WO2022142272A1 - Anticorps anti-cldn18.2 et son application - Google Patents

Anticorps anti-cldn18.2 et son application Download PDF

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WO2022142272A1
WO2022142272A1 PCT/CN2021/106899 CN2021106899W WO2022142272A1 WO 2022142272 A1 WO2022142272 A1 WO 2022142272A1 CN 2021106899 W CN2021106899 W CN 2021106899W WO 2022142272 A1 WO2022142272 A1 WO 2022142272A1
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antibody
seq
cells
present
heavy chain
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PCT/CN2021/106899
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English (en)
Chinese (zh)
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董坚
孙亚如
方捷
李文荣
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上海莱馥医疗科技有限公司
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Priority claimed from CN202011635146.5A external-priority patent/CN114685670A/zh
Priority claimed from CN202011634499.3A external-priority patent/CN114685682B/zh
Priority claimed from CN202110262448.0A external-priority patent/CN115073606A/zh
Priority claimed from CN202110262451.2A external-priority patent/CN115073596A/zh
Application filed by 上海莱馥医疗科技有限公司 filed Critical 上海莱馥医疗科技有限公司
Publication of WO2022142272A1 publication Critical patent/WO2022142272A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the field of biomedicine, in particular to a CLDN18.2 antibody and its application.
  • Gastrointestinal and pancreatic tumors are a great threat to human life. Although surgical treatment, radiotherapy and chemotherapy, and interventional therapy have certain curative effects on these tumors, the survival rate of patients has not been significantly improved.
  • Cellular immunotherapy is an emerging tumor treatment method, which constructs the expression vector of chimeric antigen receptor (CAR) through molecular biology technology and introduces the expression vector into immune cells isolated from the human body. , to express CAR on the cell surface, then expand and culture it, and then infuse it back into the human body.
  • CAR is composed of antigen recognition domain, hinge region, transmembrane region and intracellular signal domain connected in turn. Immune cells expressing CAR can specifically recognize and bind target cells, and kill them by releasing specific immune factors.
  • the first-generation CAR-T can mediate the killing effect on tumor cells, it does not transduce proliferation signals and induce cytokine production, and it does not last for a long time in vivo, and can only cause transient T cell proliferation. Therefore, anti-tumor The effect is minimal; the second-generation CAR-T prolongs its in vivo survival time by increasing the intracellular domain of costimulatory molecules (such as 4-1BB or CD28) and promotes its rapid expansion ability.
  • costimulatory molecules such as 4-1BB or CD28
  • scFv/4-1BB ( Or CD28)/CD3-z CAR-T can lyse target cells, transmit activation signals, and produce a large number of cytokines such as IFN- ⁇ and IL-2; the second generation CAR-T has better enhancement T than the first generation. Cell activation, expansion, anti-tumor and ability to promote transgene expression.
  • Gastric cancer is one of the most common cancers in the world. In China, gastric cancer is the second most common malignant tumor and is considered to be one of the most intractable cancers in the world. Despite recent advances in treatment options, gastric cancer recurrence is unavoidable, with a five-year survival rate of approximately 5–20% for patients with advanced gastric cancer, and a median overall survival of approximately 10 months. With the in-depth research on the molecular mechanism of gastric cancer occurrence and development, targeted therapy has become an effective treatment plan for advanced cancer, and the targets mainly include EGFR, HER-2, VEGF, VEGFR, etc.
  • CLDN18.2 As a highly specific expressed cell surface molecule, CLDN18.2 is only expressed on differentiated gastric mucosal epithelial cells in normal tissues. Therefore, it is necessary to develop targeted drugs with greater anti-gastric cancer potential, lower toxicity and lower dosage. Therapeutic antibody to CLDN18.2.
  • the purpose of the present invention is to provide a new CLDN18.2 antibody and its application.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1.
  • the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID Nos: 15 to 20.
  • an antibody heavy chain wherein the heavy chain has the heavy chain variable region and the heavy chain constant region described in the first aspect of the present invention.
  • the heavy chain constant region is of human or murine origin.
  • the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 13.
  • a light chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO:5.
  • the light chain variable region has the amino acid sequence shown in any one of SEQ ID NOs: 21 to 23.
  • an antibody light chain is provided, the light chain has the light chain variable region and the light chain constant region according to the third aspect of the present invention.
  • the constant region of the light chain is of human or murine origin.
  • the light chain constant region has the amino acid sequence shown in SEQ ID NO: 14.
  • an antibody having:
  • variable region of the heavy chain and the variable region of the light chain of the antibody further comprise human or murine FR regions.
  • the FR region of the antibody is a FR region obtained by humanizing a murine FR region.
  • the antibody has: the heavy chain according to the second aspect of the present invention; and the light chain according to the third aspect of the present invention.
  • the antibody of the present invention is an antibody specific for CLDN18.2 protein.
  • the antibodies include: single-chain antibodies, diabodies, monoclonal antibodies, chimeric antibodies (such as human-mouse chimeric antibodies), murine antibodies, or humanized antibodies.
  • the antibody is a murine antibody, which includes:
  • the heavy chain of the murine antibody has the variable region of the heavy chain shown in SEQ ID NO: 1 and the constant region of the heavy chain shown in SEQ ID NO: 13; the light chain has the variable region of the heavy chain shown in SEQ ID NO: 13; The light chain variable region shown in ID NO:5 and the light chain constant region shown in SEQ ID NO:14.
  • the antibody is a humanized antibody, which includes:
  • the antibody is a humanized antibody, which includes:
  • the heavy chain of the humanized antibody comprises the antibody heavy chain variable region shown in any one of SEQ ID NOs: 15 to 20; the light chain thereof comprises any one of SEQ ID NOs: 21 to 23 An antibody light chain variable region is shown.
  • the humanized antibody comprises an antibody heavy chain variable region and an antibody light chain variable region selected from the following table A:
  • the heavy chain of the humanized antibody has the antibody heavy chain variable region shown in SEQ ID NO: 18; the light chain has the antibody light chain variable region shown in SEQ ID NO: 21 .
  • the humanized antibody has the amino acid sequence shown in SEQ ID NO: 32, 29, 30, 31, 33, 34, 35, 36, 37 or 38.
  • the humanized antibody has the amino acid sequence shown in SEQ ID NO:32.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein is specific against CLDN18.2 protein.
  • a chimeric antigen receptor comprising: a single-chain antibody targeting CLDN18.2, a hinge region, a transmembrane domain and an intracellular Signaling domain, wherein the amino acid sequence of the single-chain antibody targeting CLDN18.2 is shown in SEQ ID NO: 39 or 42.
  • the chimeric antigen receptor has the structure shown in formula I:
  • L is none or a signal peptide sequence
  • scFv is a single chain antibody targeting CLDN 18.2;
  • H is no or hinge region
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • Each "-" independently represents a linking peptide or peptide bond connecting each of the above elements.
  • amino acid sequence of the single-chain antibody is shown in SEQ ID NO:39, which has the variable region of the antibody heavy chain shown in SEQ ID NO:1 and the variable region shown in SEQ ID NO:5 The variable region of the antibody light chain.
  • amino acid sequence of the single-chain antibody is shown in SEQ ID NO:42, which has the variable region of the antibody heavy chain shown in SEQ ID NO:18 and the variable region shown in SEQ ID NO:21 The variable region of the antibody light chain.
  • the H is a hinge region selected from the following histones: CD8, CD28, CD137, or a combination thereof.
  • the hinge region is CD8Hinge, and its amino acid sequence is shown in SEQ ID NO:45.
  • the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.
  • the transmembrane domain is CD8TM, and its amino acid sequence is shown in SEQ ID NO:46.
  • the C is a costimulatory signal molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • C includes a costimulatory signal molecule derived from 4-1BB, and/or a costimulatory signal molecule derived from CD28.
  • the intracellular signaling domain is composed of 4-1BB and CD3 ⁇ , and its amino acid sequence is shown in SEQ ID NO:50.
  • the intracellular signaling domain consists of CD28 and CD3 ⁇ .
  • the intracellular signaling domain consists of 4-1BB, CD28 and CD3 ⁇ .
  • amino acid sequence of CD28 is shown in SEQ ID NO:47.
  • amino acid sequence of 4-1BB is shown in SEQ ID NO:48.
  • amino acid sequence of CD3 ⁇ is shown in SEQ ID NO:49.
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:40 or SEQ ID NO:43.
  • a vector which contains the polynucleotide according to the eighth aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell which contains the vector described in the ninth aspect of the present invention or the polynucleotide described in the eighth aspect of the present invention is integrated into the genome, or expresses this The chimeric antigen receptor according to the seventh aspect of the invention.
  • the cells are immune cells.
  • the immune cells are T cells, NK cells or a combination thereof.
  • the immune cells are T cells.
  • the immune cells are chimeric antigen receptor T cells (CAR-T cells).
  • an immunoconjugate comprising:
  • variable region of the heavy chain according to the first aspect of the present invention (a) the variable region of the heavy chain according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • a pharmaceutical composition which contains:
  • variable region of the heavy chain according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention The light chain described in the aspect, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the chimeric antigen receptor according to the seventh aspect of the present invention, the The cell of the tenth aspect, or the immunoconjugate of the eleventh aspect of the present invention.
  • the pharmaceutical composition is in the form of injection.
  • the concentration of the CAR-T cells is 1 ⁇ 10 6 -5 ⁇ 10 6 cells/ml, preferably 1 ⁇ 10 6 -2 ⁇ 10 6 cells/ml.
  • the pharmaceutical composition is used to prepare a medicine for treating tumors, and the tumor is selected from the group consisting of gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer.
  • a heavy chain variable region according to the first aspect of the present invention, a heavy chain according to the second aspect of the present invention, and a light chain according to the third aspect of the present invention.
  • the reagent, detection plate or kit is used for: detecting CLDN18.2 protein in a sample;
  • the agent is used to treat or prevent tumors expressing CLDN18.2 protein.
  • the tumor includes gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer.
  • the tumor is selected from gastric cancer and pancreatic cancer.
  • the reagent includes a chip and an antibody-coated immune particle.
  • a method for preparing a recombinant polypeptide comprising:
  • the sixteenth aspect of the present invention provides a method for preventing and/or treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the heavy chain variable region according to the first aspect of the present invention, as described in the second aspect of the present invention.
  • the recombinant protein according to the sixth aspect, the chimeric antigen receptor according to the seventh aspect of the present invention, the cell according to the tenth aspect of the present invention, the immunoconjugate according to the eleventh aspect of the present invention, or the present invention The pharmaceutical composition of the twelfth aspect.
  • the disease is cancer or tumor.
  • the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestinal cancer cancer, oral cancer, or nasopharyngeal cancer.
  • the subject in need is a human or a non-human mammal.
  • Figure 1 shows the hybridoma methodology of the present invention.
  • Figure 2 shows a summary of hCLDN18.2 positive hybridomas.
  • Figure 3 shows cross-reactive FACS detection of subcloned hybridoma supernatants.
  • the abscissa is the fluorescence intensity, which detects the binding strength of the antibody and CLDN18.2;
  • the ordinate, SSC is the biased dispersion of the cell, which detects the complexity of the cell.
  • Figure 4 shows the titer assay of subclones.
  • Figure 5 shows the flow cytometry chart of the expression and identification results of the C18-16-1H1-1A8 recombinant antibody.
  • FL1-H is the fluorescence intensity, which detects the binding strength of the antibody and CLDN18.2;
  • SSC-H subset is the biased dispersion of the cell, which detects the complexity of the cell.
  • Figure 6 shows the construction map of the expression vector for the heavy chain of the A8 antibody.
  • Figure 7 shows the expression vector construction map of the A8 antibody light chain.
  • Figure 8 shows a graph showing the identification results of FACS binding of A8 antibody to 18.2-K562. Among them, IgG1 was the negative control.
  • Figure 9 shows the LF001 variable region structural model.
  • Figure 10 shows the location of the CDR regions of the LF001 sequence in the structural simulation: CDR regions are marked in grey, VH on the left and VL on the right.
  • Figure 11 shows a comparison diagram of the sequence analysis of the murine heavy chain of LF001.
  • Figure 12 shows a comparison diagram of the sequence analysis of the murine light chain of LF001.
  • Figure 13 shows the results of FACS identification of antibody binding to 18.2-K562.
  • Figure 14 shows the results of FACS identification of antibody binding to 18.1-K562.
  • FIG. 15 shows the results of FACS identification of antibody binding to K562.
  • Figure 16 shows exemplary diagrams of three structures of the chimeric antigen receptor of the present invention.
  • Figure 17 shows a schematic diagram of the structure of a lentiviral expression vector inserted with a nucleic acid fragment encoding a chimeric antigen receptor targeting CLDN 18.2 used in the examples of the present invention.
  • Figure 18 shows the restriction endonuclease XbaI digestion electrophoresis identification map of the lentiviral expression vector inserted with the nucleic acid fragment encoding the chimeric antigen receptor targeting CLDN 18.2.
  • A is the result of enzyme digestion and identification of murine CLDN18.2 chimeric antigen receptor expression vector
  • B is the result of enzyme digestion and identification of humanized CLDN18.2 chimeric antigen receptor expression vector.
  • Figure 19 shows the killing of CLDN 18.2-positive K562 tumor cells by CLDN18.2-CART cells (murine origin) and control T cells.
  • Figure 20 shows the killing of CLDN18.2-CART cells (murine origin) and control T cells to CLDN18.2-negative K562 tumor cells.
  • Figure 21 shows a graph of the results of IL-2 secretion by T cells (murine origin) expressing a chimeric antigen receptor targeting CLDN 18.2.
  • Figure 22 shows a graph of the results of IFN- ⁇ secretion by T cells (murine origin) expressing targeting the CLDN 18.2 chimeric antigen receptor.
  • Figure 23 shows the results of CAR virus detection in CAR-T cells (mouse origin) on the ninth day of lentivirus transfection.
  • Figure 24 shows the results of CAR virus detection in CAR-T cells (mouse origin) on the sixteenth day of lentivirus transfection.
  • Figure 25 shows the killing of CLDN 18.2-positive K562 tumor cells by CLDN18.2-CART cells (humanized) and control T cells.
  • Figure 26 shows the killing of CLDN18.2-negative K562 tumor cells by CLDN18.2-CART cells (humanized) and control T cells.
  • Figure 27 shows a graph of the results of IL-2 secretion by T cells (humanized) expressing a chimeric antigen receptor targeting CLDN 18.2.
  • Figure 28 shows a graph of the results of IFN- ⁇ secretion by T cells (humanized) expressing a chimeric antigen receptor targeting CLDN 18.2.
  • the present inventors found an anti-CLDN18.2 monoclonal antibody unexpectedly. Moreover, the inventors carried out humanization transformation on the basis of the murine CLDN18.2 monoclonal antibody, and designed and screened out a humanized antibody targeting cells expressing CLDN18.2. The experimental results show that the monoclonal antibody has high specificity and strong affinity, and can specifically bind to CLDN18.2.
  • the monoclonal antibody or humanized antibody can effectively and specifically target malignant cells (such as tumor cells) expressing the surface antigen of CLDN 18.2, and has high affinity, and can be expressed on the surface of immune cells such as T cells, and has a high affinity for expression. Specific killing of malignant cells with CLDN 18.2 surface antigen.
  • the present invention has been completed on this basis.
  • Claudins are a family of proteins whose role is to maintain tight junctions that control the exchange of molecules between cells. Widely distributed in the stomach, pancreas and lung tissue, can be used for diagnosis and treatment.
  • Claudin (CLDN) has four transmembrane domains and is involved in the regulation of physiological processes such as paracellular permeability and conductance. Its family contains at least 24 members, CLDN18 is a member of the Claudin protein family, encoded by the CLDN18 gene in humans.
  • the human CLDN18 gene has two different exon 1, which are alternatively spliced after transcription to generate two protein isoforms: CLDN18.1 and CLDN18.2, both of which are composed of 261 amino acids, only at the N-terminus There are 8 amino acid differences in the extracellular region.
  • CLDN18.2 subtype is a gastric-specific subtype. Under normal physiological conditions, Claudin 18.2 (CLDN18.2) is only expressed on the surface of human gastric epithelial short-lived cells; It is highly expressed in various tumors such as ovarian cancer and colon cancer. For example, the expression of this target exists in 50%-80% of gastric cancer patients.
  • CLDN18.2 is usually buried in the gastric mucosa, and is basically inaccessible to monoclonal antibodies in normal tissues. The occurrence of malignant tumors will lead to the destruction of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells and becoming specific. target. Thus, CLDN18.2 confers specificity for targeted therapy.
  • pancreatic cancer 50%
  • esophageal cancer, and lung cancer also shows potential for diagnosis and treatment of other tumors.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be grouped into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific to a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characteristically obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-CLDN18.2 protein monoclonal antibody, a monoclonal antibody having the variable region chain of the anti-CLDN18.2 protein monoclonal antibody, and these chains of other proteins or protein conjugates and fusion expression products.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having light and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules and the anti-CLDN18.2 protein monoclonal Conjugates formed by binding of antibodies or fragments thereof.
  • the present invention also includes cell surface markers or antigens that bind to the anti-CLDN18.2 protein monoclonal antibody or fragment thereof.
  • the present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs:
  • CDR1 its amino acid sequence is GYTFTSYWMH (SEQ ID NO:2);
  • CDR2 its amino acid sequence is MIHPNSGSTN (SEQ ID NO:3);
  • CDR3 the amino acid sequence of which is GGYYGNSLDF (SEQ ID NO: 4).
  • amino acid sequence of the heavy chain variable region is:
  • amino acid sequence of the heavy chain variable region is:
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • amino acid sequence of the heavy chain constant region (mIgG2c) is:
  • VL light chain variable region
  • the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the group consisting of:
  • CDR1' its amino acid sequence is KSSQSLLNSGNQKNYLT (SEQ ID NO:6);
  • CDR3' the amino acid sequence of which is QNAYSYPFT (SEQ ID NO: 8).
  • amino acid sequence of the light chain variable region is:
  • the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the group consisting of:
  • CDR1' its amino acid sequence is RASQSLLNSGNQKNYLT (SEQ ID NO:26);
  • CDR3' the amino acid sequence of which is QNAYSYPFT (SEQ ID NO: 8).
  • amino acid sequence of the light chain variable region is:
  • the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
  • amino acid sequence of the light chain constant region (IgK) is:
  • the terms "antibody of the present invention”, “protein of the present invention”, or “polypeptide of the present invention” are used interchangeably, and all refer to an antibody that specifically binds to CLDN18.2 protein, for example, having a heavy chain variable region (such as SEQ ID NO.: 1, 15-20 amino acid sequence) and/or light chain variable region (such as SEQ ID NO.: 5, 21-23 amino acid sequence) protein or polypeptide. They may or may not contain the starting methionine.
  • the antibody is a mouse or human-mouse chimeric monoclonal antibody against CLDN18.2 protein, and its heavy chain constant region and/or light chain constant region can be a humanized heavy chain constant region 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 or light chain constant region of human IgG1, IgG2 and the like.
  • single-chain antibody and “scFv” refer to an antibody formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody by a short peptide (linker) of 15-20 amino acids. scFv can better retain its affinity for antigens, and has the characteristics of small molecular weight, strong penetration and weak antigenicity.
  • the chimeric antigen receptor targeting cells expressing CLDN 18.2 of the present invention comprises a single-chain antibody targeting CLDN 18.2, and the amino acid sequence of the single-chain antibody targeting CLDN 18.2 is as shown in SEQ ID NO.:39 or 42 shown.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any proteins or protein conjugates and fusion expression products (ie, immunoconjugates and fusion expression products) having variable region-containing heavy and light chains, as long as the variable region is compatible with the antibody of the invention
  • the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the present invention includes those molecules having CDR-bearing monoclonal antibody light and heavy chain variable regions, as long as their CDRs are greater than 90% (preferably greater than 95%, optimally 98%) of the CDRs identified herein above) homology.
  • the present invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having CLDN18.2 protein-binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties generally do not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the present invention also includes fragments of the antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” 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 most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table B.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also referred to as antigen binding domains).
  • the intracellular domain includes the costimulatory signaling region and the zeta chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an efficient lymphocyte response to an antigen.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting Claudin 18.2.
  • the CAR of the present invention when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to not grow, being driven to die, or otherwise being affected, and resulting in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain.
  • the antigen binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain, and the CD3 ⁇ signaling domain.
  • antigen-binding domain As used herein, "antigen-binding domain”, “single-chain antibody fragment” both refer to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment that has antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are 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 structure required for antigen binding.
  • the antigen binding domain is usually a scFv (single-chain variable fragment). The size of scFv is generally 1/6 of that of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • the antigen-binding domain comprises an antibody that specifically recognizes CLDN18.2, preferably a single-chain antibody.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with receptor complexes interactions with other members.
  • the intracellular domains in the CAR of the present invention include the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof, or the above-mentioned chimeric antigen receptors.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • the coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO.: 9, 10, 11, 12, 41, 44, 51 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to encoding having the same amino acid sequence as a polypeptide of the present invention, but having the same amino acid sequence as SEQ ID NO.:9, 10, 11, 12, 41, 44, 51 Nucleic acid sequences that differ from the coding region sequences shown.
  • Polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences and various additional coding sequences for the mature polypeptides; coding sequences (and optional additional coding sequences) for the mature polypeptides and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • polypeptide encoded by the hybridizable polynucleotide and SEQ ID NO.: 1,5,15,16,17,18,19,20,21,22,23,29,30,31,32,33,34 , 35, 36, 37, 38, 39, 40, 42, and/or 43 have the same biological function and activity.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
  • Therapeutic agents that can be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, National Academy of Sciences Journal (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4.
  • Gold Nanoparticles / Nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. Viral particles (Peng et al, 2004 , Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT - diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT - diaphorase
  • BPHL biphenyl hydrolase-like protein
  • 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, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains the above-mentioned chimeric antigen receptor-expressing immune cells (eg, CAR-T cells).
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind CLDN18.2 protein molecules, and thus can be used to prevent and treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention also provides a heavy chain variable region according to the first aspect of the present invention, a heavy chain according to the second aspect of the present invention, a light chain variable region according to the third aspect of the present invention, such as The light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the chimeric antigen receptor according to the seventh aspect of the present invention,
  • the cell according to the tenth aspect of the present invention, or the immunoconjugate according to the eleventh aspect of the present invention, or the pharmaceutical composition according to the present invention is used in the prevention and/or treatment of tumor cells containing CLDN 18.2 expression use in cancer.
  • the cancer is a solid tumor, and the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestinal gland cancer cancer, oral cancer or nasopharyngeal cancer.
  • CAR-modified cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous or allogeneic, syngeneic, relative to the recipient.
  • the present invention provides a method of treating tumors comprising administering to a subject in need thereof an effective amount of the universal CAR-T cells of the present invention.
  • the universal CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-15, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may include target cells as described herein in association with one or more pharmaceutically or clinically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease, although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be indicated that the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight (including all integers within those ranges). value) application. The T cell composition can also be administered multiple times at these doses.
  • Cells can be administered by using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally, intrapleurally.
  • the T cell composition of the present invention is preferably administered by i.v. intravenous injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or the site of infection. (CART cell products are mainly intravenous infusion, and can be directly injected into tumors, lymph nodes or infected sites)
  • cells are activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels, in conjunction with any number of relevant therapeutic modalities (eg, before, at the same time, or after) administration to a patient including, but not limited to, treatment with an agent such as antiviral therapy, cidofovir, interleukin-2, IFN- ⁇ , arabinophage
  • an agent such as antiviral therapy, cidofovir, interleukin-2, IFN- ⁇ , arabinophage
  • cytotoxic chemotherapeutics such as glycosides (also known as ARA-C), checkpoint inhibitors such as PD-1 antibodies, anti-CTLA-4 antibodies, and cytokine storm-inhibiting drugs such as anti-IL-6 receptor bead antibodies and other treatments.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and, antibodies or other immunotherapeutics.
  • the cellular composition of the invention is administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out according to art-accepted practice. Typically, 1 ⁇ 10 6 to 1 ⁇ 10 10 general-purpose CAR-DNT cells of the present invention can be administered to a patient, eg, by intravenous infusion, per treatment or per course of treatment.
  • the present invention also provides a hybridoma cell line capable of producing the monoclonal antibody against the CLDN18.2 protein of the present invention; preferably, the present invention provides a hybridoma cell line with a high titer of the monoclonal antibody against the CLDN18.2 protein.
  • the hybridoma producing the CLDN18.2 protein monoclonal antibody of the present invention After obtaining the hybridoma producing the CLDN18.2 protein monoclonal antibody of the present invention, those skilled in the art can conveniently use the hybridoma cell line to prepare the antibody. In addition, those skilled in the art can easily know the structure of the antibody of the present invention (such as the heavy chain variable region and light chain variable region of the antibody), and then the monoclonal antibody of the present invention can be prepared by recombinant methods.
  • Antibodies of the present invention can be prepared by various techniques known to those skilled in the art.
  • the antigens of the present invention can be administered to animals to induce the production of monoclonal antibodies.
  • monoclonal antibodies hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).
  • myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to a medium (HAT medium matrix), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, CA, USA) and SP-2, NZ0, or X63-Ag8 -653 cells (available from American Type Culture Collection, Rockville, Maryland, USA).
  • HAT medium matrix including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, CA, USA) and SP-2, NZ0, or X63-Ag8 -653 cells (available from American Type Culture Collection, Rockville, Maryland, USA).
  • the medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies with the desired specificity, eg, by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • the location of antibody-expressing cells can be detected by FACS.
  • Hybridoma clones can then be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59- 103 pages).
  • Suitable media for this purpose include, for example, DMEM or RPMI-1640 media.
  • hybridoma cells can grow as ascites tumors in animals.
  • Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification processes such as, for example, protein A-Sepharose, hydroxyphosphorus Limestone chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • the present invention provides a monoclonal antibody against CLDN18.2 protein, especially a monoclonal antibody against CLDN18.2 protein.
  • the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of hybridoma cell culture was taken, and the IgG was crudely extracted by saturated ammonium sulfate precipitation, and then the crudely extracted antibody was purified by affinity chromatography (Protein G-Sephrose).
  • the monoclonal antibody is prepared by the method for producing monoclonal antibody from ascites of Balb/C mice. About 10 days after the hybridoma cells were inoculated into the abdominal cavity of the sensitized mice, the abdomen was obviously distended. The ascites fluid was extracted and crudely extracted by saturated ammonium sulfate precipitation method, and the crudely extracted antibody was purified by affinity chromatography column (Protein G-Sephrose).
  • the immunoglobulin has a detectable label. More preferably, the label is selected from the group consisting of colloidal gold label, colored label or fluorescent label.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the monoclonal antibody of the CLDN18.2 protein is labeled with colloidal gold to obtain a monoclonal antibody labeled with colloidal gold.
  • the CLDN18.2 protein monoclonal antibody of the present invention has good specificity and high titer.
  • the present invention relates to a method for the detection of tumors in a cell and/or tissue lysed sample.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of CLDN18.2 protein in the lysed sample.
  • the sample used in the method of the present invention can be any sample including cells present in a cell preservation solution, as used in liquid-based cytometry.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention or the detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer agent, etc.
  • the present invention further designs a detection kit for detecting the level of CLDN18.2, the kit includes an antibody that recognizes the CLDN18.2 protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers solution, detection label, detection substrate, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the antibody of the present invention has high specificity and strong affinity, and can be prepared in large quantities, and the quality of the monoclonal antibody is easy to control.
  • the antibodies of the present invention can be used for targeted drugs, antibody drug conjugates or multifunctional antibodies that specifically target CLDN18.2 positive tumor cells.
  • the antibodies of the present invention can be used to prepare reagents for diagnosing tumors or to prepare chimeric antigen receptor immune cells.
  • the Claudin18.2 CAR-T cells of the present invention have stronger killing and cytokine release functions than the positive control CAR-T cells, indicating that the CAR-T cells are more effective in vivo.
  • the Claudin 18.2 CAR-T cells of the present invention continued to maintain a high proportion of CAR positive expression throughout the in vitro culture process, indicating that CAR-T cells can maintain a high proportion of growth in in vitro culture, which also indicates that CAR -T cells can have a good effect in the body.
  • the present invention adopts the hybridoma method system, obtains positive hybridoma cells through DNA and cell immunization, and flow screening, and the screening system is stable.
  • the hybridoma method system is shown in Figure 1.
  • CHO cells (Cell Pool) were used to detect the cross-reaction with 18.1 by flow cytometry.
  • the antibody (C18-16-1H1-1A8) was a CLDN18.2 specific antibody and did not react with CLDN18.1 .
  • CHO-hCLDN18.2 cells Dispense an appropriate amount of CHO-hCLDN18.2 cells into 1.5ml EP tubes, centrifuge and resuspend in 50 ⁇ l PBS-diluted hybridoma supernatant (10-fold dilution), let stand at 4°C for 15 minutes, add 50 ⁇ l PBS after centrifugation 1:500 diluted goat anti-mouse IgG Fc-FITC, left at 4°C for 15 minutes, centrifuged to replace the supernatant, and then resuspended in 200 ⁇ l PBS for FACS flow analysis.
  • A8 antibody related information is as follows:
  • the C18-16-1H1-1A8 recombinant antibody in the 293T supernatant had better activity and the sequence was correct.
  • Cell plating 18.2-K562, 18.1-K562 and K562 were prepared into cell suspension, and the density was adjusted to 1 ⁇ 10 6 /mL. Take three 96-well round bottom plates and mark them as plate1, plate2, and plate3. 100 ⁇ L of the suspensions of 18.2-K562, 18.1-K562 and K562 were added to plate1, plate2 and plate3 using a 100 ⁇ L pipette. Centrifuge at 300 g for 5 min in a centrifuge. Throw away the supernatant.
  • the experimental results are shown in FIG. 8 .
  • the results show that the antibody affinity is: 0.6753 ⁇ g/ml.
  • hIgG1 was the negative control.
  • the results of FACS binding of antibody to 18.1-K562 and antibody to K562 showed that the antibody did not bind to 18.1-K562.
  • the humanized design is to use the 3D modeling method to mutate the original mouse-derived sequence to the human-derived sequence through database comparison.
  • the main method is to perform structural simulation first, select the optimal structural model, and then analyze the original mouse sequence (the A8 antibody sequence in the above embodiment) to clarify the sequence composition of different parts, and finally design the humanized sequence. , mutating the murine sequence into a humanized sequence.
  • the heavy chain IGHV1 sequence with the highest homology was designed as 6 humanized sequences (VH1, VH2, VH3, VH4, VH5, VH6), and IGKV1 was selected as the humanized design template for the antibody light chain, and 3 humanized sequences were designed.
  • the 9 designed sequences (VL1, VL2, VL3) were combined into 10 pairs of humanized antibodies for subsequent expression verification.
  • Loop region is generally modeled by homology modeling method, if the CDR amino acid sequence alignment result shows less than 50% Identity, then use de novo modeling Methods
  • the structure model of CDR3 was constructed.
  • Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence (structural resolution higher than 2.5 angstroms), compare the automatic modeling models, and select the optimal structural model.
  • Figure 9 Compare the existing antibody structure in the database, the simulated antibody structure model is shown in Figure 9 and Figure 10, the antibody variable region structure cartoon diagram (Fig. 9), the antibody Complementarity Determining Region (CDR) structure cartoon diagram (Fig. 10) Both use Made by Pymol.
  • Figure 9 shows that the identity of the LF001 antibody sequence and the antibody structure database is 90%, and the confidence of the simulation model is higher than 95%.
  • VH has the highest homology with the human Germline IGHV1 sequence, containing 24 mouse amino acid sites, and the V region comparison results are shown in Figure 11.
  • the 24 murine amino acid sites refer to the FR1, FR2 and FR3 region sequences aligned in the figure, and the CDR regions are not counted.
  • the comparison results show that: compared with human Germline IGKV1, VL contains 24 murine amino acid sites, and the results of gene comparison in the V region are shown in Figure 12.
  • the 24 murine amino acid sites refer to the FR1, FR2 and FR3 region sequences aligned in the figure, and the CDR regions are not counted.
  • the heavy chain design template selects the IGHV1 category.
  • the light chain design template adopts the IGKV1 category.
  • VH was predicted to be moderately immunogenic and VL was predicted to be low immunogenic.
  • Predicted VH immunogenic peptides include: SLDFWGQGTSL, FTSYWMHWV, QLQQPGAEL;
  • the predicted immunogenic peptides of VL are: FTFGSGTKL, KLLIYWAST.
  • the light and heavy chains are predicted to contain potential Deamidation sites: MIHPNSGST, GGYYGNSLD, KSSQSLLNSGNQ.
  • Table 1 is the LF001 heavy chain design sequence alignment table, in which the gray shading part is the mouse sequence, the bold and slanted part is the CDR region sequence, the black shading part is the humanized sequence, and the rest is the original human sequence.
  • Table 2 is the LF001 light chain design sequence alignment table, in which the gray shading part is the mouse sequence, the bold and slanted part is the CDR region sequence, the black shading part is the humanized sequence, and the rest is the original human sequence.
  • the constructed sequences are: LF001-H1, LF001-H2, LF001-H3, LF001-H4, LF001-H5, LF001-H6, LF001-L1, LF001-L2, LF001-L3.
  • Vector pcDNA3.4; Isotype: mIgG2c.
  • the expression combinations are: LF001-H1L1, LF001-H2L1, LF001-H3L1, LF001-H4L1, LF001-H5L1, LF001-H6L1, LF001-H2L2, LF001-H3L3, LF001-H5L3, LF001-H6L3.
  • RPMI1640 medium (purchased from Gibco), FBS (purchased from Bovogen), PBS (purchased from Yuanbi), Goat-Anti-Human-IgG-Fc-FITC (purchased from Jackson), flow cytometer (purchased from BD) .
  • the sample information to be tested is shown in the following table:
  • hIgG1 is a negative control antibody purified in-house by the inventor's company.
  • Cell plating 18.2-K562, 18.1-K562, and K562 were prepared into cell suspensions, and the density was adjusted to 1 ⁇ 10 6 /mL. Take three 96-well round bottom plates and mark them as plate1, plate2, and plate3. 100 ⁇ L of the suspensions of 18.2-K562, 18.1-K562 and K562 were added to plate1, plate2 and plate3 using a 100 ⁇ L pipette. Centrifuge at 300 g for 5 min in a centrifuge. Throw away the supernatant.
  • Figure 13 is a graph showing the identification results of FACS binding of the antibody to 18.2-K562.
  • the results show that the antibody affinity is ranked as follows: LF001-H5L1>LF001-H4L1>LF001-H2L1>LF001-MHL ⁇ LF001-H2L2 ⁇ LF001-H1L1 ⁇ LF001-H5L3 >LF001-H3L3, LF001-H6L3, LF001-H3L1, LF001-H6L1 have poor affinity with 18.2-K562.
  • hIgG1 was the negative control.
  • Antibody EC50 ⁇ g/mL Antibody EC50 : ⁇ g/mL LF001-MHL 0.6753 LF001-H6L1 1.3230 LF001-H1L1 0.6961 LF001-H2L2 0.6127 LF001-H2L1 0.6705 LF001-H3L3 0.7072 LF001-H3L1 0.8360 LF001-H5L3 0.5781 LF001-H4L1 0.5812 LF001-H6L3 0.8252 LF001-H5L1 0.5012 hIgG1 N.A
  • Figure 14 is a graph showing the results of FACS identification of antibody binding to 18.1-K562, and the results show that the LF001 candidate antibody does not bind to 18.1-K562. Among them hIgG1 was the negative control.
  • Fig. 15 is a graph showing the identification results of FACS binding of the antibody to K562, and the results show that the LF001 candidate antibody does not bind to K562. Among them hIgG1 was the negative control.
  • the negative control did not bind to 18.2-K562, 18.1-K562, and K562.
  • the antibodies in the experimental group did not bind to 18.2-K562, but the antibodies in the experimental group did not bind to 18.1-K562 and K562 cells. Or leaks, etc., so the experimental process conforms to the system applicability, indicating that the experimental results are valid.
  • the DNA fragments with a length of 1461 bp shown in SEQ ID NO.: 41 (murine source) and SEQ ID NO.: 44 (humanization) were artificially synthesized, wherein the nucleotides at positions 1-63 encode the leader, Nucleotides at positions 64-789 encode CLDN 18.2 scFv (scFv targeting CLDN 18.2), nucleotides at positions 790-924 encode the CD8 hinge region, and nucleotides at positions 925-996 encode the CD8 transmembrane region, Nucleotides at positions 997-1122 encode 4-1BB, and nucleotides at positions 1123-1458 encode CD3 ⁇ .
  • the positions of the above elements on the nucleotide sequence are shown in FIG. 16 .
  • FIG. 18 is a picture of restriction endonuclease Xba I digestion and electrophoresis identification of the lentiviral expression vector inserted into the chimeric antigen receptor targeting CLDN 18.2.
  • the chimeric antigen receptor expression plasmid, structural plasmid and envelope plasmid were transfected into 293T/17 cells by calcium phosphate transfection at a ratio of 3:2:1. Twelve hours after transfection, fresh DMEM medium containing 10% FBS was replaced with sodium butyrate at a final concentration of 5 mM. 48 hours after transfection, the virus-containing cell culture supernatant was sucked into a centrifuge tube, centrifuged at 1500 rpm at 4 °C for 10 min, and the supernatant was transferred to a new centrifuge tube, filtered with a 0.45 ⁇ m filter, and stored at -80 °C.
  • lymphocyte separation solution Take 10ml of fresh blood from healthy people, and separate peripheral blood mononuclear cells with lymphocyte separation solution (Yunfei Biological). The specific method is shown in the instructions. The cell density was adjusted to 2 ⁇ 10 6 /ml with T551 medium containing 4% autologous serum, and IL-2 with a final concentration of 300U/ml and CD3 monoclonal antibody of 100ng/ml were added to induce culture for 24h to obtain T cells.
  • This lentivirus solution was added to one well of a 6-well plate containing 2 ⁇ 10 6 T cells induced and cultured above at an MOI of 10, and co-cultured at 37°C in a 5% CO 2 incubator. Three days later, the cells were washed by centrifugation, and fresh T551 medium containing 300 IU/ml of IL-2, 100 ng/ml of CD3 mAb and 4% human autologous serum was added, and the cell density was adjusted to 2 ⁇ 10 6 /ml to continue the culture. Check the cell density every 2-3 days, centrifuge and adjust the cell density with fresh medium, and continue to expand the culture. This is repeated until the cells expand to a sufficient amount.
  • CAR-T cells targeting CLDN18.2 were obtained.
  • Example 12 ELISA detects the expression of IFN- ⁇ and IL-2
  • the results show that compared with the CAR-T positive control cell group and the T cell group, the humanized CAR-T cell group has a higher expression of CLDN18 in the case of an effector-target ratio of 3:1. .2 K562 cells produced a large amount of IL-2 and IFN- ⁇ , and the difference was significant (p ⁇ 0.05); K562 cells with high CLDN18.1 expression did not induce cytokines, and there was no significant difference (p>0.05).
  • the CAR-T cells of the present invention have specific cytokine-inducing effects on tumor cells that highly express CLDN18.2.
  • Figure 23 shows the flow cytometry detection chart of CAR-T cells on the ninth day of virus transfection (the upper part is the positive ratio of CLDN18.2 CAR-T cells 66.58%; the lower part is the positive ratio of positive control 35.92%).
  • Figure 24 shows the flow cytometry detection chart of CAR-T cells on the sixteenth day of virus transfection (the upper part is the positive ratio of CLDN18.2 CAR-T cells 78.94%; the lower part is the positive ratio of positive control 44.66%).

Abstract

La présente invention concerne un anticorps anti-CLDN18.2 et son application. Plus précisément, la présente invention concerne également un acide nucléique codant l'anticorps, une composition comprenant l'anticorps, une procédé de préparation de l'anticorps, un récepteur antigénique chimérique préparé sur la base de l'anticorps, et une cellule immunitaire exprimant le récepteur antigénique chimérique. L'invention concerne en outre une méthode de traitement ou de prévention de maladies telles que le cancer et/ou des maladies inflammatoires à l'aide de l'anticorps ou de la cellule.
PCT/CN2021/106899 2020-12-31 2021-07-16 Anticorps anti-cldn18.2 et son application WO2022142272A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN202011634499.3 2020-12-31
CN202011635146.5 2020-12-31
CN202011635146.5A CN114685670A (zh) 2020-12-31 2020-12-31 Cldn18.2抗体及其应用
CN202011634499.3A CN114685682B (zh) 2020-12-31 2020-12-31 一种靶向表达cldn 18.2的细胞的嵌合抗原受体
CN202110262451.2 2021-03-10
CN202110262448.0A CN115073606A (zh) 2021-03-10 2021-03-10 一种靶向表达cldn 18.2的细胞的人源化嵌合抗原受体
CN202110262448.0 2021-03-10
CN202110262451.2A CN115073596A (zh) 2021-03-10 2021-03-10 一种人源化Claudin 18.2抗体及其应用

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Publication number Priority date Publication date Assignee Title
CN111848809A (zh) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848809A (zh) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途

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