karanjawala et al., 2008). Due to the tight connection between normal cells, antibodies cannot contact Claudin18.2 expressed by cells; however, in tumor cells, due to cytopathic changes, the connection between cells becomes loose, thus exposing CLDN18.2 to the outside, and antibodies can bind to these exposed CLDN18.2 molecules.
  • CLDN18.2 has become an ideal drug target molecule (Klamp, T., Schumacher, J., Huber, G., Kuhne, C., Meissner, U., Selmi, A., Sahin, U. (2011). Highly specific auto-antibodies against claudin-18isoform 2induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases. Cancer Res, 71(2), 516-527.).
  • Zolbetuximab is the first chimeric antibody drug developed for CLDN18.2. It has not been humanized and can specifically bind to CLDN18.2 on the surface of tumor cells, thereby inducing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibiting cell proliferation (Woll, S., Schlitter, AM, Dhaene, K., Roller, M., Esposito, I., Sahin, U., & Tureci, O. (2014). Claudin 18.2 is a target for IMAB362antibody in pancreatic neoplasms. Int J Cancer, 134(3), 731-739).
  • the technical problem to be solved by the present invention is to obtain an antibody that can specifically bind to CLDN18.2 but not to CLDN18.1, and has high affinity, low immunogenicity, and good clinical prospects.
  • the inventors prepared an antibody or an antigen-binding fragment thereof against human CLDN18.2.
  • the antibody or the antigen-binding fragment thereof obtained by the present invention has higher affinity and lower immunogenicity.
  • the antibodies of the present invention can have various uses, including detecting Claudin 18.2 protein, diagnosing, treating or preventing Claudin18.2-related cancers, etc.
  • the present invention relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, which comprises: a light chain complementary determining region LC-CDR1 as shown in the amino acid sequence SEQ ID NO: 5, a light chain complementary determining region LC-CDR2 as shown in the amino acid sequence SEQ ID NO: 7, and a light chain complementary determining region LC-CDR3 as shown in the amino acid sequence SEQ ID NO: 9; and a heavy chain complementary determining region HC-CDR1 as shown in the amino acid sequence SEQ ID NO: 11, a heavy chain complementary determining region HC-CDR2 as shown in the amino acid sequence SEQ ID NO: 13, and a heavy chain complementary determining region HC-CDR3 as shown in the amino acid sequence SEQ ID NO: 15.
  • amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the present invention also provides humanized antibodies or antigen-binding fragments thereof of the above antibodies or antigen-binding fragments thereof.
  • the humanized antibody is characterized in that the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 19.
  • the invention thus provides nucleic acids encoding the antibodies or antigen-binding portions thereof.
  • the present invention also provides an expression vector and/or a host cell comprising the nucleic acid.
  • the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof, The nucleic acid, the expression vector or the host cell.
  • the present invention also provides a kit comprising the antibody or antigen-binding portion thereof, the nucleic acid, the expression vector or the host cell.
  • the present invention also provides use of the antibody or antigen-binding portion thereof, the nucleic acid, the expression vector or the host cell in preparing a drug or reagent for diagnosing, treating or preventing tumors.
  • the tumor is a tumor associated with CLDN18.2.
  • the tumor is gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer or breast cancer.
  • GEJ gastroesophageal junction
  • the antibody or antigen-binding fragment thereof of the present invention has high affinity and low immunogenicity, can bind to CLDN18.2 with high specificity, and has endocytosis activity, CDC activity, ADCC activity and ADCP phagocytosis.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
  • antibodies e.g., monoclonal antibodies
  • antigen-binding fragments thereof that specifically bind to CLDN18.2.
  • CLDN18.2 e.g., human CLDN18.2
  • the term "CLDN18.2" refers to any CLDN18.2 receptor known to those skilled in the art.
  • the CLDN18.2 may be from a mammal, for example, CLDN18.2 may be from a human or a cynomolgus monkey.
  • a full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds.
  • An "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody, but at least comprises a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and/or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived.
  • the heavy chain constant region is composed of three domains, namely CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • VL light chain variable region
  • CL light chain constant region
  • the light chain constant region is composed of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by more conservative framework regions (FRs).
  • Each VH and VL is composed of three CDRs and four FRs, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are arranged in order.
  • Each heavy chain and light chain of the antibody molecule has three CDRs (i.e., the heavy chain has HC-CDR1, HC-CDR2, HC-CDR3; the light chain has LC-CDR1, LC-CDR2, LC-CDR3).
  • CDR is also called a hypervariable region and is present in the variable region of each heavy chain and light chain of the antibody, with very high variability sites in the primary structure of the CDR.
  • the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the heavy chain, which has the same meaning as HC-CDR1, HC-CDR2, and HC-CDR3, and the CDR of the light chain is represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the light chain, which has the same meaning as LC-CDR1, LC-CDR2, and LC-CDR3.
  • These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen bound by the antibody.
  • Antibodies include antibody fragments, examples of which include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (Welschof & Krauss, 2003).
  • Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M-1) to an antigen.
  • “Functional fragments” or “analogs of anti-CLDN18.2 antibodies” are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.
  • functional fragments are generally synonymous with "antibody fragments", and with respect to antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, and the like.
  • the "Fv” fragment consists of a dimer (VH-VL dimer) formed by non-covalent binding of the variable domain of a heavy chain and the variable domain of a light chain.
  • variable domain interacts to determine the target binding site on the surface of the VH-VL dimer, as in the case of a complete antibody.
  • the six CDRs together confer target binding specificity to the complete antibody.
  • a single variable domain or half of an Fv that includes only three target-specific CDRs can still have the ability to recognize and bind to a target.
  • monoclonal antibody refers to a colony of identical antibodies, representing that each individual antibody molecule in a monoclonal antibody colony is identical to other antibody molecules. This characteristic is contrary to the characteristic of a polyclonal colony of antibodies, which comprises antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by many well-known methods. For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by merging with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal colony of host cells in vitro by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding antibodies.
  • chimeric antibody refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from mouse antibodies and the constant region sequences are derived from human antibodies.
  • Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins.
  • humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDRs complementary determining regions
  • PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Clusters typically contain chemically active surface patterns of molecules, such as amino acids or sugar side chains, and often have specific three-dimensional structural features as well as specific charge characteristics.
  • telomere binding As used herein, "specific binding” or “immunospecifically binds” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen.
  • the antigen may be an isolated antigen or present in a tumor cell.
  • an antibody that immunospecifically binds (or specifically binds) an antigen binds the antigen with an affinity constant Ka of about 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater (or a dissociation constant ( Kd ) of 1 ⁇ 10-7 M or 1 ⁇ 10-8 M or less).
  • the affinity constant can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.
  • nucleic acid molecule refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • expression refers to the process of producing a polypeptide by transcription and translation of a polynucleotide.
  • the expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • host cell is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • vector is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation.
  • Vectors also include those containing nucleic acids encoding polypeptides.
  • Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression/display of polypeptides encoded by nucleic acids.
  • Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors.”
  • Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.
  • expression vector includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA.
  • Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.
  • pharmaceutical composition refers to a pharmaceutically acceptable composition, which includes, for example, one or more, such as two, three, four, five, six, seven, eight, or more, of the therapeutic agents described herein, formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible, etc.
  • the carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
  • treating an individual suffering from a disease or condition means that the symptoms of the individual are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. Prevention refers to preventing potential diseases and/or preventing symptoms from worsening or disease development. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and any composition provided herein.
  • Diagnosis refers to an assessment of the likelihood that an individual has a disease or condition or is at risk of developing a disease or condition. In particular, as will be appreciated by those skilled in the art, such an assessment, while preferably correct for 100% of the subjects to be diagnosed, is generally not the case. In one embodiment, the term requires that a statistically significant portion of the subjects can be identified as having a disease or having a predisposition thereto.
  • therapeutic effect refers to the effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
  • EC50 also known as half maximal effect concentration, refers to the antibody concentration that elicits 50% of the maximal effect.
  • Figure 1 shows the binding activity of hybridoma supernatants on 293T-hCLDN18.2 cells.
  • Figures 2A-2G show the binding activity of H7E12-2 humanized antibody and human CLDN18.2.
  • FIG2A shows the binding activity of humanized antibody H7E12-2 on 293T-hCLDN18.2 cells
  • FIG2B shows a comparison of the expression intensity of human CLDN18.2 in different cell lines
  • Figure 2C shows the binding activity in CT-26-hCLDN18.2 cells
  • Figure 2D shows the binding activity in MC-38-hCLDN18.2-C4 cells
  • FIG2E shows the binding activity in 293T-hCLDN18.2 cells
  • FIG2F shows the binding activity in MC-38-hCLDN18.2-A11 cells
  • FIG. 2G shows the binding activity in KATOIII-hCLDN18.2 cells.
  • FIG. 3 shows the cross-binding activity of the H7E12-2 antibody to human CLDN18.1.
  • FIG. 4 shows the cross-binding activity of the H7E12-2 antibody to mouse CLDN18.2.
  • FIG5 shows the endocytic activity of the H7E12-2 antibody.
  • Fig. 6 shows the CDC activity of the H7E12-2 antibody.
  • Figures 7A-7C show the ADCC reporter cell line (Jurkat-NFAT-Luc2-CD16a-V158) activity of the H7E12-2 antibody.
  • FIG7A shows MC38-hCLDN18.2 as target cells
  • FIG7B shows 293T-hCLDN18.2-A11 as a target cell
  • FIG. 7C shows KATOIII-hCLDN18.2 as target cells.
  • FIG. 8 shows ADCC activity mediated by the H7E12-2 antibody in PBMC-derived NK cells.
  • FIG. 9 shows the macrophage-mediated ADCP activity of the H7E12-2 antibody.
  • CHO-K1 Human CLDN18.2 Cell Line (kyinno KC-1180) that stably expressed the CLDN18.2 gene (from Genbank accession number NM_001002026.3, its sequence is shown as SEQ ID NO: 1) was used as an immunogen to immunize mice. Five mice were immunized intraperitoneally, and the adjuvant used was Quick antibody 5W water-soluble adjuvant. The titer was measured 2 weeks after the booster immunization, and two mice with high titers were selected for immune shock. After 3 days, the cell fusion described below was performed.
  • mice Take two mice to be fused, collect serum, dissect and remove the spleen, separate spleen cells, fuse the spleen cells with cultured myeloma cells, plate 96-well plates, add selective culture medium for screening, change the medium after 7 days, perform ELISA test after 10 days, and select those with OD values greater than 10 times that of the negative control for flow cytometry detection.
  • Select the double positive cells perform subclone plating by cell limiting dilution method, and select monoclonal cells. Take the culture supernatant of the selected monoclonal cells, perform ELISA and flow cytometry detection, and select the double positive cells to expand the culture. keep.
  • Example 2 ELISA detection of the binding between the culture supernatant of hybridoma cells and CLDN18.2
  • Bxpc-3 Human CLDN18.2 Cell Line (purchased from Kangyuan Bochuang, cell number KC-1272) containing the CLDN18.2 gene (from Genbank accession number NM_001002026.3) was added to a 96-well cell culture plate at 1x10 4 cells/well and 100 ⁇ L/well, and cultured overnight at 37°C, 5% CO 2. The liquid in the well was discarded and washed 3 times with washing solution. 200 ⁇ L of blocking solution was added to each well and blocked overnight at 4°C for 2 hours.
  • the reaction was terminated by adding 2 mol/L H 2 SO 4 , and the OD value was read on an enzyme-linked immunosorbent reader.
  • Example 3 FACS detection of the binding between the culture supernatant of hybridoma cells and human CLDN18.2
  • the human CLDN18.2 gene (from Genbank accession number NM_001002026.3) was constructed into the PLVX virus packaging vector (clontech, virus package mix, catalog number 631275), transfected with 293T cells to package the virus, and the virus was used to infect 293T cells. Puromycin was added to screen for drug-resistant cell lines, i.e., 293T cells that stably express the human CLDN18.2 gene (hereinafter referred to as 293T-hCLDN18.2, cell number KC-0986, purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., hereinafter referred to as Kangyuan Bochuang).
  • 293T-hCLDN18.2 cells were prepared into a cell suspension with a cell concentration of 10 ⁇ 7 cells/ml in PBS containing 2% FBS. 50 ⁇ L of the cell suspension was added to each flow tube (sample tube), and then 50 ⁇ L of the culture supernatant of the hybridoma cells to be tested was added, and incubated at 4°C for 60 minutes. Add 1 ml of flow buffer to each flow tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat washing three times. At the same time, set up control tube 1 (no culture supernatant and the following secondary antibody are added, only the cell suspension is added) and control tube 2 (no culture supernatant is added, only the cell suspension and the following secondary antibody are added).
  • the corresponding mouse antibody 7E12 was obtained from the hybridoma cells, and the heavy chain and light chain variable region sequences were SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the heavy chain CDR1, CDR2, and CDR3 sequences were obtained according to the Kabat definition method.
  • the sequences of CDR1, CDR2 and CDR3 of the light chain are SEQ ID NO: 5 (KS SQSLFNSGNQKNYLA), SEQ ID NO: 7 (GASTRES) and SEQ ID NO: 9 (QNDHSFPFT), respectively.
  • mouse anti-7E12 For mouse anti-7E12, the following human germline sequences were selected as templates for the heavy chain and light chain: IGHV4-39 and IGKV1-39. Homology modeling was performed on mouse anti-7E12, and structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the 7E12 antibody was finally obtained.
  • the humanized antibody H7E12-2 was obtained by recombining the humanized heavy chain and light chain, and the heavy chain and light chain sequences were SEQ ID NO: 19 and SEQ ID NO: 17, respectively.
  • the sequence shown in SEQ ID NO: 23 was used as the heavy chain constant region, and the sequence shown in SEQ ID NO: 21 was used as the light chain constant region.
  • the mouse antibody and its humanized variable region sequences were combined in pairs to prepare chimeric antibodies and humanized antibodies.
  • the coding gene of the corresponding antibody sequence was synthesized, connected to the eukaryotic expression vector, transformed into DH5alpha competent cells, cultured in a 37°C constant temperature incubator overnight, and the monoclonal strain was selected for sequencing and identification.
  • the strain with the correct sequence was selected, the plasmid was extracted, and the mammalian expression cell HEK-293F was transfected, placed in an incubator at 37°C and 5% CO 2 , and the expression culture was carried out for 7 days.
  • H7E12-2 The expression supernatant was collected, centrifuged, filtered, and purified by protein G affinity chromatography column.
  • the purified antibody was tested for purity by SDS-PAGE electrophoresis, and the antibody concentration was tested by BCA protein detection kit.
  • the antibody was packaged and stored in a -80°C refrigerator for later use.
  • the sequences of the obtained chimeric antibody and humanized antibody (hereinafter referred to as H7E12-2) are shown in Table 1.
  • the engineered cell line 293T-hCLDN18.2 cells (cell number-KC-0986, purchased from Kangyuan Bochuang) overexpressing human CLDN18.2 membrane protein were first used. Binding activity detection was performed. 293T-hCLDN18.2 cells in the logarithmic growth phase were collected, washed once with flow cytometry buffer, and centrifuged at 1000 rpm for 4 minutes. The cells were resuspended with flow cytometry buffer, and the cell density was adjusted to 4 ⁇ 10 ⁇ 6/mL. 50 ⁇ L of the system was plated in a 96-well plate, and the wells without the test antibody and the secondary antibody were set as the blank control group and the wells with only the secondary antibody were set as the secondary antibody control group.
  • test antibody H7E12-2 and the positive control antibody Zolbetuximab were diluted 3.16 times to 10 gradient points, and 50 ⁇ L of the system was added to the plated cells, with a total system of 100 ⁇ L, and incubated at 4°C in the dark for 1 hour; the cells were washed twice with flow buffer, 200 ⁇ L each time, and centrifuged at 1500 rpm for 4 minutes.
  • the PE-labeled secondary antibody (PE anti-human IgG Fc Antibody, Biolegend) staining solution was prepared with flow buffer at a dilution ratio of 1:100, and 100 ⁇ L was added to the experimental group and the secondary antibody control group, and an equal volume of flow buffer was added to the blank control well; after mixing, it was incubated at 4°C in the dark for 1 hour; the cells were washed twice with flow buffer, 200 ⁇ L each time, and centrifuged at 1500 rpm for 4 minutes. The system was resuspended in 100 ⁇ L, and the average fluorescence intensity of the positive signal was detected by Guava easyCyte6HT (Millipore) flow cytometer.
  • CLDN18.2 compared the differences in the expression of CLDN18.2 on the cell membrane surface of 5 different genetically engineered cell lines, including the human CLDN18.2 overexpressing engineered cell line CT26-hCLDN18.2 (purchased from Nanjing Bowang), MC38-hCLDN18.2-C4 (purchased from Nanjing Bowang), MC38-hCLDN18.2-A11 (purchased from Nanjing Bowang), KATO III-hCLDN18.2 (cell number KC-1453, purchased from Kangyuan Bochuang), and 293T-hCLDN18.2 cells (cell number KC-0986, purchased from Kangyuan Bochuang).
  • CT26-hCLDN18.2 purchased from Nanjing Bowang
  • MC38-hCLDN18.2-C4 purchased from Nanjing Bowang
  • MC38-hCLDN18.2-A11 purchased from Nanjing Bowang
  • KATO III-hCLDN18.2 cell number KC-1453, purchased from
  • the H7E12-2 antibody has a significant affinity advantage. Although no effective EC 50 was obtained, it can be seen that the binding activity of H7E12-2 showed a concentration gradient-dependent increase, while the positive control antibody Zolbetuximab had no binding activity at all under low antigen density. In the cell MC38-hCLDN18.2-C4 with low antigen density, the EC 50 of H7E12-2 and Zolbetuximab binding activity were 0.2 ⁇ g/mL and 2.7 ⁇ g/mL, respectively, and the affinity was increased by 13.5 times.
  • the binding activity EC50 of H7E12-2 and Zolbetuximab was 0.7 ⁇ g/mL and 8.7 ⁇ g/mL, respectively, with an affinity increased by 12.7 times.
  • the H7E12-2 antibody in the present invention has significantly improved affinity compared with the positive control antibody Zolbetuximab in cell lines with different antigen expression intensities, especially in the case of low antigen abundance, which will provide an important theoretical basis for subsequent applications.
  • CLDN18.1 and CLDN18.2 belong to the Claudins family, with a sequence homology of about 92% (240/261), and only 21 amino acids differ in the first transmembrane region of the N-terminus (Sahin et al., 2008).
  • the binding activity of the H7E12-2 antibody to the human CLDN18.1 antigen was tested.
  • the experimental method was similar to that of Example 5, except that the cells used were engineered cell lines 293T-hCLDN18.1 cells that highly express human CLDN18.1 membrane protein (purchased from Kangyuan Bochuang, cell number-KC-0990).
  • the cross-recognition experimental method refers to Implementation Case 5, except that the cells used are 293T-mCLDN18.2 cells in the logarithmic growth phase (purchased from Kangyuan Bochuang, cell number-KC-1014), which overexpress the mouse CLDN18.2 antigen.
  • GraphPad Prism 7.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain a dose-effect curve, and the EC 50 value was calculated from this.
  • the experimental results are shown in Figure 4 and Table 5.
  • Receptor-mediated endocytosis is an important pathway for many membrane proteins to mediate the entry of antibodies and antibody-drug conjugates into cells.
  • the endocytic activity of H7E12-2 was evaluated.
  • 293T-hCLDN18.2 cells (cell number KC-0986, purchased from Kangyuan Bochuang) in the logarithmic growth phase were collected, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS, resuspended in flow buffer and counted, and the density was adjusted to 5 ⁇ 106 cells/mL and added to the flow tube.
  • the test antibody was added with a working final concentration of 10 ⁇ g/mL, and incubated at 4°C in the dark for 1 hour.
  • the cells were washed twice with pre-cooled flow buffer, and the cells were resuspended with 100 ⁇ L of washing solution and 2 ⁇ L of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody) was added, and incubated at 4°C in the dark for 40 minutes.
  • the cells were washed twice with pre-cooled flow buffer and resuspended in 200 ⁇ L complete medium (DMEM+10% FBS) with 100 ⁇ L of washing solution, and cultured in a 37°C incubator for 2h, 1h, 0.5h, and 0h, respectively.
  • DMEM+10% FBS complete medium
  • Dissociation Wash cells once with flow cytometry buffer, resuspend cells with 200ul of surface antibody dissociation buffer, and incubate at room temperature for 7min.
  • Neutralization Add neutralization buffer and centrifuge, and finally resuspend cells with 100 ⁇ L of flow cytometry buffer. Detect PE signals with NovoCyteQuanteon flow cytometer (Agilent). The experimental results are shown in Figure 5 and Table 6.
  • Complement-dependent cytotoxicity is the mechanism of action of many antibodies in causing tumor killing.
  • the Fab end of the antibody binds to the antigen
  • the Fc end of the antibody binds to complement C1q in the serum, activating the complement system to form a membrane attack complex that exerts a lytic effect on target cells. Therefore, the CDC activity of H7E12-2 and the positive control antibody Zolbetuximab was further compared.
  • 293T-hCLDN18.2 cell number KC-0986, purchased from Kangyuan Bochuang
  • Antibody-dependent cellular cytotoxicity is the main mechanism of action for many antibody drugs to kill tumor cells.
  • Fc ⁇ RIIIA CD16a
  • Fc ⁇ RIIIA CD16a
  • NK cells NK cells on tumor cells.
  • Jurkat-NFAT-Luc2-CD16a-V15 constructed by Kangyuan Bochuang
  • MC38-hCLDN18.2-A11 purchased from Nanjing Bowang
  • 293T-hCLDN18.2 cell number KC-0986, purchased from Kangyuanbo
  • KATO III-hCLDN18.2 cell number KC-1453, purchased from Kangyuan Bochuang
  • the extracellular region of this effector cell overexpresses high-affinity Fc ⁇ RIIIA CD16a-V158 was co-incubated with target cells and different concentrations of test antibodies (starting at 20 ⁇ g/mL, 5-fold dilution, 9 concentration points).
  • the antibody Fc end bound to the extracellular region with high affinity CD16a-V158 to activate the NFAT-luc2 luciferase reporter system.
  • the ADCC activity of the antibody can be detected by detecting the content of luciferase using a Plus multifunctional microplate reader (BMG LABTECH).
  • the data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was obtained by fitting the data using nonlinear S-curve regression, and the EC 50 value was calculated from this.
  • the experimental results are shown in Figures 7A-7C and Table 8.
  • the fluorescence signal of Jurkat-NFAT-Luc2-CD16a-V15 reporter cell line showed that the ADCC activity of H7E12-2 antibody and positive control antibody Zolbetuximab in high-density cell lines MC38-hCLDN18.2-A11 and H7E12-2 was significantly better than that of positive control antibody Zolbetuximab, with EC 50 of 9.6 ⁇ g/mL and 272.7 ⁇ g/mL, respectively (Figure 7A). They had similar killing activity on 293T-hCLDN18.2 cells with medium-density antigen level (Figure 7B), with EC 50 of 125.1 ⁇ g/mL and 29.7 ⁇ g/mL, respectively.
  • ADCC is caused by the Fab end of the antibody binding to the antigen epitope of the tumor cell, and its Fc end binding to the FC ⁇ R on the surface of the natural killer cell (NK cell).
  • the NK cell is activated to release cytotoxic substances such as perforin and granzyme, which mediate the killing of target cells by NK cells and cause apoptosis of target cells.
  • PBMCs peripheral blood mononuclear cells
  • 293T-hCLDN18.2 cell number KC-0986, purchased from Kangyuan Bochuang
  • the target cells 293T-hCLDN18.2 were labeled with 1.6 ⁇ M CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester, 565082, BD), and labeled for 10 minutes at room temperature in the dark.
  • the labeled cells were washed twice with 5 volumes of pre-cooled serum-free medium, and the labeled cells were resuspended in ADCC medium and counted.
  • 5 x 10 ⁇ 4 cells were added to each well of a 96-well U-shaped plate, and then the test antibody was added for gradient dilution.
  • the starting concentration was 50 ⁇ g/mL, and the gradient was diluted 3.16 times with complete medium, with a total of 10 concentration points.
  • the target cell effector cell antibody complex was incubated at 37°C for 4 hours. After the incubation, the cells were washed twice with PBS + 2% FBS, and 100 ⁇ L of PBS solution containing 1 ⁇ L 7-AAD (559925, BD) was added to each well for staining, incubated at room temperature in the dark for 10 minutes, and the activity of the target cells was detected using a NovoCyteQuanteon flow cytometer (Agilent). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was fitted using nonlinear S-curve regression, and the EC 50 value was calculated from this. The experimental results are shown in Figure 8 and Table 9.
  • H7E12-2 still showed strong ADCC activity with an EC 50 of 12 ng/mL, while the EC 50 of the positive control antibody Zolbetuximab was 74 ng/mL.
  • the ADCC activity mediated by primary NK cells of H7E12-2 increased by 6.17 times, indicating that its tumor killing activity was superior to that of Zolbetuximab.
  • Antibody-dependent cell-mediated phagocytosis is another important mechanism for antibody drugs to kill tumor cells.
  • the Fab of the antibody binds to the antigen, causing conformational changes and promoting the binding of the Fc end of the antibody to Fc ⁇ RIIA (CD32a) on the surface of macrophages, and activating the ITAM signal downstream of Fc ⁇ RIIA, promoting macrophages to phagocytose tumor antigen-positive cells.
  • mouse bone marrow-derived macrophages were used to differentiate into 293T-hCLDN18.2 (cell number KC-0986, purchased from Kangyuan Bochuang) cells were co-cultured to evaluate the ADCP activity of the H7212-2 antibody.
  • the femur and tibia of the mouse were dissected aseptically, and the surface tissue was shaved off. The two ends were cut with scissors, and the bone marrow was blown out with a syringe and collected by centrifugation. The culture medium was resuspended and counted, and mouse M-CSF was used to differentiate BMDM.
  • the induced BMDM was digested with 1mL Accutase, centrifuged at 1200 rpm for 5 minutes, and resuspended with 1640+5% FBS for counting and set aside.
  • the target cells were labeled with CFSE at a final concentration of 1.6 ⁇ M/mL, resuspended and counted in 1640+5% FBS for staining at room temperature in the dark, and plated into a low-absorption 96-well U-bottom plate at 5x10 ⁇ 4 cells/well, 80 ⁇ L per well, and added 40 ⁇ L/well of the gradient diluted test drug, followed by slow instant centrifugation.
  • the induced BMDM were plated into a low-absorption 96-well U-bottom plate according to the corresponding effector-target ratio, 80 ⁇ L per well, mixed with a shot gun, and incubated at 37 degrees for 4 hours.
  • H7E12-2 in FUT8 knockout HEK293/FUT8- cells purchased from Kangyuan Bochuang Biotechnology Co., Ltd. (KC-2300).
  • the cell line was passaged in Expi293TM expression medium for production, with a passage density of 0.2-0.3*E6/ml and a passage cycle of 2-3 days.
  • the cells were diluted to 2*E6/ml the day before transfection.
  • the cell density should be around 6E6/ml and the viability should be greater than 95%.
  • the transfection was performed according to the instructions of the transfection kit.
  • the plasmids used were 432-M147-LC and 432-M147-HC, and the molar ratio of light chain to heavy chain was 1:1.
  • Enhancer1 and Enhancer2 were added at a final concentration of 0.6% v/v 18-22 hours after transfection. After the fifth day of transfection, the cell viability was monitored. When the cell viability was less than 70%, the cells were harvested and the protein was purified and named ICP.
  • the method for detecting the ADCC activity of ICP is as described in Example 11, using 293T-hCLDN18.2 (cells purchased from Kangyuan Bochuang, cell number KC-0986) cells as target cells and co-cultured with PBMC-derived primary NK cells to detect ADCC activity mediated by the Fc end of the fusion protein.

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Abstract

本发明涉及一种新型的人源化抗人CLDN18.2抗体及其抗原结合片段。本发明还涉及所述抗体的制备方法。本发明进一步涉及所述抗体或其抗原结合片段通过靶向结合CLDN18.2治疗和/或预防肿瘤的方法。

Description

一种能够结合CLDN18.2的抗体或其抗原结合片段及其应用 技术领域
本发明属于生物医药领域，具体涉及一种能够结合CLDN18.2的抗体或其抗原结合片段，本发明还涉及所述抗体的应用。
背景技术
Claudins是紧密连接蛋白家族中重要的成员，在细胞与细胞的连接及细胞与基质的连接中起着重要作用。Claudin18由基因CLDN18编码，是一个四次跨膜蛋白，具有两个胞外区，广泛分布在胃、胰腺和肺等组织均有表达。Claudin18被认为可作为诊断标志物及治疗靶点(Krause,G.,Winkler,L.,Mueller,S.L.,Haseloff,R.F.,Piontek,J.,&Blasig,I.E.(2008).Structure and function of claudins.Biochimica et Biophysica Acta(BBA)-Biomembranes,1778(3),631-645.)。
Claudin18家族中包含Claudin18.1(CLDN18.1)与Claudin18.2(CLDN18.2)两个变体，其中，CLDN18.1特异性表达在肺泡上皮细胞上(Yasui,W.,Sentani,K.,Sakamoto,N.,Anami,K.,Naito,Y.,&Oue,N.(2011).Molecular pathology of gastric cancer:Research and practice.Pathology-Research and Practice,207(10),608-612.)，与CLDN18.2只有21个氨基酸的差别(Sahin et al.,2008)，CLDN18.2广泛存在于胃部肿瘤组织中；最新发现其在胰腺癌、食管癌和肺癌中也有表达(Jovov et al.,2007；Karanjawala et al.,2008)。正常细胞由于细胞间紧密的连接，抗体不能接触到细胞表达的Claudin18.2；而在肿瘤细胞中，由于细胞病变导致细胞间的连接变得松散，从而将CLDN18.2暴露在外，抗体可以结合这些暴露在外的CLDN18.2分子。因此，CLDN18.2已经成为一种理想的药物靶点分子(Klamp,T.,Schumacher,J.,Huber,G.,Kuhne,C.,Meissner,U.,Selmi,A.,Sahin,U.(2011).Highly specific auto-antibodies against claudin-18isoform 2induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.Cancer Res,71(2),516-527.)。
Zolbetuximab(IMAB362)是第一个针对CLDN18.2靶点开发的嵌合的抗体药物，其并没有进行人源化，能够在肿瘤细胞表面与CLDN18.2特异结合，从而引发抗体依赖性细胞毒性(ADCC)、补体依赖性细胞毒性(CDC)、凋亡以及抑制细胞增殖(Woll,S.,Schlitter,A.M.,Dhaene,K.,Roller,M.,Esposito,I.,Sahin,U.,&Tureci,O.(2014).Claudin 18.2is a  target for IMAB362antibody in pancreatic neoplasms.Int J Cancer,134(3),731-739)。临床前研究已经成功证实Zolbetuximab具有清除癌细胞和控制疾病的强大能力；随后，通过多个I/II期试验评估了其临床疗效和安全性，但以CLDN18.2为靶点的胃癌治疗中仍然存在尚未满足的临床需求(Lordick,F.,Al-Batran,S.E.,Ganguli,A.,Morlock,R.,Sahin,U.,&Tureci,O.(2021).Patient-reported outcomes from the phase II FAST trial of zolbetuximab plus EOX compared to EOX alone as first-line treatment of patients with metastatic CLDN18.2+gastroesophageal adenocarcinoma.Gastric Cancer,24(3),721-730)。因此开发特异性结合CLDN18.2而不结合CLDN18.1，同时具有高度人源化，低免疫原性抗体的单克隆抗体在胃癌等肿瘤的诊断和开发上具有重要意义。
发明内容
本发明要解决的技术问题是获得可以特异性结合CLDN18.2，而不结合CLDN18.1，并具有高亲和力、低免疫原性，以及良好的临床前景的抗体。为此，发明人制备得到了一种抗人CLDN18.2的抗体或其抗原结合片段，相比于现有技术，本发明所得的抗体或其抗原结合片段具有更高的亲和力以及更低免疫原性。
本发明的抗体可以有多种用途，包括检测Claudin 18.2蛋白，诊断、治疗或预防Claudin18.2相关癌症等。
因此，在一个方面本发明涉及一种抗CLDN18.2抗体或其抗原结合片段，其包含：如氨基酸序列SEQ ID NO：5所示的轻链互补决定区LC-CDR1、如氨基酸序列SEQ ID NO：7所示的轻链互补决定区LC-CDR2和如氨基酸序列SEQ ID NO：9所示的轻链的互补决定区LC-CDR3；以及如氨基酸序列SEQ ID NO：11所示的重链互补决定区HC-CDR1、如氨基酸序列SEQ ID NO：13所示的重链互补决定区HC-CDR2、如氨基酸序列SEQ ID NO：15所示的重链的互补决定区HC-CDR3。
在一些实施方案中，其重链、轻链可变区氨基酸序列分别为SEQ ID NO：3和SEQ ID NO：4所示。
在一些实施方案中，本发明还提供上述抗体或其抗原结合片段的人源化抗体或其抗原结合片段。
在一些实施方案中，该人源化抗体特征在于，其轻链可变区氨基酸序列为SEQ ID NO：17所示；重链可变区氨基酸序列为SEQ ID NO：19所示。
在一个方面，本发明由此提供编码所述的抗体或其抗原结合部分的核酸。
在一个方面，本发明还提供包含所述核酸的表达载体和/或宿主细胞。
在一个方面，本发明提供一种药物组合物，其包含所述的抗体或其抗原结合部分、所 述的核酸、所述的表达载体或宿主细胞。
本发明也提供一种试剂盒，其包含所述的抗体或其抗原结合部分、所述的核酸、所述的表达载体或宿主细胞。
本发明还提供其包含所述的抗体或其抗原结合部分、所述的核酸、所述的表达载体或宿主细胞在制备用于诊断、治疗或预防肿瘤的药物或试剂中的用途。
更进一步地，所述肿瘤是与CLDN18.2相关的肿瘤。
更进一步地，所述肿瘤是胃癌、胃食管交界处(GEJ)腺癌、胰腺癌、食管癌、支气管癌或乳腺癌。
本发明的抗体或其抗原结合片段具有高亲和力、低免疫原性，可以高特异性的结合CLDN18.2，具有内吞活性、CDC活性、ADCC活性和ADCP吞噬作用。
定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
本文提供的是特异性结合CLDN18.2的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合CLDN18.2(例如，人CLDN18.2)的抗体。术语“CLDN18.2”是指本领域技术人员已知的任何CLDN18.2受体。例如所述CLDN18.2可以来自哺乳动物，例如CLDN18.2可以来自人或食蟹猴。
如本文所用，全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(complementarity-determining region，CDR)的高变区，其由较为保守的骨架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、 FR3、CDR3、FR4的顺序排布。抗体分子的每个重链和轻链具有3个CDR(即重链具有HC-CDR1、HC-CDR2、HC-CDR3；轻链中具有LC-CDR1、LC-CDR2、LC-CDR3)。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，与HC-CDR1、HC-CDR2、HC-CDR3具有相同含义，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，与LC-CDR1、LC-CDR2、LC-CDR3具有相同含义。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
抗体包括抗体片段，抗体片段的实例包括但不限于Fab、Fab'、F(ab')2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(Welschof&Krauss,2003)。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约107-108M-1的Ka)抗原的抗体。“功能片段”或“抗CLDN18.2抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab')2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定VH-VL二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
在本文中，氨基酸的三字母或单字母缩写已以其常规含义使用，如下表中所示：

如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本公开所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定 簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约1×107M-1或1x108M-1或更大的亲和常数Ka(或者1x10-7M或1×10-8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用“药物组合物”是指药学上可接受的组合物，其包括例如本文所述的治疗剂中的一种或多种，例如两种、三种、四种、五种、六种、七种、八种、或更多种，连同药学上可接受的载剂一起配制。如本文所用，“药学上可接受的载剂”包括生理学上相容的任何和所有溶剂、分散介质、等渗剂和吸收延迟剂等。载剂可适用于静脉内、肌肉内、皮下、肠胃外、直肠、脊柱或表皮施用(例如通过注射或输注)。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
“诊断”是指评估个体患有疾病或病症或处于发展疾病或病症的风险的可能性。特别地，如本领域技术人员将理解的，这种评估虽然优选对于100％的待诊断受试者都是正确的，但通常并非如此。在一个实施方案中，所述术语要求统计学上显著部分的受试者可以被鉴定为患有疾病或具有其易感体质。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
术语“EC50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
附图说明
图1表示了杂交瘤上清在293T-hCLDN18.2细胞上的结合活性。
图2A-图2G表示了H7E12-2人源化抗体和人源CLDN18.2的结合活性。其中：
图2A表示了人源化抗体H7E12-2在293T-hCLDN18.2细胞上的结合活性；
图2B表示了不同细胞系人CLDN18.2表达强度的比较；
图2C表示了在CT-26-hCLDN18.2细胞的结合活性；
图2D表示了在MC-38-hCLDN18.2-C4细胞的结合活性；
图2E表示了在293T-hCLDN18.2细胞的结合活性；
图2F表示了在MC-38-hCLDN18.2-A11细胞的结合活性；
图2G表示了在KATOIII-hCLDN18.2细胞的结合活性。
图3表示了H7E12-2抗体交叉结合人CLDN18.1的活性。
图4表示了H7E12-2抗体交叉结合小鼠CLDN18.2的活性。
图5表示了H7E12-2抗体的内吞活性。
图6表示了H7E12-2抗体的CDC活性。
图7A-图7C表示了H7E12-2抗体的ADCC报告细胞系(Jurkat-NFAT-Luc2-CD16a-V158)活性。其中：
图7A表示了MC38-hCLDN18.2作为靶细胞；
图7B表示了293T-hCLDN18.2-A11作为靶细胞；
图7C表示了KATOIII-hCLDN18.2作为靶细胞。
图8表示了H7E12-2抗体在PBMC来源的NK细胞中介导的ADCC活性。
图9表示了H7E12-2抗体的巨噬细胞介导的ADCP活性。
具体实施方案
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试剂、原料等，如无特殊说明，均为市售购买产品。Zolbetuximab(IMAB362)：Ganymed Pharmaceuticals，参见NW_004504382.1。
实施例1杂交瘤细胞的制备
用稳转并表达了CLDN18.2基因(来自Genbank登记号NM_001002026.3,其序列如SEQ ID NO：1)的CHO-K1 Human CLDN18.2 Cell Line(kyinno KC-1180)作为免疫原免疫小鼠。5只小鼠腹腔免疫，佐剂采用Quick antibody 5W水溶性佐剂，加强免疫以后2周测效价，选取效价高的两只小鼠进行免疫冲击，3天之后进行下文所述的细胞融合。
取准备融合的两只小鼠，取血清，解剖后取脾脏，分离脾细胞，将该脾细胞与培养的骨髓瘤细胞进行融合，铺96孔板，同时加入选择性培养基进行筛选，7天后换液，10天后进行ELISA检测，选择OD值大于阴性对照10倍以上的再进行流式细胞仪检测。
挑选双阳的细胞，通过细胞有限稀释法进行亚克隆铺板，挑选单克隆细胞。将挑选出的单克隆细胞，取培养上清，进行ELISA检测及流式细胞仪检测，挑选双阳的细胞扩大培 养。
实施例2杂交瘤细胞的培养上清与CLDN18.2结合的ELISA检测
用包含CLDN18.2基因(来自Genbank登记号NM_001002026.3)的Bxpc-3 Human CLDN18.2 Cell Line(购自康源博创，细胞编号KC-1272)，以1x104个细胞/孔、100μL/孔加入96孔细胞培养板中，37℃，5％CO2培养过夜。弃去孔内的液体，同时用洗涤液洗3次。每孔加入200μL封闭液4℃过夜封闭2小时。
向每孔中加入50-100μL待检杂交瘤细胞的培养上清，同时设立阳性对照(加入融合小鼠的血清)、阴性对照(加入正常小鼠的血清)和空白对照(加入培养基)。在37℃孵育1-2小时，洗涤。然后向每孔中加入酶标二抗，为1:10000稀释的辣根过氧化物酶标记的羊抗小鼠IgG(SIGMA，货号A9044-2ml)，每孔50-100μL，37℃孵育0.5-1小时，洗涤。向每孔中加入新鲜配制的底物显色液TMB 50-100μL，37℃孵育10-30分钟。
加入2mol/L H2SO4终止反应，在酶联免疫阅读仪上读取OD值。
结果判定：以P/N>2:1(P代表阳性数值，N代表正常小鼠血清数值)为阳性。若阴性对照孔无色或接近无色，阳性对照孔明确显色，则可直接用肉眼观察结果。
实施例3杂交瘤细胞的培养上清与人CLDN18.2结合的FACS检测
将人CLDN18.2基因(来自Genbank登记号NM_001002026.3)构建到PLVX病毒包装载体(clontech，virus package mix，货号631275)，转染293T细胞包装病毒，用该病毒感染293T细胞，加药嘌呤霉素(puromycin)筛选得到耐药细胞株即稳定表达人CLDN18.2基因的293T细胞(后续以293T-hCLDN18.2表示，细胞编号KC-0986，购自购自康源博创生物科技(北京)有限公司，以下简称康源博创)。将293T-hCLDN18.2细胞在含2％FBS的PBS中制备成细胞浓度为10^7个细胞/ml的细胞悬液。向每个流式管(样品管)中加入50μL细胞悬液，然后加入50μL待检杂交瘤细胞的培养上清，4℃孵育60分钟。向每个流式管中加入1ml的流式缓冲液，1200转离心5分钟，弃上清，重复洗涤三次。同时设置对照管1(不加入培养上清和下文的二抗，仅加入细胞悬液)和对照管2(不加入培养上清，仅加入细胞悬液和下文的二抗)。
然后向每个流式管中加入100μL流式缓冲液进行重悬，根据实验要求加入5μL PE标记的抗鼠Fc标签的二抗(Biolegend，货号409304)，4℃避光孵育30分钟，然后加入1ml流式缓冲液，室温下1200转离心5分钟，弃上清，重复洗涤三次。再向每个流式管中加入250μL流式缓冲液，重悬混匀，上机检测。目标抗体与人CLDN18.2的结合检测结果见图1。
从上述杂交瘤细胞获得相应的鼠源抗体7E12，其重链、轻链可变区序列分别为SEQ ID NO：3和SEQ ID NO：4，根据Kabat定义方法得到重链CDR1、CDR2、CDR3序列分别 为SEQ ID NO：11(RYGVH)，SEQ ID NO：13(VIWAGGSTNYNSALMS)，SEQ ID NO：15(EGLRHTMDY)，轻链CDR1、CDR2、CDR3序列分别为SEQ ID NO：5(KS SQSLFNSGNQKNYLA)，SEQ ID NO：7(GASTRES)和SEQ ID NO：9(QNDHSFPFT)。
实施例4 7E12鼠抗的人源化改造
对鼠抗7E12，分别选取了如下人germline的序列作为重链与轻链的模板：IGHV4-39和IGKV1-39。对鼠抗7E12进行同源建模，进行Fab区域的结构模拟。经过同源建模计算，最后得到预测的7E12抗体的Fab结构。
通过将预测的Fab结构及重链与IGHV4-39序列进行比对分析，将该重链CDR区中2V、37V、48L、67L、71K、73N、76R、78V保留为原始鼠的氨基酸，其它的鼠的氨基酸均替换成相应的IGHV4-39模板人的氨基酸。
通过对预测的Fab结构及轻链与IGKV1-39序列进行比对分析，将该VL中除了CDR区保留原始鼠的氨基酸外，其它的鼠的氨基酸均替换成相应的IGKV1-39模板人的氨基酸。将人源化的重链和轻链重组后得到的人源化抗体H7E12-2，重链和轻链序列分别为SEQ ID NO：19和SEQ ID NO：17。
以SEQ ID NO：23所示序列作为重链恒定区，SEQ ID NO：21所示序列作为轻链恒定区，将鼠抗及其人源化可变区序列分别两两组合，制备嵌合抗体和人源化抗体。其中，合成相应抗体序列的编码基因，连入真核表达载体中，转化DH5alpha感受态细胞，37℃恒温培养箱培养过夜，挑单克隆菌株测序鉴定。挑选序列正确的菌株，提取质粒，转染哺乳动物表达细胞HEK-293F，置于37℃、5％CO2的培养箱中，表达培养7天。
收取表达上清，离心，过滤，选择protein G亲和层析柱进行纯化，纯化的抗体通过SDS-PAGE电泳检测纯度，用BCA蛋白检测试剂盒检测抗体浓度，分装，保存于-80℃冰箱中备用。获得的嵌合抗体和人源化抗体(以下简称H7E12-2)的序列如表1所示。
表1嵌合抗体和人源化抗体序列
实施例5 H7E12-2抗体与人CLDN18.2结合的FACS检测
为了测定人源化H7E12-2抗体与人的CLDN18.2蛋白的结合活性，首先用过表达人CLDN18.2膜蛋白的工程细胞系293T-hCLDN18.2细胞(细胞编号-KC-0986,购自康源博创) 进行结合活性检测。收集处于生长对数期的293T-hCLDN18.2细胞，用流式缓冲液洗涤一次，1000转离心4分钟。用流式缓冲液重悬细胞，调整细胞密度至4×10^6/mL，以50μL体系铺96孔板中，设置待测抗体和二抗均不加孔为空白对照组和只加二抗孔为二抗对照组。
实验组将待测抗体H7E12-2和阳性对照抗体Zolbetuximab(WO2014/146778A1)以20ug/mL的浓度起始，3.16倍稀释10个梯度点，50μL体系加入已铺板的细胞中，总体系100μL,放于4℃避光孵育1小时；使用流式缓冲液洗涤细胞2次，每次200μL，1500转/分钟速度下，离心4分钟。用流式缓冲液以1:100的稀释比配置PE标记的二抗(PE anti-human IgG Fc Antibody，Biolegend)染色液，在实验组、二抗对照组中加入100μL，空白对照孔加入等体积的流式缓冲液；混匀后，放于4℃避光孵育1小时；使用流式缓冲液洗涤细胞2次，每次200μL，1500转/分钟速度下，离心4分钟。100μL体系重悬，用Guava easyCyte6HT(Millipore)流式细胞仪检测阳性信号的平均荧光强度，使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线四参数拟合Y＝Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))回归来拟合数据得出剂量-效应曲线，其中Bottom表示荧光信号下限值，代表S曲线的下渐进线，Top表示荧光信号的上限值，代表着S曲线的上渐近线。HillSlope为吸光度增加速率参数，相当于曲线的斜率，并由此计算EC50值，实验结果见图2A和表2。
表2.不同CLDN18.2抗体与人CLDN18.2结合的FACS检测结果
实验结果显示，H7E12-2人源化单抗结合293T-hCLDN18.2细胞上过表达的人CLDN18,2抗原的EC50为0.54μg/mL,相较在三期临床的同类药物Zolbetuximab(EC50为1.71μg/mL)，亲和力提高了3.16倍。
进一步，发明者比较了5株不同基因工程改造的细胞系包括过表达人的CLDN18.2工程细胞系CT26-hCLDN18.2(购自南京博望)、MC38-hCLDN18.2-C4(购自南京博望)、MC38-hCLDN18.2-A11(购自南京博望)和KATO III-hCLDN18.2(细胞编号KC-1453，购自康源博创)和293T-hCLDN18.2细胞(细胞编号KC-0986，购自康源博创)等5株细胞其细胞膜表面CLDN18.2表达量的差异，用1μg/mL的H7E12-2抗体与8x 10^4上述不同的细胞进行孵育，按照上述结合活性的实验步骤进行流式染色和数据处理，并将不同细胞表达的H7E12-2结合平均荧光强度统计如图2B和表3所示。结果显示这5株细胞系膜表面CLDN18.2的表达水平排序为CT26-hCLDN18.2<MC38-hCLDN18.2-C4<293T-hCLDN18.2< KATOIII-hCLDN18.2<MC38-hCLDN18.2-A11。
表3.不同工程细胞系CLDN18.2表达量的比较
接下来，利用这些携带不同膜表面CLDN18.2分子密度的细胞系对H7E12-2和阳性对照抗体Zolbetuximab进行全面的结合活性检测，实验步骤如上所述，不同之处在于H7E12-2抗体的浓度从100μg/mL起始，3.16倍稀释8个浓度，阴性对照为hIgG1和空白二抗对照，用NovoCyteQuanteon流式细胞仪(安捷伦)检测PE的信号，使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值，实验结果见图2C-2G，和表4。其中，EC50的单位为μg/mL。
表4.H7E12-2在不同人CLDN18.2抗原强度细胞系的结合活性总结
在人CLDN18.2荧光强度最低的细胞CT26-hCLDN18.2中，可以看到H7E12-2抗体明显的亲和力优势，虽然没有得到有效的EC50，但可以看到H7E12-2的结合活性显示浓度梯度依赖的升高，而阳性对照抗体Zolbetuximab在低抗原密度的情况下，完全没有结合活性。在低抗原密度的细胞MC38-hCLDN18.2-C4中，H7E12-2和Zolbetuximab结合活性EC50分别为0.2μg/mL和2.7μg/mL，亲和力提高13.5倍。
在中等抗原密度的细胞293T-hCLDN18.2的两次实验中，由于流式机器激光强度不同，图2A和2E的MFI绝对值有差别，但两次实验中H7E12-2结合293T-hCLDN18.2细胞表面抗原的EC50很接近，分别为0.54μg/mL和0.8μg/mL，而阳性对照抗体Zolbetuximab的EC50分别为1.7μg/mL和1.8μg/mL，两次实验均显示H7E12-2在293T-hCLDN18.2细胞中结合活性增强了2.25～3.16倍。
在高抗原密度的细胞KATOIII-hCLDN18.2，H7E12-2和Zolbetuximab结合活性EC50 分别为0.7μg/mL和1.5μg/mL，亲和力相差2.1倍；另一株高抗原密度的细胞MC38-hCLDN18.2-A11中，H7E12-2和Zolbetuximab结合活性EC50分别为0.7μg/mL和8.7μg/mL，亲和力提高了12.7倍。
综上，本发明中的H7E12-2抗体较阳性对照抗体Zolbetuximab在不同抗原表达强度的细胞系上都有显著的亲和力提升，尤其是在低抗原丰度的情况下，将为其中后续的应用中提供重要的理论依据。
实施例6 H7E12-2抗体与人CLDN18.1结合的FACS检测
CLDN18.1和CLDN18.2同属于Claudins家族，序列同源性约92％(240/261)，仅在N端第一个跨膜区存在21个氨基酸差异(Sahin et al.,2008)，为了进一步研究H7E12-2抗体的特异性，测试了H7E12-2抗体与人的CLDN18.1抗原的结合活性，实验方法参考实施案例5，不同之处是所用细胞为工程化改造的高表达人CLDN18.1膜蛋白的细胞系293T-hCLDN18.1细胞(购买自康源博创，细胞编号-KC-0990)，使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值，实验结果见图3。如图所示，H7E12-2和阳性对照抗体Zolbetuximab在浓度高达100μg/mL的情况下，没有与人CLDN18.1抗原的交叉结合活性，提示H7E12-2具有很高的特异性。
实施例7 H7E12-2抗体与小鼠CLDN18.2结合的FACS检测
接下来验证了H7E12-2识别鼠源CLDN18.2抗原的交叉识别活性，交叉识别活性将极大的方便动物药效实验的评价。交叉识别实验方法参考实施案例5，不同之处是所用细胞为生长对数期的细胞293T-mCLDN18.2细胞(购买自康源博创，细胞编号-KC-1014)，该细胞过表达小鼠的CLDN18.2抗原，使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值，实验结果见图4和表5。
表5.H7E12-2抗体与293T-mCLDN18.2细胞结合的FACS检测结果
交叉识别的实验结果显示：H7E12-2和阳性对照抗体Zolbetuximab均具有交叉识别小鼠CLDN18.2的能力，EC50分别为0.7μg/mL和2.8μg/mL，H7E12-2结合小鼠CLDN18.2的亲和力比阳性对照抗体Zolbetuximab提高了4倍。
实施例8 H7E12-2抗体的内吞实验
受体介导的内吞是很多膜蛋白介导抗体以及抗体偶联药物进入细胞重要的途径，因此 评价了H7E12-2的内吞活性。收集对数生长期的293T-hCLDN18.2细胞(细胞编号KC-0986，购买自康源博创)，1000转/分钟速度下离心5分钟后弃上清，使用PBS洗涤细胞一次，使用流式缓冲液重悬细胞计数，调整密度为5×106细胞/mL加到流式管中，加入测试抗体，工作终浓度为10μg/mL，置于4℃避光孵育1小时，使用预冷的流式缓冲液洗涤细胞2次，并用100μL洗液重悬细胞后加入2μL的PE标记的二抗(PE anti-human IgG Fc Antibody)，置于4℃避光孵育40分钟，使用预冷的流式缓冲液洗涤细胞2次，并用100μL洗液重悬于200μL完全培养基(DMEM+10％FBS)，分别0h，1h，1.5h，2h放置于在37℃孵箱中培养2h，1h，0.5h，0h。解离：流式缓冲液洗涤细胞1次，使用200ul的表面抗体解离缓冲液-重悬细胞，室温孵育7min。中和：加入中和缓冲液离心，最后使用100μL流式缓冲液重悬细胞。用NovoCyteQuanteon流式细胞仪(安捷伦)检测PE的信号。实验结果见图5和表6。
表6.H7E12-2抗体的内吞活性总结
实验结果显示，与阳性对照抗体Zolbetuximab相比，H7E12-2显示出显著的内吞增强活性，2小时后的内吞率高达63％，而Zolbetuximab只有32.5％。
实施例9 H7E12-2人源化抗体的CDC活性检测
补体依赖的细胞毒性作用(CDC)很多抗体引起肿瘤杀伤的作用机制，当抗体的Fab端与抗原结合后，抗体Fc端与血清中的补体C1q，激活补体系统形成攻膜复合物对靶细胞发挥裂解效应。因此，进一步比较了H7E12-2和阳性对照抗体Zolbetuximab的CDC活性。将293T-hCLDN18.2(细胞编号KC-0986，购买自康源博创)细胞离心计数，以每孔1x10^5个细胞接种到U形底96孔板，加入待测抗体后分别加入56℃ 30分钟灭活的人AB血清和非灭活人AB血清，37℃孵育4小时，用流式缓冲液清洗细胞两遍，每孔加入100μL含1μL与凋亡细胞的DNA结合的核酸染料7-AAD(559925，BD)的PBS溶液染色，室温避光孵育10分钟，用NovoCyteQuanteon流式细胞仪(安捷伦)检测靶细胞的活性。使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值。实验结果见图6和表7。
表7.H7E12-2抗体的CDC活性总结

结果显示H7E12-2抗体具有较强的CDC活性，EC50为2.1μg/mL,而阳性对照抗体Zolbetuximab完全没有CDC的活性。
实施例10 H7E12-2抗体的ADCC报告细胞系活性检测
抗体依赖的细胞毒作用(ADCC)是许多抗体药物杀伤肿瘤细胞的主要作用机制。FcγRⅢA(CD16a)可以介导NK细胞对肿瘤细胞的ADCC作用，在此利用Jurkat-NFAT-Luc2-CD16a-V15(由康源博创公司构建)作为替代的效应细胞分别与三种过表达人的CLDN18.2的靶细胞MC38-hCLDN18.2-A11(购自南京博望)，293T-hCLDN18.2(细胞编号KC-0986，购自康源博)、KATO III-hCLDN18.2(细胞编号KC-1453，购自康源博创)以效靶比为1：1的比例共同孵育，此效应细胞的胞外区过表达高亲和力FcγRⅢA CD16a-V158，与靶细胞和不同浓度的测试抗体(20μg/mL起始，5倍稀释，9个浓度点)共孵育，抗体Fc端结合胞外区高亲和力CD16a-V158激活NFAT-luc2荧光素酶报告系统，通过Plus多功能酶标仪(BMG LABTECH)检测荧光素酶的含量可以检测抗体的ADCC活性的情况。使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值。实验结果见图7A-7C和表8。
表8.H7E12-2抗体的ADCC报告细胞系活性总结
Jurkat-NFAT-Luc2-CD16a-V15报告细胞系荧光信号显示，H7E12-2抗体和阳性对照抗体Zolbetuximab在高密度细胞系MC38-hCLDN18.2-A11,H7E12-2的ADCC活性明显优于阳性对照抗体Zolbetuximab，EC50分别为9.6μg/mL和272.7μg/mL(图7A),在中等密度抗原水平的293T-hCLDN18.2细胞上具有相似的杀伤活性(图7B)，EC50分别为125.1μg/mL和29.7μg/mL，在另一株高密度的细胞系KATOIII-hCLDN18.2中，H7E12-2的ADCC活性明显优于阳性对照抗体Zolbetuximab，EC50分别为5.6μg/mL和302μg/mL(图7C)。在MC38-hCLDN18.2-A11和KATOIII-hCLDN18.2两种高密度细胞系中，H7E12-2的ADCC活性较阳性对照抗体Zolbetuximab分别提高了28.4倍和53.9倍，表明H7E12-2有极强的 ADCC活性，对其潜在的临床应用提供基础。
实施例11 H7E12-2抗体的ADCC活性检测
在体内，ADCC是由抗体的Fab端结合肿瘤细胞的抗原表位，其Fc端与自然杀伤细胞(NK细胞)表面的FCγR结合，NK细胞活化释放穿孔素、颗粒酶等细胞毒物质，介导NK细胞杀伤靶细胞致使靶细胞凋亡。本实验中，我们利用外周血单核细胞PBMC来源的原代NK细胞作为效应细胞，用293T-hCLDN18.2(细胞编号KC-0986，购自康源博创)作为靶细胞建立共培养系统，在体外模拟ADCC作用。首先用1.6μM的CFSE(Carboxyfluorescein Diacetate Succinimidyl Ester，羧基荧光素二醋酸盐琥珀酰亚胺酯，565082，BD)标记靶细胞293T-hCLDN18.2，室温避光标记10分钟，用5倍体积预冷的无血清培养基洗2遍，将标记好的细胞重悬在ADCC培养液中并计数，向96孔U型板中加入每孔5x 10^4细胞，之后加入测试抗体进行梯度稀释，起始浓度为50μg/mL，用完全培养基梯度稀释3.16倍，共10个浓度点，500转离心30秒后加入1.5x 10^5PBMC细胞/孔，效靶比为30：1。将靶细胞效应细胞抗体复合物在在37℃共孵育4h。孵育结束后，用PBS+2％FBS清洗细胞两遍，每孔加入100μL含1μL 7-AAD(559925，BD)的PBS溶液染色，室温避光孵育10分钟，用NovoCyteQuanteon流式细胞仪(安捷伦)检测靶细胞的活性。使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值。实验结果见图8和表9。
表9.H7E12-2抗体的原代NK细胞介导的ADCC活性总结
实验结果显示，在原代NK细胞介导的ADCC杀伤实验中，与报告细胞系的结果相似，H7E12-2依然显示很强的ADCC活性，EC50为12ng/mL,而阳性对照抗体Zolbetuximab的EC50为74ng/mL，H7E12-2原代NK细胞介导的ADCC活性提高了6.17倍，显示其优于Zolbetuximab的肿瘤杀伤活力。
实施例12 H7E12-2抗体的ADCP活性检测
抗体依赖性细胞介导的吞噬作用(ADCP)是另一个抗体类药物杀伤肿瘤细胞的重要作用机制，抗体的Fab通过与抗原结合，引起构象变化，并促进抗体的Fc端与巨噬细胞表面的FcγRIIA(CD32a)的结合，并激活FcγRIIA下游信号ITAM信号，促进巨噬细胞对肿瘤抗原阳性细胞的吞噬，在本实验中，利用小鼠骨髓分化来源的巨噬细胞与 293T-hCLDN18.2(细胞编号KC-0986，购自康源博创)细胞共培养来评价H7212-2抗体的ADCP活性。无菌解剖小鼠取股骨和胫骨剃掉表面的组织，用剪子剪断两端将骨髓用注射器吹出并收集离心，用培养基重悬计数，用小鼠M-CSF分化BMDM。将诱导好的BMDM用1mL Accutase消化下来，1200转离心5分钟，用1640+5％FBS重悬计数备用。吞噬实验当天，将靶细胞1.6μM/mL的终浓度CFSE标记，室温避光染色用1640+5％FBS重悬计数，以5x10^4细胞/孔铺到低吸附96孔U形底板，每孔80μL，加入梯度稀释的测试药40μL/孔后低速瞬离，将诱导好的BMDM按照对应的效靶比的细胞数铺到低吸付96孔U形底板，每孔80μL，用排枪混匀后37度孵育4小时。用流式缓冲液洗两遍，加入100μL PBS中含有1μL的APC-F4/80，混匀后4度孵育30min，用流式缓冲液洗两遍，加入100μL PBS中含有1μL的7-AAD，混匀后室温孵育10分钟，用NovoCyteQuanteon流式细胞仪(安捷伦)检测ADCP的吞噬比例，结果的出后利用公式计算吞噬指数(％)＝％(F4/80+CFSE+)/％(F4/80+)×100％。使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算EC50值。实验结果见图9。虽然为得到有效的EC50,依然可以看到H7E12-2显示浓度梯度依赖的吞噬活性的增加，相比阳性对照抗体Zolbetuximab在各浓度点都有显著增强的吞噬活性。
实施例13 H7E12-2ADCC增强型抗体的制备
在FUT8基因敲除的HEK293/FUT8-细胞中的表达H7E12-2，该细胞购自康源博创生物科技有限公司(KC-2300)。细胞株在Expi293TM expression medium中传代用于生产，传代密度0.2～0.3*E6/ml，传代周期2～3天。转染前一天将细胞稀释至2*E6/ml，转染当日，检测细胞密度应在6E6/ml左右，活率大于95％。转染当日按照并使用转染试剂盒使用说明操作转染，质粒使用432-M147-LC和432-M147-HC，轻链和重链摩尔浓度比为1:1。转染后18-22小时按终浓度0.6％v/v加入Enhancer1和Enhancer2。转染第五天以后，监测细胞活率，细胞活率小于70％时收获细胞，纯化蛋白，命名为ICP。
实施例14蛋白ICP糖型分析
取500μg蛋白加入4μL PNGaseF糖苷酶，于37℃孵育16h，样品加入预冷好的300μL无水乙醇补至400μL，混匀后放如-20℃冰箱，1h。取出后12000rpm离心10min，用移液枪小心吸取上清。将上清放入真空离心浓缩仪中，室温浓缩干燥(10h～15h)，直至没有液体。加入DMSO和乙酸混合液(350:150)：2-AB:氰基硼氢化钠＝100μL:5mg:6mg 的比例混合液20μL。65℃避光环境中，反应4h。加乙腈和水混合液(8:2)200μL，振荡，离心2min，取上清检测。
色谱条件：柱温65℃；进样池温度8℃；进样量10μL。流动相A:50mmol·L-1甲酸铵溶液,流动相B:乙腈，梯度为25min内B相从70％到63％，5min内B相从63％到10％，流速为0.4mL·min-1。2-AB标记的检测波长为：激发波长330nm，发射波长330nm。实施例15蛋白ICP活性检测
ICP的ADCC活性检测方法如实施例11所述，用293T-hCLDN18.2(细胞购自康源博创，细胞编号KC-0986)细胞作为靶细胞和PBMC来源的原代NK细胞共培养，检测融合蛋白Fc端介导的ADCC活性。

Claims (10)

  1. 一种抗CLDN18.2抗体或其抗原结合片段，其包含：如氨基酸序列SEQ ID NO：5所示的轻链互补决定区LC-CDR1、如氨基酸序列SEQ ID NO：7所示的轻链互补决定区LC-CDR2和如氨基酸序列SEQ ID NO：9所示的轻链的互补决定区LC-CDR3；如氨基酸序列SEQ ID NO：11所示的重链互补决定区HC-CDR1、如氨基酸序列SEQ ID NO：13所示的重链互补决定区HC-CDR2、如氨基酸序列SEQ ID NO：15所示的重链的互补决定区HC-CDR3；
  2. 如权利要求1所述的抗CLDN18.2抗体或其抗原结合片段，其轻链可变区氨基酸序列为SEQ ID NO：3所示；重链可变区氨基酸序列为SEQ ID NO：4所示。
  3. 如权利要求1-2任一项所述的抗CLDN18.2抗体或其抗原结合片段，其为人源化的抗体或抗原结合片段，其轻链可变区氨基酸序列为SEQ ID NO：17所示；重链可变区氨基酸序列为SEQ ID NO：19所示。
  4. 编码根据权利要求1-3任一项所述的抗体或其抗原结合部分的核酸。
  5. 一种表达载体，其包含权利要求4所述的核酸。
  6. 一种宿主细胞，其包含权利要求4所述的核酸或权利要求5所述的表达载体。
  7. 一种药物组合物，其包含权利要求1-3任一项所述的抗体或其抗原结合部分、权利要求4所述的核酸、权利要求5所述的表达载体或权利要求6所述的宿主细胞。
  8. 一种试剂盒，其包含权利要求1-3任一项所述的抗体或其抗原结合部分、权利要求4所述的核酸、权利要求5所述的表达载体或权利要求6所述的宿主细胞。
  9. 如权利要求1-3任一项所述的抗体或其抗原结合部分、权利要求4所述的核酸、权利要求5所述的表达载体、权利要求6所述的重组细胞在制备用于诊断、治疗或预防肿瘤的药物或试剂中的用途。
  10. 如权利要求9所述的用途，其特征在于，所述肿瘤是与CLDN18.2相关的肿瘤；优选地，所述肿瘤是胃癌、胃食管交界处腺癌、胰腺癌、食管癌、支气管癌或乳腺癌。
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