WO2023083327A1 - 抗cldn18.2单克隆抗体及其应用 - Google Patents

抗cldn18.2单克隆抗体及其应用 Download PDF

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WO2023083327A1
WO2023083327A1 PCT/CN2022/131502 CN2022131502W WO2023083327A1 WO 2023083327 A1 WO2023083327 A1 WO 2023083327A1 CN 2022131502 W CN2022131502 W CN 2022131502W WO 2023083327 A1 WO2023083327 A1 WO 2023083327A1
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antibody
variable region
heavy chain
chain variable
light chain
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PCT/CN2022/131502
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English (en)
French (fr)
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瞿爱东
梁红远
张坤明
吴丽娜
祝婧烨
邱建华
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上海生物制品研究所有限责任公司
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Definitions

  • the invention relates to the field of medicine, in particular to an anti-CLDN18.2 humanized monoclonal antibody and its preparation.
  • TJs Tight Junctions
  • tight junction proteins claudins
  • CLDN18 is the main component of tight junctions in lung and gastric epithelial cells. CLDN18 has two isoforms, CLDN18.1 and CLDN18.2. CLDN18.1 is mainly expressed in the lung, and CLDN18.2 is mainly expressed in the stomach.
  • CLDN18.2 is highly expressed in primary gastric cancer and metastatic gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, cholangiocarcinoma, ovarian cancer and other tumors (Sahin U, et al. Clin Cancer Res, 2008; 14( 23):7624-34). Due to the characteristics of fast proliferation, easy invasion and metastasis of tumor cells, the tight junction structure is lost, and the CLDN18.2 molecules on the surface are more easily exposed. CLDN18.2 becomes an ideal target for tumor therapy (Hewitt KJ, et al. BMC Cancer, 2006 ;6:186).
  • IMAB362 targeting CLDN18.2 developed by Ganymed Pharmaceuticals AG has entered phase III clinical research.
  • IMAB362 combined with the first-line chemotherapy drug EOX (erubicin, oxaliplatin, capecitabine) significantly prolonged the PFS and OS of patients compared with chemotherapy alone, It has become a potential drug for the treatment of gastric cancer, esophageal cancer and other diseases (Lordick F, et al. Gastric Cancer, 2021; 24(3):721–730.).
  • EOX erubicin, oxaliplatin, capecitabine
  • the purpose of the present invention is to provide a CLDN18.2 antibody with high affinity and high biological activity and its application.
  • a heavy chain variable region of an antibody is provided, and the heavy chain variable region includes the following three complementarity determining regions CDRs:
  • Complementarity-determining region CDR1 the amino acid sequence of said complementarity-determining region CDR1 is shown in SEQ ID NO:3;
  • complementary determining region CDR2 the amino acid sequence of said complementary determining region CDR2 is shown in SEQ ID NO: 4 or 16;
  • Complementarity determining region CDR3 the amino acid sequence of said complementarity determining region CDR3 is shown in SEQ ID NO:5 or 17.
  • sequences of the three CDRs of the heavy chain variable region are SEQ ID Nos: 3, 4 and 5; or 3, 16 and 5; or 3, 4 and 17.
  • the heavy chain variable region includes the following four framework region FRs, wherein the four framework region FRs are from four frameworks corresponding to SEQ ID Nos: 1, 11, 12 and 13 District Fr.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 11, 12 or 13.
  • an antibody heavy chain having the heavy chain variable region of the antibody according to the first aspect of the present invention.
  • the constant region of the heavy chain is of human origin.
  • the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2 or the like.
  • a light chain variable region of an antibody is provided, and the light chain variable region includes the following three complementarity determining regions CDR':
  • Complementarity determining region CDR1' the amino acid sequence of said complementarity determining region CDR1' is shown in SEQ ID NO:8;
  • Complementarity determining region CDR3' the amino acid sequence of said complementarity determining region CDR3' is shown in SEQ ID NO:10.
  • the light chain variable region includes the following four framework regions FR, wherein the four framework regions FR are from four framework regions corresponding to SEQ ID No: 6, 14, and 15 Fr.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6, 14, or 15.
  • an antibody light chain having the light chain variable region of the antibody according to the third aspect of the present invention there is provided an antibody light chain having the light chain variable region of the antibody according to the third aspect of the present invention.
  • the constant region of the light chain is of human origin.
  • the light chain constant region is the light chain constant region of human Kappa, Lambda, etc.
  • an antibody which has:
  • the antibody has: the heavy chain as described in the second aspect of the present invention; and/or the light chain as described in the fourth aspect of the present invention.
  • the antibody also has a heavy chain constant region and a light chain constant region.
  • the antibody has a heavy chain variable region as shown in SEQ ID NO: 1, 11, 12 or 13; and/or a light chain variable region as shown in SEQ ID NO: 6, 14, and 15 chain variable region.
  • the antibody has a heavy chain variable region as shown in SEQ ID NO: 1, 11, 12 or 13; and/or a light chain variable region as shown in SEQ ID NO: 15.
  • the antibody is selected from the following group:
  • (Z2) have the heavy chain variable region shown in SEQ ID No:11 and the antibody of the light chain variable region shown in SEQ ID No:14;
  • (Z3) have the heavy chain variable region shown in SEQ ID No:11 and the antibody of the light chain variable region shown in SEQ ID No:15;
  • (Z4) have the heavy chain variable region shown in SEQ ID No:12 and the antibody of the light chain variable region shown in SEQ ID No:14;
  • (Z5) have the heavy chain variable region shown in SEQ ID No:12 and the antibody of the light chain variable region shown in SEQ ID No:15;
  • (Z6) have the heavy chain variable region shown in SEQ ID No:13 and the antibody of the light chain variable region shown in SEQ ID No:14;
  • the heavy chain of the antibody has the amino acid sequence shown in SEQ ID No: 18, 20, 21 or 22.
  • the light chain of the antibody has the amino acid sequence shown in SEQ ID No: 19.
  • the antibody is a humanized antibody.
  • the antibody specifically binds to CLDN18.2.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a bispecific antibody.
  • the antibody is in the form of a drug conjugate.
  • the present invention also provides an antibody corresponding to 624, which has a heavy chain variable region shown in SEQ ID No: 23 and a light chain variable region shown in SEQ ID No: 24.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • an antibody preparation is provided, and the antibody preparation includes:
  • carrier said carrier includes: buffer, sterile water, optional surfactant.
  • the concentration of the antibody is 5-100 mg/mL; preferably 10-70 mg/mL, more preferably 20-60 mg/mL.
  • the buffer is selected from the group consisting of citric acid buffer system, histidine buffer system, or a combination thereof.
  • the pH range of the preparation is 5.0-7.5, preferably 5.5-7.
  • the preparation is an injection preparation.
  • a kit which contains the antibody preparation according to the seventh aspect of the present invention, and a container for containing the antibody preparation.
  • a CAR construct is provided, the scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to CLDN18.2, and the scFv has a The heavy chain variable region according to one aspect and the light chain variable region according to the third aspect of the present invention.
  • a recombinant immune cell expressing the exogenous CAR construct as described in the ninth aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are from humans or non-human mammals (such as mice).
  • an antibody-drug conjugate is provided, and the antibody-drug conjugate contains:
  • an antibody part which is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the antibody part is coupled to the coupling part through a chemical bond or a linker.
  • an active ingredient selected from the following group: the heavy chain variable region as described in the first aspect of the present invention, as described in the second aspect of the present invention.
  • the heavy chain of the heavy chain, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, as described in the sixth aspect of the present invention The recombinant protein described above, the immune cell as described in the tenth aspect of the present invention, the antibody drug conjugate as described in the eleventh aspect of the present invention, or a combination thereof, the active ingredient is used for (a) preparing a detection reagent or Reagent test kit;
  • the tumor is selected from the group consisting of blood tumors, solid tumors, or a combination thereof.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse massive B cell lymphoma (DLBCL), Hodgkin lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse massive B cell lymphoma
  • Hodgkin lymphoma Hodgkin lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • the tumor is a tumor that highly expresses CLDN18.2.
  • the medicament or preparation is used to prepare a medicament or preparation for preventing and/or treating diseases related to CLDN18.2 (positive expression).
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used for diagnosing CLDN18.2 related diseases.
  • the detection reagent or kit is used to detect CLDN18.2 protein in a sample.
  • the detection reagent is a detection chip.
  • a pharmaceutical composition which contains:
  • an active ingredient selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention
  • the light chain variable region of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the recombinant protein as described in the tenth aspect of the present invention The above-mentioned immune cells, the antibody-drug conjugate according to the eleventh aspect of the present invention, or a combination thereof; and
  • the pharmaceutical composition further includes a second anti-tumor active ingredient.
  • the second active ingredient is selected from the group consisting of cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or combinations thereof.
  • the second active ingredient includes: an antibody targeting EGFR and an antibody targeting HER2.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used for treating tumors.
  • the tumor is a tumor that highly expresses CLDN18.2.
  • the fifteenth aspect of the present invention there is provided a vector containing the polynucleotide according to the fourteenth aspect of the present invention.
  • the vectors include: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector as described in the fifteenth aspect of the present invention or is integrated in the genome with the vector described in the fourteenth aspect of the present invention of polynucleotides.
  • a method for in vitro detection (including diagnostic or non-diagnostic) of CLDN18.2 protein in a sample comprising the steps of:
  • a detection board in the eighteenth aspect of the present invention, includes: a substrate (supporting board) and a test strip, and the test strip contains the antibody or antibody as described in the fifth aspect of the present invention.
  • the antibody drug conjugate according to the eleventh aspect of the present invention is provided, and the detection board includes: a substrate (supporting board) and a test strip, and the test strip contains the antibody or antibody as described in the fifth aspect of the present invention.
  • a kit comprising:
  • the kit contains the detection plate as described in the eighteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a method for CLDN18.2-related diseases comprising: administering the antibody as described in the fifth aspect of the present invention, the antibody-drug of the antibody to a subject in need A conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • Figure 1 shows the results of Elisa binding detection of specific binding hybridoma supernatant cells.
  • Figure 2 shows the results of flow cytometry detection.
  • Figure 2A is the detection result of chimeric antibody ch429
  • Figure 2B is the detection result of chimeric antibody ch623
  • Figure 2C is the detection result of chimeric antibody ch624
  • Figure 2D is the detection result of chimeric antibody ch782
  • Figure 2E is the detection result of reference antibody Ref.ch175 .
  • Figure 3 shows the detection results of the binding activity of anti-CLDN18.2 human-mouse chimeric antibody to recombinant cells.
  • Figure 3A is the result of binding activity of anti-CLDN18.2 human-mouse chimeric antibody to HEK293-hCLDN18.1 cells;
  • Figure 3B is the result of binding activity of anti-CLDN18.2 human-mouse chimeric antibody to HEK293-hCLDN18.2 cells.
  • Figure 4 shows the detection results of the binding activity of the anti-CLDN18.2 human-mouse chimeric antibody to NUGC4 cells.
  • Figure 5 shows the flow cytometric detection results of the cell-binding activity of the anti-CLDN18.2 human-mouse chimeric antibody and recombinant cells.
  • Figure 6 shows the results of ADCC activity of anti-CLDN18.2 human-mouse chimeric antibody on HEK293-hCLDN18.2 cells.
  • Figure 7 shows the binding activity of humanized antibodies to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2 and NUGC4 cells.
  • Figure 8 shows the ADCC activity of humanized antibodies.
  • Figure 9 shows the detection results of CDC activity of the humanized antibody.
  • Figure 10 shows the results of the humanized antibody's proliferation inhibitory activity on HEK293-hCLDN18.2.
  • Figure 11 shows the comparison of key binding sites between the antibody hu782 of the present invention and the reference antibody.
  • Figure 11A shows the key binding site of Ref.ch175
  • Figure 11B shows the key binding site of the antibody hu782 of the present invention.
  • Figure 12 shows the in vivo anti-tumor activity of the antibodies of the present invention.
  • the inventors unexpectedly obtained an anti-human CLDN18.2 antibody with high affinity and excellent anti-tumor activity for the first time. Specifically, the inventors obtained multiple murine antibodies with high affinity for human CLDN18.2 for the first time after extensive screening.
  • the present invention further prepares chimeric antibodies and humanized antibodies, and performs mutation screening on the obtained humanized antibodies.
  • the antibody of the present invention especially the mutated humanized antibody has an affinity similar to that of the chimeric antibody, and will be further developed into a humanized monoclonal antibody drug for targeted therapy in the future.
  • the antibody of the invention can effectively bind to human CLDN18.2, inhibit the proliferation of various tumor cells, inhibit the growth of tumors in nude mouse endosomes, and have excellent anti-tumor activity.
  • the present invention has been accomplished on this basis.
  • mice alternately by constructing various forms of hCLDN18.2 DNA as antigens, and boosted immunization with recombinantly expressed hCLDN18.2 cell lines, and obtained mouse singles that specifically recognized hCLDN18.2 through hybridoma fusion technology.
  • the chimeric antibody ch782 specifically binds to hCLDN18.2 and can recognize CLDN18.2 molecules on the surface of tumor cells NUGC4, and its affinity to CLDN18.2 is significantly higher than that of the reference antibody Ref.ch175.
  • ch782 has ADCC, CDC and proliferation inhibitory activities on hCLDN18.2 cells.
  • the chimeric antibody ch782 was humanized to obtain four IgG1-type humanized antibody combinations hu782H1.1L1, hu782H1.2L1, hu782H1.1L2, and hu782H1.2L2.
  • the four humanized antibodies specifically recognized hCLDN18.2, and ch782 has comparable affinity.
  • the S60A site mutation was carried out on the humanized antibody heavy chain hu782H1.1 to remove the potential glycosylation site, and the V101A mutation was carried out to enhance its expression level to obtain the humanized antibody heavy chain hu782H1.2-V101A.
  • the above S60A and V101A sites are defined according to the Kabat numbering rules.
  • the 239th and 332nd amino acids of the heavy chain constant region of the humanized antibody hu782 were mutated with S239D/I332E to obtain the humanized antibody hu782-DE.
  • the above S239D and I332E are defined according to the EU numbering system.
  • the ADCC activity of the humanized antibody hu782-DE on tumor cell NUGC4 was significantly better than that of the reference antibody Ref.ch175, and its EC50 value was 24 times lower than that of Ref.ch175.
  • Humanized antibodies hu782 and hu782-HV have CDC killing activity on hCLDN18.2 target cells.
  • Humanized antibodies hu782 and hu782-HV have proliferation inhibitory activity on hCLDN18.2 target cells, and their proliferation inhibitory activity is dose-dependent, which is significantly better than the reference antibody Ref.ch175.
  • the three antibodies hu782, hu782-HV, and hu782-DE can significantly grow the transplanted tumor, and are better than the reference antibody Ref.ch175, especially the two antibodies, hu782 and hu782-DE, inhibit tumor growth The effect is significantly better than the reference antibody Ref.ch175.
  • Both hu782 and hu782-DE antibody treatment groups had complete tumor regression in mice, and the tumor inhibition rates of hu782-HV, hu782 and hu782-DE were 34.68%, 62.88% and 65.05%, and the reference antibody Ref.ch175 was 15.5%.
  • the term “about” can refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprises” or “includes (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of”, or “consisting of”.
  • Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein). sequence, and determine the number of positions where the same residue occurs. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is well known to those skilled in the art.
  • VL light chain variable region
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to antibodies specifically binding to CLDN18.2, for example, having a heavy chain variable region (such as SEQ The amino acid sequence of ID NO:1, 11, 12 or 13) and/or the protein or polypeptide of the light chain variable region (such as the amino acid sequence of SEQ ID NO:6, 14 or 15). They may or may not contain starting methionine.
  • a heavy chain variable region such as SEQ The amino acid sequence of ID NO:1, 11, 12 or 13
  • protein or polypeptide of the light chain variable region such as the amino acid sequence of SEQ ID NO:6, 14 or 15. They may or may not contain starting methionine.
  • Another preferred antibody of the present invention is an antibody corresponding to 624 that specifically binds to CLDN18.2, for example having a heavy chain variable region (such as the amino acid sequence of SEQ ID NO: 23) and/or a light chain variable region (such as The protein or polypeptide of the amino acid sequence of SEQ ID NO:24).
  • a heavy chain variable region such as the amino acid sequence of SEQ ID NO: 23
  • a light chain variable region such as The protein or polypeptide of the amino acid sequence of SEQ ID NO:24.
  • human CLDN18 contains 261 amino acids. It is a tetraspanning protein with four transmembrane hydrophobic regions and two extracellular ring structures. Loop 1 is surrounded by transmembrane region 1 and transmembrane region 2; 2 is surrounded by transmembrane region 3 and transmembrane region 4.
  • Human CLDN18.1 and CLDN18.2 have differences in the amino acid composition of the N-terminus, transmembrane region 1 and extracellular loop 1, and the rest are completely identical, with a sequence similarity of 92%.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the Kabat scheme (Kabat et al., 1991) is based on the location of regions of high sequence variation between sequences of the same domain type, with different numbering of antibody heavy (VH) and light (V ⁇ and V ⁇ ) variable domains.
  • Chothia's scheme (Al-Lazikani, 1997) is identical to Kabat's scheme, but corrects the insertion of annotations around the first VH complementarity determining region (CDR) to correspond to the structural loop.
  • CDR VH complementarity determining region
  • the enhanced Chothia program (Abhinandan and Martin, 2008) made further structural corrections to the insertion position.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, and ⁇ chain.
  • IgM immunoglobulins
  • IgD immunoglobulins
  • IgG immunoglobulins
  • IgA immunoglobulin heavy chain
  • IgE immunoglobulins
  • heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively.
  • ⁇ chain, and ⁇ chain respectively.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains depending on the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the antibody light chain of the present invention may further comprise a light chain constant region, and the light chain constant region comprises human or mouse ⁇ , ⁇ chain or variants thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions, and the sequence sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
  • Example 13 of the present invention the 6 CDRs of the ch782 antibody were divided by combining kabat and Chothia methods.
  • murine antibody in the present invention refers to an anti-CLDN18.2 monoclonal antibody prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with the CLDN18.2 antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • the murine CLDN18.2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or variant thereof, or further comprise a murine IgG1 , IgG2, IgG3 or variants thereof heavy chain constant region.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in framework sequences. Humanized antibodies can overcome the heterologous reaction induced by chimeric antibodies because they carry a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. In order to avoid decreased immunogenicity and decreased activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity.
  • antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, CLDN18.2). It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody” include
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • epitope refers to a site on an antigen to which an immunoglobulin or an antibody specifically binds (eg, a specific site on a CLDN18.2 molecule).
  • An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or noncontiguous amino acids in a unique spatial conformation.
  • the terms “specifically bind”, “selectively bind”, “selectively bind” and “specifically bind” refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 "7 M, eg, about less than 10 "8 M, 10" 9 M, or 10"10 M or less.
  • KD affinity
  • the term "competing binding” refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a part of the same epitope on the extracellular region of CLDN18.2 as the monoclonal antibody of the present invention and binds to the antigen.
  • An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of CLDN18.2 recognized by the monoclonal antibody of the present invention.
  • KD or "Kd” refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • antibodies of the invention bind CLDN18.2 with a dissociation equilibrium constant (KD) of less than about 10 ⁇ 7 M, eg, less than about 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • antigenic determinant refers to a discrete three-dimensional site on an antigen recognized by an antibody or antigen-binding fragment of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, directed against a single antigenic epitope.
  • the cells may be eukaryotic, prokaryotic or phage clonal cell strains.
  • antibodies can be monospecific, bispecific, trispecific, or more multiple specific.
  • the antibody of the present invention also includes its conservative variant, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and optimally Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to the table below.
  • the present invention provides an anti-CLDN18.2 humanized antibody (hereinafter referred to as CLDN18.2 antibody). Specifically, the present invention provides a humanized antibody with high specificity and high affinity against CLDN18.2, which includes a heavy chain and a light chain, and the heavy chain contains a heavy chain variable region (VH) amino acid sequence, so The light chain contains the light chain variable region (VL) amino acid sequence.
  • CLDN18.2 antibody an anti-CLDN18.2 humanized antibody
  • the present invention provides a humanized antibody with high specificity and high affinity against CLDN18.2, which includes a heavy chain and a light chain, and the heavy chain contains a heavy chain variable region (VH) amino acid sequence, so The light chain contains the light chain variable region (VL) amino acid sequence.
  • Pedersen et al. reported the humanization of antibodies by resurfacing.
  • Hsiao et al. reported the humanization method of CDR transplantation with the framework region of human antibody Germline sequence.
  • Jespers et al. successfully constructed a humanized method using a phage library (shuffling library) method.
  • the human framework region in antibody humanization There are usually two choices for the human framework region in antibody humanization, one is the known mature antibody, and the other is the human Germline sequence.
  • Known mature antibody framework regions often contain somatic mutation sites, which may bring potential immunogenicity.
  • the framework region of human Germline sequence is theoretically less immunogenic, more flexible in structure, strong in plasticity, and easily accepts different CDR regions.
  • the frequency of use of the human antibody Germline gene in the human body is biased to a certain extent.
  • the humanized antibody of the Germline framework region with high frequency of use has the advantages of low immunogenicity, high expression level, and stable structure.
  • the Germline sequence with the highest similarity to the murine antibody was not selected during humanization, but the sequence of the preferred sequence was selected after a large number of experimental screenings taking into account the similarity and the frequency of use in the human body.
  • the framework regions were humanized.
  • the human antibody Germline framework region is selected for CDR transplantation, and the humanized antibody constructed in this way has a more stable structure, high expression, low immunogenicity, and higher druggability.
  • the heavy chain constant region and/or the light chain constant region may be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region of human IgG1, IgG2, etc. or the light chain constant region of human kappa or Lambda.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90% %, optimally at least 95% of the amino acid sequence.
  • Antibodies of the present invention may be double-chain or single-chain antibodies, and may preferably be fully humanized antibodies.
  • the antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab')2, or other known antibody derivatives in the field, etc., as well as IgA, IgD, Any one or several of IgE, IgG and IgM antibodies or other subtypes of antibodies.
  • the antibody of the present invention may be a humanized antibody targeting CLDN18.2, a CDR-grafted and/or modified antibody.
  • the number of amino acids added, deleted, modified and/or substituted is preferably no more than 40% of the total amino acid number of the original amino acid sequence, more preferably no more than 35%, more preferably 1-33% , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • any method suitable for producing monoclonal antibodies can be used to produce the CLDN18.2 antibodies of the invention.
  • animals can be immunized with linked or naturally occurring CLDN18.2 protein or fragments thereof.
  • Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, one or more routes may be used.
  • CLDN18.2 can be used as an immunogen (antigen) for producing non-human antibodies specific to CLDN18.2, and screening the biological activity of the antibodies.
  • Immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources, or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic in origin (eg cDNA).
  • DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • immunoglobulins including IgM, IgD, IgG, IgA and IgE.
  • either type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant or animal cell (eg, a mammalian cell).
  • the step of transforming host cells with recombinant DNA described in the present invention can be carried out by techniques well known in the art.
  • the obtained transformant can be cultivated by conventional methods, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.
  • transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibody of the present invention can be purified by conventional separation and purification means well known to personnel.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies have different stability in different preparation buffers, manifested as changes in charge heterogeneity, degradation of antibody molecules, aggregation, etc. These changes in quality properties are related to the physical and chemical properties of the antibody itself. Therefore, in the development of antibody drugs In the process, it is necessary to screen the suitable preparation buffer according to the physicochemical properties of different antibodies.
  • buffer systems for antibody preparations include phosphate buffer, citrate buffer, histidine buffer, etc.
  • salt ions or excipients such as sorbitol, trehalose, and sucrose are added according to the nature of the antibody, and An appropriate amount of surfactant such as Tween to maintain the stability of the antibody.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cells, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the invention can be directly used for binding CLDN18.2 protein molecules, and thus can be used for preventing and treating CLDN18.2 related diseases.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, more Preferably the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dose, which are within the skill of skilled physicians.
  • Antibodies of the invention can be used in detection applications, for example for testing a sample, thereby providing diagnostic information.
  • the sample (sample) used includes cells, tissue samples and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsies known to those skilled in the art.
  • a biopsy as used in the present invention may thus include a tissue sample prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention.
  • the kit further includes a container, instructions for use, buffer and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the antibody of the present invention has a typical tryptophan and tyrosine embedded structure.
  • the template structure of the human VH and VL framework regions screened is stable, and it is well adapted to the CDR region of the mouse monoclonal antibody.
  • the light and heavy chains can be
  • the variable regions can be well paired together, with high affinity and stable structure.
  • the humanized antibody of the present invention has excellent biological activity and specificity, and has lower immunogenicity and higher expression level while retaining an affinity comparable to CLDN18.2 .
  • the humanized antibody of the present invention has significant in vitro biological activity, and can efficiently inhibit the proliferation of tumor cells cultured in vitro, and the activity is better than that of the control antibody.
  • the humanized antibody of the present invention has significant biological activity in vivo, and has excellent inhibitory activity against tumor cells such as human gastric cancer cells, and the activity is better than that of the control antibody.
  • the sequence of the reference antibody used in the experiment of the present invention is derived from the antibody sequence of the 175D10 clone in the patent US8168427.
  • the 175D10 antibody is also known as IMAB362.
  • the variable region sequence of the 175D10 antibody was synthesized completely, constructed on a eukaryotic expression vector containing the constant region of the IgG1 chimeric antibody, and purified.
  • the prepared reference antibody is abbreviated as Ref.ch175 in the present invention.
  • the reference antibody Ref.ch175 (or IMAB362) specifically recognizes the extracellular loop 1 part of CLDN18.2, and does not bind to CLDN18.1, mainly through antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADCC) and complement dependent cytotoxicity (complement dependent cytotoxicity, CDC) kill tumor cells.
  • Example 1 Construction of cell lines stably expressing CLDN18.1 or CLDN18.2
  • cell lines stably expressing human CLDN18.1, human CLDN18.2, mouse CLDN18.1, and mouse CLDN18.2 were constructed. Methods as below:
  • HEK293 cells purchased from the Cell Bank of the Chinese Academy of Sciences, GNHu43 were inoculated into 6-well plates at 4 ⁇ 105 cells/well, and the next day, pEGFP-N2-hCLDN18.1, pEGFP -N2-hCLDN18.2 were transfected into HEK293 cells, and after 24 hours of transfection, the cells were subcultured at a ratio of 1:10. After the cells adhered to the wall, G418 (purchased from Gibco) was added at a final concentration of 300 ⁇ g/ml for pressurized screening.
  • HEK293 cells stably expressing human CLDN18.1 and human CLDN18.2 membrane proteins were cloned and named HEK293-hCLDN18.1 and HEK293-hCLDN18.2, respectively.
  • mouse CLDN18.1 (NP_062789.1) and mouse CLDN18.2 (NP_001181850.1) were synthesized and cloned into the pcDNA3.1-P2A-EGFP eukaryotic expression vector ( purchased from Nanjing GenScript Company), and named pcDNA3.1-mCLDN18.1 and pcDNA3.1-mCLDN18.1 respectively.
  • HEK293 cells were transfected by the same method as above to obtain cells stably expressing mouse CLDN18.1 and CLDN18.2 membrane proteins, which were named HEK293-mCLDN18.1 and HEK293-mCLDN18.2, respectively.
  • the nucleotides encoding human CLDN18.2ECL1 and CLDN18.2 full-length amino acids were cloned in tandem with the P2 epitope TT830-844 and P30 epitope TT947-967 of tetanus toxoid into the eukaryotic expression vector PTT5 (purchased from Invitrogen Corporation ), named as PTT5-TT-hCLDN18.2 and PTT5-TT-hCLDN18.2-ECL1 respectively.
  • CLDN18.2ECL1 is fused to the Fc-C end of human IgG1, which is named hFc-CLDN18.2-ECL1.
  • TT-hCLDN18.2-ECL1 nucleotide sequence SEQ ID No: 30
  • hFc-CLDN18.2-ECL1 nucleotide sequence SEQ ID No: 31
  • Embodiment 3 Preparation of anti-human CLDN18.2 hybridoma cell line
  • mice used in the experiment were purchased from Shanghai Xipuer-Bikai Laboratory. All mice used for immunization were 4-6 weeks old, female, standardized disease-free, healthy purebred mice.
  • CLDN18.2 is a 4-time transmembrane protein, in order to obtain specific antibodies that recognize tumor cells with high affinity, mice were immunized alternately with various plasmids, and cells stably expressing human CLDN18.2 membrane protein were used for shock immunization. Methods as below:
  • Example 4 Screening of hybridoma cells specifically binding to human CLDN18.2 but not binding to human CLDN18.1
  • the cell based-ELISA method was used to screen hybridomas that specifically bind to human CLDN18.2 but not to human CLDN18.1 hybridoma cells. Methods as below:
  • HEK293-hCLDN18.1 cells and HEK293-hCLDN18.2 cells were trypsinized and seeded on poly-lysine (P4707, Sigma)-coated 96-well plates at a cell density of 3 ⁇ 105 cells/mL. , 200 ⁇ L/well, culture overnight; when the confluence of the cells reaches more than 90%, remove the supernatant, fix with 10% neutral formalin solution at room temperature for 15 min; wash with PBS three times, and block with 5% skimmed milk at 4°C overnight.
  • poly-lysine P4707, Sigma
  • the subtype detection kit (SBA Clonotyping System-HRP kit, purchased from southernbiotech, 5300-05) was used to detect the subtype of the clones specifically binding to CLDN18.2.
  • Example 6 Cloning and identification of the coding sequence of the variable region of the anti-human CLDN18.2 mouse monoclonal antibody
  • variable region sequence of the murine monoclonal antibodies was obtained by RACE-PCR and a chimeric antibody was constructed.
  • RNA extraction kit TakaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767) was used to extract the total mRNA of the hybridoma obtained in Example 4, and the sequence of the murine antibody variable region was obtained by PCR using SMARTer RACE 5'/3' Kit (Clontech, 634858) , the result is as follows:
  • Example 7 Construction and expression of anti-CLDN18.2 human-mouse chimeric antibody
  • Primers were designed according to the primer design principles of the homologous recombination method, the heavy and light chain variable region gene fragments of the murine antibody obtained in Example 6 were amplified, respectively connected to the human heavy and light chain constant region sequences, and constructed into the expression vector PTT5 (purchased from Invitrogen).
  • Plasmids were extracted using a commercial plasmid extraction kit, and 293FV suspension cells were transfected by PEI transfection method. Seven days after transfection, the supernatant was collected, and protein A affinity chromatography was used to obtain chimeric antibodies.
  • Example 8 Specificity analysis of anti-CLDN18.2 human-mouse chimeric antibody
  • HEK293 cells stably transfected with human CLDN18.2 and HEK293 cells stably transfected with human CLDN18.1 were respectively digested with trypsin, 5 ⁇ 105 cells/100 ⁇ l were added to 100 ⁇ l of hybridoma supernatant, and incubated on ice for 1 Hours, after washing 2 times with FACS buffer, add goat anti-mouse IgG-PE secondary antibody diluted 1:400, incubate on ice for 45min, wash 2 times with FACS buffer, add 300 ⁇ l loading buffer, transfer to BD flow cytometer tubes and analyzed by BD FACS calibur. The average fluorescence intensity MFI (Median Fluorescence Intensity) was analyzed by Flowjo software.
  • MFI Median Fluorescence Intensity
  • P/N is the MFI of antibody binding to HEK293-hCLDN18.2 cells divided by the MFI of HEK293-hCLDN18.1 cells, which is used to express its specificity.
  • Example 9 Detection of binding activity of anti-CLDN18.2 human-mouse chimeric antibody to recombinant cells
  • HEK293-hCLDN18.1 and HEK293-hCLDN18.2 cells were digested with trypsin and seeded on polylysine (P4707, Sigma)-coated 96-well plates according to a certain cell density, and cultured overnight; When it reaches above 90%, remove the supernatant, use 10% formalin solution to fix at room temperature for 15min; wash once with PBS, and block with 5% skimmed milk at 4°C overnight for later use.
  • Dilute the candidate chimeric antibody and reference antibody Ref.ch175 3 times from 1 ⁇ g/ml to 10 gradients add 100 ⁇ l/well to the cell-coated 96-well plate, incubate at 37°C for 1 hour, and wash the plate 3 times with PBST , add 1:2000 diluted mouse anti-human IgG Fc-HRP, incubate at 37°C for 1 hour, wash the plate 3 times with PBST, develop color with TMB chromogenic solution for 15 minutes, and stop with 2M H 2 SO 4 .
  • Read the OD450-OD630 value and use the GraphPad Prism 8 software to fit the graph and calculate the EC50.
  • Example 10 Detection of binding activity of anti-CLDN18.2 human-mouse chimeric antibody to NUGC4 cells
  • ch782 antibody has the best binding activity to NUGC4 cells endogenously expressing human CLDN18.2 protein, which is significantly stronger than the reference antibody Ref.ch175.
  • Example 11 Detection of binding activity of anti-CLDN18.2 human-mouse chimeric antibody to HEK293-hCLDN18.2 cells
  • the chimeric antibodies ch429, ch624, ch782, and Ref.ch175 were serially diluted 3-fold from a concentration of 10 ⁇ g/ml, and flow cytometry experiments were performed. With the MFI as the ordinate, use the GraphPad Prism 8 software to fit the graph and calculate the EC50.
  • the chimeric antibodies ch429, ch782 of the present invention and the reference antibody Ref.ch175 can all bind to HEK293-hCLDN18.2 cells, with EC50 of 0.52 ⁇ g/ml, 0.81 ⁇ g/ml, and 0.85 ⁇ g/ml, respectively, but the binding activity of ch624 is obvious weaker.
  • Example 12 Detection of ADCC activity of anti-CLDN18.2 antibody human-mouse chimeric antibody on HEK293-hCLDN18.2 cells by reporter gene method
  • ADCC antibody-dependent cell-mediated cytotoxicity. Its mechanism of action is that after the antibody binds to the tumor surface antigen, the Fc region of the antibody binds to the immune effector cell receptor, mainly the Fc receptor on the NK cell. Lysis kills tumor cells.
  • the reporter gene method was used to detect the biological activity of antibody ADCC.
  • the engineered Jurkat cells stably expressing the Fc ⁇ RIIIa receptor were selected as effector cells.
  • the activation of the pathway quantitative detection of antibody Fc effector function.
  • HEK293-hCLDN18.2 cells constructed in Example 1 Dilute the HEK293-hCLDN18.2 cells constructed in Example 1 and dilute them at 2.5 ⁇ 10 5 /ml, spread 100 ⁇ l/well on a white opaque cell culture plate, and culture at 37°C for 24 hours, discard the supernatant, and add 25 ⁇ l/well assay medium containing 0.1% BSA; dilute Jurkat/NFAT-luc+Fc ⁇ RIIIa cells to 6 ⁇ 106 cells/mL, take out the 96-well plate, add 25 ⁇ l Jurkat/NFAT-luc+Fc ⁇ RIIIa cell suspension to each well , and then add 25 ⁇ L of antibody diluent to each well to make the final concentration 6 ⁇ g/mL, and then make 3-fold serial dilutions, a total of 10 dilution gradients; put the cell plate into a constant temperature incubator, induce culture for 6 hours, and place the 96-well plate in Equilibrate at room temperature for at least 15 minutes,
  • the preferred antibody ch782 was humanized. Specifically, the variable region sequence of the mab782 antibody in Example 6 was subjected to humanized sequence design. The heavy and light chain amino acid sequences were encoded according to the kabat coding system, and the sequence homology comparison was carried out through NCBIIgblast (https://www.ncbi.nlm.nih.gov/igblast/), and the human antibody sequence with higher homology was selected as the For the framework, the murine antibody CDR was transplanted, and some core amino acids in the framework region were back-mutated.
  • NCBIIgblast https://www.ncbi.nlm.nih.gov/igblast/
  • FR and CDR sequences defined by kabat in the heavy chain variable region of the 782 antibody are as follows:
  • FR and CDR sequences defined by kabat in the light chain variable region of the 782 antibody are as follows:
  • IGHV4-61 with high homology is selected as the template for the humanization of the antibody heavy chain to replace the CDR region.
  • the heavy chain CDR1 transplantation also includes the CDR1 site defined by Chothia, and the I48M/V67I/V71R3 positions Point back mutation to get hu782H1.1.
  • antibody light chain humanization uses V ⁇ I consensus sequence as a template to replace the CDR region to obtain hu782L1; uses IGKV4-1 as a template to perform CDR region replacement to obtain hu782L2
  • the whole gene is synthesized, and cloned into an expression vector containing the constant region of the heavy and light chains for expression and purification.
  • the binding activity of the humanized antibody to human CLDN18.1 and CLDN18.2, mouse CLDN18.1 and CLDN18.2, and NUGC4 cells was detected by using the chimeric antibody binding activity evaluation method in the above examples.
  • the EC50 values of the cell binding of the four humanized antibodies to the tumor cell NUGC4 were significantly better than those of the chimeric antibody ch782.
  • ADCC functional activity Human IgG1 antibody (hIgG1) mainly binds to Fc ⁇ R through the antibody hinge region and CH2 region to exert ADCC effect.
  • the S239D/I332E(DE) mutation was introduced into the Fc of the humanized antibody heavy chain hu782H1.2 to enhance its affinity for Fc ⁇ R, thereby achieving enhanced ADCC the goal of.
  • the reporter gene method was used to evaluate the ADCC of wild-type and DE mutant human antibodies.
  • the method is as follows: according to the specific operation steps in Example 12, the target cells HEK293-hCLDN18.2 and NUGC4 cells were respectively diluted at 2.5 ⁇ 10 5 /ml, and then 100 ⁇ l/well was spread on a white opaque cell culture plate, and statically placed at 37°C.
  • CDC is a complement-dependent cell-mediated cytotoxicity, and its mechanism is that the antigen-antibody mixture combined with complement eventually perforates the cell membrane, changes the osmotic pressure of the cell, and finally leads to cell lysis.
  • HEK293-hCLDN18.2 cells were used as target cells, the complement serum of young rabbits was used to provide supplementation, and the CCK-8 cell proliferation-toxicity detection kit was used to detect the CDC effect of the antibody.
  • Example 1 Use the HEK293-hCLDN18.2 cells in Example 1 as the target cells, digest and dilute to 2.0 ⁇ 10 5 cells/mL, spread them in a 96-well light-transmitting black body culture plate at a volume of 100 ⁇ L per well, and culture them statically for about 4 hours Wait for the cells to adhere completely.
  • the growth medium was aspirated dry, and 50 ⁇ L of assay medium was added to each well.
  • ch782, hu782, and hu782HV in the present invention can all induce CDC effects comparable to those of the reference antibody Ref.ch175 on HEK293-hCLDN18.2 cells (EC50 are 30.9ng/ml, 49.46ng/ml, 47.58ng/ml respectively , 40.89ng/ml).
  • HEK293-hCLDN18.2 cells in the logarithmic growth phase were taken. Digest the target cells, count and dilute the cells to 1.0 ⁇ 10 5 cells/mL, spread them in a 96-well light-transmitting black body culture plate at a volume of 100 ⁇ L per well; let them stand for about 4 hours until the cells are completely adhered to the wall.
  • Example 18 Analysis of humanized antibody and hCLDN18.2 protein binding site
  • the pEFGP-N2-hCLDN18. 2 On the plasmid, the amino acid difference site between hCLDN18.2ECL1 and hCLDN18.1ECL1 and its surrounding amino acids were mutated to alanine, in which A42 was mutated to the allelic amino acid S (Q29A, S31A, N37A, A42S, N45A, Q47A, G48A in hCLDN18.1ECL1 , S52A, E56A, G65A, L69A, G71A).
  • the mutated plasmids and wild-type plasmids were respectively transfected into 293Fv cells according to the method in Example 1, and the cells were taken for flow cytometric detection after 48 hours.
  • IsotypecontrolhIgG1 isotype control
  • Flowjo software was used to analyze the average fluorescence intensity MFI (Median Fluorescence Intensity) results, and the results determined that when the MFI of the transfected cells after mutation was reduced by more than 50% compared with the wild type, it was considered that the site was a key amino acid site that affected the binding of the antibody.
  • MFI Median Fluorescence Intensity
  • the E56A mutation has a greater effect on the binding of hu782.
  • the present invention unexpectedly found that the reference antibody Ref.ch175 and hu782 were unable to bind after the G48A mutation at the same amino acid position of hCLDN18.2ECL1 and hCLDN18.1ECL1.
  • Example 19 In vivo activity evaluation of humanized antibody
  • BALB/c nude mice were subcutaneously transplanted with tumor cells to establish a CDX animal model to evaluate the efficacy of humanized antibodies in vivo.
  • the NUGC4-hCLDN18.2 stably transfected cell line constructed according to the method in Example 1 was selected for expanded culture. Select 4-6 female SPF grade BALB/c nude mice, 4-6 weeks old, and prepare for seeding tumors after 3-4 days of adaptive feeding. Recombinant NUGC4-hCLDN18.2 cells in the logarithmic growth phase were digested and washed twice with serum-free 1640 medium, then diluted to 2.5 ⁇ 10 7 /ml, and subcutaneously inoculated 0.2ml of 5 ⁇ 10 6 cells in the right axilla of the mouse /Only.
  • the tumors were divided into groups in a randomized block design, with 6 rats in each group.
  • the blank model group was the IgG1 isotype control.
  • the experimental group was administered at a dose of 10 mg/kg, twice a week, intraperitoneal injection and intravenous injection alternately, and the data of tumor volume and mouse body weight were recorded before each administration.
  • V(mm 3 ) (W 2 x L)/2, W is the short diameter of the tumor near the midline, and L is the long diameter of the tumor near the midline.
  • mice were sacrificed by cervical dislocation when tumor volume was greater than 2000 mm 3 , body weight loss was greater than 20%, or disturbance of important physiological functions or tumor ulcer necrosis was observed.
  • TGI tumor inhibition rate
  • Vt-V0 (average tumor volume of the administration group on day t)-(average tumor volume of the group on the day of grouping)
  • Vct-Vc0 (the average tumor volume of the administration group on day t)-(the average tumor volume of the group on the grouping day)
  • the anti-tumor growth activity of the humanized antibody of the present invention and its mutants were observed, and both showed certain drug effects.
  • the tumor volumes of each group are shown in the table below.
  • the tumor inhibition rates of the humanized antibody hu782 and its mutants hu782-HV and hu782-DE groups were significantly higher than those of the Ref.ch175 group, especially the tumor inhibition rates of the hu782 and its ADCC-enhancing mutant antibody hu782-DE groups reached 62.88 % and 65.05%.
  • complete tumor regression was observed in 1 mouse out of 6 mice in the hu782 group, 2 out of 6 mice in the hu782-DE group, and complete tumor regression in 2 mice in the Ref.ch175 group This phenomenon was not observed in the hu782-HV group.

Abstract

本发明提供了抗CLDN18.2单克隆抗体及其制剂。具体地，本发明提供了一种新的抗CLDN18.2人源化抗体。本发明的抗体能够高特异性地结合抗原，具有高亲和力和高生物活性，可特异性结合人CLDN18.2抗原分子，在体外和体内均可高效地抑制肿瘤。

Description

抗CLDN18.2单克隆抗体及其应用 技术领域
本发明涉及医药领域，具体地涉及抗CLDN18.2人源化单克隆抗体及其制剂。
背景技术
紧密连接(Tight Junctions，TJs)是调节上皮组织细胞间渗透性的多分子复合体。在紧密连接复合体中，紧密连接蛋白(claudins)在邻近细胞间形成紧密通道，调控离子、溶质等物质的流通。
CLDN18是肺和胃上皮细胞中紧密连接的主要组成部分，CLDN18具有CLDN18.1和CLDN18.2两种亚型，CLDN18.1主要表达于肺部，CLDN18.2主要表达于胃部。
研究发现，CLDN18.2在原发性胃癌及转移性胃癌、胰腺癌、食管癌、肺癌、胆管癌、卵巢癌等多种肿瘤中高表达(Sahin U,et al.Clin Cancer Res,2008；14(23):7624-34)。由于肿瘤细胞具有增殖快、易侵袭转移等特性而丧失紧密连接结构，其表面的CLDN18.2分子更易暴露出来，CLDN18.2成为理想的肿瘤治疗靶点(Hewitt KJ,et al.BMC Cancer,2006；6:186)。
Ganymed Pharmaceuticals AG公司开发的针对CLDN18.2的嵌合抗体IMAB362已进入III期临床研究。在针对晚期或复发性胃食管癌II期临床研究中，IMAB362联合一线化疗药物EOX(表柔比星、奥沙利铂、卡培他滨)相比单独化疗显著延长了患者的PFS及OS，成为胃癌、食管癌等疾病治疗的潜力药物(Lordick F,et al.Gastric Cancer,2021；24(3):721–730.)。
由于CLDN18.2在肿瘤治疗中的潜力，开发高活性的抗体药物具有较大的临床需求。
人CLDN18.1与CLDN18.2的高度相似性为CLDN18.2特异性抗体的开发带来挑战。此外，小鼠CLDN18.2与人CLDN18.2胞外环1序列完全一致，在小鼠体内筛选特异性抗人CLDN18.2的抗体需打破小鼠的免疫耐受。因此，开发高活性的特异性识别人CLDN18.2抗体药物需克服众多技术难题。
因此，本领域仍然需要开发一种高亲和力和高生物活性的CLDN18.2抗体及其应用。
发明内容
本发明目的是提供了一种高亲和力和高生物活性的CLDN18.2抗体及其应用。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包 括以下三个互补决定区CDR：
(1)互补决定区CDR1，所述互补决定区CDR1的氨基酸序列如SEQ ID NO:3所示；
(2)互补决定区CDR2，所述互补决定区CDR2的氨基酸序列如SEQ ID NO:4或16所示；及
(3)互补决定区CDR3，所述互补决定区CDR3的氨基酸序列如SEQ ID NO:5或17所示。
在另一优选例中，所述的重链可变区的三个互补决定区CDR的序列为SEQ ID No:3、4和5；或3、16和5；或3、4和17。
在另一优选例中，所述重链可变区包括以下四个骨架区FR，其中，所述四个骨架区FR来自与SEQ ID No:1、11、12和13中相应的四个骨架区FR。
在另一优选例中，所述的重链可变区具有SEQ ID NO:1、11、12或13所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体重链，所述抗体重链具有如本发明第一方面所述的抗体的重链可变区。
在另一优选例中，所述重链的恒定区是人源的。
在另一优选例中，所述重链的恒定区为人IgG1、IgG2等的重链恒定区。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR'：
(1)互补决定区CDR1'，所述互补决定区CDR1'的氨基酸序列如SEQ ID NO:8所示；
(2)互补决定区CDR2'，所述互补决定区CDR2'的氨基酸序列如SEQ ID NO:9所示；及
(3)互补决定区CDR3'，所述互补决定区CDR3'的氨基酸序列如SEQ ID NO:10所示。
在另一优选例中，所述轻链可变区包括以下四个骨架区FR，其中，所述四个骨架区FR来自与SEQ ID No:6、14、和15中相应的四个骨架区FR。
在另一优选例中，所述的轻链可变区具有SEQ ID NO:6、14、或15所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体轻链，所述抗体轻链具有如本发明第三方面所述的抗体的轻链可变区。
在另一优选例中，所述轻链的恒定区是人源的。
在另一优选例中，所述轻链的恒定区为人Kappa、Lambda等的轻链恒定区。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区。
或者，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第四方面所述的轻链。
在另一优选例中，所述抗体还具有重链恒定区和轻链恒定区。
在另一优选例中，所述抗体具有如SEQ ID NO:1、11、12或13所示的重链可变区；和/或如SEQ ID NO:6、14、和15所示的轻链可变区。
在另一优选例中，所述抗体具有如SEQ ID NO:1、11、12或13所示的重链可变区；和/或如SEQ ID NO:15所示的轻链可变区。
在另一优选例中，所述抗体选自下组：
(Z1)具有SEQ ID No:1所示的重链可变区和SEQ ID No:6所示的轻链可变区的抗体：
(Z2)具有SEQ ID No:11所示的重链可变区和SEQ ID No:14所示的轻链可变区的抗体；
(Z3)具有SEQ ID No:11所示的重链可变区和SEQ ID No:15所示的轻链可变区的抗体；
(Z4)具有SEQ ID No:12所示的重链可变区和SEQ ID No:14所示的轻链可变区的抗体；
(Z5)具有SEQ ID No:12所示的重链可变区和SEQ ID No:15所示的轻链可变区的抗体；
(Z6)具有SEQ ID No:13所示的重链可变区和SEQ ID No:14所示的轻链可变区的抗体；
(Z7)具有SEQ ID No:13所示的重链可变区和SEQ ID No:15所示的轻链可变区的抗体。
在另一优选例中，所述抗体的重链具有SEQ ID No:18、20、21或22所示的氨基酸序列。
在另一优选例中，所述抗体的轻链具有SEQ ID No:19所示的氨基酸序列。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体为特异性结合CLDN18.2。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体为双特异性抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
本发明还提供了对应于624的抗体，所述的抗体具有SEQ ID No:23所示的重链可变区和SEQ ID No:24所示的轻链可变区。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在本发明的第七方面，提供了一种抗体制剂，所述的抗体制剂包括：
(a)如本发明第五方面所述的抗体；以及
(b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
在另一优选例中，在所述的制剂中，所述抗体的浓度为5-100mg/mL；优选为10-70mg/mL，更优选为20-60mg/mL。
在另一优选例中，所述缓冲剂选自下组：柠檬酸缓冲体系、组氨酸缓冲体系、或其组合。
在另一优选例中，所述的制剂pH范围为5.0-7.5，较佳地为5.5-7。
在另一优选例中，所述的制剂为注射制剂。
在本发明的第八方面，提供了一种试剂盒，所述的试剂盒含有如本发明第七方面所述的抗体制剂，以及盛装所述抗体制剂的容器。
在本发明的第九方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于CLDN18.2的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
在本发明的第十方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第九方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十一方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十二方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合，所述活性成分用于(a)制备检测试剂或试剂盒；
(b)制备预防和/或治疗CLDN18.2相关疾病的药物或制剂；和/或
(c)制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、霍奇金淋巴瘤、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中，所述肿瘤为高表达CLDN18.2的肿瘤。
在另一优选例中，所述药物或制剂用于制备预防和/或治疗与CLDN18.2(表达阳性的)相关的疾病的药物或制剂。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断CLDN18.2相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中CLDN18.2蛋白。
在另一优选例中，所述的检测试剂为检测片。
在本发明的第十三方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物还包含抗肿瘤的第二活性成分。
在另一优选例中，所述的第二活性成分选自下组：细胞毒性药物、毒素、细胞因子、酶、抗体、或其组合。
在另一优选例中，所述的第二活性成分包括：靶向EGFR的抗体、靶向HER2的抗体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于治疗肿瘤。
在另一优选例中，所述肿瘤为高表达CLDN18.2的肿瘤。
在本发明的第十四方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；和/或
(3)如本发明第九方面所述的CAR构建物。
在本发明的第十五方面，提供了一种载体，所述的载体含有如本发明第十四方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第十六方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十五方面所述的载体或基因组中整合有如本发明第十四方面所述的多核苷酸。
在本发明的第十七方面，提供了一种体外检测(包括诊断性或非诊断性)样品中 CLDN18.2蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CLDN18.2蛋白。
在本发明的第十八方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第十一方面所述的抗体药物偶联物。
在本发明的第十九方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十八方面所述的检测板。
在本发明的第二十方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
在本发明的第二十一方面，提供了一种CLDN18.2相关疾病的方法，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了特异性结合杂交瘤上清细胞Elisa结合检测结果。
图2显示了流式细胞术检测结果。图2A为嵌合抗体ch429检测结果，图2B为嵌合抗体ch623检测结果，图2C为嵌合抗体ch624检测结果，图2D为嵌合抗体ch782检测结果，图2E为参照抗体Ref.ch175检测结果。
图3显示了抗CLDN18.2人鼠嵌合抗体与重组细胞结合活性检测结果。图3A为抗CLDN18.2人鼠嵌合抗体与HEK293-hCLDN18.1细胞结合活性结果；图3B为抗CLDN18.2人鼠嵌合抗体与HEK293-hCLDN18.2细胞结合活性结果。
图4显示了抗CLDN18.2人鼠嵌合抗体与NUGC4细胞结合活性检测结果。
图5显示了抗CLDN18.2人鼠嵌合抗体与重组细胞的细胞结合活性流式细胞检测结果。
图6显示了抗CLDN18.2人鼠嵌合抗体对HEK293-hCLDN18.2细胞ADCC活性结果。
图7显示了人源化抗体对人CLDN18.1&CLDN18.2、小鼠CLDN18.1&CLDN18.2以及NUGC4细胞结合活性。
图8显示了人源化抗体ADCC活性。
图9显示了人源化抗体CDC活性检测结果。
图10显示了人源化抗体对HEK293-hCLDN18.2增殖抑制活性结果。
图11显示了本发明抗体hu782与参照抗体的关键结合位点比较。其中，图11A显示了Ref.ch175的关键结合位点，图11B显示了本发明抗体hu782的关键结合位点。
图12显示了本发明抗体的体内抗肿瘤活性。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地获得一种高亲和力优异和高抗肿瘤活性的抗人CLDN18.2抗体。具体地，本发明人经过大量筛选，首次获得了多株对人CLDN18.2具有高亲和力的鼠源抗体。在鼠源抗体基础上，本发明进一步制备了嵌合抗体和人源化抗体，并对获得的人源化抗体进行突变筛选。本发明的抗体，尤其是突变后的人源化抗体具有与嵌合抗体相近的亲和力，未来有进一步开发成靶向治疗的人源化单抗药物。本发明的抗体能有效结合人CLDN18.2，抑制多种肿瘤细胞的增殖，抑制裸鼠内体抑制肿瘤的生长，具有优良的抗肿瘤活性。在此基础上完成了本发明。
具体地，本发明人通过构建多种形式的hCLDN18.2DNA作为抗原交替免疫小鼠，并用重组表达hCLDN18.2的细胞株进行加强免疫，通过杂交瘤融合技术获得特异性识别hCLDN18.2的鼠单抗mab429、mab623、mab624、mab782。
嵌合抗体ch782与hCLDN18.2特异性结合，可以识别肿瘤细胞NUGC4细胞表面的CLDN18.2分子，与CLDN18.2的亲和力显著高于参照抗体Ref.ch175。ch782对hCLDN18.2细胞具有ADCC、CDC及增殖抑制活性。
对嵌合抗体ch782进行人源化，获得4种IgG1型人源化抗体组合hu782H1.1L1、hu782H1.2L1、hu782H1.1L2、hu782H1.2L2，4种人源化抗体特异性识别hCLDN18.2，与ch782具有相当的亲和力。
对人源化抗体重链hu782H1.1进行S60A位点突变以去除潜在糖基化位点，同时进行V101A突变以增强其表达量，获得人源化抗体重链hu782H1.2-V101A。上述S60A、V101A位点根据Kabat编号规则定义。
人源化抗体hu782(H1.2L2组合)、hu782-HV(H1.2-V101A L2组合)对肿瘤细胞的ADCC活性显著优于参照抗体Ref.ch175。
对人源化抗体hu782重链恒定区第239位、第332位氨基酸进行S239D/I332E突变获得人源化抗体hu782-DE。上述S239D、I332E按照EU编号系统定义。
人源化抗体hu782-DE对肿瘤细胞NUGC4的ADCC活性显著优于参照抗体Ref.ch175，其EC50值比Ref.ch175低24倍。
人源化抗体hu782、hu782-HV对hCLDN18.2靶细胞具有CDC杀伤活性。
人源化抗体hu782、hu782-HV对hCLDN18.2靶细胞具有增殖抑制活性，其增殖抑制活性具有剂量依赖性，显著优于参照抗体Ref.ch175。
表位分析发现人源化抗体hu782识别的关键位点包括hCLDN18.2分子的E56、G48位点，而参照抗体Ref.ch175识别的关键位点包括A42S、N45A、E56A、G48位点，证实两个抗体结合表位具有一定的差异。
在裸小鼠肿瘤移植模型中，hu782、hu782-HV、hu782-DE三个抗体均能明显移植肿瘤生长，且优于参照抗体Ref.ch175，尤其是hu782、hu782-DE两个抗体肿瘤生长抑制效果显著优于参照抗体Ref.ch175。hu782、hu782-DE两个抗体治疗组均有小鼠肿瘤发生完全消退，hu782-HV、hu782、hu782-DE肿瘤抑制率为34.68％、62.88％和65.05％，参照抗体Ref.ch175为15.5％。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文所用，术语“重链可变区”与“V H”可互换使用。
如本文所用，术语“轻链可变区”与“V L”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合CLDN18.2的抗体，例如具有重链可变区(如SEQ ID NO:1、11、12或13的氨基酸序列)和/或轻链可变区(如SEQ ID NO:6、14或15的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
另一种优选的本发明抗体是对应于624的特异性结合CLDN18.2的抗体，例如具有重链可变区(如SEQ ID NO:23的氨基酸序列)和/或轻链可变区(如SEQ ID NO:24的氨基酸序列)的蛋白或多肽。
CLDN18.2
人CLDN18全长含261个氨基酸，为四次跨膜蛋白，具有四个跨膜疏水区，胞外为两个环状结构，环1由跨膜区1和跨膜区2环绕而成；环2由跨膜区3和跨膜区4环绕而成。人CLDN18.1与CLDN18.2的N端、跨膜区1及胞外环1的氨基酸组成上存在差异，其余部分均完全一致，其序列相似度达92％。
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
已有的抗体编号方案包括：
1.Kabat方案(Kabat等，1991)是基于相同结构域类型的序列之间的高序列变异区域的位置，抗体重(VH)和轻(Vλ和Vκ)可变结构域的编号不同。
2.Chothia的方案(Al-Lazikani，1997)与Kabat的方案相同，但校正了在第一个VH互补决定区(CDR)周围插入注释的位置，使其对应于结构环。同样，增强型Chothia计划(Abhinandan和Martin，2008)对插入位置进行了进一步的结构修正。
3.与这些类似Kabat的方案相反，IMGT(Lefranc，2003)和AHo(Honegger和Plückthun，2001)都定义了抗体和T细胞受体(TCR)(Vα和Vβ)可变结构域的独特方案。因此，可以容易地在域类型之间比较等效残基位置。IMGT和AHo在他们注释的位置数量(分别为128和149)以及他们认为indel发生的位置上有所不同。
免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其较链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2、IgG3，IgG4。轻链根据恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人 源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到竣基端依次排列的顺序序为:FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。本发明的实施例13中，结合kabat及Chothia方法，对ch782抗体的6个CDR进行划分。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的抗CLDN18.2的单克隆抗体。制备时用CLDN18.2抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源CLDN18.2抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
术语“抗体的抗原结合片段”(或简称“抗体片段”)是指抗体的保持特异性结合抗原(例如，CLDN18.2)的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗体的抗原结合片段”中包含的结合片段的实例包括
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab') 2片段，包含通过较链区上的二硫桥连接的两个Fab片段的二价片段；
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段。
Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，CLDN18.2分子上的特定部位)。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于1O -8M、1O -9M或lO -10M或更小的亲和力(KD)结合。
术语“竞争结合”是指与本发明的单克隆抗体识别CLDN18.2的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述抗原结合的抗体。与本发明的单克隆抗体结合相同表位的抗体是指识别并结合于本发明的单克隆抗体所识别的CLDN18.2的氨基酸序列的抗体。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10 -7M，例如小于大约1O -8M、1O -9M或lO -10M或更小的解离平衡常数(KD)结合CLDN18.2。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗CLDN18.2人源化抗体
本发明提供抗CLDN18.2人源化抗体(以下简称CLDN18.2抗体)。具体地，本发明提供一种针对CLDN18.2的高特异性和高亲和力的人源化抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
1986年Jones等人首次将鼠单抗重链CDR移植到人抗体重链骨架区，然后与鼠单抗轻链组装成完整抗体并保持了与原鼠单抗相似的亲和力，为抗体人源化技术的发展提供了思路。1989年Queen等人通过CDR移植的方法，成功构建抗CD25人源化抗体，该方法使用的是人抗体骨架区进行人源化，在骨架区部分位点保留了鼠源抗体氨基酸以保持亲和力。1992年Presta等人报道了以人抗体亚群共有序列(consensus sequence)为模板进行CDR移植成功构建人源化的方法。1994年Pedersen等人报道了用表面重塑(resurfacing)的方法对抗体人源化。1994年Hsiao等人报道了以人抗体Germline序列骨架区进行CDR移植的人源化方法。1994年Jespers等人用噬菌体库(shuffling library)的方法成功构建人源化方法。
抗体人源化中人骨架区的选择通常有两种，一种是已知的成熟抗体，一种是 人Germline序列。已知的成熟抗体骨架区通常含有体细胞突变位点，可能带来潜在的免疫原性。相比已成熟抗体，人Germline序列骨架区理论上免疫原性更低，而且结构更灵活，可塑性强，容易接受不同的CDR区。人抗体Germline基因在人体中的使用频率具有一定的偏向性，选择使用频率高的Germline骨架区人源化后的抗体具有免疫原性低、表达量高、结构稳定等优点。
在本发明的一个优选地实施方式中，在人源化时并未选择与鼠源抗体相似度最高的Germline序列，而是兼顾相似性及人体使用频率，经过大量实验筛选，选择了优选序列的骨架区进行人源化。选用人抗体Germline骨架区进行CDR移植，这样构建的人源化抗体结构更加稳定、表达量高、免疫原性低、成药性更高。
在另一优选例中，所述的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地，所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或人kappa、Lambda轻链恒定区。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2、或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
本发明抗体可以是靶向CLDN18.2的人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的CLDN18.2抗体。例如，可以用连接或天然存在的CLDN18.2蛋白或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的CLDN18.2都可以作为免疫原(抗原)，用于产生对CLDN18.2特异的非人抗体，筛选所述抗体的生物学活性。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、杆状病毒载体、质粒和非病毒载体。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输己与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物或动物细胞(如哺乳动物细胞)。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
抗体制剂
抗体在不同的制剂缓冲液中具有不同的稳定性，表现为电荷异质性的变化、抗体分子的降解、聚合等，这些质量性质的变化与抗体本身的理化性质相关，因此，在抗体药物开发过程，需根据不同抗体的理化性质筛选适合其自身的制剂缓冲液。目前常用的抗体制剂缓冲体系有磷酸盐缓冲液、柠檬酸缓冲液、组氨酸缓冲液等，同时根据抗体性质会添加不同浓度的盐离子或山梨醇、海藻糖、蔗糖等赋形剂，以及适量的诸如吐温等表面活性剂，以维持抗体的稳定性。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合CLDN18.2蛋白分子，因而可用于预防和治疗CLDN18.2相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用 的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明的主要优点包括：
1.本发明抗体为典型的色氨酸、酪氨酸包埋较好的结构，所筛选的人VH、VL骨架区模板结构稳定，且与鼠单抗CDR区适配较好，轻重链可变区能较好的配对在一起，亲和力高、结构稳定。
2.与嵌合抗体相比，本发明人源化抗体具有优异的生物活性、特异性，在保留与CLDN18.2相当的亲合力的同时，具有更低的免疫原性和更高的表达量。
3.本发明人源化抗体具有显著的体外生物活性，可高效抑制体外培养肿瘤细胞增殖的活性，活性优于对照抗体。
4.本发明人源化抗体具有显著的体内生物活性，对人胃癌细胞等肿瘤细胞具有优异的抑制活性，活性优于对照抗体。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
材料与方法
1.参照抗体
本发明实验中所使用的参照抗体，其序列来源于专利US8168427中175D10克隆的抗体序列。175D10抗体又名为IMAB362。对175D10抗体的可变区序列进行全基因合成，并构建到含有IgG1嵌合抗体恒定区的真核表达载体上，并进行表达纯化。该制备的参照抗体在本发明中简称为Ref.ch175。
参照抗体Ref.ch175(或IMAB362)特异性识别CLDN18.2的胞外环1部分，不与CLDN18.1结合，主要通过抗体依赖的细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity，ADCC)及补体依赖的细胞毒性作用(complement dependent cytotoxicity，CDC)杀伤肿瘤细胞。
实施例1：稳定表达CLDN18.1或CLDN18.2细胞株的构建
在本实施例中，构建稳定表达人CLDN18.1、人CLDN18.2、小鼠CLDN18.1、小鼠CLDN18.2的细胞株。方法如下：
全基因合成编码人CLDN18.1(NP_057453.1)、人CLDN18.2(NP_001002026.1)全长基因片段，将全长片段通过Xhol+BamHI酶切分别克隆到pEGFP-N2真核表达载体(购自Clontech公司)中，分别命名为pEGFP-N2-hCLDN18.1和pEGFP-N2-hCLDN18.2。
将HEK293细胞(购自中国科学院细胞库，GNHu43)以4×10 5个/孔接种到6孔板中，第二天，采用脂质体转染试剂LipoMax将pEGFP-N2-hCLDN18.1、pEGFP-N2-hCLDN18.2分别转染HEK293细胞，转染24h后以1:10的比例传代细胞，待细胞贴壁后加入G418(购自Gibco)终浓度为300μg/ml进行加压筛选，经过亚克隆获得稳定表达人CLDN18.1、人CLDN18.2膜蛋白的HEK293细胞，分别命名为HEK293-hCLDN18.1和HEK293-hCLDN18.2。
全基因合成编码小鼠CLDN18.1(NP_062789.1)、小鼠CLDN18.2(NP_001181850.1)全长基因片段，通过KpnI+XbaI酶切克隆到pcDNA3.1-P2A-EGFP真核表达载体(购自南京金斯瑞公司)，分别命名为pcDNA3.1-mCLDN18.1和pcDNA3.1-mCLDN18.1。采用同上方法转染HEK293细胞，获得稳定表达小鼠CLDN18.1和CLDN18.2膜蛋白的细胞，分别命名为HEK293-mCLDN18.1和HEK293-mCLDN18.2。
Figure PCTCN2022131502-appb-000001
Figure PCTCN2022131502-appb-000002
实施例2：免疫原制备
由于人CLDN18.2胞外环1(CLDN18.2ECL1)与小鼠CLDN18.2ECL1氨基酸序列完全一致，因此较难获得与CLDN18.2ECL1结合的抗体。同时考虑到肿瘤细胞表面CLDN18.2分子构象与正常组织细胞表面可能存在差异，在本实施例中，设计了多种形式的DNA质粒作为抗原来突破免疫耐受，以获得高亲和力识别肿瘤细胞的抗体。
将编码人CLDN18.2ECL1以及CLDN18.2全长氨基酸的核苷酸分别和破伤风类毒素的P2表位TT830-844、P30表位TT947-967串联克隆到真核表达载体PTT5(购自Invitrogen公司)，分别命名为PTT5-TT-hCLDN18.2、PTT5-TT-hCLDN18.2-ECL1。
本发明将CLDN18.2ECL1融合到人IgG1Fc-C端，命名为hFc-CLDN18.2-ECL1。
TT-hCLDN18.2核苷酸序列SEQ ID No:29
Figure PCTCN2022131502-appb-000003
Figure PCTCN2022131502-appb-000004
TT-hCLDN18.2-ECL1核苷酸序列：SEQ ID No:30
Figure PCTCN2022131502-appb-000005
hFc-CLDN18.2-ECL1核苷酸序列：SEQ ID No:31
Figure PCTCN2022131502-appb-000006
实施例3：抗人CLDN18.2杂交瘤细胞株的制备
实验用Balb/c小鼠购自上海西普尔-必凯实验室，所有免疫用小鼠均为4-6周 龄、雌性，标准化无病、健康的纯种小鼠。
由于CLDN18.2是4次跨膜蛋白，为了获得高亲和力识别肿瘤细胞的特异性抗体，小鼠免疫是通过多种质粒交替免疫，并用稳转表达人CLDN18.2膜蛋白的细胞进行冲击免疫。方法如下：
在第1天和第14天：将50μg编码人CLDN18.2全长基因的真核表达载体质粒pEGFP-N2-hCLDN18.2，注射入小鼠四头肌(i.m)，佐剂为In vivo-Jet PEI-Man体内转染试剂(购至Polyplus，203-10G)。在第28天将50μg真核表达质粒hFc-CLDN18.2-ECL1DNA注射入小鼠四头肌(i.m)；在第42天将50μg真核表达载体质粒PTT5-TT-hCLDN18.2注射入小鼠四头肌(i.m)；在第56天将50μg真核表达载体质粒PTT5-TT-CLDN18.2ECL1注射入小鼠四头肌(i.m)；在第70天将50μg真核表达载体质粒PTT5-TT-hCLDN18.2以及50μg真核表达载体质粒PTT5-TT-CLDN18.2ECL1注射入小鼠四头肌(i.m)。
14天后小鼠尾静脉采血，采用cell based-elisa方法测定血清抗体滴度，在融合前3天用2×10 7个HEK293-hCLDN18.2细胞/只小鼠腹腔注射加强免疫抗体滴度最高的2只小鼠。融合当日，小鼠眼球取血并摘除脾脏，制备单细胞悬液，与SP20细胞采用电融合方式得到杂交瘤铺板。
Figure PCTCN2022131502-appb-000007
实施例4：特异性结合人CLDN18.2而不结合人CLDN18.1杂交瘤细胞的筛选
在本实施例中，采用cell based-ELISA方法进行杂交瘤的筛选特异性结合人CLDN18.2而不结合人CLDN18.1杂交瘤细胞。方法如下：
将HEK293-hCLDN18.1细胞和HEK293-hCLDN18.2细胞经胰酶消化后按照3×10 5个/mL的细胞密度分别接种于多聚赖氨酸(P4707，Sigma)包被的96孔板上，200μL/孔，过夜培养；待细胞汇合度达到90％以上时，去除上清，使用10％中性福尔马林溶液室温固定15min；PBS洗3遍后，并用5％脱脂牛奶4℃封闭过夜。检测杂交瘤时将同一孔上清分别加入到HEK293-hCLDN18.1细胞和HEK293-hCLDN18.2细胞包被板上，加入上清80μl/孔，37℃孵育1h，PBST洗3次后，加入100μl/孔1:5000稀释的羊抗鼠IgG(H+L)-HRP，37℃孵育45min，显色 30min加入50μl/孔2M H 2SO 4终止显色，酶标仪读取OD450。
经过对上万个克隆的筛选，最获得多株hCLDN18.2特异性抗体，最优的4株如下表所示：
Figure PCTCN2022131502-appb-000008
实施例5：鼠单抗亚型检测
采用亚型检测试剂盒(SBA Clonotyping System-HRP kit，购于southernbiotech，5300-05)对CLDN18.2特异性结合的克隆株进行亚型检测。
检测结果如下：
克隆编号 重链亚型 轻链亚型
mab429 IgM Kappa
mab623 IgM Kappa
mab624 IgG2b Kappa
mab782 IgG2a Kappa
实施例6：抗人CLDN18.2鼠源单抗可变区编码序列的克隆和鉴定
由于筛选到的鼠单抗中存在IgM亚型抗体，为了进一步评价其活性，采用RACE-PCR法获取鼠单抗可变区序列，并构建嵌合抗体。
采用RNA提取试剂盒TakaRa MiniBEST Universal RNA Extration Kit(Takara，9767)提取实施例4中获得杂交瘤总mRNA，采用SMARTer RACE 5'/3'Kit(Clontech，634858)PCR获得鼠源抗体可变区序列，结果如下：
624抗体重链可变区氨基酸序列(SEQ ID No:23)：
Figure PCTCN2022131502-appb-000009
624抗体轻链链可变区氨基酸序列(SEQ ID No:24)：
Figure PCTCN2022131502-appb-000010
782抗体重链可变区核苷酸序列(SEQ ID No:2)：
Figure PCTCN2022131502-appb-000011
782抗体重链可变区氨基酸序列(SEQ ID No:1)：
Figure PCTCN2022131502-appb-000012
782抗体轻链链可变区核苷酸序列(SEQ ID No:7)：
Figure PCTCN2022131502-appb-000013
782抗体轻链链可变区氨基酸序列(SEQ ID No:6)：
Figure PCTCN2022131502-appb-000014
实施例7：抗CLDN18.2人鼠嵌合抗体构建和表达
按照同源重组方法引物设计原则设计引物，扩增出实施例6中获得的鼠源抗体重轻链可变区基因片段，分别连接到人重轻链恒定区序列，构建到表达载体PTT5(购自Invitrogen公司)中。
采用商业化质粒大抽试剂盒进行质粒提取，并采用PEI转染方法转染293FV悬浮细胞，转染7天后收集上清，采用ProteinA亲和层析获得嵌合抗体。
实施例8：抗CLDN18.2人鼠嵌合抗体特异性分析
分别将稳定转染人CLDN18.2的HEK293细胞和稳定转染人CLDN18.1的HEK293细胞用胰酶消化后，将5×10 5个细胞/100μl，加入杂交瘤上清100μl，冰上孵育1小时，FACS缓冲液洗涤2遍后，加入1:400稀释的羊抗鼠IgG-PE二抗，冰上孵育45min，FACS缓冲液洗涤2遍后，加入300μl上样缓冲液，转移到BD流式管中，通过BD FACS calibur分析。采用Flowjo软件分析平均荧光强度 MFI(MedianFluorescence Intensity)。
结果见图2和下表。
抗体编号 HEK293-hCLDN18.2 HEK293-hCLDN 18.1 P/N
ch429 1219 2.26 539.4
ch623 235 2.05 114.6
ch624 1420 2.27 625.6
ch782 4179 2.86 1461.2
Ref.ch175 2618 2.48 1055.6
注：P/N为抗体与HEK293-hCLDN18.2细胞结合的MFI除以HEK293-hCLDN18.1细胞结合的MFI，用于表示其特异性。
结果表明，ch429、ch623、ch624、ch782与人CLDN18.2特异性结合，不与人CLDN18.1结合。其中，嵌合抗体ch782的MFI高于于参照抗体Ref.ch175。
实施例9：抗CLDN18.2人鼠嵌合抗体与重组细胞结合活性检测
将HEK293-hCLDN18.1和HEK293-hCLDN18.2细胞经胰酶消化后按照一定的细胞密度接种于多聚赖氨酸(P4707，Sigma)包被的96孔板上，过夜培养；待细胞汇合度达到90％以上时，去除上清，使用10％福尔马林溶液室温固定15min；PBS洗一遍后，并用5％脱脂牛奶4℃封闭过夜备用。将候选嵌合抗体及参照抗体Ref.ch175从1μg/ml起3倍稀释10个梯度，100μl/孔加入到细胞包被的96孔板中，37℃孵育1小时后，用PBST洗板3遍，加入1:2000稀释的鼠抗人IgG Fc-HRP，37℃孵育1小时后，用PBST洗板3遍，用TMB显色液显色15分钟，用2M H 2SO 4终止。读取OD450-OD630值，用GraphPad Prism 8软件拟合做图计算EC50。结果见图3，表明ch429、ch623、ch624、ch782与参照抗体Ref.ch175均不与HEK293-hCLDN18.1细胞结合，与HEK293-HCLDN18.2细胞特异性结合，并且我们发现ch429和ch782相对于参照抗体Ref.ch175的相对结合活性分别为257.5％和179.9％。结果见下表：
抗体编号 EC50(ng/ml) 相对结合活性
ch429 12.06 257.5％
ch623 91.81 33.8％
ch624 39.17 79.3％
ch782 17.26 179.9％
Ref.ch175 31.05 100.0％
实施例10：抗CLDN18.2人鼠嵌合抗体与NUGC4细胞结合活性检测
采用实施例9中同样的方法将NUGC4细胞(购自南京科佰生物科技有限公司)铺到96孔板上，将候选嵌合抗体及参照抗体Ref.ch175从100μg/ml起3倍稀释10个梯度，100μl/孔加入到细胞包被的96孔板中，37℃孵育1小时后，用PBST洗板3遍，加入1:2000稀释的鼠抗人IgG Fc-HRP，37℃孵育1小时后，用PBST洗板3遍，用TMB显色液显色15分钟，用2M H 2SO 4终止。读取OD450-OD630值，用GraphPad Prism 8软件拟合做图计算EC50。
结果见图4和下表。
抗体编号 EC50(μg/ml)
ch429 5.25
ch623 -
ch624 -
ch782 0.57
Ref.ch175 -
嵌合抗体ch782和ch429的EC50分别是0.57μg/ml和5.25μg/ml，并且ch782在高浓度时的读数值(OD450＝2.3)高于发现ch429的读数值(OD450＝1.2)；而ch623、ch624及参照抗体Ref.ch175均未能计算出EC50。
这表明，ch782抗体与内源性表达人CLDN18.2蛋白的NUGC4细胞的结合活性最好，明显强于参照抗体Ref.ch175。
实施例11：抗CLDN18.2人鼠嵌合抗体与HEK293-hCLDN18.2细胞结合活性检测
按照实施例8中的方法，将嵌合抗体ch429、ch624、ch782、Ref.ch175从10μg/ml浓度进行3倍梯度稀释，进行流式实验。以MFI为纵坐标用GraphPad Prism 8软件拟合做图计算EC50。
结果见图5和下表。
抗体编号 EC50(μg/ml)
ch429 0.52
ch624 -
ch782 0.81
Ref.ch175 0.85
本发明的嵌合抗体ch429、ch782及参照抗体Ref.ch175均能与HEK293-hCLDN18.2细胞结合，EC50分别为0.52μg/ml、0.81μg/ml、0.85μg/ml， 但ch624的结合活性明显较弱。
此外，高浓度时ch782与HEK293-hCLDN18.2细胞结合的MFI高于Ref.ch175。
实施例12：报告基因法检测抗CLDN18.2抗体人鼠嵌合抗体对HEK293-hCLDN18.2细胞ADCC活性
抗体Fc端介导的ADCC效应是评价抗体抗肿瘤活性的重要指标。ADCC是抗体依赖的细胞介导的细胞毒性作用，其作用机制是抗体与肿瘤表面抗原结合后，抗体Fc区与免疫效应细胞受体结合，主要是NK细胞上的Fc受体靶向识别，直接溶解杀死肿瘤细胞。
本实施例采用报告基因法检测抗体ADCC生物学活性，选取稳定表达FcγRIIIa受体的工程改造Jurkat细胞作为效应细胞，选用Bio-GloTM Luciferase Assay System试剂盒检测荧光素酶化学发光信号，通过测量NFAT信号通路的活化，定量检测抗体Fc效应因子功能。
将实施例1中构建的HEK293-hCLDN18.2细胞消化后按照2.5×10 5/ml稀释，然后100μl/孔铺到白色不透明细胞培养板中，37℃静置培养24h后，弃上清，加入25μl/孔含有0.1％BSA的分析培养基；将Jurkat/NFAT-luc+FcγRIIIa细胞稀释成6×10 6个/mL，取出96孔板，每孔加入25μl Jurkat/NFAT-luc+FcγRIIIa细胞悬液，再在每孔加入25μL抗体稀释液使其终浓度为6μg/mL起始做3倍系列稀释，共10个稀释梯度；将细胞板放入恒温培养箱，诱导培养6h后，96孔板置于室温条件下平衡至少15min，使用连续加样枪，向所有反应孔中每孔加入75μL Bio-GloTM Luciferase Assay Reagent(染色液需避光)，室温条件下反应5min，置于多功能酶标仪检测Luminescence光信号数值。荧光数值应与抗体浓度用GraphPad Prism 8软件拟合作图计算EC50值。
结果见图6和下表。
抗体编号 EC50(ng/ml)
ch782 47.64
Ref.ch175 36.46
结果表明，将ch782与参照抗体Ref.ch175梯度稀释后，按照效靶比E:T＝6：1，经过37℃孵育6h后发现ch782和Ref.ch175均能诱导较强的ADCC活性。
实施例13：抗CLDN18.2抗体ch782的人源化
为了降低抗体免疫原性，减少抗体给要到体内后产生的人抗鼠抗体(HAMA)效应，在本实施例中，对优选抗体ch782进行了人源化。具体地，将实施例6中mab782抗体可变区序列进行人源化序列设计。按照kabat编码系统对重轻链氨基 酸序列进行编码，经过NCBIIgblast(https://www.ncbi.nlm.nih.gov/igblast/)进行序列同源对比，选取同源性较高人源抗体序列作为骨架，进行鼠源抗体CDR移植，并骨架区的部分核心氨基酸进行回复突变。
Blast检索782抗体重链与人germline同源序列：
IGHV4-38-2*02 71.1％(69/97)
IGHV4-61*01 71.4％(70/98)
IGHV4-61*08 71.4％(70/98)
Blast检索782抗体轻链与人germline同源序列：
IGKV4-1*01 79.2％(80/101)
IGKV3-7*02 65.3％(66/101)
IGKV3-7*04 65.3％(66/101)
782抗体的重链可变区中按照kabat定义FR和CDR序列如下：
Figure PCTCN2022131502-appb-000015
782抗体的轻链可变区中按照kabat定义FR和CDR序列如下：
Figure PCTCN2022131502-appb-000016
本发明中抗体重链人源化选择同源性较高的IGHV4-61为模板进行CDR区替换，重链CDR1移植时同时包括了Chothia定义的CDR1位点，并对I48M/V67I/V71R3个位点进行回复突变得到hu782H1.1。
通过翻译后修饰分析发现S60位点存在潜在的糖基化位点，通过点突变的方式突变为S60A，命名为hu782H1.2，并将其第101位氨基酸V突变为A，以优化其表达量，并名为hu782H1.2-V101A
本发明中抗体轻链人源化分别以VκI consensus sequence为模板进行CDR区替换，得到hu782L1；以IGKV4-1为模板进行CDR区替换，得到hu782L2
以上序列通过密码子优化后，进行全基因合成，并克隆到含有重轻链恒定区的表达载体上进行表达纯化。
Figure PCTCN2022131502-appb-000017
实施例14：人源化抗体对CLDN18.2的特异性结合活性检测
采用上述实施例中嵌合抗体结合活性评价方法，对人源化后的抗体与人CLDN18.1和CLDN18.2、小鼠CLDN18.1和CLDN18.2以及NUGC4细胞的结合活性进行检测。
结果如图7和下表所示。
Figure PCTCN2022131502-appb-000018
结果表明，人源化抗体均能与人CLDN18.2特异性结合，并且结合活性相对比与嵌合抗体ch782未见到明显降低。
此外，出乎意料地，4种人源化抗体与肿瘤细胞NUGC4的细胞结合的EC50值显著优于嵌合抗体ch782。
实施例15：人源化抗体ADCC活性检测
本发明中抗体的体内活性主要机制之一是ADCC功能活性，人IgG1抗体(hIgG1)主要通过抗体铰链区及CH2区域与FcγR结合，发挥ADCC效应。为了增强人源化抗体的ADCC活性，在本实施例中，在人源化抗体重链hu782H1.2的Fc中引入S239D/I332E(DE)突变，以增强其对FcγR的亲和力，从而达到ADCC增强的目的。经表达纯化后，采用报告基因法对野生型及DE突变型人源抗体的ADCC进行评价。
  重链 轻链
hu782 hu782H1.2(SEQ ID No:18) hu782L2(SEQ ID No:19)
hu782-HV hu782H1.2-V101A(SEQ ID No:21) hu782L2(SEQ ID No:19)
hu782-DE hu782H1.2-S239D/I332E(SEQ ID No:20) hu782L2(SEQ ID No:19)
hu782-HV-DE hu782H1.2-V101A/S239D/I332E(SEQ ID No:22) hu782L2(SEQ ID No:19)
方法如下：按照实施例12中的具体操作步骤，将靶细胞HEK293-hCLDN18.2和NUGC4细胞分别按照2.5×10 5/ml稀释，然后100μl/孔铺到白色不透明细胞培养板中，37℃静置培养24h后，弃上清，加入25μl/孔含有0.1％BSA的分析培养基；将Jurkat/NFAT-luc+FcγRIIIa细胞稀释成6×10 6个/mL，取出96孔板，每孔加入25μL Jurkat/NFAT-luc+FcγRIIIa细胞悬液，再在每孔加入25μL抗体稀释液使其终浓度为6μg/mL起始做3倍系列稀释，共10个稀释梯度；将细胞板放入恒温培养箱，诱导培养6h后，96孔板置于室温条件下平衡至少15min，使用连续加样枪，向所有反应孔中每孔加入75μL Bio-GloTM Luciferase Assay Reagent(染色液需避光)，室温条件下反应5min，置于多功能酶标仪检测Luminescence光信号数值。 荧光数值应与抗体浓度用GraphPad Prism 8软件拟合作图计算EC50值。
Figure PCTCN2022131502-appb-000019
*nd：未进行检测；*NA：因信号弱，未计算出。
结果表明，对于高表达人CLDN18.2的重组细胞，嵌合抗体ch782以及人源化抗体hu782、hu782-HV均能有效产生与参照抗体相当的ADCC活性(EC50分别为：47.64ng/ml、64.03ng/ml、70.94ng/ml)，并且在Fc引物S239D/I332E突变的hu782-DE的ADCC活性得到了显著提高(图8)。
同样的结果在内源性地表达人CLDN18.2的NUGC4细胞上也观察到，并且在本发明得到的候选抗体ch782及hu782对NUGC4细胞的ADCC活性优于参照抗体Ref.ch175。
接着，采用稳转hCLDN18.2的NUGC4细胞为靶细胞进行了ADCC活性检测，结果表明，hu782-DE、hu782-HV-DE的ADCC活性显著优于对照抗体Ref.ch175，EC50值分别为6.87ng/ml和11.48ng/ml(图8C)。
实施例16：人源化抗体CDC活性检测
CDC是补体依赖的细胞介导的细胞毒性作用，其作用机制是结合补体的抗原抗体混合物最终在细胞膜上穿孔，使细胞渗透压改变，最后导致细胞裂解。在本实施例中，采用HEK293-hCLDN18.2细胞为靶细胞，以幼兔补体血清提供补提，采用CCK-8细胞增殖-毒性检测试剂盒检测抗体CDC效应。
采用实施例1中的HEK293-hCLDN18.2细胞作为靶细胞，消化后稀释至2.0×10 5个/mL，以每孔100μL的体积铺在96孔透光黑体培养板中，静置培养4h左右待细胞完全贴壁。将生长培养基吸干，每孔加入50μL分析培养基。反应孔内每孔加入50μL稀释后抗体，再加入50μL按照1:15稀释好的幼兔补体，使得每孔终体积为150μL，每孔样品体积为总体积的1/3，初始浓度是目的浓度的3倍，目的浓度为6μg/mL起始。将细胞板放入恒温培养箱，诱导培养12h后吸弃培养基，每孔加入100μL新鲜的分析培养基，室温条件下每孔加入CCK-8溶液10μL，置 于5％CO 2培养箱，37℃避光反应4h。置于多功能酶标仪450nm波长检测OD值。用GraphPad Prism 8软件拟合作图计算EC50值。
结果如下表所示，
Figure PCTCN2022131502-appb-000020
结果表明，本发明中ch782、hu782、hu782HV均能对HEK293-hCLDN18.2细胞诱导出与参照抗体Ref.ch175相当的CDC效应(EC50分别为30.9ng/ml、49.46ng/ml、47.58ng/ml、40.89ng/ml)。
实施例17人源化抗体对HEK293-hCLDN18.2细胞增殖抑制作用检测
取对数生长期的HEK293-hCLDN18.2细胞。消化靶细胞，计数后将细胞稀释至1.0×10 5个/mL，以每孔100μL的体积铺在96孔透光黑体培养板中；静置培养4h左右待细胞完全贴壁。反应孔内每孔加入100μL稀释后抗体，每孔终体积为200μL，将96孔板置于5％CO 2培养箱，37℃培养诱导培养96h后吸弃培养基，每孔加入100μL新鲜的分析培养基，室温条件下每孔加入CCK-8溶液10μL，置于5％CO 2培养箱，37℃避光反应4h。置于多功能酶标仪450nm波长检测OD值。结果计算细胞增殖抑制率＝1-(OD450 抗体/OD450 空白)。
结果表明，本发明抗体hu782在低浓度(1μg/ml)下对靶细胞的增殖抑制效果既优于高浓度(30μg/ml)的参照抗体Ref.ch175(18.3％vs.7.3％)，并且随着抗体浓度的增加，增殖抑制效果加强呈现剂量依赖效应，在30μg/ml浓度时，ch782、hu782、hu782-HV的细胞增殖抑制率分别达到了67.8％、76％、63.2％；显著优于参照抗体Ref.ch175。
实施例18:人源化抗体与hCLDN18.2蛋白结合位点分析
为探究本发明中抗体与hCLDN18.2蛋白结合为不与hCLDN18.1蛋白结合的关键氨基酸位点，在本实施例中，采用点突变的方式在实施例1中构建的pEFGP-N2-hCLDN18.2质粒上将hCLDN18.2ECL1与hCLDN18.1ECL1氨基酸差异位点及其附近氨基酸进行丙氨酸突变，其中A42突变为hCLDN18.1ECL1中等位氨基酸S(Q29A、S31A、N37A、A42S、N45A、Q47A、G48A、S52A、E56A、G65A、L69A、G71A)。将突变成功的质粒及野生型质粒按照实施例1中的方法分别转染 293Fv细胞，48小时后取细胞进行流式检测。
将hu782、Ref.ch175以及同型对照(IsotypecontrolhIgG1)稀释到10μg/ml，分别与与转染以上转染细胞冰上孵育1小时，FACS缓冲液洗涤2遍后，加入1:400稀释的羊抗鼠IgG-PE二抗，冰上孵育45min，FACS缓冲液洗涤2遍后，加入300μl上样缓冲液，转移到BD流式管中，通过BD FACS calibur分析。采用Flowjo软件分析平均荧光强度MFI(Median Fluorescence Intensity)结果，结果判定，突变后转染细胞MFI相对野生型降低达到50％以上时认为该位点是为影响抗体的结合关键氨基酸位点。
结果表明，如图11所示，本发明A42S、N45A、E56A突变后对参照抗体Ref.ch175的结合影响较大，而A42S突变对hu782的结合没有影响，N45A突变对hu782的结合影响较小，E56A突变对hu782的结合影响较大。同时本发明意外发现，hCLDN18.2ECL1与hCLDN18.1ECL1相同氨基酸位点G48A突变后导致参照抗体Ref.ch175及hu782均不能结合。
实施例19:人源化抗体体内活性评价
在本实施例中，采用BALB/c裸小鼠皮下移植肿瘤细胞建立CDX动物模型，评估人源化抗体体内药效评估。
选用按照实施例1方法构建NUGC4-hCLDN18.2稳转细胞株，进行扩大培养。选用4-6雌性SPF级BALB/c裸小鼠，4～6周龄，适应性饲养3-4天后准备种瘤。将对数生长期重组NUGC4-hCLDN18.2细胞，消化后用无血清1640培养基洗2次后，稀释到2.5×10 7/ml，小鼠右腋窝皮下接种0.2ml共5×10 6个细胞/只。接种1周后肿瘤长至70～100mm 3时进行随机区组设计分组，每组6只。空白模型组为IgG1同型对照。实验组按10mg/kg剂量给药，给药频率一周两次，腹腔注射和静脉注射交替给药，每次给药前记录肿瘤体积和小鼠体重数据。
肿瘤体积计算：V(mm 3)＝(W 2x L)/2，W为肿瘤近中线短直径，L为肿瘤近中线长直径。
当肿瘤体积大于2000mm 3，体重减轻大于20％或观察到对重要生理机能的干扰或肿瘤性溃疡坏死时颈椎脱臼法处死小鼠。
肿瘤抑制率(TGI)计算公式如下：
Figure PCTCN2022131502-appb-000021
Vt-V0＝(给药组第t天时的平均肿瘤体积)-(该组在分组当天平均肿瘤体积)
Vct-Vc0＝(给药组第t天时的平均肿瘤体积)-(该组在分组当天平均肿瘤体积)
在NUGC4-hCLDN18.2小鼠皮下移植肿瘤模型中，观察到本发明人源化抗体及其突变体抗肿瘤生长活性，均显示了一定的药效。在终点时，各组肿瘤体积见下表。
Figure PCTCN2022131502-appb-000022
*:CR，肿瘤完全消退的动物数/本组动物总数
人源化抗体hu782及其突变体hu782-HV、hu782-DE组的肿瘤抑制率均显著高于Ref.ch175组，尤其是hu782及其ADCC增强突变抗体hu782-DE组肿瘤抑制率分别达到了62.88％和65.05％。此外，在hu782组中的6只小鼠中观察到1只小鼠肿瘤完全消退，在hu782-DE组中的6只小鼠中观察到2只小鼠肿瘤完全消退，而在Ref.ch175组合hu782-HV组均未观察到此现象。
上述结果表明，本发明抗体(尤其是人源化抗体)的体内药效效果显著优于参照抗体Ref.ch175。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种抗人CLDN18.2抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    (1)互补决定区CDR1，所述互补决定区CDR1的氨基酸序列如SEQ ID NO:3所示；
    (2)互补决定区CDR2，所述互补决定区CDR2的氨基酸序列如SEQ ID NO:4或16所示；及
    (3)互补决定区CDR3，所述互补决定区CDR3的氨基酸序列如SEQ ID NO:5或17所示。
  2. 一种抗人CLDN18.2抗体重链，其特征在于，所述抗体重链具有如权利要求1所述的抗体的重链可变区。
  3. 一种抗人CLDN18.2抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR'：
    (1)互补决定区CDR1'，所述互补决定区CDR1'的氨基酸序列如SEQ ID NO:8所示；
    (2)互补决定区CDR2'，所述互补决定区CDR2'的氨基酸序列如SEQ ID NO:9所示；及
    (3)互补决定区CDR3'，所述互补决定区CDR3'的氨基酸序列如SEQ ID NO:10所示。
  4. 一种抗人CLDN18.2抗体轻链，其特征在于，所述抗体轻链具有如权利要求3所述的抗体的轻链可变区。
  5. 一种抗人CLDN18.2抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 如权利要求5所述的抗体，其特征在于，所述抗体具有如SEQ ID NO:1、11、12、或13所示的重链可变区；和/或如SEQ ID NO:6、14或15所示的轻链可变区。
  7. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  8. 一种抗体制剂，其特征在于，所述的抗体制剂包括：
    (a)如权利要求5所述的抗人CLDN18.2抗体；以及
    (b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
  9. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于CLDN18.2的结合区，并且所述scFv具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区。
  10. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求9所述的CAR构建物。
  11. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  12. 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求7所述的重组蛋白、如权利要求10所述的免疫细胞、如权利要求11所述的抗体药物偶联物、或其组合，所述活性成分用于
    (a)制备检测试剂或试剂盒；
    (b)制备预防和/或治疗CLDN18.2相关疾病的药物或制剂；和/或
    (c)制备预防和/或治疗癌症或肿瘤的药物或制剂。
  13. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求7所述的重组蛋白、如权利要求10所述的免疫细胞、如权利要求11所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  14. 一种多核苷酸，其特征在于，所述的多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；或
    (2)如权利要求7所述的重组蛋白；和/或
    (3)如权利要求9所述的CAR构建物。
  15. 一种载体，其特征在于，所述的载体含有如权利要求14所述的多核苷酸。
  16. 一种遗传工程化的宿主细胞，其特征在于，所述的宿主细胞含有如权利要 求15所述的载体或基因组中整合有如权利要求14所述的多核苷酸。
  17. 一种体外检测(包括诊断性或非诊断性)样品中CLDN18.2蛋白的方法，其特征在于，所述方法包括步骤：
    (1)在体外，将所述样品与如权利要求5所述的抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CLDN18.2蛋白。
  18. 一种检测板，其特征在于，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如权利要求5所述的抗体或如权利要求11所述的抗体药物偶联物。
  19. 一种试剂盒，其特征在于，所述试剂盒中包括：
    (1)第一容器，所述第一容器中含有如权利要求5所述的抗体；和/或
    (2)第二容器，所述第二容器中含有抗如权利要求5所述的抗体的二抗；
    或者，所述试剂盒含有如权利要求18所述的检测板。
  20. 一种预防和/或治疗CLDN18.2相关疾病的方法，其特征在于，所述方法包括：给需要的对象施用如权利要求5所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
PCT/CN2022/131502 2021-11-11 2022-11-11 抗cldn18.2单克隆抗体及其应用 WO2023083327A1 (zh)

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WO2019242505A1 (zh) * 2018-06-17 2019-12-26 上海健信生物医药科技有限公司 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
CN111836644A (zh) * 2018-03-08 2020-10-27 东莞凡恩世生物医药有限公司 抗密蛋白18.2抗体及其用途
CN112166123A (zh) * 2018-03-14 2021-01-01 北京轩义医药科技有限公司 抗紧密连接蛋白18.2抗体
CN112707965A (zh) * 2019-09-30 2021-04-27 和铂医药(苏州)有限公司 靶向cldn18.2的抗体及其制备方法和应用
CN112770723A (zh) * 2018-07-25 2021-05-07 阿克罗斯生物科学公司 新型cldn 18.2特异性单克隆抗体及其使用方法

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CN112166123A (zh) * 2018-03-14 2021-01-01 北京轩义医药科技有限公司 抗紧密连接蛋白18.2抗体
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