WO2022142272A1 - Cldn18.2 antibody and application thereof - Google Patents

Cldn18.2 antibody and application thereof Download PDF

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Publication number
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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French (fr)
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 CN202011634499.3A external-priority patent/CN114685682B/en
Priority claimed from CN202011635146.5A external-priority patent/CN114685670A/en
Priority claimed from CN202110262448.0A external-priority patent/CN115073606A/en
Priority claimed from CN202110262451.2A external-priority patent/CN115073596A/en
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/en

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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

The present invention provides a CLDN18.2 antibody and an application thereof. Specifically, the present invention also describes a nucleic acid encoding the antibody, a composition comprising the antibody, a method for preparing the antibody, a chimeric antigen receptor prepared on the basis of the antibody, and an immune cell expressing the chimeric antigen receptor. A method for treating or preventing diseases such as cancer and/or inflammatory diseases by using the antibody or cell.

Description

CLDN18.2抗体及其应用CLDN18.2 antibody and its application 技术领域technical field
本发明属于生物医药领域，具体地涉及一种CLDN18.2抗体及其应用。The invention belongs to the field of biomedicine, in particular to a CLDN18.2 antibody and its application.
背景技术Background technique
胃肠道和胰腺肿瘤对于人类生命是很大的威胁，虽然手术治疗、放化疗、介入治疗等治疗手段对于该类肿瘤有一定疗效，但患者生存率仍无显著改善。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.
细胞免疫治疗是一种正在兴起的肿瘤治疗方法，其通过分子生物学技术构建嵌合抗原受体(chimeric antigen receptor，CAR)的表达载体，并将该表达载体导入到从人体分离的免疫细胞中，使其细胞表面表达CAR后进行扩增培养，将其回输至人体。CAR由抗原识别域、铰链区和跨膜区及胞内信号域依次连接组成,表达CAR的免疫细胞能够特异性识别并结合靶细胞，通过释放特异的免疫因子对其进行杀伤。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.
第一代CAR-T虽然能够介导对肿瘤细胞的杀伤作用，但是不转导增殖信号和诱导细胞因子产生，并且体内持续作用时间不长，只能引起短暂的T细胞增殖，因此，抗肿瘤效果甚微；第二代CAR-T通过增加共刺激分子(例如4-1BB或CD28)的胞内结构域以延长其体内存活时间，促进其迅速扩增能力，通过构建scFv/4-1BB(或CD28)/CD3-z的CAR-T可以裂解靶细胞，传递活化信号，产生大量的IFN-γ、IL-2等细胞因子；第二代CAR-T较第一代具有更好的增强T细胞活化、扩增、抗肿瘤以及促进转基因表达的能力。Although 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. By constructing 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-γ 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.
胃癌是世界上最常见的癌症之一，在中国，胃癌是第二大常见恶性肿瘤,被认为是全世界最难治愈的癌症之一。尽管近年来在治疗选择方面取得了进步，但是胃癌复发难以避免，晚期胃癌患者的五年生存率约为5–20％，中位总体生存期约为10个月。随着胃癌发生、发展分子机制研究的深入，靶向治疗成为一种有效的晚期癌症的治疗方案，靶点主要包括EGFR、HER-2、VEGF、VEGFR等。CLDN18.2作为一个高度特异性表达的细胞表面分子，在正常的组织中仅表达在分化的胃粘膜上皮细胞上，因此需要开发具有更大抗胃癌潜力、更低毒性、更低用药剂量的针对CLDN18.2的治疗性抗体。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. 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.
综上所述，本领域急需开发一种新的CLDN18.2抗体用于肿瘤治疗。In conclusion, there is an urgent need in the art to develop a new CLDN18.2 antibody for tumor therapy.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种新的CLDN18.2抗体及其应用。The purpose of the present invention is to provide a new CLDN18.2 antibody and its application.
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：In a first aspect of the present invention, a heavy chain variable region of an antibody is provided, wherein the heavy chain variable region comprises the following three complementarity determining region CDRs:
SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
SEQ ID NO:3或24所示的CDR2，和CDR2 shown in SEQ ID NO: 3 or 24, and
SEQ ID NO:4或25所示的CDR3。CDR3 shown in SEQ ID NO:4 or 25.
在另一优选例中，所述重链可变区具有SEQ ID NO:1所示的氨基酸序列。In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1.
在另一优选例中，所述重链可变区具有SEQ ID NO:15至20中任一所示的氨基酸序列。In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NOs: 15 to 20.
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有本发明第一方面所述的重链可变区和重链恒定区。In the second aspect of the present invention, there is provided 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.
在另一优选例中，所述的重链恒定区为人源或鼠源的。In another preferred embodiment, the heavy chain constant region is of human or murine origin.
在另一优选例中，所述的重链恒定区具有SEQ ID NO:13所示的氨基酸序列。In another preferred embodiment, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 13.
在本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区具有选自下组的互补决定区CDR：In a third aspect of the present invention, there is provided a light chain variable region of an antibody, the light chain variable region having a complementarity determining region CDR selected from the group consisting of:
SEQ ID NO:6或26所示的CDR1’，CDR1' shown in SEQ ID NO: 6 or 26,
SEQ ID NO:7，27或28所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, 27 or 28, and
SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
在另一优选例中，所述的轻链可变区具有SEQ ID NO:5所示的氨基酸序列。In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO:5.
在另一优选例中，所述轻链可变区具有SEQ ID NO:21至23中任一所示的氨基酸序列。In another preferred embodiment, the light chain variable region has the amino acid sequence shown in any one of SEQ ID NOs: 21 to 23.
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有本发明第三方面所述的轻链可变区和轻链恒定区。In the fourth aspect of the present invention, 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.
在另一优选例中，所述轻链的恒定区为人源或鼠源的。In another preferred embodiment, the constant region of the light chain is of human or murine origin.
在另一优选例中，所述的轻链恒定区具有SEQ ID NO:14所示的氨基酸序列。In another preferred embodiment, the light chain constant region has the amino acid sequence shown in SEQ ID NO: 14.
在本发明的第五方面，提供了一种抗体，所述抗体具有：In a fifth aspect of the present invention, there is provided an antibody having:
(1)如本发明第一方面所述的重链可变区；和/或(1) the heavy chain variable region according to the first aspect of the present invention; and/or
(2)如本发明第三方面所述的轻链可变区。(2) The light chain variable region according to the third aspect of the present invention.
在另一优选例中，所述抗体的重链可变区和轻链可变区内还包含人源或鼠源的FR区。In another preferred embodiment, the variable region of the heavy chain and the variable region of the light chain of the antibody further comprise human or murine FR regions.
在另一优选例中，所述抗体的FR区是将鼠源的FR区人源化得到的FR区。In another preferred embodiment, the FR region of the antibody is a FR region obtained by humanizing a murine FR region.
在另一优选例中，所述抗体具有：如本发明第二方面所述的重链；以及如本发明第三方面所述的轻链。In another preferred embodiment, 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.
在另一优选例中，所述本发明的抗体为特异性抗CLDN18.2蛋白的抗体。In another preferred embodiment, the antibody of the present invention is an antibody specific for CLDN18.2 protein.
在另一优选例中，所述的抗体包括：单链抗体、双链抗体、单克隆抗体、嵌合抗体(如人鼠嵌合抗体)、鼠源抗体、或人源化抗体。In another preferred embodiment, the antibodies include: single-chain antibodies, diabodies, monoclonal antibodies, chimeric antibodies (such as human-mouse chimeric antibodies), murine antibodies, or humanized antibodies.
在另一优选例中，所述抗体为鼠源抗体，其包括：In another preferred embodiment, the antibody is a murine antibody, which includes:
(1)重链可变区，所述重链可变区具有以下互补决定区CDR：(1) a heavy chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
SEQ ID NO:3所示的CDR2，和CDR2 shown in SEQ ID NO: 3, and
SEQ ID NO:4所示的CDR3；以及CDR3 shown in SEQ ID NO:4; and
(2)轻链可变区，所述轻链可变区具有以下互补决定区CDR：(2) a light chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:6所示的CDR1’，CDR1' shown in SEQ ID NO:6,
SEQ ID NO:7所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, and
SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
在另一优选例中，所述鼠源抗体的重链具有如SEQ ID NO:1所示的重链可变区和SEQ ID NO:13所示的重链恒定区；其轻链具有如SEQ ID NO:5所示的轻链可变区和SEQ ID NO:14所示的轻链恒定区。In another preferred embodiment, 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.
在另一优选例中，所述抗体为人源化抗体，其包括：In another preferred embodiment, the antibody is a humanized antibody, which includes:
(1)重链可变区，所述重链可变区具有以下互补决定区CDR：(1) a heavy chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
SEQ ID NO:3或24所示的CDR2，和CDR2 shown in SEQ ID NO: 3 or 24, and
SEQ ID NO:4或25所示的CDR3；以及CDR3 shown in SEQ ID NO: 4 or 25; and
(2)轻链可变区，所述轻链可变区具有以下互补决定区CDR：(2) a light chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:26所示的CDR1’，CDR1' shown in SEQ ID NO: 26,
SEQ ID NO:7，27或28所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, 27 or 28, and
SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
在另一优选例中，所述抗体为人源化抗体，其包括：In another preferred embodiment, the antibody is a humanized antibody, which includes:
(1)重链可变区，所述重链可变区具有以下互补决定区CDR：(1) a heavy chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
SEQ ID NO:3所示的CDR2，和CDR2 shown in SEQ ID NO: 3, and
SEQ ID NO:4所示的CDR3；以及CDR3 shown in SEQ ID NO:4; and
(2)轻链可变区，所述轻链可变区具有以下互补决定区CDR：(2) a light chain variable region having the following complementarity determining region CDRs:
SEQ ID NO:26所示的CDR1’，CDR1' shown in SEQ ID NO: 26,
SEQ ID NO:7所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, and
SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
在另一优选例中，所述人源化抗体的重链包含SEQ ID NO:15至20中任一所示的抗体重链可变区；其轻链包含SEQ ID NO:21至23中任一所示的抗体轻链可变区。In another preferred embodiment, 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.
在另一优选例中，所述人源化抗体包含选自下表A的抗体重链可变区和抗体 轻链可变区：In another preferred embodiment, the humanized antibody comprises an antibody heavy chain variable region and an antibody light chain variable region selected from the following table A:
表ATable A
Figure PCTCN2021106899-appb-000001
Figure PCTCN2021106899-appb-000001
在另一优选例中，所述人源化抗体的重链具有SEQ ID NO:18所示的抗体重链可变区；其轻链具有SEQ ID NO:21所示的抗体轻链可变区。In another preferred embodiment, 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 .
在另一优选例中，所述人源化抗体具有如SEQ ID NO:32、29、30、31、33、34、35、36、37或38所示的氨基酸序列。In another preferred embodiment, the humanized antibody has the amino acid sequence shown in SEQ ID NO: 32, 29, 30, 31, 33, 34, 35, 36, 37 or 38.
在另一优选例中，所述人源化抗体具有如SEQ ID NO:32所示的氨基酸序列。In another preferred embodiment, the humanized antibody has the amino acid sequence shown in SEQ ID NO:32.
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：In the sixth aspect of the present invention, a recombinant protein is provided, and the recombinant protein has:
(i)如本发明第一方面所述的重链可变区的序列、如本发明第二方面所述的重链的序列、如本发明第三方面所述的轻链可变区的序列、如本发明第四方面所述的轻链的序列、或如本发明第五方面所述的抗体的序列；以及(i) The sequence of the heavy chain variable region according to the first aspect of the present invention, the sequence of the heavy chain according to the second aspect of the present invention, the sequence of the light chain variable region according to the third aspect of the present invention , the sequence of the light chain according to the fourth aspect of the invention, or the sequence of the antibody according to the fifth aspect of the invention; and
(ii)任选的协助表达和/或纯化的标签序列。(ii) Optional tag sequences to facilitate expression and/or purification.
在另一优选例中，所述的标签序列包括6His标签。In another preferred embodiment, the tag sequence includes a 6His tag.
在另一优选例中，所述的重组蛋白特异性抗CLDN18.2蛋白。In another preferred embodiment, the recombinant protein is specific against CLDN18.2 protein.
在本发明的第七方面，提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含：靶向CLDN18.2的单链抗体、铰链区、跨膜结构域和细胞内信号结构域，其中所述靶向CLDN18.2的单链抗体的氨基酸序列如SEQ ID NO:39或42所示。In a seventh aspect of the present invention, there is provided a chimeric antigen receptor (CAR), the 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.
在另一优选例中，所述嵌合抗原受体具有如式I所示的结构：In another preferred embodiment, the chimeric antigen receptor has the structure shown in formula I:
L-scFv-H-TM-C-CD3ζ    (式I)L-scFv-H-TM-C-CD3ζ (Formula I)
其中，in,
L为无或信号肽序列；L is none or a signal peptide sequence;
scFv为靶向CLDN 18.2的单链抗体；scFv is a single chain antibody targeting CLDN 18.2;
H为无或铰链区；H is no or hinge region;
TM为跨膜结构域；TM is the transmembrane domain;
C为共刺激信号分子；C is a costimulatory signal molecule;
CD3ζ为源于CD3ζ的胞浆信号传导序列；CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;
各“-”独立地表示连接上述各元件的连接肽或肽键。Each "-" independently represents a linking peptide or peptide bond connecting each of the above elements.
在另一优选例中，所述单链抗体的氨基酸序列如SEQ ID NO:39所示，其具有如SEQ ID NO:1所示的抗体重链可变区和如SEQ ID NO:5所示的抗体轻链可变区。In another preferred embodiment, the 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.
在另一优选例中，所述单链抗体的氨基酸序列如SEQ ID NO:42所示，其具有如SEQ ID NO:18所示的抗体重链可变区和如SEQ ID NO:21所示的抗体轻链可变区。In another preferred example, the 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.
在另一优选例中，所述的H为选自下组蛋白的铰链区：CD8、CD28、CD137、或其组合。In another preferred embodiment, the H is a hinge region selected from the following histones: CD8, CD28, CD137, or a combination thereof.
在另一优选例中，所述铰链区为CD8Hinge，其氨基酸序列如SEQ ID NO:45所示。In another preferred embodiment, the hinge region is CD8Hinge, and its amino acid sequence is shown in SEQ ID NO:45.
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。In another preferred embodiment, 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.
在另一优选例中，所述跨膜结构域为CD8TM，其氨基酸序列如SEQ ID NO:46所示。In another preferred embodiment, the transmembrane domain is CD8TM, and its amino acid sequence is shown in SEQ ID NO:46.
在另一优选例中，所述的C为选自下组的蛋白的共刺激信号分子：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、或其组合。In another preferred embodiment, 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.
在另一优选例中，C包括4-1BB来源的共刺激信号分子，和/或CD28来源的共刺激信号分子。In another preferred embodiment, C includes a costimulatory signal molecule derived from 4-1BB, and/or a costimulatory signal molecule derived from CD28.
在另一优选例中，所述细胞内信号结构域由4-1BB和CD3ζ组成，其氨基酸序列如SEQ ID NO:50所示。In another preferred example, the intracellular signaling domain is composed of 4-1BB and CD3ζ, and its amino acid sequence is shown in SEQ ID NO:50.
在另一优选例中，所述细胞内信号结构域由CD28和CD3ζ组成。In another preferred embodiment, the intracellular signaling domain consists of CD28 and CD3ζ.
在另一优选例中，所述细胞内信号结构域由4-1BB、CD28和CD3ζ组成。In another preferred embodiment, the intracellular signaling domain consists of 4-1BB, CD28 and CD3ζ.
在另一优选例中，所述CD28的氨基酸序列如SEQ ID NO:47所示。In another preferred embodiment, the amino acid sequence of CD28 is shown in SEQ ID NO:47.
在另一优选例中，所述4-1BB的氨基酸序列如SEQ ID NO:48所示。In another preferred embodiment, the amino acid sequence of 4-1BB is shown in SEQ ID NO:48.
在另一优选例中，所述CD3ζ的氨基酸序列如SEQ ID NO:49所示。In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:49.
在另一优选例中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:40或SEQ ID NO:43所示。In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:40 or SEQ ID NO:43.
在本发明的第八方面，提供了一种多核苷酸，它编码选自下组的多肽：In an eighth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(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(1) The heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the fourth aspect of the present invention The light chain of aspect, or the antibody of the fifth aspect of the invention; or
(2)如本发明第六方面所述的重组蛋白；或(2) the recombinant protein according to the sixth aspect of the present invention; or
(3)如本发明第七方面的嵌合抗原受体。(3) The chimeric antigen receptor according to the seventh aspect of the present invention.
在本发明的第九方面，提供了一种载体，它含有本发明第八方面所述的多核苷酸。In the ninth aspect of the present invention, a vector is provided, which contains the polynucleotide according to the eighth aspect of the present invention.
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。In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
在本发明的第十方面，提供了一种遗传工程化的宿主细胞，它含有本发明第九方面所述的载体或基因组中整合有本发明第八方面所述的多核苷酸，或表达本发明第七方面所述的嵌合抗原受体。In the tenth aspect of the present invention, a genetically engineered host cell is provided, 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.
在另一优选例中，所述细胞为免疫细胞。In another preferred embodiment, the cells are immune cells.
在另一优选例中，所述免疫细胞为T细胞、NK细胞或其组合。In another preferred example, the immune cells are T cells, NK cells or a combination thereof.
在另一优选例中，所述免疫细胞为T细胞。In another preferred embodiment, the immune cells are T cells.
在另一优选例中，所述免疫细胞为嵌合抗原受体T细胞(CAR-T细胞)。In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).
在本发明的第十一方面，提供了一种免疫偶联物，该免疫偶联物含有：In an eleventh aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:
(a)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、如本发明第五方面所述的抗体、或本发明第六方面所述的重组蛋白；和(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 The light chain of aspect, the antibody of the fifth aspect of the present invention, or the recombinant protein of the sixth aspect of the present invention; and
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。(b) a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
在本发明的第十二方面，提供了一种药物组合物，它含有：In the twelfth aspect of the present invention, there is provided a pharmaceutical composition, which contains:
(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、如本发明第六方面所述的重组蛋白、如本发明第七方面所述的嵌合抗原受体、如本发明第十方面所述的细胞，或如本发明第十一方面所述的免疫偶联物；以及(i) 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 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; and
(ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
在另一优选例中，所述的药物组合物为注射剂型。In another preferred embodiment, the pharmaceutical composition is in the form of injection.
在另一优选例中，所述药物组合物中，所述CAR-T细胞的浓度为1×10 6-5×10 6个细胞/ml，较佳地为1×10 6-2×10 6个细胞/ml。 In another preferred example, in the pharmaceutical composition, 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.
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自下组：胃癌、食道癌、胆管癌、胰腺癌、肺癌、卵巢癌、结肠癌。In another preferred embodiment, 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.
在本发明的第十三方面，提供了一种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、如本发明第七方面所述的嵌合抗原受体、如本发明第十方面所述的细胞，或如本发明第十一方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；In the thirteenth aspect of the present invention, there is provided 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. Chain variable region, 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, or the seventh aspect of the present invention. Use of the chimeric antigen receptor, the cell according to the tenth aspect of the present invention, or the immunoconjugate according to the eleventh aspect of the present invention, for preparing a medicament, a reagent, a detection plate or a kit;
所述试剂、检测板或试剂盒用于：检测样品中CLDN18.2蛋白；The reagent, detection plate or kit is used for: detecting CLDN18.2 protein in a sample;
所述药剂用于治疗或预防表达CLDN18.2蛋白的肿瘤。The agent is used to treat or prevent tumors expressing CLDN18.2 protein.
在另一优选例中，所述肿瘤包括：胃癌、食道癌、胆管癌、胰腺癌，肺癌,卵巢癌、结肠癌。In another preferred embodiment, the tumor includes gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer.
在另一优选例中，所述肿瘤选自：胃癌和胰腺癌。In another preferred embodiment, the tumor is selected from gastric cancer and pancreatic cancer.
在另一优选例中，所述的试剂包括芯片、包被抗体的免疫微粒。In another preferred embodiment, the reagent includes a chip and an antibody-coated immune particle.
在本发明的第十四方面，提供了一种检测样品中CLDN18.2蛋白的方法，所述方法包括步骤：In the fourteenth aspect of the present invention, there is provided a method for detecting CLDN18.2 protein in a sample, the method comprising the steps of:
(1)将样品与本发明第五方面所述的抗体接触；(1) contacting the sample with the antibody described in the fifth aspect of the present invention;
(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。(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CLDN18.2 protein in the sample.
在本发明的第十五方面，提供了一种重组多肽的制备方法，所述方法包含：In a fifteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:
(a)在适合表达的条件下，培养本发明第十方面所述的宿主细胞；(a) under conditions suitable for expression, culturing the host cell described in the tenth aspect of the present invention;
(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。(b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
本发明第十六方面，提供了一种预防和/或治疗疾病的方法，包括给需要的对象施用治疗有效量的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、如本发明第七方面所述嵌合抗原受体、如本发明第十方面所述细胞、如本发明第十一方面 所述的免疫偶联物、或本发明第十二方面所述的药物组合物。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 heavy chain according to the third aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth 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, 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.
在另一优选例中，所述疾病为癌症或肿瘤。In another preferred embodiment, the disease is cancer or tumor.
在另一优选例中，所述癌症选自下组：肺癌、结直肠癌、乳腺癌、胃癌、卵巢癌、肝癌、胰腺癌、膀胱癌、宫颈癌、子宫内膜癌、前列腺癌、小肠腺癌、口腔癌或鼻咽癌。In another preferred embodiment, 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.
在另一优选例中，所述需要的对象为人或非人哺乳动物。In another preferred embodiment, the subject in need is a human or a non-human mammal.
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。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
附图说明Description of drawings
图1显示了本发明的杂交瘤方法体系。Figure 1 shows the hybridoma methodology of the present invention.
图2显示了hCLDN18.2阳性杂交瘤汇总。Figure 2 shows a summary of hCLDN18.2 positive hybridomas.
图3显示了亚克隆的杂交瘤上清交叉反应FACS检测。其中，横坐标为荧光强度，检测抗体和CLDN18.2的结合强度；纵坐标SSC为细胞的偏向色散，检测细胞的复杂程度。Figure 3 shows cross-reactive FACS detection of subcloned hybridoma supernatants. Among them, 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.
图4显示了亚克隆的效价检测。Figure 4 shows the titer assay of subclones.
图5显示了C18-16-1H1-1A8重组抗体表达鉴定结果的流式检测图。其中，FL1-H为荧光强度，检测抗体和CLDN18.2的结合强度；SSC-H subset为细胞的偏向色散，检测细胞的复杂程度。Figure 5 shows the flow cytometry chart of the expression and identification results of the C18-16-1H1-1A8 recombinant antibody. Among them, 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.
图6显示了A8抗体重链的表达载体构建图谱。Figure 6 shows the construction map of the expression vector for the heavy chain of the A8 antibody.
图7显示了A8抗体轻链的表达载体构建图谱。Figure 7 shows the expression vector construction map of the A8 antibody light chain.
图8显示了A8抗体与18.2-K562的FACS结合的鉴定结果图。其中，IgG1为阴性对照。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.
图9显示了LF001可变区结构模型。Figure 9 shows the LF001 variable region structural model.
图10显示了LF001序列CDR区域在结构模拟中的位置：灰色标记为CDR区域，左边为VH，右边为VL。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.
图11显示了LF001的鼠源重链序列分析对比图。Figure 11 shows a comparison diagram of the sequence analysis of the murine heavy chain of LF001.
图12显示了LF001的鼠源轻链序列分析对比图。Figure 12 shows a comparison diagram of the sequence analysis of the murine light chain of LF001.
图13显示了抗体与18.2-K562结合FACS鉴定结果图。Figure 13 shows the results of FACS identification of antibody binding to 18.2-K562.
图14显示了抗体与18.1-K562结合FACS鉴定结果图。Figure 14 shows the results of FACS identification of antibody binding to 18.1-K562.
图15显示了抗体与K562结合FACS鉴定结果图。Figure 15 shows the results of FACS identification of antibody binding to K562.
图16显示了本发明的嵌合抗原受体的3种结构的示例图。Figure 16 shows exemplary diagrams of three structures of the chimeric antigen receptor of the present invention.
图17显示了本发明的实施例采用的插入有编码靶向CLDN 18.2的嵌合抗原 受体的核酸片段的慢病毒表达载体的结构示意图。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.
图18显示了插入有编码靶向CLDN 18.2的嵌合抗原受体的核酸片段的慢病毒表达载体的限制性内切酶XbaI酶切电泳鉴定图。其中，A为鼠源CLDN18.2嵌合抗原受体表达载体的酶切鉴定结果；B为人源化CLDN18.2嵌合抗原受体表达载体的酶切鉴定结果。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. Among them, 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.
图19显示了CLDN18.2-CART细胞(鼠源)和对照T细胞对CLDN 18.2阳性的K562肿瘤细胞的杀伤情况。Figure 19 shows the killing of CLDN 18.2-positive K562 tumor cells by CLDN18.2-CART cells (murine origin) and control T cells.
图20显示了CLDN18.2-CART细胞(鼠源)和对照T细胞对CLDN 18.2阴性的K562肿瘤细胞的杀伤情况。Figure 20 shows the killing of CLDN18.2-CART cells (murine origin) and control T cells to CLDN18.2-negative K562 tumor cells.
图21显示了表达靶向CLDN 18.2嵌合抗原受体的T细胞(鼠源)分泌IL-2的结果图。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.
图22显示了表达靶向CLDN 18.2嵌合抗原受体的T细胞(鼠源)分泌IFN-γ的结果图。Figure 22 shows a graph of the results of IFN-γ secretion by T cells (murine origin) expressing targeting the CLDN 18.2 chimeric antigen receptor.
图23显示了慢病毒转染第九天的CAR-T细胞(鼠源)的CAR病毒检测结果图。Figure 23 shows the results of CAR virus detection in CAR-T cells (mouse origin) on the ninth day of lentivirus transfection.
图24显示了慢病毒转染第十六天的CAR-T细胞(鼠源)的CAR病毒检测结果图。Figure 24 shows the results of CAR virus detection in CAR-T cells (mouse origin) on the sixteenth day of lentivirus transfection.
图25显示了CLDN18.2-CART细胞(人源化)和对照T细胞对CLDN 18.2阳性的K562肿瘤细胞的杀伤情况。Figure 25 shows the killing of CLDN 18.2-positive K562 tumor cells by CLDN18.2-CART cells (humanized) and control T cells.
图26显示了CLDN18.2-CART细胞(人源化)和对照T细胞对CLDN 18.2阴性的K562肿瘤细胞的杀伤情况。Figure 26 shows the killing of CLDN18.2-negative K562 tumor cells by CLDN18.2-CART cells (humanized) and control T cells.
图27显示了表达靶向CLDN 18.2嵌合抗原受体的T细胞(人源化)分泌IL-2的结果图。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.
图28显示了表达靶向CLDN 18.2嵌合抗原受体的T细胞(人源化)分泌IFN-γ的结果图。Figure 28 shows a graph of the results of IFN-γ secretion by T cells (humanized) expressing a chimeric antigen receptor targeting CLDN 18.2.
具体实施方式Detailed ways
本发明人经过广泛而深入地研究，经过大量的筛选，意外发现一种抗CLDN18.2单克隆抗体。并且，本发明人在鼠源CLDN18.2单克隆抗体基础上进行了人源化改造，设计并筛选出一种靶向表达CLDN 18.2的细胞的人源化抗体。实验结果表明，该单克隆抗体特异性高，亲和力强，能够特异性结合CLDN18.2。所述单克隆抗体或人源化抗体能够有效并且特异性地靶向表达CLDN 18.2表面抗原的恶性细胞(例如肿瘤细胞)，且具有高亲和力，并可表达于免疫细胞例如T细胞表面，对表达CLDN 18.2表面抗原的恶性细胞进行特异性杀伤。在此基础上完成了本发明。After extensive and in-depth research and extensive screening, 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.
术语the term
CLDN18.2CLDN18.2
Claudins是一个蛋白质家族，其作用是维持控制细胞间分子交换的紧密连接。广泛分布于胃、胰腺和肺组织，可用于诊断和治疗。Claudin(CLDN)具有4个跨膜结构域，参与机体生理过程如细胞旁通透性和电导的调节。其家族包含至少24个成员，CLDN18是Claudin蛋白家族成员，在人体内由CLDN18基因编码。人CLDN18基因具有两个不同的1号外显子，经过转录后可变剪接生成两个蛋白亚型：CLDN18.1和CLDN18.2，这两个亚型均由261个氨基酸组成，只在N端胞外区存在8个氨基酸的差别。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亚型是一种胃特异性亚型，正常生理状态下，Claudin 18.2(CLDN18.2)仅在人胃黏膜上皮短寿细胞表面表达；但在胃癌、食管癌、胰腺癌、肺癌、卵巢癌、结肠癌等多种肿瘤中高表达，比如在50％-80％的胃癌患者存在该靶点的表达。CLDN18.2通常埋藏在胃粘膜中，正常组织中的单克隆抗体基本上接触不到，恶性肿瘤的发生会导致紧密连接的破坏，使肿瘤细胞表面的CLDN18.2表位暴露出来，成为特定的靶点。因此，CLDN18.2赋予靶向治疗的特异性。最近发现CLDN18.2在胰腺癌(50％)、食管癌和肺癌中的表达也显示了诊断和治疗其他肿瘤的潜力。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. The recent discovery of CLDN18.2 expression in pancreatic cancer (50%), esophageal cancer, and lung cancer also shows potential for diagnosis and treatment of other tumors.
抗体Antibody
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。As used herein, the term "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. 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 . Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗 体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。As used herein, the term "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). The variable regions of native heavy and light chains each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a β-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.
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。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。The "light chains" of vertebrate antibodies (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.
如本文所用，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。As used herein, 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.
本发明还包括具有所述的抗CLDN18.2蛋白单克隆抗体的相应氨基酸序列的单克隆抗体、具有所述的抗CLDN18.2蛋白单克隆抗体可变区链的单克隆抗体，以及具有这些链的其他蛋白质或蛋白质偶联物及融合表达产物。具体地，本发明包括具有含超变区(互补决定区，CDR)的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该超变区与本发明的轻链和重链的超变区相同或至少90％同源性，较佳地至少95％同源性。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. Specifically, 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.
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与所述的抗CLDN18.2蛋白单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗CLDN18.2蛋白单克隆抗体或其片段结合的细胞表面标记物或抗原。As known to those skilled in the art, 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.
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab’) 2片段；抗体重链；抗体轻链。 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.
如本文所用，术语“重链可变区”与“VH”可互换使用。As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.
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。As used herein, the term "variable region" is used interchangeably with "complementarity determining region (CDR)".
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括以下三个互补决定区CDR：In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs:
CDR1，其氨基酸序列为GYTFTSYWMH(SEQ ID NO:2)；CDR1, its amino acid sequence is GYTFTSYWMH (SEQ ID NO:2);
CDR2，其氨基酸序列为MIHPNSGSTN(SEQ ID NO:3)；CDR2, its amino acid sequence is MIHPNSGSTN (SEQ ID NO:3);
CDR3，其氨基酸序列为GGYYGNSLDF(SEQ ID NO:4)。CDR3, the amino acid sequence of which is GGYYGNSLDF (SEQ ID NO: 4).
在另一优选例中，所述重链可变区的氨基酸序列为：In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:
MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNGKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGGYYGNSLDFWGQGTSLTVSS(SEQ ID NO.:1)；其编码核苷酸序列为：MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNGKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGGYYGNSLDFWGQGTSLTVSS(SEQ ID NO.:1); its coding nucleotide sequence is:
Figure PCTCN2021106899-appb-000002
Figure PCTCN2021106899-appb-000002
在另一优选例中，所述重链可变区的氨基酸序列为：In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:
Figure PCTCN2021106899-appb-000003
Figure PCTCN2021106899-appb-000003
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区，所述重链恒定区可以为鼠源或人源。In a preferred embodiment of the present invention, 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.
在另一优选例中，所述重链恒定区(mIgG2c)的氨基酸序列为：In another preferred embodiment, the amino acid sequence of the heavy chain constant region (mIgG2c) is:
Figure PCTCN2021106899-appb-000004
Figure PCTCN2021106899-appb-000004
如本文所用，术语“轻链可变区”与“V L”可互换使用。 As used herein, the terms "light chain variable region" and " VL " are used interchangeably.
在本发明的一个优选的实施方式中，根据本发明的抗体的轻链可变区，具有选自下组的互补决定区CDR：In a preferred embodiment of the present invention, 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’，其氨基酸序列为KSSQSLLNSGNQKNYLT(SEQ ID NO:6)；CDR1', its amino acid sequence is KSSQSLLNSGNQKNYLT (SEQ ID NO:6);
CDR2’，其氨基酸序列为WASTRES(SEQ ID NO:7)；CDR2', its amino acid sequence is WASTRES (SEQ ID NO:7);
CDR3’，其氨基酸序列为QNAYSYPFT(SEQ ID NO:8)。CDR3', the amino acid sequence of which is QNAYSYPFT (SEQ ID NO: 8).
在另一优选例中，所述的轻链可变区的氨基酸序列为：In another preference, the amino acid sequence of the light chain variable region is:
Figure PCTCN2021106899-appb-000005
Figure PCTCN2021106899-appb-000006
Figure PCTCN2021106899-appb-000005
Figure PCTCN2021106899-appb-000006
其编码核苷酸序列为：Its coding nucleotide sequence is:
Figure PCTCN2021106899-appb-000007
Figure PCTCN2021106899-appb-000007
在本发明的一个优选的实施方式中，根据本发明的抗体的轻链可变区，具有选自下组的互补决定区CDR：In a preferred embodiment of the present invention, 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’，其氨基酸序列为RASQSLLNSGNQKNYLT(SEQ ID NO:26)；CDR1', its amino acid sequence is RASQSLLNSGNQKNYLT (SEQ ID NO:26);
CDR2’，其氨基酸序列为WASTRES(SEQ ID NO:7)；CDR2', its amino acid sequence is WASTRES (SEQ ID NO:7);
CDR3’，其氨基酸序列为QNAYSYPFT(SEQ ID NO:8)。CDR3', the amino acid sequence of which is QNAYSYPFT (SEQ ID NO: 8).
在另一优选例中，所述的轻链可变区的氨基酸序列为：In another preference, the amino acid sequence of the light chain variable region is:
DIQMTQSPSSLSASVGDRVTMTCRASQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQNAYSYPFTFGQGTKLEIK(SEQ ID NO.:21)。在本发明的一个优选的实施方式中，所述抗体的轻链包括上述轻链可变区和轻链恒定区，所述轻链恒定区可以为鼠源或人源。DIQMTQSPSSLSASVGDRVTMTCRASQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQNAYSYPFTFGQGTKLEIK (SEQ ID NO.:21). In a preferred embodiment of the present invention, 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.
在另一优选例中，所述的轻链恒定区(IgK)的氨基酸序列为：In another preference, the amino acid sequence of the light chain constant region (IgK) is:
Figure PCTCN2021106899-appb-000008
Figure PCTCN2021106899-appb-000008
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蛋白的抗体，例如具有重链可变区(如SEQ ID NO.:1,15-20的氨基酸序列)和/或轻链可变区(如SEQ ID NO.:5,21-23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。In the present invention, 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.
在另一优选例中，所述的抗体为抗CLDN18.2蛋白的鼠或人鼠嵌合单克隆抗体，它的重链恒定区和/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等的重链恒定区或轻链恒定区。In another preferred example, 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.
如本文所用，术语“单链抗体”、“scFv”，是由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成的抗体。scFv能较好地保留其对抗原的亲和活性,并具有分子量小、穿透力强和抗原性弱等特点。As used herein, the terms "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.
本发明所述的靶向表达CLDN 18.2的细胞的嵌合抗原受体其包含靶向CLDN  18.2的单链抗体，所述的靶向CLDN 18.2的单链抗体的氨基酸序列如SEQ ID NO.:39或42所示。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.
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90％同源性，较佳地至少95％同源性。The present invention also provides other protein or fusion expression products with the antibodies of the present invention. Specifically, 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.
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which are separated into four framework regions (FRs), four The amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the β-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.
本发明抗体的重链和/或轻链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。The 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. Thus, 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.
本发明不仅包括完整的单克隆抗体，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。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.
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。As used herein, the terms "fragment," "derivative," and "analog" refer 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), or (iv) 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). These fragments, derivatives and analogs are well known to those skilled in the art in light of the teachings herein.
本发明抗体指具有CLDN18.2蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通 常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。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.
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。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.
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, 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.
在本发明中，“本发明抗体的保守性变异体”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表B进行氨基酸替换而产生。In the present 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.
表BForm B
最初的残基initial residue 代表性的取代representative substitution 优选的取代Preferred substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Lys；ArgGln; His; Lys; Arg GlnGln
Asp(D)Asp(D) GluGlu GluGlu
Cys(C)Cys(C) SerSer SerSer
Gln(Q)Gln(Q) AsnAsn AsnAsn
Glu(E)Glu(E) AspAsp AspAsp
Gly(G)Gly(G) Pro；AlaPro; Ala AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe LeuLeu
Leu(L)Leu(L) Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr LeuLeu
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) SerSer SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala LeuLeu
嵌合抗原受体(CAR)Chimeric Antigen Receptor (CAR)
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。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.
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。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. As used herein, 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.
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向Claudin 18.2的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。In a preferred embodiment of the present invention, 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. Preferably, the antigen binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain, and the CD3ζ signaling domain.
如本文所用，“抗原结合结构域”、“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一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。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述抗原结合结构域包含特异性识别CLDN18.2的抗体，较佳地为单链抗体。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. As a preferred mode of the present invention, the antigen-binding domain comprises an antibody that specifically recognizes CLDN18.2, preferably a single-chain antibody.
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain naturally associated with one of the domains in the CAR is used. In some instances, 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.
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。The intracellular domains in the CAR of the present invention include the signaling domain of 4-1BB and the signaling domain of CD3ζ.
多核苷酸分子和载体Polynucleotide molecules and vectors
本发明还提供了编码上述抗体或其片段或其融合蛋白，或上述嵌合抗原受体的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:9、10、11、12、41、44、51所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有与本发明的多肽相同的氨基酸序列，但与SEQ ID NO.:9、10、11、12、41、44、51所示的编码区序列有差别的核酸序列。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. As used herein, 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.
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。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 .
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与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,和/或43所示的成熟多肽有相同的生物学功能和活性。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. In particular, the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention. In 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%. And, the 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.
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是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。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。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. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
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。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. Biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。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。此外，还可通过化学合成将突变引入本发明蛋白序列 中。At present, the DNA sequences encoding the proteins of the present invention (or fragments thereof, or derivatives thereof) 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. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
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。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.
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，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。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. 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.
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。 Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When 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 . If desired, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。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.
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, 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.
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。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.
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。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.
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American  Chemical Society)128，2115)；5.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；6.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。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.
药物组合物pharmaceutical composition
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，以及药学上可接受的载体。在另一优选例中，所述药物组合物含有上述表达嵌合抗原受体的免疫细胞(例如CAR-T细胞)。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。The present invention also provides a composition. In a preferred example, 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. In another preferred embodiment, the pharmaceutical composition contains the above-mentioned chimeric antigen receptor-expressing immune cells (eg, CAR-T cells). Generally, 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.
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肿瘤。此外，还可同时使用其他治疗剂。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. In addition, other therapeutic agents may also be used concomitantly.
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。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. Such carriers 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. Pharmaceutical 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. In addition, the polypeptides of the present invention may also be used with other therapeutic agents.
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，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8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1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的。When using the pharmaceutical composition, 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. Of course, 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.
本发明还提供了一种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、如本发明第七方面所述的嵌合抗原受体、如本发明第十方面所述的细胞，或如本发明第十一方面所述的免疫偶联物，或如本发明所述的药物组合物在预防和/或治疗含有表达CLDN 18.2的肿瘤细胞的癌症中的用途。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.
所述癌症为实体瘤，所述癌症选自下组：肺癌、结直肠癌、乳腺癌、胃癌、卵巢癌、肝癌、胰腺癌、膀胱癌、宫颈癌、子宫内膜癌、前列腺癌、小肠腺癌、 口腔癌或鼻咽癌。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.
对于离体免疫细胞制备，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。For ex vivo immune cell preparation, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing the nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.
离体细胞处理程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体对上述细胞进行基因修饰(即体外转导或转染)。CAR-修饰的细胞可被施用于哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，CAR-修饰的细胞相对于接受者可为自体，也可为同种异基因的、同基因的(syngeneic)。Ex vivo cell processing procedures are well known in the art and are discussed more fully below. Briefly, 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.
本发明提供了治疗肿瘤的方法，其包括施用给需要的对象以有效量的本发明的通用型CAR-T细胞。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.
本发明的通用型CAR-T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-15、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞，与一种或多种药学或临床上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。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. Briefly, 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. Such 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.
本发明的药物组合物可以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症的特征、疾病的类型和严重度,尽管适当的剂量可由临床试验确定。The pharmaceutical 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.
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 7个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，New Eng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象，治疗方案由医学领域技术人员确定。 When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", 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.
对象组合物的施用可以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内、胸膜内施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.静脉内注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。(CART细胞产品以静脉输注为主，可采用直接注入肿瘤，淋巴结或感染位置)Administration of the composition to a subject can be carried out in any convenient manner, including by nebulization, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally, intrapleurally. In another embodiment, 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)
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩增至治疗性水平的方法活化和扩增细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦、白细胞介素-2、IFN-γ、阿糖胞苷(也已知为ARA-C)等其它细胞毒化疗药物、检验点抑制剂，如PD-1抗体、抗CTLA-4抗体和抑制细胞因子风暴的药物，如抗IL-6受体的托珠抗体等其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩增的免疫细胞的注入。在一个额外的实施方式中，扩增的细胞在外科手术前或外科手术后施用。In certain embodiments of the invention, 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-γ, arabinophage Other 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. In a further embodiment, 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. In a further embodiment, 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. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明的通用型CAR-DNT细胞，通过例如静脉回输的方式，施用于患者。 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.
杂交瘤细胞株Hybridoma cell line
本发明还提供了可生产本发明针对CLDN18.2蛋白单克隆抗体的杂交瘤细胞株；优选的，本发明提供了高效价的针对CLDN18.2蛋白单克隆抗体的杂交瘤细胞株。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.
在获得生产本发明的CLDN18.2蛋白单克隆抗体的杂交瘤之后，本领域技术人员可以方便地利用该杂交瘤细胞株制备抗体。此外，本领域技术人员还可很方便地获知本发明的抗体的结构(比如抗体的重链可变区和轻链可变区)，然后可通过重组方法来制备本发明的单克隆抗体。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.
单克隆抗体的制备Preparation of monoclonal antibodies
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如，本发明抗原，可被施用于动物以诱导单克隆抗体的产生。对于单克隆抗体，可利用杂交瘤技术来制备(见Kohler等人,Nature 256；495,1975；Kohler等人,Eur.J.Immunol.6:511,1976；Kohler等人,Eur.J.Immunol.6:292,1976；Hammerling等人,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981)或可用重组DNA法(美国专利号4,816,567)制备。Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the antigens of the present invention can be administered to animals to induce the production of monoclonal antibodies. For 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).
代表性的骨髓瘤细胞是有效融合、通过选择的抗体产生细胞支持抗体的稳定高水平产生、且对培养基(HAT培养基基质)敏感的那些骨髓瘤细胞，包括骨髓瘤细胞株，例如鼠类的骨髓瘤细胞株，包括衍生自MOPC-21和MPC-11小鼠肿瘤的骨髓瘤细胞株(可购自Salk Institute Cell Distribution Center，圣地亚哥，加利福尼亚，美国)以及SP-2、NZ0或X63-Ag8-653细胞(可购自American Type Culture Collection，洛克维尔，马里兰，美国)。人骨髓瘤和小鼠-人杂合骨髓瘤细胞株也已被描述用于产生人单克隆抗体[Kozbor，J.Immunol.，133：3001(1984)；Brodeur等，单克隆抗体的生产技术和应用(Monoclonal Antibodies Production Techniques and Applications)，51-63页(Marcel Dekker，Inc.，纽约，1987)]。Representative 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). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Production Techniques for Monoclonal Antibodies and Applications (Monoclonal Antibodies Production Techniques and Applications), pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].
对杂交瘤细胞生长于其中的培养基进行分析以检测具有所需特异性的单克隆抗体的产生，如，通过体外结合分析例如，酶联免疫吸附分析(ELISA)或放射免疫分析(RIA)。表达抗体的细胞的位置可用FACS进行检测。然后，可将杂交瘤克隆通过有限稀释步骤形成亚克隆(subcloned)，并通过标准方法生长(Goding，单克隆抗体(Monoclonal Antibodies)：原则和实践(Principles and Practice)，Academic Press(1986)59-103页)。为了达到这一目的而使用的适合的培养基包括，例如，DMEM或RPMI-1640培养基。此外，杂交瘤细胞可在动物体内作为腹水瘤生长。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). 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. In addition, hybridoma cells can grow as ascites tumors in animals.
由亚克隆分泌的单克隆抗体从培养基、腹水或血清中通过常规的免疫球蛋白纯化工艺适当地得到分离，这些纯化工艺为例如，蛋白A-琼脂糖法(protein A-Sepharose)、羟基磷灰石层析、凝胶电泳、透析或亲和层析。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.
本发明提供了一种针对CLDN18.2蛋白的单克隆抗体，特别是针对CLDN18.2蛋白的单克隆抗体。在本发明的一个优选的方案中，单克隆抗体采用培养杂交瘤细胞方法制备。取杂交瘤细胞培养的上清液，经饱和硫酸铵沉淀法粗提出IgG,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。The present invention provides a monoclonal antibody against CLDN18.2 protein, especially a monoclonal antibody against CLDN18.2 protein. In a preferred embodiment of the present invention, 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).
本发明的一个优选的方案中，单克隆抗体采用Balb/C小鼠腹水生产单克隆抗体的方法制备。将约杂交瘤细胞接种到致敏的小鼠腹腔内，10天左右可见腹部明显胀大。抽取腹水，经饱和硫酸铵沉淀法粗提后,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。In a preferred solution of the present invention, 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).
标记的免疫球蛋白labeled immunoglobulin
在本发明的一个优选例中，所述免疫球蛋白带有可检测标记物。更佳地，所述的标记物选自下组：胶体金标记物、有色标记物或荧光标记物。In a preferred embodiment of the present invention, 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.
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，CLDN18.2蛋白的单克隆抗体用胶体金标记，得到胶体金标记的单克隆抗体。Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred solution of the present invention, the monoclonal antibody of the CLDN18.2 protein is labeled with colloidal gold to obtain a monoclonal antibody labeled with colloidal gold.
本发明的CLDN18.2蛋白单克隆抗体具有很好的特异性，很高的效价。The CLDN18.2 protein monoclonal antibody of the present invention has good specificity and high titer.
方法和样本Methods and samples
本发明涉及用于在以细胞和/或组织溶解的样本检测肿瘤的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中CLDN18.2蛋白的水平。本发明方法所使用的样本可以是存在于细胞保存液中的包括细胞的任何样本，正如在液基细胞检测法中所使用的。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.
试剂盒Reagent test kit
本发明还提供了一种指含有本发明的抗体(或其片段)或本发明的检测板的试剂盒，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。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. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer agent, etc.
本发明进一步设计用于检测CLDN18.2水平的检测试剂盒，该试剂盒包括识别CLDN18.2蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。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.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明的抗体特异性高，亲和力强，并且可以大量制备、单克隆抗体质量容易控制。(1) 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.
(2)本发明的抗体可以用于特异性靶向CLDN18.2阳性肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体。(2) 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.
(3)本发明的抗体可以用于制备诊断肿瘤的试剂或用于制备嵌合抗原受体免疫细胞。(3) The antibodies of the present invention can be used to prepare reagents for diagnosing tumors or to prepare chimeric antigen receptor immune cells.
(4)本发明的Claudin18.2 CAR-T细胞拥有比阳性对照CAR-T细胞更强的杀伤和细胞因子释放功能，预示着CAR-T细胞在体内更有效的效果。(4) 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.
(5)本发明的Claudin 18.2 CAR-T细胞在整个体外培养过程中，持续保持着很高比例的CAR阳性表达，表明CAR-T细胞在体外培养中能够保持着高比例增长，同样预示着CAR-T细胞能够在体内有很好的疗效。(5) 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.
下面结合具体实施例，进一步陈述本发明。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详细条件的实验方法，通常按照常规条件如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，否则百分比和份数按重量计算。The present invention is further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer the proposed conditions. Percentages and parts are by weight unless otherwise indicated.
本发明的实验体系Experimental system of the present invention
本发明采用杂交瘤方法体系，通过DNA和细胞免疫，流式筛选获得阳性杂交瘤细胞，筛选体系稳定。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.
杂交瘤方法体系如图1所示。The hybridoma method system is shown in Figure 1.
实施例1 CLDN18.2抗体的制备Example 1 Preparation of CLDN18.2 antibody
1.免疫小鼠1. Immunization of Mice
2.血清检测2. Serum testing
3.融合3. Fusion
4.融合后筛选4. Post-Fusion Screening
5.亚克隆建株5. Subcloning
6.抗体序列分析6. Antibody Sequence Analysis
7.抗体序列的表达验证7. Expression Verification of Antibody Sequences
8.抗体的亲和力检测8. Antibody Affinity Detection
实施例2 hCLDN18.2阳性杂交瘤Example 2 hCLDN18.2 positive hybridoma
如图2所示，本发明中的hCLDN18.2阳性杂交瘤汇总。As shown in Fig. 2, hCLDN18.2-positive hybridomas in the present invention are summarized.
实施例3 杂交瘤上清交叉反应检测Example 3 Cross-reaction detection of hybridoma supernatant
CHO-hCLDN18.1+杂交瘤上清+羊抗鼠IgGFc-FITCCHO-hCLDN18.1+hybridoma supernatant+goat anti-mouse IgGFc-FITC
分装适量的CHO-hCLDN18.1细胞于1.5ml EP管内，离心后重悬于50μl PBS 1:100稀释的杂交瘤上清，4℃静置15分钟，离心后加入50μl由PBS 1:500稀释的羊抗鼠IgG Fc-FITC，4℃静置15分钟，离心置换上清之后重悬于200μl PBS，进行FACS流式分析。Dispense an appropriate amount of CHO-hCLDN18.1 cells into 1.5ml EP tubes, centrifuge and resuspend in 50μl of hybridoma supernatant diluted 1:100 in PBS, let stand at 4°C for 15 minutes, and add 50μl of 1:500 dilution in PBS after centrifugation The goat anti-mouse IgG Fc-FITC was left at 4°C for 15 minutes, centrifuged to replace the supernatant, and then resuspended in 200 μl PBS for FACS flow analysis.
如图3所示，使用CHO稳转细胞(Cell Pool)通过流式方法检测与18.1的交叉反应，抗体(C18-16-1H1-1A8)为CLDN18.2特异性抗体，不与CLDN18.1反应。As shown in Figure 3, 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 .
实施例4 杂交瘤上清效价检测Example 4 Hybridoma supernatant titer detection
10倍倍比稀释：10-fold dilution:
CHO-hCLDN18.2+杂交瘤上清(10倍倍比稀释)+羊抗鼠IgGFc-FITCCHO-hCLDN18.2+hybridoma supernatant (10-fold dilution)+goat anti-mouse IgGFc-FITC
分装适量的CHO-hCLDN18.2细胞于1.5ml EP管内，离心后重悬于50μl PBS稀释的杂交瘤上清(10倍倍比稀释)，4℃静置15分钟，离心后加入50μl由PBS 1:500稀释的羊抗鼠IgG Fc-FITC，4℃静置15分钟，离心置换上清之后重悬于200μl PBS，进行FACS流式分析。Dispense an appropriate amount of CHO-hCLDN18.2 cells into 1.5ml EP tubes, centrifuge and resuspend in 50μl PBS-diluted hybridoma supernatant (10-fold dilution), let stand at 4°C for 15 minutes, add 50μl 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 μl PBS for FACS flow analysis.
其中16号小鼠阳性克隆的杂交瘤上清效价检测如图4所示。选择C18-16-1H1-1A8(A8)进行抗体序列的表达验证。The titer detection of the hybridoma supernatant of the positive clone of mouse No. 16 is shown in Figure 4 . C18-16-1H1-1A8(A8) was selected for expression verification of the antibody sequence.
A8抗体相关信息如下：A8 antibody related information is as follows:
细胞株C18-16-1H1-1A8Cell line C18-16-1H1-1A8
来源SP2/0 细胞与C57 小鼠B 细胞融合Source SP2/0 cells fused with C57 mouse B cells
亚型IgG2c,kappaSubtype IgG2c, kappa
重链V 基因mIgHV1-64Heavy chain V gene mIgHV1-64
轻链V 基因MIgKV8-19。Light chain V gene MIgKV8-19.
实施例5 抗体序列的表达验证Example 5 Expression Verification of Antibody Sequences
将各10μg轻重链表达载体(如图6和7所示)使用磷酸钙方法共转进入10厘米培养皿中的293T细胞，三天后收集上清在CHO-hCLDN18.2细胞中进行FACS验证。10 μg of each light and heavy chain expression vector (shown in Figures 6 and 7) were co-transfected into 293T cells in a 10 cm dish using the calcium phosphate method, and the supernatant was collected three days later for FACS verification in CHO-hCLDN18.2 cells.
流式检测实验结果如图5所示。The results of the flow detection experiment are shown in Figure 5.
293T上清中C18-16-1H1-1A8重组抗体活性较好，序列正确。The C18-16-1H1-1A8 recombinant antibody in the 293T supernatant had better activity and the sequence was correct.
实施例6 A8抗体的活性检测Example 6 Activity detection of A8 antibody
6.1.细胞铺板：将18.2-K562、18.1-K562、K562制成细胞悬液，将密度调整为1×10 6/mL。取3块96孔圆底板，标记为plate1、plate2、plate3。采用100μL的移液排枪在plate1、plate2、plate3中分别加入100μL的18.2-K562、18.1-K562、K562的悬液。在离心机中采用300g的转速离心5min。甩去上清。 6.1. 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 μL of the suspensions of 18.2-K562, 18.1-K562 and K562 were added to plate1, plate2 and plate3 using a 100 μL pipette. Centrifuge at 300 g for 5 min in a centrifuge. Throw away the supernatant.
6.2.加抗体稀释液：将抗体用FACS Buffer稀释成20μg/mL、6.667μg/mL、2.222μg/mL、0.741μg/mL、0.247μg/mL、0.082μg/mL、0.027μg/mL、0.002μg/mL等8个浓度梯度的稀释液。采用100μL的移液排枪将抗体稀释液分别加到18.2-K562、18.1-K562、K562中，每孔100μL，4℃孵育60min。洗板：FACS Buffer洗板2次。6.2. Add antibody diluent: Dilute the antibody with FACS Buffer to 20μg/mL, 6.667μg/mL, 2.222μg/mL, 0.741μg/mL, 0.247μg/mL, 0.082μg/mL, 0.027μg/mL, 0.002μg /mL and other 8 concentration gradient dilutions. The antibody diluent was added to 18.2-K562, 18.1-K562, and K562 using a 100 μL pipette, respectively, 100 μL per well, and incubated at 4°C for 60 min. Wash the plate: Wash the plate twice with FACS Buffer.
6.3.加二抗：采用FACS Buffer 1：150稀释二抗Anti-Human IgG FITC，100μL/孔加入到各孔中，4℃孵育30min。洗板：FACS buffer洗板3次。6.3. Add secondary antibody: Dilute the secondary antibody Anti-Human IgG FITC with FACS Buffer 1:150, add 100 μL/well to each well, and incubate at 4°C for 30 min. Wash the plate: wash the plate 3 times with FACS buffer.
6.4.上机：在流式细胞仪上进行样品检测。6.4. On the machine: carry out sample detection on the flow cytometer.
实验结果如图8所示，在抗体与18.2-K562的FACS结合的鉴定结果图中，结果显示抗体亲和力为：0.6753μg/ml。其中，hIgG1为阴性对照。抗体与18.1-K562以及抗体与K562的FACS结合的鉴定结果都显示抗体与18.1-K562无结合。The experimental results are shown in FIG. 8 . In the graph of the identification results of FACS binding of the antibody to 18.2-K562, the results show that the antibody affinity is: 0.6753 μg/ml. Among them, 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.
实施例7 人源化Claudin 18.2抗体制备Example 7 Preparation of humanized Claudin 18.2 antibody
7.1技术方案：7.1 Technical solution:
本人源化设计是利用3D建模的方法，经数据库比对，将原始鼠源序列突变成人源序列。主要方法是先进行结构模拟，选出最优结构模型，下一步对原始鼠源 序列(上述实施例中的A8抗体序列)进行分析，明确不同部分的序列组成，最后进行人源化序列的设计，将鼠源序列突变成人源化序列。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.
本设计结果将同源度最高的重链IGHV1序列设计为6个人源化序列(VH1、VH2、VH3、VH4、VH5、VH6)，抗体轻链选择IGKV1为人源化设计模板，设计为3个人源化序列(VL1、VL2、VL3)，将设计的9条序列组合为10对人源化抗体进行后续表达验证。As a result of this design, 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.
7.2实验步骤：7.2 Experimental steps:
7.2.1模拟结构模型7.2.1 Simulated structural model
(1)结构模拟方法(1) Structural simulation method
分别使用Discovery Studio和
Figure PCTCN2021106899-appb-000009
Antibody Modeling，采用同源建模方法选取5-10个最优结构解，Loop区域一般使用同源建模方法建模，如CDR氨基酸序列比对结果显示低于50％Identity，则使用从头建模方法搭建CDR3结构模型。使用PDB BLAST调取序列最接近的10个抗体晶体结构模型(结构分辨率高于2.5埃)，对比自动建模模型，选取最优的结构模型。 Using Discovery Studio and
Figure PCTCN2021106899-appb-000009
Antibody Modeling, use homology modeling method to select 5-10 optimal structural solutions, 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.
(2)结构模型结果(2) Structural model results
比对数据库已有抗体结构，模拟的抗体结构模型如图9和图10所示，抗体可变区结构卡通图(图9)、抗体互补决定区(Complementarity Determining Region，CDR)结构卡通图(图10)均使用
Figure PCTCN2021106899-appb-000010
Pymol制作。图9显示LF001抗体序列与抗体结构数据库同源度(Identity)为90％，模拟模型可信度(Confidence)高于95％。 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
Figure PCTCN2021106899-appb-000010
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%.
7.2.2鼠源抗体序列分析7.2.2 Murine antibody sequence analysis
(1)LF001抗体鼠源重链序列分析(1) Sequence analysis of mouse heavy chain of LF001 antibody
使用IgBLAST与人Germline序列比对，结果如图11所示。比对结果显示：VH与人Germline IGHV1大类序列同源度最高，含24个鼠源氨基酸位点，V区比对结果如图11所示。其中24个鼠源氨基酸位点是指图中比对的FR1，FR2和FR3区序列，CDR区没有计算进去。Using IgBLAST to align with human Germline sequences, the results are shown in Figure 11. The comparison results show that: 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.
(2)LF001抗体鼠源轻链序列分析(2) Sequence analysis of mouse light chain of LF001 antibody
使用IgBLAST与人Germline序列比对，结果如图12所示。比对结果显示：VL与人Germline IGKV1对比，含24个鼠源氨基酸位点，V区基因比对结果如图12所示。其中24个鼠源氨基酸位点是指图中比对的FR1，FR2和FR3区序列，CDR区没有计算进去。Using IgBLAST to align with human Germline sequences, the results are shown in Figure 12. 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.
7.2.3人源化设计结果7.2.3 Humanization Design Results
(1)人源化模板的选择(1) Selection of humanized template
重链设计模板选取IGHV1大类。The heavy chain design template selects the IGHV1 category.
轻链设计模板采用IGKV1大类。The light chain design template adopts the IGKV1 category.
(2)人源化突变策略与原则(2) Humanization mutation strategy and principle
1)不影响抗体结构稳定性；2)不影响抗体与抗原结合；3)不引入糖基化、磷酸化等蛋白修饰位点；4)不引入易被氧化、氨基化等位点；5)增强结构稳定性。1) It does not affect the structural stability of the antibody; 2) It does not affect the binding of the antibody to the antigen; 3) It does not introduce protein modification sites such as glycosylation and phosphorylation; 4) It does not introduce sites that are easily oxidized and aminated; 5) Enhance structural stability.
(3)免疫原性分析(3) Immunogenicity analysis
预测VH为中免疫原性，预测VL为低免疫原性。VH was predicted to be moderately immunogenic and VL was predicted to be low immunogenic.
预测VH具有免疫原性肽段包括：SLDFWGQGTSL、FTSYWMHWV、QLQQPGAEL；Predicted VH immunogenic peptides include: SLDFWGQGTSL, FTSYWMHWV, QLQQPGAEL;
预测VL具有免疫原性肽段为：FTFGSGTKL、KLLIYWAST。The predicted immunogenic peptides of VL are: FTFGSGTKL, KLLIYWAST.
预测轻重链含潜在Deamidation位点：MIHPNSGST、GGYYGNSLD、KSSQSLLNSGNQ。The light and heavy chains are predicted to contain potential Deamidation sites: MIHPNSGST, GGYYGNSLD, KSSQSLLNSGNQ.
(4)人源化抗体序列(4) Humanized antibody sequence
表1为LF001重链设计序列比对表，其中灰色底纹部分是鼠源序列，加粗倾斜部分是CDR区序列，黑色底纹部分是人源化后序列，其余部分为原始人源序列。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.
表1.LF001重链设计序列比对表Table 1. LF001 heavy chain design sequence alignment table
Figure PCTCN2021106899-appb-000011
Figure PCTCN2021106899-appb-000011
表2为LF001轻链设计序列比对表，其中灰色底纹部分是鼠源序列，加粗倾斜部分是CDR区序列，黑色底纹部分是人源化后序列，其余部分为原始人源序列。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.
表2.LF001轻链设计序列比对表Table 2. LF001 light chain design sequence alignment table
Figure PCTCN2021106899-appb-000012
Figure PCTCN2021106899-appb-000012
(5)人源化抗体人源化程度计算(5) Calculation of Humanization Degree of Humanized Antibody
将设计好的人源化序列轻重链进行配对组合后与人源Germline序列进行比对，计算出每个部分及全长抗体的人源化程度百分比，汇总结果如下：The light and heavy chains of the designed humanized sequences were paired and combined with the human Germline sequences, and the percentage of humanization degree of each partial and full-length antibody was calculated. The summary results are as follows:
表3.人源化程度信息汇总表Table 3. Summary of Humanization Degree Information
蛋白名称protein name 回复突变back mutation 人源化比率(ScFv-Fc)Humanization ratio (ScFv-Fc)
LF001-H1L1LF001-H1L1 1616 96.55％96.55%
LF001-H2L1LF001-H2L1 1313 97.20％97.20%
LF001-H3L1LF001-H3L1 1111 97.63％97.63%
LF001-H4L1LF001-H4L1 1010 97.84％97.84%
LF001-H5L1LF001-H5L1 77 98.49％98.49%
LF001-H6L1LF001-H6L1 77 98.49％98.49%
LF001-H2L2LF001-H2L2 1010 97.84％97.84%
LF001-H3L3LF001-H3L3 66 98.71％98.71%
LF001-H5L3LF001-H5L3 22 99.57％99.57%
LF001-H6L3LF001-H6L3 22 99.57％99.57%
(6)构建表达方案(6) Construction of expression scheme
构建序列为：LF001-H1、LF001-H2、LF001-H3、LF001-H4、LF001-H5、LF001-H6、LF001-L1、LF001-L2、LF001-L3。载体：pcDNA3.4；亚型：mIgG2c。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.
表达组合为：LF001-H1L1、LF001-H2L1、LF001-H3L1、LF001-H4L1、LF001-H5L1、LF001-H6L1、LF001-H2L2、LF001-H3L3、LF001-H5L3、LF001-H6L3。The expression combinations are: LF001-H1L1, LF001-H2L1, LF001-H3L1, LF001-H4L1, LF001-H5L1, LF001-H6L1, LF001-H2L2, LF001-H3L3, LF001-H5L3, LF001-H6L3.
实施例8 人源化Claudin 18.2抗体亲和力检测Example 8 Humanized Claudin 18.2 antibody affinity detection
采用FACS方法对LF001项目11个抗体蛋白(PcDNA3.4-LF001-MHL，PcDNA3.4-LF001-H1L1、PcDNA3.4-LF001-H2L1、PcDNA3.4-LF001-H3L1、PcDNA3.4-LF001-H4L1、PcDNA3.4-LF001-H5L1、PcDNA3.4-LF001-H6L1、PcDNA3.4-LF001-H2L2、PcDNA3.4-LF001-H3L3、PcDNA3.4-LF001-H5L3、pcDNA3.4-LF001-H6L3)与细胞(18.2-K562，18.1-K562，K562)的亲和力测定及排序。11 antibody proteins of LF001 project (PcDNA3.4-LF001-MHL, PcDNA3.4-LF001-H1L1, PcDNA3.4-LF001-H2L1, PcDNA3.4-LF001-H3L1, PcDNA3.4-LF001-H4L1 , PcDNA3.4-LF001-H5L1, PcDNA3.4-LF001-H6L1, PcDNA3.4-LF001-H2L2, PcDNA3.4-LF001-H3L3, PcDNA3.4-LF001-H5L3, pcDNA3.4-LF001-H6L3) and Affinity determination and ranking of cells (18.2-K562, 18.1-K562, K562).
8.1试剂与仪器8.1 Reagents and instruments
RPMI1640培养基(购自Gibco)，FBS(购自Bovogen)，PBS(购自源培)，Goat-Anti-Human-IgG-Fc-FITC(购自Jackson)，流式细胞仪(购自BD)。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) .
8.2待检测样品8.2 Samples to be tested
待检测样品信息如下表所示：The sample information to be tested is shown in the following table:
表4.样品信息表Table 4. Sample Information Sheet
样品名称sample name 浓度(mg/mL)Concentration (mg/mL)
LF001-MHLLF001-MHL 2.352.35
LF001-H1L1LF001-H1L1 1.571.57
LF001-H2L1LF001-H2L1 1.331.33
LF001-H3L1LF001-H3L1 2.722.72
LF001-H4L1LF001-H4L1 2.942.94
LF001-H5L1LF001-H5L1 2.232.23
LF001-H6L1LF001-H6L1 2.002.00
LF001-H2L2LF001-H2L2 1.571.57
LF001-H3L3LF001-H3L3 2.482.48
LF001-H5L3LF001-H5L3 2.132.13
LF001-H6L3LF001-H6L3 2.252.25
hIgG1hIgG1 1.151.15
备注：hIgG1为发明人所在公司内部纯化的阴性对照抗体。Remarks: hIgG1 is a negative control antibody purified in-house by the inventor's company.
8.3实验方法8.3 Experimental methods
1)细胞铺板：将18.2-K562、18.1-K562、K562制成细胞悬液，将密度调整为1×10 6/mL。取3块96孔圆底板，标记为plate1、plate2、plate3。采用100μL的移液排枪在plate1、plate2、plate3中分别加入100μL的18.2-K562、18.1-K562、K562的悬液。在离心机中采用300g的转速离心5min。甩去上清。 1) 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 μL of the suspensions of 18.2-K562, 18.1-K562 and K562 were added to plate1, plate2 and plate3 using a 100 μL pipette. Centrifuge at 300 g for 5 min in a centrifuge. Throw away the supernatant.
2)加抗体稀释液：将抗体用FACS Buffer稀释成20μg/mL、6.667μg/mL、2.222μg/mL、0.741μg/mL、0.247μg/mL、0.082μg/mL、0.027μg/mL、0.002μg/mL等8个浓度梯度的稀释液。采用100μL的移液排枪将抗体稀释液分别加到18.2-K562、18.1-K562、K562中，每孔100μL，4℃孵育60min。洗板：FACS Buffer洗板2次。2) Add antibody diluent: Dilute the antibody with FACS Buffer to 20μg/mL, 6.667μg/mL, 2.222μg/mL, 0.741μg/mL, 0.247μg/mL, 0.082μg/mL, 0.027μg/mL, 0.002μg /mL and other 8 concentration gradient dilutions. The antibody diluent was added to 18.2-K562, 18.1-K562, and K562 using a 100 μL pipette, respectively, 100 μL per well, and incubated at 4°C for 60 min. Wash the plate: Wash the plate twice with FACS Buffer.
3)加二抗：采用FACS Buffer 1：150稀释二抗Anti-Human IgG FITC，100μL/孔加入到各孔中，4℃孵育30min。洗板：FACS buffer洗板3次。3) Add secondary antibody: Dilute the secondary antibody Anti-Human IgG FITC with FACS Buffer 1:150, add 100 μL/well to each well, and incubate at 4°C for 30 min. Wash the plate: wash the plate 3 times with FACS buffer.
4)上机：打开流式细胞仪及软件，清洗完成之后，建立本实验的文件夹，在该文件夹下进行样品检测。4) On the computer: Open the flow cytometer and software, and after the cleaning is completed, create a folder for this experiment, and perform sample detection in this folder.
8.4实验结果与分析8.4 Experimental results and analysis
图13是抗体与18.2-K562的FACS结合的鉴定结果图，结果显示抗体亲和力排序为：LF001-H5L1>LF001-H4L1>LF001-H2L1>LF001-MHL≈LF001-H2L2≈LF001-H1L1≈LF001-H5L3>LF001-H3L3，LF001-H6L3、LF001-H3L1、LF001-H6L1 三个分子与18.2-K562亲和力较差。其中hIgG1为阴性对照。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. Among them hIgG1 was the negative control.
与18.2-K562结合的各个抗体的EC 50值如下表所示： The EC50 values of each antibody binding to 18.2-K562 are shown in the table below:
表5.抗体与18.2-K562结合的EC 50值 Table 5. EC50 values for antibody binding to 18.2-K562
抗体Antibody EC 50：μg/mL EC50 : μg/mL 抗体Antibody EC 50：μg/mL EC50 : μg/mL
LF001-MHLLF001-MHL 0.67530.6753 LF001-H6L1LF001-H6L1 1.32301.3230
LF001-H1L1LF001-H1L1 0.69610.6961 LF001-H2L2LF001-H2L2 0.61270.6127
LF001-H2L1LF001-H2L1 0.67050.6705 LF001-H3L3LF001-H3L3 0.70720.7072
LF001-H3L1LF001-H3L1 0.83600.8360 LF001-H5L3LF001-H5L3 0.57810.5781
LF001-H4L1LF001-H4L1 0.58120.5812 LF001-H6L3LF001-H6L3 0.82520.8252
LF001-H5L1LF001-H5L1 0.50120.5012 hIgG1hIgG1 N.AN.A
图14是抗体与18.1-K562的FACS结合的鉴定结果图，结果显示LF001候选抗体与18.1-K562无结合。其中hIgG1为阴性对照。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.
图15是抗体与K562的FACS结合的鉴定结果图，结果显示LF001候选抗体与K562无结合。其中hIgG1为阴性对照。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.
8.5实验结果判定8.5 Determination of experimental results
阴性对照(hIgG1)与18.2-K562、18.1-K562、K562均无结合，实验组抗体与18.2-K562都有结合，实验组抗体与18.1-K562、K562细胞无结合，无人为操作导致试剂加错或漏孔等，所以该实验过程符合系统适用性，说明此实验结果有效。The negative control (hIgG1) 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.
实施例9 含有靶向CLDN 18.2的scFv的嵌合抗原受体表达质粒的构建Example 9 Construction of Chimeric Antigen Receptor Expression Plasmid Containing scFv Targeting CLDN 18.2
通过人工合成SEQ ID NO.:41(鼠源)和SEQ ID NO.:44(人源化)所示的长度为1461bp的DNA片段，其中，第1-63位的核苷酸编码leader，第64-789位的核苷酸编码CLDN 18.2scFv(靶向CLDN 18.2的scFv)，第790-924位的核苷酸编码CD8铰链区，第925-996位的核苷酸编码CD8跨膜区，第997-1122位的核苷酸编码4-1BB，第1123-1458位的核苷酸编码CD3ζ。以上元件在核苷酸序列上的位置如图16所示。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 CD3ζ. The positions of the above elements on the nucleotide sequence are shown in FIG. 16 .
将上述DNA片段插入慢病毒表达载体的EF1alpha启动子下游，得到靶向CLDN 18.2的嵌合抗原受体表达质粒(pLV-CLDN 18.2-scFv-BBz)，如图17所示。图18为插入靶向CLDN 18.2的嵌合抗原受体的慢病毒表达载体的限制性内切酶Xba I酶切电泳鉴定图。The above DNA fragment was inserted downstream of the EF1alpha promoter of the lentiviral expression vector to obtain a chimeric antigen receptor expression plasmid (pLV-CLDN 18.2-scFv-BBz) targeting CLDN 18.2, as shown in Figure 17. Figure 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.
实施例10 靶向CLDN 18.2的嵌合抗原受体表达质粒转染T细胞Example 10 Transfection of T cells with chimeric antigen receptor expression plasmid targeting CLDN 18.2
(1)慢病毒的包装制备(1) Packaging and preparation of lentivirus
将嵌合抗原受体表达质粒和结构质粒、包膜质粒以3:2:1的比例用磷酸钙转法转染293T/17细胞。转染12小时后，更换含10％FBS的新鲜DMEM培养基，同时加入终浓度为5mM的丁酸钠。转染48小时后，将含有病毒的细胞培养上清吸入离心 管中，4℃1500rpm离心10min，转移上清至新的离心管中，0.45μm滤器过滤后-80℃保存.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 μm filter, and stored at -80 °C.
(2)T细胞的制备(2) Preparation of T cells
取10ml健康人的新鲜血液，用淋巴细胞分离液(韵飞生物)分离外周血单核细胞，具体方法见说明书。用含4％自体血清的T551培养基调整细胞密度至2x10 6/ml并加入终浓度为300U/ml的IL-2、100ng/ml的CD3单抗诱导培养24h，得到T细胞。 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.
(3)慢病毒感染T细胞及感染后T细胞的扩增培养(3) Lentiviral infection of T cells and expansion of T cells after infection
用该慢病毒液以MOI 10加入含有2×10 6个上述诱导培养的T细胞的6孔板的1个孔中，在37℃，5％CO 2培养箱内共培养。三天之后，离心洗涤细胞，加入新鲜的含有300IU/ml的IL-2、100ng/ml的CD3单抗和4％人自体血清的T551培养基，并调整细胞密度至2x10 6/ml继续培养。每2-3天检测一次细胞密度，离心并用新鲜培养基调整细胞密度，持续扩大培养。如此反复，直至细胞扩增至足够的用量。 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.
检验嵌合抗原受体在T细胞中有效表达后，即获得针对CLDN18.2的CAR-T细胞。After testing the effective expression of chimeric antigen receptor in T cells, CAR-T cells targeting CLDN18.2 were obtained.
实施例11 CAR-T细胞对CLDN18.2阳性的恶性细胞的特异性杀伤活性检测Example 11 Detection of specific killing activity of CAR-T cells on CLDN18.2 positive malignant cells
取5×10 4个高表达CLDN18.2的K562细胞(CLDN18.2阳性，以CLDN18.2-K562表示)和对照高表达CLDN18.1的K562细胞(CLDN18.2阴性，以CLDN18.1-K562表示)接种于96孔板，分别向两种细胞培养孔中按效应细胞(Effector)：靶细胞(Target)＝10:1、3:1、1:1三个比例加入CAR-T细胞(CLDN18.2-CART)、CAR-T阳性对照细胞(CLDN18.2-CART阳性对照)和未加修饰的T细胞(T)。孵育18h后，按照LDH细胞毒性分析试剂盒(碧云天)说明书进行操作，检测CAR-T细胞对高表达CLDN18.2的K562细胞的特异杀伤活性。 Take 5×10 4 K562 cells with high expression of CLDN18.2 (positive for CLDN18.2, represented by CLDN18.2-K562) and control K562 cells with high expression of CLDN18.1 (negative for CLDN18.2, represented by CLDN18.1-K562) Indicated) were seeded in a 96-well plate, and CAR-T cells (CLDN18 .2-CART), CAR-T positive control cells (CLDN18.2-CART positive control) and unmodified T cells (T). After 18 hours of incubation, operate according to the instructions of the LDH cytotoxicity assay kit (Biyuntian) to detect the specific killing activity of CAR-T cells on K562 cells highly expressing CLDN18.2.
结果显示,鼠源CAR-T细胞组与CAR-T阳性对照细胞组和未加修饰的T细胞组相比，对高表达CLDN18.2的K562细胞具有更强的杀伤作用，且这种差异具有显著性(p<0.05)(图19)；同样地，人源化CAR-T细胞组与CAR-T阳性对照细胞组和未加修饰的T细胞组相比，对高表达CLDN18.2的K562细胞具有更强的杀伤作用，且这种差异具有显著性(p<0.05)(图25)；鼠源CAR-T细胞组或人源化CAR-T细胞组与CAR-T阳性对照细胞组和未加修饰的T细胞组相比，对表达CLDN18.1的K562细胞的杀伤作用无显著性差异(p>0.05)(图20和图26)。The results showed that compared with the CAR-T positive control cell group and the unmodified T cell group, the mouse-derived CAR-T cell group had a stronger killing effect on K562 cells with high expression of CLDN18. Significant (p<0.05) (Fig. 19); similarly, the humanized CAR-T cell group was more sensitive to K562 highly expressing CLDN18.2 than the CAR-T positive control cell group and the unmodified T cell group. The cells had stronger killing effect, and this difference was significant (p<0.05) (Figure 25); the mouse-derived CAR-T cell group or the humanized CAR-T cell group and the CAR-T positive control cell group and Compared with the unmodified T cell group, there was no significant difference in the killing effect on K562 cells expressing CLDN18.1 (p>0.05) (Figure 20 and Figure 26).
实施例12 ELISA检测IFN-γ，IL-2的表达Example 12 ELISA detects the expression of IFN-γ and IL-2
取5×10 4个高表达CLDN18.2的K562靶细胞(CLDN18.2阳性)和对照CLDN18.1-K562靶细胞(CLDN18.2阴性)接种于96孔板，分别向两种细胞培养孔中按效应细胞(Effector)：靶细胞(Target)＝3:1的比例加入CAR-T细胞 (CLDN18.2-CART)、CLDN18.2-CART阳性对照细胞(CLDN18.2-CART阳性对照)和未加修饰的T细胞(T)等效应细胞。孵育18h后，按照Human IL-2 ELISA Kit(达科为)说明书进行操作，取上清检测IL-2和IFN-γ含量。 Take 5×10 4 K562 target cells (CLDN18.2 positive) and control CLDN18.1-K562 target cells (CLDN18.2 negative) highly expressing CLDN18.2 in a 96-well plate, and inoculate them into two cell culture wells respectively. Add CAR-T cells (CLDN18.2-CART), CLDN18.2-CART positive control cells (CLDN18.2-CART positive control) and untreated cells at the ratio of Effector:Target=3:1 Effector cells such as modified T cells (T). After 18 hours of incubation, the operation was performed according to the instructions of Human IL-2 ELISA Kit (Daktronics), and the supernatant was collected to detect the content of IL-2 and IFN-γ.
如图21和图22所示，结果显示鼠源CAR-T细胞组与CAR-T阳性对照细胞组和T细胞组相比，在效靶比为3:1的情况下，对高表达CLDN18.2的K562细胞产生大量的IL-2和IFN-γ，且这种差异具有显著性(p<0.05)；高表达CLDN18.1的K562的细胞对细胞因子没有诱导作用，而且无显著性差异(p>0.05)。As shown in Figure 21 and Figure 22, the results showed that compared with the CAR-T positive control cell group and the T cell group, the mouse-derived CAR-T cell group had an effect on the high expression of CLDN18. 2 K562 cells produced a large amount of IL-2 and IFN-γ, 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).
如图27和图28所示，结果显示人源化CAR-T细胞组与CAR-T阳性对照细胞组和T细胞组相比，在效靶比为3:1的情况下，对高表达CLDN18.2的K562细胞产生大量的IL-2和IFN-γ，且这种差异具有显著性(p<0.05)；高表达CLDN18.1的K562的细胞对细胞因子没有诱导作用，而且无显著性差异(p>0.05)。As shown in Figure 27 and Figure 28, 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-γ, 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).
因此本发明的CAR-T细胞对高表达CLDN18.2的肿瘤细胞具有特异细胞因子诱导的作用。Therefore, the CAR-T cells of the present invention have specific cytokine-inducing effects on tumor cells that highly express CLDN18.2.
实施例13 流式细胞术检测CAR-T细胞表面的CAR的表达Example 13 Detection of CAR expression on the surface of CAR-T cells by flow cytometry
在慢病毒感染T细胞的第九天和第十六天，分别取3×10 5本发明的鼠源Claudin18.2 CAR-T细胞和阳性对照细胞，使用FITC-labeled goat anti-mouse-F(ab)2 antibody在流式细胞仪上检测细胞表面的CAR的表达。 On the ninth and sixteenth days of lentivirus infection of T cells, 3×10 5 murine Claudin18.2 CAR-T cells of the present invention and positive control cells were taken, respectively, and FITC-labeled goat anti-mouse-F ( ab) 2 antibody to detect the expression of CAR on the cell surface on a flow cytometer.
图23显示了病毒转染第九天的CAR-T细胞的流式细胞仪检测图(上方是CLDN18.2 CAR-T细胞的阳性比例66.58％；下方是阳性对照的阳性比例35.92％)。图24显示了病毒转染第十六天的CAR-T细胞的流式细胞仪检测图(上方是CLDN18.2 CAR-T细胞的阳性比例78.94％；下方是阳性对照的阳性比例44.66％)。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%).
结果显示CAR-T细胞始终维持着恒定的高表达，表明CAR-T细胞始终维持着恒定的高比例。The results showed that CAR-T cells always maintained a constant high expression, indicating that CAR-T cells always maintained a constant high proportion.
本发明的序列信息Sequence information of the present invention
Figure PCTCN2021106899-appb-000013
Figure PCTCN2021106899-appb-000013
Figure PCTCN2021106899-appb-000014
Figure PCTCN2021106899-appb-000014
Figure PCTCN2021106899-appb-000015
Figure PCTCN2021106899-appb-000015
Figure PCTCN2021106899-appb-000016
Figure PCTCN2021106899-appb-000016
Figure PCTCN2021106899-appb-000017
Figure PCTCN2021106899-appb-000017
Figure PCTCN2021106899-appb-000018
Figure PCTCN2021106899-appb-000018
Figure PCTCN2021106899-appb-000019
Figure PCTCN2021106899-appb-000019
Figure PCTCN2021106899-appb-000020
Figure PCTCN2021106899-appb-000020
Figure PCTCN2021106899-appb-000021
Figure PCTCN2021106899-appb-000021
Figure PCTCN2021106899-appb-000022
Figure PCTCN2021106899-appb-000022
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。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (16)

  1. 一种靶向CLDN18.2的抗体，其特征在于，所述抗体包含重链和轻链，其中An antibody targeting CLDN18.2, wherein the antibody comprises a heavy chain and a light chain, wherein
    (1)所述重链的重链可变区包括以下三个互补决定区CDR：(1) The heavy chain variable region of the heavy chain includes the following three complementarity determining region CDRs:
    SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
    SEQ ID NO:3或24所示的CDR2，和CDR2 shown in SEQ ID NO: 3 or 24, and
    SEQ ID NO:4或25所示的CDR3；和/或CDR3 shown in SEQ ID NO: 4 or 25; and/or
    (2)所述轻链的轻链可变区包括以下三个互补决定区CDR：(2) the light chain variable region of the light chain includes the following three complementarity determining region CDRs:
    SEQ ID NO:6或26所示的CDR1’，CDR1' shown in SEQ ID NO: 6 or 26,
    SEQ ID NO:7，27或28所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, 27 or 28, and
    SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
  2. 如权利要求1所述的抗体，其特征在于，所述的抗体包括：单链抗体、双链抗体、单克隆抗体、嵌合抗体(如人鼠嵌合抗体)、鼠源抗体、或人源化抗体。The antibody of claim 1, wherein the antibody comprises: a single-chain antibody, a diabody, a monoclonal antibody, a chimeric antibody (such as a human-mouse chimeric antibody), a mouse-derived antibody, or a human-derived antibody Antibodies.
  3. 如权利要求2所述的抗体，其特征在于，所述抗体为鼠源抗体，其包括：The antibody of claim 2, wherein the antibody is a murine antibody, comprising:
    (1)重链可变区，所述重链可变区具有以下互补决定区CDR：(1) a heavy chain variable region having the following complementarity determining region CDRs:
    SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
    SEQ ID NO:3所示的CDR2，和CDR2 shown in SEQ ID NO: 3, and
    SEQ ID NO:4所示的CDR3；以及CDR3 shown in SEQ ID NO:4; and
    (2)轻链可变区，所述轻链可变区具有以下互补决定区CDR：(2) a light chain variable region having the following complementarity determining region CDRs:
    SEQ ID NO:6所示的CDR1’，CDR1' shown in SEQ ID NO:6,
    SEQ ID NO:7所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, and
    SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
  4. 如权利要求2所述的抗体，其特征在于，所述抗体为人源化抗体，其包括：The antibody of claim 2, wherein the antibody is a humanized antibody, comprising:
    (1)重链可变区，所述重链可变区具有以下互补决定区CDR：(1) a heavy chain variable region having the following complementarity determining region CDRs:
    SEQ ID NO:2所示的CDR1，CDR1 shown in SEQ ID NO:2,
    SEQ ID NO:3或24所示的CDR2，和CDR2 shown in SEQ ID NO: 3 or 24, and
    SEQ ID NO:4或25所示的CDR3；以及CDR3 shown in SEQ ID NO: 4 or 25; and
    (2)轻链可变区，所述轻链可变区具有以下互补决定区CDR：(2) a light chain variable region having the following complementarity determining region CDRs:
    SEQ ID NO:26所示的CDR1’，CDR1' shown in SEQ ID NO: 26,
    SEQ ID NO:7，27或28所示的CDR2’，和CDR2' shown in SEQ ID NO: 7, 27 or 28, and
    SEQ ID NO:8所示的CDR3’。CDR3' shown in SEQ ID NO:8.
  5. 如权利要求3所述的抗体，其特征在于，所述抗体的重链具有如SEQ ID NO:1所示的重链可变区和重链恒定区；所述抗体的轻链具有如SEQ ID NO:5所示的轻链可变区和轻链恒定区。The antibody of claim 3, wherein the heavy chain of the antibody has a heavy chain variable region and a heavy chain constant region as shown in SEQ ID NO: 1; the light chain of the antibody has a heavy chain as shown in SEQ ID NO: 1 The light chain variable region and light chain constant region shown in NO:5.
  6. 如权利要求4所述的抗体，其特征在于，所述抗体的重链包含SEQ ID NO:15至20中任一所示的抗体重链可变区；所述抗体的轻链包含SEQ ID NO:21至23中任一所示的抗体轻链可变区。The antibody of claim 4, wherein the heavy chain of the antibody comprises the antibody heavy chain variable region shown in any one of SEQ ID NOs: 15 to 20; the light chain of the antibody comprises SEQ ID NO: : the antibody light chain variable region shown in any one of 21 to 23.
  7. 一种重组蛋白，其特征在于，所述的重组蛋白具有：A kind of recombinant protein, it is characterized in that, described recombinant protein has:
    (i)如权利要求1-6任一项所述的抗体的序列；以及(i) the sequence of the antibody of any one of claims 1-6; and
    (ii)任选的协助表达和/或纯化的标签序列。(ii) Optional tag sequences to facilitate expression and/or purification.
  8. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体包含：靶向CLDN18.2的单链抗体、铰链区、跨膜结构域和细胞内信号结构域，其中所述靶向CLDN18.2的单链抗体的氨基酸序列如SEQ ID NO:39或42所示。A chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor comprises: a single-chain antibody targeting CLDN18.2, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the The amino acid sequence of the single-chain antibody targeting CLDN18.2 is shown in SEQ ID NO: 39 or 42.
  9. 如权利要求8所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体具有如式I所示的结构：The chimeric antigen receptor of claim 8, wherein the chimeric antigen receptor has the structure shown in formula I:
    L-scFv-H-TM-C-CD3ζ  (式I)L-scFv-H-TM-C-CD3ζ (Formula I)
    其中，in,
    L为无或信号肽序列；L is none or a signal peptide sequence;
    scFv为靶向CLDN 18.2的单链抗体；scFv is a single chain antibody targeting CLDN 18.2;
    H为无或铰链区；H is no or hinge region;
    TM为跨膜结构域；TM is the transmembrane domain;
    C为共刺激信号分子；C is a costimulatory signal molecule;
    CD3ζ为源于CD3ζ的胞浆信号传导序列；CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;
    各“-”独立地表示连接上述各元件的连接肽或肽键。Each "-" independently represents a linking peptide or peptide bond connecting each of the above elements.
  10. 一种多核苷酸，其特征在于，它编码选自下组的多肽：A polynucleotide characterized in that it encodes a polypeptide selected from the group consisting of:
    (1)如权利要求1-6任一项所述的抗体；或(1) The antibody of any one of claims 1-6; or
    (2)如权利要求7所述的重组蛋白；或(2) recombinant protein as claimed in claim 7; Or
    (3)如权利要求8或9所述的嵌合抗原受体。(3) The chimeric antigen receptor according to claim 8 or 9.
  11. 一种载体，其特征在于，所述载体含有权利要求10所述的多核苷酸。A vector, characterized in that the vector contains the polynucleotide of claim 10 .
  12. 一种遗传工程化的宿主细胞，其特征在于，它含有权利要求11所述的载体或基因组中整合有权利要求10所述的多核苷酸，或表达如权利要求8或9所述的嵌合抗原受体。A genetically engineered host cell, characterized in that it contains the vector of claim 11 or the polynucleotide of claim 10 integrated in the genome, or expresses the chimera of claim 8 or 9 antigen receptors.
  13. 如权利要求11所述细胞，其特征在于，所述宿主细胞为免疫细胞，所述免疫细胞选自：NK细胞、T细胞或其组合。The cell of claim 11, wherein the host cell is an immune cell, and the immune cell is selected from the group consisting of NK cells, T cells, or a combination thereof.
  14. 一种免疫偶联物，其特征在于，该免疫偶联物含有：An immunoconjugate, characterized in that the immunoconjugate contains:
    (a)如权利要求1-6任一项所述的抗体、或权利要求7所述的重组蛋白；和(a) the antibody of any one of claims 1-6, or the recombinant protein of claim 7; and
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。(b) a coupling moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes.
  15. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-6任一 项所述的抗体、如权利要求7所述的重组蛋白、如权利要求8或9所述的嵌合抗原受体、如权利要求10所述的多核苷酸、如权利要求11所述的载体或如权利要求12或13所述的细胞，以及药学上可接受的载体、赋形剂或稀释剂。A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody according to any one of claims 1-6, the recombinant protein according to claim 7, the chimeric protein according to claim 8 or 9 antigen receptor, the polynucleotide of claim 10, the carrier of claim 11, or the cell of claim 12 or 13, and a pharmaceutically acceptable carrier, excipient or diluent .
  16. 一种如权利要求1-6任一项所述的抗体、如权利要求7所述的重组蛋白、如权利要求8或9所述的嵌合抗原受体、如权利要求10所述的多核苷酸、如权利要求11所述的载体或如权利要求12或13所述的细胞的用途，其特征在于，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。A kind of antibody as claimed in any one of claim 1-6, recombinant protein as claimed in claim 7, chimeric antigen receptor as claimed in claim 8 or 9, polynucleoside as claimed in claim 10 Use of the acid, the carrier according to claim 11 or the cell according to claim 12 or 13, characterized in that it is used to prepare a medicament or preparation for preventing and/or treating cancer or tumor.
PCT/CN2021/106899 2020-12-31 2021-07-16 Cldn18.2 antibody and application thereof WO2022142272A1 (en)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN202011634499.3A CN114685682B (en) 2020-12-31 2020-12-31 Chimeric antigen receptor of cell targeted to express CLDN 18.2
CN202011634499.3 2020-12-31
CN202011635146.5A CN114685670A (en) 2020-12-31 2020-12-31 CLDN18.2 antibodies and uses thereof
CN202011635146.5 2020-12-31
CN202110262448.0A CN115073606A (en) 2021-03-10 2021-03-10 Humanized chimeric antigen receptor targeting cells expressing CLDN18.2
CN202110262448.0 2021-03-10
CN202110262451.2 2021-03-10
CN202110262451.2A CN115073596A (en) 2021-03-10 2021-03-10 Humanized Claudin18.2 antibody and application thereof

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Publication number Priority date Publication date Assignee Title
CN111848809A (en) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 CAR molecule targeting Claudin18.2, immune cell modified by same and application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848809A (en) * 2019-04-08 2020-10-30 上海健信生物医药科技有限公司 CAR molecule targeting Claudin18.2, immune cell modified by same and application

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