WO2020239005A1 - 靶向Claudin18.2的抗体或嵌合抗原受体 - Google Patents

靶向Claudin18.2的抗体或嵌合抗原受体 Download PDF

Info

Publication number
WO2020239005A1
WO2020239005A1 PCT/CN2020/092849 CN2020092849W WO2020239005A1 WO 2020239005 A1 WO2020239005 A1 WO 2020239005A1 CN 2020092849 W CN2020092849 W CN 2020092849W WO 2020239005 A1 WO2020239005 A1 WO 2020239005A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
seq
binding fragment
cells
Prior art date
Application number
PCT/CN2020/092849
Other languages
English (en)
French (fr)
Inventor
宋德勇
周立
董创创
宁振飞
Original Assignee
山东博安生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东博安生物技术有限公司 filed Critical 山东博安生物技术有限公司
Priority to BR112021023897A priority Critical patent/BR112021023897A2/pt
Priority to AU2020281380A priority patent/AU2020281380B2/en
Priority to CN202080000840.1A priority patent/CN114127109B/zh
Priority to EP20815562.2A priority patent/EP3929214A4/en
Priority to US17/601,765 priority patent/US20220204609A1/en
Priority to KR1020217039106A priority patent/KR20220006085A/ko
Priority to JP2021560532A priority patent/JP7266117B2/ja
Priority to CA3136281A priority patent/CA3136281C/en
Publication of WO2020239005A1 publication Critical patent/WO2020239005A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/51—Stomach
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention belongs to the field of biomedicine or biopharmaceutical technology, and relates to an antibody or its antigen binding fragment or chimeric antigen receptor targeting Claudin 18.2, as well as its preparation method and its use for preparing pharmaceutical compositions, treatment, prevention, and detection Or the purpose of diagnosing disease.
  • Claudin 18.2 is only briefly expressed in gastric epithelial cells, and there is almost no expression in other normal tissues. However, the expression of Claudin 18.2 will increase abnormally after canceration in many tissues (Niimi, Mol. Cell. Biol. 21: 7380-90, 2001). Claudin 18.2 is expressed in gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer and other tumors. Antibodies targeting Claudin 18.2 can mediate specific killing of tumor cells through ADCC, CDC, inducing apoptosis and directly inhibiting proliferation. Therefore, Claudin 18.2 is currently the most potential target in the treatment of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, and ovarian cancer.
  • Claudin 18.1 is selectively expressed in the epithelium of normal lung and stomach, and the presence of two different variants adds complexity to the Claudin 18 molecule (Niimi, Mol. Cell. Biol. 21: 7380-90, 2001). How to further improve the effectiveness and safety is a question that needs to be considered in this field.
  • the IMAB362 project (Chinese application number CN201380026898.3) developed by Ganymed, Germany is the first Claudin 18.2 antibody to enter clinical trials.
  • the combination of this antibody and chemotherapy significantly prolonged the survival time than standard chemotherapy ( 13.2 vs. 8.4 months), it has a more obvious therapeutic effect in patients with high expression of Claudin 18.2, and patients have a longer median survival time (16.7 months) (NCT01630083).
  • the present invention provides an antibody or antigen binding fragment or chimeric antigen receptor T cell targeting Claudin 18.2 with good anti-Claudin 18.2 positive tumor effect, which brings new hope to patients with advanced gastric cancer and pancreatic cancer .
  • VL light chain variable region
  • VH heavy chain variable region
  • LCDR light chain complementarity determining region
  • HCDR heavy chain complementarity determining region
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 The various embodiments of HCDR3 and HCDR3 can be implemented individually or in any combination.
  • an antibody or antigen-binding fragment thereof comprising:
  • the antibody or antigen-binding fragment thereof comprises any combination of the following:
  • the three light chain complementarity determining regions include the LCDR1 amino acid sequence shown in SEQ ID NO: 5, the LCDR2 amino acid sequence shown in SEQ ID NO: 6, and the LCDR3 amino acid sequence shown in SEQ ID NO: 7, and/or
  • the three heavy chain complementarity determinations include the HCDR1 amino acid sequence shown in SEQ ID NO: 8, the HCDR2 amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 amino acid sequence shown in SEQ ID NO: 10;
  • the three light chain complementarity determining regions include the LCDR1 amino acid sequence shown in SEQ ID NO: 11, the LCDR2 amino acid sequence shown in SEQ ID NO: 12, and the LCDR3 amino acid sequence shown in SEQ ID NO: 13, and/or
  • the three heavy chain complementarity determining regions include the HCDR1 amino acid sequence shown in SEQ ID NO: 14, the HCDR2 amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 amino acid sequence shown in SEQ ID NO: 16;
  • the antibody or antigen-binding fragment thereof comprises any combination of the following:
  • the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, scFv, or dsFv fragments and so on.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof comprises a light chain constant region of the amino acid sequence shown in SEQ ID NO: 28.
  • the antibodies or antigen-binding fragments provided by the present invention have one or more of the following advantages: stronger affinity to cells secreting Claudin 18.2, enhanced ability to mediate ADCC effects, and better tumor suppressive effects.
  • any one of the aforementioned antibodies or antigen-binding fragments thereof binds Claudin 18.2.
  • the present invention relates to chimeric antigen receptors and related CAR-T cells containing the above-mentioned antibodies and antigen-binding fragments thereof, as well as preparation methods and uses thereof.
  • the present invention relates to a chimeric antigen receptor (CAR), which includes any one of the above-mentioned antibodies or antigen-binding fragments thereof, wherein the three light chain complementarity determining regions of the antibody or the antigen-binding fragment thereof
  • CAR chimeric antigen receptor
  • the LCDR1 amino acid sequence shown in SEQ ID NO: 5, the LCDR2 amino acid sequence shown in SEQ ID NO: 6 and the LCDR3 amino acid sequence shown in SEQ ID NO: 7 are included; and the antibody or its antigen-binding fragment has 3 multiples
  • the chain complementarity determining region includes the HCDR1 amino acid sequence shown in SEQ ID NO: 8, the HCDR2 amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 amino acid sequence shown in SEQ ID NO: 10.
  • the present invention relates to a chimeric antigen receptor (CAR), which includes an antibody or antigen-binding fragment thereof, wherein the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise SEQ ID NO: 11.
  • the LCDR1 amino acid sequence shown in SEQ ID NO: 12, the LCDR2 amino acid sequence shown in SEQ ID NO: 13 and the LCDR3 amino acid sequence shown in SEQ ID NO: 13; and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include SEQ ID
  • the present invention relates to a chimeric antigen receptor, wherein the sequence of the VL of the antibody or antigen-binding fragment thereof is SEQ ID NO: 1, and the VH is SEQ ID NO: 2.
  • the present invention relates to a chimeric antigen receptor, wherein the sequence of the VL of the antibody or antigen-binding fragment thereof is SEQ ID NO: 3, and the VH is SEQ ID NO: 4.
  • the VH and VL of the antibody or antigen-binding fragment thereof are connected by a linker; preferably, they are connected by a GGGGSGGGGSGGGGS linker; preferably, the connection sequence is VH-GGGGSGGGGSGGGGS-VL from N end to C end.
  • the present invention relates to a chimeric antigen receptor, which in turn includes the antibody or antigen-binding fragment thereof, an extracellular hinge region, a transmembrane region, and an intracellular signal region as described in any of the foregoing aspects.
  • the present invention relates to a chimeric antigen receptor whose antibody or antigen-binding fragment thereof is directed by a signal peptide.
  • the present invention relates to a chimeric antigen receptor, wherein the signal peptide can be a CD8 ⁇ signal peptide, a VH3 signal peptide or an IL2 signal peptide, etc., and the extracellular hinge region can be a CD8 hinge region or a CD28 hinge region, etc.
  • the transmembrane region can be CD8 transmembrane region, CD28 transmembrane region or 4-1BB transmembrane region, etc.
  • the intracellular signal region can be CD28 signal region, 4-1BB signal region, OX40 signal region or CD3 ⁇ signal District etc.
  • the present invention relates to a chimeric antigen receptor, wherein the extracellular hinge region is a CD8 hinge region, the transmembrane region is a CD8 transmembrane region, and the intracellular signal region is 4-1BB and CD3 ⁇ , The antibody or its antigen-binding fragment is guided by the CD8 ⁇ signal peptide.
  • the CD8 ⁇ signal peptide is the CD8 ⁇ signal peptide shown in the sequence SEQ ID NO: 21, the extracellular hinge region is the CD8 hinge region shown in the sequence SEQ ID NO: 22, and the transmembrane region is the sequence SEQ ID
  • the CD8 transmembrane region shown in NO: 23 and the intracellular signal region are 4-1BB shown in the sequence SEQ ID NO: 24 and CD3 ⁇ shown in the sequence SEQ ID NO: 25.
  • the present invention relates to nucleic acid, which encodes the antibody or antigen-binding fragment or chimeric antigen receptor of any one of the foregoing aspects.
  • the present invention relates to a vector comprising the nucleic acid described in the previous aspect, or it can express the antibody or antigen-binding fragment or chimeric antigen receptor described in any of the foregoing aspects.
  • the vector can be a viral vector; preferably, the viral vector includes, but is not limited to, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector; preferably, the vector can be a non-viral vector A vector; preferably, the non-viral vector can be a transposon vector; preferably, the transposon vector can be a Sleeping Beauty vector or a Piggybac vector; preferably, the vector can be a mammalian expression vector; preferably Preferably, the expression vector may be a bacterial expression vector; preferably, the expression vector may be a fungal expression vector.
  • the vector is a lentiviral vector.
  • the lentiviral vector is the plasmid pRRLSIN-Claudin18.2CAR-P2A-EGFRt as shown in FIG. 11.
  • the vector is a piggyBac (PB) transposon vector.
  • PB piggyBac
  • the PB transposon vector is the plasmid PB CN02CAR as shown in FIG. 24.
  • the present invention relates to a cell which can express the antibody or antigen-binding fragment or chimeric antigen receptor of any one of the foregoing aspects.
  • the cell is a bacterial cell; preferably, the bacterial cell is an E. coli cell, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast
  • the cells are Pichia pastoris cells, etc.; preferably, the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the present invention relates to CAR-T cells, which include the chimeric antigen receptor described in any of the foregoing aspects.
  • the present invention relates to a method for preparing the CAR-T cell described in the previous aspect, which comprises transfecting the T cell with a vector containing the chimeric antigen receptor described in any of the foregoing aspects.
  • the vector is a non-viral vector.
  • the vector is a PB transposon vector.
  • the PB transposon vector is the plasmid PB CN02CAR as shown in Figure 24.
  • the present invention relates to a method for preparing the CAR-T cell described in the previous aspect, which includes transfecting the T cell with a vector containing a transposase.
  • the transposase is PB transposase.
  • the present invention relates to a method for preparing the CAR-T cell described in the previous aspect, comprising transfecting T with a transposon vector containing the chimeric antigen receptor described in any one of the foregoing aspects and a transposase vector. cell.
  • the transposon vector is a PB transposon vector.
  • the PB transposon vector is the plasmid PB CN02CAR as shown in Figure 24.
  • the transposase is PB transposase.
  • the present invention relates to a method for preparing CAR-T cells as described in the previous aspect, which comprises transducing T cells with a lentivirus containing the chimeric antigen receptor vector as described in any of the foregoing aspects to obtain CAR-T cells. T cells.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the CAR-T cell described in any of the foregoing aspects.
  • the present invention relates to a method of treating cancer, which comprises administering the CAR-T cell of any one of the foregoing aspects to a subject in need thereof.
  • the present invention relates to the use of the CAR-T cell of any one of the foregoing aspects for the treatment of cancer.
  • the present invention relates to the use of the CAR-T cell described in any one of the foregoing aspects for preparing a pharmaceutical composition for treating cancer.
  • the present invention relates to CAR-T cells, which have one or more of the following advantages: good killing ability to cells expressing Claudin 18.2; low killing ability to cells expressing Claudin 18.1.
  • a pharmaceutical composition which comprises the antibody or antigen-binding fragment thereof, or chimeric antigen receptor of the present invention, or comprises an encoded antibody or antigen-binding fragment thereof or chimeric antigen receptor.
  • the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plasticizers, and pharmaceutical excipients.
  • kits comprising the antibody or antigen-binding fragment thereof of the present invention, or a chimeric antigen receptor, or a coding antibody or antigen-binding fragment thereof, or a chimeric antigen receptor.
  • Body nucleic acid In one aspect of the present invention, there is provided a kit comprising the antibody or antigen-binding fragment thereof of the present invention, or a chimeric antigen receptor, or a coding antibody or antigen-binding fragment thereof, or a chimeric antigen receptor.
  • the pharmaceutical composition may also include other therapeutic agents.
  • other therapeutic agents include chemotherapeutic agents, immunotherapeutic agents, or hormone therapy agents. The combined administration of the antibody or antigen-binding fragment and other therapeutic agents can enhance the therapeutic effect.
  • the "enhancement of the therapeutic effect” refers to enhancing the therapeutic effect of other therapeutic agents or therapies.
  • the antibody or antigen-binding fragment provided by the present invention can be administered alone or in combination with other therapeutic agents or therapies.
  • other therapeutic agents or therapies include chemotherapeutic agents, immunotherapeutic agents, hormone therapy agents, radiation therapy, surgical treatment.
  • the present invention relates to the use of the antibody or antigen-binding fragment, chimeric antigen receptor, nucleic acid, vector or cell of any one of the foregoing aspects in the preparation of a pharmaceutical composition for treating or preventing diseases.
  • the present invention relates to the use of the antibody or antigen-binding fragment, chimeric antigen receptor, and nucleic acid of any one of the foregoing aspects in the preparation of diagnostic and detection kits.
  • a method for treating or preventing diseases comprises administering the antibody or antigen-binding fragment, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition of the present invention to a subject in need.
  • a method for diagnosis and detection which includes administering the antibody or antigen-binding fragment, chimeric antigen receptor, nucleic acid or kit of the present invention to a subject or sample in need.
  • the use of the antibody or antigen-binding fragment, chimeric antigen receptor, nucleic acid, or kit of any one of the foregoing aspects for detection and diagnosis is provided.
  • the disease of the present invention is cancer.
  • the cancer is Claudin 18.2 positive cancer.
  • the cancer includes gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, colon cancer, kidney cancer, colorectal cancer, liver cancer, melanoma , Breast cancer, myeloma, glioma, leukemia and lymphoma.
  • Figures 1 to 2 show the binding sensitivity of the ELSIA detection candidate antibody to Claudin 18.2 protein.
  • Figures 3A-3B show the flow cytometric binding of candidate antibodies to 293T-Claudin 18.2 cells.
  • Figures 4A-4B show the flow cytometric binding of candidate antibodies to 293T-Claudin 18.1 cells.
  • Figures 5A-5B show the flow cytometric binding of candidate antibodies to NUGC4 cells.
  • Figures 6A-6B show the ADCC effect of candidate antibodies on 293T-Claudin 18.2 cells.
  • Figures 7A-7B show the ADCC effect of candidate antibodies on NUGC4 cells.
  • Figure 8 shows the ADCC effect of candidate antibodies on 293T-Claudin 18.1 cells.
  • Figure 9 shows the pharmacodynamic results of the candidate antibody.
  • Figure 10 shows a schematic diagram of the structure of plasmid pRRLSIN-EGFRt.
  • Figure 11 shows a schematic diagram of the structure of the recombinant plasmid pRRLSIN-Claudin18.2CAR-P2A-EGFRt.
  • Figure 12 shows the lentiviral activity titers of different chimeric antigen receptors.
  • Figure 13 shows the positive rate of T lymphocytes expressing different chimeric antigen receptors.
  • Figure 14 shows the results of in vitro experiments on the specific killing of Claudin 18.2 CAR-T cells with different scFvs.
  • Figure 15 shows the results of IFN-gamma cytokine release in the supernatant of different scFv Claudin 18.2 CAR-T and different 293T cells.
  • Figure 16 shows the experimental results of the specific killing of Claudin 18.2 CAR-T cells with different scFv on Claudin 18.2 positive tumor cells.
  • Figure 17 shows the release results of IFN-gamma cytokine in the supernatant of Claudin 18.2 CAR-T co-cultured with Claudin 18.2 positive tumor cells of different scFvs.
  • Figure 18 shows the test results of Claudin 18.2 CAR-T cells in female NOG mice carrying NUGC4-Claudin 18.2 tumor cells: tumor volume.
  • Figure 19 shows the test results of Claudin 18.2 CAR-T cells in female NOG mice carrying NUGC4-Claudin 18.2 tumor cells: mouse body weight.
  • FIG. 20 shows the test results of Claudin 18.2 CAR-T cells in female NOG mice carrying NUGC4-Claudin 18.2 tumor cells: tumor inhibition rate (TGI).
  • Figure 21 shows the test results of Claudin 18.2 CAR-T cells in female NOG mice carrying NUGC4-Claudin 18.2 tumor cells: mouse survival rate.
  • Figure 22 shows the positive rate of T lymphocytes expressing different chimeric antigen receptors.
  • Figure 23 shows the results of in vitro experiments on the specific killing of CN02 CAR-T cells prepared by different methods.
  • Figure 24 shows a schematic diagram of the structure of plasmid PB CN02 CAR.
  • the plasmid with the Claudin 18.2 gene inserted (Kangyuan Bochuang) and the CHO cell line (Kangyuan Bochuang) stably expressing the Claudin 18.2 protein were used to immunize Shandong Boan Biological fully human antibody transgenic mice BoAn-hMab (according to Chinese patent Prepared by the method described in CN103571872B). Plasmid immunization was used for the first immunization, and plasmids and cell lines were used for immunization across the second to seventh immunity. This time, 10 mice were immunized. Five mice with higher serum titers were selected for booster immunization. After 4 days, the mice were sacrificed, and the spleens were removed for processing and frozen for later use.
  • variable regions of the heavy and light chains are obtained from the cDNA by PCR, and then the heavy and light chains Single-chain antibody (scFv) was obtained by overlap extension PCR method for the variable region, and the scFv was digested with SfiI enzyme (NEB, catalog number R0123L) for 5h (50°C), and the digested scFv was digested by T4DNA ligase (sense Alice Shenzhou) was ligated with plasmid pCOMB3x (China Plasmid Vector Strain Cell Line Gene Collection, BIOVECTOR510837), and then the ligation product was electrotransfected into E.
  • coli TG1 competent cells (Lucigen, catalog number: A96595-2), after transfection After the TG1 was cultured on a shaker at 37°C and 220 rpm, phage infection was added, and then the culture supernatant was recovered, concentrated and purified to obtain a phage library.
  • variable region gene amplification 2*Phanta Max Master Mix manufacturer: Vazyme article number: P515-AA batch number: 7E211GB
  • signal peptide and variable region overlap extension homologous recombination (ClonExpress II One Step)
  • Cloning Kit manufacturer Vazyme Item No.: C112-01 Lot No.: 7E211L8
  • other methods finally insert the nucleotide sequence encoding VH into the nucleotide sequence with the amino acid sequence encoding the constant region of the antibody heavy chain (SEQ ID NO: 17)
  • the vector pCDNA3.4 (Life Technology) is inserted into the vector pCDNA3.4 (Life Technology) with the nucleotide sequence encoding the amino acid sequence of the constant region of the antibody light chain (SEQ ID NO: 28).
  • transfected into HEK293 cells transfected into HEK293 cells and cultured in a shaker at 37°C ⁇ 8%CO 2 ⁇ 125rpm. After 6-7 days, the transient expression supernatant was purified by Protein A affinity chromatography to obtain Claudin18.2 antibody, and bound by UV280 The extinction coefficient determines the antibody concentration.
  • the IMAB362 antibody was selected as the control antibody.
  • the combination of this antibody and chemotherapy significantly prolonged survival (13.2 vs. 8.4 months) than standard chemotherapy;
  • IMAB362 has a more obvious treatment in patients with high expression of Claudin 18.2 In effect, the patient has a longer median survival time (16.7 months).
  • IMAB362 is also one of the first Claudin 18.2 antibodies to undergo clinical trials.
  • Control antibody production The amino acid sequence of the Claudin 18.2 antibody IMAB362 from Ganymed, Germany was determined by IMGT data and patent CN201380026898.
  • the heavy chain sequence of IMAB362 is shown in SEQ ID NO: 19, and the light chain sequence of IMAB362 is shown in SEQ ID NO: 20.
  • the inserted vector pCDNA3.4 was expressed by HEK293 cells, and the antibody produced was named IMAB362.
  • Coated antigen Claudin 18.2 (GenScript) with different concentrations (0.2 ⁇ g/ml, 0.05 ⁇ g/ml, 0.0125 ⁇ g/ml), 100 ⁇ L/well at 4°C overnight; blocked with 3% skimmed milk powder at 37°C for 1h; Add 100 ⁇ L each of 1 ⁇ g/mL candidate antibody to the well, and incubate at 37°C for 1 h; then add goat anti-human IgG/HRP, incubate at 37°C for 1 h, develop color for 10 minutes, and read OD450 on the microplate reader. The results are shown in Figure 1, Figure 2, Table 2, Table 3.
  • the candidate antibody CLDQMIX-CA808.1-IgG1 when the target cell is 293T-Claudin18.2 cells, the candidate antibody CLDQMIX-CA808.1-IgG1 has an average fluorescence at the concentrations of 3 ⁇ g/ml, 1.5 ⁇ g/ml, 0.75 ⁇ g/ml, and 0.375 ⁇ g/ml. The intensity is greater than the average fluorescence intensity of IMAB362 corresponding concentration. It shows that the candidate antibody CLDQMIX-CA808.1-IgG1 has stronger affinity with 293T-Claudin18.2 cells at the concentrations of 3 ⁇ g/ml, 1.5 ⁇ g/ml, 0.75 ⁇ g/ml, and 0.375 ⁇ g/ml.
  • the average fluorescence intensity of candidate antibody CLDQMIX-CA808.1-IgG1 at the concentrations of 3 ⁇ g/ml, 1.5 ⁇ g/ml, and 0.75 ⁇ g/ml is greater than the average fluorescence of the corresponding concentration of IMAB362 strength. It indicates that the candidate antibody CLDQMIX-CA808.1-IgG1 has stronger affinity with NUGC4 cells expressing Claudin 18.2 at the concentrations of 3 ⁇ g/ml, 1.5 ⁇ g/ml, and 0.75 ⁇ g/ml.
  • the average fluorescence intensity of candidate antibody CLDQ1-CA841-IgG1 at the concentrations of 3 ⁇ g/ml and 1.5 ⁇ g/ml is greater than the average fluorescence intensity of the corresponding concentration of IMAB362. It shows that the candidate antibody CLDQ1-CA841-IgG1 has stronger affinity with 293T-Claudin18.2 cells at the concentration of 3 ⁇ g/ml and 1.5 ⁇ g/ml.
  • the candidate antibodies CLDQMIX-CA808.1-IgG1, CLDQ1-CA841-IgG1 and IMAB362 have similar average fluorescence intensity at each concentration and are all at a low level.
  • CLDQMIX-CA808.1-IgG1 and CLDQ1-CA841-IgG1 have a stronger ability to bind to cells that secrete Claudin 18.2, but have poor binding to Claudin 18.1. It indicates that it is easier to bind to cells secreting Claudin 18.2 in clinical practice, and it is not prone to non-Claudin 18.2 specific binding, which has better pharmaceutical effects.
  • fetal bovine serum Take aliquots of heat-inactivated fetal bovine serum and add it to RPMI1640 medium at a ratio of 1:99 after thawing, which is ADCC buffer. Resuscitate PBMC cells and culture overnight at 37°C in a 5% CO 2 incubator. Adjust the density of target cells (293T-Claudin 18.1 or 18.2) with ADCC Buffer to 2 ⁇ 10 5 cells/mL, and pave the target cells on a 96-well circular bottom plate with 50 ⁇ L per well.
  • the antibody concentration to be tested is 10 times diluted with ADCC Buffer starting from 10 ⁇ g/mL or 50 ⁇ g/mL, 50 ⁇ L per well is added to a 96-well circular bottom plate with target cells, and placed in a 37°C, 5% CO 2 incubator and incubated for 30-60 minutes .
  • Collect PBMC cells dilute the PBMC density with ADCC buffer from 2x10 6 cells/mL to 5x10 6 cells/mL, add 100 ⁇ L per well to 96-well circular bottom plate with target cells and samples to be tested, 37°C, 5% CO 2 incubator Incubate for 4h-6h.
  • the candidate antibody CLDQMIX-CA808.1-IgG1 inhibits the target cell at the concentration of 1 ⁇ g/ml and 0.1 ⁇ g/ml The rates are all greater than the inhibition rate of the corresponding concentration of the control antibody IMAB362. It shows that the candidate antibody CLDQMIX-CA808.1-IgG1 has a better ability to mediate ADCC effect than the control antibody IMAB362 at the concentration of 1 ⁇ g/ml and 0.1 ⁇ g/ml. It indicates that CLDQMIX-CA808.1-IgG1 can better kill the target cells that secrete Claudin 18.2 and have better pharmaceutical effects.
  • Collect cell counts add ADCC buffer and dilute to 4x10 5 cells/mL; take appropriate samples and dilute gradiently; collect effector cells Jurkat (G7011, Promega), centrifuge at 1500rpm, remove the supernatant, add 1% FBS RPMI-1640 medium and pipette repeatedly Resuspend the cells, add ADCC buffer and dilute to 8x10 5 cells/mL after counting the cells; add 25 ⁇ L/well of target cells to a white 96-well plate (3917, Costar), and add gradient dilutions of antibodies to the wells covered with target cells.
  • NUGC4 is used as the target cell, and the EC50 value of CLDQMIX-CA808.1-IgG1 is 1.264 ⁇ g/ml, which is lower than the EC50 value of the control antibody IMAB362, 2.154 ⁇ g/ml, indicating that the candidate antibody CLDQMIX-CA808.1-IgG1 is more
  • the control antibody IMAB362 has a better ability to mediate ADCC effects. It indicates that CLDQMIX-CA808.1-IgG1 can better kill the target cells that secrete Claudin 18.2 and have better pharmaceutical effects.
  • Antibody ID EC50( ⁇ g/mL) Antibody ID EC50( ⁇ g/mL) CLD389-279-IgG1 0.81 IMAB362 2.56 CLD389-CA802-IgG1 15.73 / /
  • the Fc terminal of the antibody was mutated.
  • the amino acid sequence of the constant region of the heavy chain after the mutation is shown in SEQ ID NO: 18.
  • the final antibody was named CLDQMix-CA808.1-IgG1-VLPLL.
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling human FcRn (Bepress, FCM-H82W4) (1 ⁇ g/mL) to Streptavidin(SA)Dip and Read TM Biosensors, the loading height is 0.2nm, and the antibody is diluted 2-fold serially with PBST, 33.3nM Start and set the concentration to 0, the Association time to 150s, and the Dissociation time to 100s. Steady State Analysis was used to calculate the equilibrium dissociation constant (kD) after the detection.
  • KD equilibrium dissociation constant
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling human CD32a(H) (Yiqiao Shenzhou, 10374-H27H1-B) (1 ⁇ g/mL) to Streptavidin(SA) Dip and Read TM Biosensors, the loading height is 0.2nm, and the antibody is continuously doubled with PBST Dilute, start at 1000nM, and set the concentration to 0, the Association time is set to 150s, and the Dissociation time is set to 100s. Steady State Analysis was used to calculate the equilibrium dissociation constant (kD) after the detection.
  • KD equilibrium dissociation constant
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling human CD16a(V) (Yiqiao Shenzhou, 10389-H27H1-B) (0.5 ⁇ g/mL) to Streptavidin(SA) Dip and Read TM Biosensors, the loading height is 0.5nm, and the antibody is doubled with PBST Serial dilution, starting at 166.7nM, and setting the concentration to 0, the Association time is set to 30s, and the Dissociation time is set to 100s. After the detection, use 1:1 Model Curve Fitting to calculate the binding constant (kon) and dissociation constant (kdis), and the equilibrium dissociation constant (kD) is calculated as the ratio kd/ka.
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling human CD16a(F) (Yiqiao Shenzhou, 10389-H27H-B) (0.5 ⁇ g/mL) to Streptavidin(SA) Dip and Read TM Biosensors, the loading height is 0.5nm, the antibody is doubled with PBST Serial dilution, starting at 333.3nM, and setting the concentration to 0, the Association time is set to 30s, and the Dissociation time is set to 100s. After the detection, use 1:1 Model Curve Fitting to calculate the binding constant (kon) and dissociation constant (kdis), and the equilibrium dissociation constant (kD) is calculated as the ratio kd/ka.
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling human CD32a(H) (Yiqiao Shenzhou, 10374-H27H1-B) (1 ⁇ g/mL) to Streptavidin(SA) Dip and Read TM Biosensors, the loading height is 0.2nm, and the antibody is continuously doubled with PBST Dilute, start at 2000nM, and set the concentration to 0, the Association time is set to 40s, and the Dissociation time is set to 50s. Steady State Analysis was used to calculate the equilibrium dissociation constant (kD) after the detection.
  • KD equilibrium dissociation constant
  • NUGC4 cells Human gastric cancer NUGC4 cells (JCRB Cell Bank, article number: JCRB0834) were cultured in a monolayer in vitro.
  • the culture conditions were RPMI1640 medium with 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin, 37°C with 5% CO 2 culture incubator.
  • pancreatin-EDTA for routine digestion and passage twice a week.
  • the cells are collected, counted, and inoculated with BALB/c nude mice, female, 6-8 weeks old, weighing 18-22 grams (Shanghai Lingchang Biotechnology Co., Ltd. ).
  • CLDQMIX-CA808.1-IgG1-VLPLL had the same tumor suppressive effect as 10 mg/kg of IMAB362.
  • 10mg/kg CLDQMIX-CA808.1-IgG1-VLPLL has a better tumor suppressive effect than 10mg/kg IMAB362.
  • the anti-tumor ability of CLDQMIX-CA808.1-IgG1-VLPLL antibody is better than IMAB362.
  • the inhibitory effect of CLDQMIX-CA808.1-IgG1-VLPLL on tumors has a significant dose-dependent relationship. The higher the dose, the better the tumor-inhibiting effect.
  • Antibody ID End point tumor volume (mm 3 ) Vehicle 1536.5 ⁇ 195.8 IMAB362 10mg/kg 1301.0 ⁇ 177.2 CLDQMIX-CA808.1-IgG1-VLPLL 5mg/kg 1307.8 ⁇ 186.4 CLDQMIX-CA808.1-IgG1-VLPLL 10mg/kg 1103.3 ⁇ 186.5 CLDQMIX-CA808.1-IgG1-VLPLL 20mg/kg 1006.4 ⁇ 207.1
  • the present invention designs fusion gene fragments according to the sequence of the following coding genes: CD8 ⁇ signal peptide, CA841scFv VH-linker-CA841scFv VL, CD8 hinge region, CD8 transmembrane region, and 4-1BB and CD3 ⁇ intracellular signal regions, which are directly synthesized by gene synthesis technology
  • the fusion gene makes the expressed chimeric antigen receptor have the amino acid structure of scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3 ⁇ .
  • the linker sequence is GGGGSGGGGSGGGGS
  • the CD8 ⁇ signal peptide sequence is SEQ ID NO: 21
  • the CD8 hinge region (CD8hinge) sequence is SEQ ID NO: 22
  • the CD8 transmembrane region (CD8TM) sequence is SEQ ID NO: 23
  • 4-1BB sequence is SEQ ID NO: 24
  • CD3 ⁇ sequence is SEQ ID NO: 25.
  • the pRRLSIN lentiviral vector was synthesized through the whole gene, which contains the human EF1a promoter, and the GFP green fluorescent protein sequence was replaced with the EGFRt marker protein sequence to obtain the pRRLSIN-EGFRt vector (see Figure 10).
  • the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third-generation self-inactivating lentiviral vector system.
  • the system has 3 plasmids, which are respectively the pMDLg-pRRE packaging plasmid encoding the protein Gag/Pol (Youbao Bio, VT1449), the pRSV-rev packaging plasmid encoding Rev protein (Ubao Bio, VT1445) and the envelope plasmid PMD2.G (Youbao Bio, VT1443) encoding VSV-G protein.
  • a lentiviral expression vector that co-expressed specific CAR and EGFRt (SEQ ID NO: 27) linked by P2A (SEQ ID NO: 26) was constructed, and the target gene obtained in step 6.1 was linked to On the pRRLSIN-EGFRt vector, a recombinant plasmid was formed and named pRRLSIN-Claudin18.2CAR-P2A-EGFRt (see Figure 11).
  • the specific structure is pRRLSIN-CD8 ⁇ -scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3 ⁇ -P2A-EGFRt.
  • CAR-P2A-EGFRt is transcribed into one mRNA, but finally translated into two EGFRt and anti-Claudin18.2 chimeric antigen receptors
  • the peptide chain, where the anti-Claudin 18.2 CAR will be located on the cell membrane under the guidance of the CD8 ⁇ signal peptide.
  • scFv CA808.1-CD8hinge-CD8TM-4-1BB-CD3 ⁇ -P2A-EGFRt (abbreviated as CN01)
  • scFv CA841-CD8hinge-CD8TM-4-1BB-CD3 ⁇ -P2A-EGFRt (abbreviated as CN02)
  • the cell culture supernatant containing the virus was collected and centrifuged at 3000 rpm for 5 min at 4°C. After the supernatant was filtered through a 0.45 ⁇ m filter, it was mixed with PEG8000/NaCl in a 4:1 volume, allowed to stand at 4°C for 2 to 3 hours, and then centrifuged at high speed for 30 minutes. The supernatant was discarded, and the pellet was resuspended and dissolved in pre-cooled T cell culture medium X-VIVO 15 (Lonza, 04-418Q) or PBS to obtain a virus concentrate, which was stored at -80°C for later use.
  • the cell infection method was used to determine the biological activity titer of the lentivirus.
  • the medium is DMEM medium containing 10% FBS, and 1 mL of fresh medium is added to each well.
  • the activity titers of the lentiviral concentrates of the above CARs (CN01, CN02, CN03, CN04) packaged by the PEI transfection method were all greater than 1 ⁇ 10 8 TU/mL ( Figure 12).
  • PBMC Peripheral blood mononuclear cells
  • CD3MicroBeads human-lyophilized Kit purchased from Miltenyi Biotech
  • high-purity CD3+T lymphocytes were positively sorted Cells, the proportion of CD3 positive T cells after sorting is more than 95%.
  • purified T cells use human CD3CD28T cell activator (Dynabeads Human T-Activator CD3/CD28, Thermo Fisher, 11132D) for T lymphocyte activation and proliferation.
  • CAR-T cells are obtained by transducing T cells with the lentivirus prepared in section 6.3. 6.5 After partial stimulus activation for 24-48h, observe whether T lymphocytes are activated under microscope. After activation, the volume of T lymphocytes becomes larger and the shape is elongated or irregular. The activated T lymphocytes were collected, centrifuged and resuspended in T cell medium X-VIVO 15 (Lonza, 04-418Q) containing a final concentration of 10ng/mL IL-7 and 5ng/mL IL15, the final volume was 1mL, and Into a 12-well culture plate.
  • T lymphocytes were respectively infected with lentiviruses packaging different chimeric antigen receptors, and cultured to the 9th day, there was about 300-fold amplification, indicating that T lymphocytes expressing different chimeric antigen receptors can perform a certain number of in vitro Amplification provides a guarantee for subsequent in vitro functional studies and in vivo animal efficacy studies.
  • Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, and there are only eight amino acid differences between the two sequences.
  • Claudin 18.1 is selectively expressed in normal lung cells
  • Claudin 18.2 is highly restricted in normal cells, but it is frequently ectopically activated and overexpressed in a variety of tumors (gastric cancer, lung cancer, pancreatic cancer, etc.).
  • 293T cells purchased from Kangyuan Bochuang, KC-0990/KC-0986
  • overexpressing Claudin 18.1 protein and Claudin 18.2 protein were used as target cells, and Claudin 18 with different scFv prepared above was used.
  • Claudin 18.2 CAR-T (CN01, CN02, CN03, CN04) and 293T-hClaudin 18.1 with different scFv
  • the differential expression of IFN-gamma cytokine released from the co-culture supernatant of cells is consistent with the results of the killing test ( Figure 15, Table 18).
  • the release of IFN-gamma cytokine released by the supernatant of 293T-hClaudin18.1 cells in the CN02 group was significantly lower than that in the CN01, CN03, and CN04 groups.
  • LDH Release Assay Kit (LDH Release Assay Kit) (Nippon Tongjin Chemical, CK12) is used. It is an INT color reaction based on diaphorase catalysis. When cytotoxicity is detected by colorimetry Released lactate dehydrogenase activity. Its principle is that the destruction of cell membrane structure caused by apoptosis or necrosis will cause the enzymes in the cytoplasm to be released into the culture medium, including lactate dehydrogenase (LDH), which has relatively stable enzyme activity. The quantitative analysis of cytotoxicity can be achieved by detecting the activity of LDH released from the cells with ruptured plasma membrane into the culture medium. LDH release is regarded as an important indicator of cell membrane integrity, and is widely used in cytotoxicity testing.
  • Cytokine detection method using human IFN-gamma enzyme-linked immunoassay kit (R&D Systems, SIF50), which is based on the immobilization of antigen or antibody and the enzyme labeling of antigen or antibody.
  • the antigen or antibody bound on the surface of the solid carrier still maintains its immunological activity, and the enzyme-labeled antigen or antibody retains its immunological activity and enzyme activity.
  • the test substance (antigen or antibody) in the sample binds to the immobilized antibody or antigen.
  • the non-binding substances are removed by washing the plate, and then the enzyme-labeled antigen or antibody is added.
  • the amount of the enzyme that can be fixed is related to the amount of the test substance in the sample.
  • an in vitro drug efficacy test was established by simulating the mechanism of action (MOA) of the product.
  • MOA mechanism of action
  • the present inventors used plvx vector to construct a Claudin 18.2 protein overexpression plasmid, and then proceeded with lentivirus preparation, and infected gastric cancer cell line NUGC4 with lentivirus And AGS, after subsequent positive cell screening, NUGC4 cells and AGS cells with high expression of Claudin 18.2 protein were obtained, which were used as target cells for CAR-T cell function verification.
  • Claudin 18.2CAR-T with different scFv prepared above as effector cells according to different E:T (effector cells: target cells) ratios, a co-culture system of CAR-T cells and targeted tumor cells was established, and tumor cells were detected by The killing rate is used to evaluate the biological efficacy of CAR-T, and a control system for co-culture of untransduced T cells and tumor cells is established.
  • T lymphocytes expressing different chimeric antigen receptors can have a good killing effect on Claudin 18.2 positive tumor cells, which provides a basis for animal in vivo drug efficacy research.
  • an immunodeficiency mouse pharmacodynamic model of gastric cancer tumor cell burden was established, and based on in vitro studies, female NOG mice (purchased from Beijing Weitong Lihua) were injected with 1 ⁇ 10 7 NUGC4- Claudin 18.2 cells were administered on the 11th day of inoculation (the tumor volume was about 80-100mm 3 ), the solvent control group was 0.9% saline, and the Mock-T (untransfected T cells) group was 1 ⁇ 10 7 Cells, CN02 low-dose and high-dose groups (positive cells) were 5.00 ⁇ 10 6 and 1.00 ⁇ 10 7 respectively , and the administration volume was 100 ⁇ L. The number of animals in all condition groups is 6.
  • TGI Tumor inhibition rate
  • the gene insulator sequence cHS4 was found, placed at both ends of the multiple cloning site, and the 5'ITR and 3'ITR sequences of PB transposon were found to construct Enter the inside of the cHS4 sequence of the vector, insert the EF1a promoter at the 5 end of the ITR, insert the polyA signal at the 3 end, and retain the polyclonal sequence in the middle, insert the CAR-P2A-EGFRt sequence inside the polyclonal sequence to form the PBCN02CAR plasmid
  • the structure is shown in Figure 24.
  • PBMC Peripheral blood mononuclear cells
  • CD3MicroBeads human-lyophilized Kit purchased from Miltenyi Biotech
  • high purity CD3 was positively sorted + T lymphocytes, the proportion of CD3 positive T cells after sorting is above 95%.
  • purified T cells use human CD3CD28T cell activator (Dynabeads Human T-Activator CD3/CD28, Thermo Fisher, 11132D) for T lymphocyte activation and proliferation.
  • DPBS DPBS resuspended cells were washed, room temperature Centrifuge at 300x g for 10 minutes, aspirate the supernatant as much as possible to avoid contact with the cell pellet, add 100 ⁇ L of electroporation buffer Entranster-E (Engreen, 98668-20) to resuspend the cells, and transfer the cell suspension to a 1.5mL centrifuge tube .
  • Entranster-E Engreen, 98668-20
  • anti-human IgG (Fab) 2 antibody was used to detect the expression of chimeric antigen receptor by flow cytometry, and untransduced T lymphocytes were used as a negative control to express T lymphocytes of different chimeric antigen receptors.
  • the positive rate of cells is shown in Table 22 ( Figure 22):
  • an in vitro drug efficacy test was established by simulating the mechanism of action (MOA) of the product, and the constructed gastric cancer cell line NUGC4 and AGS cells with high expression of Claudin 18.2 were used as target cells, prepared by the above non-viral (PB) CN02CAR-T cells and CN02CAR-T cells prepared by lenti virus (lenti) are used as effector cells.
  • PB non-viral
  • CN02CAR-T cells and CN02CAR-T cells prepared by lenti virus (lenti) are used as effector cells.
  • E:T (effector cells: target cells) ratios a co-culture system of CAR-T cells and targeted tumor cells is established .
  • To evaluate the biological efficacy of CAR-T by detecting the killing rate of tumor cells, and to establish a control system for co-culture of untransduced T cells and tumor cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

公开了一种结合Claudin18.2的抗体或其抗原结合片段或嵌合抗原受体，以及其制备方法和用途。所述嵌合抗原受体依次包括本发明的结合Claudin18.2抗原的抗体或其抗原结合片段、胞外铰链区、跨膜区和胞内信号区。本发明所提供的抗体或其抗原结合片段及嵌合抗原受体对分泌Claudin18.2的细胞有更强的亲和力和杀伤能力，以及更好的肿瘤抑制效果。

Description

靶向Claudin18.2的抗体或嵌合抗原受体 技术领域
本发明属于生物医学或生物制药技术领域，涉及一种靶向Claudin18.2的抗体或其抗原结合片段或嵌合抗原受体，以及其制备方法和用于制备药物组合物、治疗、预防、检测或诊断疾病的用途。
背景技术
Claudin18.2仅在胃上皮细胞短暂表达，其它正常组织几乎没有表达，但是很多组织癌变后Claudin18.2表达会异常升高(Niimi，Mol.Cell.Biol.21：7380-90，2001)。Claudin18.2在胃癌、食道癌、胰腺癌、肺癌、卵巢癌等多种肿瘤中均有表达。靶向Claudin18.2的抗体可以通过ADCC、CDC、诱导凋亡以及直接抑制增殖来介导对肿瘤细胞特异性的杀伤。因此，Claudin18.2是目前胃癌、食道癌、胰腺癌、肺癌、卵巢癌等治疗领域中最具潜力的靶点。
Claudin18.1在正常肺和胃的上皮中选择性表达，两种不同变体的存在给Claudin18分子增添了复杂性(Niimi，Mol.Cell.Biol.21：7380-90，2001)。如何进一步提高有效性和安全性是这一领域需要思考的问题。
德国Ganymed公司开发的IMAB362项目(中国申请号CN201380026898.3)是最早进入临床试验的Claudin18.2抗体，在其胃癌临床II期试验中，该抗体和化疗的联用比标准化疗显著延长生存期(13.2对8.4个月)，在Claudin18.2高表达患者中具有更为明显的治疗效果，患者出现更长的中位生存期(16.7个月)(NCT01630083)。
本发明提供了具有良好的抗Claudin18.2阳性肿瘤的效果的靶向Claudin18.2的抗体或其抗原结合片段或嵌合抗原受体T细胞，为晚期胃癌、胰腺癌等患者带来新的希望。
发明内容
本发明全文中关于VL(轻链可变区)、VH(重链可变区)、LCDR(轻链互补决定区)、HCDR(重链互补决定区)、LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3的各个实施方式可以各自单独实施，也可以任意组合实施。
在本发明的一个方面中，提供一种抗体或其抗原结合片段，该抗体或其抗原结合片段包含：
在一些实施方式中，所述抗体或其抗原结合片段包含如下的任意一种组合：
(1)3个轻链互补决定区包含SEQ ID NO:5所示的LCDR1氨基酸序列、SEQ ID NO:6所示的LCDR2氨基酸序列和SEQ ID NO:7所示的LCDR3氨基酸序列，和/或3个重链互补决定去包含SEQ ID NO:8所示的HCDR1氨基酸序列、SEQ ID NO:9所示的HCDR2氨基酸序列、和SEQ ID NO:10所示的HCDR3氨基酸序列；
(2)3个轻链互补决定区包含SEQ ID NO:11所示的LCDR1氨基酸序列、SEQ ID NO:12所示的LCDR2氨基酸序列和SEQ ID NO:13所示的LCDR3氨基酸序列，和/或3个重链互补决定区包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:15所示的HCDR2氨基酸序列、和SEQ ID NO:16所示的HCDR3氨基酸序列；
在一些实施方式中，所述抗体或其抗原结合片段包含如下的任意一种组合：
(1)SEQ ID NO:1所示氨基酸序列的轻链可变区，和/或SEQ ID NO:2所示氨基酸序列的 重链可变区；
(2)SEQ ID NO:3所示氨基酸序列的轻链可变区，和/或SEQ ID NO:4所示氨基酸序列的重链可变区。
在本发明的一个方面中，所述抗体或其抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
在本发明的一个方面中，所述抗体或其抗原结合片段包含SEQ ID NO:17所示氨基酸序列的重链恒定区。
在本发明的一个方面中，所述抗体或其抗原结合片段包含SEQ ID NO:18所示氨基酸序列的重链恒定区。
在本发明的一个方面中，所述抗体或其抗原结合片段包含SEQ ID NO:28所示氨基酸序列的轻链恒定区。
本发明提供的抗体或抗原结合片段具有以下中的一种或多种优势：对分泌Claudin18.2的细胞有更强的亲和力、增强的介导ADCC效应的能力、更好的肿瘤抑制效果。
在本发明的一个方面中，上述任意一种抗体或其抗原结合片段结合Claudin18.2。
本发明涉及包含上述抗体及其抗原结合片段的嵌合抗原受体及相关的CAR-T细胞，以及其制备方法和用途。
具体而言，在一方面，本发明涉及嵌合抗原受体(CAR)，其包括上述任意一种抗体或其抗原结合片段，其中所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:5所示的LCDR1氨基酸序列、SEQ ID NO:6所示的LCDR2氨基酸序列和SEQ ID NO:7所示的LCDR3氨基酸序列；并且所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:8所示的HCDR1氨基酸序列、SEQ ID NO:9所示的HCDR2氨基酸序列、和SEQ ID NO:10所示的HCDR3氨基酸序列。
在另一方面，本发明涉及嵌合抗原受体(CAR)，其包括抗体或其抗原结合片段，其中所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:11所示的LCDR1氨基酸序列、SEQ ID NO:12所示的LCDR2氨基酸序列和SEQ ID NO:13所示的LCDR3氨基酸序列；并且所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:14所示的HCDR1氨基酸序列、SEQ ID NO:15所示的HCDR2氨基酸序列、和SEQ ID NO:16所示的HCDR3氨基酸序列。
在另一方面，本发明涉及嵌合抗原受体，其中所述抗体或其抗原结合片段的VL的序列为SEQ ID NO:1，并且VH为SEQ ID NO:2。
在另一方面，本发明涉及嵌合抗原受体，其中所述抗体或其抗原结合片段的VL的序列为SEQ ID NO:3，并且VH为SEQ ID NO:4。
在另一方面，所述抗体或其抗原结合片段的VH和VL是通过接头连接；优选地，通过GGGGSGGGGSGGGGS接头连接；优选地，其连接顺序由N端到C端依次为VH-GGGGSGGGGSGGGGS-VL。
在另一方面，本发明涉及嵌合抗原受体，其依次包括前述任一方面所述的抗体或其抗原结合片段、胞外铰链区、跨膜区和胞内信号区。
在另一方面，本发明涉及嵌合抗原受体，其抗体或其抗原结合片段由信号肽引导。
在另一方面，本发明涉及嵌合抗原受体，其中所述信号肽可以是CD8α信号肽、VH3信号肽或IL2信号肽等，所述胞外铰链区可以是CD8铰链区或CD28铰链区等，所述跨膜区可 以是CD8跨膜区、CD28跨膜区或4-1BB跨膜区等，所述胞内信号区可以是CD28信号区、4-1BB信号区、OX40信号区或CD3ζ信号区等。
在另一方面，本发明涉及嵌合抗原受体，其中所述胞外铰链区为CD8铰链区、所述跨膜区为CD8跨膜区、所述胞内信号区为4-1BB和CD3ζ，抗体或其抗原结合片段由CD8α信号肽引导。优选地，其中所述CD8α信号肽为序列SEQ ID NO:21所示的CD8α信号肽、胞外铰链区为序列SEQ ID NO:22所示的CD8铰链区、所述跨膜区为序列SEQ ID NO:23所示的CD8跨膜区、所述胞内信号区为序列SEQ ID NO:24所示的4-1BB和序列SEQ ID NO:25所示的CD3ζ。
在另一方面，本发明涉及核酸，其编码前述任一方面所述的抗体或其抗原结合片段或嵌合抗原受体。
在另一方面，本发明涉及载体，其包含前一方面所述的核酸，或其可表达前述任一方面所述的抗体或其抗原结合片段或嵌合抗原受体。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述非病毒载体可以是转座子载体；优选地，所述转座子载体可以是Sleeping Beauty载体或Piggybac载体等；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优选地，所述表达载体可以是真菌表达载体。
在另一方面，所述载体为慢病毒载体。
在另一方面，所述慢病毒载体为如图11所示的质粒pRRLSIN-Claudin18.2CAR-P2A-EGFRt。
在另一方面，所述载体为piggyBac(PB)转座子载体。
在另一方面，所述PB转座子载体为如图24所示的质粒PB CN02CAR。
在另一方面，本发明涉及一种细胞，其可表达前述任一方面所述的抗体或其抗原结合片段或嵌合抗原受体。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。
在另一方面，本发明涉及CAR-T细胞，其包括前述任一方面所述的嵌合抗原受体。
在另一方面，本发明涉及制备前一方面所述的CAR-T细胞的方法，包括使用包含前述任一方面所述的嵌合抗原受体的载体转染T细胞。在一个优选的实施方案中，所述载体为非病毒载体。在一个优选的实施方案中，所述载体为PB转座子载体。在一个优选的实施方案中，所述PB转座子载体为如图24所示的质粒PB CN02CAR。
在另一方面，本发明涉及制备前一方面所述的CAR-T细胞的方法，包括使用包含转座酶的载体转染T细胞。在另一个优选的实施方案中，所述转座酶为PB转座酶。
在另一方面，本发明涉及制备前一方面所述的CAR-T细胞的方法，包括使用包含前述任一方面所述的嵌合抗原受体的转座子载体和转座酶载体转染T细胞。在一个优选的实施方案中，所述转座子载体为PB转座子载体。在一个优选的实施方案中，所述PB转座子载体为如图24所示的质粒PB CN02CAR。在一个优选的实施方案中，所述转座酶为PB转座酶。
在另一方面，本发明涉及制备前一方面所述的CAR-T细胞的方法，包括使用包含前 述任一方面所述的嵌合抗原受体载体的慢病毒转导T细胞，从而获得CAR-T细胞。
在另一方面，本发明涉及包括前述任一方面所述的CAR-T细胞的药物组合物。
在另一方面，本发明涉及治疗癌症的方法，包括将前述任一方面所述的CAR-T细胞给予有此需要的受试者。
在另一方面，本发明涉及前述任一方面所述的CAR-T细胞用于治疗癌症的用途。
在另一方面，本发明涉及前述任一方面所述的CAR-T细胞用于制备治疗癌症的药物组合物的用途。
在另一方面，本发明涉及CAR-T细胞，其具有以下一种或几种优势：对表达Claudin18.2的细胞有良好的杀伤能力；对表达Claudin18.1的细胞的杀伤能力低。
在本发明的一个方面中，提供一种药物组合物，其包含本发明所述的抗体或其抗原结合片段，或嵌合抗原受体，或包含编码抗体或其抗原结合片段或嵌合抗原受体的核酸，或包含表达抗体或其抗原结合片段或嵌合抗原受体的细胞，以及药学上可接受的载体。所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂、药物辅料。
在本发明的一个方面中，提供一种试剂盒，其包含本发明所述的抗体或其抗原结合片段，或包含嵌合抗原受体，或包含编码抗体或其抗原结合片段或嵌合抗原受体的核酸。
在一些实施方式中，所述药物组合物还可以包含其他治疗剂。在一些实施方式中，其他治疗剂包括化疗剂、免疫治疗剂或激素治疗剂。所述抗体或抗原结合片段与其他治疗剂的联合施用能够增强治疗效果。
在一些实施方式中，所述“增强治疗效果”是指增强其他治疗剂或疗法的治疗效果。本发明提供的所述抗体或抗原结合片段可以单独施用，也可以与其他治疗剂或疗法联合施用。在一些实施方式中，其他治疗剂或疗法包括化疗剂、免疫治疗剂、激素治疗剂、放射治疗、手术治疗。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、嵌合抗原受体、核酸、载体或细胞在制备治疗或预防疾病的药物组合物中的应用。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、嵌合抗原受体、核酸在制备诊断、检测试剂盒中的应用。
在另一方面，提供一种治疗或预防疾病的方法，包括将本发明的抗体或抗原结合片段、嵌合抗原受体、核酸、载体、细胞或药物组合物给予有需要的受试者。
在另一方面，提供一种诊断、检测的方法，包括将本发明的抗体或抗原结合片段、嵌合抗原受体、核酸或试剂盒给予有需要的受试者或样本。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、嵌合抗原受体、核酸、载体、细胞或药物组合物用于治疗、预防疾病的用途。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、嵌合抗原受体、核酸、或试剂盒用于检测、诊断的用途。
在另一方面，本发明所述疾病为癌症。
在另一方面，所述癌症为Claudin18.2阳性癌症。
在另一方面，所述癌症包括胃癌、胰腺癌、食道癌、肺癌、卵巢癌、头颈癌、膀胱癌、宫颈癌、肉瘤、细胞瘤、结肠癌、肾脏癌、结肠直肠癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤等。
附图说明
图1-图2示出了ELSIA检测候选抗体对Claudin18.2蛋白的结合灵敏度。
图3A-图3B示出了候选抗体与293T-Claudin18.2细胞的流式结合。
图4A-图4B示出了候选抗体与293T-Claudin18.1细胞的流式结合。
图5A-图5B示出了候选抗体与NUGC4细胞的流式结合。
图6A-图6B示出了候选抗体对293T-Claudin18.2细胞的ADCC效应。
图7A-图7B示出了候选抗体对NUGC4细胞的ADCC效应。
图8示出了候选抗体对293T-Claudin18.1细胞的ADCC效应。
图9示出了候选抗体药效学结果。
图10示出了质粒pRRLSIN-EGFRt的结构示意图。
图11示出了重组质粒pRRLSIN-Claudin18.2CAR-P2A-EGFRt的结构示意图。
图12示出了不同嵌合抗原受体的慢病毒活性滴度。
图13示出了表达不同嵌合抗原受体的T淋巴细胞的阳性率。
图14示出了不同scFv的Claudin18.2 CAR-T细胞的特异性杀伤的体外实验的结果。
图15示出了不同scFv的Claudin18.2 CAR-T与不同293T细胞共培养上清中IFN-gamma细胞因子释放结果。
图16示出了不同scFv的Claudin18.2 CAR-T细胞对Claudin18.2阳性肿瘤细胞的特异性杀伤的实验结果。
图17示出了不同scFv的Claudin18.2 CAR-T与Claudin18.2阳性肿瘤细胞共培养上清中IFN-gamma细胞因子释放结果。
图18示出了携带NUGC4-Claudin18.2肿瘤细胞的雌性NOG小鼠中Claudin18.2 CAR-T细胞的测试结果：肿瘤体积。
图19示出了携带NUGC4-Claudin18.2肿瘤细胞的雌性NOG小鼠中Claudin18.2 CAR-T细胞的测试结果：小鼠体重。
图20示出了携带NUGC4-Claudin18.2肿瘤细胞的雌性NOG小鼠中Claudin18.2 CAR-T细胞的测试结果：肿瘤抑制率(TGI)。
图21示出了携带NUGC4-Claudin18.2肿瘤细胞的雌性NOG小鼠中Claudin18.2 CAR-T细胞的测试结果：小鼠存活率。
图22示出了表达不同嵌合抗原受体的T淋巴细胞的阳性率。
图23示出了不同方法制备的CN02 CAR-T细胞的特异性杀伤的体外实验的结果。
图24示出了质粒PB CN02 CAR的结构示意图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1.抗Claudin18.2单克隆抗体的产生
1.1免疫方案
用插入了Claudin18.2基因的质粒(康源博创)和稳定表达Claudin18.2蛋白的CHO细胞株(康源博创)免疫山东博安生物全人抗体转基因小鼠BoAn-hMab(按照中国专利CN103571872B记载的方法制备)。首次免疫使用质粒免疫，二免至七免交叉使用质粒和细胞株进行免疫，本次共免疫10只小鼠。选取血清滴度较高的5只小鼠进行加强免疫，4天后处死小鼠，取出脾脏处理并冷冻备用。
1.2噬菌体库的建立
取免疫小鼠的脾脏细胞，加入Trizol(Thermo Scientific，目录货号15596-026)，待裂解充分后加入1/5体积的氯仿，充分混匀，室温放置20min后在4℃下以12000rpm离心20min，取上层水溶液，并加入等体积的异丙醇，室温放置20min，在4℃下以12000rpm离心20min，弃去上清水溶液，加入75％乙醇洗涤两次，在4℃下以12000rpm离心5min，弃去水溶液，保留沉淀，室温风干后加入DEPC水重悬沉淀获得RNA，使用罗氏反转录试剂盒Transcriptor First Strand cDNA Synthesis Kit(Roche Applied Science，目录货号4897030001)按照说明书将RNA反转录成cDNA。
噬菌体库的建立步骤参照Carlos F.Barbas III，Phage display：A laboratory manual中记载的方法进行，首先用PCR的方法从cDNA中获得重链和轻链的可变区，再将重链和轻链的可变区通过重叠延伸PCR的方法获得单链抗体(scFv)，用SfiI酶(NEB,目录货号R0123L)对scFv进行酶切5h(50℃)，酶切后的scFv通过T4DNA连接酶(义翘神州)与质粒pCOMB3x(中国质粒载体菌株细胞株基因保藏中心,BIOVECTOR510837)连接，然后将连接产物电转染至大肠杆菌TG1感受态细胞(Lucigen，目录号：A96595-2)中，转染后的TG1经37℃、220rpm摇床培养后加入噬菌体侵染，然后回收培养物上清，浓缩纯化获得噬菌体库。
1.3以两种方法进行筛选
1.3.1平板筛选：用Claudin18.2蛋白(金斯瑞)以0.3μg/孔包被平板，4℃放置过夜，第二天通过2％BSA封闭平板1h，加入噬菌体库(2x10 12)孵育2h，洗涤4-10次后用洗脱缓冲液(pH 2.2)(向500mL超纯水中加入4.2mL浓盐酸(科密欧)，用甘氨酸粉末(Biotopped，BG0617-500)调节pH至2.2)洗脱Claudin18.2特异性结合的噬菌体。
1.3.2细胞筛选：将噬菌体库(2x10 12)与293T-Claudin18.1细胞(3x10 6细胞/库)在室温下旋转混合并孵育1h，用2％BSA封闭1h，再与293T-Claudin18.2细胞(2x10 6细胞/库)在室温下旋转混合并孵育2h，洗涤4-10次后用洗脱缓冲液(pH 2.2)洗脱Claudin18.2特异性结合的噬菌体。细胞筛选完成后可以继续投入进行平板筛选。
实施例2.完整抗体的分子构建与生产
将克隆CLD387-C115、CLD389-C279\CA802\CA852、CLDQMix-CA808.1\CA811\CA818、CLDQ1-CA841\CA843、CLD393-C1002\C1024送Invitrogen生物技术有限公司测序。各克隆可变区氨基酸序列如表1。
表1.活性克隆可变区的氨基酸序列
Figure PCTCN2020092849-appb-000001
Figure PCTCN2020092849-appb-000002
Figure PCTCN2020092849-appb-000003
通过常规的分子生物学技术可变区基因扩增(2*Phanta Max Master Mix厂家：Vazyme货号：P515-AA批号：7E211GB)、信号肽与可变区重叠延伸、同源重组(ClonExpress Ⅱ One Step Cloning Kit厂家：Vazyme货号：C112-01批号：7E211L8)等方法，最终把编码VH的核苷酸序列片段插入带有编码抗体重链恒定区氨基酸序列(SEQ ID NO:17)的核苷酸序列的载体pCDNA3.4(Life Technology)，把编码VL的核苷酸序列片段插入带有编码抗体轻链恒定区氨基酸序列(SEQ ID NO:28)的核苷酸序列的载体pCDNA3.4(Life Technology)，转染进入HEK293细胞在37℃\8％CO 2\125rpm摇床中培养，6-7天后的瞬时表达上清通过Protein A亲和层析，纯化获得Claudin18.2抗体，并通过UV280结合消光系数确定抗体浓度。
对照抗体选用IMAB362抗体，在胃癌二期试验中，该抗体和化疗的联用比标准化疗显著延长生存期(13.2对8.4个月)；IMAB362在Claudin18.2高表达患者中具有更为明显的治疗效果，患者出现更长的中位生存期(16.7个月)。IMAB362也是最先进行临床试验的Claudin18.2抗体之一。
对照抗体生产：通过IMGT数据及专利CN201380026898确定德国Ganymed公司Claudin18.2抗体IMAB362的氨基酸序列，IMAB362重链序列如SEQ ID NO:19所示，IMAB362轻链序列如SEQ ID NO:20所示，全基因合成后插入载体pCDNA3.4通过HEK293细胞表达，生产的抗体命名为IMAB362。
实施例3.Anti-Claudin18.2抗体分子的表征
3.1 Elisa检测抗体与Claudin18.2蛋白的结合
包被不同浓度(0.2μg/ml、0.05μg/ml、0.0125μg/ml)的抗原Claudin18.2(金斯瑞)，100 μL/孔4℃过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1μg/mL候选抗体各100μL，37℃孵育1h；然后加入山羊抗人IgG/HRP，37℃孵育1h，显色10min后，酶标仪上读取OD450。结果见图1、图2、表2、表3。
表2.ELSIA检测候选抗体对Claudin18.2蛋白的结合灵敏度数据
Figure PCTCN2020092849-appb-000004
表3.ELSIA检测候选抗体对Claudin18.2蛋白的结合灵敏度数据
Figure PCTCN2020092849-appb-000005
3.2流式检测抗体与293T-Claudin18.1/18.2细胞、NUGC4细胞的结合
96孔圆底板中，加入50μL 293T-Claudin18.1或18.2细胞(北京康源博创)，或NUGC4细胞，细胞数为1x10 5细胞/孔，用FACS buffer(无菌PBS，0.2％BSA)梯度稀释各候选抗体，按50μL/孔加入96孔圆底板中，4℃孵育1h。2000rpm离心3min后弃上清，用FACS buffer洗2次，加入100μL/孔荧光二抗(Southern Biotech，2040-09)，终浓度1μg/mL，4℃孵育1h，2000rpm离心3min后弃上清，用FACS buffer洗2次后用100μL/孔FACS buffer重悬，用流式细胞仪(艾森，NovoCyte 2060)进行检测。结果见图3A-5B及表4-9。
如图3A所示，靶细胞为293T-Claudin18.2细胞时，候选抗体CLDQMIX-CA808.1-IgG1在3μg/ml、1.5μg/ml、0.75μg/ml、0.375μg/ml浓度下，平均荧光强度均大于IMAB362对应浓度的平均荧光强度。说明候选抗体CLDQMIX-CA808.1-IgG1在3μg/ml、1.5μg/ml、0.75μg/ml、0.375μg/ml浓度下与293T-Claudin18.2细胞有更强的亲和力。
如图5B所示，靶细胞为NUGC4细胞时，候选抗体CLDQMIX-CA808.1-IgG1在3μg/ml、1.5μg/ml、0.75μg/ml浓度下，平均荧光强度均大于IMAB362对应浓度的平均荧光强度。说明候选抗体CLDQMIX-CA808.1-IgG1在3μg/ml及1.5μg/ml、0.75μg/ml浓度下与表达Claudin18.2的NUGC4细胞有更强的亲和力。
如图3A所示，靶细胞为293T-Claudin18.2细胞时，候选抗体CLDQ1-CA841-IgG1在3μg/ml和1.5μg/ml浓度下，平均荧光强度大于IMAB362对应浓度的平均荧光强度。说明候选抗体CLDQ1-CA841-IgG1在3μg/ml和1.5μg/ml浓度下与293T-Claudin18.2细胞有更强的亲和力。
如图4A所示，靶细胞为293T-Claudin18.1细胞时，候选抗体CLDQMIX-CA808.1-IgG1 及CLDQ1-CA841-IgG1与IMAB362在各浓度下平均荧光强度相似且均处于较低水平。
以上结果说明CLDQMIX-CA808.1-IgG1、CLDQ1-CA841-IgG1有更强的结合分泌Claudin18.2的细胞的能力，对Claudin18.1的结合较差。预示临床中更易结合分泌Claudin18.2的细胞，不易发生非Claudin18.2特异性结合，有更好的药学效果。
表4.候选抗体与293T-Claudin18.2细胞的流式结合数据(对应图3A)
Figure PCTCN2020092849-appb-000006
表5.候选抗体与293T-Claudin18.2细胞的流式结合数据(对应图3B)
Figure PCTCN2020092849-appb-000007
表6.候选抗体与293T-Claudin18.1细胞的流式结合数据(对应图4A)
Figure PCTCN2020092849-appb-000008
表7.候选抗体与293T-Claudin18.1细胞的流式结合数据(对应图4B)
Figure PCTCN2020092849-appb-000009
表8.候选抗体与NUGC4细胞的流式结合数据(对应图5A)
Figure PCTCN2020092849-appb-000010
表9.候选抗体与NUGC4细胞的流式结合数据(对应图5B)
Figure PCTCN2020092849-appb-000011
3.3检测抗体的ADCC效应
取分装好的热灭活胎牛血清，融化后以1：99的比例加入RPMI1640培养基中，即为ADCC buffer。复苏PBMC细胞，37℃，5％CO 2培养箱培养过夜。用ADCC Buffer调整靶细胞(293T-Claudin18.1或18.2)密度为2x10 5细胞/mL，每孔50μL铺靶细胞于96孔圆底板。待测抗体浓度从10μg/mL或50μg/mL开始用ADCC Buffer进行10倍稀释，每孔50μL加入铺有靶细胞的96孔圆底板，放入37℃，5％CO 2培养箱孵育30～60min。收集PBMC细胞，用ADCC buffer稀释PBMC密度为2x10 6细胞/mL到5x10 6细胞/mL，每孔100μL加入铺有靶细胞和待测样品的96孔圆底板，37℃，5％CO 2培养箱孵育4h-6h。孵育结束后，300g离心2-5min，小心吸取50μL上清至新的96孔平底板，再加入50μL LDH检测液(Promega，G1780)，37℃，5％CO 2培养箱孵育30min。孵育完成后加入终止液。酶标仪测量490nm处OD值，背景波长为650nm。结果见图6A-6B、表10-表11。
如图6A所示，在293T-Claudin18.2作为靶细胞，PBMC作为效应细胞的条件下，候选抗体CLDQMIX-CA808.1-IgG1在1μg/ml、0.1μg/ml浓度下，对靶细胞的抑制率均大于对照抗体IMAB362对应浓度的抑制率。说明候选抗体CLDQMIX-CA808.1-IgG1在1μg/ml及0.1μg/ml浓度下，较对照抗体IMAB362有更好的介导ADCC效应的能力。预示CLDQMIX-CA808.1-IgG1能更好的杀伤分泌Claudin18.2的靶细胞，有更好的药学效果。
表10.候选抗体对293T-Claudin18.2细胞的ADCC效应数据(对应图6A)
Figure PCTCN2020092849-appb-000012
Figure PCTCN2020092849-appb-000013
表11.候选抗体对293T-Claudin18.2细胞的ADCC效应数据(对应图6B)
Figure PCTCN2020092849-appb-000014
收集细胞计数，加入ADCC buffer稀释为4x10 5细胞/mL；取适当样品梯度稀释；收集效应细胞Jurkat(G7011，Promega)，1500rpm离心，去上清，加入1％FBS RPMI-1640培养基反复吹吸重悬细胞，细胞计数后加入ADCC buffer稀释为8x10 5细胞/mL；在白色96孔板(3917，Costar)中加入靶细胞25μL/孔，在铺有靶细胞的孔中加入梯度稀释的抗体，25μL/孔，加入效应细胞(Jurkat)25μL/孔，使效应细胞:靶细胞比例为20000：10000，将96孔板放入细胞培养箱培养5h；取出96孔板，放置室温，使其温度平衡到室温；加入Bio-Glo显色液(G7940，Promega)75μL/孔，反应15min后Tecan酶标仪读数(化学发光)。结果见图7A-图8、表12-表13。
如图7A所示，NUGC4作为靶细胞，CLDQMIX-CA808.1-IgG1的EC50值为1.264μg/ml，小于对照抗体IMAB362的EC50值2.154μg/ml，说明候选抗体CLDQMIX-CA808.1-IgG1较对照抗体IMAB362有更好的介导ADCC效应的能力。预示CLDQMIX-CA808.1-IgG1能更好的杀伤分泌Claudin18.2的靶细胞，有更好的药学效果。
表12.候选抗体对NUGC4细胞的ADCC效应数据(对应图7A)
抗体ID EC50(μg/mL) 抗体ID EC50(μg/mL)
CLDQMIX-CA808.1-IgG1 1.264 CLD387-115-IgG1 14.27
CLDQ1-CA841-IgG1 17.33 IMAB362 2.154
表13.候选抗体对NUGC4细胞的ADCC效应数据(对应图7B)
抗体ID EC50(μg/mL) 抗体ID EC50(μg/mL)
CLD389-279-IgG1 0.81 IMAB362 2.56
CLD389-CA802-IgG1 15.73 / /
实施例4.Anti-Claudin18.2抗体分子Fc端的改造
4.1改造抗体分子的Fc端
为了增强抗体的ADCC作用，改变抗体与FC受体的亲和力，将抗体Fc端进行突变，突变后重链恒定区氨基酸序列如SEQ ID NO:18所示。将最终抗体命名为CLDQMix-CA808.1-IgG1-VLPLL。
4.2检测改造抗体的Fc受体亲和力
4.2.1抗体与human FcRn亲和力检测
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器 测量。将human FcRn(百普赛斯，FCM-H82W4)(1μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.2nm，将抗体用PBST进行2倍连续稀释，33.3nM起始，并设置0浓度，Association时间设置为150s，Dissociation时间设置为100s。检测结束后使用Steady State Analysis计算平衡解离常数(kD)。
4.2.2抗体与human CD32a(H)亲和力检测
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器测量。将human CD32a(H)(义翘神州，10374-H27H1-B)(1μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.2nm，将抗体用PBST进行2倍连续稀释，1000nM起始，并设置0浓度，Association时间设置为150s，Dissociation时间设置为100s。检测结束后使用Steady State Analysis计算平衡解离常数(kD)。
4.2.3抗体与human CD16a(V)亲和力检测
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器测量。将human CD16a(V)(义翘神州，10389-H27H1-B)(0.5μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.5nm，将抗体用PBST进行2倍连续稀释，166.7nM起始，并设置0浓度，Association时间设置为30s，Dissociation时间设置为100s。检测结束后使用1:1Model的Curve Fitting计算结合常数(kon)、解离常数(kdis)，平衡解离常数(kD)以比率kd/ka计算。
4.2.4抗体与human CD16a(F)亲和力检测
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器测量。将human CD16a(F)(义翘神州，10389-H27H-B)(0.5μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.5nm，将抗体用PBST进行2倍连续稀释，333.3nM起始，并设置0浓度，Association时间设置为30s，Dissociation时间设置为100s。检测结束后使用1:1Model的Curve Fitting计算结合常数(kon)、解离常数(kdis)，平衡解离常数(kD)以比率kd/ka计算。
4.2.5抗体与human CD32b亲和力检测
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器测量。将human CD32a(H)(义翘神州，10374-H27H1-B)(1μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.2nm，将抗体用PBST进行2倍连续稀释，2000nM起始，并设置0浓度，Association时间设置为40s，Dissociation时间设置为50s。检测结束后使用Steady State Analysis计算平衡解离常数(kD)。
如表14所示，CLDQMIX-CA808.1-IgG1-VLPLL对激动性受体尤其是Human CD16a(F)受体的亲和力大幅度提高，因此在患者体内可以更好促进的ADCC效应。
表14.候选抗体的Fc受体亲和力数据
Figure PCTCN2020092849-appb-000015
实施例5.Anti-Claudin18.2抗体的药效学实验
将人胃癌NUGC4细胞(JCRB Cell Bank,货号：JCRB0834)体外单层培养，培养条件为RPMI1640培养基中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃5％CO 2孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种BALB/c裸小鼠，雌性，6～8周龄，体重18～22克(上海灵畅生物科技有限公司)。将0.2mL(1x10 6个)NUGC4细胞(加基质胶，体积比为1:1)皮下接种于每只小鼠的右后背，肿瘤平均体积达到约60-70mm 3时开始分组给药。给药前称重动物，测量瘤体积。根据瘤体积随机分组(随机区组设计)，每组8只。每周测量两次体重。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。实验结果见图9及表15。
如图9所示，5mg/kg的CLDQMIX-CA808.1-IgG1-VLPLL相比于10mg/kg的IMAB362肿瘤抑制效果相当。10mg/kg的CLDQMIX-CA808.1-IgG1-VLPLL相比于10mg/kg的IMAB362有更好的肿瘤抑制效果。说明CLDQMIX-CA808.1-IgG1-VLPLL抗体的抑瘤能力优于IMAB362。并且CLDQMIX-CA808.1-IgG1-VLPLL对肿瘤的抑制效果具有明显剂量依赖关系，剂量越高，抑瘤效果越好。
表15.候选抗体药效学数据(对应图9)
抗体ID 终点肿瘤体积(mm 3)
Vehicle 1536.5±195.8
IMAB362 10mg/kg 1301.0±177.2
CLDQMIX-CA808.1-IgG1-VLPLL 5mg/kg 1307.8±186.4
CLDQMIX-CA808.1-IgG1-VLPLL 10mg/kg 1103.3±186.5
CLDQMIX-CA808.1-IgG1-VLPLL 20mg/kg 1006.4±207.1
实施例6 Claudin18.2特异性嵌合抗原受体修饰的T细胞的制备
6.1嵌合抗原受体基因片段制备
本发明按以下编码基因的顺序设计融合基因片段：CD8α信号肽、CA841scFv VH-linker-CA841scFv VL、CD8铰链区、CD8跨膜区以及4-1BB和CD3ζ胞内信号区，通过基因合成技术直接合成该融合基因，使表达的嵌合抗原受体具有scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3ζ的氨基酸结构。其中接头(linker)序列为GGGGSGGGGSGGGGS，CD8α信号肽的序列为SEQ ID NO:21，CD8铰链区(CD8hinge)的序列为SEQ ID NO:22，CD8跨膜区(CD8TM)的序列为SEQ ID NO:23，4-1BB序列为SEQ ID NO:24，且CD3ζ序列为SEQ ID NO:25。
通过全基因合成pRRLSIN慢病毒载体，该载体包含人EF1a启动子，将GFP绿色荧光蛋白序列替换为EGFRt标记蛋白序列从而获得pRRLSIN-EGFRt载体(参见图10)。
6.2嵌合抗原受体慢病毒表达载体的构建
在本实施例中，构建本发明的慢病毒质粒载体使用的载体系统属于第三代自灭活慢病毒载体系统，该系统共有3个质粒，分别是编码蛋白Gag/Pol的pMDLg-pRRE包装质粒(优宝生物，VT1449)、编码Rev蛋白的pRSV-rev包装质粒(优宝生物，VT1445)和编码VSV-G蛋白的包膜质粒PMD2.G(优宝生物，VT1443)。
在本实施例中，构建了共表达由P2A(SEQ ID NO:26)连接的特异性CAR和EGFRt(SEQ ID NO:27)的慢病毒表达载体，通过将步骤6.1中获得的目的基因连接在pRRLSIN-EGFRt载体上，形成重组质粒，命名为pRRLSIN-Claudin18.2CAR-P2A-EGFRt (参见图11)。具体结构为pRRLSIN-CD8α-scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt。构建成功的载体经酶切鉴定及序列测定为正确后，可以准备用于慢病毒包装，CAR-P2A-EGFRt转录为一条mRNA，但最终翻译成EGFRt和抗Claudin18.2嵌合抗原受体两条肽链，其中在CD8α信号肽的引导下抗Claudin18.2CAR将定位在细胞膜上。
本实施例得到的四种含目的CAR结构如下
scFv CA808.1-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt(简称CN01)
scFv CA841-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt(简称CN02)
scFv C279-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt(简称CN03)
scFv C115-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt(简称CN04)
6.3嵌合抗原受体慢病毒制备
提取pRRLSIN-Claudin18.2CAR-P2A-EGFRt表达质粒和pMDLg-pRRE、pRSV-rev及pMD2.G辅助质粒，通过一定比例与转染试剂聚乙烯亚胺(PEI)混合，共转染293T细胞，主要步骤如下：
(1)以7x10 6的细胞密度接种培养至5-8代的293T细胞(ATCC CRL-3216)于75cm 3的细胞培养瓶中，培养基为含有10％FBS(购自GIBCO)的DMEM培养基(购自GIBCO)，混匀并放入CO 2培养箱中培养24h准备用于转染，培养条件为37℃和5％CO 2。次日观察细胞汇合度在70-80％左右，即可进行转染。
(2)24h后，将目的表达质粒和pMDLg-pRRE、pRSV-rev及pMD2.G辅助质粒按照质量比4：3：2：2混合，稀释液为Opti-MEM培养基(购自GIBCO)，设置为A液。按照总质粒：PEI＝3：1的比例配置PEI稀释液，稀释液为Opti-MEM培养基，设置为B液。将A液和B液混匀后室温孵育15min。
(3)取出铺板好的293T细胞，将质粒-PEI混合液缓慢加入到细胞上清培养基中，轻轻摇匀，放入CO 2培养箱中培养4-6h，培养条件为37度和5％CO 2。培养后更换为新鲜的含10％FBS的DMEM培养基。
(4)转染48h和96h后，收集含有病毒的细胞培养上清，在4℃下以3000rpm离心5min。上清经0.45μm滤器过滤后，与PEG8000/NaCl按4:1体积混匀，4℃下静置2～3h后高速离心30min。弃上清，沉淀用预冷的T细胞培养基X-VIVO 15(Lonza,04-418Q)或者PBS重悬溶解，即获得病毒浓缩液，-80℃保存备用。
6.4慢病毒滴度检测
在本实施例中，采用细胞感染方法来测定慢病毒的生物活性滴度。以293T细胞作为慢病毒活性的检测细胞，按1x10 5/孔接种24孔培养板，培养基为含10％FBS的DMEM培养基，每孔添加新鲜培养基1mL。加入助转染添加剂Polybrene，使其终浓度为6μg/mL。再加入1μL/孔的慢病毒浓缩液，3倍稀释，共5个梯度，每个梯度2个重复，加入后充分混匀，放入CO 2培养箱中培养24h，培养条件为37度和5％CO 2。24h后，将细胞消化下来，通过流式细胞仪，采用抗人IgG(Fab)2(Jackson ImmunoResearch，109-065-006)或者抗人EGFRt(Biolegend，352904)流式染料检测CAR或者EGFRt的蛋白表达阳性率，滴度计算公式为：慢病毒活性滴度(TU/mL)＝阳性率×稀释倍数×100×10 5。PEI转染法包装的上述CAR(CN01、CN02、CN03、CN04)的慢病毒浓缩液的活性滴度均大于1×10 8TU/mL(图12)。
6.5 T淋巴细胞的制备
采用购自澳赛尔斯生物技术公司的外周血单核细胞(PBMC)，通过CD3MicroBeads human-lyophilized Kit(购自Miltenyi Biotech)对细胞进行磁珠标记，阳性分选出高纯度的CD3+T淋巴细胞，分选后的CD3阳性T细胞比例在95％以上。对于纯化后的T细胞，再利用人CD3CD28T cell激活剂(Dynabeads Human T-Activator CD3/CD28，Thermo Fisher，11132D)进行T淋巴细胞激活及增殖。
6.6慢病毒转导T淋巴细胞
CAR-T细胞是通过用6.3部分制备的慢病毒转导T细胞而获得。6.5部分刺激活化24-48h后，镜检观察T淋巴细胞是否被激活，活化后的T淋巴细胞体积变大、形态呈现拉长形或不规则形态。收集激活的T淋巴细胞，离心重悬在含有终浓度为10ng/mL IL-7和5ng/mL IL15的T细胞培养基X-VIVO 15(Lonza,04-418Q)中，终体积为1mL，加到12孔培养板中。用相同培养基将慢病毒稀释至MOI＝3-5，感染1x10 6个活化的T淋巴细胞，将细胞和慢病毒混合液充分混匀加在24孔板中，置37℃、5％CO 2培养箱中孵育过夜。第二天，再次离心并换新鲜培养基，每隔2天检测一次细胞密度，并对细胞进行继续扩大培养，使细胞密度不要超过2x10 6细胞/mL。在慢病毒感染T细胞48-72h后，通过流式细胞技术检测不同嵌合抗原受体表达，以未转导的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的T淋巴细胞的阳性率如表16所示(图13)：
表16.表达不同嵌合抗原受体的T淋巴细胞的阳性率
转染有下列CAR的细胞 CAR的阳性率
CN01 30.5％
CN02 19.1％
CN03 24.3％
CN04 13.2％
T淋巴细胞分别在感染包装不同嵌合抗原受体的慢病毒后，培养至第9天约有300倍的扩增，表明表达不同嵌合抗原受体的T淋巴细胞能在体外进行一定数量的扩增，为后续体外功能研究和动物体内药效研究提供了保证。
6.7体外毒性试验
6.7.1靶点特异性试验
Claudin-18具有两个剪接变体，分别为Claudin 18.1和Claudin 18.2，两者序列之间仅有八个氨基酸的差异。Claudin 18.1在正常肺的细胞中选择性表达，Claudin 18.2在正常细胞中表达高度受限，但在多种肿瘤(胃癌、肺癌和胰腺癌等)中频繁异位激活和过表达。在本实施例中，分别采用过表达Claudin18.1蛋白和Claudin18.2蛋白的293T细胞(购自康源博创，KC-0990/KC-0986)作为靶细胞，通过上述制备的不同scFv的Claudin18.2CAR-T作为效应细胞，以293T细胞、过表达Claudin18.2蛋白的293T细胞及过表达Claudin18.1蛋白的293T细胞，按照不同的E:T(效应细胞：靶细胞)比，建立CAR-T细胞与靶向肿瘤细胞的共培养体系，通过检测肿瘤细胞杀伤率来评估CAR-T对两个蛋白的特异性反应。体外实验结果(图14)所示，在肿瘤细胞数目固定的情况下，不同scFv制备的Claudin18.2CAR-T(CN01、CN02、CN03、CN04)与293T-hClaudin18.2细胞以效靶比1:1、3:1共孵育时，24h时杀伤肿瘤细胞的效率可达20％-50％(表17)，与293T-hClaudin18.1细胞以效靶比1:1、3:1共孵育时，不同scFv制备的Claudin18.2 CAR-T对其特异性杀伤有显著性差 异，其中CN02CAR-T对293T-hClaudin18.1细胞无明显特异性杀伤，CN01、CN03、CN04CAR-T对293T-hClaudin18.1细胞均有不同程度的特异性杀伤。同时通过检测分泌至培养基上清中细胞因子(INF-gamma)含量来评估CAR-T特异性反应，不同scFv的Claudin18.2CAR-T(CN01、CN02、CN03、CN04)与293T-hClaudin18.1细胞共培养上清释放的IFN-gamma细胞因子释放表达差异和杀伤试验结果一致(图15、表18)。CN02组中293T-hClaudin18.1细胞共培养上清释放的IFN-gamma细胞因子释放显著低于CN01、CN03、CN04组。
特异性杀伤检测方法：采用乳酸脱氢酶释放检测试剂盒(LDH Release Assay Kit)(日本同仁化学,CK12)进行，是一种基于diaphorase催化的INT显色反应，通过比色法检测细胞毒性时释放的乳酸脱氢酶活性。它的原理是采用细胞凋亡或坏死而造成的细胞膜结构的破坏会导致细胞浆内的酶释放到培养液里，其中包括酶活性较为稳定的乳酸脱氢酶(lactate dehydrogenase,LDH)。通过检测从质膜破裂的细胞中释放到培养液中的LDH的活性，就可以实现对细胞毒性的定量分析。LDH释放被看做细胞膜完整性的重要指标，并被广泛用于细胞毒性检测。
细胞因子检测方法：采用人IFN-gamma酶联免疫检测试剂盒(R&D Systems,SIF50)进行，是基于抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。进行检测时，样品中的受检物质(抗原或抗体)与固定的抗体或抗原结合。通过洗板除去非结合物，再加入酶标记的抗原或抗体，此时，能固定下来的酶量与样品中被检物质的量相关。通过加入与酶反应的底物后显色，根据颜色的深浅可以判断样品中物质的含量，进行定性或定量的分析。
表17.不同scFv的Claudin18.2CAR-T细胞的特异性杀伤的体外实验的结果
Figure PCTCN2020092849-appb-000016
表18.不同scFv的Claudin18.2CAR-T与不同293T细胞共培养上清中IFN-gamma细胞因子释放结果
Figure PCTCN2020092849-appb-000017
Figure PCTCN2020092849-appb-000018
6.7.2靶点毒性试验
在本实施例中，通过模拟产品的作用机制(MOA)建立体外药效试验，本发明人以plvx载体构建Claudin18.2蛋白过表达质粒，然后进行慢病毒制备，通过慢病毒感染胃癌细胞株NUGC4和AGS，再经过后续阳性细胞筛选获得高表达Claudin18.2蛋白的NUGC4细胞和AGS细胞，以此作为CAR-T细胞功能验证的靶细胞。通过上述制备的不同scFv的Claudin18.2CAR-T作为效应细胞，按照不同的E:T(效应细胞：靶细胞)比，建立CAR-T细胞与靶向肿瘤细胞的共培养体系，通过检测肿瘤细胞杀伤率来评估CAR-T生物学效力，同时设立未经转导的T细胞与肿瘤细胞共培养对照体系。
体外实验结果(图16、表19)显示，CAR-T与Claudin18.2阳性肿瘤细胞(AGS-claudin18.2和NUGC-4)共孵育时，24h时杀伤肿瘤细胞的效率优异，显著高于T细胞，对Claudin18.2表达阴性的细胞株AGS杀伤效果一般。同时通过检测分泌至培养基上清中细胞因子(INF-gamma)含量来评估CAR-T生物学效力，Claudin18.2CAR-T与Claudin18.2阳性肿瘤细胞(AGS-claudin18.2和NUGC-4)共培养后，细胞因子IFN-gamma表达量显著高于T细胞组(图17、表20)。
表19.不同scFv的Claudin18.2CAR-T细胞对Claudin18.2阳性肿瘤细胞的特异性杀伤的实验结果
Figure PCTCN2020092849-appb-000019
表20.不同scFv的Claudin18.2 CAR-T与Claudin18.2阳性肿瘤细胞共培养上清中IFN-gamma细胞因子释放结果
Figure PCTCN2020092849-appb-000020
基于体外细胞毒性试验，表明表达不同嵌合抗原受体的T淋巴细胞能对Claudin18.2 阳性肿瘤细胞均有良好的杀伤作用，这为动物体内药效研究提供了依据。
6.8动物模型试验
在本实施例中，建立胃癌肿瘤细胞负荷的免疫缺陷型小鼠药效模型，同时基于体外研究以雌性NOG小鼠(购自北京维通利华)经背皮注射1×10 7个NUGC4-Claudin18.2细胞，接种第11天(肿瘤体积在80-100mm 3大小左右)给药，溶剂对照组为0.9％生理盐水，Mock-T(未转染质粒的T细胞)组为1×10 7细胞，CN02低剂量和高剂量组(阳性细胞)分别为5.00×10 6、1.00×10 7，给药体积均为100μL。所有条件组动物数量均为6只。给药后每周2次量瘤；绘制肿瘤生长曲线，计算TGI、T/C，试验终点对所有肿瘤进行拍照。CAR-T给药前(day-2)、给药后第2、9和28天取血，通过qPCR的方法检测小鼠外周血中CAR的拷贝数(VCN)，从而确认CART细胞的扩增情况。结果表明CAR-T给药36天,各个药物组疗效显著，其中Claudin18.2 CAR-T(CN02)低剂量组6只小鼠肿瘤完全消退(6/6)，高剂量组5只小鼠肿瘤完全消退(5/6)(图18)。
(1)体重：与溶剂对照组及Mock比较，Claudin18.2 CAR-T(CN02)低剂量组和高剂量组体重无显著性差异(图19)。
(2)肿瘤抑制率(TGI)统计：药后14天，Claudin18.2 CAR-T(CN02)低剂量和高剂量组TGI分别为100％、88.33％(图20)。
(3)死亡率：至药后31天，溶剂对照组动物发现死亡1只，Claudin18.2 CAR-T(CN02)低剂量组和高剂量组未出现小鼠死亡的现象(图21)。
实施例7非病毒方法制备CAR-T细胞
7.1非病毒piggyBac(PB)转座子载体构建
在本实施例中，以pBluescirpt载体(通用生物合成)为起始骨架，寻找到基因绝缘子序列cHS4，安置在多克隆位点两端，寻找到PB transposon的5’ITR和3’ITR序列，构建进入载体的cHS4序列的内侧，在ITR内侧，5端插入EF1a启动子，在3端插入polyA信号，中间保留多克隆序列，在多克隆序列内部接入CAR-P2A-EGFRt序列，形成PB CN02CAR质粒结构，如图24所示。
7.2非病毒PB转座子载体制备CAR-T细胞
7.2.1使用购自澳赛尔斯生物技术公司的外周血单核细胞(PBMC)，通过CD3MicroBeads human-lyophilized Kit(购自Miltenyi Biotech)对细胞进行磁珠标记，阳性分选出高纯度的CD3+T淋巴细胞，分选后的CD3阳性T细胞比例在95％以上。对于纯化后的T细胞，再利用人CD3CD28T cell激活剂(Dynabeads Human T-Activator CD3/CD28，Thermo Fisher，11132D)进行T淋巴细胞激活及增殖。
7.2.2在刺激活化第3天开始进行电转。将用于电转的细胞进行重悬，使用吸头或移液管吹散细胞团块，对细胞重悬液进行计数，使用5x 10 6细胞用于单次电转实验。使用DPBS(GIBCO，14190-144)将5x 10 6细胞稀释至4mL，室温条件下300x g离心10分钟，尽可能吸弃上清，避免接触到细胞沉淀，加入5mL DPBS重悬清洗细胞，室温条件下300x g离心10分钟，尽可能吸弃上清，避免接触到细胞沉淀，加入100μL电转缓冲液Entranster-E(Engreen,98668-20)重悬细胞，将细胞悬液转移到1.5mL离心管中。
将表21中的组分加入到离心管中，混匀:
表21.电转体系
组分 体积(μL)
PB CN02CAR质粒(1μg/μL) 5
PB转座酶质粒(1μg/μL) 5
细胞悬液 100
总体积 110
采用Lonza公司电转仪进行电转，将细胞/质粒悬液迅速转移至电击杯中，并轻轻磕击电击杯使细胞悬液充分在电击杯中形成平衡液面，使用程序EO115进行电转。电转后，将电击杯小心取出。加入500μL预热T细胞培养基X-VIVO 15(Lonza,04-418Q)并在37℃培养箱中平衡5分钟，使用微孔上样吸头重悬细胞，轻轻吹打2-3次。将细胞转移至加有2mL预热培养基的12孔板中，37℃培养。电转后4-6小时对细胞进行换液有利于增加细胞存活率。小心吸弃上清，加入预热的新鲜培养基。在37℃,5％CO 2培养箱中培养48小时直至检测。
同时设置慢病毒制备CAR-T对照组，制备方法与6.6部分相同。
电转48-72h后，采用抗人IgG(Fab)2抗体通过流式细胞技术检测嵌合抗原受体表达，以未转导的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的T淋巴细胞其阳性率如表22所示(图22)：
表22.表达不同嵌合抗原受体的T淋巴细胞的阳性率
细胞名称 CAR阳性率％
未转导的T细胞 0.56
非病毒PB CN02CAR-T 35.4
慢病毒Lenti CN02CAR-T 38
7.3靶点毒性试验
在本实施例中，通过模拟产品的作用机制(MOA)建立体外药效试验，以构建的高表达Claudin18.2的胃癌细胞株NUGC4细胞和AGS细胞作为靶细胞，通过上述非病毒(PB)制备的CN02CAR-T细胞和慢病毒(lenti)制备的CN02CAR-T细胞作为效应细胞，按照不同的E:T(效应细胞：靶细胞)比，建立CAR-T细胞与靶向肿瘤细胞的共培养体系，通过检测肿瘤细胞杀伤率来评估CAR-T生物学效力，同时设立未经转导的T细胞与肿瘤细胞共培养对照体系。
体外实验结果(图23、表23)显示，在肿瘤细胞数目固定的情况下，对肿瘤细胞杀伤率与效应细胞数目呈线性相关；CAR-T与Claudin18.2阳性肿瘤细胞(AGS-claudin18.2和NUGC-4)共孵育时，24h时杀伤肿瘤细胞的效率优异，显著高于未转导的T细胞，对Claudin18.2表达阴性的细胞株AGS杀伤效果一般。
表23.不同方法制备的CN02CAR-T细胞的特异性杀伤的体外实验的结果
Figure PCTCN2020092849-appb-000021
Figure PCTCN2020092849-appb-000022

Claims (10)

  1. 一种抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，其中
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:5所示的LCDR1、SEQ ID NO:6所示的LCDR2和SEQ ID NO:7所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:8所示的HCDR1、SEQ ID NO:9所示的HCDR2和SEQ ID NO:10所示的HCDR3，
    或者
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:11所示的LCDR1、SEQ ID NO:12所示的LCDR2和SEQ ID NO:13所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:14所示的HCDR1、SEQ ID NO:15所示的HCDR2和SEQ ID NO:16所示的HCDR3。
  2. 一种抗体或其抗原结合片段，其中
    所述抗体或其抗原结合片段包含SEQ ID NO:1所示的轻链可变区，和/或所述抗体或其抗原结合片段包含SEQ ID NO:2所示的重链可变区，
    或者
    所述抗体或其抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或所述抗体或其抗原结合片段包含SEQ ID NO:4所示的重链可变区。
  3. 根据权利要求1或2所述抗体或其抗原结合片段，其包含SEQ ID NO:17所示的重链恒定区，或者包含SEQ ID NO:18所示的重链恒定区。
  4. 一种嵌合抗原受体(CAR)，其包含根据权利要求1或2所述抗体或其抗原结合片段。
  5. 根据权利要求4所述的嵌合抗原受体，其依次包括所述抗体或其抗原结合片段、胞外铰链区、跨膜区和胞内信号区；优选地，其中所述胞外铰链区为CD8铰链区、所述跨膜区为CD8跨膜区、所述胞内信号区为4-1BB和CD3ζ。
  6. 一种核酸或细胞，所述核酸编码根据权利要求1-3任一项所述的抗体或其抗原结合片段或根据权利要求4-5任一项所述的嵌合抗原受体；所述细胞表达根据权利要求1-3任一项所述的抗体或其抗原结合片段或根据权利要求4-5任一项所述的嵌合抗原受体。
  7. 一种药物组合物，所述药物组合物包含根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4-5任一项所述的嵌合抗原受体，或根据权利要求6所述的核酸或细胞。
  8. 一种试剂盒，所述试剂盒包含根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的核酸。
  9. 根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4-5任一项所述的嵌合抗原受体，或根据权利要求6所述的核酸用于治疗、预防、检测或诊断疾病的用途，优选地，所述疾病是癌症。
  10. 根据权利要求9所述的用途，其中所述癌症为Claudin18.2阳性癌症；优选地，所述癌症包括胃癌、胰腺癌、食道癌、肺癌、卵巢癌、头颈癌、膀胱癌、宫颈癌、肉瘤、细胞瘤、结肠癌、肾脏癌、结肠直肠癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
PCT/CN2020/092849 2019-05-30 2020-05-28 靶向Claudin18.2的抗体或嵌合抗原受体 WO2020239005A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
BR112021023897A BR112021023897A2 (pt) 2019-05-30 2020-05-28 Anticorpo ou receptor de antígeno quimérico que visa claudina18.2
AU2020281380A AU2020281380B2 (en) 2019-05-30 2020-05-28 Antibody or chimeric antigen receptor which targets claudin 18.2
CN202080000840.1A CN114127109B (zh) 2019-05-30 2020-05-28 靶向Claudin18.2的抗体或嵌合抗原受体
EP20815562.2A EP3929214A4 (en) 2019-05-30 2020-05-28 ANTIBODY OR CHIMERIC ANTIGEN RECEPTOR TARGETING CLAUDIN 18.2
US17/601,765 US20220204609A1 (en) 2019-05-30 2020-05-28 Antibody or chimeric antigen receptor which targets claudin 18.2
KR1020217039106A KR20220006085A (ko) 2019-05-30 2020-05-28 클라우딘(Claudin) 18.2를 표적화하는 항체 또는 키메라 항원 수용체
JP2021560532A JP7266117B2 (ja) 2019-05-30 2020-05-28 クローディン１８．２を標的とする抗体又はキメラ抗原受容体
CA3136281A CA3136281C (en) 2019-05-30 2020-05-28 Antibody or chimeric antigen receptor which targets claudin 18.2

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201910459622 2019-05-30
CN201910459129 2019-05-30
CN201910459129.1 2019-05-30
CN201910459622.3 2019-05-30

Publications (1)

Publication Number Publication Date
WO2020239005A1 true WO2020239005A1 (zh) 2020-12-03

Family

ID=73553501

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/092849 WO2020239005A1 (zh) 2019-05-30 2020-05-28 靶向Claudin18.2的抗体或嵌合抗原受体

Country Status (9)

Country Link
US (1) US20220204609A1 (zh)
EP (1) EP3929214A4 (zh)
JP (1) JP7266117B2 (zh)
KR (1) KR20220006085A (zh)
CN (1) CN114127109B (zh)
AU (1) AU2020281380B2 (zh)
BR (1) BR112021023897A2 (zh)
CA (1) CA3136281C (zh)
WO (1) WO2020239005A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022135578A1 (zh) * 2020-12-25 2022-06-30 信达生物制药(苏州)有限公司 Claudin18.2嵌合抗原受体以及其用途

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3128502A1 (en) 2019-02-01 2020-08-06 Novarock Biotherapeutics, Ltd. Anti-claudin 18 antibodies and methods of use thereof
CN113416260B (zh) * 2021-04-14 2022-02-01 南京凯地医疗技术有限公司 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
CN116789822A (zh) * 2022-03-18 2023-09-22 广东东阳光药业股份有限公司 Claudin18.2人源化抗体及其应用
US20240216430A1 (en) * 2022-11-28 2024-07-04 Allogene Therapeutics, Inc. Claudin 18.2 targeting chimeric antigen receptors and binding agents and uses thereof
CN117777306A (zh) * 2023-07-04 2024-03-29 深圳豪石生物科技有限公司 一种靶向cldn18.2的增强型嵌合抗原受体及其用途

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1749029A2 (en) * 2004-05-27 2007-02-07 Crucell Holland B.V. Binding molecules capable of neutralizing rabies virus and uses thereof
WO2011031695A1 (en) * 2009-09-09 2011-03-17 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human protease-activated receptor-2
CN103571872A (zh) 2012-08-09 2014-02-12 山东国际生物科技园发展有限公司 一种能够表达人抗体的转基因动物的制备方法
WO2014127785A1 (en) * 2013-02-20 2014-08-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2014146672A1 (en) * 2013-03-18 2014-09-25 Ganymed Pharmaceuticals Ag Therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2016165762A1 (en) * 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Drug conjugates comprising antibodies against claudin 18.2
WO2018091661A1 (en) * 2016-11-18 2018-05-24 Symphogen A/S Anti-pd-1 antibodies and compositions
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006017538A2 (en) * 2004-08-03 2006-02-16 Dyax Corp. Hk1-binding proteins
WO2013167153A1 (en) * 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Antibodies useful in cancer diagnosis
WO2013174404A1 (en) * 2012-05-23 2013-11-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
AU2013347184B2 (en) * 2012-11-13 2018-06-14 Astellas Pharma Inc. Agents for treatment of claudin expressing cancer diseases
WO2014075697A1 (en) * 2012-11-13 2014-05-22 Biontech Ag Agents for treatment of claudin expressing cancer diseases
CN112979828A (zh) * 2014-07-17 2021-06-18 恺兴生命科技(上海)有限公司 靶向cld18a2的t淋巴细胞及其制备方法和应用
WO2016180468A1 (en) * 2015-05-11 2016-11-17 Biontech Cell & Gene Therapies Gmbh Claudin-18.2-specific immunoreceptors and t cell epitopes
CN106519037B (zh) * 2015-09-11 2019-07-23 科济生物医药（上海）有限公司 可活化的嵌合受体
EP3448987A4 (en) * 2016-04-29 2020-05-27 Voyager Therapeutics, Inc. COMPOSITIONS FOR THE TREATMENT OF DISEASES
EP3483182B1 (en) * 2016-07-08 2024-05-08 CRAGE medical Co., Limited Antibody for anti-claudin 18a2 and use thereof
CN109206524B (zh) * 2018-09-25 2022-04-05 山东兴瑞生物科技有限公司 抗Claudin18A2嵌合抗原受体、其修饰的T细胞及T细胞制备方法和用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1749029A2 (en) * 2004-05-27 2007-02-07 Crucell Holland B.V. Binding molecules capable of neutralizing rabies virus and uses thereof
WO2011031695A1 (en) * 2009-09-09 2011-03-17 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human protease-activated receptor-2
CN103571872A (zh) 2012-08-09 2014-02-12 山东国际生物科技园发展有限公司 一种能够表达人抗体的转基因动物的制备方法
WO2014127785A1 (en) * 2013-02-20 2014-08-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2014146672A1 (en) * 2013-03-18 2014-09-25 Ganymed Pharmaceuticals Ag Therapy involving antibodies against claudin 18.2 for treatment of cancer
WO2016165762A1 (en) * 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Drug conjugates comprising antibodies against claudin 18.2
WO2018091661A1 (en) * 2016-11-18 2018-05-24 Symphogen A/S Anti-pd-1 antibodies and compositions
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NIIMI, MOL. CELL BIOL., vol. 21, 2001, pages 7380 - 90
See also references of EP3929214A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022135578A1 (zh) * 2020-12-25 2022-06-30 信达生物制药(苏州)有限公司 Claudin18.2嵌合抗原受体以及其用途

Also Published As

Publication number Publication date
CN114127109A (zh) 2022-03-01
JP2022527129A (ja) 2022-05-30
AU2020281380A1 (en) 2021-10-07
CA3136281A1 (en) 2020-12-03
CN114127109B (zh) 2022-06-21
EP3929214A1 (en) 2021-12-29
EP3929214A4 (en) 2022-06-22
CA3136281C (en) 2024-06-25
JP7266117B2 (ja) 2023-04-27
KR20220006085A (ko) 2022-01-14
AU2020281380B2 (en) 2024-04-11
BR112021023897A2 (pt) 2022-01-25
US20220204609A1 (en) 2022-06-30

Similar Documents

Publication Publication Date Title
WO2020239005A1 (zh) 靶向Claudin18.2的抗体或嵌合抗原受体
RU2748281C2 (ru) Полностью человеческие антитела к мезотелину и иммунные эффекторные клетки, нацеленные на мезотелин
US20220017625A1 (en) Tumor-specific anti-egfr antibody and application thereof
CN108373504B (zh) Cd24特异性抗体和抗cd24-car-t细胞
WO2022012097A1 (zh) 抗人msln抗体以及靶向msln的免疫效应细胞
CN111465616B (zh) 特异性识别恶性b细胞的抗体或其抗原结合片段、包含其的嵌合抗原受体及其用途
CN113788894A (zh) 靶向人Claudin18.2蛋白的单克隆抗体及其应用
WO2022135578A1 (zh) Claudin18.2嵌合抗原受体以及其用途
WO2023060922A1 (zh) 一种特异性结合cd276的抗体或其抗原结合片段及其制备方法和应用
US20220127343A1 (en) Antigen Binding Regions Against Fibronectin Type III Domains and Methods of Using the Same
CA3201582A1 (en) Compositions of guanylyl cyclase c (gcc) antigen binding agents and methods of use thereof
WO2022002112A1 (en) Anti-gpc3 antibody, anti-gpc3 chimeric antigen receptor and gpc3/cd3 bispecific antibody
WO2024002252A1 (zh) 抗Trop2纳米抗体及其用途
WO2023072307A1 (zh) 一种靶向cd70的抗原结合片段、单链抗体和嵌合抗原受体及其应用
CN109970859B (zh) Glypican-3特异性抗体及其特异性CAR-T细胞
CN116284389A (zh) 抗afp/hla02 tcr样抗体及其用途
WO2021139755A1 (zh) 工程改造的t细胞、其制备及应用
WO2020248938A1 (zh) 抗cd25抗体及其应用
WO2024120418A1 (zh) 抗ccr8抗体及其应用
CN116457463B (zh) 抗ox40抗体、其药物组合物及应用
US20240034790A1 (en) Antibody specific for cd47 and uses thereof
WO2021121383A1 (zh) 工程改造的t细胞、其制备及应用
TW202423987A (zh) 抗ccr8抗體及其應用
EP3904383A1 (en) Anti-ox40 monoclonal antibody and application thereof
CN116589584A (zh) 靶向人Nectin4蛋白的抗体或其片段和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20815562

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3136281

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2021560532

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020281380

Country of ref document: AU

Date of ref document: 20200528

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020815562

Country of ref document: EP

Effective date: 20210924

ENP Entry into the national phase

Ref document number: 20217039106

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021023897

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112021023897

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20211126