WO2022007808A1 - 一种结合紧密连接蛋白-18.2的抗体及其用途 - Google Patents

一种结合紧密连接蛋白-18.2的抗体及其用途 Download PDF

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WO2022007808A1
WO2022007808A1 PCT/CN2021/104819 CN2021104819W WO2022007808A1 WO 2022007808 A1 WO2022007808 A1 WO 2022007808A1 CN 2021104819 W CN2021104819 W CN 2021104819W WO 2022007808 A1 WO2022007808 A1 WO 2022007808A1
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antibody
claudin
antigen
variant
cancer
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French (fr)
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陈博
王莹
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康诺亚生物医药科技（成都）有限公司
上海岺樾生物医药科技有限公司
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Publication of WO2022007808A1 publication Critical patent/WO2022007808A1/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
    • 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
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to an antibody that binds to Claudin-18 and uses thereof, particularly an antibody that binds to Claudin-18.2 and uses thereof.
  • Claudin is a family of cell surface proteins that build paracellular barriers and control molecular flow between cells, first reported by Shorichiro Tsukita et al in 1998 (Furuse et al., (1998) J Cell Biol 141 (7 ): 1539-1550). Claudin is an essential component of tight binding and plays an important role in maintaining epithelial cell polarity, controlling paracellular spreading, and regulating cell growth and differentiation.
  • Claudin-18 belongs to the Claudin family of claudin proteins, with four transmembrane domains, the N- and C-termini are intracellular, and there are two extracellular domains. Due to the alternative splicing of exon 1, Claudin 18 has two splicing isoforms, Claudin 18.1 and Claudin 18.2, both of which consist of 261 amino acids. Claudin 18.2 has different sequences at the N-terminus. 8 amino acids differ.
  • Claudin 18.1 and Claudin 18.2 are tissue-specific, and Claudin 18.1 is mainly expressed in normal tissues such as lung, stomach and bladder; Claudin 18.2, as a highly specific cell surface molecule, is only expressed in normal tissues It is not expressed on gastric stem cells on differentiated epidermal cells of gastric mucosa, but is highly expressed in primary gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer and its metastases. Therefore, Claudin 18.2 is frequently expressed on tumors. Overexpression qualifies this molecule as a highly potential new tumor therapy target for the development of therapeutics (eg, vaccine therapeutics and therapeutic antibodies) against Claudin 18.2.
  • therapeutics eg, vaccine therapeutics and therapeutic antibodies
  • the present invention utilizes hybridoma technology to obtain a plurality of monoclonal antibodies that recognize claudin-18, in particular, monoclonal antibodies that bind claudin-18.2 (Claudin 18.2) with high affinity.
  • monoclonal antibodies that bind to Claudin-18.2 with high affinity can be used for IHC detection, which can provide a basis for judging the distribution, cell expression and targeted therapy effect of Claudin-18.2 positive tumor cells.
  • the present disclosure provides antibodies or antigen-binding portions thereof that bind the Claudin-18.2 protein.
  • the present disclosure provides bispecific or multispecific molecules comprising the antibodies or antigen-binding portions thereof of the preceding aspects
  • the present disclosure provides nucleic acids encoding antibodies, or antigen-binding portions thereof, or bispecific or multispecific molecules according to the preceding aspects.
  • the present disclosure provides vectors comprising the nucleic acids of the preceding aspects.
  • the present disclosure provides cells comprising the vector of the preceding aspects.
  • An antibody or antigen-binding portion thereof according to any preceding aspect, wherein the antibody or antigen-binding portion thereof is humanized.
  • the present disclosure provides a pharmaceutical composition or kit comprising an antibody, or antigen-binding portion thereof, or nucleic acid encoding thereof, according to any of the preceding aspects, and a pharmaceutically acceptable carrier.
  • the present disclosure provides antibody-drug conjugates comprising an antibody, or antigen-binding portion, bispecific or multispecific molecule thereof, of any of the preceding aspects covalently attached to a therapeutic moiety.
  • the present disclosure provides a method of treating a Claudin-18.2-related disorder comprising the step of: administering to said mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector thereof of any of the preceding aspects , cells and/or pharmaceutical compositions.
  • the present disclosure provides an antibody or antigen-binding fragment, nucleic acid, vector, cell, and/or pharmaceutical composition of any of the preceding aspects in the manufacture of a medicament or reagent for the treatment of a Claudin-8.2-related disorder in a mammal Use in the box.
  • the antibodies of the present disclosure can be used in a variety of applications, including detection of Claudin-18.2 protein, and diagnosis, treatment, or prevention of Claudin-18.2-related diseases.
  • Figure 1 shows the results of RT-PCR detection of Claudin 18 stably transfected cell lines.
  • Figure 2 shows the results of FACS detection of HEK293-Claudin 18.2 stably transfected cell line, in which the black shade is the negative control, and the gray shade is the HEK293-Claudin 18.2 stably transfected cell.
  • Figure 3 shows the results of FACS detection of NIH3T3-Claudin 18.2 stable cells, in which the black line is the negative control, and the gray is the NIH3T3-Claudin 18.2 stable cells.
  • Figure 4 shows the results of immunohistochemical detection of paraffin sections.
  • Figure 5 shows the affinity of recombinant antibodies 3H2-C5 and 4F6-G11 to cell surface Claudin 18.2 detected by flow cytometry.
  • Figure 6 shows the immunohistochemical detection results of antibody 3H2-C5 on some gastric cancer tissue sections.
  • antibodies eg, monoclonal antibodies
  • antigen-binding fragments thereof that specifically bind Claudin.
  • monoclonal anti-Claudin antibodies that specifically bind Claudin, wherein the anti-Claudin antibodies comprise variants of the parent antibody.
  • antibodies that specifically bind Claudin eg, human Claudin.
  • anti-Claudin antibodies comprising modifications in one or more amino acid residues (eg, 5-13 amino acid substitutions in the framework regions of the heavy chain variable regions), and no such modifications Compared to the parent antibody, it retains its affinity for the antigen.
  • Claudin refers to claudin and includes Claudin 18.2.
  • the claudin is human claudin.
  • Claudin 18 refers to Claudin 18 and includes any variant, including Claudin 18 splice variant 1 (Claudin 18.1 (Claudin 18.1)) and Claudin 18 splice variant 2 (Claudin 18.2 (Claudin 18.2)).
  • Claudin 18.2 preferably refers to human Claudin 18.2.
  • disease associated with cells of Claudin-18.2 or similar expressions means that according to the present disclosure, Claudin 18.2 is expressed in cells of a diseased tissue or organ.
  • cell surface is used according to its normal meaning in the art and thus includes the exterior of the cell accessible by binding to proteins and other molecules.
  • Claudin 18.2 is on the cell surface, then Claudin 18.2 is expressed on the cell surface and is accessible by binding of Claudin 18.2-specific antibodies added to the cell.
  • the term “about” or “approximately” means within plus or minus 10% of the given value or range. Where a whole number is required, the term refers to within plus or minus 10% of the given value or range, rounded up or down to the nearest whole number.
  • the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains of 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequences the term is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 A nucleotide sequence of %, 99% or greater sequence identity.
  • sequence identity or “identity” has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. While there are many methods of measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled artisan.
  • substitutional variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites.
  • one amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions.
  • An “insertional” variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.
  • variable domains of antibodies refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs; ie CDR1, CDR2 and CDR3) or hypervariable regions, all located within the variable domains of light and heavy chains. The more conserved portions of the variable domains are referred to as framework (FR) regions or framework sequences.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of native heavy and light chains includes four FR regions, predominantly in a beta-sheet configuration, linked by three CDRs that form loops that connect the beta-sheet structure and Partial ⁇ -sheet structures are formed in some cases.
  • the CDRs of each chain are usually linked in proximity by FR regions and, with the aid of CDRs from other chains, contribute to the formation of antibody target binding sites (epitopes or determinants).
  • the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • a CDR can have the ability to specifically bind to the cognate epitope.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable regions of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments and other fragments, including modified fragments.
  • the fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any fragment of an antibody, which antibody binding is inserted into the antibody framework (e.g., by replacing the corresponding region) is obtained specifically immunization (i.e., Ka exhibits at least or at least about 10 7 -10 8 M-1) of the antigen .
  • a "functional fragment” or “analog of an anti-Claudin antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2, and so on.
  • Dimer (V H -V L dimer) "Fv" fragments consisting of the variable domain of a heavy chain and a light chain variable domain membership non-covalently manner to form a composition.
  • the three CDRs of each variable domain interact to define the target binding site on the surface of the VH- VL dimer, as is the case with intact antibodies.
  • the six CDRs collectively confer the target-binding specificity of the intact antibody.
  • a single variable domain or half of an Fv that includes only 3 target-specific CDRs
  • BsAb Bispecific antibody
  • a bispecific antibody and/or antigen-binding molecule Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • hybridomas refers to a cell or cell line (usually myeloma or lymphoma cells) produced by fusing antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and provide continuous supply to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art.
  • hybridomas When referring to the term “hybridoma” or “hybridoma cell”, it also includes subclones and progeny cells of hybridomas.
  • a full-length antibody is one that has two full-length heavy chains (eg, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region antibodies, such as those naturally produced by antibody-secreting B cells and those produced synthetically with the same domains.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • CDR refers to a complementarity-determining region
  • each of the heavy and light chains of antibody molecules is known to have 3 CDRs.
  • the CDRs are also referred to as hypervariable regions, and are present in the variable regions of each heavy and light chain of antibodies, with sites of very high variability in the primary structure of the CDRs.
  • the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically comprise chemically active surface profiles of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • immunospecifically binds or “immunospecifically binds” in reference to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the passage of an antibody or antigen-binding fragment between the antibody and antigen's antibody binding sites The ability of non-covalent interactions to form one or more non-covalent bonds with alloantigens.
  • the antigen may be an isolated antigen or present in tumor cells.
  • immunospecifically bind (or specifically binds) an antibody or antigen is from about 1 ⁇ 10 7 M -1 or 1x10 8 M -1 or greater affinity constant Ka (or 1x10 -7 M or 1 ⁇ A dissociation constant (Kd) of 10 ⁇ 8 M or lower binds the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody responses, eg, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art; see also U.S. Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating binding affinity of antibodies No).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • nucleic acid molecules refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked in reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • conservative sequence modifications of the sequences described in the Sequence Listing described herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody to the antigen encoded by the nucleotide sequence or containing the amino acid sequence.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the Sequence Listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative sequence modifications include conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • amino acids with acidic side chains eg, aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains amino acids e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids with non-polar side chains e.g. alanine, valine
  • leucine, isoleucine, proline, phenylalanine, methionine amino acids with beta branched side chains
  • a predicted non-essential amino acid residue in an anti-Claudin antibody is preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12 ( 10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • mutations can be introduced randomly along all or a portion of the anti-Claudin antibody coding sequence, eg, by saturation mutagenesis, and the resulting modified anti-Claudin antibodies can be screened for improved binding activity.
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • a vector also includes a "viral vector” or “viral vector.”
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • treating an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any provided antibodies or antigen-binding fragments thereof and any pharmaceutical uses of the compositions provided herein.
  • therapeutic effect refers to an effect resulting from treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds Claudin 18.2, comprising a heavy chain CDR selected from the group consisting of amino acid sequences SEQ ID NOs: 2-4, 12-14, or any variant thereof, and/or selected Light chain CDRs from the amino acid sequence SEQ ID NO: 7-9, 17-19 or any variant.
  • An antibody or antigen-binding portion thereof comprising a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO: 2, 12 or any variant thereof, selected from the amino acid sequence SEQ ID NO: 3, 13 or any variant thereof
  • the heavy chain CDR2 of the body selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 4, 14 or any variant thereof; and/or selected from the light chain CDR1 of the amino acid sequence SEQ ID NO: 7, 17 or any variant thereof , selected from the light chain CDR2 of amino acid sequence SEQ ID NO: 8, 18 or any variant thereof, selected from the light chain CDR3 of amino acid sequence SEQ ID NO: 9, 19 or any variant thereof.
  • An antibody or antigen-binding portion thereof comprising a combination of CDRs of heavy and light chains selected from the group consisting of:
  • (1) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 2-4, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7-9;
  • An antibody or antigen-binding portion thereof comprising a heavy chain variable region selected from the amino acid sequences of SEQ ID NO: 1, 11 or any variant thereof, and/or selected from the amino acid sequences of SEQ ID NO: 6, 16 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds Claudin 18.2, comprising the heavy chain variable region of the amino acid sequence SEQ ID NO: 1 or any variant thereof, and the amino acid sequence SEQ ID NO: 6 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds Claudin 18.2, comprising the heavy chain variable region of the amino acid sequence SEQ ID NO: 11 or any variant thereof, and the amino acid sequence SEQ ID NO: 16 or the light chain variable region of any variant thereof.
  • the present disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen-binding portion thereof of any of the preceding aspects.
  • nucleic acid molecule encoding an antibody or antigen-binding portion thereof or bispecific or multispecific molecule according to any of the preceding aspects.
  • the nucleic acid molecule comprises an antibody heavy chain nucleic acid sequence selected from SEQ ID NO: 5, 15 or any variant thereof, and/or an antibody light chain selected from SEQ ID NO: 10, 20 or any variant thereof nucleic acid sequence.
  • a vector comprising a nucleic acid according to any of the preceding aspects.
  • a cell comprising a vector according to any of the preceding aspects.
  • a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, or nucleic acid encoding thereof, according to any of the preceding aspects, and a pharmaceutically acceptable carrier.
  • the antibodies of the present disclosure are useful as therapeutic or diagnostic tools in various diseases in which Claudin 18.2 is unfavorably expressed or found.
  • the expression of Claudin 18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ.
  • To increase means to increase by at least 10%, especially at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.
  • expression is found only in diseased tissue, whereas expression in corresponding healthy tissue is suppressed.
  • the diseases associated with Claudin-18.2 include tumors, preferably, the tumors are cancer diseases.
  • cancer diseases are preferably those in which the cancer cells express Claudin 18.2.
  • a “cancer disease” is characterized by cells expressing Claudin 18.2 and cancer cells expressing Claudin 18.2.
  • Said cancer diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer and their metastases, especially gastric cancer metastases such as ovarian Klugenberg Tumor, peritoneal metastasis and lymph node metastasis.
  • cancer diseases are adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the pancreatic duct, adenocarcinoma of the bile duct, adenocarcinoma of the lung and adenocarcinoma of the ovary.
  • Cells expressing Claudin 18.2 are preferably cancer cells, preferably cancer cells of the cancers described herein.
  • Methods of treating diseases and symptoms with the Claudin 18.2 antibodies of the present disclosure comprise the steps of: administering to said mammal a therapeutically effective amount of an antibody or antigen-binding fragment or nucleic acid molecule or vector or cell or pharmaceutical composition of any of the preceding aspects .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding Claudin 18.2, wherein the antibody is administered to provide a serum level of at least 40 ⁇ g/ml.
  • the antibody is administered to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml.
  • the antibody is administered to provide serum levels of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • the term "serum level" as used in this specification means the concentration of the substance in question in serum.
  • the serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering an antibody dose of at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding Claudin 18.2 at at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 2.
  • the antibody is administered at a dose of up to 1200 mg/m 2 or up to 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding Claudin 18.2, wherein at least 50%, preferably 60%, 70%, 80% or 90% of the patient % of the cancer cells are positive for Claudin 18.2 and/or at least 40%, preferably 50% or 60% of the cancer cells of the patient are positive for surface expression of Claudin 18.2.
  • the present disclosure also provides a method of treating or preventing a cancer disease, the method comprising: a.
  • Claudin 18.2 positive cancer cells exhibiting at least 50%, preferably 60%, 70%, 80% or 90% and/or or at least 40%, preferably 50% or 60% of a patient with cancer cells positive for surface expression of Claudin 18.2; and b. administering to said patient an antibody capable of binding Claudin 18.2.
  • at least 95% or at least 98% of the cancer cells of the patient are Claudin 18.2 positive.
  • at least 70%, at least 80% or at least 90% of the cancer cells of the patient are positive for surface expression of Claudin 18.2.
  • the outcome of the treatment of the cancer disease is the achievement of stable disease.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the present disclosure provides methods of achieving stable disease in a cancer patient comprising administering to the patient an antibody capable of binding Claudin 18.2.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the antibody is administered in a single dose or multiple doses.
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding Claudin 18.2, wherein the antibody is administered in multiple doses.
  • the antibody is administered in multiple doses according to the present disclosure, preferably at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses
  • the antibody is administered in at least 10 doses and preferably at most 30 doses, 25 doses, 20 doses, 15 doses or 10 doses.
  • Doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days.
  • Doses of the antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.
  • the antibody is administered so as to provide serum levels of at least 40 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide a serum level of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • the serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering a dose of the antibody of at least 300 mg/m 2 , such as at least 600 mg/m 2 and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the antibody is conjugated to other drugs, such as labeled or cytotoxic conjugates.
  • the present disclosure also includes kits, eg, the kits comprising the antibodies of the present disclosure, fragments, homologues, derivatives thereof, etc., eg, labeled or cytotoxic conjugates, and instructions for use of the antibodies , conjugates that kill specific types of cells, and more.
  • the instructions may include instructions for using the antibody, conjugate, etc. in vitro, in vivo or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Packaged reagent combinations in predetermined quantities are contemplated along with instructions for their use, eg, for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore e.g., a substrate precursor that provides a detectable chromophore or fluorophore.
  • other additives such as stabilizers, buffers (eg, blocking buffers or lysis buffers), etc., may also be included.
  • the relative amounts of the various reagents can be varied to provide concentrates of reagent solutions, which provides user flexibility, space savings, reagent savings, and the like.
  • These reagents can also be provided in dry powder form, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagents of appropriate concentrations.
  • antibodies of the present disclosure can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • compositions comprising an anti-Claudin 18.2 antibody of the present disclosure, or a fragment thereof, conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is routine in the art practice.
  • the term "pharmaceutical composition” refers to a formulation of various preparations. Formulations containing a therapeutically effective amount of the multivalent antibody are in sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally containing stabilizers or excipients.
  • the antibodies of the present disclosure can be used as compositions for administration alone, or can be used in combination with other active agents.
  • the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (ie, a drug).
  • Therapeutic moieties can be, for example, cytotoxins, chemotherapeutic agents, cytokines, immunosuppressive agents, immunostimulatory agents, lytic peptides, or radioisotopes.
  • conjugates are referred to herein as "antibody-drug conjugates" or "ADCs".
  • the antibody is conjugated to a cytotoxic moiety.
  • Cytotoxic moieties may, for example, be selected from the following: paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; ipecine; mitomycin; etoposide; teniposide; vincristine; vinblastine ; Colchicine; Doxorubicin; Daunorubicin; F or its analogues or derivatives; Dolastatin 10 or 15 or its analogues; Irinotecan or its analogues; Mitoxantrone; corticosteroids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine , 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebadiazine, hydroxy
  • the antibody is conjugated to auristatin or a peptide analog, derivative or prodrug thereof.
  • Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division and has anticancer and antifungal activity.
  • auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E).
  • Suitable auristatin and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatin to the Ab are described, for example, in US Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publication WO02088172 , WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.
  • the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or a peptide analog, derivative or prodrug thereof.
  • PDB pyrrolo[2,1-c][1,4]-benzodiazepine
  • Suitable PDBs and PDB derivatives and related techniques are described, for example, in Hartley JA et al, Cancer Res 2010; 70(17):6849-6858; Antonow D. et al, Cancer J 2008; 14(3):154-169; Howard PW et al, Bioorg Med Chem Lett 2009; 19:6463-6466 and Sagnou et al, Bioorg Med Chem Lett 2000; 10(18):2083-2086.
  • the antibody is conjugated to a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • Dolastatin 15 irinotecan, monomethylauristatin E, monomethylauristatin F, PDB, or any analog, derivative or prodrug thereof.
  • the antibody is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Dokamycin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof.
  • the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.
  • the antibody is conjugated with a cytokine (eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNFa) couple link.
  • a cytokine eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNFa
  • the antibody is conjugated to a radioisotope or radioisotope-containing chelate.
  • the antibody can be conjugated to a chelator linker (eg, DOTA, DTPA, or tiracetam) that allows complexation of the antibody with the radioisotope.
  • chelator linker eg, DOTA, DTPA, or tiracetam
  • Antibodies may also or alternatively contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples include radioactive isotopes 3 H, 14 C, 15 N , 35 S, 90 Y, 99 Tc, 125 I, 131 I, 186 Re, 213 Bi, 225 Ac and 227 Th.
  • radioisotope ⁇ particle radiation e.g. 131 I, 90 Y, 211 At , 212 Bi, 67 Cu, 186 Re, 188 Re , and 212 Pb.
  • nucleic acid molecules are covalently linked to lysine or cysteine on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.
  • Conjugation methods using engineered cysteines or incorporating unnatural amino acids have been reported to improve the homogeneity of the conjugates.
  • acyl-donor glutamine-containing tags eg Gin peptide-containing tags or Q-tags
  • polypeptide engineering eg by amino acid deletions, insertions, substitutions or mutations on polypeptides
  • Transglutaminase can then be covalently cross-linked with an amine-donating agent (eg, a small molecule comprising or linked to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, wherein Amine donor agents are site-specifically coupled to Fc-containing polypeptides via an acyl-donor glutamine-containing tag or an accessible/exposed/reactive endogenous glutamine (WO2012059882).
  • an amine-donating agent eg, a small molecule comprising or linked to a reactive amine
  • the therapeutic agents according to the described embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable formulations can be found in the pharmacopoeia known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), especially Chapter 87 in Blaug, Seymour.
  • Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (eg, Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.
  • the antibody can be used as a therapeutic agent.
  • agents will typically be used to treat, alleviate and/or prevent a disease or pathology associated with aberrant Claudin 18.2 expression, activity and/or signaling in a subject.
  • Treatment regimens can be implemented using standard methods by identifying a subject, such as a human patient having (or at risk or developing) a disease or disorder associated with aberrant Claudin 18.2 expression, activity and/or signaling, such as a Claudin 18.2-related disorder .
  • An antibody preparation preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target.
  • the administered antibody may abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (eg, Claudin 18.2).
  • Administered antibodies can abolish or inhibit or prevent the target (eg, Claudin 18.2) from binding to the endogenous ligand to which it naturally binds.
  • an antibody binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes and/or otherwise interferes with Claudin 18.2 expression, activity and/or signaling.
  • an antibody having heavy and light chain CDRs can be administered to a subject.
  • antibodies to Claudin 18.2 can be used in methods known in the art related to the localization and/or quantification of Claudin 18.2 (eg, for the determination of Claudin 18.2 and/or Claudin 18.2 in appropriate physiological samples) levels, for diagnostic methods, for protein imaging, etc.).
  • an antibody specific for Claudin 18.2 or a derivative, fragment, analog or homolog thereof, comprising an antibody-derived antigen binding domain is used as a pharmaceutically active compound (hereinafter referred to as as "therapeutic agent").
  • Claudin 18.2 polypeptides can be isolated by standard techniques such as immunoaffinity, chromatography or immunoprecipitation using antibodies specific for Claudin 18.2.
  • Antibodies to the Claudin 18.2 protein (or fragments thereof) can be used to detect the protein in biological samples.
  • Claudin 18.2 can be detected in biological samples as part of a clinical testing procedure, eg, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase;
  • suitable prosthetic complexes include streptavidin/biotin and avidin/ Biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, or phycoerythrin;
  • luminescent materials include minocycline;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • antibodies according to the present disclosure can be used as reagents for detecting the presence of Claudin 18.2 or a protein fragment thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Whole antibodies or fragments thereof (eg Fab, scFv or F(ab') 2 ) are used.
  • labeling in reference to an antibody is intended to include direct labeling of the antibody by conjugating (ie, physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody, and end-labeling the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject.
  • biological sample is used to include blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection methods of the described embodiments can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • Analyte protein in vitro detection techniques include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence.
  • In vitro detection techniques for analyte genomic DNA include Southern hybridization.
  • in vivo detection techniques for analyte proteins include introducing into a subject a labeled anti-analyte protein antibody. For example, an antibody can be labeled with a radiolabel, and the presence and location of the radiolabel in a subject can then be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • the principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art.
  • compositions typically comprise the antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain may be preferred.
  • peptide molecules can be designed that retain the ability to bind target protein sequences. Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard bibliography in the art, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or reagents that are incompatible with the antibody, its use in compositions is contemplated.
  • compositions of the embodiments are formulated to be compatible with their intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile injectable diluents such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate Or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin to maintain the desired particle size in the case of dispersions, and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation are vacuum drying and freeze-drying to obtain a powder containing the active ingredient and any additional desired ingredient from a sterile-filtered solution of those previously enumerated. .
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a gas of a suitable propellant, such as carbon dioxide, or a nebulizer.
  • a gas of a suitable propellant such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the permeation barrier are used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • one or more of the antibodies may be formulated into an ointment, ointment, gel, or cream as generally known in the art.
  • the compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • the antibody may be prepared with a carrier that will prevent its rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.
  • Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier one or more of the antibodies.
  • the specifications for dosage unit forms of the embodiments are dictated by and directly dependent upon the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulation of such antibodies for the treatment of individuals.
  • the pharmaceutical composition can be placed in a container, pack, or dispenser with instructions for administration.
  • compositions described herein may also contain more than one such antibody, preferably those having complementary activities but not negatively affecting each other, depending on the particular condition to be treated.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • agents that enhances its function such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • Such molecules are suitably combined in amounts effective for the intended purpose. For example, it can be combined in a kit, and can also be combined in use.
  • one or more of the antibodies may be administered in combination therapy, ie, with other agents such as therapeutic agents (which are useful in the treatment of pathological conditions or disorders, eg, various forms of cancer, autoimmune disorders and inflammatory diseases).
  • agents such as therapeutic agents (which are useful in the treatment of pathological conditions or disorders, eg, various forms of cancer, autoimmune disorders and inflammatory diseases).
  • the term "combination” as used herein refers to the administration of the agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, at the start of administration of the second compound, the first of the two compounds is still preferably detected at an effective concentration at the treatment site. In one instance, “combination” can also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.
  • a combination therapy can comprise one or more antibodies described herein with one or more additional therapeutic agents (eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors) , enzyme inhibitors, and/or cytotoxins or cytostatics, as described in more detail below) are co-formulated and/or co-administered.
  • additional therapeutic agents eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors
  • enzyme inhibitors e.g., enzyme inhibitors, and/or cytotoxins or cytostatics
  • Claudin 18.1 (UniProtKB-P56856, SynbioTech, its amino acid sequence is shown as SEQ ID NO: 21) as template, use upstream primer PRIMER 18.1-FO (SEQ ID NO: 23) and downstream primer PRIMER 18BA (SEQ ID NO: 23) :24) Amplify human Claudin 18.1 full-length fragment (Met1-Val261), and the PCR product was subcloned into pCMV3 eukaryotic expression plasmid (self-constructed eukaryotic expression vector, containing CAG promoter, multiple cloning site, prokaryotic selection marker Amp , eukaryotic selection marker puromycin and replication site and other originals, which were synthesized in SynbioTech sequence) to obtain pCMV3-Claudin 18.1 plasmid.
  • pCMV3 eukaryotic expression plasmid self-constructed eukaryotic expression vector, containing CAG promoter, multiple cloning site, prokaryotic
  • the upstream primer PRIMER 18.2 FO SEQ ID NO: 25
  • the downstream primer PRIMER was used as the template.
  • the full-length fragment of human Claudin 18.2 (Met1-Val261) was amplified by 18BA, and the PCR product was subcloned into the pCMV3 eukaryotic expression plasmid to obtain the pCMV3-Claudin 18.2 plasmid.
  • HEK293 and NIH-3T3 cells were transfected with pCMV3-Claudin 18.1 and pCMV3-Claudin 18.2 plasmids, respectively, and 1-10 ⁇ g/mL puromycin (Gibco#A1113803) was used for stepwise pressure screening to obtain HEK293-Claudin 18.1 stably transfected cells, HEK293-Claudin 18.2 stably transfected cells, NIH3T3-Claudin 18.1 stably transfected cells, and NIH3T3-Claudin 18.2 stably transfected cells.
  • KATO III cells (ATCC HTB-103) expressing Claudin 18.2 were used as positive controls.
  • the results showed that both HEK293-Claudin 18.2 stably transfected cells and KATO III cells could amplify a Claudin 18.2-specific 780 bp characteristic band and a 504 bp Claudin 18 common fragment band, while HEK293-Claudin 18.1 stably transfected cells could only amplify A Claudin 18 common fragment of 504 bp was obtained (Fig. 1).
  • the constructed HEK293-Claudin 18.2 and NIH3T3-Claudin 18.2 cells were stably transfected and incubated with 1:200 rabbit anti-Claudin 18.2 antibody (Abcam, #ab222512) for 1 hour at 4°C, and the excess primary antibody was washed away with 0.5% BSA/PBS. solution, and then add 50 ⁇ L of goat anti-rabbit IgGFc-AF647 (Jackson ImmunoResearch, #111-606-046), and incubate at 4°C for 45 minutes.
  • FACS test showed that the screened HEK293-Claudin 18.2 stably transfected cells highly expressed Claudin 18.2 on the surface ( Figure 2). 3).
  • HEK293-Claudin 18.2 stably transfected cells were mixed with an equal volume of TiterMax (Sigma, #T2684) and injected into the thigh root and footpad of female Balb/c mice for 6-8 weeks, followed by 20 ⁇ g pCMV3-Claudin two weeks later. 18.2 After the plasmid was mixed with gold powder (Biorad, #1652264), the mouse abdomen was directly bombarded with a gene gun, once a week, for 3 weeks. Before fusion, the cells were stably transfected with NIH3T3-Claudin 18.2 high expression, and pulsed by tail vein injection (1 ⁇ 10 6 cells/50 ⁇ L/cell).
  • lymph node and spleen were collected, ground in DMEM and centrifuged to obtain a cell suspension.
  • An appropriate amount of lymph node and spleen cell suspension was mixed with SP2/0 cells, and the hybridoma was prepared by cell fusion using an electrofusion apparatus.
  • HEK293-Claudin 18.2 stably transfected cell line, HEK293-Claudin 18.1 stably transfected cell line, and HEK293 cells were used for FACS positive and negative screening. Live cells were pre-stained with HEK293-Claudin 18.1, HEK293-Claudin 18.2, and HEK293 at 5 ⁇ M, 0.5 ⁇ M, and 0 ⁇ M Cell Tracker Green CMFDA Dye (Thermo, #C2925), respectively, according to the manufacturer’s instructions. mix 1 and added to 96-well plates (2x10 5 cells / well) and combined with hybridoma supernatants, the ice bath for 1 hour.
  • the secondary antibody AlexaFluro647-labeled goat anti-mouse IgG Fc was added, and FACS detection (iQue Screener) was performed after incubation on ice for 45 minutes. According to the fluorescence intensity of the FL1 channel, HEK293-Claudin 18.1, HEK293-Claudin 18.2 and HEK293 were circled respectively After three different cell populations, the binding of the antibody to be tested in the FL4 channel to each cell population was detected.
  • the above positive clones were further screened for antibodies that could be used for histochemical detection.
  • the cells for screening were pretreated as follows: collect HEK293-Claudin 18.2 cells in logarithmic growth phase stably, wash off the medium with PBS, add neutral formalin for 2 hours at 4°C, centrifuge at 1000 rpm for 5 minutes, Discard the supernatant; use siliconized EP tubes to dehydrate the cells sequentially from 75% ethanol, 85% ethanol, 90% ethanol and 100% ethanol for 40 minutes each time; soak the dehydrated cells in xylene for 40 minutes each time min, 2 times in total; finally, cells were hydrated in gradients of 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and double distilled water for 2 min each.
  • the treated cells were subjected to FACS detection according to the aforementioned method, and multiple strains of
  • the positive clones 3H2-C5, 4F6-G11 and 7E8-F3 were expanded into 6-well plates, cultured with low IgG serum (Gibco, #16250), and the supernatant was collected after the cells were confluent, and protein A/G filler was used for Purification, and the purified antibody was verified on paraffin sections of Claudin 18 stably transfected cells.
  • the cut paraffin sections were baked at 65°C for 1 hour, then deparaffinized in xylene for 2 times for 10 minutes each, and finally hydrated in 100%, 95%, 85%, and 75% ethanol gradients in sequence for 2 minutes each.
  • 3H2-C5 and 4F6-G11 can be used for immunohistochemistry of paraffin sections, wherein 3H2-C5 specifically recognizes Claudin 18.2 but not Claudin 18.1, while 4F6-G11 recognizes both Claudin 18.1 and Claudin 18.2 ( Figure 4).
  • TRNzol to lyse the hybridoma cells and extract total RNA, use this as a template to synthesize the first-strand cDNA, use the first-strand cDNA as a subsequent template to amplify the variable region sequence, and sequence the amplified product to obtain candidate hybridoma cells Heavy and light chain variable region sequences of secreted cells.
  • the heavy chain variable region is inserted into the vector containing the murine heavy chain constant region
  • the light chain variable region is inserted into the vector containing the murine light chain constant region, and mixed.
  • the light and heavy chain expression plasmids were transfected into HEK293EBNA cells and cultured at 37°C with 5% CO 2 for 5-7 days. The supernatant was collected and purified by Protein A chromatography.
  • Sample serial number IHC % positive cells Sample serial number IHC % positive cells 1802945F ++ 51.9 D190279 ++ 29.9 1812502E ++ 51.3 D190655 ++ 38.5 1814387 ++ 33.5 1805278I +++ 39.5 1816264 ++ 50.0 1806648R + 43.6

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Abstract

本公开涉及一种结合紧密连接蛋白-18.2的抗体或其抗原结合部分及其用途。该抗体可高亲和性地结合紧密连接蛋白-18.2，可用于检测紧密连接蛋白-18.2蛋白，诊断、治疗或预防与紧密连接蛋白-18.2的细胞相关的疾病。

Description

一种结合紧密连接蛋白-18.2的抗体及其用途 技术领域
本公开涉及一种结合紧密连接蛋白-18的抗体及其用途，特别是结合紧密连接蛋白-18.2的抗体及其用途。
背景技术
紧密连接蛋白(Claudin)是一类建立细胞旁屏障并控制细胞间分子流动的细胞表面蛋白家族，由Shorichiro Tsukita等人在1998年首次报道(Furuse et al.，(1998)J Cell Biol 141(7):1539-1550)。Claudin是紧密结合的必需成分，在保持上皮细胞极性、控制细胞旁扩散以及调控细胞生长分化方面起到重要作用。
紧密连接蛋白-18(Claudin 18)属于紧密连接蛋白Claudin家族，有四个跨膜结构域，N端和C端位于胞内，具有两个胞外区。由于外显子1的选择性剪切，Claudin 18存在两种剪接异构体Claudin 18.1和Claudin 18.2，均由261个氨基酸组成，Claudin 18.2在N端有不同序列，其中第一胞外区中仅8个氨基酸不同。在人体中，Claudin 18.1和Claudin 18.2的表达具有组织特异性，Claudin 18.1主要表达在肺、胃和膀胱等正常组织中；Claudin 18.2作为一个高度特异性的细胞表面分子，在正常的组织中仅表达在分化的胃粘膜上表皮细胞上，不表达在胃干细胞上，而在原发性胃癌、胰腺癌、食管癌、卵巢癌、肺癌及其转移灶中高表达，因此，Claudin 18.2在肿瘤上的频繁过表达使该分子有资格成为用于开发针对Claudin 18.2的治疗剂(如疫苗治疗剂和治疗性抗体)的极具潜力的肿瘤治疗新靶点。
实现肿瘤样本免疫组化(immunohistochemistry，IHC)染色的准确检测需要高度特异的肿瘤标志物抗体。在早期研究中，曾使用识别Claudin 18中段序列的兔抗体，检测到原发性胃癌中56％为中等至强阳性(≥IHC 2+)(Sahin et al.,(2008)Clinical Cancer Research,14(23),7624-7634)；而使用商业化抗体(克隆EPR19202,Abcam)检测，Claudin 18.2中等至强阳性(≥IHC 2+)仅约14.1％，弱阳性(≥IHC 1+)为13.3％(Dottermusch et al.,(2019)Virchows Archiv,475(5):563-571)。另一个在临床研究中使用的是鼠单抗41-14A，但该抗体针对Claudin 18C端共同序列的抗体，不能区分临床样本Claudin 18.2和Claudin 18.1的表达(US9512232B2)。
发明内容
本发明利用杂交瘤技术，获得多个识别紧密连接蛋白-18的单克隆抗体，特别是高亲和力结合紧密连接蛋白-18.2(Claudin 18.2)的单克隆抗体。高亲和力结合紧密连接蛋白-18.2的抗体可用于IHC检测，可对紧密连接蛋白-18.2阳性肿瘤细胞的分布、细胞表达和靶向治疗效果提供判断依据。
因此，在一方面，本公开提供了结合紧密连接蛋白-18.2蛋白的抗体或其抗原结合部分。
在一方面，本公开提供了包含前述方面的抗体或其抗原结合部分的双特异性或多特异性分子
在一方面，本公开提供了编码如前述方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸。
在一方面，本公开提供了包含前述方面的核酸的载体。
在一方面，本公开提供了包含前述方面的载体的细胞。
根据前述任一方面的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分是人源化的。
在一方面，本公开提供了包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物或试剂盒。
在一方面，本公开提供了包含共价附着至治疗部分的前述任一方面所述的抗体或其抗原结合部分、双特异性或多特异性分子的抗体-药物偶联物。
在一方面，本公开提供了治疗紧密连接蛋白-18.2相关病症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段、核酸、载体、细胞和/或药物组合物。
在一方面，本公开提供了前述任一方面的抗体或其抗原结合片段、核酸、载体、细胞和/或药物组合物在制备用于治疗哺乳动物中紧密连接蛋白-8.2相关病症的药物或试剂盒中的用途。
本公开的抗体可以用于多种应用，包括检测紧密连接蛋白-18.2蛋白，诊断、治疗或预防与紧密连接蛋白-18.2相关的疾病。
附图说明
图1示出了Claudin 18稳转细胞株RT-PCR检测结果。
图2示出了HEK293-Claudin 18.2稳转细胞株FACS检测结果，其中黑色的阴影为阴性对照，灰色的阴影为HEK293-Claudin 18.2稳转细胞。
图3示出了NIH3T3-Claudin 18.2稳转细胞FACS检测结果，其中黑色的线为阴性对照，灰色为NIH3T3-Claudin 18.2稳转细胞。
图4示出了石蜡切片免疫组化检测结果。
图5示出了流式细胞检测重组抗体3H2-C5和4F6-G11与细胞表面Claudin 18.2的亲和力。
图6示出了抗体3H2-C5对部分胃癌组织切片的免疫组化检测结果。
具体实施方式
I.定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
在一个方面，本文提供的是特异性结合Claudin的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合Claudin的单克隆抗Claudin抗体，其中所述抗Claudin抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合Claudin(例如，人Claudin)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗Claudin抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。
本文使用的术语“Claudin”是指紧密连接蛋白，并包括Claudin 18.2。优选地，紧密连接蛋白是人类紧密连接蛋白。
术语“Claudin 18”涉及紧密连接蛋白18并包括任何变体，这包括紧密连接蛋白18剪接变体1(紧密连接蛋白18.1(Claudin 18.1))和紧密连接蛋白18剪接变体2(紧密连接蛋白18.2(Claudin 18.2))。
术语“Claudin 18.2”优选涉及人类Claudin 18.2。
术语“与紧密连接蛋白-18.2的细胞相关的疾病”或相似表达意指根据本公开，Claudin 18.2在患病组织或器官的细胞中表达。
术语“细胞表面”的使用是根据其在本领域的正常含义，因此包括可通过与蛋白和其它分子结合而接近的细胞外部。
如果Claudin 18.2位于细胞表面，则Claudin 18.2在细胞表面表达，并且可通过加入细胞的Claudin 18.2特异性抗体进行结合而接近。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M-1的Ka)抗原的抗体。“功能片段”或“抗Claudin抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab') 2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(V H-V L二聚体)组成。在该构型 中，每个可变结构域的三个CDRs相互作用，以确定V H-V L二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab') 2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本公开所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x10 8M -1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7，229，619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
亦提供本文所述序列表中所述序列的“保守序列修饰”，即不消除由核苷酸序列编码或含有氨基酸序列的抗体 与抗原的结合的核苷酸及氨基酸序列修饰。这些保守序列修饰包括保守核苷酸及氨基酸取代以及核苷酸及氨基酸添加及缺失。例如，可通过本领域已知的标准技术(例如定点诱变及PCR介导的诱变)将修饰引入本文所述的序列表中。保守序列修饰包括保守氨基酸取代，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族是本领域中已有定义的。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、具有β分枝侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗Claudin抗体中的预测的非必需氨基酸残基优选被来自同一侧链家族的另一氨基酸残基替代。鉴定不消除抗原结合的核苷酸及氨基酸保守取代的方法为本领域所熟知(例如，参见Brummell et al.,Biochem.32:1180-1187(1993)；Kobayashi et al.,Protein Eng.12(10):879-884(1999)；Burks et al.,Proc.Natl.Acad.Sci.USA 94:412-417(1997))。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗Claudin抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗Claudin抗体。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
II.具体实施方式
在一方面，本公开提供了结合Claudin 18.2的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2-4、12-14或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:7-9、17-19或任何变体的轻链CDR。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2、12或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:3、13或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:4、14或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:7、17或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:8、18或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:9、19或其任何变体的轻链CDR3。
根据前一方面的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
(1)分别包含SEQ ID NO:2-4的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:7-9的轻链CDR1、CDR2及CDR3序列；
(2)分别包含SEQ ID NO:12-14的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:17-19的轻 链CDR1、CDR2及CDR3序列。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:1、11或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:6、16或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合Claudin 18.2的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:1或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:6或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合Claudin 18.2的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:11或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:16或其任何变体的轻链可变区。
在一方面，本公开提供了一种双特异性或多特异性分子，其包含前述任一方面的抗体或其抗原结合部分。
编码根据前述任一方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸分子。优选地，所述核酸分子包含选自SEQ ID NO:5、15或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:10、20或其任何变体的抗体轻链核酸序列。
结合Claudin 18.2的抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
编码如前述任一方面的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子。
包含如前述任一方面的核酸的载体。
包含如前述任一方面的载体的细胞。
包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物。
本公开的抗体在各种Claudin 18.2被不利地表达或发现的疾病中可用作治疗或诊断工具。
在一个与紧密连接蛋白-18.2相关的疾病实施方案中，Claudin 18.2在患病组织或器官的细胞中的表达与在健康组织或器官中的状态相比有所增加。增加是指增加至少10％、特别是至少20％、至少50％、至少100％、至少200％、至少500％、至少1000％、至少10000％或甚至更多。在一个实施方案中，表达仅在患病组织中发现，而在相应健康组织中的表达受阻抑。根据本公开，与紧密连接蛋白-18.2相关的疾病包括肿瘤，优选地，所述肿瘤为癌症疾病。此外，根据本公开，癌症疾病优选为其中癌细胞表达Claudin 18.2的那些疾病。“癌症疾病”的特征为表达Claudin 18.2的细胞并且癌细胞表达Claudin 18.2。所述癌症疾病如胃癌、食管癌、胰腺癌、肺癌如非小细胞肺癌(NSCLC)、卵巢癌、结肠癌、肝癌、头颈癌以及胆囊癌及其转移，特别是胃癌转移如卵巢克鲁根勃氏瘤、腹膜转移和淋巴结转移。特别优选的癌症疾病为胃的腺癌、食道的腺癌、胰腺管的腺癌、胆管的腺癌、肺的腺癌和卵巢的腺癌。表达Claudin 18.2的细胞优选为癌细胞，优选为本文所述癌症的癌细胞。
用本公开的Claudin 18.2抗体治疗的疾病和症状方法包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合Claudin 18.2的抗体，其中施用所述抗体以提供至少40μg/ml的血清水平。在不同实施方案中，施用所述抗体以提供至少50μg/ml、至少150μg/ml、至少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用所述抗体以提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或者300μg/ml至500μg/ml。如本说明书中使用的术语“血清水平”，其意指所讨论的物质在血清中的浓度。在一个实施方案中，提供所述血清水平至少7天或至少14天。在一个实施方案中，所述方法包括施用至少300mg/m 2的抗体剂量，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合Claudin 18.2的抗体，其中以至少300mg/m 2，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2的剂量施用所述抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合Claudin 18.2的抗体，其中所述患者的至少50％，优选为60％、70％、80％或90％的癌细胞为Claudin 18.2阳性和/或所述患者的至少40％，优选为50％或60％的癌细胞为Claudin 18.2的表面表达阳性。在该方面，本公开还提供治疗或预防癌症疾病的方法，所述方法包括：a.鉴定显示至少50％，优选为60％、70％、80％或90％的Claudin 18.2阳性癌细胞和/或至少40％，优选为50％或60％的癌细胞的患者，所述癌细胞为Claudin 18.2的表面表达阳性；以及b.向所述患者施用能够结合Claudin 18.2的抗体。在一个实施方案中，所述患者的至少95％或至少98％的癌细胞为Claudin 18.2阳性的。在一个实施方案中，所述患者的至少70％、至少80％或至少90％的癌细胞为Claudin 18.2的表面表达阳性。
在本文所述的任何方面的方法的一个实施方案中，癌症疾病的治疗结果是实现病情稳定。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在一些实施方案中，本公开提供实现癌症患者的病情稳定的方法，其包括向所述患者施用能够结合Claudin 18.2的抗体。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在本文所述的任何方面的方法的一个实施方案中，以单剂量或多剂量施用抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合Claudin 18.2的抗体，其中以多剂量施用所述抗体。
如果根据本公开以多次剂量施用所述抗体，则优选以至少3次剂量、至少4次剂量、至少5次剂量、至少6次 剂量、至少7次剂量、至少8次剂量、至少9次剂量或至少10次剂量且优选至多30次剂量、25次剂量、20次剂量、15次剂量或10次剂量施用所述抗体。优选以至少7天、至少10天、至少14天或至少20天的时间间隔施用所述抗体的剂量。优选以7至30天、10至20天且优选为约14天的时间间隔施用所述抗体的剂量。
在一个实施方案中，施用所述抗体以便提供至少40μg/ml的血清水平。在不同实施方案中，施用所述抗体以便提供至少50μg/ml、至少150μg/ml、至少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用所述抗体以便提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或300μg/ml至500μg/ml。在一个实施方案中，提供所述血清水平至少7天或至少14天。在一个实施方案中，所述方法包括施用至少300mg/m 2，如至少600mg/m 2且优选至多1500mg/m 2、至多1200mg/m 2或至多1000mg/m 2的抗体的剂量。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中Claudin 18.2相关病症的药物中的用途。
根据前述任一方面，任选地，所述抗体偶联其他药物，例如带标记或具有细胞毒性的缀合物。
在一方面，本公开还包括试剂盒，例如所述试剂盒包括本公开的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备抑制Claudin 18.2结合的试剂中的用途。
此外，本公开的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本公开还提供包含本公开的抗Claudin 18.2的抗体或其片段的组合物，所述抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本公开所使用的术语“药物组合物”系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
本公开的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
在一些实施方案中，本公开的人源化抗体与治疗部分(即药物)偶联。治疗部分可以是例如细胞毒素、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素。此类偶联物在本文中称为“抗体-药物偶联物”或“ADC”。
在一些实施方案中，抗体与细胞毒性部分偶联。细胞毒性部分可以例如选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素。
在一些实施方案中，抗体与奥瑞他汀或其肽类似物、衍生物或前药偶联。已经表明，奥瑞他汀干扰微管动力学、GTP水解以及核和细胞分裂并具有抗癌和抗真菌活性。例如，奥瑞他汀E可以与对乙酰基苯甲酸或苯甲酰基戊酸反应，分别产生AEB和AEVB。其他典型的奥瑞他汀衍生物包括AFP、MMAF(单甲基奥瑞他汀F)和MMAE(单甲基奥瑞他汀E)。合适的奥瑞他汀和奥瑞他汀的类似物、衍生物和前药，以及用于将奥瑞他汀与Ab偶联的合适接头描述于例如美国专利号5,635,483、5,780,588和6,214,345以及国际专利申请公开WO02088172、WO2004010957、 WO2005081711、WO2005084390、WO2006132670、WO03026577、WO200700860、WO207011968和WO205082023。
在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)或其肽类似物、衍生物或前药偶联。合适的PDB以及PDB衍生物和相关技术描述于例如Hartley J.A.等,Cancer Res 2010；70(17):6849-6858；Antonow D.等,Cancer J 2008；14(3):154-169；Howard P.W.等,Bioorg Med Chem Lett 2009；19:6463-6466和Sagnou等,Bioorg MedChem Lett2000；10(18):2083-2086。
在一些实施方案中，抗体与选自以下的细胞毒性部分偶联：蒽环类抗生素、美登素、卡奇霉素、多卡霉素、雷切霉素(CC-1065)、海兔毒素10、海兔毒素15、伊立替康、单甲基奥瑞他汀E、单甲基奥瑞他汀F、PDB，或它们任何的类似物、衍生物或前药。
在一些实施方案中，抗体与蒽环类抗生素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与美登素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与卡奇霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与多卡霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与雷切霉素(CC-1065)或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素10或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素15或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀E或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀F或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与伊立替康或其类似物、衍生物或前药偶联。
在一些实施方案中，抗体与细胞因子(例如IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、IL-28b、IL-29、KGF、IFNa、IFN3、IFNy、GM-CSF、CD40L、Flt3配体、干细胞因子、安西司亭和TNFa)偶联。
在一些实施方案中，抗体与放射性同位素或含放射性同位素的螯合物偶联。例如，抗体可以与允许抗体与放射性同位素络合的螯合剂接头(例如DOTA、DTPA或噻西坦)偶联。抗体还可以或可选地包含一个或多个放射性标记的氨基酸或其他放射性标记的分子或与之偶联。放射性同位素的非限制性实例包括 3H、 14C、 15N、 35S、 90Y、 99Tc、 125I、 131I、 186Re、 213Bi、 225Ac和 227Th。为了治疗目的，可以使用发射β或α颗粒辐射的放射性同位素，例如 131I、 90Y、 211At、 212Bi、 67Cu、 186Re、 188Re和 212Pb。
将分子与抗体偶联的技术是本领域熟知的。通常，核酸分子分别通过N-羟基琥珀酰亚胺酯或马来酰亚胺官能团与抗体上的赖氨酸或半胱氨酸共价连接。据报道，使用工程化半胱氨酸或整合非天然氨基酸的偶联方法可改进偶联物的同质性。特别地，本领域技术人员还可以预期用含酰基供体谷氨酰胺的标签(例如含有Gin肽的标签或Q-标签)或通过多肽工程(例如通过多肽上的氨基酸缺失、插入、取代或突变)产生反应性的内源性谷氨酰胺工程化的含Fc多肽。然后，转谷氨酰胺酶可与胺供体剂(例如，包含或连接于反应性胺的小分子)共价交联，以形成稳定且均质的工程化含Fc多肽偶联物群，其中胺供体剂通过含酰基供体谷氨酰胺标签或可接近/暴露/反应性的内源性谷氨酰胺位点特异性地与含Fc多肽偶联(WO2012059882)。
应当理解，根据所述实施方案的治疗剂将与合适的药学上可接受的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。大量适当的制剂可见于所有药物化学工作者已知的药典中：Remington's Pharmaceutical Sciences(第15版，Mack Publishing Company，Easton，Pa.(1975))，特别是其中Blaug、Seymour的第87章。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如Lipofectin TM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本公开的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
在一个实施方案中，可将所述抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常Claudin 18.2表达、活性和/或信号传导相关的疾病或病理。可使用标准方法通过鉴定受试者，例如患有(或处于风险或发展)与异常Claudin 18.2表达、活性和/或信号传导相关的疾病或障碍，例如Claudin 18.2相关病症的人患者来实施治疗方案。将抗体制剂，优选对其靶抗原有高特异性和高亲和性的抗体制剂施用给受试者并且将通常因其与靶标结合而产生效应。施用的抗体可消除或抑制或妨碍靶标(例如Claudin 18.2)的表达、活性和/或信号传导功能。施用的抗体可消除或抑制或妨碍靶标(例如Claudin 18.2)与其所天然结合的内源性的配体结合。例如，抗体与靶标结合并调节、阻断、抑制、减少、拮抗、中和/或以其它方式妨碍Claudin 18.2表达、活性和/或信号传导。在一些实施方案中，为治疗与异常Claudin 18.2表达相关的疾病或障碍，可将具有重链和轻链CDR的抗体施用给受试者。
在另一个实施方案中，针对Claudin 18.2的抗体可用于本领域中已知的与Claudin 18.2定位和/或定量相关的方法(例如，用于测定适当生理样品中的Claudin 18.2和/或Claudin 18.2的水平，用于诊断方法，用于蛋白成像等等)。在一个给定实施方案中，对Claudin 18.2或其衍生物、片段、类似物或同系物具有特异性的、包含源于抗体的抗原结合结构域的抗体，被用作药物学活性化合物(下文称为“治疗剂”)。
在另一个实施方案中，可通过标准技术例如免疫亲和、色谱或免疫沉淀，使用对Claudin 18.2具有特异性的抗体来分离Claudin 18.2多肽。针对Claudin 18.2蛋白质的抗体(或其片段)可用于检测生物样品中的蛋白质。在一些实施方案中，在生物样品中可检测Claudin 18.2作为临床测试过程的一部分，例如，用于确定给定治疗方案的功效。将抗体偶联(即物理连接)到可检测物质可有利于检测。可检测物质的示例包括各种酶、辅基、荧光材料、发光材料、生物发光材料和放射性材料。合适的酶的示例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；合适的辅基复合物的示例包括链霉亲和素/生物素和亲和素/生物素；合适的荧光材料的示例包括伞形酮、荧光 素、异硫氰酸荧光素、罗丹明、二氯三嗪氨荧光素、丹磺酰氯或藻红蛋白；发光材料的一个示例包括鲁米诺；生物发光材料的示例包括荧光素酶、荧光素和水母蛋白，并且合适放射性材料的示例包括 125I、 131I、 35S或 3H。
在另一个实施方案中，根据本公开的抗体可用作检测样品中Claudin 18.2或其蛋白质片段存在的试剂。在一些实施方案中，抗体包含可检测标记。抗体为多克隆抗体，或更优选单克隆抗体。使用完整的抗体或其片段(例如Fab、scFv或F(ab') 2)。关于抗体的术语“标记”旨在包括通过将可检测物质偶联(即物理连接)到该抗体来直接标记该抗体，以及通过与直接标记的另一种试剂反应来间接标记该抗体。间接标记的示例包括使用荧光标记的第二抗体检测第一抗体，以及用生物素进行末端标记抗体，以便能够用荧光标记的链霉亲和素进行检测。术语“生物样品”旨在包括从受试者分离的组织、细胞和生物学流体，以及受试者体内存在的组织、细胞和流体。因此，使用的术语“生物样品”包括血液和血液中的级分或组分，包括血清、血浆、或淋巴液。换言之，所述实施方案的检测方法可用于在体外及体内检测生物样品中的分析物mRNA、蛋白质或基因组DNA。例如，分析物mRNA体外检测技术包括Norhtern杂交和原位杂交。分析物蛋白质体外检测技术包括酶联免疫吸附测定(ELISA)、Western印迹、免疫沉淀、以及免疫荧光。分析物基因组DNA体外检测技术包括Southern杂交。此外，分析物蛋白质的体内检测技术包括向受试者体内导入标记的抗分析物蛋白抗体。例如，可以用放射性标记标记抗体，然后可以通过标准成像技术检测受试者体内该放射性标记物的存在和位置。
可将本文所述抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA，90:7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适的药学上可接受的载体描述于最新版的Remington's Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将所述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的药学上可接受的载体包括生理盐水、抑菌水、Cremophor EL TM(BASF，Parsippany，N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在所述组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥，该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种所述抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，所述抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指 用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种所述抗体。所述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
所述药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种所述抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种所述抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本公开的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1构建Claudin 18稳转细胞株
以合成基因Claudin 18.1(UniProtKB-P56856，SynbioTech，其氨基酸序列如SEQ ID NO:21所示)为模板，用上游引物PRIMER 18.1-FO(SEQ ID NO:23)和下游引物PRIMER 18BA(SEQ ID NO:24)扩增人Claudin 18.1全长片段(Met1-Val261)，PCR产物亚克隆到pCMV3真核表达质粒(自主构建的真核表达载体，含CAG启动子、多克隆位点、原核筛选标记Amp、真核筛选标记puromycin和复制位点等原件，在SynbioTech顺序合成得到)，获得pCMV3-Claudin 18.1质粒。与此类似，以合成基因Claudin 18.2(UniProtKB-P56856-2，SynbioTech，其氨基酸序列如SEQ ID NO:22所示)为模板，用上游引物PRIMER 18.2 FO(SEQ ID NO:25)和下游引物PRIMER 18BA扩增人Claudin 18.2全长片段(Met1-Val261)，PCR产物亚克隆到pCMV3真核表达质粒，获得pCMV3-Claudin 18.2质粒。以pCMV3-Claudin 18.1和pCMV3-Claudin 18.2质粒分别转染HEK293和NIH-3T3细胞，使用1-10μg/mL puromycin(Gibco#A1113803)逐步加压筛选，获得HEK293-Claudin 18.1稳转细胞、HEK293-Claudin 18.2稳转细胞、NIH3T3-Claudin 18.1稳转细胞以及NIH3T3-Claudin 18.2稳转细胞。
从生长起来的抗性细胞中提取总RNA，用SuperScript First-Strand Synthesis System逆转录试剂盒(Thermofisher，#18080051)，逆转录得到cDNA，进行RT-PCR验证。设计Claudin 18.1和Claudin 18.2共同引物KNB14(SEQ ID NO:26)和KNB15(SEQ ID NO:27)，可以扩增第二跨膜区到C端之间区域，特征条带为504bp；设计Claudin 18.2N端特异性引物KNB16(SEQ ID NO:28)，与C端特异引物KNB15可扩增Claudin 18.2全长片段，特征条带为780bp，其中，各引物的序列如表1所示。以表达Claudin 18.2的KATO III细胞(ATCC HTB-103)作为阳性对照。结果显示，HEK293-Claudin 18.2稳转细胞和KATO III细胞均可扩增出Claudin 18.2特异性的780bp特征条带和504bp的Claudin 18共同片段条带，而HEK293-Claudin 18.1稳转细胞只能扩增出504bp的Claudin 18共同片段(图1)。
表1
Figure PCTCN2021104819-appb-000001
将构建的HEK293-Claudin 18.2和NIH3T3-Claudin 18.2稳转细胞，分别与1:200兔抗Claudin 18.2抗体(Abcam，#ab222512)4℃孵育1小时，用0.5％BSA/PBS洗去多余的一抗溶液，再加入50μL山羊抗兔IgGFc-AF647(Jackson ImmunoResearch，#111-606-046)，4℃孵育45分钟。FACS检测显示，筛选得到的HEK293-Claudin 18.2稳转细胞表面高表达Claudin 18.2(图2)，筛选得到的多株NIH3T3-Claudin 18.2稳转细胞分别具有Claudin 18.2低表达、中表达和高表达(图3)。
实施例2抗Claudin18.2单克隆抗体的制备
1)小鼠免疫
将1×10 6HEK293-Claudin 18.2稳转细胞与TiterMax(Sigma，#T2684)等体积混合后，注射6～8周雌性Balb/c小鼠大腿根部和足垫，两周后采用20μg pCMV3-Claudin 18.2质粒与金粉(Biorad，#1652264)混合后，用基因枪直接轰击小鼠腹部，每周一次，持续免疫3周。融合前，用NIH3T3-Claudin 18.2高表达稳转细胞，进行尾静脉注射冲击免疫(1×10 6细胞/50μL/只)。3天后断颈处死，收集腘淋巴结、腹股沟淋巴结、髂淋巴结和脾脏，在DMEM中研磨后离心，得到细胞悬浮液。取适量的淋巴结和脾细胞的混悬液和SP2/0细胞混合后，采用电融合仪进行细胞融合制备杂交瘤。
2)杂交瘤筛选
首轮使用HEK293-Claudin 18.2稳转细胞株、HEK293-Claudin 18.1稳转细胞株、HEK293细胞进行FACS正负筛选。将HEK293-Claudin 18.1、HEK293-Claudin 18.2和HEK293分别以5μM、0.5μM和0μM Cell Tracker Green CMFDA Dye(Thermo，#C2925)按使用说明进行活细胞预染，洗去染料后，按1:1:1混合，加至96孔板(2x10 5细胞/孔)，并与杂交瘤上清结合，冰浴孵育1小时。加入二抗AlexaFluro647标记的山羊抗鼠IgG Fc(Jackson ImmunoResearch)，冰上孵育45分钟后进行FACS检测(iQue Screener)，根据FL1通道荧光强度，分别圈出HEK293-Claudin 18.1、HEK293-Claudin 18.2和HEK293三种不同的细胞群后，检测FL4通道待测抗体与各细胞群的结合情况。
将HEK293-Claudin 18.2稳转细胞经过甲醛固定并脱水化-水化处理后，从上述阳性克隆中进一步筛选可用于组化检测的抗体。筛选用的细胞预处理如下：收集处于对数生长期的HEK293-Claudin 18.2稳转细胞，以PBS洗去培养基，加入中性福尔马林4℃固定2个小时，1000转离心5分钟，弃上清；使用硅化EP管，将细胞从75％乙醇、85％乙醇、90％乙醇和100％乙醇依次脱水处理，每次40分钟；将脱水后的细胞在二甲苯中浸泡，每次40分钟，共2次；最后将细胞在100％乙醇、95％乙醇、85％乙醇、75％乙醇和双蒸水中进行梯度水化，每次2分钟。处理好的细胞按前述方法进行FACS检测，筛选得到多株可识别Claudin 18.2的抗体。
实施例3细胞石蜡切片验证
将阳性克隆3H2-C5、4F6-G11和7E8-F3扩大至6孔板中，使用低IgG血清(Gibco，#16250)进行培养，待细胞长满后收集上清，使用protein A/G填料进行纯化，纯化得到的抗体在Claudin 18稳转细胞的石蜡切片上进行验证。
收集对数生长期的HEK293-Claudin 18.2稳转细胞、HEK293-Claudin 18.1稳转细胞和HEK293细胞，1000转离心5分钟，弃上清，并用PBS洗3遍后，加入10mL的中性福尔马林溶液，置于4℃固定过夜；1000转离心5分钟，弃去上清，从20μl开始逐渐加入HistoGel(Thermo，#HG-4000-012)直至细胞粘稠并凝固，然后对细胞团进行脱水处理：75％乙醇1小时，85％乙醇1小时，90％乙醇40分钟，95％乙醇40分钟，100％乙醇40分钟(3次)脱水结束后进行二甲苯浸泡40分钟，2次；石蜡浸泡，1小时，3次；最后将细胞块放入包埋盒中包埋，待包埋盒中的蜡块自然冷却后，剥离。将蜡块切4～5μm薄片，在42℃水浴中摊片，用载玻片捞片，烘干。
将切好的石蜡切片65℃烘1小时，然后二甲苯中脱蜡2次，每次10分钟，最后100％、95％、85％、75％乙醇依次梯度水化，每个梯度2分钟。使用pH9.0的Tris-EDTA缓冲液微波加热法修复抗原，于室温放冷，加3％H 2O 2阻断内源性过氧化氢酶，然后加5％山羊血清封闭1小时，滴加5μg/mL的一抗，4℃湿盒孵育过夜；第二天取出湿盒室温恢复1小时后，加入HRP标记的二抗山羊抗鼠IgG Fc(Jackson ImmunoResearch，#115-035-071)湿盒孵育半小时，DAB显色15分钟，苏木素复染5分钟，自来水中反蓝10分钟，80％、95％、100％酒精梯度脱水，每个梯度2分钟，二甲苯透明2次，每次5分钟，置于通风橱中自然风干后封片拍照。筛选得到的阳性抗体中，3H2-C5和4F6-G11可用于石蜡切片的免疫组化，其中3H2-C5特异性识别Claudin 18.2而不识别Claudin 18.1，而4F6-G11同时识别Claudin 18.1和Claudin 18.2(图4)。
实施例4杂交瘤序列克隆和重组抗体表达
以TRNzol裂解杂交瘤细胞并提取总RNA，以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板扩增可变区序列，将扩增产物测序后，得到候选杂交瘤细胞所分泌细胞的重链和轻链可变区序列。
3H2-C5：
重链可变区(SEQ ID NO:1)
Figure PCTCN2021104819-appb-000002
核苷酸序列(SEQ ID NO:5)
Figure PCTCN2021104819-appb-000003
轻链可变区(SEQ ID NO:6)
Figure PCTCN2021104819-appb-000004
Figure PCTCN2021104819-appb-000005
核苷酸序列(SEQ ID NO:10)
Figure PCTCN2021104819-appb-000006
4F6-G11：
重链可变区(SEQ ID NO:11)
Figure PCTCN2021104819-appb-000007
核苷酸序列(SEQ ID NO:15)
Figure PCTCN2021104819-appb-000008
轻链可变区(SEQ ID NO:16)
Figure PCTCN2021104819-appb-000009
核苷酸序列(SEQ ID NO:20)
Figure PCTCN2021104819-appb-000010
上述重链和轻链可变区序列片段进行PCR扩增后，将重链可变区插入含有鼠重链恒定区的载体，将轻链可变区插入含有鼠轻链恒定区的载体，混合轻重链表达质粒，转染HEK293EBNA细胞，37℃5％CO 2培养5-7天后，收集上清，采用Protein A层析进行纯化。
实施例5重组表达的抗体可识别细胞表面Claudin 18.2
取对数生长期的HEK293-Claudin 18.2稳转细胞，以5×10 4细胞/孔加入96孔U型板，1100转，离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从600nM起，3倍稀释，共12个梯度)，4℃孵育1小时；孵育结束后，每孔加入140μL 0.5％BSA，洗涤3次；加50μL/孔二抗AlexaFluro647抗鼠IgG(Jackson ImmunoResearch，#109-606-170)，4℃孵育40分钟；最后将每孔细胞重悬于50μL PBS进行流式细胞检测。结果显示，3H2-C5和4F6-G11均高亲和力结合Claudin 18.2稳转细胞，EC 50分别为1.01nM和1.18nM(图5)。
实施例6胃癌组织石蜡切片免疫组化评价
使用重组表达的3H2-C5抗体对35例胃癌组织切片(上海精勤)进行免疫组化评价。将切好的石蜡切片65℃烘1小时，然后二甲苯中脱蜡2次，每次10分钟，最后100％、95％、85％、75％乙醇依次梯度水化，每个梯度2分钟。使用pH9.0的Tris-EDTA缓冲液微波加热法修复抗原，于室温放冷，加3％H 2O 2阻断内源性过氧化氢酶，然后加5％山羊血清封闭1个小时后滴加5μg/mL的3H2-C5 4℃湿盒孵育过夜。第二天取出湿盒室温恢复1小时后，加入IHC二抗(Gene Tech，#GK500710)湿盒孵育半小时，DAB显色5分钟，苏木素复染1分钟，自来水中反蓝10分钟，80％、95％、100％酒精梯度脱水，每个梯度2分钟，二甲苯透明2次，每次5分钟，置于通风橱中自然风干后封片。分别由2位实验人员独立对35例胃癌组织染色结果进行判断，判断标准为：阴性，无着色；+(弱阳性)，任何比例的细胞呈现微弱、不完整膜着色；++(中等阳性)：>10％的细胞呈现弱至中等强度、完整但不均匀膜棕黄着色；+++(强阳性)，>30％的细胞呈现强、完整膜棕褐着色。结果见图6和表2，显示3H2-C5可特异性识别胃癌组织石蜡切片，呈现不同等级的阳性信号。
表2、35例胃癌组织IHC染色结果
样品编号 IHC ％阳性细胞 样品编号 IHC ％阳性细胞
1802945F ++ 51.9 D190279 ++ 29.9
1812502E ++ 51.3 D190655 ++ 38.5
1814387 ++ 33.5 1805278I +++ 39.5
1816264 ++ 50.0 1806648R + 43.6
1901214 ++ 41.7 1807641C ++ 44.8
1903629 +++ 31.1 1811075D ++ 46.9
1906789 ++ 50.4 1812196E +++ 50.4
1917023E +++ 42.0 1816522F ++ 61.0
1917135E +++ 50.3 1903168F ++ 57.9
1917338J + 30.7 1905572J +++ 37.8
1918445H ++ 45.3 1910665F ++ 46.3
1918944F ++ 49.8 1911156F ++ 51.4
1925086 + 32.7 00157 + 52.2
1925087 ++ 50.8 00296 +++ 52.5
1925088 +++ 50.6 00297 +++ 34.3
1925870 ++ 59.4 00444 ++ 34.1
1926155 +++ 55.7 17422 + 33.5
1926369 ++ 52.2      

Claims (13)

  1. 一种结合紧密连接蛋白-18.2的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2-4、12-14或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:7-9、17-19或任何变体的轻链CDR。
  2. 根据权利要求1的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2、12或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:3、13或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:4、14或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:7、17或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:8、18或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:9、19或其任何变体的轻链CDR3。
  3. 根据前一方面的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO:2-4的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:7-9的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO:12-14的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:17-19的轻链CDR1、CDR2及CDR3序列。
  4. 根据权利要求1或2的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:1、11或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:6、16或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:1或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:6或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:11或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:16或其任何变体的轻链可变区。
  5. 一种双特异性或多特异性分子，其包含权利要求1-4任一项所述的结合紧密连接蛋白-18.2的抗体或其抗原结合部分。
  6. 根据权利要求1-4任一项所述的抗体或其抗原结合部分或权利要求5所述的双特异性或多特异性分子，所述抗体或其抗原结合部分是人源化的。
  7. 编码根据前述任一方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸；优选地，所述核酸分子包含选自SEQ ID NO:5、15或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:10、20或其任何变体的抗体轻链核酸序列。
  8. 包含权利要求7所述核酸的载体。
  9. 包含权利要求7所述的核酸或权利要求8所述载体的细胞。
  10. 一种组合物，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸、权利要求8所述的载体和/或权利要求9所述的细胞。
  11. 一种抗体-药物偶联物，其包含共价附着至治疗部分的权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子；
    优选地，所述治疗部分选自细胞毒性部分、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素；
    优选地，所述细胞毒性部分选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素；
    优选地，所述细胞因子选自IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、IL-28b、IL-29、KGF、IFNa、IFN3、IFNy、GM-CSF、CD40L、Flt3配体、干细胞因子、安西司亭和TNFa；
    优选地，所述放射性同位素选自 3H、 14C、 15N、 35S、 67Cu、 90Y、 99Tc、 125I、 131I、 186Re、 188Re、 211At、 212Bi、 212Pb、 213Bi、 225Ac和 227Th。
  12. 一种试剂盒，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸分子、 权利要求8所述的载体、权利要求9所述的细胞、权利要求10所述的组合物和/或权利要求11的抗体-药物偶联物。
  13. 权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸、权利要求8所述的载体、权利要求9所述的细胞、权利要求10所述的组合物和/或权利要求11的抗体-药物偶联物在制备用于诊断、治疗或预防与紧密连接蛋白-18.2相关病症的药物或试剂盒中的用途；优选地，与紧密连接蛋白-18.2相关的疾病包括肿瘤，优选地，所述肿瘤为癌症疾病；优选地，所述癌症疾病选自胃癌、食管癌、胰腺癌、肺癌如非小细胞肺癌(NSCLC)、卵巢癌、结肠癌、肝癌、头颈癌以及胆囊癌及其转移，特别是胃癌转移如卵巢克鲁根勃氏瘤、腹膜转移和淋巴结转移；特别优选的癌症疾病选自胃的腺癌、食道的腺癌、胰腺管的腺癌、胆管的腺癌、肺的腺癌和卵巢的腺癌。
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