WO2022179039A1 - 抗人cd73抗体及其应用 - Google Patents

抗人cd73抗体及其应用 Download PDF

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WO2022179039A1
WO2022179039A1 PCT/CN2021/106917 CN2021106917W WO2022179039A1 WO 2022179039 A1 WO2022179039 A1 WO 2022179039A1 CN 2021106917 W CN2021106917 W CN 2021106917W WO 2022179039 A1 WO2022179039 A1 WO 2022179039A1
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antibody
seq
heavy chain
light chain
variable region
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PCT/CN2021/106917
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English (en)
French (fr)
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李德彬
宋作伟
路娜
于福涛
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苏州近岸蛋白质科技股份有限公司
上海近岸科技有限公司
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Priority claimed from CN202110202193.9A external-priority patent/CN112552406B/zh
Priority claimed from CN202110292998.7A external-priority patent/CN113045659B/zh
Application filed by 苏州近岸蛋白质科技股份有限公司, 上海近岸科技有限公司 filed Critical 苏州近岸蛋白质科技股份有限公司
Priority to JP2023563043A priority Critical patent/JP2024516581A/ja
Priority to EP21927468.5A priority patent/EP4299589A1/en
Publication of WO2022179039A1 publication Critical patent/WO2022179039A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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
    • C07K2317/70—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
    • C07K2317/80—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to an anti-human CD73 antibody and its application.
  • CD73 cluster of differentiation 73, also called 5'-nucleotidase, is anchored to the cell membrane by GPI (glycosylphosphatidylinositol), and the structure is a dimer.
  • GPI glycosylphosphatidylinositol
  • CD73 plays an important role in the tumor microenvironment and can convert AMP into adenosine, which binds to specific G protein-coupled receptors, leading to abnormal infiltration of immune cells in tumor sites, and regulating the proliferation and migration of various cancers. It mainly inhibits the function of the immune system related to T cells and is a marker of poor tumor prognosis.
  • CD73 is expressed on the surface of a variety of tumor cells, including lung cancer, melanoma, colon cancer, pancreatic cancer and other tumor cells.
  • a variety of immune cells are also affected by CD73.
  • CD73 is expressed on the surface of Treg and participates in the inhibitory regulation of Treg on immune activation; the secreted adenosine can bind to adenosine receptors on DC cells, thereby inhibiting the function of DCs ; CD73 is highly expressed on M2-type macrophages and enhances tumor metastasis.
  • the CD73 target has great potential in tumor therapy, and CD73 antibody can also enhance the effect of PD1 antibody and CTLA4 antibody.
  • CD73-specific targeting antibody there is currently no CD73-specific targeting antibody on the market.
  • mouse monoclonal antibody can cause human anti-mouse antibody (HAMA) in clinical treatment, it is limited in clinical treatment.
  • Antibody humanization technology can greatly reduce the immunogenicity of murine mAbs.
  • the purpose of the present invention is to provide an anti-human CD73 antibody with high affinity and high biological activity and its application.
  • a heavy chain variable region of an anti-CD73 antibody wherein the heavy chain variable region has VH-CDRs selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.: 15 or 21,
  • VH-CDR3 independently selected from the group consisting of: SEQ ID NO.:17, SEQ ID NO.:22, SEQ ID NO.:23, SEQ ID NO.:24, SEQ ID NO.:25, SEQ ID NO.:25, SEQ ID NO.:25 ID NO.:26, SEQ ID NO.:27.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:2.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:5.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:7.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:9.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:34.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:36.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:37.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:38.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:39.
  • any one of the above amino acid sequences also includes a derivative sequence that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain CD73 binding affinity.
  • a heavy chain of an anti-CD73 antibody the heavy chain has the heavy chain variable region according to the first aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human origin, mouse origin or rabbit origin, preferably human origin.
  • the heavy chain constant region is a human antibody heavy chain IgG1 constant region.
  • a light chain variable region of an anti-CD73 antibody wherein the light chain variable region has VL-CDRs selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.: 18,
  • VL-CDR3 independently selected from the group consisting of: SEQ ID NO.:20, SEQ ID NO.:28, SEQ ID NO.:29, SEQ ID NO.:30, SEQ ID NO.:31, SEQ ID NO.:31 ID NO.:32, SEQ ID NO:33.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 1.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:6.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:8.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:10.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:40.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:41.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:42.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:43.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:44.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:45.
  • any one of the above amino acid sequences also includes a derivative sequence that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain CD73 binding affinity.
  • a light chain of an anti-CD73 antibody the light chain has the light chain variable region according to the third aspect of the present invention.
  • the light chain of the antibody further includes a light chain constant region.
  • the light chain constant region is of human origin, mouse origin or rabbit origin, preferably human origin.
  • the light chain constant region is a human antibody light chain kappa constant region.
  • an anti-CD73 antibody is provided, and the antibody has:
  • the antibody has: a heavy chain as described in the second aspect of the invention; and/or a light chain as described in the fourth aspect of the invention,
  • any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity of CD73.
  • the number of added, deleted, modified and/or substituted amino acids is 1-5 (eg 1-3, preferably 1-2, more preferably 1).
  • the antibody is a humanized antibody.
  • the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibodies include monospecific, bispecific, or trispecific antibodies.
  • variable region of the heavy chain of the antibody further includes a human framework region
  • variable region of the light chain of the antibody further includes a human framework region
  • the heavy chain variable region of the antibody further includes a murine framework region, and/or the light chain variable region of the antibody further includes a murine framework region.
  • the antibody has the variable region of the heavy chain as described in the first aspect of the present invention and the variable region of the light chain as described in the third aspect of the present invention;
  • described heavy chain variable region comprises the VH-CDR that is selected from the following group:
  • VH-CDR1 shown in SEQ ID NO.: 15 or 21,
  • VH-CDR3 independently selected from the group consisting of: SEQ ID NO.:17, SEQ ID NO.:22, SEQ ID NO.:23, SEQ ID NO.:24, SEQ ID NO.:25, SEQ ID NO.:25, SEQ ID NO.:26 or SEQ ID NO.:27; and
  • the light chain variable region includes a VL-CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.: 18,
  • VL-CDR3 independently selected from the group consisting of: SEQ ID NO.:20, SEQ ID NO.:28, SEQ ID NO.:29, SEQ ID NO.:30, SEQ ID NO.:31, SEQ ID NO.:31 ID NO.:32 or SEQ ID NO:33.
  • the antibody has the variable region of the heavy chain according to the first aspect of the present invention and the variable region of the light chain according to the third aspect of the present invention; wherein,
  • the three light chain CDRs of the light chain variable region and the three heavy chain CDRs of the heavy chain variable region are selected from the group consisting of:
  • amino acid sequence of the light chain variable region (VL) of the anti-human CD73 antibody is as shown in SEQ ID NO: 1, 6, 8, 10, 40, 41, 42, 43, 44 or 45 and the amino acid sequence of the heavy chain variable region (VH) of the anti-human CD73 antibody is shown in SEQ ID NO: 2, 5, 7, 9, 34, 35, 36, 37, 38 or 39.
  • the antibody is selected from the following group:
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:2; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:1 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:5; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:6 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:7; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:8 sequence.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 9; and the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 10.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:34; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:40 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:35; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:41 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:36; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:42 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:37; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:43 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:38; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:44 sequence.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:39; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.:45 sequence.
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains CD73 binding affinity.
  • amino acid sequence of the heavy chain variable region is the same as the amino acid shown in SEQ ID NO.: 2, 5, 7, 9, 34, 35, 36, 37, 38 or 39 in the sequence listing
  • the sequences have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • amino acid sequence of the light chain variable region is the same as the amino acid shown in SEQ ID NO.: 1, 6, 8, 10, 40, 41, 42, 43, 44 or 45 in the sequence listing
  • the sequences have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the anti-CD73 antibody includes a light chain and a heavy chain, and the light chain variable region of the light chain includes the following three light chain CDRs:
  • VL-CDR1 shown in SEQ ID NO.: 18,
  • VL-CDR2 shown in SEQ ID NO.: 19, and
  • VL-CDR3 shown in SEQ ID NO.: 20, 28, 29, 30, 31, 32 or 33; wherein, the heavy chain variable region of the heavy chain includes the following three heavy chain CDRs:
  • VH-CDR1 shown in SEQ ID NO.:3915 or 21,
  • VH-CDR2 shown in SEQ ID NO.: 16, and
  • the light chain of the antibody includes the three light chain CDRs and the light chain framework region for connecting the light chain CDRs; and the heavy chain of the antibody includes the three light chain CDRs. Heavy chain CDRs and heavy chain framework regions for linking heavy chain CDRs.
  • the antibody is a humanized antibody.
  • the antibody specifically binds to CD73.
  • the KD value (M) of the affinity of the antibody to human CD73 is 1.0E-10 to 2.0E-12.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibodies include monospecific, bispecific, or trispecific antibodies.
  • a recombinant protein is provided, and the recombinant protein has:
  • variable region of the heavy chain according to the first aspect of the present invention the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention
  • the light chain of aspect, or the antibody of the fifth aspect of the invention and (ii) an optional tag sequence to facilitate expression and/or purification.
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused with the element (i).
  • an antibody preparation comprising:
  • carrier includes: buffer, sterile water, and optional surfactant.
  • kits contains the antibody preparation according to the seventh aspect of the present invention, and a container for holding the antibody preparation.
  • a CAR construct is provided, wherein the scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to CD73, and the scFv segment has the The heavy chain variable region according to the first aspect and the light chain variable region according to the third aspect of the present invention.
  • a recombinant immune cell is provided, and the immune cell expresses the exogenous CAR construct according to the fifth aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • an antibody-drug conjugate is provided, and the antibody-drug conjugate contains:
  • an antibody portion selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a heavy chain as described in the third aspect of the invention
  • the light chain variable region of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, or the recombinant protein of the sixth aspect of the present invention, or a combination thereof and
  • a coupling moiety coupled to the antibody moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • an active ingredient selected from the group consisting of the antibody according to the fifth aspect of the present invention, or the recombinant protein according to the sixth aspect of the present invention
  • the CAR construct according to the seventh aspect of the present invention, the immune cell according to the tenth aspect of the present invention, the antibody drug conjugate according to the eleventh aspect of the present invention, or a combination thereof, the active ingredient is At:
  • the cancer or tumor is selected from the group consisting of lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, Liver cancer, lymphoma, myeloma, leukemia.
  • a pharmaceutical composition in the thirteenth aspect of the present invention, contains:
  • an active ingredient selected from the group consisting of an antibody as described in the fifth aspect of the present invention, or a recombinant protein as described in the sixth aspect of the present invention, or a CAR construct as described in the seventh aspect of the present invention , the immune cell according to the tenth aspect of the present invention, the antibody drug conjugate according to the eleventh aspect of the present invention, or a combination thereof; and
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to treat tumors.
  • the tumor is a tumor that highly expresses CD73.
  • the fifteenth aspect of the present invention there is provided a vector containing the polynucleotide according to the fourteenth aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector according to the fifteenth aspect of the present invention or the fourteenth aspect of the present invention is integrated into the genome the polynucleotide.
  • an in vitro non-diagnostic method for detecting (including diagnostic or non-diagnostic) CD73 protein in a sample comprising the steps of:
  • an in vitro method for detecting (including diagnostic or non-diagnostic) CD73 protein in a sample comprising the steps of:
  • a method for treating CD73-related diseases comprising:
  • the antibody of the fifth aspect of the present invention the drug conjugate of the antibody, or the CAR-T cell expressing the antibody, or a combination thereof is administered to a subject in need thereof.
  • Figure 1 shows a graph of the affinity of CQ137 for rhCD73 as determined by Biacore.
  • Fig. 2 shows the affinity test graph of CQ137 and recombinant monkey CD73 protein.
  • Figure 3 shows the affinity detection graph of CQ137 and SK-OV-3 cells.
  • Figure 4 shows a graph of the affinity detection of CQ137 with CHOK1-cynoCD73 cells.
  • Figure 5 shows a comparison of the inhibition of rhCD73 enzymatic activity by two CD73 antibodies (CQ137 and MEDI-9447).
  • Figure 6 shows a graph of the activity of CQ137 to inhibit CD73 on SK-OV-3 cells.
  • Figure 7 shows a graph of the activity of CQ137 to inhibit CD73 on CHOK1-cynoCD73 cells.
  • Figure 8A shows the binding activity of humanized CD73 antibodies (137-1, 137-2, 137-3) to rhCD73.
  • Figure 8B shows the binding activity of humanized CD73 antibodies (137-1, 137-2, 137-3) to recombinant monkey CD73 protein.
  • Figure 9 shows the graph of the affinity detection of humanized CD73 antibodies (137-1, 137-2, 137-3) with SK-OV-3 cells.
  • Figure 10 shows the graph of affinity detection of humanized CD73 antibodies (137-1, 137-2, 137-3) with CHOK1-cynoCD73 cells.
  • Figure 11 shows the comparison of humanized CD73 antibodies (137-1, 137-2, 137-3) inhibiting rhCD73 enzymatic activity (RLU response fold), RLU is relative light unit.
  • Figure 12 shows a comparison of the inhibition of CD73 enzymatic activity on SK-OV-3 cells by humanized CD73 antibodies (137-1, 137-2, 137-3).
  • Figure 13 shows the results of humanized CD73-mediated CD73 internalization in SK-OV-3 cells, and MFI is the relative fluorescence intensity.
  • Figure 14 shows that humanized 137 antibodies (137-1, 137-2, 137-3) inhibit tumor cells in the NSG-A375 model.
  • Figure 15 shows the binding of humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to recombinant human CD73 protein.
  • Figure 16 shows the binding of humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to recombinant monkey CD73 protein.
  • Figure 17 shows the affinity of CD73 antibodies (humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 and MEDI-9447) with SK-OV-3 cells picture.
  • Figure 18 shows affinity graphs of humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 and CHOK1-cynoCD73 cells.
  • Figure 19 shows the inhibitory effect of humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 on rhCD73 enzymatic activity.
  • Figure 20 shows the inhibitory effect of humanized CD73 antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 on CD73 enzymatic activity on SK-OV-3 cells.
  • Figure 21 shows the results of humanized CD73 anti-137-4, 137-5, 137-6, 137-7, 137-8, 137-9 body-mediated CD73 internalization by SK-OV-3 cells.
  • the present invention utilizes phage display technology to obtain a highly specific CD73 antibody through screening, and performs humanization while taking into account the similarity and the frequency of human use.
  • the antibody specifically binds to human and monkey CD73 protein and CD73 positive cells, and has the effect of inhibiting CD73 protease activity.
  • Antibodies of the invention can also induce tumor cell internalization of CD73.
  • the antibodies of the present invention showed better antitumor activity than MEDI-9447 of AstraZeneca (AZ)/Medimmune. The present invention has been completed on this basis.
  • administer refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells.
  • administering and “treating” also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, animal or research subject, means therapeutic treatment, prophylactic or preventive measures, research and diagnosis; chamber or contact with physiological fluids.
  • treatment refers to the administration of an internal or external therapeutic agent, comprising any one of the anti-CD73 antibodies and compositions thereof of the invention, to a patient with one or more disease symptoms for which the treatment is known Drugs have a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance can, but need not, occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may, but does not necessarily have, one, two or three.
  • Sequence identity as used in the present invention means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequences described in the present invention and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • antibody refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called ⁇ , respectively. , ⁇ , ⁇ , ⁇ , and ⁇ . IgG represents the most important class of immunoglobulins.
  • Light chains are classified into kappa or lambda chains by differences in the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 FR regions (FR) with relatively conserved sequences. The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction.
  • Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions (framework regions).
  • the three CDR regions of the light chain namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3;
  • the three CDR regions of the heavy chain namely the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3.
  • the number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the numbering rules of Kabat and Chothia (HCDR1). ).
  • the four FR regions in the variable regions of native heavy and light chains are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a linking loop, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are brought together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • the term "antigen-binding fragment” refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • antigenic determinant refers to a discrete three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "monoclonal antibody” refers to a cloned secreted antibody derived from a single cell. Monoclonal antibodies are highly specific, directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic or phage clonal cell strains.
  • chimeric antibody is an antibody molecule expressed by a murine antibody V region gene and a human antibody C region gene spliced into a chimeric gene, which is then inserted into a vector and transfected into a host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effector functions.
  • humanized antibody is a variable region engineered form of the murine antibody of the present invention having CDR regions derived from (or substantially derived from) a non-human antibody, preferably a mouse monoclonal antibody , and FR and constant regions derived substantially from human antibody sequences; that is, grafting the CDR region sequences of the murine antibody onto different types of human germline antibody framework sequences. Because the CDR sequences are responsible for most antibody-antigen interactions, expression vectors can be constructed to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies.
  • antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • CD73 generally refers to native or recombinant human CD73, as well as non-human homologs of human CD73.
  • the present invention provides a high-specificity and high-affinity antibody against CD73, comprising a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, and the light chain containing a light chain variable Region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable Region
  • a human-mouse chimeric anti-CD73 full-length antibody obtained is a CQ137 antibody protein.
  • the obtained CQ137 antibody can bind to the CD73 molecule expressed by the tumor and inhibit the CD73 activity on human ovarian adenocarcinoma cells. Has the potential to treat a variety of CD73-overexpressing tumors.
  • the CDRs of the heavy chain variable region (VH) are selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.: 15,
  • VH-CDR2 shown in SEQ ID NO.: 16, and
  • VH-CDR3 shown in SEQ ID NO.: 17; and/or
  • the CDRs of the light chain variable region (VL) are selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.: 18,
  • VL-CDR2 shown in SEQ ID NO.: 19, and
  • VL-CDR3 shown in SEQ ID NO.:20.
  • any amino acid sequence in the above-mentioned amino acid sequence also includes at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) after addition, deletion, modification and/or substitution Derivative sequences of amino acids with CD73 binding affinity.
  • the sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90% %, optimally at least 95% of the amino acid sequence.
  • the antibodies of the present invention may be double-chain or single-chain antibodies, and may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully human Antibody.
  • the antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the animal is preferably a mammal, such as a mouse.
  • the antibodies of the invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting human CD73.
  • VH heavy chain variable region
  • VL light chain variable region
  • VH CDR1, CDR2 and CDR3 are independently selected from any one or more of SEQ ID NO.:15, SEQ ID NO.:16 and SEQ ID NO.:17 Sequences, or their sequences with CD73 binding affinity in which at least one amino acid has been added, deleted, modified and/or substituted;
  • VL CDR1, CDR2, CDR3 are independently selected from SEQ ID NO.: 18, SEQ ID NO.: 19 and Any one or several sequences in SEQ ID NO.:20, or their sequences with CD73 binding affinity to which at least one amino acid has been added, deleted, modified and/or substituted.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33% , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, 1 is optimally.
  • an antibody against human CD73 (CQ137) is provided:
  • hIgG1 constant region amino acid sequence (SEQ ID NO.: 3):
  • amino acid sequence of kappa chain constant region (SEQ ID NO.: 4):
  • the present invention also provides a nucleotide sequence encoding the above amino acid: CQ137VH nucleotide sequence (SEQ ID NO.: 11):
  • the selected hIgG1 constant region nucleotide sequence is (SEQ ID NO.: 13):
  • the selected kappa chain constant region nucleotide sequence is (SEQ ID NO.: 14):
  • the present invention also provides a humanized antibody with high specificity and high affinity against CD73.
  • the sequence also includes a sequence formed by adding, deleting, modifying and/or replacing at least one amino acid sequence, preferably the homology is at least 80%, preferably at least 85%, more preferably Preferably at least 90%, and optimally at least 95% of the amino acid sequence.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.
  • the antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab') 2 , or other known antibody derivatives in the field, etc., as well as IgA, IgD, Any one or more of IgE, IgG and IgM antibodies or other subtypes of antibodies.
  • the antibodies of the invention may be humanized antibodies targeting CD73, CDR-grafted and/or modified antibodies.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33% , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • any method suitable for producing monoclonal antibodies can be used to produce the CD73 antibodies of the invention.
  • animals can be immunized with linked or naturally occurring CD73 protein or fragments thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • CD73 can be used as an immunogen (antigen) for generating non-human antibodies specific for CD73 and screening the antibodies for biological activity.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources, or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic in origin (eg, cDNA).
  • the DNA encoding the immunogen can be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculovirus vectors, plasmids and non-viral vectors.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.
  • sequences of the DNA molecules of the antibodies or fragments thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments followed by ligation. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (eg, mammalian cells).
  • the steps of transforming host cells with recombinant DNA described in the present invention can be performed using techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the genes of the present invention. Depending on the host cell used, it is cultured with conventional media under appropriate conditions.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • the antibody of the present invention can be obtained by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • ADCs Antibody-Drug Conjugates
  • the present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg, ethylene glycol polymers
  • therapeutic agents eg, ethylene glycol polymers
  • Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the linker Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (eg, iodo, bromo, or chloro); haloesters (eg, iodo, bromo, or chloro) ); halogenated methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines).
  • Antibodies have different stability in different formulation buffers, which are manifested as changes in charge heterogeneity, degradation of antibody molecules, polymerization, etc. These changes in quality properties are related to the physicochemical properties of the antibody itself. Therefore, in the development of antibody drugs During the process, it is necessary to screen the suitable preparation buffer according to the physicochemical properties of different antibodies.
  • the commonly used buffer systems for antibody preparations include phosphate buffer, citrate buffer, histidine buffer, etc.
  • the antibody-drug combination preparation of the present invention can effectively inhibit side reactions such as aggregation, precipitation, hydrolysis, oxidation and deamidation of the humanized antibody of the present invention, and at the same time can effectively improve the product's performance under pressure (high temperature, strong light irradiation, freezing and thawing, etc.), Stability under accelerated and long-term refrigerated conditions.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cells, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention can also be expressed in a cell by a nucleotide sequence for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used to bind CD73 protein molecules, and thus can be used to prevent and treat CD73-related diseases.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, compared to Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration, the patient's health status, etc., which are all within the skill of the skilled physician.
  • Antibodies of the invention can be used in detection applications, eg, in detection of samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsies known to those skilled in the art. Biopsy used in the present invention may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the humanized antibody of the present invention has better safety compared with murine and chimeric antibodies
  • the antibody of the present invention has higher affinity through competitive screening
  • the antibody of the present invention is derived from phage display technology, which is more diverse than hybridoma technology.
  • This example mainly describes the sequence of anti-human CD73 single chain antibody obtained by mouse immunization and phage display.
  • Balb/C mice were immunized with recombinant human CD73 protein (C446, Novoprotein). After the first immunization, booster immunization was performed every 14 days for a total of 4 immunizations.
  • the mouse serum was collected for antibody titer evaluation.
  • B cells were taken from the effective mice, RNA was extracted and reverse transcribed, and a phage display library was constructed.
  • CQ137VH and VL were respectively constructed into eukaryotic expression vectors containing hIgG1-kappa constant region, and transfected into freestyle 293F cells. After culturing for 3-7 days, the supernatant was collected and purified with protein A column to obtain CQ137 antibody protein.
  • hIgG1 constant region amino acid sequence (SEQ ID NO.: 3)
  • Amino acid sequence of kappa chain constant region (SEQ ID NO.:4)
  • This example mainly describes the affinity detection of CQ137 with human and monkey recombinant CD73 protein, as well as the affinity detection of CQ137 with human CD73 positive cells (SK-OV-3, human ovarian adenocarcinoma cells) and monkey CD73 cell lines (CHOK1-cynoCD73). Affinity testing situation.
  • the affinity of CQ137 and recombinant hCD73 protein was determined by Biacore, CQ137 was immobilized, and the CD73 protein was diluted by a two-fold gradient (2.5nM-40nM).
  • the recombinant monkey CD73 protein was coated at 3 ⁇ g/ml, and CQ137 diluted in a gradient manner was added, and the binding of CQ137 to the recombinant monkey CD73 was detected by ELISA. The results are shown in FIG. 2 .
  • the full-length monkey CD73 was constructed into a eukaryotic expression vector containing NEO resistance, transfected into CHO-K1 cells, and the CHOK1 -cynoCD73 cell line was obtained after screening with G418.
  • the CQ137 protein was incubated for 1 h, washed three times with PBS, added Anti-hFC-APC (purchased from Jackson immunology), and detected by flow cytometry. The resulting S-curve is shown in Figure 4.
  • This example mainly demonstrates that CQ137 inhibits CD73 protein and cellular enzymatic activity.
  • the CD73 protein was diluted to a working concentration of 5 ⁇ g/ml, and three-fold gradient (0.001-10 ⁇ g/ml) diluted CD73 antibody was added, incubated at 37°C for 15 min, added a mixture of 1 mM ATP and AMP, incubated at 37°C for 30 min, and added CellTiter- Glo detection reagent (purchased from promega), the autoluminescence value was read by a microplate reader, and the enzyme activity without antibody was calculated as 100%, and the change in the activity of rhCD73 was obtained as shown in Figure 5.
  • the humanized framework closest to CQ137 was analyzed to obtain a series of humanized antibodies.
  • the VH and VL of the humanized sequences were constructed into vectors containing hIgG1 and kappa light chain constant regions, respectively, and transfected into Freestyle 293F cells. The supernatant was collected, and purified by protein A column to obtain the desired antibody protein.
  • Humanized CQ137 mAb (137-1) heavy chain VH amino acid sequence (SEQ ID NO.: 5)
  • Humanized CQ137 monoclonal antibody (137-1) light chain VL amino acid sequence is (SEQ ID NO.: 6)
  • Humanized CQ137 monoclonal antibody (137-2) heavy chain VH amino acid sequence is (SEQ ID NO.: 7)
  • Humanized CQ137 monoclonal antibody (137-2) light chain VL amino acid sequence is (SEQ ID NO.: 8)
  • Humanized CQ137 monoclonal antibody (137-3) heavy chain VH amino acid sequence is (SEQ ID NO.: 9)
  • Humanized CQ137 monoclonal antibody (137-3) light chain VL amino acid sequence is (SEQ ID NO.: 10)
  • Humanized CQ137 monoclonal antibody (137-4) heavy chain VH amino acid sequence is (SEQ ID NO.: 34)
  • Humanized CQ137 monoclonal antibody (137-4) light chain VL amino acid sequence is (SEQ ID NO.: 40)
  • Humanized CQ137 monoclonal antibody (137-5) heavy chain VH amino acid sequence is (SEQ ID NO.: 35)
  • Humanized CQ137 monoclonal antibody (137-5) light chain VL amino acid sequence is (SEQ ID NO.: 41)
  • Humanized CQ137 monoclonal antibody (137-6) heavy chain VH amino acid sequence is (SEQ ID NO.: 36)
  • Humanized CQ137 monoclonal antibody (137-6) light chain VL amino acid sequence is (SEQ ID NO.: 42)
  • Humanized CQ137 monoclonal antibody (137-7) heavy chain VH amino acid sequence is (SEQ ID NO.: 37)
  • Humanized CQ137 monoclonal antibody (137-7) light chain VL amino acid sequence is (SEQ ID NO.: 43)
  • Humanized CQ137 monoclonal antibody (137-8) heavy chain VH amino acid sequence is (SEQ ID NO.: 38)
  • Humanized CQ137 monoclonal antibody (137-9) heavy chain VH amino acid sequence is (SEQ ID NO.: 39)
  • Humanized CQ137 monoclonal antibody (137-9) light chain VL amino acid sequence is (SEQ ID NO.: 45)
  • the underlined area is CDRs (IMGT definition), 137-1, 137-2, 137-3, 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 are nine people respectively Protein numbering of the CQ137 antibody.
  • the third amino acid (Thr) in the heavy chain VH-CDR1 of humanized antibody 137-1: GYTFTGYY (SEQ ID NO.: 21) and the heavy chain VH-CDR1 of the CQ137 antibody: GYSFTGYY (SEQ ID NO.: 15 ) in the third amino acid (Ser) is different.
  • the CDR regions of the humanized antibodies 137-2 and 137-3 are the same as those of the CQ137 antibody.
  • the FR regions of the humanized antibody were replaced on the basis of the CQ137 antibody.
  • the CDRs of the partially humanized antibody were replaced on the basis of the CQ137 antibody.
  • This example mainly shows the affinity of the humanized CD73 antibody with recombinant human and monkey CD73 protein, and the affinity with CD73 positive cells SK-OV-3 cells and CHOK1-cynoCD73 cells.
  • the recombinant human CD73 protein was coated at 3 ⁇ g/ml, and the humanized CD73 antibodies (137-1, 137-2, 137-3) diluted in series were added, and the binding of the antibody to the recombinant human CD73 was detected by ELISA.
  • the results are shown in Figure 8A.
  • the recombinant monkey CD73 protein was coated at 3 ⁇ g/ml, and the humanized CD73 antibodies (137-1, 137-2, 137-3) diluted in gradients were added, and the binding of the antibody to the recombinant monkey CD73 was detected by ELISA. The results are shown in Figure 8B.
  • Humanized CD73 antibody number 137-1 137-2 137-3 EC50( ⁇ g/ml) 3.763 0.3132 0.2016
  • the full-length monkey CD73 was constructed into a eukaryotic expression vector containing NEO resistance, transfected into CHO-K1 cells, and the CHOK1 -cynoCD73 cell line was obtained after screening with G418.
  • the humanized CD73 antibodies (137-1, 137-2, 137-3) were incubated for 1 h, washed three times with PBS, added Anti-hFC-APC (purchased from Jackson immunology), and detected by flow cytometry. The resulting S-curve is shown in Figure 10.
  • This example mainly demonstrates that humanized 137 antibody inhibits CD73 protein and cellular enzyme activity.
  • the CD73 protein was diluted to a working concentration of 5 ⁇ g/ml, and the humanized CD73 antibodies (137-1, 137-2, 137-3) diluted in series were added, incubated at 37°C for 15 minutes, and a mixture of 1mM ATP and AMP was added, and incubated at 37°C.
  • an equal volume of Cell Titer-Glo detection reagent purchased from promega
  • the autoluminescence value was read by a microplate reader
  • the enzyme activity without antibody was calculated as 1, and the change in rhCD73 activity was obtained in Figure 11.
  • Figure 19 shows that the six humanized CD73 antibodies can inhibit the enzymatic activity of recombinant human CD73.
  • the experimental results show that the nine humanized CD73 antibodies of the present invention can also induce the internalization of CD73 in tumor cells.
  • Example 8 Humanized 137 antibody inhibits the proliferation of human melanoma cell A375 in mice
  • a mouse model of human melanoma cells (A375), NSG-A375, was constructed with NSG mice, and experimental mice inoculated with human PBMC and control mice without human PBMC were prepared, administered every other day, and the tumor volume was measured.
  • the experimental mice were given the administration blank control of solvent PBS, MEDI-9447 low dose (low dose is 0.5mpk), 137-2 high dose (3mpk) and low dose, 137-3 high, medium and low dose (medium dose is 1mpk) ), and the experimental results are shown in Figure 14. Where mpk (Milligrams Per Kilograms) is the milligram kilogram.
  • VL is light chain variable region
  • VH is heavy chain variable region
  • CH is heavy chain constant region
  • CL is light chain constant region
  • VH-CDR1, VH-CDR2, VH-CDR3 are heavy chain variable region CDR1, CDR2, CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are CDR1, CDR2, and CDR3 of the light chain variable region.

Abstract

提供了一种新的抗人CD73的人源化抗体，能够特异性地结合CD73抗原，具有较高的亲和力和生物活性。

Description

抗人CD73抗体及其应用 技术领域
本发明涉及生物医药领域，具体地，本发明涉及一种抗人CD73抗体及其应用。
背景技术
CD73，分化簇73，也叫5'-核苷酸酶，通过GPI(糖基磷脂酰肌醇)锚定在细胞膜上，结构为二聚体。CD73在肿瘤微环境中发挥重要作用，可以将AMP转化成腺苷，腺苷通过与特异的G蛋白偶联受体结合，导致免疫细胞在肿瘤部位浸润异常，调控多种癌症的增殖和迁移，主要抑制T细胞相关的免疫***的功能，是肿瘤预后不良的标志。
在过去的大量研究中表明，抑制CD73可以阻止肿瘤的发生和转移。不管是CD73的小分子酶抑制剂还是大分子抗体，都表现出了抗肿瘤效果。CD73表达在多种肿瘤细胞表面，包括肺癌、黑色素瘤、结肠癌、胰腺癌等肿瘤细胞。除此之外，多种免疫细胞也受到CD73影响，CD73表达在Treg表面，参与Treg对免疫活化的抑制性调节；分泌的腺苷可以与DC细胞上腺苷受体结合，从而抑制DC的功能；CD73在M2型巨噬细胞上高表达，增强了肿瘤的转移。
CD73靶点在肿瘤治疗中的潜力巨大，CD73的抗体还可以增强PD1抗体和CTLA4抗体的作用。但是目前还没有CD73的特异性靶向抗体上市。此外，由于鼠单抗在临床治疗时会引起人抗鼠抗体反应(human anti-mouse antibody，HAMA)，因此在临床治疗上受到限制。抗体人源化技术可以很大程度的降低鼠单抗的免疫原性。
因此，鉴于CD73在各类相关疾病中作用和功能，本领域需要开发适于治疗患者的抗CD73人源化抗体。
发明内容
本发明的目的在于提供一种高亲和力高生物活性的抗人CD73抗体及其应用。
在本发明的第一方面，提供了一种抗CD73抗体的重链可变区，所述的重链可变区具有选自下组的VH-CDR：
(1)SEQ ID NO.:15或21所示的VH-CDR1，
(2)SEQ ID NO.:16所示的VH-CDR2，和
(3)独立地选自下组的VH-CDR3：SEQ ID NO.:17、SEQ ID NO.:22、SEQ ID NO.:23、SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26、SEQ ID NO.:27。
在另一优选例中，所述重链可变区具有SEQ ID NO.:2所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:5所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:9所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:34所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:36所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:37所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:38所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:39所示的氨基酸序列。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在本发明的第二方面，提供了一种抗CD73抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的，较佳地为人源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1恒定区。
在本发明的第三方面，提供了一种抗CD73抗体的轻链可变区，所述的轻链可变区具有选自下组的VL-CDR：
(1)SEQ ID NO.:18所示的VL-CDR1，
(2)SEQ ID NO.:19所示的VL-CDR2，和
(3)独立地选自下组的VL-CDR3：SEQ ID NO.:20、SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30、SEQ ID NO.:31、SEQ ID NO.:32、SEQ ID NO：33。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:6所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:10所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:40所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:41所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:42所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:43所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:44所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:45所示的氨基酸序列。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺 失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在本发明的第四方面，提供了一种抗CD73抗体的轻链，所述的轻链具有如本发明的第三方面所述的轻链可变区。
在另一优选例中，所述抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的，较佳地为人源的。
在另一优选例中，所述轻链恒定区为人源抗体轻链kappa恒定区。
在本发明的第五方面，提供了一种抗CD73抗体，所述的抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第四方面所述的轻链，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体包括单特异性、双特异性、或三特异性抗体。
在另一优选例中，所述抗体的重链可变区还包括人源的框架区，和/或所述抗体的轻链可变区还包括人源的框架区。
在另一优选例中，所述抗体的重链可变区还包括鼠源的框架区，和/或所述抗体的轻链可变区还包括鼠源的框架区。
在另一优选例中，所述的抗体具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区；
其中，所述的重链可变区包括选自下组的VH-CDR：
(1)SEQ ID NO.:15或21所示的VH-CDR1，
(2)SEQ ID NO.:16所示的VH-CDR2，和
(3)独立地选自下组的VH-CDR3：SEQ ID NO.:17、SEQ ID NO.:22、SEQ ID NO.:23、SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26或SEQ ID NO.:27；和
所述的轻链可变区包括选自下组的VL-CDR：
(1)SEQ ID NO.:18所示的VL-CDR1，
(2)SEQ ID NO.:19所示的VL-CDR2，和
(3)独立地选自下组的VL-CDR3：SEQ ID NO.:20、SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30、SEQ ID NO.:31、SEQ ID NO.:32或SEQ ID NO：33。
在另一优选例中，所述的抗体具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区；其中，
所述轻链可变区的3个轻链CDR和所述重链可变区的3个重链CDR选自下组：
(Z1)SEQ ID NO：18、19、20所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、17所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z2)SEQ ID NO：18、19、20所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：21、16、17所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z3)SEQ ID NO：18、19、28所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、22所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z4)SEQ ID NO：18、19、29所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、23所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z5)SEQ ID NO：18、19、30所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、24所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z6)SEQ ID NO：18、19、31所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、25所述的VH-CDR1、VH-CDR2和VH-CDR3；
(Z7)SEQ ID NO：18、19、32所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、26所述的VH-CDR1、VH-CDR2和VH-CDR3；或
(Z8)SEQ ID NO：18、19、33所示的VL-CDR1、VL-CDR2和VL-CDR3；以及SEQ ID NO：15、16、27所述的VH-CDR1、VH-CDR2和VH-CDR3。
在另一优选例中，所述的抗人CD73抗体的轻链可变区(VL)的氨基酸序列如SEQ ID NO：1、6、8、10、40、41、42、43、44或45所示；和所述的抗人CD73抗体的重链可变区(VH)的氨基酸序列如SEQ ID NO：2、5、7、9、34、35、36、37、38或39所示。
在另一优选例中，所述的抗体选自下组：
Figure PCTCN2021106917-appb-000001
Figure PCTCN2021106917-appb-000002
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:2所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:5所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:6所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:7所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:9所示的氨基酸序列；且所述轻链可变区具有SEQ ID NO.:10所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:34所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:40所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:35所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:41所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:36所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:42所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:37所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:43所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:38所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:44所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区具有SEQ ID NO.:39所示的氨基酸序列；且所述抗体的轻链可变区具有SEQ ID NO.:45所示的氨基酸序列。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述重链可变区的氨基酸序列与如序列表中SEQ ID NO.:2、5、7、9、34、35、36、37、38或39所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO.:1、6、8、10、40、41、42、43、44或45所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述抗CD73的抗体包括轻链和重链，并且，所述的轻链的轻链 可变区包括以下三个轻链CDR：
SEQ ID NO.:18所示的VL-CDR1，
SEQ ID NO.:19所示的VL-CDR2，和
SEQ ID NO.:20、28、29、30、31、32或33所示的VL-CDR3；其中，所述的重链的重链可变区包括以下三个重链CDR：
SEQ ID NO.:3915或21所示的VH-CDR1，
SEQ ID NO.:16所示的VH-CDR2，和
SEQ ID NO.:17、22、23、24、25、26或27所示的VH-CDR3。在另一优选例中，所述的抗体的轻链包括所述的三个轻链CDR以及用于连接轻链CDR的轻链框架区；和所述的抗体的重链包括所述的三个重链CDR以及用于连接重链CDR的重链框架区。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体特异性结合CD73。
在另一优选例中，所述抗体对人CD73的亲和力的KD值(M)为1.0E-10～2.0E-12。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体包括单特异性、双特异性、或三特异性抗体。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面中所述的抗体；以及以及(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述的重组蛋白还包括与所述元件(i)融合在一起的额外的融合元件(或融合多肽片段)。
在本发明的第七方面，提供了一种抗体制剂，所述的抗体制剂包括：
(a)如本发明第五方面所述的抗体；以及
(b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
在本发明的第八方面，提供了一种试剂盒，所述的试剂盒含有本发明第七方面所述的抗体制剂，以及盛装所述抗体制剂的容器。
在本发明的第九方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区 域的scFv区段为特异性结合于CD73的结合区，并且所述scFv区段具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。在本发明的第十方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明的第五方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十一方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面中所述的抗体、或本发明第六方面所述的重组蛋白、或其组合；和(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在本发明的第十二方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第五方面所述的抗体、或本发明第六方面所述的重组蛋白、如本发明第七方面所述的CAR构建物、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合，所述活性成分用于：
(a)制备检测试剂或试剂盒；
(b)制备预防和/或治疗CD73相关疾病的药物或制剂；和/或
(c)制备预防和/或治疗CD73相关疾病的癌症或肿瘤的药物或制剂。
在另一优选例中，所述癌症或肿瘤选自下组：肺癌、黑色素瘤、结肠癌、胰腺癌、膀胱癌、乳腺癌、卵巢癌、***癌、睾丸癌、食道癌、胃肠癌、肝癌、淋巴瘤、骨髓瘤、白血病。
在本发明的第十三方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第五方面所述的抗体、或本发明第六方面所述的重组蛋白、如本发明第七方面所述的CAR构建物、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于***。
在另一优选例中，所述肿瘤为高表达CD73的肿瘤。
在本发明的第十四方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多 肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面中所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；
(3)如本发明第九方面所述的CAR构建物。
在本发明的第十五方面，提供了一种载体，所述的载体含有如本发明的第十四方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第十六方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明的第十五方面所述的载体或基因组中整合有如本发明的第十四方面所述的多核苷酸。
在本发明的第十七方面，提供了一种体外非诊断的检测(包括诊断性或非诊断性)样品中CD73蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD73蛋白。
在本发明的第十八方面，提供了一种体外的检测(包括诊断性或非诊断性)样品中CD73蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD73蛋白。
在本发明的第十九方面，提供了一种治疗CD73相关疾病的方法，所述方法包括：
给需要的对象施用如本发明第五方面的抗体、所述的抗体的药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Biacore测定的CQ137与rhCD73的亲和力图。
图2显示了CQ137与重组猴CD73蛋白的亲和力检测图。
图3显示了CQ137与SK-OV-3细胞的亲和力检测图。
图4显示了CQ137与CHOK1-cynoCD73细胞的亲和力检测图。
图5显示了两种CD73抗体(CQ137与MEDI-9447)抑制rhCD73酶活比较。
图6显示了CQ137抑制SK-OV-3细胞上CD73的活性图。
图7显示了CQ137抑制CHOK1-cynoCD73细胞上CD73的活性图。
图8A显示了人源化CD73抗体(137-1、137-2、137-3)与rhCD73的结合活性。
图8B显示了人源化CD73抗体(137-1、137-2、137-3)与重组猴CD73蛋白的结合活性。
图9显示了人源化CD73抗体(137-1、137-2、137-3)与SK-OV-3细胞的亲和力检测图。
图10显示了人源化CD73抗体(137-1、137-2、137-3)与CHOK1-cynoCD73细胞的亲和力检测图。
图11显示了人源化CD73抗体(137-1、137-2、137-3)抑制rhCD73酶活(RLU响应倍数)比较，RLU为相对光单位。
图12显示了人源化CD73抗体(137-1、137-2、137-3)抑制SK-OV-3细胞上CD73酶活比较。
图13显示了人源化CD73介导的SK-OV-3细胞CD73内化结果图，MFI为相对荧光强度。
图14显示了人源化137抗体(137-1、137-2、137-3)抑制NSG-A375模型中的肿瘤细胞。
图15显示了人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9与重组人CD73蛋白的结合情况。
图16显示了人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9与重组猴CD73蛋白的结合情况。
图17显示了CD73抗体(人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9和MEDI-9447)与SK-OV-3细胞的亲和图。
图18显示了人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9与CHOK1-cynoCD73细胞亲和图。
图19显示了人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9对rhCD73酶活的抑制作用。
图20显示了人源化CD73抗体137-4、137-5、137-6、137-7、137-8、137-9对SK-OV-3细胞上CD73酶活的抑制作用。
图21显示了人源化CD73抗137-4、137-5、137-6、137-7、137-8、137-9体介导的 SK-OV-3细胞的CD73内化结果。
具体实施方式
本发明人经过广泛而深入地研究，经过大量筛选，意外地获得一种具有优异亲和力的抗CD73人源化抗体。具体地，本发明利用噬菌体展示技术，通过筛选获得一株高特异性的CD73抗体，兼顾相似性及人体使用频率的同时进行人源化。通过对人源化的抗体的结合实验和亲和力的检测，所述抗体与人和猴的CD73蛋白、CD73阳性细胞特异性结合，并具有抑制CD73蛋白酶活的效果。本发明的抗体还可以诱导CD73的肿瘤细胞内化。在小鼠实验中，本发明的抗体表现出比AstraZeneca(AZ)/Medimmune的MEDI-9447更优的抗肿瘤活性。在此基础上完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括抗CD73抗体与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种抗CD73抗体及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、***或缺失等突变的情况下 最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的不同种型，即IgM、IgD、IgG、IgA和IgE，对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。IgG代表免疫球蛋白中最重要的一类，由于化学结构和生物功能差异，它又可以分为4个子类：IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的FR区(FR)。4个FR的氨基酸序列相对比较保守，不直接参与结合反应。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区(框架区)组成，从氨基端到羧基端依次排列的顺序为FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区，即轻链高变区(LCDR)，指LCDR1、LCDR2和LCDR3；重链的3个CDR区，即重链高变区(HCDR),指HCDR1、HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。天然重链和轻链可变区中的四个FR区大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
如本文所用，术语“嵌合抗体”是由鼠源性抗体的V区基因与人抗体的C区基因拼接为嵌合基因，然后***载体，转染宿主细胞表达的抗体分子。既保留了亲本鼠抗体的高特异性和亲和力，又使其人源Fc段能有效介导生物学效应功能。
如本文所用，术语“人源化抗体”，是本发明鼠抗的一种可变区改造形式，具有源自(或基本上源自)非人类抗体(优选小鼠单克隆抗体)的CDR区，和基本源自人源抗体序列的FR区和恒定区；即将鼠抗的CDR区序列嫁接到不同类型的人种系抗体构架序列上。因为CDR序列负责大部分的抗体-抗原相互作用，所以可以通过构建表达载体来表达模拟特定天然存在的抗体性质的重组抗体。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A.
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗CD73抗体
如本文所用，术语“CD73”一般是指天然的或重组的人CD73，以及人CD73的非人同源物。
本发明提供一种针对CD73的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
在本发明中，通过选择优质的人CD73抗原免疫小鼠，取小鼠免疫细胞构建噬菌体库，采用特殊的噬菌体展示技术，将单链抗体(scfv)展示到噬菌体表面，通过CD73抗原多轮筛选，获得抗体序列，通过重组构建方式构建到hIgG1框架的真核表达载体上，哺乳动物细胞表达获得抗CD73全长抗体(即获得人鼠嵌合抗体)。
在本发明的一个优选实施例中，所获得的一个人鼠嵌合的抗CD73全长抗体为CQ137抗体蛋白。所获得的CQ137抗体可以和肿瘤表明的CD73分子结合，并且抑制人卵巢腺癌细胞上的CD73活性。具有治疗多种CD73过表达肿瘤的潜力。
优选地，重链可变区(VH)的CDR选自下组：
SEQ ID NO.:15所示的VH-CDR1，
SEQ ID NO.:16所示的VH-CDR2，和
SEQ ID NO.:17所示的VH-CDR3；和/或
轻链可变区(VL)的CDR选自下组：
SEQ ID NO.:18所示的VL-CDR1，
SEQ ID NO.:19所示的VL-CDR2，和
SEQ ID NO.:20所示的VL-CDR3。
其中，上述氨基酸序列中任意一种氨基酸序列还包括经过添加、缺失、修饰和/或取代至少一个(如1-5、1-3个，较佳地1-2个，更佳地1个)氨基酸的具有CD73结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地 至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人CD73的鼠源抗体、嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID NO.:15、SEQ ID NO.:16和SEQ ID NO.:17中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID NO.:18、SEQ ID NO.:19和SEQ ID NO.:20中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:15、SEQ ID NO.:16和SEQ ID NO.:17中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:18、SEQ ID NO.:19和SEQ ID NO.:20中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
在本发明的一种优选实施例中，提供了一种抗人CD73(CQ137)的抗体：
CQ137轻链可变区(SEQ ID NO.:1)：
Figure PCTCN2021106917-appb-000003
CQ137重链可变区(SEQ ID NO.:2)：
Figure PCTCN2021106917-appb-000004
Figure PCTCN2021106917-appb-000005
hIgG1恒定区氨基酸序列(SEQ ID NO.:3)：
Figure PCTCN2021106917-appb-000006
kappa链恒定区氨基酸序列(SEQ ID NO.:4)：
Figure PCTCN2021106917-appb-000007
本发明还提供了编码上述氨基酸的核苷酸序列：CQ137VH核苷酸序列(SEQ ID NO.:11)：
Figure PCTCN2021106917-appb-000008
CQ137VL核苷酸序列(SEQ ID NO.:12)：
Figure PCTCN2021106917-appb-000009
所选hIgG1恒定区核苷酸序列为(SEQ ID NO.:13)：
Figure PCTCN2021106917-appb-000010
Figure PCTCN2021106917-appb-000011
所选kappa链恒定区核苷酸序列为(SEQ ID NO.:14)：
Figure PCTCN2021106917-appb-000012
人源化抗CD73抗体
1986年Jones等人首次将鼠单抗重链CDR移植到人抗体重链骨架区，然后与鼠单抗轻链组装成完整抗体并保持了与原鼠单抗相似的亲和力，为抗体人源化技术的发展提供了思路。1989年Queen等人通过CDR移植的方法，成功构建抗CD25人源化抗体，该方法使用的是人抗体Eu骨架区进行人源化，在骨架区部分位点保留了鼠源抗体氨基酸以保持亲和力。1992年Presta等人报道了以人抗体亚群共有序列(consensus sequence)为模板进行CDR移植成功构建人源化的方法。1994年Pedersen等人报道了用表面重塑(resurfacing)的方法对抗体人源化。1994年Hsiao等人报道了以人抗体Germline序列骨架区进行CDR移植的人源化方法。1994年Jespers等人用噬菌体库(shuffling library)的方法成功构建人源化方法。
本发明还提供一种针对CD73的高特异性和高亲和力的人源化抗体。
在另一优选例中，所述序列还包括经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列，优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab') 2、或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
本发明抗体可以是靶向CD73的人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的CD73抗体。例如，可以用连接或天然存在的CD73蛋白或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的CD73都可以作为免疫原(抗原)，用于产生对CD73特异的非人抗体，筛选所述抗体的生物学活性。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、杆状病毒载体、质粒和非病毒载体。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
制备本发明CD73抗体的示例性方法描述于实施例1。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过 先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输己与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物或动物细胞(如哺乳动物细胞)。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基 团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
抗体制剂
抗体在不同的制剂缓冲液中具有不同的稳定性，表现为电荷异质性的变化、抗体分子的降解、聚合等，这些质量性质的变化与抗体本身的理化性质相关，因此，在抗体药物开发过程，需根据不同抗体的理化性质筛选适合其自身的制剂缓冲液。目前常用的抗体制剂缓冲体系有磷酸盐缓冲液、柠檬酸缓冲液、组氨酸缓冲液等，同时根据抗体性质会添加不同浓度的盐离子或山梨醇、海藻糖、蔗糖等赋形剂，以及适量的吐温20或吐温80等表面活性剂，以维持抗体的稳定性。
本发明的抗体药物组合制剂可有效抑制本发明人源化抗体的聚集沉淀、水解、氧化及脱酰胺等副反应，同时能有效提高产品在加压(高温、强光照射及冻融等)、加速及长期冷藏条件下的稳定性。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合CD73蛋白分子，因而可用于预防和治疗CD73相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物 制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明的主要优点包括
(1)本发明的人源化抗体，相较于鼠源和嵌合抗体有更好的安全性；
(2)本发明抗体通过竞争性筛选，具有更高的亲和力；
(3)本发明抗体来源于噬菌体展示技术，比杂交瘤技术多样性更强。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1 抗人CD73单克隆抗体的制备
本实施例主要描述小鼠免疫及噬菌体展示获得抗人CD73单链抗体序列。使用重组人CD73蛋白(C446，Novoprotein)免疫Balb/C小鼠，首次免疫后，每个14天做加强免疫，共免疫4次，取小鼠血清进行抗体效价评价。评价有效的小鼠取B细胞，提取RNA 反转录，构建噬菌体展示库。将重组人CD73蛋白按照1-5μg/ml包板，加入展示的噬菌体，进行噬菌体库筛选，筛选后的未被洗掉的噬菌体收集后感染宿主菌，获得第一轮筛选文库，按照此法进行二轮和三轮筛选。筛选完成后，进行phage-ELISA鉴定，鉴定阳性的送测序获得Anti-CD73候选序列。将CQ137VH和VL分别构建到含有hIgG1-kappa恒定区的真核表达载体上，转染freestyle 293F细胞，培养3-7天后收集上清，用protein A柱纯化，得到CQ137抗体蛋白。
CQ137轻链可变区(SEQ ID NO.:1)
Figure PCTCN2021106917-appb-000013
CQ137重链可变区(SEQ ID NO.:2)
Figure PCTCN2021106917-appb-000014
hIgG1恒定区氨基酸序列(SEQ ID NO.:3)
Figure PCTCN2021106917-appb-000015
kappa链恒定区氨基酸序列(SEQ ID NO.:4)
Figure PCTCN2021106917-appb-000016
实施例2 CQ137抗体的亲和力检测
本实施例主要描述CQ137与人和猴重组CD73蛋白的亲和检测，以及与人的CD73阳性细胞(SK-OV-3，人卵巢腺癌细胞)以及猴的CD73细胞株(CHOK1-cynoCD73)的亲和检测情况。
(1)与人CD73蛋白亲和
采用Biacore测定CQ137与重组hCD73蛋白的亲和，将CQ137固化，两倍梯度(2.5nM-40nM)稀释CD73蛋白，所得亲和力图见图1。
亲和力数据如下表1：
表1.CQ137亲和力
KD(M) ka(1/Ms) kd(1/s)
8.653E-11 6.977E+5 6.037E-5
结果表明：测得CQ137与rhCD73的亲和力为8.653×10 -11M。
(2)与猴CD73蛋白亲和
将重组猴CD73蛋白按照3μg/ml包板，加入梯度稀释的CQ137，用ELISA的方法检测CQ137与重组猴CD73结合情况，结果见图2。
结果表明：计算CQ137与重组猴CD73结合的EC50为0.217μg/ml。
(3)与人CD73阳性细胞结合
取4×10 5个细胞SK-OV-3，加入三倍梯度(0.009-20μg/ml)稀释的CQ137蛋白，孵育1h后，用PBS清洗3次，加入Anti-hFC-APC(购自Jackson immunology)，流式上机检测。结果绘制S曲线见图3。
结果表明：CQ137与SK-OV-3细胞结合的EC50为0.5883ng/ml，MEDI-9447与SK-OV-3细胞结合的EC50为9.627ng/ml。因此，CQ137与SK-OV-3细胞的结合优于MEDI-9447。
(4)与猴CD73阳性细胞结合
将猴CD73全长构建到含有NEO抗性的真核表达载体上，转染CHO-K1细胞，用G418筛选后获得CHOK1-cynoCD73细胞株，取4×10 5个CHOK1-cynoCD73细胞，加入梯度稀释的CQ137蛋白，孵育1h后，用PBS清洗3次，加入Anti-hFC-APC(购自Jackson immunology)，流式上机检测。结果绘制S曲线见图4。
结果表明：CQ137的EC50＝0.09991ng/ml，MEDI-9447的EC50＝4.119ng/ml。因此，CQ137与CHOK1-CynoCD73细胞的结合优于MEDI-9447。
实施例3 CQ137介导的CD73酶活性抑制检测
本实施例主要展示CQ137抑制CD73蛋白和细胞酶活性。具体的，
(1)CD73蛋白酶活抑制
CD73蛋白稀释到工作浓度5μg/ml，加入三倍梯度(0.001-10μg/ml)稀释的CD73抗体，37℃孵育15min，加入1mM ATP和AMP的混合液，37℃孵育30min，等体积加入CellTiter-Glo检测试剂(购自promega)，酶标仪读取自发光数值，以未加抗体为酶活性100％计算，获得rhCD73活性变化见图5。
结果表明：CQ137在抑制CD73蛋白酶活上优于对照分子MEDI-9447。
(2)人SK-OV-3细胞上CD73酶活抑制
取5×10 4个SK-OV-3细胞，加入三倍梯度(0.009-20μg/ml)稀释的抗体，37℃孵育15min，加入1mM AMP 37℃孵育2h，加入1mM ATP，立即加入CellTiter-Glo检测试剂，酶标仪读取自发光数值，以未加抗体为酶活性100％计算，获得细胞上CD73活性变化见图6。
结果表明：CQ137可以抑制SK-OV-3细胞上的CD73酶活性，CQ137的IC50为0.3708μg/ml。
(3)CHOK1-cynoCD73细胞上CD73酶活抑制
取5×10 4个CHOK1-cynoCD73细胞，加入三倍梯度(0.003-20μg/ml)稀释的抗体，37℃孵育15min，加入1mM AMP 37℃孵育2h，加入1mM ATP，立即加入CellTiter-Glo检测试剂，酶标仪读取自发光数值，以未加抗体为酶活性100％计算，获得细胞上CD73活性变化见图7。
结果表明：CQ137可以抑制CHOK1-cynoCD73细胞上CD73酶活性，CQ137的IC50为0.9899μg/ml。
实施例4 CQ137人源化
利用结构模拟与理性设计，分析与CQ137最接近的人源框架，获得一系列人源化抗体。将人源化序列的VH和VL分别构建到含有hIgG1和kappa轻链恒定区的载体上，转染Freestyle 293F细胞，收集上清，protein A柱纯化后获得所需抗体蛋白。
获得的9个人源化CQ137单抗(137-1、137-2、137-3、137-4、137-5、137-6、137-7、137-8、137-9)的序列如下所示：
人源化CQ137单抗(137-1)重链VH氨基酸序列(SEQ ID NO.:5)
Figure PCTCN2021106917-appb-000017
人源化CQ137单抗(137-1)轻链VL氨基酸序列为(SEQ ID NO.:6)
Figure PCTCN2021106917-appb-000018
人源化CQ137单抗(137-2)重链VH氨基酸序列为(SEQ ID NO.:7)
Figure PCTCN2021106917-appb-000019
人源化CQ137单抗(137-2)轻链VL氨基酸序列为(SEQ ID NO.:8)
Figure PCTCN2021106917-appb-000020
人源化CQ137单抗(137-3)重链VH氨基酸序列为(SEQ ID NO.:9)
Figure PCTCN2021106917-appb-000021
人源化CQ137单抗(137-3)轻链VL氨基酸序列为(SEQ ID NO.:10)
Figure PCTCN2021106917-appb-000022
人源化CQ137单抗(137-4)重链VH氨基酸序列为(SEQ ID NO.:34)
Figure PCTCN2021106917-appb-000023
人源化CQ137单抗(137-4)轻链VL氨基酸序列为(SEQ ID NO.:40)
Figure PCTCN2021106917-appb-000024
人源化CQ137单抗(137-5)重链VH氨基酸序列为(SEQ ID NO.:35)
Figure PCTCN2021106917-appb-000025
人源化CQ137单抗(137-5)轻链VL氨基酸序列为(SEQ ID NO.:41)
Figure PCTCN2021106917-appb-000026
人源化CQ137单抗(137-6)重链VH氨基酸序列为(SEQ ID NO.:36)
Figure PCTCN2021106917-appb-000027
人源化CQ137单抗(137-6)轻链VL氨基酸序列为(SEQ ID NO.:42)
Figure PCTCN2021106917-appb-000028
人源化CQ137单抗(137-7)重链VH氨基酸序列为(SEQ ID NO.:37)
Figure PCTCN2021106917-appb-000029
Figure PCTCN2021106917-appb-000030
人源化CQ137单抗(137-7)轻链VL氨基酸序列为(SEQ ID NO.:43)
Figure PCTCN2021106917-appb-000031
人源化CQ137单抗(137-8)重链VH氨基酸序列为(SEQ ID NO.:38)
Figure PCTCN2021106917-appb-000032
人源化CQ137单抗(137-8)轻链VL氨基酸序列为(SEQ ID NO.:44)
Figure PCTCN2021106917-appb-000033
人源化CQ137单抗(137-9)重链VH氨基酸序列为(SEQ ID NO.:39)
Figure PCTCN2021106917-appb-000034
人源化CQ137单抗(137-9)轻链VL氨基酸序列为(SEQ ID NO.:45)
Figure PCTCN2021106917-appb-000035
其中，下划线区为CDRs(IMGT定义)，137-1、137-2、137-3、137-4、137-5、137-6、137-7、137-8、137-9分别为九个人源化CQ137抗体的蛋白编号。人源化抗体137-1的重链VH-CDR1：GYTFTGYY(SEQ ID NO.:21)中的第三位氨基酸(Thr)与CQ137抗体的重链VH-CDR1：GYSFTGYY(SEQ ID NO.:15)中第三位氨基酸(Ser)不同。人源化抗体137-2、137-3的CDR区与CQ137抗体相同。人源化抗体的FR区在CQ137抗体的基础上进行了替换。部分人源化抗体的CDR在CQ137抗体的基础上进行了替换。
实施例5 CQ137人源化后亲和力评价
本实施例主要展示人源化后的CD73抗体分别与重组人和猴CD73蛋白亲和，与CD73阳性细胞SK-OV-3细胞以及CHOK1-cynoCD73细胞的亲和情况。具体的，
(1)与重组人CD73蛋白结合
将重组人CD73蛋白按照3μg/ml包板，加入梯度稀释的人源化CD73抗体(137-1,137-2,137-3)，用ELISA的方法检测抗体与重组人CD73结合情况，结果见图8A。
结果如表2所示。
表2.人源化CD73抗体与重组人CD73蛋白结合的EC50
人源化CD73抗体编号 137-1 137-2 137-3
EC50(μg/ml) 0.2581 0.2746 0.3307
人源化抗体137-4、137-5、137-6、137-7、137-8、137-9抗体与重组人CD73结合情况结果如表3和图15所示。
表3.人源化CD73抗体与重组人CD73亲和力结果
Figure PCTCN2021106917-appb-000036
(2)与重组猴CD73蛋白结合
将重组猴CD73蛋白按照3μg/ml包板，加入梯度稀释的人源化CD73抗体(137-1，137-2，137-3)，用ELISA的方法检测抗体与重组猴CD73结合情况，结果见图8B。
结果如图8B、表4所示。
表4.人源化CD73抗体与重组猴CD73蛋白结合的EC50
人源化CD73抗体编号 137-1 137-2 137-3
EC50(μg/ml) 3.763 0.3132 0.2016
人源化抗体137-4、137-5、137-6、137-7、137-8、137-9抗体与重组猴CD73蛋白结合情况如表5和图16所示。
表5：人源化CD73抗体与重组猴CD73亲和力结果
Figure PCTCN2021106917-appb-000037
(3)与人CD73阳性细胞结合
取4×10 5个细胞SK-OV-3，加入梯度稀释的人源化CD73抗体(137-1，137-2，137-3)，孵育1h后，用PBS清洗3次，加入Anti-hFC-APC(购自Jackson immunology)，流式上机检测。结果绘制S曲线见图9。
结果如表6和图9所示。
表6.人源化CD73抗体与SK-OV-3细胞结合的EC50
抗体名称 137-1 137-2 137-3 MEDI-9447
EC50(ng/ml) 2.135 3.264 7.026 23.486
结果表明，人源化CD73抗体(137-1，137-2，137-3)与SK-OV-3细胞的结合优于MEDI-9447。
人源化抗体137-4、137-5、137-6、137-7、137-8、137-9抗体与SK-OV-3细胞结合情况结果如表7和图17所示。
表7：人源化CD73抗体与SK-OV-3细胞亲和力结果
Figure PCTCN2021106917-appb-000038
(4)与猴CD73阳性细胞结合
将猴CD73全长构建到含有NEO抗性的真核表达载体上，转染CHO-K1细胞，用G418筛选后获得CHOK1-cynoCD73细胞株，取4×10 5个CHOK1-cynoCD73细胞，加入梯度稀释的人源化CD73抗体(137-1，137-2，137-3)，孵育1h后，用PBS清洗3次，加入Anti-hFC-APC(购自Jackson immunology)，流式上机检测。结果绘制S曲线见图10。
结果如表8所示。
表8.人源化CD73抗体与猴CD73阳性细胞结合的EC50
人源化CD73抗体编号 137-1 137-2 137-3
EC50(μg/ml) 0.08622 0.0233 0.04027
结果表明，人源化CD73抗体与猴CD73阳性细胞具有很高的结合活性。
人源化抗体137-4、137-5、137-6、137-7、137-8、137-9抗体与猴CD73阳性细胞结合情况结果如表9和图18所示。
表9：人源化CD73抗体与CHOK1-cynoCD73细胞亲和力结果
编号 137-4 137-5 137-6 137-7 137-8 137-9
EC50(μg/ml) 0.008310 0.01538 0.007690 0.02392 0.007819 0.01934
实施例6 CQ137人源化后酶活性抑制评价
本实施例主要展示人源化137抗体抑制CD73蛋白和细胞酶活性。具体的，
(1)CD73蛋白酶活抑制
CD73蛋白稀释到工作浓度5μg/ml，加入梯度稀释的人源化CD73抗体(137-1，137-2，137-3)，37℃孵育15min，加入1mM ATP和AMP的混合液，37℃孵育30min，等体积加入Cell Titer-Glo检测试剂(购自promega)，酶标仪读取自发光数值，以未加抗体为酶活性为1计算，获得rhCD73活性变化图11。
图19结果表明，人源化的6个CD73抗体均能抑制重组人CD73的酶活性。
(2)人SK-OV-3细胞上CD73酶活抑制
取5×10 4个SK-OV-3细胞，加入梯度稀释的人源化CD73抗体(137-1、137-2、137-3、137-4、137-5、137-6、137-7、137-8、137-9)，37℃孵育15min，加入1mM AMP 37℃孵育2h，加入1mM ATP，立即加入CellTiter-Glo检测试剂，酶标仪读取自发光数值，以未加抗体为酶活性1计算，获得细胞上CD73活性变化如图12和图20所示。
实验结果表明，9个人源化137的抗体可以抑制CD73蛋白和细胞上的CD73酶活性。
实施例7 人源化137抗体介导的细胞内化
取3×10 5个SK-OV-3细胞，加入0.2μg CD73抗体37℃孵育，按照时间0、1、2、3h和过夜孵育，孵育完成后再加入等量的相应人源化CD73抗体(137-1、,137-2、,137-3、137-4、137-5、137-6、137-7、137-8、137-9)，孵育1h后加入Anti-Hfc-APC流式抗体，孵育洗脱后上流式细胞仪检测，以0h时的相对荧光强度(MFI)为1，计算后结果见图13和图21。
实验结果表明，本发明的9个人源化CD73抗体还可以诱导CD73的肿瘤细胞内化。
实施例8 人源化137抗体抑制人黑色素瘤细胞A375在小鼠体内的增殖
用NSG小鼠构建人黑色素瘤细胞(A375)的小鼠模型NSG-A375，制备接种人PBMC实验鼠和未接种人PBMC对照鼠，隔天给药，测量肿瘤体积。实验小鼠设置溶剂PBS的给药空白对照、MEDI-9447低剂量(低剂量为0.5mpk)给药、137-2高剂量(3mpk)和低剂量、137-3高中低剂量(中剂量为1mpk)，实验结果见图14。其中mpk(Milligrams Per Kilograms)为毫克公斤。
结果表明：低剂量时137-2和137-3抑瘤效果均明显高于MEDI-9447。
本发明的序列信息
CQ137及其人源化抗体的CDR总结于下表10。
表10.
  CQ137 137-1 137-2 137-3 137-4 137-5 137-6 137-7 137-8 137-9
VH CDR1 15 21 15 15 15 15 15 15 15 15
VH CDR2 16 16 16 16 16 16 16 16 16 16
VH CDR3 17 17 17 17 22 23 24 25 26 27
VL CDR1 18 18 18 18 18 18 18 18 18 18
VL CDR2 19 19 19 19 19 19 19 19 19 19
VL CDR3 20 20 20 20 28 29 30 31 32 33
本发明的序列信息如下表所示：
Figure PCTCN2021106917-appb-000039
Figure PCTCN2021106917-appb-000040
Figure PCTCN2021106917-appb-000041
注：VL为轻链可变区，VH为重链可变区，CH为重链恒定区，CL为轻链恒定区，VH-CDR1、VH-CDR2、VH-CDR3为重链可变区的CDR1、CDR2、CDR3，VL-CDR1、VL-CDR2、VL-CDR3为轻链可变区的CDR1、CDR2、CDR3。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种抗人CD73抗体，其特征在于，所述抗体包括轻链和重链，
    并且，所述的轻链的轻链可变区包括以下三个轻链CDR：
    SEQ ID NO.:18所示的VL-CDR1，
    SEQ ID NO.:19所示的VL-CDR2，和
    SEQ ID NO.:20、28、29、30、31、32或33所示的VL-CDR3；
    其中，所述的重链的重链可变区包括以下三个重链CDR：
    SEQ ID NO.:15或21所示的VH-CDR1，
    SEQ ID NO.:16所示的VH-CDR2，和
    SEQ ID NO.:17、22、23、24、25、26或27所示的VH-CDR3。
  2. 如权利要求1所述的抗体，其特征在于，所述的抗体的轻链包括所述的三个轻链CDR以及用于连接轻链CDR的轻链框架区；和所述的抗体的重链包括所述的三个重链CDR以及用于连接重链CDR的重链框架区。
  3. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)轻链和/或重链，或者所述轻链和所述重链形成的抗CD73的抗体，
    其中，所述轻链的轻链可变区包括
    SEQ ID NO.:18所示的VL-CDR1，
    SEQ ID NO.:19所示的VL-CDR2，和
    SEQ ID NO.:20、28、29、30、31、32或33所示的VL-CDR3；
    而所述重链的重链可变区包括
    SEQ ID NO.:15或21所示的VH-CDR1，
    SEQ ID NO.:16所示的VH-CDR2，和
    SEQ ID NO.:17、22、23、24、25、26或27所示的VH-CDR3；
    以及(ii)任选的协助表达和/或纯化的标签序列。
  4. 一种抗体制剂，其特征在于，所述的抗体制剂包括：
    (a)如权利要求1所述的抗体；以及
    (b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
  5. 一种试剂盒，其特征在于，所述的试剂盒含有权利要求4所述的抗体制剂，以及盛装所述抗体制剂的容器。
  6. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合区域的scFv区段为特异性结合于CD73的结合区，并且所述scFv区段具有轻链可变区和重链可变区，其 中，轻链可变区包括
    SEQ ID NO.:18所示的VL-CDR1，
    SEQ ID NO.:19所示的VL-CDR2，和
    SEQ ID NO.:20、28、29、30、31、32或33所示的VL-CDR3；而重链可变区包括
    SEQ ID NO.:15或21所示的VH-CDR1，
    SEQ ID NO.:16所示的VH-CDR2，和
    SEQ ID NO.:17、22、23、24、25、26或27所示的VH-CDR3。
  7. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求6所述的CAR构建物。
  8. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的抗体、或权利要求3所述的重组蛋白、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  9. 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求1所述的抗体、如权利要求3所述的重组蛋白、如权利要求6所述的CAR构建物、如权利要求7所述的免疫细胞、如权利要求8所述的抗体药物偶联物、或其组合，所述活性成分用于：
    (a)制备检测试剂或试剂盒；
    (b)制备预防和/或治疗CD73相关疾病的药物或制剂；和/或
    (c)制备预防和/或治疗CD73相关疾病的癌症或肿瘤的药物或制剂。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的抗体、如权利要求3所述的重组蛋白、如权利要求6所述的CAR构建物、如权利要求7所述的免疫细胞、如权利要求8所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  11. 一种多核苷酸，其特征在于，所述的多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的抗体；或
    (2)如权利要求3所述的重组蛋白；
    (3)如权利要求6所述的CAR构建物。
  12. 一种载体，其特征在于，所述的载体含有如权利要求11所述的多核苷酸。
  13. 一种遗传工程化的宿主细胞，其特征在于，所述的宿主细胞含有如权利要求12所述的载体或基因组中整合有如权利要求11所述的多核苷酸。
  14. 一种体外非诊断的检测样品中CD73蛋白的方法，其特征在于，所述方法包括步骤：
    (1)在体外，将所述样品与如权利要求1所述的抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD73蛋白。
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