WO2021227306A1 - 抗cd73抗体及其用途 - Google Patents

抗cd73抗体及其用途 Download PDF

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WO2021227306A1
WO2021227306A1 PCT/CN2020/112271 CN2020112271W WO2021227306A1 WO 2021227306 A1 WO2021227306 A1 WO 2021227306A1 CN 2020112271 W CN2020112271 W CN 2020112271W WO 2021227306 A1 WO2021227306 A1 WO 2021227306A1
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antibody
antigen
binding fragment
sequence
human
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PCT/CN2020/112271
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French (fr)
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杜靓
张红艳
金理娜
陈亚莉
袁纪军
张振清
缪小牛
黄威锋
贾云莉
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普米斯生物技术(珠海)有限公司
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Priority to CN202080100831.XA priority Critical patent/CN115551888A/zh
Publication of WO2021227306A1 publication Critical patent/WO2021227306A1/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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • 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

Definitions

  • the present invention relates to the field of disease treatment and immunology. Specifically, the present invention relates to anti-CD73 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding them, immunoconjugates, bispecific molecules and pharmaceutical compositions containing them, And their use for enhancing immune response and/or treating tumors.
  • the tumor microenvironment is a dynamic microenvironment, including cancer cells, immune cells, fibroblasts, myofibroblasts, cytokines, blood vessels, and extracellular matrix. Tumors are often under hypoxic conditions, and the environment is also lacking in glucose and other nutrients. In order to survive, cancer cells will reorganize their metabolic mechanisms in such an environment. Among them, adjusting purine metabolism is a very critical step, especially to increase the expression of cluster of differentiation 73 (CD73, also known as extracellular-5'-nucleotidase).
  • CD73 cluster of differentiation 73
  • CD73 is a glycosylphosphatidylinositol-anchored cell surface protein usually expressed on subgroups of endothelial cells and hematopoietic cells (Misumi Y et al. European Journal of Biochemistry 1990; 191(3):563-9). Outside the cell, CD73 and CD39 together regulate the conversion of adenosine triphosphate into adenosine, and the step of dephosphorylation of adenosine monophosphate catalyzed by CD73 into adenosine is the speed determining step on the above-mentioned conversion axis (Resta R et al. Immunological Reviews 1998; 161:95–109.).
  • Adenosine is a signal transduction molecule that mediates its biological effects through several receptors (including A1, A2A, A2B, and A3) that have been extensively studied. It is known that adenosine can regulate the proliferation and migration of many cancers, and extracellular adenosine accumulates in cancerous tissues and constitutes an important mechanism for tumor immune escape (Bin Z. Cancer Research 2010; 70: 6407-6411). Among other effects, adenosine of tumor origin deeply inhibits infiltrating effector T cells through A2A receptors activated by adenylate cyclase.
  • CD73 is expressed on many different tumors, including melanoma, colon cancer, lung cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer and breast cancer.
  • CD73 is an effective prognostic biomarker in solid tumors, and the overexpression of CD73 is associated with shorter patient survival or shorter disease progression-free survival (Rong W et al. Oncotarget 2017; 8(34): 57327-57336) .
  • the expression of CD73 in cancer is associated with increased proliferation, migration, new blood vessel formation, invasiveness, and metastasis.
  • CD73-/- mice are protected from transplanted tumors and spontaneous tumors (John S et al. Cancer Research 2010; 71: 2892-2900).
  • CD73 expression and activity are also associated with weakened T cell responses (Dachuan J et al. Cancer Res 2010; 70:2245-55).
  • CD73 also involves resistance to chemotherapeutics, such as anthracycline antibiotics (Loi, S et al.
  • CD73 can directly and indirectly regulate cancer progression, highlighting its potential as a novel therapeutic target.
  • cancer immune checkpoint suppression drugs have shown good efficacy in a variety of cancer patients.
  • CD73 has shown its potential as a target for anti-tumor therapy.
  • the antibody of the present invention can specifically bind to the membrane-bound CD73 on the surface of tumor cells and the non-membrane surface of CD73, inhibit its enzymatic activity, enhance the immune response, have good anti-tumor activity, and is comparable to known anti-CD73 antibodies. It has better functional characteristics than. Therefore, the antibody of the present invention has the potential to be used in the prevention and/or treatment of tumors, and provides a choice for clinical tumor immunotherapy drugs.
  • the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or Added) sequence;
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 4 or SEQ ID NO: 11, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 A sequence of amino acid substitutions, deletions or additions);
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 5, or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or Added) sequence;
  • VL The light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs):
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 6, or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or Added) sequence;
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 7, or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or Added) sequence; and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 8, or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or Add) sequence.
  • substitutions described in any one of (i)-(vi) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the present invention is capable of binding to human CD73, such as membrane-bound human CD73 and/or soluble human CD73.
  • the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, the antibody or antigen-binding fragment thereof comprising:
  • the following three heavy chain variable region CDRs the VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in any one of SEQ ID NOs: 1, 9, 12, and 13, and
  • VLCDR1, VLCDR2 and VLCDR3 contained in the light chain variable region shown in SEQ ID NO: 2 or 10.
  • the 3 CDRs contained in the heavy chain variable region and the 3 CDRs contained in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system.
  • the antibody or antigen-binding fragment thereof comprises: the VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 1, and the light chain shown in SEQ ID NO: 2 VLCDR1, VLCDR2 and VLCDR3 contained in the variable region.
  • the antibody or antigen-binding fragment thereof comprises: the VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 9, and the light chain shown in SEQ ID NO: 10 VLCDR1, VLCDR2 and VLCDR3 contained in the variable region.
  • the antibody or antigen-binding fragment thereof comprises: the VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 2 VLCDR1, VLCDR2 and VLCDR3 contained in the variable region.
  • the antibody or antigen-binding fragment thereof comprises: the VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 13, and the light chain shown in SEQ ID NO: 10 VLCDR1, VLCDR2 and VLCDR3 contained in the variable region.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or is similar to it.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or has at least about 85%, 90% %, 95%, or 99% sequence identity.
  • the heavy chain variable region includes VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO: 3-5, respectively, and the light chain variable region includes each of SEQ ID NO: VL CDR1, VL CDR2 and VL CDR3 shown in 6-8.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12 or is compared A sequence with at least about 85%, 90%, 95%, or 99% sequence identity
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or has at least about 85%, 90% compared with the amino acid sequence shown in SEQ ID NO: 2. , 95% or 99% sequence identity.
  • the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 shown in SEQ ID NOs: 3, 11, and 5, respectively, and the light chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 shown in SEQ ID NO: 3, 11, and 5, respectively.
  • the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin, and the framework region optionally comprises one or more (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to corresponding murine residues.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence (ie, an amino acid sequence encoded by a human heavy chain germline gene), and a sequence derived from The light chain framework region sequence of the human light chain germline sequence (i.e., the amino acid sequence encoded by the human light chain germline gene), the heavy chain framework region and/or the light chain framework region optionally comprising one or more ( For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to corresponding murine residues.
  • a heavy chain framework region sequence derived from a human heavy chain germline sequence ie, an amino acid sequence encoded by a human heavy chain germline gene
  • the light chain framework region sequence of the human light chain germline sequence i.e., the amino acid sequence encoded by the human light chain germline gene
  • the heavy chain framework region and/or the light chain framework region optionally comprising one or more ( For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a heavy chain germline sequence, and a light chain framework region sequence derived from a light chain germline sequence, the heavy chain
  • the framework region and/or light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Back mutation from human residue to corresponding murine residue.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or is compared A sequence having at least about 85%, 90%, 95%, or 99% sequence identity
  • the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 or having at least about 85%, 90% compared with the amino acid sequence shown in SEQ ID NO: 10 , 95% or 99% sequence identity.
  • the heavy chain variable region includes VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO: 3-5, respectively, and the light chain variable region includes each of SEQ ID NO: VL CDR1, VL CDR2 and VL CDR3 shown in 6-8.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or is compared A sequence having at least about 85%, 90%, 95%, or 99% sequence identity
  • the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 or having at least about 85%, 90% compared with the amino acid sequence shown in SEQ ID NO: 10 , 95% or 99% sequence identity.
  • the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 shown in SEQ ID NOs: 3, 11, and 5, respectively, and the light chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 shown in SEQ ID NO: 3, 11, and 5, respectively.
  • the antibody or antigen-binding fragment thereof of the present invention may further comprise a constant region derived from a mammalian (e.g., murine or human) immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a mammalian (e.g., murine or human) immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), so
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a mammalian (e.g., murine or human) immunoglobulin (e.g., kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has a One or more amino acid substitutions, deletions, or additions (for example, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; for example, 1, 2, 3, 4 Or 5 amino acid substitutions, deletions or additions); and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has conservative substitutions of up to 20 amino acids compared to the sequence from which it is derived (For example, conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, or 5 amino acids).
  • CL light chain constant region
  • the constant region may contain amino acid mutations to alter one or more of the following characteristics of the antibodies of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cells Function or complement function, etc.
  • the function can be changed by replacing at least one amino acid residue in the constant region of the antibody with a different residue, for example, changing the affinity of the antibody to the effector ligand (such as FcR or complement C1q), thereby changing the effector function (such as reducing ).
  • Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC and so on. In some cases, these effector functions are required for therapeutic antibodies; but in other cases, these effector functions may be unnecessary or even harmful, depending on the intended purpose.
  • the antibodies of the present invention or antigen-binding fragments thereof have reduced or even eliminated effector functions (e.g., ADCC and/or CDC activity).
  • the antibody or antigen-binding fragment thereof of the present invention may comprise a variant of the constant region of a human IgG heavy chain that has at least one of the following substitutions compared to the wild-type sequence from which it is derived: At least two or all three: L234F, L235E, P331S (the amino acid positions mentioned above are based on the EU numbering system), see, for example, Acta Cryst. (2008). D64, 700-704.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a human wild-type IgG1 heavy chain constant region.
  • the antibodies of the invention or antigen-binding fragments thereof have ADCC and CDC activities.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the constant region of a human IgG1 heavy chain, which has the following substitutions compared to the wild-type sequence from which it is derived: L234F, L235E , P331S (according to the position of the EU numbering system), such as the heavy chain constant region shown in SEQ ID NO: 19.
  • the antibodies of the invention or antigen-binding fragments thereof have eliminated or reduced ADCC and/or CDC activity.
  • the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, and the variant and the wild-type sequence from which it is derived Compared with basically unchanged effector function.
  • the variant may have up to 20 amino acid conservative substitutions compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions; for example, 1, 2, 3, 4 or 5 conservative substitutions).
  • the antibody or antigen-binding fragment thereof of the present invention comprises a human kappa light chain constant region, such as the light chain constant region shown in SEQ ID NO:20.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain constant region (CH) shown in SEQ ID NO: 19; and/or, the light chain constant region shown in SEQ ID NO: 20 District (CL).
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibodies of the invention are murine antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.
  • the antigen-binding fragment of the present invention is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics:
  • membrane-bound human CD73 (a) Combined with membrane-bound human CD73 or soluble human CD73 or both; for example, the membrane-bound human CD73 is expressed on the surface of tumor cells;
  • CD73 for example, membrane-bound human CD73 or soluble human CD73
  • AMP adenosine monophosphate
  • CD73 Internalization of CD73 into cells (e.g., tumor cells) through antibody-mediated receptor internalization, the cells expressing CD73 on their surface; for example, by FACS or flow cytometry (e.g., Example 7) Said method) an internalization level of at least 10% (for example at least 15%, at least 20% or more) measured;
  • Stimulating an immune response for example, stimulating an immune response against a tumor (such as a tumor expressing CD73);
  • tumors e.g., tumors expressing CD73.
  • the antibody or antigen-binding fragment thereof is 72GB or its antigen-binding fragment, its chimeric antibody, or its humanized antibody, or a functional variant thereof, and the variant substantially retains its The biological function of the derived antibody or its antigen-binding fragment.
  • the antibody or antigen-binding fragment thereof of the present invention may include a variant that differs from the antibody or antigen-binding fragment from which it is derived by only one or more (for example, up to 20 Conservative substitutions of 1, at most 15, at most 10, or at most 5 amino acids) conservative substitutions of amino acid residues, or at least 85%, at least 90%, or at least 95% of the antibody or antigen-binding fragment from which it is derived , At least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and basically retains the above-mentioned biological functions of the antibody or antigen-binding fragment from which it is derived.
  • Conservative substitutions of 1, at most 15, at most 10, or at most 5 amino acids conservative substitutions of amino acid residues, or at least 85%, at least 90%, or at least 95% of the antibody or antigen-binding fragment from which it is derived , At least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and basically retain
  • the antibody of the present invention can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present invention is expressed.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium.
  • the present invention provides an isolated nucleic acid molecule comprising nucleotides encoding the antibody of the present invention or its antigen-binding fragment, or its heavy chain variable region and/or light chain variable region. sequence.
  • the isolated nucleic acid molecule encodes an antibody of the invention or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
  • the isolated nucleic acid molecule comprises the first nucleotide sequence encoding the heavy chain variable region of the antibody of the invention or its antigen-binding fragment, and/or encoding the antibody of the invention or its antigen binding The second nucleotide sequence of the light chain variable region of the fragment.
  • the first nucleotide sequence comprises a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 14, or the sequence described in (b) and (a) The nucleotide sequence is substantially the same sequence (for example, compared with the nucleotide sequence described in (a), a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having One or more nucleotide substitutions), or (c) a sequence that does not differ from the nucleotide sequence described in (a) by more than 3, 6, 15, 30 or 45 nucleotides;
  • the dinucleotide sequence comprises a sequence selected from the group consisting of: (d) the nucleotide sequence shown in SEQ ID NO: 15, or (e) a sequence substantially identical to the nucleotide sequence described in (d) ( For example, compared with the nucleotide sequence described in (d), a sequence having at least about 85%, 90%, 95%, 99%
  • the first nucleotide sequence comprises a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO: 16, or the sequence described in (b) and (a) The nucleotide sequence is substantially the same sequence (for example, compared with the nucleotide sequence described in (a), a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having One or more nucleotide substitutions), or (c) a sequence that does not differ from the nucleotide sequence described in (a) by more than 3, 6, 15, 30 or 45 nucleotides;
  • the dinucleotide sequence includes a sequence selected from the group consisting of: (d) the nucleotide sequence shown in SEQ ID NO: 17, or (e) a sequence substantially identical to the nucleotide sequence described in (d) ( For example, compared with the nucleotide sequence described in (d), a sequence having at least about 85%, 90%, 95%, 99% or higher
  • the first nucleotide sequence comprises a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO: 18, or the sequence described in (b) and (a) The nucleotide sequence is substantially the same sequence (for example, compared with the nucleotide sequence described in (a), a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having One or more nucleotide substitutions), or (c) a sequence that does not differ from the nucleotide sequence described in (a) by more than 3, 6, 15, 30 or 45 nucleotides;
  • the dinucleotide sequence includes a sequence selected from the group consisting of: (d) the nucleotide sequence shown in SEQ ID NO: 17, or (e) a sequence substantially identical to the nucleotide sequence described in (d) ( For example, compared with the nucleotide sequence described in (d), a sequence having at least about 85%, 90%, 95%, 99% or higher
  • the isolated nucleic acid molecule comprises the first nucleotide sequence encoding the heavy chain of the antibody or antigen-binding fragment thereof of the present invention, and/or the light chain encoding the antibody or antigen-binding fragment thereof of the present invention.
  • the second nucleotide sequence of the chain is the first nucleotide sequence encoding the heavy chain of the antibody or antigen-binding fragment thereof of the present invention.
  • the present invention provides a vector (e.g., cloning vector or expression vector) comprising the isolated nucleic acid molecule of the present invention.
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages and the like.
  • the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (such as a human).
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44).
  • a method for preparing the antibody or antigen-binding fragment thereof of the present invention comprises culturing the host cell of the present invention under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and obtaining the host cell from the cultured host The antibody or antigen-binding fragment thereof is recovered from the cell culture.
  • the antibody or antigen-binding fragment thereof of the present invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein).
  • another molecule e.g., another polypeptide or protein.
  • the derivatization (eg, labeling) of the antibody or its antigen-binding fragment will not adversely affect its binding to CD73. Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.
  • the antibody or antigen-binding fragment thereof of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (for example, to form Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (for example, avidin or polyhistidine tag).
  • the antibody or antigen-binding fragment thereof of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • the antibody or antigen-binding fragment thereof of the present invention bears a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radioactive nuclear (E.g., 3 H, 125 I, 35 S, 14 C, or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , Phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridine ester compounds), Magnetic beads (for example, ), calorimetric markers such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex,
  • the detectable label as described above can be detected by methods known in the art.
  • radioactive labels can be detected using photographic film or a scintillation calculator
  • fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting reaction products produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the antibodies of the present invention or antigen-binding fragments thereof can be used to form bispecific or multispecific molecules.
  • the antibody or antigen-binding fragment thereof of the present invention may be a part of a bispecific or multispecific molecule comprising a binding specificity that is different from that of the antibody or antigen-binding fragment thereof of the present invention.
  • the second functional module (for example, the second antibody) can thus bind to at least two different binding sites and/or target molecules.
  • the antibody or antigen-binding fragment thereof of the present invention can be linked to a second antibody or antigen-binding fragment thereof that can specifically bind to any protein that can be used as a potential target for combination therapy.
  • the antibody or antigen-binding fragment thereof of the present invention can be linked (for example, by chemical coupling, gene fusion, non-covalent association or other means) to one or more other Binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).
  • Binding molecules e.g., additional antibodies, antibody fragments, peptides, or binding mimetics.
  • the present invention provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof of the present invention.
  • the bispecific or multispecific molecule specifically binds to CD73 (e.g., membrane-bound human CD73 and/or soluble human CD73), and additionally specifically binds to one or more other targets.
  • CD73 e.g., membrane-bound human CD73 and/or soluble human CD73
  • the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (e.g., a second antibody).
  • a second target e.g., a second antibody
  • the antibody or antigen-binding fragment thereof of the present invention can be linked to a therapeutic agent to form an immunoconjugate. Since immunoconjugates have the ability to selectively deliver one or more therapeutic agents to target tissues (for example, tumor-associated antigens, such as tumors expressing CD73), immunoconjugates can improve the antibody of the present invention or its The therapeutic efficacy of antigen-binding fragments in the treatment of diseases such as cancer.
  • target tissues for example, tumor-associated antigens, such as tumors expressing CD73
  • the present invention provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the therapeutic agent is a cytotoxic agent.
  • the cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells).
  • the therapeutic agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • alkylating agents examples include, but are not limited to, nitrogen mustards (such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethylene imine Classes (such as cetepa, etc.), sulfates and polyols (such as busulfan, dibromomannitol), nitrosoureas (such as carmustine, lomustine, etc.), platinum anti-tumor Agents (such as cisplatin, oxaliplatin, carboplatin, etc.) and so on.
  • nitrogen mustards such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.
  • ethylene imine Classes such as cetepa, etc.
  • sulfates and polyols such as busulfan, dibromomannitol
  • nitrosoureas such as carmustine, lomustine,
  • mitosis inhibitors examples include, but are not limited to, maytansinoids (e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.), taxanes (e.g., Docetaxel, paclitaxel or nanoparticle paclitaxel, etc.), vinca alkaloids (e.g. vindesine sulfate, vincristine, vinblastine or vinorelbine, etc.)
  • maytansinoids e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.
  • taxanes e.g., Docetaxel, paclitaxel or nanoparticle paclitaxel, etc.
  • vinca alkaloids e.g. vindesine sulfate, vincristine, vinblastine or vinorelbine, etc.
  • anti-tumor antibiotics examples include, but are not limited to, actinomycin, anthracycline antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.) , Calicheamicin, becinomycin, etc.
  • antimetabolites examples include, but are not limited to, folate antagonists (e.g. methotrexate, etc.), pyrimidine antagonists (e.g. 5-fluorouracil, fluorouridine, cytarabine, carbohydrate Pecitabine, gemcitabine, etc.), purine antagonists (e.g. 6-mercaptopurine, 6-thioguanine, etc.), adenosine deaminase inhibitors (e.g. cladribine, fludarabine, nelarabine, pens Tatin etc.).
  • folate antagonists e.g. methotrexate, etc.
  • pyrimidine antagonists e.g. 5-fluorouracil, fluorouridine, cytarabine, carbohydrate Pecitabine, gemcitabine, etc.
  • purine antagonists e.g. 6-mercaptopurine, 6-thioguanine, etc.
  • adenosine deaminase inhibitors e.
  • topoisomerase inhibitors examples include but are not limited to (camptothecins and their derivatives (e.g., irinotecan, topotecan, etc.), amsacrine, daunor Amycin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, propylimine, teniposide, etc.
  • tyrosine kinase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitin Ni, imatinib, lapatinib, letutinib, nilotinib, simazanib, sunitinib, vandetanib, etc.
  • radionuclide agents examples include, but are not limited to, I 131 , In 111 , Y 90 , Lu 177 and the like.
  • the therapeutic agent is selected from platinum anti-tumor agents, anthracycline antibiotics, taxanes, nucleoside analogs, camptothecins, and analogs or homologs thereof , And any combination thereof.
  • the antibodies of the invention or antigen-binding fragments thereof are optionally conjugated to the therapeutic agent via a linker.
  • the cytotoxic agent can be coupled to the antibody or antigen-binding fragment thereof of the present invention by using linker technology available in the art.
  • linker technology available in the art.
  • Examples of the types of linkers that have been used to couple cytotoxic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.
  • a linker can be selected that is easy to be cleaved by low pH in the lysosomal compartment or easy to be cleaved by a protease (e.g., a protease that is preferentially expressed in tumor tissues, such as cathepsin, such as cathepsin B, C, D).
  • the antibody or antigen-binding fragment thereof of the present invention can modulate (for example, enhance, stimulate, increase, inhibit, decrease, or neutralize) one or more biological activities of CD73.
  • the antibodies or antigen-binding fragments of the present invention result in one or more of the following: inhibit or reduce the enzymatic activity of CD73; inhibit or reduce the conversion of adenosine monophosphate (AMP) to adenosine; and In the presence of adenosine monophosphate (AMP), it increases the proliferation of anti-CD3/anti-CD28 stimulated T cells (such as CD4+ T cells). Therefore, the antibody or antigen-binding fragment thereof of the present invention can be used as a single agent to inhibit or reduce the enzymatic activity of CD73, thereby achieving the purpose of preventing and/or treating tumors.
  • AMP adenosine monophosphate
  • CD73 may exhibit a synergistic effect with other anticancer drugs.
  • high levels of CD73 gene expression were significantly associated with poor clinical outcomes.
  • HER2/ErbB2-driven breast cancer immunocompetent mouse model The expression of CD73 in tumor cells and host cells significantly inhibited the immune-mediated response mediated by anti-ErbB2 monoclonal antibodies (Martin T et al. Cancer Research 2017; 77(20); 5652–63).
  • anti-CD73 antibody significantly enhances the activity of anti-CTLA-4 antibody and anti-PD-1 antibody in a variety of mouse tumor models, and single-agent therapy and combination therapy both rely on host interferon gamma and cytotoxic T cells;
  • the effect of extracellular adenosine on tumor-infiltrating T cells shows that the receptor activation of adenosine can enhance the expression of PD-1 on tumor-specific cytotoxic T cells and helper T cells (Bertrand A et al. Clin Cancer Res 2013 ; 19(20):5626-5635).
  • Clinical studies have found that the increase in CD73 levels in melanoma patients treated with Pembrolizumab (anti-PD-1) is positively correlated with disease progression.
  • the expression level of CD73 and the expression level of PD-L1 were complementary to each other. It can be seen that the antibodies or antigen-binding fragments of the present invention can also be combined with immune checkpoint inhibitors or tumor-specific antibodies for the prevention and treatment of tumors.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention, and a pharmaceutically acceptable Carriers and/or excipients.
  • the pharmaceutical composition may also include additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers (e.g. gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), anti-angiogenesis agents, cytokines (e.g.
  • GM-CSF GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.
  • molecular targeted drugs for example, CD20 antibody such as rituximab, Her2 antibody such as trastuzumab, VEGF antibody such as bevacizumab, EGFR antibody Such as cetuximab, etc.
  • immune checkpoint inhibitors for example, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, etc.
  • oncolytic virus etc.
  • the additional pharmaceutically active agent is selected from immune checkpoint inhibitors (e.g., PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors), anti- CD39 antibody, anti-A2AR antibody or anti-HER2/ErbB2 antibody.
  • immune checkpoint inhibitors e.g., PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors
  • anti- CD39 antibody e.g., anti-A2AR antibody or anti-HER2/ErbB2 antibody.
  • the antibody or antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention and the additional pharmaceutically active agent are separated Components or provided as a mixture. Therefore, the antibody or antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the pharmaceutical composition comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), glucose Solutions (e.g. 5% glucose), solutions containing surfactants (e.g. 0.01% polysorbate 20), pH buffered solutions (e.g. phosphate buffered solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • glucose Solutions e.g. 5% glucose
  • solutions containing surfactants e.g. 0.01% polysorbate 20
  • pH buffered solutions e.
  • the present invention provides a method for preventing and/or treating tumors in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of the present invention Antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates or pharmaceutical compositions.
  • an antibody or antigen-binding fragment, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention is provided for use in prevention and/or in a subject (e.g., human) Use for treating tumors, or use in preparing a medicament for preventing and/or treating tumors in subjects (such as humans).
  • the tumor expresses CD73.
  • the CD73 may be membrane-bound human CD73 and/or soluble human CD73.
  • the tumor involves tumor cells that express CD73.
  • the CD73 is expressed on the surface of the tumor cell.
  • the tumor is selected from melanoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer And breast cancer.
  • the antibodies of the invention or antigen-binding fragments thereof are administered in combination with a second therapeutic agent or treatment.
  • the second therapeutic agent or treatment may be administered before, at the same time or after the administration of the antibody or antigen-binding fragment thereof of the present invention.
  • the second therapeutic agent is selected from drugs with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • drugs with anti-tumor activity such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • the antibody or antigen-binding fragment thereof of the present invention is administered in combination with a therapeutic agent selected from the group consisting of immune checkpoint inhibitors (eg, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 Inhibitor, LAG-3 inhibitor), anti-CD39 antibody, anti-A2AR antibody or anti-HER2/ErbB2 antibody.
  • a therapeutic agent selected from the group consisting of immune checkpoint inhibitors (eg, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 Inhibitor, LAG-3 inhibitor), anti-CD39 antibody, anti-A2AR antibody or anti-HER2/ErbB2 antibody.
  • the PD-1 inhibitor is selected from PDR001, nivolumab, pembrolizumab, pidclizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP- 224.
  • the PD-L1 inhibitor is selected from FAZ053, Atezolizumab, Aviruzumab, Duvaluzumab, and BMS-936559.
  • the CTLA-4 inhibitor is selected from apilimumab or tramelimumab.
  • the LAG-3 inhibitor is selected from LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767.
  • the second treatment may be any therapy known to be used for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • the present invention provides a method for stimulating an immune response in a subject, the method comprising administering an effective amount of the antibody or antigen-binding fragment thereof of the present invention to a subject in need thereof, Bispecific or multispecific molecules, immunoconjugates or pharmaceutical compositions.
  • Bispecific or multispecific molecules, immunoconjugates or pharmaceutical compositions there is provided the use of the antibody or antigen-binding fragment, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention for stimulating an immune response in a subject, or Use in the preparation of a medicament for stimulating an immune response in a subject.
  • the immune response is a T cell-mediated immune response.
  • the immune response is an immune response against a tumor (e.g., a tumor expressing CDD73).
  • a tumor e.g., a tumor expressing CDD73
  • the subject has a tumor (e.g., a tumor expressing CDD73).
  • the immune response is an immune response to an immunogen.
  • the method further comprises administering an immunogen to the subject.
  • the immunogen is selected from tumor-related antigens (eg, proteins, polypeptides, or carbohydrate molecules), tumor cells, dendritic cells sensitized by the antigen, and any combination thereof.
  • the immunogen is selected from antigens (e.g., proteins, polypeptides, or carbohydrate molecules) associated with pathogens (e.g., viruses), inactivated or attenuated pathogens, and trees sensitized by the antigens. Distinctive cells, and any combination thereof.
  • the present invention provides a method for reducing the level of adenosine in tumor cells expressing CD73, which comprises combining the cells with the antibody or antigen-binding fragment, bispecific or multispecific molecule of the present invention , Immunoconjugate, or pharmaceutical composition contact.
  • the method is used to reduce adenosine levels in CD73-expressing tumor cells in vitro for non-therapeutic purposes.
  • the antibody or its antigen-binding fragment, bispecific or multispecific molecule, immunoconjugate, and pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions , Solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage.
  • a preferred dosage form is injection. Such injection may be a sterile injection solution.
  • a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody of the present invention in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusting agent). , Surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization.
  • the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), Glucose solution (e.g. 5% dextrose), solution containing surfactant (e.g. 0.01% polysorbate 20), pH buffer solution (e.g. phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • Glucose solution e.g. 5% dextrose
  • surfactant e.g. 0.01% polysorbate 20
  • pH buffer solution e.g. phosphate buffer solution
  • Ringer's solution e.g. phosphate buffer solution
  • antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention may be present in the pharmaceutical composition in a unit dosage form for easy administration.
  • the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, Sublingual, eyeball, topical, parenteral, rectal, intralobular, intracytoplasmic reticulum, groin, intravesical, topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral administration (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • the route and/or manner of administration will vary according to the intended purpose.
  • the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention is administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, and pharmaceutical composition of the present invention.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who have already suffered from the disease.
  • the therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, and drug Mode of administration, and other treatments administered at the same time, and so on.
  • the dosage regimen can be adjusted to obtain the best objective response (for example, therapeutic or preventive response).
  • the best objective response for example, therapeutic or preventive response
  • it can be administered as a single dose, it can be administered multiple times over a period of time, or the dose can be reduced or increased proportionally to the urgency of the treatment situation.
  • the subject may be a mammal, such as a human.
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to CD73, and thus can be used to detect the presence or level of CD73 in a sample.
  • the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the present invention bears a detectable label.
  • the kit further includes a second antibody, which specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof.
  • the second antibody further includes a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • an enzyme such as horseradish peroxidase
  • a radionuclide such as a radionuclide
  • a fluorescent dye such as a fluorescent dye
  • a luminescent substance such as a chemiluminescent substance
  • the present invention provides a method for detecting the presence or amount of CD73 in a sample, which includes the following steps:
  • the formation of the complex indicates the presence of CD73 or CD73-expressing cells.
  • the sample is a cell sample, that is, a sample containing cells (eg, tumor cells).
  • the complex is formed between the antibody or antigen-binding fragment thereof and CD73 expressed by the cells in the sample.
  • the antibody or antigen-binding fragment thereof of the present invention also bears a detectable label.
  • a reagent with a detectable label is used to detect the antibody or antigen-binding fragment thereof of the present invention.
  • the method can be used for diagnostic purposes, or for non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).
  • the CD73 is human CD73, such as membrane-bound and/or soluble human CD73.
  • the CD73 is human CD73, such as membrane-bound and/or soluble human CD73.
  • CD73 cluster of differentiation 73
  • CD73 is also called extracellular-5'-nucleotidase, which can convert extracellular 5'monophosphate nucleosides into nucleosides, that is, monophosphate Adenosine (AMP) is converted to adenosine.
  • AMP monophosphate Adenosine
  • CD73 includes membrane-bound forms (also called membrane-bound CD73) or soluble forms (also called soluble or non-membrane-bound CD73). CD73 can be isolated from cells or tissues that naturally express them, or produced recombinantly using techniques well known in the art. The sequence of CD73 is well known in the art, please refer to NCBI database accession number NM_002526.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • the assignment of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 -917; Definition of Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • Each of the variable regions of the heavy chain and the light chain contains three CDRs, named CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat, Chothia or IMGT numbering system.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), and such polypeptides, which contain enough to confer specific antigen binding to the polypeptide At least part of an antibody that is capable.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody composed of two “full-length heavy chains” and two “full-length light chains.”
  • full-length heavy chain refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • HR hinge region
  • heavy chain constant region CH3 domain are composed; and, when the full-length antibody is of the IgE isotype, it optionally also includes the heavy chain constant region CH4 domain.
  • the "full-length heavy chain” is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention includes two antigen binding sites formed by a pair of VH and VL respectively, and the two antigen binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment composed of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment composed of VH domains (Ward et al., Nature 341:544 546 ( 1989))
  • the term “Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means two fragments connected by a disulfide bridge on the hinge region
  • the term “Fab'fragment” means a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment. Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment composed of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind antigen, although its affinity may be lower than the complete binding site.
  • Fc means a disulfide bond formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions, but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain, Thereby forcing the domain to pair with the complementary domain of the other chain and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to the structure of a single monomer variable antibody domain (e.g., a single heavy chain variable antibody). Region), which retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the above-mentioned antibody fragments maintains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of antibodies e.g., the aforementioned antibody fragments
  • a given antibody e.g., the antibody provided by the present invention
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the terms “monoclonal antibody”, “monoclonal antibody”, and “mAb” have the same meaning and are used interchangeably, which refers to a group of highly homologous antibody molecules.
  • An antibody or a fragment of an antibody that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a group of highly homologous antibodies, and cannot be understood as requiring any specific method to prepare the antibody.
  • the monoclonal antibody of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495,1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage Antibody library technology (see, for example, Clackson et al. Nature352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).
  • Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or as an alternative, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography.
  • the purification of immunoglobulin can refer to, for example, D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
  • chimeric antibody refers to an antibody whose light chain or/and part of its heavy chain is derived from an antibody (which may be derived from a specific species or belong to a certain species).
  • a specific antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but no matter However, it still retains the binding activity to the target antigen (USP 4,816,567 to Capability et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from the first antibody (e.g., murine antibody), and The heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).
  • first antibody e.g., murine antibody
  • second antibody e.g., a human antibody
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with the sequence of a human antibody.
  • CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from human source.
  • Immunoglobulin receptor antibody
  • at least one or two but usually all three (heavy and/or light immunoglobulin chains) acceptor CDRs of the humanized antibody are replaced by donor CDRs.
  • the immunoglobulin that provides the CDR is called the "donor", and the immunoglobulin that provides the framework is called the “acceptor”.
  • the donor immunoglobulin is a non-human (e.g., murine) antibody, and the acceptor framework can be a naturally occurring human framework, or has about 85%, 90%, 95%, 99% or more compared to it. Sequence of identity.
  • Humanized antibodies generally retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with desired properties (e.g., antigen specificity, affinity, reactivity, etc.).
  • the expected properties of the antibody of the present invention include: (1) specifically binding to CD73 (for example, membrane-bound human CD73 or soluble human CD73); (2) inhibiting or reducing CD73 (for example, membrane-bound human CD73 or soluble human CD73) enzyme activity; (3) in the presence of adenosine monophosphate (AMP), increase the proliferation of anti-CD3/anti-CD28 stimulated T cells (such as CD4+T cells); (4) mediate CD73 internalization; ( 5) Reduce the level of adenosine in tumor cells expressing CD73; (6) Stimulate an immune response (e.g., immune response against tumors or immunogens); (7) Prevent and/or treat tumors (e.g., tumors expressing CD73).
  • the antibody of the present invention has one or more of the aforementioned expected properties.
  • the chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared above.
  • DNA encoding the heavy and light chains can be obtained from the target murine hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.
  • the murine immunoglobulin variable region can be linked to the human immunoglobulin constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.).
  • the DNA encoding VH is operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene.
  • the sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region.
  • DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene).
  • the sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.
  • transgenic animals can also be used, which can produce no endogenous immunoglobulin after immunization and can produce a complete human antibody library.
  • JH antibody heavy chain joining region
  • Non-limiting examples of the above-mentioned transgenic animals include HuMAb mice (Medarex, Inc.), which contain human immunoglobulin genes encoding unrearranged human heavy chain ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences. Locus (miniloci), plus targeted mutations that inactivate the endogenous mu and kappa chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or carrying human heavy chain transgenes and humans Light chain transchromosome "KM MouseTM " (see patent application WO02/43478). Other methods of antibody humanization also include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996 , Nature Biotech 14:309).
  • germline antibody gene or “germline antibody gene segment (germline antibody gene segment)” refers to a sequence encoding immunoglobulin that exists in the genome of an organism , It has not experienced the maturation process that can lead to genetic rearrangement and mutation of expressing specific immunoglobulins.
  • the expression “heavy chain germline gene” refers to the germline antibody gene or gene fragment encoding the heavy chain of immunoglobulin, which includes V gene (variable), D gene (diversity), and J gene (joining) And C gene (constant); similarly, the expression “light chain germline gene” refers to germline antibody genes or gene fragments encoding immunoglobulin light chains, including V genes (variable), J genes (joining) and C gene (constant).
  • the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also called “germline sequence”
  • the amino acid sequence encoded by the heavy chain germline gene is called the heavy chain germline gene.
  • the chain germline sequence the amino acid sequence encoded by the light chain germline gene is called the light chain germline sequence.
  • Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or inquired from professional databases (for example, IMGT, UNSWIg, NCBI or VBASE2).
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • an antibody that specifically binds to a certain antigen refers to an antibody with a concentration of less than about 10 -9 M, for example, less than about 10 -9 M, 10 -10 M, The affinity (K D ) of 10 -11 M or 10 -12 M or less binds to the antigen.
  • the specific binding properties between two molecules can be measured using methods known in the art, for example, using surface plasmon resonance (SPR) in a BIACORE instrument.
  • cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells), such as chemotherapeutic drugs, bacterial toxins, phytotoxins, or radioisotopes.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids 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) ; And Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancement Agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include but are not limited to sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate).
  • the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), glucose Solutions (e.g. 5% glucose), solutions containing surfactants (e.g. 0.01% polysorbate 20), pH buffered solutions (e.g. phosphate buffered solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • glucose Solutions e.g. 5% glucose
  • surfactants e.g. 0.01% polysorbate 20
  • pH buffered solutions e.g. phosphate buffered solution
  • Ringer's solution e.g. phosphate buffered solution
  • prevention refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., tumor) in a subject.
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (ie, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the disease State, and relief of symptoms (whether partial or full), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to expected survival (if not receiving treatment).
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject e.g., human
  • has a tumor e.g., a tumor expressing CD73
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent a disease
  • the patient s disease and the amount of its complications. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • antibody-mediated internalization refers to the phenomenon in which antibodies pass through cell membranes after binding to cell surface antigens. Internalization includes antibody-mediated receptor (eg CD73) internalization.
  • the term "immune response” refers to immune cells (such as lymphocytes, antigen-presenting cells, phagocytes or granulocytes) and soluble macromolecules (including antibodies, cytokines) produced by immune cells or the liver. , And complement), which results in the selective damage, destruction or destruction of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues under autoimmune or pathological inflammation Cleared from the human body.
  • the immune response refers to a T cell-mediated immune response that occurs when the T cell-specific antigen stimulates the T cell.
  • Non-limiting examples of the response produced by T cells upon antigen-specific stimulation include the proliferation of T cells and the production of cytokines.
  • the antibody of the present invention can specifically bind to membrane-bound CD73 and soluble CD73 on the surface of tumor cells, significantly inhibit its enzymatic activity, and enhance immune response. Therefore, the antibody of the present invention has the potential for preventing and/or treating tumors (especially tumors expressing CD73).
  • the humanized antibody of the present invention not only retains the functions and properties of the murine parent antibody, but also has a high degree of humanization, so that it can be safely administered to human subjects without triggering an immunogenic response. It is particularly surprising that the antibody of the present invention can more significantly restore the CD4+ T cell suppression mediated by AMP and enhance the killing of CD73-expressing tumor cells than the known anti-CD73 antibody. Therefore, the antibodies (especially humanized antibodies) of the present invention have great clinical value.
  • Figure 1 shows the binding curve of mouse antibody 72G8 to CD73 on the surface of tumor cells.
  • Figures 2A-2B respectively show the binding of humanized antibody 7002-04 to MDA-MB-231 and A375 tumor cells that naturally express human CD73.
  • ISO is an isotype control antibody.
  • Figure 3 shows the binding of humanized antibody 7002-04 to soluble recombinant CD73.
  • Figure 4 shows the inhibition of humanized antibody 7002-04 on the enzymatic activity of CD73 on the surface of tumor cells.
  • Figures 5A-5C show the inhibition of CD73 enzyme activity in the serum of patients with colorectal cancer (A), liver cancer (B), and melanoma (C) by humanized antibody 7002-04.
  • Figures 6A-6B show the alleviating effect of humanized antibody 7002-04 on AMP-mediated CD4+ T cell suppression.
  • Figures 7A-7B show the recovery effect of humanized antibody 7002-04 on the killing of PBMC on tumor cells.
  • Figures 8A-8C respectively show the binding ability of CDR mutant antibody 7002-04-N56Q to CD73 on the cell surface (A), the ability to inhibit CD73 enzyme activity on the cell surface (B), and the ability to inhibit serum CD73 enzyme activity in tumor patients ( C) The evaluation results.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer.
  • the embodiments describe the present invention by way of example, and are not intended to limit the scope of protection claimed by the present invention.
  • the antigens used include: CD73 protein (recombinantly expressed human CD73, whose sequence is shown in SEQ ID NO: 21) and CHOS-human CD73 (that is, the CHOS cell line that overexpresses CD73, and the expressed CD73 sequence is shown in SEQ ID NO: 21); adjuvants include: complete Freund's adjuvant CFA (InvivoGen company, product number vac-cfa-60), IFA (InvivoGen company, product number vac-ifa-60), QuickAntibody (Beijing Boaolong Immunology Technology Co., Ltd., catalog number KX0210041); routes of administration include: intraperitoneal (ip) and subcutaneous (sc).
  • the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method to obtain B cells that can express antibodies and proliferate immortally in vitro, and select in HAT Cultivate in medium.
  • the fused hybridoma cells were plated in a 96-well cell culture plate, and positive clones were selected through primary screening for 2-3 rounds of subcloning.
  • the mouse monoclonal antibody 72G8 was isolated and purified from the culture supernatant of the positive hybridoma monoclonal cell line finally obtained.
  • MDA-MB-231 endogenously expressing human CD73; human breast cancer cell line
  • SK-ME-S endogenously expressing human CD73; human lung squamous cell line
  • H2030 endogenously expressing human CD73; human non-small cell lung cancer cell line
  • SKLU1 endogenously expressing human CD73; human lung adenocarcinoma cell line
  • BT549 endogenously expressing human CD73; human breast tube carcinoma cell line
  • A375 endogenously expressing human CD73 ; Human melanoma cell line
  • Calu6 endogenously expressing human CD73; human degenerative cancer cell line
  • 4T1 endogenously expressing murine CD73; murine breast cancer cell line
  • CHOS-human CD73 transfected with human CD73
  • CHOS CHOS negative
  • the lentivirus was provided by Shanghai Jikai Gene Chemical Technology Co., Ltd. 72 hours after infection, the cells were cultured with corresponding resistance for 2-4 weeks, amplified and frozen for subsequent experiments.
  • Experimental method spread 10,000 cells in 100 ⁇ L DMEM+10%FBS/well, use a flat-bottomed 96-well plate, make the cells adhere to the wall or sink to the bottom of the well overnight, and remove the supernatant the next day.
  • the 8-point continuous 3-fold dilution was performed by diluting 1/3 volume (100 ⁇ L) in 200 ⁇ LDMEM.
  • Add 100 ⁇ L of diluted antibody to each well of the cell plate (use fusion clone supernatant or subclonal supernatant when screening), add 100 ⁇ LDMEM to the corresponding negative control well, and incubate for 1 hour at room temperature.
  • the imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescence-labeled cell morphology and fluorescence intensity setting parameters.
  • the imaging obtained by the bright field channel counts the adherent cells according to the cell morphology setting parameters, and then the two sets of data are compared. Divide the percentage of cells displaying fluorescence that are bound to the antibody to the total number of cells. Based on this ratio, the binding effect of the anti-CD73 antibody with the cell line expressing CD73 was determined.
  • Data analysis uses GraphPad, the abscissa uses the logarithm of the antibody concentration, the ordinate uses the percentage of the total number of cells with green fluorescence and the total number of living cells that bind to CD73 antibody, and the EC50 value of the anti-CD73 antibody bound on each cell is calculated according to the curve. .
  • the binding curve of 72G8 to each tumor cell is shown in Figure 1, and its EC50 value is shown in Table 2-1 and Table 2-2. N.B. means that it is not detected in the measured concentration range.
  • the results showed that 72G8 can bind to cells expressing CD73 naturally and CHOS cells recombinantly expressing human CD73, but the antibody does not bind to cells that do not express CD73 (CHOS), nor does it bind to cells expressing murine CD73 (4T1).
  • Table 2-2 Binding EC50 of antibodies to CHOS cells expressing recombinant human CD73 and other cells not expressing human CD73
  • PBMC peripheral blood mononuclear cells
  • Example 3 Determination of variable region sequence of murine anti-CD73 antibody and preparation of chimeric antibody
  • the hybridoma cells were collected by centrifugation. Add 1ml TRIzol and 0.2ml chloroform to every 5-10 ⁇ 10 6 cells, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes. Centrifuge the water phase and add 0.5ml isopropanol. After standing at room temperature for 10 minutes, collect the precipitate. After washing with ethanol and drying, RNA is obtained. Add template RNA and primers to the ice-bath centrifuge tube, make the primers and template correctly paired, and then perform the reverse transcription process, and then perform PCR amplification. Add 2.5 ⁇ l of the dNTP/ddNTP mixture to each of the 4 microcentrifuge tubes, and incubate the mixture at 37°C for 5 minutes for use.
  • VH and VL sequences of the murine antibody 72G8 are shown in the table below. Furthermore, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669 Page), the CDR sequence of the mouse monoclonal antibody was determined.
  • the DNA sequences (SEQ ID NOs: 14-15) encoding the heavy chain and light chain variable regions of the murine antibody described above were respectively compared with the heavy chain constant region (SEQ ID NO: 19) and the light chain constant region (SEQ ID NO: 19) and light chain constant region (SEQ ID NO: 19) encoding the human antibody.
  • the sequence of SEQ ID NO: 20) was linked and expressed in HEK293 cells (ATCC).
  • the cell supernatant containing antibody clones grown in culture flasks was harvested, purified by protein A column, and 100mM acetic acid pH3.0 was used. Elute the antibody protein. Then the purified antibody protein is loaded onto a molecular exclusion chromatography column for further separation and purification.
  • the antibody protein corresponding to the monomer was formulated in PBS buffer, and the formulation buffer was supplemented with 20% glycerol.
  • the corresponding chimeric antibody ch72G8 was obtained.
  • the murine antibody provided in the above examples can be humanized design and prepared, using methods known in the art.
  • Mouse CDR regions are inserted into human framework sequences (see US Patent No. 5,225,539 of Winter; US Patent Nos. 5,530,101 of Queen et al.; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols ,volume 248, Humana Press, New Jersey, 2004).
  • the heavy chain and light chain CDR regions of the mouse antibody 72G8 were grafted onto the FR framework of the corresponding humanized template, and a series of back mutations were made to the amino acid residues in the FR region of the humanized template. , So that the humanized antibody retains the antigen-binding ability of the murine antibody as much as possible.
  • the present inventors prepared a humanized antibody of the murine antibody 72G8, named 7002-04 (the variable region of the heavy chain and the variable region of the light chain are shown in SEQ ID NO: 9 and 10, respectively) .
  • the constant region of the heavy chain of the antibody is SEQ ID NO: 19
  • the constant region of the light chain is SEQ ID NO: 20.
  • 500,000 CD73 expressing cells were placed in 100 ⁇ L FACS buffer (PBS+2% FBS)/well for use, and a round-bottomed low-adsorption 96-well plate was used.
  • the antibody sample is diluted by 1/2 volume (100 ⁇ L) in 200 ⁇ L FACS buffer to perform 12-point serial 3-fold dilution of 3 times the concentration.
  • the plate was washed 3 times in washing buffer, and HRP-conjugated goat anti-human or goat anti-mouse IgG Fc fragment secondary antibody was added at room temperature for 1 hr to detect the bound anti-CD73 antibody.
  • the plate was washed 3 times in washing buffer, and the bound secondary antibody was revealed by adding TMB (HRP substrate) and incubating the plate in the dark at room temperature for 5 to 10 minutes.
  • the enzyme reaction was terminated by adding sulfuric acid solution 1M, and the light absorption was measured at 450 nm. Draw a graph with the light absorption value as the ordinate and the antibody concentration log as the abscissa, and use GraphPad Prism software to calculate the EC50.
  • the results are shown in Figure 3, the humanized antibody 7002-04 has good binding activity to soluble recombinant CD73, and its EC50 is 0.0047 ⁇ g/ml.
  • CD73 which cleaves AMP into adenosine + inorganic phosphate, restores luciferase activity and light emission by reducing AMP. Therefore, antibodies that block the enzymatic activity of CD73 will reduce light emission.
  • Human CD73 positive cells were harvested and counted. Seed 20,000 cells per well in a flat-bottom 96 plate in 100 ⁇ L of complete medium. The antibody sample was diluted by 1/3 volume (100 ⁇ L) in 200 ⁇ L DMEM to perform 8-point continuous 3-fold dilution, and 100 ⁇ L of the diluted sample was added to the corresponding wells. The negative control was isotype control antibody (ISO). Incubate at 37°C for 1 hour, remove the supernatant and wash the cells twice with PBS. Prepare a 125 ⁇ M AMP solution in incomplete medium, add 100 ⁇ L AMP to each well, and incubate the plate at 37°C for another two hours.
  • ISO isotype control antibody
  • FIG. 4 shows the inhibition curve of humanized antibody 7002-04 and the corresponding chimeric antibody ch72GB on CD73 enzyme activity on the surface of A375 cells. The results show that the humanized antibody 7002-04 can significantly inhibit the enzymatic activity of CD73 on the surface of tumor cells.
  • Table 7 IC50 of antibodies inhibiting CD73 enzyme activity on the surface of different human tumor cells
  • Dilute tumor patient serum in phosphoric acid buffer Tris 125mM, MgCl2 25mM, NaCl125mM
  • the antibody sample is diluted by 1/1.5 volume (100 ⁇ L) in 50 ⁇ L phosphoric acid buffer and 1/10 volume (10 ⁇ L) is diluted in 90 ⁇ L phosphoric acid buffer for 10 points continuous 2-10 times dilution, in each well of the cell plate
  • Add 12.5 ⁇ L of diluted antibody add 12.5 ⁇ L of phosphoric acid buffer to negative control, incubate at 37°C for 1.5 hours after centrifugation.
  • AMP was diluted into a 20 ⁇ M solution with phosphoric acid buffer, 25 ⁇ L of AMP was added to each well (except for the positive control), centrifuged and incubated at 37°C for another 1 hour. After the reaction is over, add 25 ⁇ L of AMP to the positive control. Immediately add 25 ⁇ L of AMP-Glo TM Reagent I (Promag, Product No. V5012) to each well, centrifuge the reaction plate, and incubate the plate at room temperature for 1 hour. Add 50 ⁇ L of AMP Detection Solution (Promag, Product No. V5012) to each well, and incubate for 1 hour at room temperature after centrifugation. Use a microplate reader to measure the fluorescence (Lum). Data analysis uses GraphPad, the abscissa uses the logarithm of the antibody concentration, and the ordinate is the inhibition rate. The calculation method of the inhibition rate is as follows:
  • Example 7 CD73 internalization mediated by anti-CD73 antibody
  • Example 8 Anti-CD73 antibody alleviates AMP-mediated CD4+ T cell suppression
  • the bright-field channel is selected to perform high-speed scanning and imaging of the cells in the well.
  • the anti-CD73 antibody alleviated the AMP-mediated CD4+T cell inhibitory effect.
  • MEDI9447 MedImmune
  • BMS986179 BMS
  • the cell growth on the 5th and 4th day of T cell proliferation is shown in Figure 6A-6B; #20 and #18 used in the figure are the numbers of internal donors for PBMC; the initial antibody concentration used in the figure is 100 ⁇ g /mL, a total of nine points in four-fold dilution.
  • the humanized antibody 7002-04 can effectively alleviate the AMP-mediated inhibition of CD4+ T cells, and T cell proliferation has been significantly restored, and the effect is better than the reference antibodies MEDI9447 and BMS986179.
  • PBMC cells that were stimulated with CD3/CD28 for 24 hours one day in advance (fresh apheresis separated by Ficoll), and resuspended with AIMV containing 40 ⁇ M EHNA and 120IU/ml IL2 (final concentration of EHNA is 20 ⁇ M, final concentration of IL2 is 60IU/ml) , Add 5,000/100 ⁇ L to each well. Prepare a 400 ⁇ M AMP solution with AIMV, and add 50 ⁇ L to each well (final AMP concentration 100 ⁇ M). After centrifugation, incubate at 37°C for 72 hours. Add 10 ⁇ L CCK8 kit (Nippon Tongren Co., Product No.
  • Example 10 Antibodies containing CDR mutations and their activity evaluation
  • the N56 in the HCDR2 of the 7002-04 heavy chain variable region was mutated to Q, and the obtained variant was named 7002-04-N56Q (the VH and VL are shown in SEQ ID NOs: 13 and 10, respectively).
  • Flow cytometry was used to determine the binding of antibodies to CD73-expressing human tumor cells MDA-MB-231 using the method described in 5.1. The results are shown in Figure 8A. 04 consistent.
  • the method in 6.1 was used to measure the antibody's inhibition of CD73 enzymatic activity in tumor cells.
  • the inhibitory ability of 7002-04-N56Q on the CD73 enzymatic activity on the surface of MDA-MB-231 is basically the same as 7002-04.

Abstract

本发明属于疾病治疗及免疫学领域，具体而言，本发明公开了抗CD73的抗体或其抗原结合片段，编码其的核酸分子，包含其的免疫缀合物、双特异性分子及药物组合物，以及其用于增强免疫应答和/或治疗肿瘤的用途。

Description

抗CD73抗体及其用途 技术领域
本发明涉及疾病治疗及免疫学领域，具体而言，本发明涉及抗CD73的抗体或其抗原结合片段，编码其的核酸分子，包含其的免疫缀合物、双特异性分子及药物组合物，以及它们用于增强免疫应答和/或治疗肿瘤的用途。
背景技术
近年来，癌症免疫治疗的飞速发展使科学界对肿瘤生物学和免疫学有了更好的了解。肿瘤微环境是一个动态的微环境，包括了癌细胞、免疫细胞、成纤维细胞、成肌纤维细胞、细胞因子、血管和细胞外基质。肿瘤往往处在缺氧的条件下，且环境中葡萄糖和其它营养物质也很缺乏。为了生存，癌细胞在这样的环境下会重组它们的代谢机制。其中，调整嘌呤代谢是非常关键的一步，尤其是增加分化簇73(CD73，亦称为胞外-5'-核苷酸酶)的表达。CD73是通常在内皮细胞和造血细胞的亚群上表达的糖基磷脂酰肌醇锚定的细胞表面蛋白(Misumi Y等人.European Journal of Biochemistry 1990；191(3):563–9)。在胞外，CD73与CD39一起调节腺苷三磷酸转化成为腺苷，而CD73催化的腺苷单磷酸盐去磷酸化成腺苷的这一步是上述转化轴上的速度决定步骤(Resta R等人.Immunological Reviews 1998；161:95–109.)。
作为对细胞死亡和细胞压力的响应，细胞释放ATP激活免疫响应反应。与此相对，ATP水解为腺苷则充当了一种反向的反馈机制，并导致了抑制免疫反应。腺苷是经广泛研究的经由若干受体(包括A1、A2A、A2B及A3)介导其生物效应的信号传导分子。已知腺苷可调控许多癌症的增殖及迁移，细胞外腺苷在癌性组织中积累并且构成肿瘤免疫逃逸的重要机制(Bin Z.Cancer Research 2010；70:6407-6411)。在其他作用中，肿瘤起源的腺苷通过腺苷酸环化酶激活的A2A受体深度抑制浸润性效应T细胞。
已有报导CD73在许多不同肿瘤上表达，包括黑色素瘤、结肠癌、肺癌、卵巢癌、膀胱癌、神经胶质瘤、神经胶母细胞瘤、甲状腺癌、食道癌、前列腺癌及乳腺癌。CD73是实体瘤中有效的预后生物标志物，并且CD73的过度表达与较短患者存活期或较短无疾病进展生存期相关(Rong W等人.Oncotarget 2017；8(34):57327-57336)。癌症中的CD73表达与增加的增殖、迁移、新血管形成、侵袭力、转移相关，已经显示，使用siRNA的敲低或CD73在肿瘤细胞上的过量表达可以调节肿瘤生长和转移(Paul B等人. PNAS 2013；110(36):14711-14716)；CD73-/-小鼠被保护免患移植性肿瘤和自发性肿瘤(John S等人.Cancer Research 2010；71:2892-2900)。除了已报道CD73可调控肿瘤细胞上的细胞-细胞及细胞-基质相互作用，CD73表达及活性亦与减弱的T细胞反应相关(Dachuan J等人.Cancer Res 2010；70:2245-55)。CD73同时也涉及到对化疗药物的抗性，例如蒽环类抗生素(Loi,S等人.PNAS 2013；110:11091–11096)，以及对由肿瘤坏死因子相关凋亡诱导配体(TRAIL)所引发的细胞凋亡的抗性。因此，CD73可以直接及间接方式调控癌症进展，凸显其作为新颖治疗靶的潜能。
此外，近年来癌症免疫检查点抑制药物在多种癌症患者身上显示出了良好的疗效，然而相当大比例的患者对这些治疗仍然没有反应，并且三分之一的患者在初始反应后出现复发(适应性耐药)，这表明多种非冗余免疫抑制机制共存于肿瘤微环境中，可以与上述药物协同或合用的药物靶点是目前癌症免疫学研究的热点。
因此，无论作为单药或联合疗法，CD73已显示出其作为抗肿瘤治疗靶点的潜力。
发明内容
本发明的抗体能够特异性结合肿瘤细胞表面的膜结合CD73以及非膜表面形式的CD73，并且抑制其酶学活性，增强免疫应答，具有良好的抗肿瘤活性，且与已知的抗CD73抗体相比具有更优的功能特性。因此，本发明的抗体具有用于预防和/或治疗肿瘤的潜力，为临床上肿瘤免疫治疗用药提供了选择。
本发明的抗体
因此，在一个方面，本发明提供了能够特异性结合CD73的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:4或SEQ ID NO:11，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或，
(b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(v)VL CDR2，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:8，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
在某些实施方案中，(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段能够结合人CD73，例如膜结合人CD73和/或可溶性人CD73。
在另一方面，本发明提供了能够特异性结合CD73的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
如下的三个重链可变区CDRs：SEQ ID NO:1、9、12、13任一项所示的重链可变区中含有的VHCDR1、VHCDR2和VHCDR3，以及
如下的三个轻链可变区CDRs：SEQ ID NO:2或10所示的轻链可变区中含有的VLCDR1、VLCDR2和VLCDR3。
在某些实施方案中，所述重链可变区中含有的3个CDR和所述轻链可变区中含有的3个CDR由Kabat、Chothia或IMGT编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：SEQ ID NO:1所示的重链可变区中含有的VHCDR1、VHCDR2和VHCDR3，以及SEQ ID NO:2所示的轻链可变区中含有的VLCDR1、VLCDR2和VLCDR3。在某些实施方案中，所述抗体或其抗原结合片段包含：SEQ ID NO:9所示的重链可变区中含有的VHCDR1、VHCDR2和VHCDR3，以及SEQ ID NO:10所示的轻链可变区中含有的VLCDR1、VLCDR2和VLCDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含：SEQ ID NO:12所示的重链可变区中含有的VHCDR1、VHCDR2和VHCDR3，以及SEQ ID NO:2所示的轻链可变区中含有的VLCDR1、VLCDR2和VLCDR3。在某些实施方案中，所述抗体或其抗原结合片段包含：SEQ ID NO:13所示的重链可变区中含有的VHCDR1、VHCDR2和VHCDR3，以及SEQ ID NO:10所示的轻链可变区中含有的VLCDR1、VLCDR2和VLCDR3。
在某些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:1所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列，所述轻链可变区包含SEQ ID NO:2所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。在某些实施方案中，所述重链可变区包含分别如SEQ ID NO:3-5所示的VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含分别如SEQ ID NO:6-8所示的VL CDR1、VL CDR2和VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:12所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列,所述轻链可变区包含SEQ ID NO:2所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。在某些实施方案中，所述重链可变区包含分别如SEQ ID NO:3、11、5所示的VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含分别如SEQ ID NO:6-8所示的VL CDR1、VL CDR2和VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列,所述框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至相应的鼠源残基的回复突变。在某些实施方案中，所述抗体或其抗原结合片段包含：来源于人重链胚系序列(即，人重链胚系基因所编码的氨基酸序列)的重链框架区序列，以及来源于人轻链胚系序列(即，人轻链胚系基因所编码的氨基酸序列)的轻链框架区序列,所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至相应的鼠源残基的回复突变。
在某些实施方案中，所述抗体或其抗原结合片段包含：来源于重链胚系序列的重链框架区序列，和来源于轻链胚系序列的轻链框架区序列,所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至相应的鼠源残基的回复突变。
在某些实施方案中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:9所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列，所述轻链可变区包含SEQ ID NO:10所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。在某些实施方案中，所述重链可变区包含分别如SEQ ID NO:3-5所示的VH CDR1、VH CDR2和VH CDR3，所述 轻链可变区包含分别如SEQ ID NO:6-8所示的VL CDR1、VL CDR2和VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:13所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列，所述轻链可变区包含SEQ ID NO:10所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。在某些实施方案中，所述重链可变区包含分别如SEQ ID NO:3、11、5所示的VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含分别如SEQ ID NO:6-8所示的VL CDR1、VL CDR2和VL CDR3。
在某些实施方案中，本发明的抗体或其抗原结合片段可以进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区。在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于哺乳动物(例如，鼠或人)免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于哺乳动物(例如，鼠或人)免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，本发明的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
本发明的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。
在某些实施方案中，恒定区可以包含氨基酸突变以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低)。
替换抗体的Fc区中的氨基酸残基以改变其效应子功能的方法是本领域已知的。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用、CDC等。在某些情况下，这些效应子功能对于治疗性抗体是需要的；但在其他情况下，这些效应子功能可能是不必要的或甚至是有害的，这取决于预期目的。
因此，在某些实施方案中，本发明的抗体或其抗原结合片段具有降低或甚至消除的效应子功能(例如ADCC和/或CDC活性)。在此类实施方案中，本发明的抗体或其抗原结合片段可以包含人IgG重链恒定区的变体，所述变体与其所源自的野生型序列相比具有以下置换中的至少一个、至少两个或全部三个：L234F、L235E、P331S(以上提及的氨基酸位置是根据EU编号系统的位置),参见例如Acta Cryst.(2008).D64,700–704。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段包含人野生型IgG1重链恒定区。在此类实施方案中，本发明的抗体或其抗原结合片段具有ADCC和CDC活性。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段包含人IgG1重链恒定区的变体，所述变体与其所源自的野生型序列相比具有以下置换：L234F、L235E、P331S(根据EU编号系统的位置)，例如SEQ ID NO:19所示的重链恒定区。在此类实施方案中，本发明的抗体或其抗原结合片段具有消除或降低的ADCC和/或CDC活性。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)的变体，所述变体与其所源自的野生型序列相比具有基本不变的效应子功能。在此类实施方案中，所述变体与其所源自的野生型序列相比可以具有至多20个氨基酸保守置换(例如至多15个、至多10个、或至多5个保守置换；例如1个，2个，3个，4个或5个保守置换)。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段包含人κ轻链恒定区，例如SEQ ID NO:20所示的轻链恒定区。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO:19所示的重链恒定区(CH)；和/或，SEQ ID NO:20所示的轻链恒定区(CL)。
在某些实施方案中，本发明的抗体是鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。在某些实施方案中，本发明的抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
在某些实施方案中，本发明的抗体或其抗原结合片段具备以下特征中的一项或多项：
(a)与膜结合人CD73或可溶性人CD73或两者结合；例如，所述膜结合人CD73表达于肿瘤细胞表面；
(b)抑制或降低CD73(例如，膜结合人CD73或可溶性人CD73)的酶活性；例如抑制或降低人CD73介导的腺苷单磷酸(AMP)向腺苷的转化，例如通过CellTiter Glo(CTG)测 定(例如实施例6所述的方法)确定；
(c)在腺苷单磷酸(AMP)存在下，增加抗CD3/抗CD28刺激的T细胞(例如CD4+T细胞)的增殖；例如通过实施例8所述的方法确定；
(d)通过抗体介导的受体内化使CD73内化至细胞(例如，肿瘤细胞)中，所述细胞在其表面表达CD73；例如以如通过FACS或流式细胞术(例如实施例7所述的方法)所测量的至少10％(例如至少15％，至少20％或更多)的内化水平；
(e)以小于约0.01μg/ml或更小的EC50结合可溶性人CD73；所述EC50通过ELISA技术测得；
(f)以小于约0.5nM或更低的KD结合可溶性人CD73；所述KD通过Biacore测得；
(g)降低表达CD73的肿瘤细胞中的腺苷水平；
(h)刺激免疫应答；例如，刺激针对肿瘤(例如表达CD73的肿瘤)的免疫应答；
(i)预防和/或治疗肿瘤(例如表达CD73的肿瘤)。
在某些实施方案中，所述抗体或其抗原结合片段是72GB或其抗原结合片段、其嵌合抗体、或其人源化抗体，或它们的功能变体，所述变体基本保留了其所源自的抗体或其抗原结合片段的生物学功能。
在本发明中，本发明的抗体或其抗原结合片段可以包括这样的变体，所述变体与其所源自的抗体或其抗原结合片段相比差异仅在于一个或多个(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的保守置换)氨基酸残基的保守置换，或者与其所源自的抗体或其抗原结合片段具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，且基本保留了其所源自的抗体或其抗原结合片段的上述生物学功能。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al., J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些实施方案中，所述分离的核酸分子编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链可变区的第一核苷酸序列，和/或编码本发明的抗体或其抗原结合片段的轻链可变区的第二核苷酸序列。
在某些实施方案中，所述第一核苷酸序列包含选自下列的序列：(a)如SEQ ID NO:14所示的核苷酸序列，或(b)与(a)所述的核苷酸序列基本上相同的序列(例如，与(a)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列，或具有一个或更多个核苷酸取代的序列)，或(c)与(a)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列；所述第二核苷酸序列包含选自下列的序列：(d)如SEQ ID NO:15所示的核苷酸序列，或(e)与(d)所述的核苷酸序列基本上相同的序列(例如，与(d)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列，或具有一个或更多个核苷酸取代的序列)，或(f)与(d)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列。
在某些实施方案中，所述第一核苷酸序列包含选自下列的序列：(a)如SEQ ID NO:16所示的核苷酸序列，或(b)与(a)所述的核苷酸序列基本上相同的序列(例如，与(a)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列，或具有一个或更多个核苷酸取代的序列)，或(c)与(a)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列；所述第二核苷酸序列包含选自下列的序列：(d)如SEQ ID NO:17所示的核苷酸序列，或(e)与(d)所述的核苷酸序列基本上相同的序列(例如，与(d)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列， 或具有一个或更多个核苷酸取代的序列)，或(f)与(d)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列。
在某些实施方案中，所述第一核苷酸序列包含选自下列的序列：(a)如SEQ ID NO:18所示的核苷酸序列，或(b)与(a)所述的核苷酸序列基本上相同的序列(例如，与(a)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列，或具有一个或更多个核苷酸取代的序列)，或(c)与(a)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列；所述第二核苷酸序列包含选自下列的序列：(d)如SEQ ID NO:17所示的核苷酸序列，或(e)与(d)所述的核苷酸序列基本上相同的序列(例如，与(d)所述的核苷酸序列相比，具有至少大约85％、90％、95％、99％或更高同一性的序列，或具有一个或更多个核苷酸取代的序列)，或(f)与(d)所述的核苷酸序列相差不超过3、6、15、30或45个核苷酸的序列。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链的第一核苷酸序列，和/或编码本发明的抗体或其抗原结合片段的轻链的第二核苷酸序列。
在另一个方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些实施方案中，所述载体能够在受试者(例如人)体内表达本发明的抗体或其抗原结合片段。
在另一个方面，本发明提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DG44)。
在另一个方面，提供了制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
衍生的抗体
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另 一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对CD73的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。此外，本发明的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
因此，在某些实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记，例如酶、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，
Figure PCTCN2020112271-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
双特异性或多特异性分子
本发明的抗体或其抗原结合片段可用于形成双特异性或多特异性分子。本发明的抗体或其抗原结合片段可以是双特异性或多特异性分子的一部分，所述双特异性或多特异性分子包含具有不同于本发明的抗体或其抗原结合片段的结合特异性的第二功能 模块(例如第二抗体)，从而能够结合至少两个不同的结合位点和/或靶分子。例如，本发明的抗体或其抗原结合片段可连接至能够特异性结合可用作联合治疗的潜在靶标的任何蛋白质的第二抗体或其抗原结合片段。为产生所述双特异性或多特异性分子，可以将本发明的抗体或其抗原结合片段连接(例如通过化学偶联、基因融合、非共价缔合或其他方式)至一个或多个其他结合分子(例如另外的抗体、抗体片段、肽或结合模拟物)。
因此，在另一个方面，本发明提供了一种双特异性或多特异性分子，其包含本发明的抗体或其抗原结合片段。
在某些实施方案中，所述双特异性或多特异性分子特异性结合CD73(例如膜结合人CD73和/或可溶性人CD73)，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
免疫缀合物
本发明的抗体或其抗原结合片段可以与治疗剂连接以形成免疫缀合物。由于免疫缀合物具有选择性递送一种或多种治疗剂至靶组织(例如与肿瘤相关的抗原，如表达CD73的肿瘤)的能力，因此，免疫缀合物可以提高本发明的抗体或其抗原结合片段在治疗疾病(例如癌症)中的治疗效力。
因此，在另一个方面，本发明提供了免疫缀合物，其包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
在某些实施方案中，所述治疗剂是细胞毒剂。在本发明中，所述细胞毒剂包括对细胞有害(例如杀伤细胞)的任何试剂。
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。
可用于本发明的免疫缀合物的烷化剂的实例包括但不限于氮芥类(如双氯乙基甲胺、苯丁酸氮芥、美法仑、环磷酰胺等)、乙烯亚胺类(如塞替哌等)、硫酸酯及多元醇类(如白消安、二溴甘露醇)、亚硝基脲类(如卡莫司汀、洛莫司汀等)、铂类抗肿瘤剂(如顺铂、奥沙利铂、卡铂等)等。
可用于本发明的免疫缀合物的有丝分裂抑制剂的实例包括但不限于美登素类(例如 美登素、美登醇、美登醇的C-3酯等)、紫杉烷类(例如多西他赛、紫杉醇或纳米颗粒紫杉醇等)、长春花生物碱类(例如硫酸长春地辛、长春新碱、长春花碱或长春瑞滨等)
可用于本发明的免疫缀合物的抗肿瘤抗生素的实例包括但不限于放线菌素、蒽环类抗生素(例如柔红霉素、阿霉素、表柔比星、伊达比星等)、卡利奇霉素、倍癌霉素等。
可用于本发明的免疫缀合物的抗代谢物的实例包括但不限于叶酸拮抗剂(例如甲氨蝶呤等)、嘧啶拮抗剂(例如5-氟尿嘧啶、氟尿苷、阿糖胞苷、卡培他滨、吉西他滨等)、嘌呤拮抗剂(例如6-巯基嘌呤、6-硫鸟嘌呤等)、腺苷脱氨酶抑制剂(例如克拉屈滨、氟达拉滨、奈拉滨、喷司他丁等)。
可用于本发明的免疫缀合物的拓扑异构酶抑制剂的实例包括但不限于(喜树碱类及其衍生物(例如伊立替康、托泊替康等)、安吖啶、道诺霉素、阿霉素、表鬼臼毒素类、玫瑰树碱类、表柔比星、依托泊苷、丙亚胺、替尼泊苷等。
可用于本发明的免疫缀合物的酪氨酸激酶抑制剂的实例包括但不限于阿西替尼、博舒替尼、西地尼布、达沙替尼、厄洛替尼、吉非替尼、伊马替尼、拉帕替尼、来妥替尼、尼洛替尼、司马沙尼、舒尼替尼、凡德他尼等。
可用于本发明的免疫缀合物的放射性核素剂的实例包括但不限于于I 131、In 111、Y 90、Lu 177等。
在某些实例性实施方案中，所述治疗剂选自铂类抗肿瘤剂、蒽环类抗生素、紫杉烷类化合物、核苷类似物、喜树碱类化合物，及其类似物或同系物，及其任意组合。
在某些实施方案中，本发明的抗体或其抗原结合片段任选地通过接头与所述治疗剂缀合。
在本发明中，使用本领域现有的接头技术可以将细胞毒剂偶联至本发明的抗体或其抗原结合片段。已经用于将细胞毒剂偶联至抗体的接头类型的实例包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。可以选择例如易于在溶酶体隔室内被低pH切割或易于被蛋白酶(例如优先在肿瘤组织中表达的蛋白酶，如组织蛋白酶，诸如组织蛋白酶B、C、D)切割的接头。
关于细胞毒剂的类型、接头及将治疗剂偶联至抗体的方法的进一步讨论还可参见Sai to，G.等人(2003)Adv.Drug Deliv.Rev.55：199-215；Trail，P.A.等人(2003)Cancer I mmunol.Immunother.52：328-337；Payne，G.(2003)Cancer Cell 3：207-212；Allen，T.M.(2002)Nat.Rev.Cancer 2：750-763；Pastan，I.and Kreitman，R.J.(2002)Curr.O pin.Investig.Drugs 3：1089-1091；Senter，P.D.and Springer，C.J.(2001)Adv.Drug  Deliv.Rev.53：247-264。
治疗用途和药物组合物
本发明的抗体或其抗原结合片段可以调节(例如，增强、刺激、增加、抑制、减少或中和)CD73的一种或多种生物学活性。在某些情况下，本发明的抗体或其抗原结合片段导致以下中的一种或多种：抑制或减少CD73的酶活性；抑制或减少腺苷单磷酸(AMP)转化为腺苷；以及在腺苷单磷酸(AMP)存在下，增加抗CD3/抗CD28刺激的T细胞(例如CD4+T细胞)的增殖。因此，本发明的抗体或其抗原结合片段可作为单药，通过抑制或减少CD73的酶活性，从而达到预防和/或治疗肿瘤的目的。
此外，已有报道靶向CD73可能与其他抗癌药物表现出协同效应。在一项评估曲妥珠单抗活性的前瞻性随机III期临床试验中，高水平的CD73基因表达与不良的临床结果显着相关，在HER2/ErbB2驱动的乳腺癌免疫活性小鼠模型中，肿瘤细胞和宿主细胞的CD73表达显著抑制由抗ErbB2单克隆抗体介导的免疫介导的应答(Martin T等人.Cancer Research 2017；77(20)；5652–63)。此外，已有体外实验表明A2A受体的激活会调控肿瘤浸润的毒杀性T细胞上的PD-1的上调，相对的使用抗PD-1抗体阻断PD-1信号通路会上调A2A受体在肿瘤浸润的毒杀性T细胞上的表达(Cekic C等人.Cancer Res 2014；74:7239-49)。已有报道抗CD73抗体显著增强抗CTLA-4抗体和抗PD-1抗体在多种小鼠肿瘤模型中的活性，且单药治疗和组合治疗皆依赖于宿主干扰素γ和细胞毒性T细胞；细胞外腺苷对肿瘤浸润T细胞的作用显示出腺苷的受体活化会增强PD-1在肿瘤特异性细胞毒性T细胞和辅助性T细胞上的表达(Bertrand A等人.Clin Cancer Res 2013；19(20):5626-5635)。临床研究发现在使用Pembrolizumab(抗PD-1)治疗的黑色素瘤病人体内CD73水平的增高和疾病进展正相关，CD73的动态上调与对于抗PD-1抗体的适应性抵抗之间的关系值得注意(Reinhardt J等人.Cancer Research 2017；77:4697–4709)。另有研究表明在接受nivolumab的转移性黑素瘤患者中高水平的可溶性CD73酶活性与较差的的总体生存和无进展生存期显著相关。在多变量分析中，CD73酶活性成为总体存活和无进展生存的最强预后因素，并且在开始nivolumab治疗之前，CD73酶活性的较高的基础水平与治疗反应率较低相关(Silvana M等人.J Transl Med 2017；15:244)。相应的在非小细胞肺癌病人肿瘤样本中，亦发现CD73的表达水平和PD-L1的表达水平互为互补。由此可见，本发明的抗体或其抗原结合片段亦可与免疫检查点抑制剂或肿瘤特异性抗体联合，用于肿瘤的防治。
因此，在另一个方面，本发明提供了一种药物组合物，其含有本发明的抗体或其抗原结合片段、双特异性或多特异性分子、或免疫缀合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂(例如吉西他滨、5-氟尿嘧啶、紫杉烷、顺铂等)、抗血管生成剂、细胞因子(例如GM-CSF、IL-7、IL-12、IL-15、IL-18、IL-21等)、分子靶向药物(例如，CD20抗体如利妥昔单抗、Her2抗体如曲妥珠单抗、VEGF抗体如贝伐珠单抗、EGFR抗体如西妥昔单抗等)、免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗体、CTLA-4抗体、LAG-3抗体等)、溶瘤病毒等。
在某些实施方案中，所述另外的药学活性剂选自免疫检查点抑制剂(例如，PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、LAG-3抑制剂)、抗CD39抗体、抗A2AR抗体或抗HER2/ErbB2抗体。
在某些实施方案中，在所述药物组合物中，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为混合物提供。因此，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、或免疫缀合物与所述另外的药学活性剂可以同时、分开或相继施用。
在某些示例性实施方案中，所述药物组合物包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在另一个方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗肿瘤的方法，所述方法包括向有此需要的受试者施用有效量的本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物或药物组合物。在另一个方面，提供了本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、或药物组合物用于在受试者(例如人)中预防和/或治疗肿瘤的用途，或在制备用于在受试者(例如人)中预防和/或治疗肿瘤的药物中的用途。
在某些实施方案中，所述肿瘤表达CD73。在某些实施方案中，所述CD73可以是膜结合人CD73和/或可溶性人CD73。
在某些实施方案中，所述肿瘤涉及表达CD73的肿瘤细胞。在某些实施方案中，所述CD73在所述肿瘤细胞表面上表达。
在某些实施方案中，所述肿瘤选自黑色素瘤、结肠癌、肺癌、肝癌、胰腺癌、卵巢癌、膀胱癌、神经胶质瘤、神经胶母细胞瘤、甲状腺癌、食道癌、前列腺癌及乳腺癌。
在某些实施方案中，将本发明的抗体或其抗原结合片段与第二治疗剂或治疗术组合施用。所述第二治疗剂或治疗术可以在施用本发明的抗体或其抗原结合片段之前、同时或之后施用。
在某些实施方案中，所述第二治疗剂选自具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，将本发明的抗体或其抗原结合片段与选自下列的治疗剂组合施用：免疫检查点抑制剂(例如，PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、LAG-3抑制剂)、抗CD39抗体、抗A2AR抗体或抗HER2/ErbB2抗体。
在某些示例性实施方案中，所述PD-1抑制剂选自PDR001、纳武单抗、派姆单抗、匹地利珠单抗、MEDI0680、REGN2810、TSR-042、PF-06801591和AMP-224。
在某些示例性实施方案中，所述PD-L1抑制剂选自FAZ053、阿特珠单抗、阿维鲁单抗、度伐鲁单抗和BMS-936559。
在某些示例性实施方案中，所述CTLA-4抑制剂选自艾匹利木单抗或曲美利木单抗。
在某些示例性实施方案中，所述LAG-3抑制剂选自由LAG525、BMS-986016、TSR-033、MK-4280和REGN3767。
在某些实施方案中，所述第二治疗术可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。
在另一个方面，本发明提供了一种用于刺激受试者中的免疫应答的方法，所述方法包括向有此需要的受试者施用有效量的本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物或药物组合物。在另一个方面，提供了本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、或药物组合物用于刺激受试者中的 免疫应答的用途，或在制备用于刺激受试者中的免疫应答的药物中的用途。
在某些实施方案中，所述免疫应答是T细胞介导的免疫应答。
在某些实施方案中，所述免疫应答是针对肿瘤(例如表达CDD73的肿瘤)的免疫应答。在某些实施方案中，所述受试者患有肿瘤(例如表达CDD73的肿瘤)。
在某些实施方案中，所述免疫应答是针对免疫原的免疫应答。在此类实施方案中，所述方法还包括向所述受试者施用免疫原。在一些实施方案中，所述免疫原选自与肿瘤相关的抗原(例如蛋白、多肽或糖类分子)、肿瘤细胞、由所述抗原致敏的树突状细胞，及其任意组合。在另一些实施方案中，所述免疫原选自与病原体(例如，病毒)相关的抗原(例如蛋白、多肽或糖类分子)、灭活或减毒的病原体、由所述抗原致敏的树突状细胞，及其任意组合。
在另一个方面，本发明提供了一种降低表达CD73的肿瘤细胞中的腺苷水平的方法，其包括将所述细胞与本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、或药物组合物接触。在某些实施方案中，所述方法用于在体外降低表达CD73的肿瘤细胞中的腺苷水平，用于非治疗目的。在另一个方面，提供了本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、或药物组合物用于降低表达CD73的肿瘤细胞中的腺苷水平的用途，或在制备用于降低表达CD73的肿瘤细胞中的腺苷水平的药物中的用途。
本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如 0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
此外，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、药物组合物可以以单位剂量形式存在于药物组合物中，以便于施用。
本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中，本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、药物组合物通过静脉注射或推注给予。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物、药物组合物。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的抗体或其抗原结合片段的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本发明中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
在本发明中，所述受试者可以为哺乳动物，例如人。
检测用途和试剂盒
本发明的抗体或其抗原结合片段能够特异性结合CD73，从而可用于检测CD73在样品中的存在或其水平。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。在一些实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记。在另一些实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结 合片段。优选地，所述第二抗体还包括可检测的标记。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在另一个方面，本发明提供了检测CD73在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与本发明的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与CD73之间复合物的形成或检测所述复合物的量。
所述复合物的形成表明存在CD73或表达CD73的细胞。
在某些实施方案中，所述样品是细胞样品，即包含细胞(例如肿瘤细胞)的样品。在此类实施方案中，优选地，所述复合物是由所述抗体或其抗原结合片段与所述样品中的细胞所表达的CD73之间形成的。
在一些实施方案中，本发明的抗体或其抗原结合片段还带有可检测的标记。在另一些实施方案中，在步骤(2)中，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。
所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。在某些实施方案中，所述CD73是人CD73，例如膜结合和/或可溶性人CD73。
在另一个方面，提供了本发明的抗体或其抗原结合片段用于测定CD73在样品中的存在或其量的用途，或在制备用于测定CD73在样品中的存在或其量的检测试剂中的用途。在某些实施方案中，所述CD73是人CD73，例如膜结合和/或可溶性人CD73。
术语定义
在本发明中，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，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“分化簇73”或“CD73”也称为胞外-5'-核苷酸酶，其能够将细胞外5’单磷酸核苷转化为核苷，即将单磷酸腺苷(AMP)转化为腺苷。术语CD73包括膜结合形式(也称为膜结合CD73)或可溶形式(也称为可溶性或非膜结合 CD73)。CD73可以分离自天然表达它们的细胞或组织，或使用本领域熟知的技术以重组方式产生。CD73的序列均是本领域公知的，可参见NCBI数据库登录号NM_002526。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的，术语“构架区(framework region)”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger  P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人.Nature,256:495,1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析进行纯化。随后或作为替代，可将特异性抗原(该抗体识别的靶分子)或其抗原表位固定在柱上，并通过免疫亲合层析法来纯化免疫特异性抗体。免疫球蛋白的纯化可参考例如D.Wilkinson(The Scientist,published by The Scientist,Inc.,Philadelphia Pa.,Vol.14,No.8(Apr.17,2000),pp.25-28)。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。典型地，人源化抗体的至少一个或两个但通常所有三个(重和/或轻免疫球蛋白链的)受体CDR被供体CDR替换。提供CDR的免疫球蛋白称为“供体”，提供框架的免疫球蛋白称为“受体”。在一个实施例中，供体免疫球蛋白是非人源(例如鼠)抗体，受体框架可以天然存在的人框架，或与其相比具有约85％、90％、95％、99％或更高同一性的序列。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
在本申请中，本发明抗体的预期性质包括：(1)特异性结合CD73(例如，膜结合人CD73或可溶性人CD73)；(2)抑制或降低CD73(例如，膜结合人CD73或可溶性人CD73)的酶活性；(3)在腺苷单磷酸(AMP)存在下，增加抗CD3/抗CD28刺激的T细胞(例如CD4+T细胞)的增殖；(4)介导CD73内化；(5)降低表达CD73的肿瘤细胞中的腺苷水平；(6)刺激免疫应答(例如针对肿瘤或免疫原的免疫应答)；(7)预防和/或治疗肿瘤(例如表达CD73的肿瘤)。本发明的抗体具有上述预期性质中的一项或多项。
本发明的嵌合抗体或人源化抗体可以根据上述制备的鼠单克隆抗体的序列进行制备。编码重链和轻链的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
为制备嵌合抗体，可使用本领域已知的方法将鼠免疫球蛋白可变区连接至人免疫球蛋白恒定区(参见例如Cabilly等人的美国专利No.4,816,567)。例如，将编码VH 的DNA可操作的连接至编码重链恒定区的另一DNA分子以获得全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是通常优选为IgG1或IgG4恒定区。例如，将编码VL的DNA可操作的连接至编码轻链恒定区CL的另一DNA分子以获得全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的(参见例如Kabat，E.A.等人(1991)Sequences of Proteins of Immunological Interest，Fifth Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区，但通常优选为κ恒定区。
为制备人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。或者，还可以利用转基因动物，其能够在免疫后不产生内源性免疫球蛋白、并且能够产生完整人抗体库。例如，已有报道在嵌合和种系突变小鼠中抗体重链连接区(JH)基因的纯合缺失可以完全抑制了内源性抗体产生，然后将人种系免疫球蛋白基因阵列转移到所述种系突变小鼠中将导致该小鼠在遇到抗原刺激时产生人抗体(参见例如，Jakobovits等，1993，Proc.Natl.Acad.Sci.USA 90：2551；Jakobovits等，1993，Nature362：255-258；Bruggermann等，1993，Year in Immunology 7：33；和Duchosal等，1992，Nature 355：258)。上述转基因动物的非限制性实例包括，HuMAb小鼠(Medarex,Inc.)，其含有编码未重排的人重链(μ和γ)和κ轻链免疫球蛋白序列的人免疫球蛋白基因微型基因座(miniloci)，加之使内源μ和κ链基因座失活的靶向突变(参见例如Lonberg等人(1994)Nature 368(6474):856-859)；或携带人重链转基因和人轻链转染色体的“KM小鼠 TM”(参见专利申请WO02/43478)。其他抗体人源化改造的方法还包括噬菌体展示技术(Hoogenboom等，1991，J.Mol.Biol.227：381；Marks等，J.Mol.Biol.1991，222：581-597；Vaughan等，1996，Nature Biotech 14：309)。
如本文中所使用的，术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指，存在于生物体的基因组中的编码免疫球蛋白的序列，其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中，表述“重链胚系基因”是指，编码免疫球蛋白重链的胚系抗体基因或基因片段，其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant)；类似地，表述“轻链胚系基因”是指，编码免疫球蛋白轻链的胚系抗体基因或基因片段，其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中，由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germline sequence)”，由重链胚系基因所编码的氨基酸序列称为重链胚系序列，由轻链胚系基因所编码的氨基酸序列称为轻链胚系序列。胚系抗体基因或胚系抗体基因片段及其相应的胚系序列是本领域技术人员熟知的，并且可从专业数据库(例如，IMGT、UNSWIg、NCBI或VBASE2)获得或查询。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于约10 -9M，例如小于约10 -9M、10 -10M、10 -11M或10 -12M或更小的亲和力(K D)结合该抗原。两分子间的特异性结合性质可使用本领域公知的方法进行测定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“细胞毒剂”包括对细胞有害(例如杀死细胞)的任何试剂，例如化疗药物、细菌毒素、植物毒素或放射性同位素等。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒 (如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族 包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(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)、非极性侧链(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)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或 酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤(例如表达CD73的肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“抗体介导的内化”是指抗体在与细胞表面抗原结合后穿过细胞膜的现象。内化包括抗体介导的受体(例如CD73)内化。
如本文中所使用的，术语“免疫应答”是指，免疫细胞(例如淋巴细胞、抗原呈递细胞、吞噬细胞或粒细胞)以及由免疫细胞或肝脏所产生的可溶性大分子(包括抗体、细胞因子、以及补体)的作用，该作用导致对侵入性病原体、被病原体感染的细胞或组织、癌细胞、或者在自身免疫或病理炎症情况下的正常人类细胞或组织的选择性损害、破坏或将它们从人体中清除。在某些实施方案中，所述免疫应答是指T细胞 介导的免疫应答，该应答产生于当该T细胞特异的抗原对该T细胞的刺激之时。由T细胞在抗原特异性刺激时产生的反应的非限制性实例包括T细胞的增殖以及细胞因子的产生。
发明的有益效果
本发明的抗体能够特异性结合肿瘤细胞表面的膜结合CD73以及可溶性CD73，显著抑制其酶学活性，增强免疫应答。因此，本发明的抗体具有用于预防和/或治疗肿瘤(特别是表达CD73的肿瘤)的潜力。此外，本发明的人源化抗体不仅保留了鼠源亲本抗体的功能和性质，而且具有较高的人源化程度，从而可安全地施用给人受试者，而不引发免疫原性反应。尤其令人意外的是，本发明的抗体相比于已知抗CD73抗体能够更为显著地恢复AMP所介导的CD4+T细胞抑制，增强对表达CD73肿瘤细胞的杀伤。因此，本发明的抗体(特别是人源化抗体)具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
附图说明
图1显示了鼠源抗体72G8对肿瘤细胞表面CD73的结合曲线。
图2A-2B分别显示了人源化抗体7002-04对天然表达人CD73的MDA-MB-231及A375肿瘤细胞的结合情况。ISO为同型对照抗体。
图3显示了人源化抗体7002-04对可溶性重组CD73的结合情况。
图4显示了人源化抗体7002-04对肿瘤细胞表面CD73的酶活性的抑制情况。
图5A-5C显示了人源化抗体7002-04对结直肠癌病人(A)、肝癌病人(B)、黑色素瘤(C)病人的血清中CD73酶活性的抑制情况。
图6A-6B显示了人源化抗体7002-04对AMP介导的CD4+T细胞抑制的缓解效果。
图7A-7B显示人源化抗体7002-04对PBMC对于肿瘤细胞的杀伤的恢复效果。
图8A-8C分别显示了CDR突变抗体7002-04-N56Q与细胞表面CD73的结合能力(A)、对细胞表面CD73酶活性的抑制能力(B)和对肿瘤病人血清CD73酶活性的抑制能力(C) 的评价结果。
序列信息
本发明涉及的部分序列的信息提供如下。
Figure PCTCN2020112271-appb-000002
Figure PCTCN2020112271-appb-000003
Figure PCTCN2020112271-appb-000004
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：鼠源抗人CD73抗体的产生
为了获得鼠源抗人CD73抗体，使用不同的免疫策略(表1)来免疫小鼠(Balb/c,上海灵畅生物)。所使用的抗原包括：CD73蛋白(即重组表达的人源CD73，其序列如SEQ ID NO:21所示)和CHOS-人CD73(即过表达CD73的CHOS细胞系，表达的CD73序列如SEQ ID NO:21所示)；佐剂包括：完全弗氏佐剂CFA(InvivoGen公司，货号vac-cfa-60)、IFA(InvivoGen公司，货号vac-ifa-60)、QuickAntibody(北京博奥龙免疫技术有限公司，货号KX0210041)；施用途径包括：腹膜内(ip)及皮下(sc)。在加强免疫3天后将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0使用聚乙二醇法进行融合，得到既能表达抗体又能在体外无限增殖的B细胞融合，并且在HAT选择培养基中培养。将融合后的杂交瘤细胞铺在96孔细胞培养板中，并且通过初级筛选挑选出阳性克隆进行2-3轮亚克隆。
表1：免疫策略
Figure PCTCN2020112271-appb-000005
Figure PCTCN2020112271-appb-000006
初级筛选：在初级筛选中通过使用人源CD73表达的肿瘤细胞或过表达细胞系测试生长克隆的上清液对细胞表面CD73结合能力。通过用DyLight488山羊抗鼠IgG(Abcam目录编号ab97015)第二抗体揭示出在上清液中的反应抗体的存在，在全视野细胞扫描分析仪上进行结合能力的评估(详细实验步骤可参见实施例2)。次级筛选：在CD73表达细胞上以及进行CD73酶活阻断能力的筛选，以评估抗体的细胞膜表面CD73酶活性阻断性质；使用人血清可溶性CD73进行CD73酶活阻断能力的筛选，以评估抗体的可溶性CD73酶活性阻断性质(详细实验步骤可参见实施例6)。
从最终获得的阳性杂交瘤单克隆细胞株的培养上清中分离纯化得到鼠源单克隆抗体72G8。
实施例2：鼠源抗CD73抗体的抗原结合活性评价
2.1通过细胞扫描分析仪检测鼠源抗体与CD73阳性细胞的结合情况
所使用的细胞：MDA-MB-231(内源表达人CD73；人乳腺癌细胞系)、SK-ME-S(内源表达人CD73；人肺鳞癌细胞系)、H2030(内源表达人CD73；人非小细胞肺癌细胞系)、SKLU1(内源表达人CD73；人肺腺癌细胞系)、BT549(内源表达人CD73；人乳腺管癌细胞系)、A375(内源表达人CD73；人黑色素瘤细胞系)、Calu6(内源表达人CD73；人退行性癌细胞系)、4T1(内源表达鼠CD73；鼠乳腺癌细胞系)、CHOS-人CD73(经人CD73转染)及CHOS(CD73阴性)细胞。
表达CD73的CHOS细胞的构建：通过慢病毒感染和抗性筛选的方法(MOI＝3-10，5μg/ml polybrene)在CHOS细胞(Invitrogen)上过表达人源CD73(SEQ ID NO:21)。慢病毒由上海吉凯基因化学技术有限公司提供，细胞感染72小时后加相应抗性继续培养2-4周，扩增并冻存，以用于后续实验。
实验方法：将10000个细胞平铺于100μL DMEM+10％FBS/孔中，使用平底96孔板，过夜使细胞贴壁或沉于孔底，第二天去掉上清。通过将1/3体积(100μL)稀释于200μLDMEM中来实施8点连续3倍稀释。细胞板的每一孔中加入100μL经稀释的抗体(筛选时 使用融合克隆上清或亚克隆上清)，相应阴性对照孔加入100μLDMEM，室温孵育1小时。去掉上清后，每孔加入100μL第二抗体(DyLight488山羊抗鼠IgG(Abcam目录编号ab97015)，浓度为5μg/mL(稀释于DMEM中)，室温孵育0.5小时。染色完成后去掉上清，用PBS+2％FBS清洗一次后每孔加入100100μLFBS+2％FBS，然后上机器进行读数。使用全视野细胞扫描分析仪(Nexcelom公司，型号
Figure PCTCN2020112271-appb-000007
Image Cytometer)对实验板进行测定读数。测定时选择第二抗体对应荧光通道和明场通道同时对孔内细胞进行高速扫描成像。荧光通道得到的成像根据有荧光标记的细胞形态和荧光强度设定参数对抗体结合的细胞进行计数，明场通道得到的成像根据细胞形态设定参数对贴壁细胞进行计数，然后两组数据相除得到和抗体结合的显示荧光的细胞占细胞总数的百分比。根据该比例判定抗CD73抗体与表达CD73的细胞株的结合效果。数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标使用和CD73抗体有结合显示绿色荧光的细胞和活细胞总数的百分比，根据曲线拟合出抗CD73抗体在各细胞上结合的EC50值。
72G8对于各肿瘤细胞的结合曲线如图1所示，其EC50值如表2-1和表2-2所示，N.B.表示在测定浓度范围内没有检测到。结果显示，72G8可结合天然表达CD73的细胞及重组表达人CD73的CHOS细胞，但该抗体不结合不表达CD73的细胞(CHOS)，也不结合表达鼠CD73的细胞(4T1)。
表2-1：抗体对内源性表达人CD73的肿瘤细胞结合EC50
Figure PCTCN2020112271-appb-000008
表2-2：抗体对重组表达人CD73的CHOS细胞以及其他不表达人CD73细胞的结合EC50
Figure PCTCN2020112271-appb-000009
2.2鼠源抗体与食蟹猴T细胞的结合
自美迪西获得来自两个供体(1132F与1300M)的猴血液。使用Ficoll密度梯度离心分离系统分离外周血单个核细胞(PBMC)。将PBMC与待测抗体一起孵育，再用荧光染料标记的二抗(DyLight488山羊抗鼠IgG，Abcam目录编号ab97015；DyLight488山羊抗人IgG，Abcam目录编号ab97003)染色细胞上结合的抗体，并使用荧光染料标记的针对CD3+ 及CD8+的抗体来识别T细胞。汇集未经染色对照样品及荧光补偿对照样品，上流式细胞仪运行样品，检测抗体对于食蟹猴T细胞的结合。
结果如表3所示，72G8能够结合食蟹猴CD8+T细胞。
表3：抗体对猴PBMC的结合
Figure PCTCN2020112271-appb-000010
实施例3：鼠源抗CD73抗体的可变区序列测定及嵌合抗体制备
离心收集杂交瘤细胞，每5-10×10 6细胞加入1ml TRIzol和0.2ml氯仿，剧烈振荡15秒，室温放置3分钟，离心取水相加入0.5ml异丙醇，室温放置10分钟后收集沉淀，乙醇洗涤后干燥得到RNA。在冰浴离心管里面加入模板RNA和引物，使引物和模板正确配对后进行反转录过程，在进行PCR扩增。4个微量离心管中各加入dNTP/ddNTP混合物2.5μl,混合物37℃温浴5min,备用。在一个空的微量离心管中加入1pmol的PCR扩增双链DNA,10pmol测序引物,2μl 5×测序缓冲液,加双蒸水至总体积10μl,96℃加热8min,冰浴泠却1min,4℃10000g离心10s。加入2μl预冷的标记混合物(dCTP、dGTP、dTTP各0.75μmol/L),α-32P-dATP 5μCi,1μl 0.1mol/L DDT,测序酶2U,加水至15μl,混匀后置冰上2min,标记新合成的DNA链。3.5μl标记反应混合物加入到准备好的4个微量离心管中,37℃温浴5min.每管各加入4μl终止液。样品在80℃的水浴中热变性5min,每一泳道加2μl加到测序胶上,电泳分离这些片段，收集序列信息。
鼠源抗体72G8的VH和VL序列如下表所示。进一步，还使用Kabat等人描述的方法(Kabat等，Sequences of Proteins of Immunological Interest,第五版，Public Health Service，美国国立卫生研究院，贝塞斯达、马里兰州(1991)，第647-669页)，确定了该鼠源单抗的CDR序列。
表4：鼠源抗体的序列信息
Figure PCTCN2020112271-appb-000011
将编码上述鼠源抗体的重链和轻链可变区的DNA序列(SEQ ID NOs:14-15)分别与 编码人抗体的重链恒定区(SEQ ID NO:19)和轻链恒定区(SEQ ID NO:20)的序列相连接，并在HEK293细胞(ATCC)中进行重组表达，收获培养瓶生长的含有抗体克隆的细胞上清液，使用protein A柱纯化，并且使用100mM醋酸pH3.0洗脱抗体蛋白。然后将纯化的抗体蛋白上样到分子排阻层析柱上进一步分离纯化。将对应于单体的抗体蛋白配制于PBS缓冲液中，配制品缓冲液补充有20％甘油。从而获得相应的嵌合抗体ch72G8。
实施例4：鼠源抗CD73抗体的人源化
为提高候选抗体与人源抗体的序列的同源性，减少抗体对人的免疫原性，可以对以上实施例提供的鼠源抗体进行人源化设计和制备，使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。
具体而言，将鼠源抗体72G8的重链和轻链CDR区分别移植到对应的人源化模板的FR框架上，并对人源化模板的FR区氨基酸残基进行了一系列的回复突变，以使人源化抗体尽可能保留鼠源抗体的抗原结合能力。根据以上方法，本发明人制备获得了鼠源抗体72G8的人源化抗体，命名为7002-04(其重链可变区及轻链可变区分别如SEQ ID NO:9和10所示)。抗体的重链恒定区为SEQ ID NO:19，轻链恒定区为SEQ ID NO:20。
实施例5：人源化抗CD73抗体的抗原结合活性评价
5.1通过流式细胞术测定抗体与CD73表达细胞的结合。
将500,000个CD73表达细胞(参见实施例2)置于100μL FACS buffer(PBS+2％FBS)/孔中待用，使用圆底低吸附96孔板。抗体样本通过将1/2体积(100μL)稀释于200μL FACS buffer中来实施3倍浓度的12点连续3倍稀释。细胞板的每一孔中加入100μL经稀释的抗体，相应阴性对照孔加入100μL FACS buffer，4摄氏度孵育1小时。离心去掉上清后，使用FACS buffer清洗两遍，每孔加入100μL第二抗体(DyLight488山羊抗鼠IgG，Abcam目录编号ab97015；DyLight488山羊抗人IgG，Abcam目录编号ab97003)5μg/mL，稀释于FACS buffer中)，4摄氏度再孵育0.5小时。染色完成后离心去掉上清，用FACS buffer清洗两次后每孔加入100μL FACS buffer重悬细胞，然后上机器进行读数。使用流式细胞分析仪(BD公司，型号ACCURI C6 PLUS)对实验 板中细胞进行测定读数。测定时先根据FCS和SSC圈定细胞位置，然后选择第二抗体对应绿色荧光通道(FITC)和SSC对细胞进行分析，数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标使用平均荧光强度数值，根据曲线拟合出抗CD73抗体的EC50。人源化抗体7002-04及相应的嵌合抗体ch72G8对天然表达人CD73的MDA-MB-231及A375细胞的结合情况分别如图2A-2B所示。表5显示了人源化抗体7002-04对于天然表达CD73的细胞及重组表达人CD73的细胞的结合情况，N.D.表示没有进行检测。结果表明，人源化抗体7002-04对膜结合CD73具备良好的结合活性，甚至与嵌合抗体相当。
表5：抗体对表达人CD73的肿瘤细胞结合EC50
Figure PCTCN2020112271-appb-000012
5.2通过ELISA测定抗体与可溶性人CD73蛋白的结合
将1μg/ml重组人CD73蛋白(百英生物，重组人源CD73蛋白)包被在PBS中的ELISA板上，在4℃下过夜。将板在洗涤缓冲液(PBS，0.05％吐温20)中洗涤3次，并且通过添加200μl/w PBS+2％BSA阻断使非特异性位点饱和。将剂量范围的抗CD73抗体梯度稀释100μL加入包被好抗原的ELISA板中在37℃下孵育1h。将板在洗涤缓冲液中洗涤3次，并且在室温下添加HRP偶联的山羊抗人或山羊抗小鼠IgG Fc片段第二抗体持续1hr以检测结合的抗CD73抗体。将板在洗涤缓冲液中洗涤3次，通过添加TMB(HRP底物)并且在室温下在黑暗中孵育板5至10分钟来揭示结合的第二抗体。通过添加硫酸溶液1M终止酶反应，并且在450nm处测量光吸收。以光吸收值为纵坐标，以抗体浓度log值为横坐标绘制曲线图，并且使用图板棱柱(GraphPad Prism)软件计算EC50。结果如图3所示，人源化抗体7002-04对可溶性重组CD73具有良好的结合活性，其EC50为0.0047μg/ml。
5.3通过Biacore测定人源化抗体与重组人源CD73蛋白的亲和力
在25℃下在Biacore T200(GE)上进行SPR测量抗体亲和力。将抗体用运行缓冲液1*HBS-EP+稀释至1ug/ml，并以10ul/min的流速分别捕获到芯片表面(Protein A chip，GE,Cat#29127556)上30s。随后，将一系列浓度的CD73蛋白(百英生物，重组人源CD73蛋白)以30ul/min的流速注入相应抗体通道，缔合阶段为180s，随后解 离900s。再生使用10mM pH1.5 Gly-HCL。使用1:1动态结合模型拟合整个传感图组。二价亲和力和动态结合和解离速率常数显示于下表中。
表6：抗体对重组CD73结合的亲和力常数
抗体 ka(1/Ms) kd(1/s) KD(M)
7002-04 1.32E+06 2.75E-04 2.073E-10
实施例6：抗CD73抗体对CD73酶活性的抑制活性评价
6.1肿瘤细胞中CD73酶活性的抑制试验
已知过量的AMP阻断ATP依赖性荧光素酶活性。将AMP切割为腺苷+无机磷酸的CD73通过减少AMP恢复荧光素酶活性和光发射。因此，阻断CD73的酶活性的抗体将减少光发射。
收获人CD73阳性细胞且计数。播种平底96板每孔20，000细胞于100μL完全培养基中。抗体样品通过将1/3体积(100μL)稀释于200μL DMEM中来实施8点连续3倍稀释，将稀释样品100μL加入相应板孔，阴性对照为同种型对照抗体(ISO)。于37℃孵育1小时，去掉上清后并用PBS清洗细胞两次。在不完全培养基中制备浓度为125μM AMP溶液，向每个孔中添加100μL AMP，并且将板在37℃下孵育另外两小时。离心反应板，取出50μL加入另一块96荧光板(OptiPlate-96,Perkin Elmer，#6005290)中，并加入同体积浓度为50μM的ATP溶液，并加入CTG试剂(普洛麦格公司，G7572)50μL于每孔，将板在室温下于黑暗中孵育15分钟，并且使用酶标仪测量荧光(Lum)。数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标为抑制率，抑制率计算方法如下：
抑制率＝(Lum 阳性对照-Lum 抗体)/(Lum 阳性对照-Lum 阴性对照)*100
抗体在不同人源肿瘤细胞系中阻断内源细胞CD73的IC50如下表所示。图4显示了人源化抗体7002-04及相应的嵌合抗体ch72GB对在A375细胞表面CD73酶活性抑制曲线。结果表明，人源化抗体7002-04能够显著抑制肿瘤细胞表面CD73的酶活性。
表7：抗体抑制不同人源肿瘤细胞表面CD73酶活的IC50
Figure PCTCN2020112271-appb-000013
6.2肿瘤病人血清CD73酶活性的抑制试验
将肿瘤病人血清稀释于磷酸buffer(Tris 125mM，MgCl2 25mM，NaCl125mM)，每孔 加入12.5μL待用，使用白色平底96孔板。抗体样品通过将1/1.5体积(100μL)稀释于50μL磷酸buffer中和将1/10体积(10μL)稀释于90μL磷酸buffer中来实施10点连续2～10倍稀释，细胞板的每一孔中加入12.5μL经稀释的抗体，阴性对照加入12.5μL磷酸buffer，离心后37℃孵育1.5小时。AMP使用磷酸buffer稀释成20μM溶液，每孔加入25μL AMP(阳性对照除外)，离心后37℃下孵育另外1小时。反应结束后在阳性对照加入25μL AMP。立即向每个孔中添加25μL AMP-Glo TM Reagent I(普洛麦格公司，货号V5012)，离心反应板，并且将板在室温下孵育1小时。向每个孔中添加50μL AMP Detection Solution(普洛麦格公司，货号V5012)，离心后室温下孵育1小时。使用酶标仪测量荧光(Lum)。数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标为抑制率，抑制率计算方法如下：
抑制率＝100-(Lum 阳性对照-Lum 抗体)/(Lum 阳性对照-Lum 阴性对照)*100
根据曲线拟合出抗CD73抗体的IC50。结果如图5A-5C所示，抗CD73抗体可有效抑制结直肠癌病人(A)、肝癌病人(B)、黑色素瘤(C)病人血清中CD73对于AMP的去磷酸化，抑制CD73酶活性。
实施例7：抗CD73抗体介导的CD73内化
通过流式细胞术测试抗CD73抗体介导的CD73内化。当测定抗体引起的内化和时间的关系的时候，在37℃下将所指示细胞与10μg/mL抗体一起孵育不同时间。用含2％FBS的PBS洗涤若干次后，加入10μg/mL第二抗体4℃下染色30分钟，然后通过流式细胞术分析细胞的CD73表达。当比较抗体引起内化程度的不同的时候，在4℃和37℃两种温度下平行将所指示细胞与10μg/mL抗体一起孵育20小时。用含2％FBS的PBS洗涤若干次后，加入10μg/mL第二抗体4℃下染色30分钟,然后通过流式细胞术分析细胞的CD73表达。
MFI 37为37℃条件下孵育的样品的MFI；MFI 4为4℃条件下孵育的样品的MFI，该条件下只发生结合而不发生内吞，MFI 背景为仅有第二抗体的MFI，抗体介导的细胞表面CD73内吞百分比由以下公式计算：
内化CD73的百分比＝100–100×(MFI 4-MFI 37)/MFI 4
结果如表8所示，这些抗体介导了不同程度的肿瘤细胞表面上CD73的内化。
表8：抗体介导肿瘤细胞表面上CD73的内化比例
Figure PCTCN2020112271-appb-000014
Figure PCTCN2020112271-appb-000015
实施例8：抗CD73抗体缓解AMP介导的CD4+T细胞抑制
实验前一天用抗CD3/抗CD28刺激PBMC细胞24小时，收集PBMC细胞(新鲜单采血经Ficoll分离所得)，使用CD4+T Cell Isolation Kit human(美天旎，货号130-096-533)分选出CD4+T细胞，细胞离心去上清，用含40μM EHNA和120IU/ml IL2的AIMV培养基将CD4+T细胞重悬(EHNA终浓度20μM，IL2终浓度60IU/ml)，将200,000 CD4+T细胞置于100μL/孔中，使用96孔低吸附圆底板。通过将1/3体积(100μL)稀释于200μL AIMV培养基中来实施10点连续2～10倍稀释，每孔加入50μL经稀释的抗体，相应阴性对照孔加入50μL AIMV培养基，37摄氏度孵育0.5小时。用AIMV制备400μM AMP(终浓度100μM)，每孔加入50μL配好的AMP溶液(对照孔加入不含AMP培养基)。离心后上机器进行读数。之后将板在37℃下孵育72小时然后上机器进行再次读数。读数使用全视野细胞扫描分析仪(Nexcelom公司，型号
Figure PCTCN2020112271-appb-000016
Image Cytometer)对实验板内细胞进行测定。测定时选择明场通道对孔内细胞进行高速扫描成像。根据该克隆团大小情况判定抗CD73抗体缓解AMP介导的CD4+T细胞抑制效果。其中，以MEDI9447(MedImmune)和BMS986179(BMS)为参比抗体，由吉凯表达纯化。
T细胞增殖第5天和第4天的细胞增长情况如图6A-6B所示；图中所使用#20和#18为内部使用PBMC捐供者编号；图中所使用抗体浓度起始为100μg/mL,四倍稀释共九个点。人源化抗体7002-04能够有效缓解AMP介导的CD4+T细胞抑制，T细胞增殖得到明显恢复，且效果优于参比抗体MEDI9447和BMS986179。
实施例9：抗CD73抗介导的肿瘤细胞杀伤
将5000个A375细胞平铺于100μL DMEM+10％FBS/孔中，使用平底96孔板，过夜使细胞贴壁，第二天去掉上清。通过将1/3体积(100μL)稀释于200μL AIMV中来实施10 点连续2～10倍稀释。细胞板的每一孔中加入50μL经稀释的抗体，相应阴性对照孔加入50μL AIMV，37摄氏度孵育0.5小时。收集提前一天用CD3/CD28刺激24小时后的PBMC细胞(新鲜单采血经Ficoll分离所得)，用含40μM EHNA和120IU/ml IL2的AIMV重悬(EHNA终浓度20μM，IL2终浓度60IU/ml)，按5,000/100μL加入每孔。用AIMV制备浓度为400μM AMP溶液，向每个孔中添加50μL(AMP终浓度100μM)。离心后37℃孵育72小时。每孔加入10μL CCK8试剂盒(日本同仁公司，货号CK04)，37度孵育4小时后使用酶标仪测量OD450。根据对照孔数值将OD值分别换算成抑制百分比，据此判定抗CD73抗体抗介导的肿瘤细胞杀伤大小。百分比越大代表抗CD73抗介导的肿瘤细胞杀伤效果越好，反之百分比越小代表抗CD73抗介导的肿瘤细胞杀伤效果越差。数据分析使用GraphPad，横坐标使用抗体浓度的对数，纵坐标使用抑制百分比，根据曲线拟合出抗CD73抗体在A375细胞上的IC50值。
结果如图7A-7B所示，图中所使用#13和#22为内部使用PBMC捐供者编号，结果显示人源化抗体7002-04能够有效恢复PBMC对于肿瘤细胞的杀伤，并且显著优于参比抗体MEDI9447和BMS986179。
实施例10:含有CDR突变的抗体及其活性评价
将7002-04重链可变区HCDR2中的N56突变为Q，所获得的变体命名为7002-04-N56Q(其VH和VL分别如SEQ ID NO:13和10所示)。使用5.1中方法通过流式细胞术测定抗体与CD73表达的人源肿瘤细胞MDA-MB-231的结合，结果如图8A所示，7002-04-N56Q与细胞表面的CD73的结合能力与7002-04一致。使用6.1中方法测量抗体对肿瘤细胞中CD73酶活性的抑制，结果如图8B所示，7002-04-N56Q对MDA-MB-231表面CD73酶活性的抑制能力与7002-04基本一致。使用6.2中方法测量抗体对肿瘤病人血清CD73酶活性的抑制，结果如图8C所示，7002-04-N56Q对结直肠癌病人血清中CD73酶活性的抑制能力与7002-04一致。由此可见，包含CDR突变的7002-04-N56Q基本保持了亲本抗体7002-04优良的生物学活性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (25)

  1. 能够特异性结合CD73的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:4或SEQ ID NO:11，或与前述任一序列相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    和/或，
    (b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:8，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换。
  2. 权利要求1所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:1所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列；所述轻链可变区包含SEQ ID NO:2所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。
  3. 权利要求1所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:12所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列；所述轻链可变区包含SEQ ID NO:2所示的氨基酸 序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。
  4. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列；
    优选地，所述抗体或其抗原结合片段包含：来源于人重链胚系序列的重链框架区序列，以及来源于人轻链胚系序列的轻链框架区序列。
  5. 权利要求4所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:9所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列；所述轻链可变区包含SEQ ID NO:10所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。
  6. 权利要求4所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:13所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列；所述轻链可变区包含SEQ ID NO:10所示的氨基酸序列或与其相比具有至少约85％、90％、95％或99％序列同一性的序列。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，所述抗体或其抗原结合片段包含选自下列的重链恒定区：
    (1)人IgG1重链恒定区；
    (2)人IgG1重链恒定区的变体，所述变体与其所源自的野生型序列相比具有以下置换：L234F、L235E、P331S，以上提及的氨基酸位置是根据EU编号系统的位置；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:19所示的重链恒定区(CH)；
    优选地，所述抗体或其抗原结合片段包含人κ轻链恒定区；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:20所示的轻链恒定区(CL)。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段具备以下特征中的一项或多项：
    (a)与膜结合人CD73或可溶性人CD73或两者结合；
    (b)抑制或降低CD73(例如，膜结合人CD73或可溶性人CD73)的酶活性；
    (c)在腺苷单磷酸(AMP)存在下，增加抗CD3/抗CD28刺激的T细胞(例如CD4+T细胞)的增殖；
    (d)降低表达CD73的肿瘤细胞中的腺苷水平；
    (e)通过抗体介导的受体内化使CD73内化至细胞(例如，肿瘤细胞)中。
  10. 分离的核酸分子，其编码权利要求1-9任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
  11. 载体，其包含权利要求10所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  12. 宿主细胞，其包含权利要求10所述的分离的核酸分子或权利要求11所述的载体。
  13. 制备权利要求1-9任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求12所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  14. 双特异性或多特异性分子，其包含权利要求1-9任一项所述的抗体或其抗原结合片段；
    优选地，所述双特异性或多特异性分子特异性结合CD73，并且额外地特异性结合一 个或多个其他靶标；
    优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
  15. 免疫缀合物，其包含权利要求1-9任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
    优选地，所述治疗剂选自细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  16. 药物组合物，其含有权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求14所述的双特异性或多特异性分子或者权利要求15所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述另外的药学活性剂选自免疫检查点抑制剂(例如，PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、LAG-3抑制剂)、抗CD39抗体、抗A2AR抗体或抗HER2/ErbB2抗体。
  17. 试剂盒，其含有权利要求1-9任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-9任一项所述的抗体或其抗原结合片段；优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  18. 一种用于在受试者(例如人)中预防和/或治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求14所述的双特异性或多特异性分子、权利要求15所述的免疫缀合物或权利要求16所述的药物组合物；
    优选地，所述肿瘤表达CD73；
    优选地，所述肿瘤选自黑色素瘤、结肠癌、肺癌、肝癌、胰腺癌、卵巢癌、膀胱癌、神经胶质瘤、神经胶母细胞瘤、甲状腺癌、食道癌、前列腺癌及乳腺癌；
    优选地，所述受试者为人。
  19. 权利要求18所述的方法，其中，所述方法还包括施用第二治疗剂，所述第二治疗剂选自具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述方法包括将权利要求1-9任一项所述的抗体或其抗原结合片段与选自下列的药剂组合施用：免疫检查点抑制剂(例如，PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、LAG-3抑制剂)、抗CD39抗体、抗A2AR抗体或抗HER2/ErbB2抗体。
  20. 权利要求18所述的方法，其中，所述方法还包括施用第二治疗术，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。
  21. 权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求14所述的双特异性或多特异性分子、权利要求15所述的免疫缀合物或权利要求16所述的药物组合物，用于在受试者中预防和/治疗肿瘤的药物的用途，或者在制备用于在受试者中预防和/治疗肿瘤的药物中的用途；
    优选地，所述的抗体或其抗原结合片段、双特异性或多特异性分子、免疫缀合物或药物组合物药物与另外的药学活性剂的联合施用；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述另外的药学活性剂选自免疫检查点抑制剂(例如，PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、LAG-3抑制剂)、抗CD39抗体、抗A2AR抗体或抗HER2/ErbB2抗体；
    优选地，所述肿瘤表达CD73；
    优选地，所述肿瘤选自黑色素瘤、结肠癌、肺癌、肝癌、胰腺癌、卵巢癌、膀胱癌、神经胶质瘤、神经胶母细胞瘤、甲状腺癌、食道癌、前列腺癌及乳腺癌；
    优选地，所述受试者为人。
  22. 一种刺激受试者中的免疫应答的方法，所述方法包括向所述受试者施用有效量的权利要求1-9任一项所述的抗体或其抗原结合片段、或权利要求16所述的药物组合物。
  23. 权利要求1-9任一项所述的抗体或其抗原结合片段或权利要求16所述的药物组合物，用于刺激受试者中的免疫应答的用途，或在制备用于刺激受试者中的免疫应答的药物中的用途。
  24. 一种检测CD73(例如人CD73)在样品中的存在或其量的方法，其包括以下步骤：
    (1)将所述样品与权利要求1-9任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与CD73之间复合物的形成或检测所述复合物的量；
    优选地，所述抗体或其抗原结合片段带有可检测的标记。
  25. 权利要求1-9任一项所述的抗体或其抗原结合片段在制备用于测定CD73(例如人CD73)在样品中的存在或其量的检测试剂中的用途。
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