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WO2021254481A9 - 抗Claudin18.2抗体以及其用途 - Google Patents

抗Claudin18.2抗体以及其用途 Download PDF

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WO2021254481A9
WO2021254481A9 PCT/CN2021/100870 CN2021100870W WO2021254481A9 WO 2021254481 A9 WO2021254481 A9 WO 2021254481A9 CN 2021100870 W CN2021100870 W CN 2021100870W WO 2021254481 A9 WO2021254481 A9 WO 2021254481A9
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amino acid
acid sequence
seq
antibody
identity
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PCT/CN2021/100870
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English (en)
French (fr)
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WO2021254481A1 (zh
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周帅祥
李莉
管哲
王杰
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信达生物制药(苏州)有限公司
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Priority to CN202180043345.3A priority Critical patent/CN115943162A/zh
Priority to US18/010,944 priority patent/US20230235047A1/en
Priority to CA3178855A priority patent/CA3178855A1/en
Priority to JP2022577518A priority patent/JP2023530003A/ja
Priority to KR1020227045059A priority patent/KR20230023664A/ko
Priority to EP21827108.8A priority patent/EP4169947A4/en
Priority to AU2021291259A priority patent/AU2021291259A1/en
Publication of WO2021254481A1 publication Critical patent/WO2021254481A1/zh
Publication of WO2021254481A9 publication Critical patent/WO2021254481A9/zh

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • 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

Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind to Claudin 18.2 and compositions containing said antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, and related uses. Furthermore, the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments.
  • Claudins are a family of proteins that are important building blocks for the formation of tight junctions in cells. They establish intercellular barriers that control the flow of molecules between cells, which can control the flow of molecules between cells.
  • the Claudins family proteins have a four-transmembrane domain whose N- and C-termini are both included in the cytoplasm. Different Claudin proteins are expressed in different tissues, and their functional changes are related to the formation of cancer in various tissues. For example, Claudin-1 is expressed in colon cancer and has prognostic value, Claudin-18 is expressed in gastric cancer, and Claudin-10 is expressed in gastric cancer. Hepatocellular carcinoma. Claudins, as cell membrane surface proteins, are useful targets for various therapeutic strategies.
  • Isoform 2 of Claudin-18 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker whose expression in normal tissues is strictly restricted to gastric mucosal differentiated epithelial cells, but not in the gastric stem cell region.
  • CLDN18.2 is expressed in a considerable part of primary gastric cancer, and its expression level is retained in gastric metastatic cancer tissue.
  • pancreatic cancer which is an ideal target molecule for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https://doi.org/10.1186/s13045-017-0473-4 ).
  • the present invention relates to an antibody or antigen-binding fragment thereof that binds CLDN18.2, comprising the 3 heavy chain variable region CDRs and the 3 light chain variable region CDRs described herein.
  • the CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and/or a light chain variable region as described herein.
  • the CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region and/or a light chain constant region as described herein.
  • the present invention also relates to the following embodiments:
  • CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1, IgG2, IgG3, or IgG4, preferably, an antibody or antigen in the form of IgG1 Combine fragments.
  • CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention wherein the antibody is a monoclonal antibody.
  • the CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.
  • the CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (eg, scFv) , (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
  • the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (eg, scFv) , (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
  • CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof has one or more of the following properties:
  • (ii) binds to human CLDN18.2 with an equilibrium dissociation constant (K D ) of less than about 15 nM, preferably less than or equal to about 10 nM, 9.5 nM, 9 nM, 8.5 nM, 8 nM, 7.5 nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4.5nM, 4nM, 3.5nM, 3nM;
  • K D equilibrium dissociation constant
  • ADCC activity or CDC activity such as ADCC activity or CDC activity comparable to known antibodies (eg Zmab), or higher ADCC activity or CDC activity;
  • tumor inhibition rate is greater than or equal to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.
  • An isolated nucleic acid encoding a light chain variable region or a heavy chain variable region, or a light chain or a heavy chain in the CLDN18.2 binding antibody or antigen-binding fragment thereof of the invention.
  • a vector comprising the nucleic acid of the present invention, preferably the vector is an expression vector.
  • a host cell comprising the nucleic acid or vector of the present invention
  • the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO- S cells or HEK293 cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • a method for preparing an antibody or antigen-binding fragment thereof that binds CLDN18.2 comprising culturing a host of the present invention under conditions suitable for expressing a nucleic acid encoding a CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention cell, optionally isolating said antibody or antigen-binding fragment thereof, optionally said method further comprising recovering said antibody or antigen-binding fragment thereof that binds CLDN18.2 from said host cell.
  • An immunoconjugate comprising an antibody or antigen-binding fragment thereof of the invention that binds CLDN18.2 and other substances, such as a cytotoxic agent.
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention that binds CLDN18.2 or an immunoconjugate of the invention, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, cytokines , cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
  • therapeutic agents such as chemotherapeutic agents, cytokines , cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
  • a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof of the invention that binds CLDN18.2 or an immunoconjugate of the invention, and one or more other therapeutic agents such as chemotherapeutic agents, cytokines, cytotoxic agents , other antibodies, small molecule drugs or immunomodulators.
  • a method of preventing or treating a tumor in a subject comprising administering to the subject an effective amount of a CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention, or an immunoconjugate of the present invention , a pharmaceutical composition or a pharmaceutical combination.
  • a method of inducing ADCC and/or CDC in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to CLDN18.2 of the present invention, or an immunization of the present invention Conjugates, pharmaceutical compositions or pharmaceutical combinations.
  • the tumor is a cancer
  • the cancer has elevated levels (eg nucleic acid or protein levels) of CLDN18.2.
  • the method further comprises administering to the patient one or more therapies, such as a therapeutic modality and/or other therapeutic agents, preferably, the therapeutic modality includes surgical treatment and/or radiotherapy, other therapeutic agents Selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • therapies such as a therapeutic modality and/or other therapeutic agents
  • the therapeutic modality includes surgical treatment and/or radiotherapy, other therapeutic agents Selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • the drug or drug combination can be administered to a subject in combination with one or more therapies, such as a therapeutic modality and/or other therapeutic agents, preferably the therapeutic modality includes surgical treatment and/or Radiation therapy, other therapeutic agents are selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents.
  • therapies such as a therapeutic modality and/or other therapeutic agents
  • the therapeutic modality includes surgical treatment and/or Radiation therapy
  • other therapeutic agents are selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents.
  • a method for detecting CLDN18.2 in a sample comprising
  • Figure 1 shows that anti-CLDN18.2 antibodies specifically bind to CLDN18.2 on the cell surface.
  • Figure 2 shows that anti-CLDN18.2 antibodies do not bind to cell surface CLDN18.1.
  • Figure 3 shows reporter gene-based ADCC activity of anti-CLDN18.2 antibody on CHO-hCLDN18.2.
  • Figure 4 shows reporter gene-based ADCC activity of humanized anti-CLDN18.2 antibody on CHO-hCLDN18.2.
  • Figure 5 shows the binding of anti-CLDN18.2 antibody to gastric cancer cell line NUGC-4, gastric cancer cell line KATO III-hCLDN18.2 and pancreatic cancer cell line DAN-G-hCLDN18.2.
  • Figure 6 shows the detection of CDC activity of anti-CLDN18.2 antibodies.
  • Figure 7 shows ADCC activity detection of anti-CLDN18.2 antibodies.
  • Figure 8 shows the anti-tumor effect of anti-CLDN18.2 antibody in a mouse model of pancreatic cancer.
  • Figure 9 shows the anti-tumor effect of anti-CLDN18.2 antibody in gastric cancer mouse model.
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the terms “comprising” or “comprising” are used, unless otherwise indicated, combinations of the stated elements, integers or steps are also encompassed.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • Claudin protein is the most important cytoskeletal protein that determines the structure of tight junctions between cells, is involved in adhesion junctions, and plays an important role in the metastasis and invasion of tumor cells.
  • Claudin protein is widely present in mammalian epithelial and endothelial cells, and its distribution is mainly on the side of epithelial cells and the plasma membrane of basal cells. Different Claudin proteins have their own specific expressions in different tissues.
  • the Claudin18 (CLDN18) gene is located at 3q22.3, with a molecular weight of 24kDa and 261 amino acid residues.
  • CLDN18 or Claudin18 protein contains 2 extracellular Ring and 4 transmembrane zones.
  • the two isoforms of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1), and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2), respectively.
  • Loop1 extracellular loop 1
  • CLDN18.1 and CLDN18.2 only 8 amino acids differ.
  • the interspecies sequence homology of the two isoforms of CLDN18 is also very high.
  • CLDN18.2 the extracellular loop 1 of CLDN18.2 is completely identical in sequence in different species such as human, mouse, and macaque, and the homology of CLDN18.2 protein between human and mouse reaches 84%, indicating that the sequence of CLDN18.2 protein is extremely conserved (O. Tureci. et al., Gene 481:83-92, 2011).
  • CLDN18.2, or any variants and isoforms thereof, can be isolated from cells or tissues in which they are naturally expressed, or recombinantly produced using techniques well known in the art and/or those described herein.
  • the CLDN18.2 described herein is human CLDN18.2.
  • anti-CLDN18.2 antibody refers to an antibody capable of binding with sufficient affinity (human) CLDN18.2 so that the antibody can be used as a therapeutic targeting (human) CLDN18.2.
  • the (human) CLDN18.2 antibody binds (human) CLDN18.2 with high affinity in vitro or in vivo.
  • the (human) CLDN18.2 antibody does not bind CLDN18.1.
  • the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1.
  • the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.
  • antibody fragment includes a portion of an intact antibody.
  • the antibody fragment is an antigen-binding fragment.
  • an "antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibody; single chain antibody (eg, scFv); single domain antibody such as VHH ; a diabody or fragment thereof; or a camelid antibody.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen (eg, CLDN18.2) that specifically interacts with an antibody molecule.
  • An antibody that binds the same or overlapping epitope as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of said reference antibody in a competition assay with Binding of an antigen, conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% more of the binding of the antibody to its antigen in a competition assay.
  • An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of said reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen in a competition assay.
  • Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Determination.
  • An antibody that inhibits (eg competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of said reference antibody to its antigen . Conversely, the reference antibody inhibits more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured by affinity (eg, equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or 95% or more of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • CDR regions are loops in the variable domains of antibodies that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ( "antigen contact point”).
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including Example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules
  • VH CDR1 was determined according to AbM rules; and VH CDR2 and 3 were determined according to Kabat rules.
  • the light chain variable region CDRs of the antibodies of the invention are determined according to Kabat's rules.
  • the heavy chain variable region CDRs of the antibodies of the invention are determined according to the following rules: VH CDR1 is determined according to the AbM rules; and VH CDR2 and 3 are both determined according to the Kabat rules; and the light chain variable region CDRs are determined according to the Kabat rules. .
  • the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.
  • Antibodies with different specificities have different binding sites for different antigens
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • Fc region is used herein to define the CH2 and CH3 constant regions of immunoglobulin heavy chains and includes native sequence Fc regions and variant Fc regions.
  • the Fc region can bind to different Fc receptors on the surface of immune cells, which can cause CDC ⁇ ADCC ⁇ ADCP effector functions.
  • effector functions generally require the association of an Fc region with a binding domain (eg, an antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.
  • an antibody in the IgG format refers to the IgG format to which the heavy chain constant region of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in the IgG4 format means that its heavy chain constant region is derived from IgG4, or an antibody in the IgG1 format means that its heavy chain constant region is derived from IgG1.
  • a “humanized” antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of the non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a human Either human cells are generated or derived from non-human sources using human antibody repertoires or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • binding means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm interferometry or MSD method or surface plasmon resonance (SPR).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD biofilm interferometry
  • SPR surface plasmon resonance
  • an “immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (eg, immunosuppressive agents) ).
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of diseases of the immune system.
  • small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecule is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less susceptible to eliciting an immune response than macromolecules.
  • immunomodulator refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressants.
  • an “immunosuppressant,” “immunosuppressive drug,” or “immunosuppressant” is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.
  • an effective amount refers to an amount or dose of an antibody or fragment or conjugate or composition or combination of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention .
  • a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result, at the required dose and for the required period of time.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition or combination thereof are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject .
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because a prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • the label can itself be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze a detectable chemical change of a substrate compound or composition.
  • the term is intended to encompass direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody is one that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed phase
  • isolated nucleic acid encoding an anti-CLDN18.2 antibody or fragment thereof refers to one or more nucleic acid molecules that encode an antibody heavy or light chain (or a fragment thereof, eg, a heavy chain variable region or a light chain variable region) , including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using the E. Meyers and W.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • hybridizes under stringent conditions eg, under conditions of low stringency, moderate stringency, high stringency, or very high stringency
  • stringent conditions eg, under conditions of low stringency, moderate stringency, high stringency, or very high stringency
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous methods are described in the references and either method can be used.
  • the preferred hybridization conditions referred to herein are as follows: 1) Low stringency hybridization conditions are at about 45°C in 6X sodium chloride/sodium citrate (SSC) followed by at least 50°C (for low stringency conditions, you can Increase the temperature of the wash to 55°C) twice in 0.2X SSC, 0.1% SDS; 2) Moderate stringency hybridization conditions are about 45°C in 6X SSC followed by 0.2X SSC, 0.1% SDS at 60°C 3) high stringency hybridization conditions are one or more washes in 6X SSC at about 45°C followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) extremely high Stringent hybridization conditions were one or more washes in 0.5M sodium phosphate, 7% SDS at 65°C followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the one that should be used unless otherwise specified.
  • anti-tumor effect refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to or describe a physiological disorder in mammals that is usually characterized by unregulated cell growth.
  • cancers suitable for treatment by the antibodies of the invention include gastric or pancreatic cancer, including metastatic forms of those cancers.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • pharmaceutical adjuvant refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.
  • composition refers to a composition that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients.
  • non-fixed combination means that the active ingredients (eg, (i) an anti-CLDN18.2 antibody or fragment thereof, and (ii) other therapeutic agents) are concomitantly, without specific time constraints, or in the same or different entities as separate entities.
  • the patients are administered sequentially at intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient.
  • the anti-CLDN18.2 antibody or fragment thereof and other therapeutic agent used in a pharmaceutical combination are administered at levels no greater than when they are used alone.
  • the term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity.
  • the doses and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts produces a greater effect in the treatment of a disease or condition than either component alone can achieve.
  • the ingredients may each be in separate formulations, which may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat the diseases described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule having a fixed ratio of active ingredients.
  • administration includes co-administration of the individual active ingredients in multiple or separate containers such as tablets, capsules, powders and liquids. Powders and/or liquids can be reconstituted or diluted to the desired dose prior to administration.
  • such administration also includes the sequential use of each type of therapeutic agent at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.
  • treating refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • a "subject/patient/individual sample” refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of a tissue or cell sample can be solid tissue like from a fresh, frozen and/or preserved organ or tissue sample or biopsy or biopsy; blood or any blood component; bodily fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid); ), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention binds CLDN18.2 (eg, human CLDN18.2) with high affinity.
  • the anti-CLDN18.2 antibodies of the invention specifically bind CLDN18.2 (eg, human CLDN18.2), but not CLDN18.1 (eg, human CLDN18.1).
  • the antibodies of the invention or antigen-binding fragments thereof have higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, such as the Zolbetuximab (Zmab for short) antibody.
  • the affinity of the antibody is determined by biofilm interferometry or surface plasmon resonance.
  • the anti-CLDN18.2 antibodies of the invention bind to human CLDN18.2 with an equilibrium dissociation constant (K D ) that is less than about 15 nM, preferably, less than or equal to about 10 nM, 9.5 nM , 9nM, 8.5nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4.5nM, 4nM, 3.5nM , 3nM, in some embodiments, the KD is between the above values (including endpoint).
  • K D equilibrium dissociation constant
  • an antibody or antigen-binding fragment thereof of the invention binds to CLDN18.2 on the cell surface. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention do not bind to CLDN18.1 on the cell surface. In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface. In some embodiments, the cells are CLDN18.2 expressing CHO cells or 293 cells, eg, CHO-S cells or HEK293 cells.
  • the cell is a cancer cell that expresses CLDN18.2, eg, a cell that expresses CLDN18.2 naturally or is artificially transfected to express CLDN18.2 or has an increased level of expression, eg, expresses Gastric or pancreatic cancer cell lines of CLDN18.2, such as cell lines NUGC-4, KATO III and DAN-G cell lines, such as KATO III and DAN-G cell lines overexpressing CLDN18.2.
  • CLDN18.2 gastric or pancreatic cancer cell lines of CLDN18.2, such as cell lines NUGC-4, KATO III and DAN-G cell lines, such as KATO III and DAN-G cell lines overexpressing CLDN18.2.
  • the binding is detected using flow cytometry.
  • the antibodies of the invention bind to CLDN18.2 overexpressed on CHO cells with an EC50 of less than or equal to about 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5.5 nM.
  • the antibodies of the invention are expressed in binding with an EC50 of less than or equal to about 4nM, 3.7nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM Cancer cells of CLDN18.2, such as gastric cancer cell lines or pancreatic cancer cell lines expressing CLDN18.2, such as cell lines NUGC-4, KATO III and DAN-G cell lines, such as KATO III and DAN-G cells overexpressing CLDN18.2 G cell line.
  • CLDN18.2 such as gastric cancer cell lines or pancreatic cancer cell lines expressing CLDN18.2, such as cell lines NUGC-4, KATO III and DAN-G cell lines, such as KATO III and DAN-G cells overexpressing CLDN18.2 G cell line.
  • the binding of an antibody of the invention to cells expressing CLDN18.2 has an EC50 comparable to, or less than, or less than 75% of, the EC50 of, a known antibody, such as Zmab, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%.
  • a known antibody such as Zmab, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%.
  • the antibodies or antigen-binding fragments thereof of the invention have ADCC activity or CDC activity, eg, ADCC activity or CDC activity comparable to known antibodies (eg, Zmab), or higher ADCC activity or CDC activity.
  • the antibodies or antigen-binding fragments thereof of the invention are capable of inhibiting tumor cells, eg, tumor cells expressing CLDN18.2.
  • the antibodies or antigen-binding fragments thereof of the invention can be used to treat cancer.
  • the antibody or antigen-binding fragment thereof of the present invention can effectively inhibit tumor growth, and the tumor inhibition rate is greater than or equal to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.
  • the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.
  • HCDRs complementarity determining regions
  • the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.
  • LCDRs complementarity determining regions
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region ).
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VH). In some aspects, an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.
  • CDRs complementarity determining regions
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention further comprise an antibody light chain constant region LC. In some embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention further comprise a heavy chain constant region HC and a light chain constant region LC.
  • the heavy chain variable regions of the invention are identical to one another.
  • amino acid sequence selected from the group consisting of SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80 or 85 having The amino acid sequence of 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) consists of the amino acid sequence , preferably, the amino acid changes do not occur in the CDR regions.
  • the light chain variable regions of the invention are identical to each other.
  • amino acid sequence selected from the group consisting of SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83 or 86 having
  • amino acid sequence of 1 or more preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1
  • amino acid changes preferably amino acid substitutions, more preferably amino acid conservative substitutions
  • consists of said amino acid sequence preferably, the amino acid changes do not occur in the CDR regions.
  • the three complementarity determining regions (HCDRs) from the heavy chain variable region of the invention, HCDR1, HCDR2 and HCDR3 are selected from
  • (x) comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution) in the three HCDR regions in total relative to the sequence of any one of (i)-(ix). , preferably conservative substitutions) sequences.
  • the three complementarity determining regions (LCDRs) from the light chain variable region of the invention, LCDR1, LCDR2 and LCDR3 are selected from
  • (x) at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution) in the three LCDR regions in total relative to the sequence of any one of (i)-(ix) , preferably conservative substitutions) sequences.
  • the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, 12, 23, 33, 42, 50, 58, 69, or 77, or the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 , 12, 23, 33, 42, 50, 58, 69 or 77 amino acid sequences compared to amino acid sequences with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 13, 20, 24, 34, 43, 51, 59, 70, 78, 84, 93 or 94, or HCDR2 comprises one, two or three changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 13, 20, 24, 34, 43, 51, 59, 70, 78, 84, 93 or 94 , preferably conservative substitution) amino acid sequence, wherein
  • SEQ ID NO: 93 is YIAPFXGDSRYNQKFKG, wherein X is any amino acid, preferably N or Q or a conservative amino acid substitution thereof; or
  • SEQ ID NO: 94 is VIWGDXSTNYHSVLIS wherein X is any amino acid, preferably G or V or conservative amino acid substitutions thereof.
  • the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, 14, 25, 35, 44, 52, 60, 71 or 79, or the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 , 14, 25, 35, 44, 52, 60, 71 or 79 amino acid sequences compared to amino acid sequences with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, 16, 28, 38, 62, 67 or 96, or the LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, 16, 28 , 38, 62, 67 or 11 amino acid sequences compared to amino acid sequences with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions);
  • SEQ ID NO: 11 is KSSQSLLXGGNQKNYLT, wherein X is any amino acid, preferably N or Q or conservative amino acid substitutions thereof.
  • the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, 17, 29, 63 or 81, or the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, 17, 29, 63 or 81
  • the amino acid sequence has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence.
  • the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8, 18, 30, 39, 46, 54, 64, 74 or 82, or the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 , 18, 30, 39, 46, 54, 64, 74 or 82 amino acid sequences compared to amino acid sequences with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the antibody heavy chain constant region HC of the invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.
  • the antibody light chain constant region LC of the invention is a lambda or Kappa light chain constant region, preferably a Kappa light chain constant region.
  • the antibody heavy chain constant region HC of the present invention is provided.
  • amino acid sequence comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 5
  • the amino acid sequence that is altered is or consists of said amino acid sequence.
  • the amino acid change occurs in the Fc region. In one embodiment, the amino acid change in the Fc region increases the CDC or ADCC activity of the antibody.
  • the antibody light chain constant region LC of the invention is provided.
  • (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10; or
  • amino acid sequence comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 10
  • the amino acid sequence that is altered is or consists of said amino acid sequence.
  • the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention comprise:
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the invention comprises:
  • the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 9 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 15 or 21 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 19 or 22 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 19 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 22 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 27 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 31 or 32 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 31 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 32 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 36 or 37 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 40 or 41 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 40 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • (x) a VH comprising the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 41 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 45 or 48 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 47 or 49 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 90% identity thereto or consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 47 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 48 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 49 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 53 or 56 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 55 or 57 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • (xv) a VH comprising the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 55 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 57 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 61 or 66 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 65 or 68 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 90% identity thereto or consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 65 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 72 or 73 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 75 or 76 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 75 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 90% identity thereto or consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 76 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 80 or 85 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid shown in SEQ ID NO: 83 or 86 an amino acid sequence with which the sequence is at least 90% identical or a VL consisting of said amino acid sequence;
  • VH comprising the amino acid sequence shown in SEQ ID NO: 80 or an amino acid sequence having at least 90% identity thereto or consisting of the amino acid sequence, and a VH comprising the amino acid sequence shown in SEQ ID NO: 83 having at least 90% identity therewith an amino acid sequence of 90% identity or a VL consisting of said amino acid sequence;
  • a VH comprising the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence having at least 90% identity thereto or consisting of said amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 86 having at least 90% identity therewith An amino acid sequence of 90% identity or a VL consisting of said amino acid sequence.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain. In some embodiments, the amino acid changes described herein occur in the Fc region of the heavy chain constant region of an antibody, and in preferred embodiments, the amino acid changes in the Fc region increase the ADCC and/or CDC effects of the antibody.
  • substitutions are conservative substitutions.
  • Conservative substitutions refer to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in the following table:
  • the substitutions occur in the CDR regions of the antibody.
  • the variant obtained has a modification (eg, improvement) in certain biological properties (eg, increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody.
  • exemplary substitutional variants are affinity matured antibodies.
  • the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the carbohydrate attached to it can be varied. In some applications, modifications to remove unwanted glycosylation sites may be useful, such as removal of fucose motifs to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733) . In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants, to alter one or more functional properties of the antibody, such as serum half-life, serum half-life, Complement fixation, complement dependent cytotoxicity, Fc receptor binding and/or antibody dependent cytotoxicity.
  • Fc region variants can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid changes (eg, substitutions) at one or more amino acid positions.
  • the antibodies described herein introduce changes in the Fc region to increase the ADCC activity or CDC activity of the antibody.
  • cysteine-engineered antibodies eg, "thioMAbs”
  • one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:
  • the anti-CLDN18.2 antibody of the present invention is an antibody in the form of IgGl or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4, preferably an antibody in the form of IgGl.
  • the anti-CLDN18.2 antibody is a monoclonal antibody.
  • the anti-CLDN18.2 antibody is humanized.
  • the anti-CLDN18.2 antibody is a human antibody.
  • the anti-CLDN18.2 antibody is a chimeric antibody.
  • At least a portion of the framework sequences of the anti-CLDN18.2 antibody are human consensus framework sequences.
  • the anti-CLDN18.2 antibodies of the invention also encompass antibody fragments thereof (eg, antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibodies (eg scFv), (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
  • antibody fragments thereof eg, antigen-binding fragments
  • Fab' fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibodies (eg scFv), (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
  • the present invention provides nucleic acids encoding any of the above anti-CLDN18.2 antibodies or fragments thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector such as pcDNA3.1.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells (eg, CHO-S) or 293 cells (eg, 293F or HEK293 cells)), or other cells suitable for the production of antibodies or fragments thereof.
  • the host cell is prokaryotic.
  • the invention provides nucleic acids encoding any of the anti-CLDN18.2 antibodies or fragments thereof described herein.
  • the nucleic acid may comprise nucleic acid encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or nucleic acid encoding the amino acid sequence of the light chain and/or heavy chain of the antibody.
  • the nucleic acid of the present invention comprises an encoding selected from the group consisting of SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80 or 85, or ID NO:
  • the amino acid sequence shown in any one has at least 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to
  • the present invention also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids that hybridize under stringent conditions or have one or more substitutions (e.g. conservative substitutions), deletions or insertions with a nucleic acid comprising an encoding selected from the group consisting of SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80, or 85, or SEQ ID NO: 9, 19, 22, 31, 32, 40, 41 , 47, 49, 55, 57, 65, 68, 75, 76, 83 or 86.
  • substitutions e.g. conservative substitutions
  • one or more vectors are provided comprising the nucleic acid.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • YACs yeast artificial chromosomes
  • the vector is pcDNA3.1.
  • a host cell comprising the vector.
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which the glycosylation pathway has been "humanized” result in antibodies with partially or fully human glycosylation patterns.
  • Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered for growth in suspension can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (HEK293, 293F or 293T cells) and the like.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2/0. Suitable mammalian host cell lines for the production of antibodies are known in the art.
  • the present invention provides a method of making an antibody molecule of the present invention or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises a method suitable for expressing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) encoding ) under conditions of culturing the host cell and optionally isolating the antibody or fragment thereof (eg, antigen-binding fragment).
  • the method further comprises recovering an antibody molecule of the invention or a fragment thereof (eg, an antigen-binding fragment) from the host cell.
  • a method of making an antibody molecule of the invention comprises, under conditions suitable for expression of the antibody, culturing a compound encoding the antibody (eg, any polypeptide chain and/or polypeptide chains) A nucleic acid or a host cell comprising an expression vector of the nucleic acid, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • a compound encoding the antibody eg, any polypeptide chain and/or polypeptide chains
  • a nucleic acid or a host cell comprising an expression vector of the nucleic acid, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding the antibody eg, the antibodies described above, eg, any polypeptide chain and/or polypeptide chains
  • nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains).
  • Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the anti-CLDN18.2 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the antibodies of the invention are tested for their antigen-binding activity, eg, by known methods such as ELISA, Western blotting, and the like.
  • Binding to CLDN18.2 can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, measured using radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR) or flow cytometry.
  • RIA radioimmunoassay
  • MSD assay surface plasmon resonance
  • competition assays can be used to identify antibodies that compete with any of the anti-CLDN18.2 antibodies disclosed herein for binding to CLDN18.2.
  • competing antibodies bind to the same or overlapping epitope (eg, a linear or conformational epitope) as bound by any of the anti-CLDN18.2 antibodies disclosed herein.
  • the present invention also provides assays for identifying biologically active anti-CLDN18.2 antibodies.
  • Biological activities can include, for example, binding to CLDN18.2 (eg, binding to human CLDN18.2), binding to cells expressing CLDN18.2, CDC or ADCC activity on cells, inhibition of tumor cells, and the like.
  • Antibodies having such biological activities in vivo and/or in vitro are also provided.
  • antibodies of the invention are tested for such biological activity.
  • Cells for use in any of the above in vitro assays include cell lines that naturally express CLDN18.2 or are engineered to express or overexpress CLDN18.2. Such cells also include cell lines transfected with DNA encoding CLDN18.2 that express CLDN18.2 and that do not normally express CLDN18.2. In some embodiments, such cells are gastric cancer cells or pancreatic cancer cells. In some embodiments, the cells are CHO cells expressing CLDN18.2. In some embodiments, the cells are cell lines NUGC-4, KATO III and DAN-G cell lines, eg, KATO III and DAN-G cell lines overexpressing CLDN18.2.
  • any of the above assays can be performed using a combination of anti-CLDN18.2 antibodies and other active agents.
  • the present invention provides immunoconjugates comprising any of the anti-CLDN18.2 antibodies provided herein and other agents, such as therapeutic agents, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small Molecular drugs or immunomodulators (eg, anti-inflammatory or immunosuppressive agents).
  • the other agent is, for example, a cytotoxic agent, which includes any agent that is detrimental to cells.
  • the immunoconjugate is used to prevent or treat cancer.
  • the present invention provides compositions comprising any of the anti-CLDN18.2 antibodies or fragments thereof described herein (preferably antigen-binding fragments thereof) or immunoconjugates thereof, preferably the compositions are pharmaceutical compositions.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises a combination of an anti-CLDN18.2 antibody or fragment thereof or immunoconjugate thereof of the invention, and one or more other therapeutic agents.
  • the present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-CLDN18.2 antibodies or immunoconjugates thereof, or compositions comprising polynucleotides encoding anti-CLDN18.2 antibodies (including pharmaceutical compositions or pharmaceutical preparations).
  • the composition comprises one or more antibodies or fragments thereof that bind CLDN18.2, or one or more polynucleotides encoding one or more antibodies or fragments thereof that anti-CLDN18.2 .
  • These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the present invention may be in a variety of forms.
  • forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes, and suppositories.
  • liposomes e.g., liposomes, and suppositories.
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • compositions comprising the antibodies described herein can be prepared by admixing an antibody of the invention of the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.
  • compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • active ingredients such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs or immunomodulatory agents, and the like.
  • the active ingredients are suitably combined in amounts effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.
  • the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-CLDN18.2 antibody or fragment thereof (preferred antigen-binding fragment) of the invention, or an immunoconjugate thereof, and one or Various other therapeutic agents (eg, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents, etc.).
  • chemotherapeutic agents e.g, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents, etc.
  • Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit. Dosage units can thus be provided according to the dosing regimen or the interval between drug administrations.
  • kit of parts of the present invention contains in the same package:
  • One aspect of the present invention provides a method of preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of an anti-CLDN18.2 antibody or fragment thereof (preferably antigen-binding fragment) of the present invention , immunoconjugate, pharmaceutical composition, pharmaceutical combination or kit.
  • a tumor eg, cancer
  • an anti-CLDN18.2 antibody or fragment thereof preferably antigen-binding fragment
  • the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) CLDN18.2.
  • the tumor eg, cancer
  • the tumor includes solid and hematological tumors and metastatic lesions.
  • examples of solid tumors include malignant tumors. Cancer can be in early, intermediate or advanced stages or metastatic.
  • the tumor therapy will benefit from inhibition of CLDN18.2 at the nucleic acid or protein level.
  • the tumor treatment benefits from ADCC or CDC effects elicited by the antibodies of the invention.
  • the anti-CLDN18.2 antibodies of the invention are capable of inhibiting the proliferation of tumor cells, such as tumor cells expressing CLDN18.2, such as gastric cancer cells or pancreatic cancer cells.
  • the anti-CLDN18.2 antibodies of the invention have strong ADCC or CDC activity.
  • the tumor is tumor immune escape.
  • the tumor is cancer, such as gastric or pancreatic cancer.
  • the subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual having or at risk of having a disease described herein).
  • the subject has or is at risk of having a disease described herein (e.g., cancer).
  • the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the subject has previously received or is receiving immunotherapy.
  • the invention provides the use of an antibody molecule or fragment thereof or immunoconjugate or pharmaceutical composition or pharmaceutical combination or kit in the manufacture or manufacture of a medicament for the use described herein, for example with For the prevention or treatment of the related diseases or conditions mentioned herein.
  • the antibody molecule or fragment thereof or immunoconjugate or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of the disorder and/or symptoms associated with the disorder.
  • the antibody molecules or fragments thereof or immunoconjugates or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, eg, therapeutic modalities and/or other therapeutic agents, for use as described herein For example, for the prevention and/or treatment of the related diseases or conditions mentioned herein.
  • therapies eg, therapeutic modalities and/or other therapeutic agents
  • the treatment modality includes surgery; radiation therapy, localized or focused radiation, and the like.
  • the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulatory agents.
  • immunomodulatory agents include immunosuppressive or anti-inflammatory agents.
  • the antibody combinations described herein can be administered separately, eg, as separate antibodies.
  • Such combination therapy encompasses combined administration (eg, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the additional therapeutic agents and/or agents may be administered Administration of the antibodies of the invention occurs before, simultaneously, and/or after.
  • the route of administration of the pharmaceutical composition is according to known methods, eg, orally, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Routes; via sustained release systems or via implanted devices.
  • the compositions can be administered by bolus injection or by continuous infusion or by implanted devices.
  • composition may also be administered topically via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated.
  • an implanted device when used, the device can be implanted into any suitable tissue or organ, and the desired molecule can be delivered via diffusion, timed-release bolus, or continuous administration.
  • any of the anti-CLDN18.2 antibodies or fragments thereof (preferably antigen-binding fragments) provided herein can be used to detect the presence of CLDN18.2 in a biological sample.
  • the term "detection" includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg RT-PCR).
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion associated lesion.
  • an anti-CLDN18.2 antibody or fragment thereof for use in a diagnostic or detection method is provided.
  • a method of detecting the presence of CLDN18.2 in a biological sample comprises detecting the presence of CLDN18.2 protein in a biological sample.
  • the CLDN18.2 is human CLDN18.2.
  • the method comprises contacting a biological sample with an anti-CLDN18.2 antibody or fragment thereof as described herein under conditions that permit binding of the anti-CLDN18.2 antibody or fragment thereof to CLDN18.2, and detecting Whether complexes are formed between anti-CLDN18.2 antibodies or fragments thereof and CLDN18.2.
  • Formation of the complex indicates the presence of CLDN18.2.
  • the method can be in vitro or in vivo.
  • an anti-CLDN18.2 antibody or fragment thereof is used to select subjects suitable for treatment with an anti-CLDN18.2 antibody or fragment thereof, eg, wherein CLDN18.2 is used to select said subjects Biomarkers.
  • the antibodies of the invention can be used to diagnose tumors, eg, cancers, eg, to evaluate (eg, monitor) treatment or progression, diagnosis and/or staging of a disease described herein in an individual.
  • labeled anti-CLDN18.2 antibodies or fragments thereof are provided.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.
  • the sample is obtained prior to treatment with an anti-CLDN18.2 antibody or fragment thereof. In some embodiments, the sample is obtained prior to other therapy. In some embodiments, the sample is obtained during or after treatment with the other therapy.
  • the sample is formalin-fixed, paraffin-coated (FFPE).
  • FFPE formalin-fixed, paraffin-coated
  • the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, a specimen from a surgical resection), or a fine needle aspirate.
  • CLDN18.2 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment following a treatment interval.
  • a method of treating a disease of the present invention comprising: testing a subject (eg, a sample) (eg, a subject sample) for the presence of CLDN18.2, thereby determining CLDN18.2. 2 values, compare the CLDN18.2 value to a control value, and if the CLDN18.2 value is greater than the control value, administer to the subject a therapeutically effective amount of an anti-CLDN18.2 antibody, optionally in combination with one or more other therapies or a fragment thereof (eg, an anti-CLDN18.2 antibody or fragment thereof described herein), thereby treating the disease.
  • the kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, the same below).
  • CLDN18.2 human Claudin18.2
  • the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pCHO1.0, and the constructed plasmids were transferred into CHO-S cells ( In Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527), the transfected cells were subjected to two rounds of pressure screening to obtain cell pools expressing CLDN18.2 respectively.
  • the kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.1 (abbreviated as CLDN18.1, the same below).
  • CLDN18.1 human Claudin18.1
  • the full-length gene of human CLDN18.1 (UniProt ID: P56856-1) was constructed into the vector pCHO1.0 (Invitrogen, A1369601), and the constructed plasmid was transferred into CHO-S cells (Invitrogen, A1369601) by chemical transfection method. A1369601), the transfected cells were subjected to two rounds of pressurized screening to obtain cell pools expressing CLDN18.1 respectively.
  • the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255), the GFP sequence was replaced, and the lentiviral packaging vector psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) was co-transfected into HEK293T (ATCC, CRL-3216) cells for virus packaging. The culture supernatants after culturing for 48 hours and 72 hours were collected, and the lentivirus was concentrated using PEG8000.
  • Pancreatic cancer DAN-G cells and gastric cancer KATO III cells were transfected with the concentrated virus, and then the cells expressing CLDN18.2 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter) to obtain stable transfection of CLDN18 .2 tumor cell lines DAN-G-CLDN18.2 and KATO III-CLDN18.2.
  • the present invention adopts hybridoma technology, immunizes mice with the cells (CHO-huClaudin18.2) obtained in Example 1 (see Table 1 for the method and process), and then obtains the spleen cells of the mice and fuses them with myeloma cells to obtain cells capable of secreting CLDN18 .2 Antibody hybridoma cells.
  • mice were sacrificed, the abdominal cavity was opened aseptically to remove the spleen, and the surrounding attached connective tissue was removed. Squeeze with a needle to fully release the spleen cells to prepare a spleen cell suspension.
  • the cell suspension was filtered through a 70 ⁇ M cell mesh, washed with RPMI-1640 medium, and centrifuged at 1200 rpm for 6 min. The supernatant was removed, the red blood cells were lysed after resuspending the cells in RBC lysis buffer (GIBCO), and after centrifuging to remove the supernatant, the cells were resuspended in RPMI-1640 medium and counted.
  • RBC lysis buffer GBC lysis buffer
  • SP2/0 and spleen cells after lysing red blood cells were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removal of the supernatant, the mixed cells were resuspended in fusion buffer. Add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, add an appropriate volume of fusion buffer to resuspend the cells, and adjust the mixed cell density to 1 ⁇ 10 7 cells/ml. After fusion by an electrofusion apparatus, the cells were placed in an electrofusion dish for 5 min at room temperature. Transfer cells into centrifuge tubes and dilute cells to 1-2 x 104 cells/ml.
  • hybridoma cells that specifically expressed anti-CLDN18.2 antibody were screened by flow cytometry (FACS), and the secreted antibody did not bind to CLDN18.1.
  • the cells to be detected (HEK293-hCLDN18.2) obtained in Example 1 were counted, diluted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate. 500g for 5min, centrifuge to remove cell culture medium. The culture supernatant of the hybridoma 96-well plate was added to the U-shaped plate and the cells were resuspended, adding 100 ⁇ l to each well, and standing on ice for 30 min. Remove the supernatant at 500 g for 5 min, and wash the cells once with PBS.
  • the positive clones were re-screened with CHO-hCLDN18.1 by the same method as above, and a total of 9 hybridomas that bound to human CLDN18.2 but did not bind to human CLDN18.1 were obtained.
  • 8 strains were derived from Bal b/c mice, and 1 strain was derived from H2L2 fully human transgenic mice, as shown in Table 2.
  • mice 3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, 51H10 H2L2 fully human transgenic mice HB37A6
  • RNA extraction kit Biomiga, R6311-02
  • total RNA was reverse transcribed with PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6110A) to obtain cDNA, and degenerate primers were used respectively.
  • the heavy chain and light chain variable region sequences of the antibody were amplified, inserted into the pMD20-T vector (Takara, 6028), and cloned and sequenced after ligation and transformation.
  • the 9 antibodies (3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, 51H10 and HB37A6) screened in Example 2 and the control antibody zolbetuximab (Zmab for short, sequence derived from INN117) were in the form of full-length monoclonal antibodies.
  • HEK293 cells Invitrogen, A14527.
  • 9 paired heavy chain variable regions and light chain variable regions were placed in the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively.
  • the N-terminus of was constructed into the pcDNA3.1 expression vector with the N-terminal signal peptide to obtain the light and heavy chain expression vector.
  • the obtained light and heavy chain expression vectors were co-transfected with PEI (Polysciences Inc, 23966) to transiently transfect HEK293 cells, and the culture supernatant was collected after 7 days of culture.
  • the supernatant was purified by Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), then ultrafiltered and exchanged into PBS (Gibco, 70011-044), the concentration was detected by A280 method and the purity was determined by SEC-HPLC method , to obtain an antibody solution with a purity greater than 95%.
  • the equilibrium dissociation constant (K D ) of the antibody of the present invention binding to human CLDN18.2 was determined by Biolayer Interferometry (BLI). Affinity measurement by BLI method was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8). The AHC (18-5060, Fortebio) sensor was soaked in SD buffer (1x PBS, BSA 0.1%, Tween-200.05%).
  • the above 9 antibodies were determined by flow cytometry (FACS) respectively with the CHO-S cell lines stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 (that is, prepared as described in the example). Binding of CHO-hCLDN18.2 and CHO-hCLDN18.1).
  • the experimental method is with reference to Example 2, and GraphPad Prism software is used to analyze the experimental data to obtain Figure 1 and Figure 2. As shown in Figures 1 and 2, all the 9 antibodies specifically bound to human CLDN18.2, but not to human CLDN18.1.
  • the ADCC activity of the above 9 antibodies was detected using ADCC effector cells (Promega, G7102) containing a luciferase reporter gene.
  • the target cells CHO-hCLDN18.2 and ADCC effector cells in the logarithmic growth phase were taken and counted respectively.
  • a total of 50uL was added to a 96-well white bottom plate at a ratio of 1:6, with a total of 1.75 ⁇ 10 5 cells in each well.
  • the above-mentioned antibodies were added to the wells and incubated at 37°C, 5% CO2 for 8-10 hours.
  • the respective concentrations of the antibodies were as follows: the starting point of the concentration was 3nM, and then it was diluted 3 times.
  • Example 3 Eight murine antibodies (3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, and 51H10) in Example 3 were selected for humanization.
  • the CDR region of the antibody was determined, the heavy chain CDR1 was named according to Abm, and the remaining CDRs were named according to Kabat.
  • the human germline gene sequence with the highest sequence similarity to the murine antibody variable region sequence was selected as the framework region template of the humanized antibody, and then the CDR regions were transplanted into each template.
  • the three-dimensional structure of each antibody was constructed by Discovery Studio software, and the amino acids in the framework region that affected the CDR region were backmutated to obtain the humanized antibody sequence. See Table A for the sequences of the humanized antibodies Hz3G3, Hz14A5, Hz25C7, Hz38F8, Hz69H9, Hz46F6, Hz59C1 and Hz51H10.
  • humanized recombinant antibodies were prepared and purified according to the method described in Example 4 to obtain antibody solutions in PBS with greater than 95% purity.
  • Affinity and kinetic constants were obtained by detecting the binding and dissociation between the chip surface antigen and the antibody in the mobile phase by Biacore (GE Healthcare, T200) according to the manufacturer's instructions.
  • the serially diluted antibody (0-100nM) was flowed over the chip surface sequentially from low concentration to high concentration, the binding time was 180s, and the dissociation time was 600s.
  • the chip was finally regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54).
  • Data Results Kinetic analysis was performed using Biacore T200 analysis software in a 1:1 binding model. As shown in Table 4: Except for Hz25C7 and Hz3G3, the affinity of other antibodies was better than that of the control antibody Zmab.
  • Example 10 Detection of ADCC activity of humanized antibody based on reporter gene
  • Hz14A5, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10 were selected for reporter gene-based ADCC activity assays.
  • the humanized antibodies Hz14A5, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10 all showed ADCC activity comparable to the control antibody.
  • one humanized antibody Hz69H9 and one fully human antibody HB37A6 were selected for further detection.
  • the binding of the two antibodies to gastric cancer cell line NUGC-4, gastric cancer cell line KATO III-hCLDN18.2 and pancreatic cancer cell line DAN-G-hCLDN18.2 was determined by FACS, respectively.
  • Figure 5 shows that both the humanized antibody Hz69H9 and the fully humanized antibody HB37A6 have better tumor cell specific binding, which is better than that of the control antibody Zmab.
  • CDC activity of antibody molecules was performed using the KATO III-CLDN18.2 cell line. Add 1 ⁇ 10 5 target cells KATO III-hCLDN18.2 to the 96-well plate, then add antibody molecules of appropriate concentration, and incubate at 37°C for 30 minutes, add the prepared human serum complement (Sigma, S1764), the antibody The respective concentrations are as follows: serial dilution concentrations, the initial concentration is 150ug/ml, 3-fold dilution, a total of 9 concentration points. The reaction system was placed in a 37°C incubator for 3 hours, and then the data was read using a microplate reader (ThermoFisher, MULTISKAN FC) using the CCK-8 kit (Tongren Chemical, CK04). The results are shown in Figure 6, Hz69H9 And HB37A6 has CDC activity comparable to the control antibody Zmab.
  • PBMC peripheral blood mononuclear cells
  • FBS fetal bovine serum
  • NUGC-4 or DAN-G tumor target cells DAN-G-CLDN18.2 overexpressing Claudin18.2 were labeled with Far-Red (Invitrogen) for 10 min, washed twice and resuspended in complete medium RPMI-1640 (Hyclone, In SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), the cell concentration was adjusted to 2 ⁇ 10 5 cells/ml.
  • PBMC peripheral blood mononal cells
  • anti-claudin18.2 monoclonal antibodies Hz69H9 and HB37A6 and control antibody Zmab the initial concentration of each antibody was 30nM, 3-fold serial dilution, a total of 12 concentration points
  • 50ul of tumor target cells 1 x 104 were added to 50ul of PBMC effector cells.
  • Anti-Claudin18.2 monoclonal antibody-dependent cell-mediated cytotoxicity of NUGC-4 tumors is shown in Figure 7A and B. Both Hz69H9 and HB37A6 and the control antibody Zmab can dose-dependently mediate NK cells against NUGC-4 cells. Cytotoxic killing, the maximum killing of tumor cells by Hz69H9 and HB37A6 was stronger than that of the control antibody Zmab. It indicated that Hz69H9 and HB37A6 have stronger cell-mediated tumor cytotoxicity.
  • Anti-Claudin18.2 monoclonal antibody-dependent cell-mediated DAN-G tumor cytotoxicity results are shown in Figure 7C and D.
  • Both Hz69H9 and HB37A6 and the control antibody Zmab can dose-dependently mediate the cytotoxicity of NK cells to tumor cells kill.
  • the maximal killing mediated by HB37A6 and Hz69H9 in PBMCs of two different donors was higher than that of the control antibody Zmab.
  • HZ69H9 and HB37A6 antibodies were selected to test their resistance to NOD-SCID mice with human pancreatic cancer (female NOD-SCID mice (15-18g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., number 60) tumor effect.
  • the human pancreatic cancer cell DAN-G-CLDN18.2 constructed in Example 1 was routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and DAN-G-CLDN18.2 was dispersed in PBS (1 ⁇ ) to obtain a suspension with a cell density of 12 ⁇ 10 ⁇ 5 cells/ml. The cell suspension was mixed with matrigel gel 1:1 to prepare a cell suspension with a cell concentration of 6 ⁇ 10 ⁇ 5 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.
  • mice with tumor volume ranging from 43.36 mm 3 to 89.47 mm 3 were selected and grouped according to the size of the tumor volume (8 mice in each group).
  • hIgG (Equitech-Bio, batch number 160308-02), Hz69H9 and HB37A6 and control antibody Zmab were administered to each mouse at a dose of 10 mg/kg each time, on the 5th, 9th, 12th, and 16th days after inoculation, respectively.
  • HZ69H9 and HB37A6 antibodies were selected to test their antitumor effects in NOG mice with human gastric cancer (female NOG mice (15-18g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., number 100).
  • the PBMC cells (Allcells) were recovered, centrifuged to collect the cells, and the PBMC cells were dispersed with PBS (1 ⁇ ), the cell density was 2.5 ⁇ 10 ⁇ 6 cells/ml cell suspension, and 0.2ml of the cell suspension was taken on the 0th day for NOG Intravenous injection of mouse eyes to establish NOG humanized mouse model.
  • NUGC-4 cells were routinely recovered and subcultured for subsequent in vivo experiments. Cells were collected by centrifugation, NUGC-4 cells were dispersed with PBS (1 ⁇ ), the cell density was 12 ⁇ 10 ⁇ 6 cells/ml, and the cells were mixed with matrigel gel 1:1 to prepare a cell suspension with a cell concentration of 6 ⁇ 10 ⁇ 6 cells/ml . On day 5, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.
  • mice in each group were randomly divided into groups, and each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), Hz69H9 and HB37A6 and control antibody Zmab at a dose of 10 mg/kg each time , respectively, on the 1st, 5th, 8th, and 12th days after inoculation, and the tumor volume and body weight of mice were monitored 2-3 times a week.
  • TGI% The relative tumor inhibition rate (TGI%) was calculated on the 26th day of inoculation with the following formula:
  • TGI% 100%*(tumor volume of control group-tumor volume of treatment group)/(tumor volume of control group-tumor volume of control group before administration).
  • the TGI of Hz69H9 and HB37A were 70% and 28%, respectively, and the TGI of Zmab was 24%;
  • the TGI of Hz69H9 and HB37A were 46% and 31%, respectively, and the TGI of Zmab was 0%.

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Abstract

提供特异性结合Claudin18.2的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外，提供编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，提供这些抗体和抗体片段的治疗和诊断用途。

Description

抗Claudin18.2抗体以及其用途
本申请是以CN申请号为202010570517.X，申请日为2020年6月19日的申请为基础，并主张其优先权，该CN申请的公开内容在此次作为整体引入本申请中。
本发明涉及特异性结合Claudin18.2的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外，本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，本发明涉及这些抗体和抗体片段的治疗和诊断用途。
发明背景
Claudins是一个蛋白质家族，是构成细胞紧密连接的重要组成部分。它们建立了控制细胞间分子流动的细胞间屏障，可控制细胞之间分子的流动。Claudins家族蛋白具有四次跨膜结构域，其N端和C端均包括在细胞质中。不同的Claudins蛋白在不同的组织上表达，其功能的改变与各个组织的癌症形成有关，比如Claudin-1表达在结肠癌中并且具有预后价值，Claudin-18表达在胃癌中，Claudin-10表达在肝细胞癌肿。Claudins作为细胞膜表面蛋白，是各种治疗策略的有用靶标。
Claudin-18的同种型2(Claudin 18.2或CLDN18.2)是高度选择性的细胞谱系标记物，其在正常组织中的表达严格限于胃粘膜分化的上皮细胞，但不表达于胃干细胞区。CLDN18.2在相当一部分原发性胃癌中表达，并且在胃转移的癌组织中保留了其表达水平。除胃癌以外，在胰腺癌中也发现有CLDN18.2的表达，是治疗这些癌症的理想的靶标分子(Singh，P.，Toom，S.& Huang，Y.Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer.J Hematol Oncol 10，105(2017). https：//doi.org/10.1186/s13045-017-0473-4)。
由于存在大量的未被满足的临床肿瘤治疗的需求，开发靶向Claudin18.2的药物十分必要。虽然，现有技术已经开发了一些针对Claudin18.2的单克隆抗体，但是，这些抗体大多为嵌合抗体，具有潜在较高的免疫原性风险，且亲和力较低，特异性差，ADCC活性一般。
因此，还需要开发新的针对Claudin18.2的抗体，其具有较低的免疫原性，与Claudin18.2的结合亲和力更强，特异性好，ADCC和CDC活性强且具有更好的抗肿瘤活性。
发明概述
在一些方面，本发明涉及结合CLDN18.2的抗体或其抗原结合片段，其包含本发明所述的3个重链可变区CDR和3个轻链可变区CDR。
在一些方面，本发明的结合CLDN18.2的抗体或其抗原结合片段包含本发明所述的重链可变区和/或轻链可变区。
在一些方面，本发明的结合CLDN18.2的抗体或其抗原结合片段还包含本发明所述的重链恒定区和/或轻链恒定区。
在一些方面，本发明还涉及以下实施方案：
1、本发明的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是IgG1形式或IgG2形式或IgG3形式或IgG4形式的抗体或抗原结合片段，优选的，是IgG1形式的抗体或抗原结合片段。
2、本发明的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
3、本发明的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是人源化的抗体或人抗体或嵌合抗体。
4、本发明的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’) 2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体。
5、本发明的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段具有以下一种或多种性质：
(i)以高亲和力结合CLDN18.2(例如人CLDN18.2)，而不结合CLDN18.1(例如人CLDN18.1)；
(ii)以以下平衡解离常数(K D)与人CLDN18.2结合，所述K D小于大约15nM，优选地，小于或等于大约10nM、9.5nM、9nM、8.5nM、8nM、7.5nM、7nM、6.5nM、6nM、5.5nM、5nM、4.5nM、4nM、3.5nM、3nM；
(iii)与细胞表面的CLDN18.2结合，不与细胞表面的CLDN18.1结合；
(iv)具有ADCC活性或CDC活性，例如与已知抗体(例如Zmab)相当的ADCC活性或CDC活性，或者更高的ADCC活性或CDC活性；
(v)抑制肿瘤细胞，例如表达CLDN18.2的肿瘤细胞；
(vi)能够有效抑制肿瘤生长，肿瘤抑制率大于或等于约25％、30％、35％、40％、45％、50％、55％、60％、65％或70％。
6、分离的核酸，其编码本发明的结合CLDN18.2的抗体或其抗原结合片段中的轻链可变区或重链可变区，或轻链或重链。
7、包含本发明的核酸的载体，优选地所述载体是表达载体。
8、包含本发明的核酸或载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞(例如293细胞或CHO细胞，例如CHO-S细胞或HEK293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
9、制备结合CLDN18.2的抗体或其抗原结合片段的方法，所述方法包括在适于表达编码本发明的结合CLDN18.2的抗体或其抗原结合片段的核酸的条件下培养本发明的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述结合CLDN18.2的抗体或其抗原结合片段。
10、免疫缀合物，其包含本发明的结合CLDN18.2的抗体或其抗原结合片段和其它物质，例如细胞毒性剂。
11、药物组合物，其包含本发明的结合CLDN18.2的抗体或其抗原结合片段或本发明的免疫缀合物，以及任选地一种或多种其它治疗剂，例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂，以及任选地药用辅料。
12、药物组合，其包含本发明的结合CLDN18.2的抗体或其抗原结合片段或本发明的免疫缀合物，以及一种或多种其它治疗剂，例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
13、预防或治疗受试者中肿瘤的方法，所述方法包括向所述受试者施用有效量的本发明的结合CLDN18.2的抗体或其抗原结合片段、或本发明的免疫缀合物、药物组合物或药物组合。
14、在受试者中引发ADCC和/或CDC的方法，所述方法包括向所述受试者施用有效量的本发明的结合CLDN18.2的抗体或其抗原结合片段、或本发明的 免疫缀合物、药物组合物或药物组合。
15、本发明的抗体或免疫缀合物在制备用于治疗或预防肿瘤和/或引发ADCC和/或CDC的药物或药物组合中的用途。
16、本发明的方法或用途，其中所述肿瘤为癌症，优选的，所述癌症具有升高水平的(例如核酸或蛋白质水平的)CLDN18.2。
17、本发明的方法，其中所述方法还包括向患者施用一种或多种疗法，例如治疗方式和/或其它治疗剂，优选地，治疗方式包括手术治疗和/或放射疗法，其它治疗剂选自化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
18、本发明的用途，其中所述药物或药物组合能与一种或多种疗法，例如治疗方式和/或其它治疗剂联合施用给受试者，优选地，治疗方式包括手术治疗和/或放射疗法，其它治疗剂选自化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
19、检测样品中CLDN18.2的方法，所述方法包括
(a)将样品与本发明的抗体或其抗原结合片段接触；以及
(b)检测抗体或其抗原结合片段与CLDN18.2间的复合物的形成；任选地，抗体是被可检测地标记的。
附图说明：
图1显示了抗CLDN18.2抗体特异性结合细胞表面的CLDN18.2。
图2显示了抗CLDN18.2抗体不结合细胞表面的CLDN18.1。
图3显示了抗CLDN18.2抗体对CHO-hCLDN18.2的基于报告基因的ADCC活性。
图4显示了人源化抗CLDN18.2抗体对CHO-hCLDN18.2的基于报告基因的ADCC活性。
图5显示了抗CLDN18.2抗体与胃癌细胞系NUGC-4、胃癌细胞系KATO III-hCLDN18.2和胰腺癌细胞系DAN-G-hCLDN18.2的结合。
图6显示了抗CLDN18.2抗体的CDC活性检测。
图7显示了抗CLDN18.2抗体的ADCC活性检测。
图8显示了抗CLDN18.2抗体在胰腺癌小鼠模型的抗肿瘤效果。
图9显示了抗CLDN18.2抗体在胃癌小鼠模型的抗肿瘤效果。
发明详述
I.定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
本文所用的术语“CLAUDIN”或“CLDN”是决定细胞间紧密连接结构的最重要的骨架蛋白，其参与粘附连接，并且在肿瘤细胞的转移和侵袭中起到重要作用。Claudin蛋白广泛存在于哺乳动物上皮和内皮细胞中，其分布主要是上皮细胞侧面以及基底细胞质膜上。不同的Claudin蛋白在不同组织中具有各自特异性表达，其中Claudin18(CLDN18)基因定位于3q22.3，分子量24kDa，包含261个氨基酸残基，属Claudins超家族成员，其蛋白结构包含2个细胞外环和4个穿膜区。人CLDN18或Claudin18蛋白的两个亚型分别是Claudin18.1或CLDN18.1(UniProt ID：P56856-1)，和Claudin18.2或CLDN18.2(UniProt ID：P56856-2)，在二者蛋白的一级结构序列中，仅N端信号肽至细胞外环1(Loop1)结构的某些位置的氨基酸残基上有所区别，尤其是在细胞外环1上，CLDN18.1和CLDN18.2仅8个氨基酸不同。CLDN18的两种亚型蛋白的种属间序列同源性也非常高。其中CLDN18.2的细胞外环1在人、小鼠、猕猴等不同物种上序列完全一致，而人与小鼠的CLDN18.2蛋白同源性达到84％，表明CLDN18.2蛋白序列极端保守性(O.Tureci.等人.，Gene 481：83-92，2011)。CLDN18.2或其任何变体和同种型可以从天然表达它们的细胞或组织中分离，或者使用本领域熟知的技术和/或本文所述的那些技术重组产生。在一个实施方案中，本文所述的CLDN18.2是人CLDN18.2。
本文所用的术语“抗CLDN18.2抗体”、“抗CLDN18.2”、“CLDN18.2抗体”或“结合CLDN18.2的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合(人)CLDN18.2以致所述抗体可以用作靶向(人)CLDN18.2的治疗剂。在一个实施方案中，所述(人)CLDN18.2抗体在体外或体内以高亲和力结合(人)CLDN18.2。在一个实施方案中，所述(人)CLDN18.2抗体不结合CLDN18.1。在一个实施方案中，所述(人)CLDN18.2抗体与表达CLDN18.2的细胞结合而不结合表达CLDN18.1的细胞。在一些实施方案中，所述结合例如通过放射性免疫测定(RIA)、生物膜薄层干涉测定法(BLI)、MSD测定法或表面等离子体共振法(SPR)或流式细胞术测量的。
术语“抗体片段”包括完整抗体的一部分。在优选的实施方案中，抗体片段 为抗原结合片段。
“抗原结合片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；dAb(domain antibody)；线性抗体；单链抗体(例如scFv)；单结构域抗体例如VHH；双价抗体或其片段；或骆驼科抗体。
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。如本文所用，术语“表位”指抗原(例如，CLDN18.2)中与抗体分子特异性相互作用的部分。
与参照抗体“结合相同或重叠表位的抗体”是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合，反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。
与参照抗体竞争结合其抗原的抗体是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。众多类型的竞争性结合测定可用于确定一种抗体是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定。
抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat (Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
Figure PCTCN2021100870-appb-000001
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的重链可变区CDR按照如下规则确定
VH CDR1按照AbM规则确定；并且VH CDR2和3均按照Kabat规则确定。
在一个实施方案中，本发明抗体的轻链可变区CDR按照Kabat规则确定。
在一个实施方案中，本发明抗体的重链可变区CDR按照如下规则确定：VH CDR1按照AbM规则确定；并且VH CDR2和3均按照Kabat规则确定；且轻链可变区CDR按照Kabat规则确定。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。 因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR(在同一指派系统下)。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat，Chothia，AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“Fc区”在本文中用于定义免疫球蛋白重链的CH2和CH3的恒定区域，该术语包括天然序列Fc区和变体Fc区。Fc区域能够和免疫细胞表面的不同的Fc受体结合，从而能够引起CDC\ADCC\ADCP效应功能。此类效应器功能一般要求Fc区与结合结构域(例如抗体可变域)联合，而且可以使用多种测定法来评估，例如本文所公开的那些。
“IgG形式的抗体”是指抗体的重链恒定区所属的IgG形式。所有同一型的抗体的重链恒定区都是相同的，不同型的抗体之间的重链恒定区不同。例如，IgG4形式的抗体是指其重链恒定区来自IgG4，或者IgG1形式的抗体是指其重链恒定区来自IgG1。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
“人抗体”或“全人抗体”或“全人源抗体”可以互换使用，其指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗原结合位点与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。
“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记) 缀合的抗体。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。
“化疗剂”包括在治疗免疫系统疾病中有用的化学化合物。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包含免疫抑制剂。
本文使用的“免疫抑制剂”、“免疫抑制药物”或“免疫抑制物”是在免疫抑制治疗中用于抑制或阻止免疫系统活性的治疗剂。
术语“有效量”指本发明的抗体或片段或缀合物或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤体积)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。
“分离的编码抗CLDN18.2抗体或其片段的核酸”是指一个或多个核酸分子，其编码抗体重链或轻链(或其片段，例如重链可变区或轻链可变区)，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48：444-453)算法(在http：//www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http：//www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法，((1989)CABIOS，4：11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
如本文所用，术语“在严格条件下(例如在低严格性、中等严格性、高严格性或极高严格性条件下)杂交”描述了杂交和洗涤条件。进行杂交反应的指导可以在通过引用方式并入的Current Protocols in Molecular Biology，John Wiley & Sons，N.Y.(1989)，6.3.1-6.3.6中找到。参考文献中描述了含水方法和非含水方法并且可以使用任一方法。本文中提及的优选的杂交条件如下：1)低严格性杂交条件是在约45℃于6X氯化钠/柠檬酸钠(SSC)中，随后至少在50℃(对于低严格性条件，可以增加洗涤的温度至55℃)于0.2X SSC，0.1％SDS中洗涤两次；2)中等严格性杂交条件是在约45℃于6X SSC中、随后在60℃在0.2X SSC、0.1％SDS中洗涤一次或多次；3)高严格性杂交条件是在约45℃在6X SSC中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次；并且优选地4)极高严格性杂交条件是在65℃于0.5M磷酸钠、7％SDS中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次。极高严格性条件(4)是优选的条件和除非另外说明，否则应当使用的一个条件。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括胃癌或胰腺癌，包括那些癌症的转移性形式。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)抗CLDN18.2抗体或其片段、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。在一些实施方案中，药物组合中使用的抗CLDN18.2抗体或其片段和其他治疗剂以不超过它们单独使用时的水平施用。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细 胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
II.抗体
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段以高亲和力结合CLDN18.2(例如人CLDN18.2)。在一些实施方案中，本发明的抗CLDN18.2抗体特异性结合CLDN18.2(例如人CLDN18.2)，而不结合CLDN18.1(例如人CLDN18.1)。在一些实施方案中，本发明的抗体或其抗原结合片段与人CLDN18.2的结合亲和力高于已知的CLDN18.2抗体，例如Zolbetuximab(简称Zmab)抗体。在一些实施方案中，通过生物膜薄层干涉测定技术或表面等离子共振法测定抗体的亲和力。
在一些实施方案中，本发明的抗CLDN18.2抗体，以下平衡解离常数(K D)与人CLDN18.2结合，所述K D小于大约15nM，优选地，小于或等于大约10nM、9.5nM、9nM、8.5nM、8nM、7.5nM、7nM、6.5nM、6nM、5.5nM、5nM、4.5nM、4nM、3.5nM、3nM，在一些实施方案中，所述K D在上述数值之间(包括端点)。
在一些实施方案中，本发明的抗体或其抗原结合片段与细胞表面的CLDN18.2结合。在一些实施方案中，本发明的抗体或其抗原结合片段不与细胞表面的CLDN18.1结合。在一些实施方案中，CLDN18.2在细胞表面表达或过表达。在一些实施方案中，细胞为表达CLDN18.2的CHO细胞或293细胞，例如CHO-S细胞或HEK293细胞。在一些实施方案中，细胞为表达CLDN18.2的癌细胞，例如天然表达CLDN18.2或经人工转染表达CLDN18.2或经人工转染具有增加的表达水平的CLDN18.2的细胞，例如表达CLDN18.2的胃癌细胞或胰腺癌细胞系，例如细胞系NUGC-4、KATO III和DAN-G细胞系，例如过表达CLDN18.2的KATO III和DAN-G细胞系。
在一些实施方案中，利用流式细胞术检测所述结合。在一些实施方案中，本发明的抗体以小于或等于大约15nM、10nM、9nM、8nM、7nM、6nM、5.5nM的EC50结合CHO细胞上过表达的CLDN18.2。在一些实施方案中，本发明的抗体以小于或等于大约4nM、3.7nM、3.5nM、3nM、2.5nM、2nM、1.5nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM的EC50结合表达CLDN18.2的癌细胞，例如表达CLDN18.2的胃癌细胞系或胰腺癌细胞系，例如细胞系NUGC-4、KATO III和DAN-G细胞系，例如过表达CLDN18.2的KATO III和DAN-G细胞系。在一些实施方案中，本发明的抗体与表达CLDN18.2的细胞的结合的EC50与已知抗体例如Zmab相当，或小于已知抗体Zmab的EC50，或小于已知抗体Zmab的EC50的75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％。
在一些实施方案中，本发明的抗体或其抗原结合片段具有ADCC活性或CDC活性，例如与已知抗体(例如Zmab)相当的ADCC活性或CDC活性，或者更高的ADCC活性或CDC活性。
在一些实施方案中，本发明的抗体或其抗原结合片段能够抑制肿瘤细胞，例如表达CLDN18.2的肿瘤细胞。
在一些实施方案中，本发明的抗体或其抗原结合片段能够用于治疗癌症。在一些实施方案中，本发明的抗体或其抗原结合片段，能够有效抑制肿瘤生长，肿瘤抑制率大于或等于约25％、30％、35％、40％、45％、50％、55％、60％、65％ 或70％。
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。
在一些方面中，本发明的抗CLDN18.2抗体或其抗原结合片段包含重链可变区(VH)。在一些方面中，本发明的抗CLDN18.2抗体或其抗原结合片段包含轻链可变区(VH)。在一些方面中，本发明的抗CLDN18.2抗体或其抗原结合片段包含重链可变区和轻链可变区(VH)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段还包含抗体重链恒定区HC。在一些实施方案中，本发明抗CLDN18.2抗体或其抗原结合片段还包含抗体轻链恒定区LC。在一些实施方案中，本发明抗CLDN18.2抗体或其抗原结合片段还包含重链恒定区HC和轻链恒定区LC。
在一些实施方案中，本发明的重链可变区
(i)包含与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的轻链可变区
(i)包含与选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3选自
(i)如SEQ ID NO：4所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(ii)如SEQ ID NO：15或21所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(iii)如SEQ ID NO：26或27所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(iv)如SEQ ID NO：36或37所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(v)如SEQ ID NO：45或48所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(vi)如SEQ ID NO：53或56所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(vii)如SEQ ID NO：61或66所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(viii)如SEQ ID NO：72或73所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，
(ix)如SEQ ID NO：80或85所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(x)相对于(i)-(ix)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，本发明的3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3选自
(i)如SEQ ID NO：9所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(ii)如SEQ ID NO：19或22所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(iii)如SEQ ID NO：31或32所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(iv)如SEQ ID NO：40或41所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(v)如SEQ ID NO：47或49所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(vi)如SEQ ID NO：55或57所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(vii)如SEQ ID NO：65或68所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(viii)如SEQ ID NO：75或76所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，
(ix)如SEQ ID NO：83或86所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(x)相对于(i)-(ix)中任一项的序列，在所述三个LCDR区上共包含至少一个 且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，HCDR1包含SEQ ID NO：1、12、23、33、42、50、58、69或77的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO：1、12、23、33、42、50、58、69或77的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO：2、13、20、24、34、43、51、59、70、78、84、93或94的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO：2、13、20、24、34、43、51、59、70、78、84、93或94的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列，其中
SEQ ID NO：93为YIAPFXGDSRYNQKFKG，其中X为任何氨基酸，优选的N或Q或其保守氨基酸取代；或者
SEQ ID NO：94为VIWGDXSTNYHSVLIS其中X为任何氨基酸，优选的G或V或其保守氨基酸取代。
在一些实施方案中，HCDR3包含SEQ ID NO：3、14、25、35、44、52、60、71或79的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO：3、14、25、35、44、52、60、71或79的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO：6、16、28、38、62、67或96的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO：6、16、28、38、62、67或11的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；
其中SEQ ID NO：11为KSSQSLLXGGNQKNYLT，其中X为任何氨基酸，优选的N或Q或其保守氨基酸取代。
在一些实施方案中，LCDR2包含SEQ ID NO：7、17、29、63或81的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO：7、17、29、63或81的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO：8、18、30、39、46、54、64、74或82的氨基酸序列，或由所述氨基酸序列组成，或者LCDR3包含与SEQ ID NO：8、18、30、39、46、54、64、74或82的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，本发明的抗体重链恒定区HC为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。在一些实施方案中，本发明的抗体轻链恒定区LC为lambda或Kappa轻链恒定区，优选的Kappa轻链恒定区。
在一些优选的实施方案中，本发明的抗体重链恒定区HC
(i)包含与选自SEQ ID NO：5的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO：5的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO：5的氨基酸序列相比具有1个或多个(优选不 超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，所述氨基酸改变发生在Fc区。在一个实施方案中，所述Fc区的氨基酸改变增加抗体的CDC或ADCC活性。
在一些实施方案中，本发明的抗体轻链恒定区LC
(i)包含与选自SEQ ID NO：10的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含选自SEQ ID NO：10的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与选自SEQ ID NO：10的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段包含：
(i)如SEQ ID NO：4所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：9所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(ii)如SEQ ID NO：15或21所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：19或22所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(iii)如SEQ ID NO：26或27所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：31或32所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(iv)如SEQ ID NO：36或37所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：40或41所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(v)如SEQ ID NO：45或48所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：47或49所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(vi)如SEQ ID NO：53或56所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：55或57所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(vii)如SEQ ID NO：61或66所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：65或68所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(viii)如SEQ ID NO：72或73所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：75或76所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
(ix)如SEQ ID NO：80或85所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：83或86所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
在本发明的一些具体实施方案中，本发明的抗CLDN18.2抗体或其抗原结合 片段包含：
(i)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：1、2和3，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：6、7和8；
(ii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、13和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：16、17和18；
(iii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、20和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：16、17和18；
(iv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、93和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：16、17和18；
(v)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：23、24和25，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、29和30；
(vi)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：33、34和35，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和39；
(vii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：42、43和44，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和46；
(viii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：50、51和52，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、17和54；
(ix)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：62、63和64；
(x)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：67、63和64；
(xi)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：11、63和64；
(xii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：69、70和71，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和74；
(xiii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、78和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82；
(xiv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、84和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82；
(xv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、94和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82。
在本发明的一些具体实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段包含：
(i)包含SEQ ID NO：4所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：9所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(ii)包含SEQ ID NO：15或21所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：19或22所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(iii)包含SEQ ID NO：15所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：19所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(iv)包含SEQ ID NO：21所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：22所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(v)包含SEQ ID NO：26或27所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：31或32所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(vi)包含SEQ ID NO：26所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：31所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(vii)包含SEQ ID NO：27所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：32所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(viii)包含SEQ ID NO：36或37所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：40或41所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(ix)包含SEQ ID NO：36所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：40所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(x)包含SEQ ID NO：37所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：41 所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xi)包含SEQ ID NO：45或48所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：47或49所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xii)包含SEQ ID NO：45所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：47所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xiii)包含SEQ ID NO：48所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：49所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xiv)包含SEQ ID NO：53或56所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：55或57所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xv)包含SEQ ID NO：53所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：55所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xvi)包含SEQ ID NO：56所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：57所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xvii)包含SEQ ID NO：61或66所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：65或68所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xviii)包含SEQ ID NO：61所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：65所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xix)包含SEQ ID NO：66所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：68所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xx)包含SEQ ID NO：72或73所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：75或76所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xxi)包含SEQ ID NO：72所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：75 所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xxii)包含SEQ ID NO：73所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：76所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xxiii)包含SEQ ID NO：80或85所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：83或86所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xxiv)包含SEQ ID NO：80所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：83所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
(xxv)包含SEQ ID NO：85所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：86所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL。
在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。在一些实施方案中，本发明所述的氨基酸改变发生在抗体重链恒定区的Fc区上，在优选的实施方案中，所述Fc区上的氨基酸改变增加抗体的ADCC和/或CDC作用。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表所示：
Figure PCTCN2021100870-appb-000002
Figure PCTCN2021100870-appb-000003
在某些实施方案中，置换发生在抗体的CDR区。通常，获得的变体相对于亲本抗体在某些生物学特性方面(例如，增加的亲和力)具有修饰(例如，改善)和/或将具有亲本抗体的基本上保留的某些生物学特性。示例性置换变体是亲和力成熟抗体。
在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体经糖基化的程度。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。当抗体包含Fc区时，可以改变附着于其的糖类。在一些应用中，除去不想要的糖基化位点的修饰可以是有用的，例如除去岩藻糖基序以提高抗体依赖性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277：26733)。在其它应用中，可以进行半乳糖苷化修饰以修饰补体依赖性细胞毒性(CDC)。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体，以改变抗体的一种或多种功能特性，例如血清半衰期、补体结合、补体依赖性细胞毒性、Fc受体结合和/或抗体依赖性细胞毒性。Fc区变体可包括在一或多个氨基酸位置处包含氨基酸改变(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在本发明的一个实施方案中，本文所述抗体在Fc区引入改变，来提高抗体的ADCC活性或CDC活性。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
在一些实施方案中，本发明的抗CLDN18.2抗体或其抗原结合片段具有以下一个或多个特性：
(i)显示与本发明抗体对CLDN18.2相同或相似的结合亲和力和/或特异性；
(ii)抑制(例如，竞争性抑制)本发明抗体与CLDN18.2的结合；
(iii)与本发明抗体结合相同或重叠的表位；
(iv)与本发明抗体竞争结合CLDN18.2；
(v)具有本发明抗体的一个或多个生物学特性。
在一些实施方案中，本发明的抗CLDN18.2抗体是IgG1形式的抗体或IgG2形式的抗体或IgG3形式的抗体或IgG4形式的抗体，优选的，为IgG1形式的抗体。
在一些实施方案中，抗CLDN18.2抗体是单克隆抗体。
在一些实施方案中，抗CLDN18.2抗体是人源化的。
在一些实施方案中，抗CLDN18.2抗体是人抗体。
在一些实施方案中，抗CLDN18.2抗体是嵌合抗体。
在一些实施方案中，至少部分的抗CLDN18.2抗体的框架序列是人共有框架序列。
在一个实施方案中，本发明的抗CLDN18.2抗体还涵盖其抗体片段(例如抗原结合片段)，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’) 2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体。
III.本发明的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何抗CLDN18.2抗体或其片段的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体，例如pcDNA3.1。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞(例如CHO-S)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
在一方面，本发明提供了编码本文所述任何抗CLDN18.2抗体或其片段的核酸。所述核酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的核酸，或包含编码抗体的轻链和/或重链的氨基酸序列的核酸。
例如，本发明的核酸包含编码选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85，或者SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85，或者SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
本发明还涵盖与下述核酸在严格性条件下杂交的核酸或与下述核酸具有一个或多个置换(例如保守性置换)、缺失或插入的核酸：包含编码选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85，或者SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86中任一项所示氨基酸序列的核酸序列的核酸；或包含编码与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85，或者SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83或86中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸序列的核酸。
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是pcDNA3.1。
在一个实施方案中，提供包含所述载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。在表达后，抗体可以从可溶级分中的细菌细胞糊状物分离，并且可以进一步纯化。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293、293F或293T细胞)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞、CHO-S细胞、ExpiCHO等；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。本领域已知适合产生抗体的哺乳动物宿主细胞系。
IV.本发明的抗体分子的生产和纯化
在一个实施方案中，本发明提供制备本发明抗体分子或其片段(优选的抗原结合片段)的方法，其中所述方法包括在适于表达编码本发明抗体分子或其片段(优选的抗原结合片段)的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体或其片段(例如抗原结合片段)。在某个实施方案中，所述方法还包括从宿主细胞回收本发明抗体分子或其片段(例如抗原结合片段)。
在一个实施方案中，提供了制备本发明抗体分子的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体(例如任意一条多肽链和/或多条多肽链)的核酸或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。
为了重组产生本发明抗体分子，分离编码抗体(例如上文所描述的抗体，例如任意一条多肽链和/或多条多肽链)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
如本文所述制备的抗体分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。
V.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗CLDN18.2抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹，等来进行。可使用本领域已知方法来测定对CLDN18.2的结合，本文中公开了例示性方法。在一些实施方案中，使用放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)或流式细胞术测量的。
另一方面，可使用竞争测定法来鉴定与本文中公开的任何抗CLDN18.2抗体竞争对CLDN18.2的结合的抗体。在某些实施方案中，此类竞争性抗体结合与本文中公开的任何抗CLDN18.2抗体所结合表位相同或重叠的表位(例如线性或构象表位)。
本发明还提供了用于鉴定具有生物学活性的抗CLDN18.2抗体的测定法。生物学活性可以包括例如结合CLDN18.2(例如结合人CLDN18.2)，对表达CLDN18.2的细胞的结合，对细胞的CDC或ADCC的活性、对肿瘤细胞的抑制作用等。还提供在体内和/或在体外具有此类生物学活性的抗体。
在某些实施方案中，对本发明的抗体测试此类生物学活性。
供任何上述体外测定法使用的细胞包括天然表达CLDN18.2或经改造而表达或过表达CLDN18.2细胞系。此类细胞还包括表达CLDN18.2和并非正常情况下表达CLDN18.2的编码CLDN18.2DNA转染的细胞系。在一些实施方案中，这类细胞是胃癌细胞或胰腺癌细胞。在一些实施方案中，这些细胞是表达CLDN18.2的CHO细胞。在一些实施方案中，这些细胞是细胞系NUGC-4、KATO III和DAN-G细胞系，例如过表达CLDN18.2的KATO III和DAN-G细胞系。
可以理解的是，能够使用本发明的免疫缀合物替换或补充抗CLDN18.2抗体来进行任何上述测定法。
可以理解的是，能够使用抗CLDN18.2抗体和别的活性剂的组合来进行任何上述测定法。
VI.免疫缀合物
在一些实施方案中，本发明提供了免疫缀合物，其包含本文中提供的任何抗CLDN18.2抗体和其它物质，例如治疗剂，包括化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如抗炎剂或免疫抑制剂)。在一个实施方案中，所述其它物质例如细胞毒性剂，其包括任何对细胞有害的药剂。
在一些实施方案中，所述免疫缀合物用于预防或治疗癌症。
VII.药物组合物和药物制剂
在一些实施方案中，本发明提供包含本文所述的任何抗CLDN18.2抗体或其片段(优选地其抗原结合片段)或其免疫缀合物的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的抗CLDN18.2抗体或其片段或其免疫缀合物，以及一种或多种其它治疗剂的组合。
本发明还包括包含抗CLDN18.2抗体或其免疫缀合物的组合物(包括药物组合物或药物制剂)，或包含编码抗CLDN18.2抗体的多核苷酸的组合物(包括药物组合物或药物制剂)。在某些实施方案中，组合物包含一种或多种结合CLDN18.2的抗体或其片段，或一种或多种编码一种或多种抗CLDN18.2的抗体或其片段的多核苷酸。这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”，第八版，R.C.Rowe，P.J.Seskey和S.C.Owen，Pharmaceutical Press，London，Chicago。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。
可以通过将具有所需纯度的本发明的抗体与一种或多种任选的药用辅料混合来制备包含本文所述的抗体的药物制剂，优选地以冻干制剂或水溶液的形式。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂，例如化疗剂、细胞因子、细胞毒性剂、疫苗、其它抗体、小分子药物或免疫调节剂等。所述活性成分以对于目的用途有效的量合适地组合存在。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
VIII.药物组合和药盒
在一些实施方案中，本发明还提供了药物组合或药物组合产品，其包含本发明的抗CLDN18.2抗体或其片段(优选的抗原结合片段)，或其免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂等)。
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式。由此可以依据给药方案或药物施用间隔提供剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
-含有包含抗CLDN18.2抗体或其片段的药物组合物的第一容器；
-含有包含其它治疗剂的药物组合物的第二容器。
IX.用途和方法
本发明一方面提供了在受试者中预防或治疗肿瘤(例如癌症)的方法，包括向受试者施用有效量的本发明的抗CLDN18.2的抗体或其片段(优选的抗原结合片段)、免疫缀合物、药物组合物、药物组合或药盒。
在一些实施方案中，所述肿瘤(例如癌症)患者中具有(例如升高水平的，例如核酸或蛋白质水平的)CLDN18.2。
在一些实施方案中，所述肿瘤例如癌症包括实体肿瘤和血液肿瘤以及转移性病灶。在一个实施方案中，实体瘤的例子包括恶性肿瘤。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，所述肿瘤治疗将受益于抑制核酸或蛋白质水平的CLDN18.2。在一些实施方案中，所述肿瘤治疗受益于本发明抗体所引发的ADCC或CDC作用。
在一个具体的实施方案中，本发明的抗CLDN18.2抗体能够抑制肿瘤细胞增殖，例如表达CLDN18.2的肿瘤细胞，例如胃癌细胞或胰腺癌细胞。
在一个具体的实施方案中，本发明的抗CLDN18.2抗体具有强的ADCC或CDC活性。
在一些实施方案中，肿瘤是肿瘤免疫逃逸。
在一些实施方案中，所述肿瘤是癌症，例如胃癌或胰腺癌。
受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类(例如，患有本文所述疾病或具有患有本文所述疾病的风险的个体)。在一个实施 方案中，受试者患有本文所述疾病(例如，癌症)或具有患有本文所述疾病的风险。在某些实施方案中，受试者接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法。在一些实施方案中，受试者之前已经接受过或正在接受免疫疗法。
在其他方面，本发明提供抗体分子或其片段或其免疫缀合物或药物组合物或药物组合或药盒在生产或制备药物中的用途，所述药物用于本文所述的用途，例如用于预防或治疗本文提及的相关疾病或病症。
在一些实施方案中，本发明的抗体分子或其片段或其免疫缀合物或药物组合物或药物组合或药盒会延迟病症和/或与病症相关的症状的发作。
在一些实施方案中，本发明的抗体分子或其片段或其免疫缀合物或药物组合物还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。
在一些实施方案中，治疗方式包括外科手术；放射疗法、局部照射或聚焦照射等。
在一些实施方案中，治疗剂选自化疗剂、细胞因子、细胞毒性剂、疫苗、其它抗体、小分子药物或免疫调节剂。
示例性的免疫调节剂包括免疫抑制剂或抗炎剂。
在一些实施方案中，本文中描述的抗体组合可以分别施用，例如，作为单独的抗体分别施用。
此类组合疗法涵盖组合施用(例如两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的治疗剂和/或药剂之前，同时，和/或之后发生本发明的抗体的施用。
药物组合物的施用途径是根据已知方法，例如，经口、通过静脉内注射、腹膜内、脑内(实质内)、脑室内、肌内、眼内、动脉内、门脉内或病灶内途径；通过持续释放系统或通过植入装置。在某些实施方案中，组合物可通过弹丸注射或通过连续输注或通过植入装置施用。
组合物还可经由植入膜、海绵或其上吸收或胶囊包封所需分子的另一种适当的材料被局部施用。在某些实施方案中，当使用植入装置时，所述装置可被植入到任何合适的组织或器官中，并且可经由扩散、定时释放的大丸剂、或连续施用递送所需分子。
X.用于诊断和检测的方法和组合物
在某些实施方案中，本文中提供的任何抗CLDN18.2抗体或其片段(优选的抗原结合片段)可以用于检测CLDN18.2在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免 疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶相关病灶。
在一个实施方案中，提供用于诊断或检测方法的抗CLDN18.2抗体或其片段。在另一个方面中，提供检测CLDN18.2在生物样品中的存在的方法。在某些实施方案中，方法包含检测CLDN18.2蛋白在生物样品中的存在。在某些实施方案中，CLDN18.2是人CLDN18.2。在某些实施方案中，所述方法包括将生物样品与如本文所述的抗CLDN18.2抗体或其片段在允许抗CLDN18.2抗体或其片段与CLDN18.2结合的条件下接触，并检测在抗CLDN18.2抗体或其片段和CLDN18.2之间是否形成复合物。复合物的形成表示存在CLDN18.2。该方法可以是体外或体内方法。在一个实施方案中，抗CLDN18.2抗体或其片段被用于选择适合利用抗CLDN18.2抗体或其片段的治疗的受试者，例如其中CLDN18.2是用于选择所述受试者的生物标记物。
在一个实施方案中，可以使用本发明抗体诊断肿瘤，例如癌症，例如评价(例如，监测)个体中本文所述疾病的治疗或进展、其诊断和/或分期。在某些实施方案中，提供标记的抗CLDN18.2抗体或其片段。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。
在本文中提供的一些实施方案中，样品是在用抗CLDN18.2抗体或其片段治疗之前获得的。在一些实施方案中，样品是在用其他疗法之前获得的。在一些实施方案中，样品是在用其他疗法治疗过程中，或者用其他疗法治疗后获得的。
在一些实施方案中，样品是福尔马林固定、石蜡包膜(FFPE)的。在一些实施方案中，样品是活检(例如芯活检)，手术标本(例如来自手术切除的标本)，或细针吸出物。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测CLDN18.2。
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验CLDN18.2的存在，因而确定CLDN18.2值，将CLDN18.2值与对照值比较，并且如果CLDN18.2值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的抗CLDN18.2抗体或其片段(例如，本文所述的抗CLDN18.2抗体或其片段)，因而治疗所述疾病。
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明 本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例
Figure PCTCN2021100870-appb-000004
实施例1、稳定表达细胞株的构建
人源CLDN18.2的过表达细胞株的制备
根据制造商的说明书，使用
Figure PCTCN2021100870-appb-000005
试剂盒(Invitrogen，A1369601)构建稳定表达人源Claudin18.2(简称CLDN18.2，以下同)的细胞系。首先将人源CLDN18.2(UniProt ID：P56856-2)的全长基因构建到载体pCHO1.0中，采用化学转染法和电转染法将构建好的质粒分别转入CHO-S细胞(Invitrogen，A1369601)和HEK293细胞(Invitrogen，A14527)中，转染后的细胞经过两轮加压筛选得到分别表达CLDN18.2的细胞池(pool)。然后利用流式细胞分选仪(MoFlo XDP，Beckman Coulter)将高表达CLDN18.2的细胞分选出来，通过稀释法获得稳定表达CLDN18.2的单克隆细胞系CHO-hCLDN18.2和HEK293-hCLDN18.2。
人源CLDN18.1的过表达细胞株的制备
根据制造商的说明书，使用
Figure PCTCN2021100870-appb-000006
试剂盒(Invitrogen，A1369601)构建稳定表达人源Claudin18.1(简称CLDN18.1，以下同)的细胞系。首先将人源CLDN18.1(UniProt ID：P56856-1)全长基因构建到载体pCHO1.0(Invitrogen，A1369601)中，采用化学转染法将构建好的质粒转入CHO-S细胞(Invitrogen，A1369601)中，转染后的细胞经过两轮加压筛选得到分别表达CLDN18.1的细胞池(pool)。然后利用流式细胞分选仪(MoFlo XDP，Beckman Coulter)将高表达CLDN18.1的细胞分选出来，通过稀释法获得稳定表达CLDN18.1的单克隆细胞系CHO-hCLDN18.1。
过表达CLDN18.2的肿瘤细胞系的构建
将人源CLDN18.2(UniProt ID：P56856-2)的全长基因构建到载体pWPT-GFP(Addgene，12255)中，替换其中的GFP序列，与慢病毒的包装载体psPAX2(Addgene，12260)和pMD2.G(Addgene，12259)共同转染到HEK293T(ATCC，CRL-3216)细胞中，进行病毒的包装。分别收集培养了48小时和72小时后的培养上清，使用PEG8000进行慢病毒的浓缩。用浓缩后的病毒转染胰腺癌DAN-G细胞和胃癌KATO III细胞，然后利用流式细胞分选仪(MoFlo XDP，Beckman Coulter)将表达CLDN18.2的细胞分选出来，获得稳定转染CLDN18.2的肿瘤细胞系DAN-G-CLDN18.2和KATO III-CLDN18.2。
实施例2、杂交瘤筛选抗hCLDN18.2的单克隆抗体
本发明采用杂交瘤技术，通过实施例1获得的细胞(CHO-huClaudin18.2)免疫小鼠(方法和过程见表1)，然后获取小鼠的脾细胞与骨髓瘤细胞融合，获得能够分泌CLDN18.2抗体的杂交瘤细胞。
小鼠免疫
表1：实验动物及免疫信息
Figure PCTCN2021100870-appb-000007
杂交瘤融合
将小鼠处死，无菌打开腹腔取出脾脏，去掉周围附着的结缔组织。用针头挤压使脾细胞充分释放，制成脾细胞悬液。细胞悬液经70μM细胞筛网过滤后用RPMI-1640培养基洗一遍，1200rpm离心6min。去除上清，用RBC裂解缓冲液(GIBCO)重悬细胞后将红细胞裂解，离心去除上清后，细胞重悬于RPMI-1640培养基中并计数。将SP2/0和裂解红细胞后的脾细胞以1∶2～1∶1的比例混合，1000rpm离心6min。去除上清后将混合的细胞重悬于融合缓冲液中。再加入15ml融合缓冲液，1000rpm离心5min，去除上清。重复上述步骤一遍后，加入适量体积的融合缓冲液重悬细胞，调整混合细胞密度至1×10 7个细胞/ml。通过电融合仪融合后，细胞于电融合皿中室温静置5min。将细胞转移入离心管中，稀释细胞至1～2×10 4个细胞/ml。96孔板中每孔加入100μl细胞悬液。融合后第5天更换培养基。培养第10天(或更久，根据细胞生长状态)后收集上清进行流式细胞仪(FACS)检测，筛选阳性克隆。
杂交瘤细胞高通量筛选
通过流式细胞仪(FACS)筛选出特异性表达抗CLDN18.2抗体的杂交瘤细胞，且分泌的抗体不与CLDN18.1结合。
将实施例1获得的待检测细胞(HEK293-hCLDN18.2)计数，并稀释至1×10 6个细胞/ml，向U型底96孔板中加入100μl/孔。500g 5min，离心，去除细胞培养基。将杂交瘤96孔板培养上清加入U型板并重悬细胞，每孔加100μl，冰上静置30min。500g 5min去除上清，PBS洗细胞1遍。每孔加入100μl抗鼠Fab的FITC标记的二抗(1∶500稀释于PBS中)，阳性对照抗体加入100μl抗人Fab的FITC标记的二抗。冰上避光孵育30min。500g 5min去除上清，PBS洗细胞1遍。用50μl 1×PBS重悬细胞，FACS上机检测。
将阳性克隆，再用上述同样方法进行CHO-hCLDN18.1的复筛，共获得9株与人CLDN18.2结合且不与人CLDN18.1结合的杂交瘤细胞。其中8株来源于Bal b/c小鼠，1株来源于H2L2全人源转基因小鼠，具体见表2。
表2.杂交瘤高通量筛选获得的候选抗体克隆
小鼠类型 克隆名称
Bal b/c小鼠 3G3，14A5，25C7，38F8，69H9，46F6，59C1，51H10
H2L2全人源转基因小鼠 HB37A6
实施例3、抗体基因的获取
利用分子生物学技术，对实施例2中获得的9株杂交瘤细胞(3G3，14A5，25C7，38F8，69H9，46F6，59C1，51H10和HB37A6)进行抗体轻重链基因序列的调取。通过RNA提取试剂盒(Biomiga，R6311-02)抽提9个杂交瘤细胞的总RNA，然后用PrimeScript II 1st Strand cDNA Synthesis Kit(Takara，6110A)反转录总RNA获得cDNA，分别用简并引物扩增抗体的重链和轻链可变区序列，插入pMD20-T载体(Takara，6028)，连接转化后挑克隆测序。
抗体的序列参见表A。
实施例4、重组CLDN18.2抗体的制备
将实施例2筛选得到的9个抗体(3G3，14A5，25C7，38F8，69H9，46F6，59C1，51H10和HB37A6)以及对照抗体zolbetuximab(简称Zmab，序列来源于INN117)以全长单克隆抗体的形式表达在HEK293细胞(Invitrogen，A14527)中。首先构建表达载体，分别将9个配对重链可变区和轻链可变区放置在人源IgG1重链恒定区(SEQ ID NO：5)和轻链kappa恒定区(SEQ ID NO：10)的N端，构建到带有N端信号肽的pcDNA3.1表达载体中，获得轻重链表达载体。将所获得的轻、重链表达载体通过PEI(Polysciences Inc，23966)共转染对HEK293细胞进行瞬时转染，培养7天后收集培养基上清。上清通过Protein A柱(Hitrap Mabselect Sure，GE 11-0034-95)进行纯化，之后超滤并换液到PBS(Gibco，70011-044)中，采用A280法检测浓度并用SEC-HPLC法测定纯度，获得纯度大于95％的抗体溶液。
实施例5、BLI法测定重组CLDN18.2抗体与抗原的亲和力
采用生物膜干涉技术(Biolayer Interferometry，BLI)测定本发明抗体结合人CLDN18.2的平衡解离常数(K D)。BLI法亲和力测定按照现有的方法(Estep，P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs，2013.5(2)：第270-8页)进行。将AHC(18-5060，Fortebio)传感器浸泡于SD缓冲液(1x PBS，BSA0.1％，Tween-200.05％)中。取100μl的SD缓冲液、各个抗体、人Claudin18.2蛋白(P50251802，GenScript)分别加入到96孔黑色聚苯乙烯半量微孔板(Greiner，675076)中。使用Fortebio Octet Red96进行检测，使用ForteBio Octet分析软件分析K D值。抗体的亲和力数据如表3所示：9个抗体均具有高亲和力，其中除3G3与对照抗体Zmab亲和力相当，其他均优于对照抗体Zmab。
表3：CLDN18.2抗体的亲和力常数
Figure PCTCN2021100870-appb-000008
Figure PCTCN2021100870-appb-000009
实施例6、CLDN18.2抗体与CLDN18细胞的结合特异性
通过流式细胞术(FACS)测定上述9个抗体分别与实施例1获得的稳定转染了人源CLDN18.2和人源CLDN18.1的CHO-S细胞系(即如实施例所述制备的CHO-hCLDN18.2和CHO-hCLDN18.1)的结合。实验方法参照实施例2，使用GraphPad Prism软件分析实验数据，得到图1和图2。如图1和图2所示，所述9个抗体均特异地结合人CLDN18.2，而不与人CLDN18.1结合。
实施例7、CLDN18.2抗体的基于报告基因的ADCC活性检测
使用含有荧光素酶报告基因的ADCC效应细胞(Promega，G7102)检测上述9个抗体的ADCC活性。取对数生长期的靶细胞CHO-hCLDN18.2和ADCC效应细胞，分别进行计数，以1∶6的比例，共50uL加入96孔白底板中，每孔共有细胞1.75×10 5个，然后每孔分别加入上述抗体，37℃，5％CO 2孵育8-10小时，所述抗体各自浓度如下：浓度起始点为3nM，然后3倍稀释，共有9个浓度点，即每种抗体使用的浓度有：3nM、1nM、0.333nM、0.111nM、0.037nM、0.123nM、0.0041nM、0.0014nM、0.00046nM。根据制造商的说明，使用Bio-Glo TM Luciferase reagent(Promega，G7940)进行荧光的检测。如图3结果显示，9个抗体均具有与对照抗体Zmab相当的ADCC活性。
实施例8、鼠源抗体的人源化改造
选取实施例3中的8个鼠源抗体(3G3，14A5，25C7，38F8，69H9，46F6，59C1，和51H10)进行人源化改造。确定抗体的CDR区域，重链CDR1采用Abm命名规则，其余CDR采用Kabat的命名规则。通过序列相似性比对，选取与鼠源抗体可变区序列相似度最高的人胚系(germline)基因序列作为人源化抗体的框架区模板，然后将CDR区域移植到各模板中。利用Discovery Studio软件构建各抗体的三维结构，并将对CDR区有影响的框架区氨基酸进行回复突变，得到人源化的抗体序列。人源化抗体Hz3G3、Hz14A5、Hz25C7、Hz38F8、Hz69H9、Hz46F6、Hz59C1和Hz51H10的序列参见表A。
根据实施例4所述的方法对这些人源化重组抗体进行制备和纯化，获得大于95％纯度的溶于PBS的抗体溶液。
实施例9、SPR法测定CLDN18.2抗体亲和力
采用表面等离子共振法(Surface plasmon resonance，SPR)测定8个人源化抗体(Hz3G3、Hz14A5、Hz25C7、Hz38F8、Hz69H9、Hz46F6、Hz59C1和Hz51H10) 和1个全人源抗体(HB37A6)结合人CLDN18.2的平衡解离常数(K D)。根据制造商的说明，使用氨基偶联试剂盒(GE Healthcare，BR-1006-33)，将人Claudin18.2(GenScrip，P50251802)偶联在CM5芯片(GE Healthcare，29-1496-03)表面，偶联后注入1M乙醇胺，对剩余的活化位点进行封闭。根据制造商的说明，通过Biacore(GE Healthcare，T200)检测芯片表面抗原与流动相中的抗体之间的结合与解离获得亲和力及动力学常数。将梯度稀释后的抗体(0-100nM)，由低浓度到高浓度的顺序流过芯片表面，结合时间180s，解离时间600s。最后使用10mM Glycine pH 1.5(GE Healthcare，BR-1003-54)对芯片进行再生。数据结果使用Biacore T200分析软件，以1∶1结合模型进行动力学的分析。如表4所示：除了Hz25C7和Hz3G3外，其他抗体的亲和力均优于对照抗体Zmab。
表4.SPR法检测CLDN18.2抗体的亲和力常数(平衡解离常数)
Figure PCTCN2021100870-appb-000010
实施例10、人源化抗体基于报告基因的ADCC活性检测
基于SPR亲和力数据，选择Hz14A5，Hz38F8，Hz69H9，Hz46F6，Hz59C1，和Hz51H10进行基于报告基因的ADCC活性检测。实验过程参照实施例7。如图4所示，人源化抗体Hz14A5，Hz38F8，Hz69H9，Hz46F6，Hz59C1，和Hz51H10均显示了与对照抗体相当ADCC活性。
实施例11、CLDN18.2抗体与肿瘤细胞系的结合
基于以上结果选择了1个人源化抗体Hz69H9和1个全人源抗体HB37A6进行下一步检测。参照实施例4，通过FACS测定两个抗体分别与胃癌细胞系NUGC-4、胃癌细胞系KATO III-hCLDN18.2和胰腺癌细胞系DAN-G-hCLDN18.2的结合。图5显示人源化抗体Hz69H9和全人源抗体HB37A6均具有较好的肿瘤细胞特异性结合，优于对照抗体Zmab。
实施例12、CLDN18.2抗体的CDC活性检测
使用KATO III-CLDN18.2细胞系进行抗体分子的CDC活性。96孔板中加入 1×10 5个靶细胞KATO III-hCLDN18.2，然后加入合适浓度的抗体分子，37℃培养30分钟后，加入现配置的人血清补体(Sigma，S1764)，所述抗体的分别浓度如下：系列稀释浓度，起始浓度为150ug/ml，3倍稀释，共9个浓度点。将反应体系再次置于37℃培养箱中孵育3小时，然后使用CCK-8试剂盒(同仁化学，CK04)利用酶标仪(ThermoFisher，MULTISKAN FC)读取数据，结果如图6所示，Hz69H9和HB37A6具有和对照抗体Zmab相当的CDC活性。
实施例13、CLDN18.2抗体的ADCC活性检测
用完全培养基RPMI-1640(Hyclone，SH30809.01)+10％胎牛血清(FBS，Hyclone，SH30084.03)重悬人外周血单个核细胞(PBMC，Allcells or Saily)，将PBMC调整为1×10 7个/ml。将NUGC-4或过表达Claudin18.2的DAN-G肿瘤靶细胞DAN-G-CLDN18.2用Far-Red(Invitrogen)标记10min，洗涤两次后重悬在完全培养基RPMI-1640(Hyclone，SH30809.01)+10％胎牛血清(FBS，Hyclone，SH30084.03)中，细胞浓度调整为2×10 5个/ml。将PBMC与抗claudin18.2单克隆抗体Hz69H9和HB37A6及对照抗体Zmab混合(每种抗体的起始浓度为30nM，3倍系列稀释，共计12个浓度点)，37度孵育30min，然后按照50∶1的效靶比，将50ul肿瘤靶细胞(1×10 4个)加入到50μl PBMC效应细胞中。37度孵育8小时，离心，用终浓度为10μg/ml的碘化丙啶(propidium iodide，PI，Invitrogen)重悬细胞，用流式细胞仪(BD，FACSCELESTA)检测Far-Red和PI双阳的细胞。通过FACSDiva software(BD，Celestsa)计算肿瘤靶细胞杀伤比例。
抗Claudin18.2单克隆抗体依赖的细胞介导的NUGC-4肿瘤细胞毒性结果如图7A和B所示，Hz69H9和HB37A6及对照抗体Zmab均可剂量依赖性介导NK细胞对NUGC-4细胞的细胞毒性杀伤，Hz69H9和HB37A6对肿瘤细胞的最大杀伤强于对照抗体Zmab。表明Hz69H9和HB37A6具有更强的细胞介导的肿瘤细胞毒性作用。抗Claudin18.2单克隆抗体依赖的细胞介导的DAN-G肿瘤细胞毒性结果如图7C和D所示，Hz69H9和HB37A6及对照抗体Zmab均可剂量依赖性介导NK细胞对肿瘤细胞的细胞毒性杀伤。其中HB37A6和Hz69H9在2个不同供者的PBMC中介导的最大杀伤均高于对照抗体Zmab。以上结果表明，相比对照抗体Zmab，Hz69H9和HB37A6具有更强的细胞介导的肿瘤细胞毒性作用。
实施例14、CLDN18.2抗体的体内抗肿瘤效果
1、抗体对DAN-G-CLDN18.2荷瘤小鼠模型的活性
选取HZ69H9和HB37A6抗体测试其在带有人胰腺癌的NOD-SCID小鼠(雌性NOD-SCID小鼠(15-18g)购自北京维通利华实验动物技术有限公司，数量60只)内的抗肿瘤效果。将实施例1构建的人胰腺癌细胞DAN-G-CLDN18.2进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)分散DAN-G-CLDN18.2，获得细胞密度为12×10^5个/ml的悬液。将细胞悬液与matrigel胶1∶1混合制备成细胞浓度为6×10^5个/ml细胞悬液。在第0天取0.2ml细胞悬液皮下接种至NOD-SCID小鼠右侧腹部区域中来建立DAN-G-CLDN18.2荷瘤小鼠模型。
肿瘤细胞接种5天后检测各只小鼠瘤体积，挑选出瘤体积在43.36mm 3～89.47mm 3范围内的小鼠按瘤体积大小蛇形分组(每组8只小鼠)分组。
对每只小鼠施用hIgG(Equitech-Bio，批号160308-02)、Hz69H9和HB37A6及对照抗体Zmab，给药剂量为每次10mg/kg，分别在接种后第5、9、12、16天 给药，每周2-3次监测小鼠瘤体积。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
2、抗体对NUCG-4荷瘤小鼠模型的活性
选取HZ69H9和HB37A6抗体测试其在带有人胃癌的NOG小鼠(雌性NOG小鼠(15-18g)购自北京维通利华实验动物技术有限公司，数量100只)内的抗肿瘤效果。将PBMC细胞(Allcells)复苏，离心收集细胞，以PBS(1×)分散PBMC细胞，细胞密度为2.5×10^6个/ml细胞悬液，在第0天取0.2ml细胞悬液用于NOG小鼠眼静脉注射，建立NOG人源化小鼠模型。
将NUGC-4细胞进行常规复苏传代培养用于后续体内实验。离心收集细胞，以PBS(1×)分散NUGC-4细胞，细胞密度为12×10^6个/ml与matrigel胶1∶1混合制备成细胞浓度为6×10^6个/ml细胞悬液。在第5天取0.2ml细胞悬液皮下接种至NOG人源化小鼠右侧腹部区域中来建立NUCG-4荷瘤小鼠模型。
肿瘤细胞接种后第1天随机分组，每组7只，对每只小鼠施用hIgG(Equitech-Bio，批号160308-02)、Hz69H9和HB37A6及对照抗体Zmab，给药剂量为每次10mg/kg，分别在接种后第1、5、8、12天给药，每周2-3次监测小鼠瘤体积与体重。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
3、结果
结果如图8所示，Hz69H9和HB37A6及对照抗体Zmab在人胰腺癌DAN-G-hCLDN18.2小鼠模型上都能抑制肿瘤生长。如图9所示，Hz69H9和HB37A6在人胃癌NUGC-4小鼠模型上比对照抗体Zmab展示出更佳的抗肿瘤效果。
在接种第26天计算相对肿瘤抑制率(TGI％)，计算公式如下：
TGI％＝100％*(对照组肿瘤体积-治疗组肿瘤体积)/(对照组肿瘤体积-对照组给药前肿瘤体积)。
其中在DAN-G-CLDN18.2荷瘤小鼠模型中，Hz69H9和HB37A的TGI分别为70％和28％，Zmab的TGI为24％；
在NUGC-4荷瘤小鼠模型中，Hz69H9和HB37A的TGI分别为46％和31％，Zmab的TGI为0％。
序列信息：
Figure PCTCN2021100870-appb-000011
Figure PCTCN2021100870-appb-000012
Figure PCTCN2021100870-appb-000013
Figure PCTCN2021100870-appb-000014
Figure PCTCN2021100870-appb-000015
Figure PCTCN2021100870-appb-000016
Figure PCTCN2021100870-appb-000017
Figure PCTCN2021100870-appb-000018
Figure PCTCN2021100870-appb-000019

Claims (24)

  1. 抗CLDN18.2抗体或其抗原结合片段，其包含：
    (i)如SEQ ID NO：4所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：9所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (ii)如SEQ ID NO：15或21所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：19或22所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (iii)如SEQ ID NO：26或27所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：31或32所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (iv)如SEQ ID NO：36或37所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：40或41所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (v)如SEQ ID NO：45或48所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：47或49所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (vi)如SEQ ID NO：53或56所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：55或57所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (vii)如SEQ ID NO：61或66所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：65或68所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (viii)如SEQ ID NO：72或73所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：75或76所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3；
    (ix)如SEQ ID NO：80或85所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，和如SEQ ID NO：83或86所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  2. 抗CLDN18.2抗体或其抗原结合片段，其包含：
    (i)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：1、2和3，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：6、7和8；
    (ii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、13和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：16、17和18；
    (iii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、20和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：16、17和18；
    (iv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：12、93和14，以及分别如以下氨基酸序列所示的LCDR1、LCDR2 和LCDR3：SEQ ID NO：16、17和18；
    (v)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：23、24和25，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、29和30；
    (vi)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：33、34和35，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和39；
    (vii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：42、43和44，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和46；
    (viii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：50、51和52，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、17和54；
    (ix)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：62、63和64；
    (x)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：67、63和64；
    (xi)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：58、59和60，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：11、63和64；
    (xii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：69、70和71，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：38、17和74；
    (xiii)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、78和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82；
    (xiv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、84和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82；
    (xv)分别如以下氨基酸序列所示的HCDR1、HCDR2、HCDR3：SEQ ID NO：77、94和79，以及分别如以下氨基酸序列所示的LCDR1、LCDR2和LCDR3：SEQ ID NO：28、81和82。
  3. 权利要求1或2的抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中重链可变区
    (i)包含与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列或由所述氨基酸序列组成；或者
    (iii)包含与选自SEQ ID NO：4、15、21、26、27、36、37、45、48、53、56、61、66、72、73、80或85的氨基酸序列相比具有1个或多个(优选不超过10 个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中；和/或
    轻链可变区
    (i)包含与选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83、86的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83、86的氨基酸序列或由所述氨基酸序列组成；或者
    (iii)包含与选自SEQ ID NO：9、19、22、31、32、40、41、47、49、55、57、65、68、75、76、83、86的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
  4. 权利要求1或2的抗体或其抗原结合片段，其包含：
    (i)包含SEQ ID NO：4所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：9所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (ii)包含SEQ ID NO：15或21所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：19或22所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (iii)包含SEQ ID NO：15所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：19所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (iv)包含SEQ ID NO：21所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：22所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (v)包含SEQ ID NO：26或27所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：31或32所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (vi)包含SEQ ID NO：26所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：31所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (vii)包含SEQ ID NO：27所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：32所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (viii)包含SEQ ID NO：36或37所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：40或41所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (ix)包含SEQ ID NO：36所示的氨基酸序列或与其具有至少90％同一性 的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：40所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (x)包含SEQ ID NO：37所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：41所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xi)包含SEQ ID NO：45或48所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：47或49所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xii)包含SEQ ID NO：45所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：47所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xiii)包含SEQ ID NO：48所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：49所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xiv)包含SEQ ID NO：53或56所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：55或57所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xv)包含SEQ ID NO：53所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：55所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xvi)包含SEQ ID NO：56所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：57所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xvii)包含SEQ ID NO：61或66所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：65或68所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xviii)包含SEQ ID NO：61所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：65所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xix)包含SEQ ID NO：66所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：68所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xx)包含SEQ ID NO：72或73所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：75或76所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xxi)包含SEQ ID NO：72所示的氨基酸序列或与其具有至少90％同一 性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：75所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xxii)包含SEQ ID NO：73所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：76所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xxiii)包含SEQ ID NO：80或85所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：83或86所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xxiv)包含SEQ ID NO：80所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：83所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL；
    (xxv)包含SEQ ID NO：85所示的氨基酸序列或与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VH，和包含SEQ ID NO：86所示的氨基酸序列与其具有至少90％同一性的氨基酸序列或由所述氨基酸序列组成的VL。
  5. 权利要求1-4中任一项的抗体或其抗原结合片段，其还包含重链恒定区和/或轻链恒定区。
  6. 权利要求5的抗体或其抗原结合片段，其中
    重链恒定区HC
    (i)包含与选自SEQ ID NO：5的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
    (ii)包含选自SEQ ID NO：5的氨基酸序列或由所述氨基酸序列组成；或者
    (iii)包含与选自SEQ ID NO：5的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成；和/或
    轻链恒定区LC
    (i)包含与选自SEQ ID NO：10的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
    (ii)包含选自SEQ ID NO：10的氨基酸序列或由所述氨基酸序列组成；或者
    (iii)包含与选自SEQ ID NO：10的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
  7. 权利要求5或6的抗体或其抗原结合片段，其中所述氨基酸改变发生在重链恒定区的Fc区。
  8. 权利要求1至7中任一项的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是IgG1形式或IgG2形式或IgG3形式或IgG4形式的抗体或抗原结合片段，优选地，所述抗体是IgG1形式的抗体或抗原结合片段。
  9. 权利要求1至8中任一项的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
  10. 权利要求1至9中任一项的结合CLDN18.2的抗体或其抗原结合片段，其中所述抗体是人源化的抗体或人抗体或嵌合抗体。
  11. 权利要求1至10中任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’) 2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体。
  12. 权利要求1至11中任一项的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段具有一种或多种以下性质：
    (i)以高亲和力结合CLDN18.2(例如人CLDN18.2)，而不结合CLDN18.1(例如人CLDN18.1)；
    (ii)以以下平衡解离常数(K D)与人CLDN18.2结合，所述K D小于大约15nM，优选地，小于或等于大约10nM、9.5nM、9nM、8.5nM、8nM、7.5nM、7nM、6.5nM、6nM、5.5nM、5nM、4.5nM、4nM、3.5nM、3nM；
    (iii)与细胞表面的CLDN18.2结合，不与细胞表面的CLDN18.1结合；
    (iv)具有ADCC活性或CDC活性，例如与已知抗体(例如Zmab)相当的ADCC活性或CDC活性，或者更高的ADCC活性或CDC活性；
    (v)抑制肿瘤细胞，例如表达CLDN18.2的肿瘤细胞；
    (vi)能够有效抑制肿瘤生长，肿瘤抑制率大于或等于约25％、30％、35％、40％、45％、50％、55％、60％、65％或70％。
  13. 分离的核酸，其编码权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段中的轻链可变区或重链可变区，或轻链或重链。
  14. 包含权利要求13的核酸的载体，优选地所述载体是表达载体。
  15. 包含权利要求13的核酸或权利要求14的载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞(例如293细胞或CHO细胞，例如CHO-S细胞或HEK293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
  16. 制备结合CLDN18.2的抗体或其抗原结合片段的方法，所述方法包括在适于表达编码权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段的核酸的条件下培养权利要求15的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述结合CLDN18.2的抗体或其抗原结合片段。
  17. 免疫缀合物，其包含权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段和其它物质，例如细胞毒性剂。
  18. 药物组合物，其包含权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段或权利要求17的免疫缀合物，以及任选地一种或多种其它治疗剂，例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂，以及任选地药用辅料。
  19. 药物组合，其包含权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段或权利要求17的免疫缀合物，以及一种或多种其它治疗剂，例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
  20. 预防或治疗受试者中肿瘤的方法，所述方法包括向所述受试者施用有效量的权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段、或权利要求17的免疫缀合物、或权利要求18的药物组合物、或权利要求19的药 物组合。
  21. 在受试者中引发ADCC和/或CDC的方法，所述方法包括向所述受试者施用有效量的权利要求1至12中任一项的结合CLDN18.2的抗体或其抗原结合片段、或权利要求17的免疫缀合物、或权利要求18的药物组合物、或权利要求19的药物组合，优选地，所述受试者患有肿瘤。
  22. 权利要求20或21的方法，其中所述肿瘤为癌症，优选的，所述癌症具有升高水平的(例如核酸或蛋白质水平的)CLDN18.2，例如所述癌症为胰腺癌或胃癌。
  23. 权利要求20-22中任一项的方法，其中所述方法还包括向患者施用一种或多种疗法，例如治疗方式和/或其它治疗剂，优选地，治疗方式包括手术治疗和/或放射疗法，其它治疗剂选自化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
  24. 检测样品中CLDN18.2的方法，所述方法包括
    (a)将样品与根据权利要求1至12中任一项所述的抗体或其抗原结合片段或权利要求17的免疫缀合物接触；以及
    (b)检测抗体或其抗原结合片段与CLDN18.2间的复合物的形成；任选地，抗体是被可检测地标记的。
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JP2023530003A (ja) 2023-07-12

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