WO2023143227A1 - 抗cd73抗体及应用 - Google Patents

抗cd73抗体及应用 Download PDF

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Publication number
WO2023143227A1
WO2023143227A1 PCT/CN2023/072527 CN2023072527W WO2023143227A1 WO 2023143227 A1 WO2023143227 A1 WO 2023143227A1 CN 2023072527 W CN2023072527 W CN 2023072527W WO 2023143227 A1 WO2023143227 A1 WO 2023143227A1
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Prior art keywords
seq
sequence
sequence shown
identity
antibody
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PCT/CN2023/072527
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English (en)
French (fr)
Chinese (zh)
Inventor
丁焕弟
惠希武
姚兵
淡墨
靳子怡
郭青娟
袁灿
李文宾
王超
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CSPC Megalith Biopharmaceutical Co Ltd
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CSPC Megalith Biopharmaceutical Co Ltd
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Priority to AU2023212216A priority Critical patent/AU2023212216A1/en
Priority to JP2024543357A priority patent/JP2025504496A/ja
Priority to CN202380018508.1A priority patent/CN118591559A/zh
Priority to EP23746109.0A priority patent/EP4471062A4/en
Priority to KR1020247024203A priority patent/KR20240139053A/ko
Publication of WO2023143227A1 publication Critical patent/WO2023143227A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application generally relates to the field of biopharmaceuticals, in particular, the present application relates to novel anti-CD73 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising said antibodies or antigen-binding fragments thereof, and pharmaceuticals for said antibodies or antigen-binding fragments thereof learning purposes.
  • CD73 is an extracellular-5'-nucleotidase encoded by the NT5E gene, which includes two parts, the N-terminal and the C-terminal, and is anchored to the outer surface of the cell membrane by glycosylphosphatidylinositol (GPI) to form a homodimer.
  • GPI glycosylphosphatidylinositol
  • CD73 is widely expressed on the surface of human endothelial cells, lymphocytes and other cells. It is highly expressed in various tumor tissues such as gastric cancer, renal cancer, prostate cancer, breast cancer (such as triple-negative breast cancer), non-small cell adenocarcinoma, and indicates poor prognosis.
  • CD73 can hydrolyze extracellular adenosine monophosphate (AMP) to adenosine.
  • AMP extracellular adenosine monophosphate
  • Adenosine is a powerful immunosuppressive molecule that inhibits the function of protective immune cells (such as effector T cells, NK cells, DCs, and B cells) while maintaining regulatory immune cells (such as Treg, MDSC, TAM and CAF) to weaken anti-tumor immunity and participate in tumor immune escape.
  • protective immune cells such as effector T cells, NK cells, DCs, and B cells
  • regulatory immune cells such as Treg, MDSC, TAM and CAF
  • CD73 can also directly stimulate tumor cell proliferation, migration, invasion and tumor angiogenesis, and inhibition of extracellular nucleotidase CD73 can reverse adenosine-mediated immunosuppression. Studies have found that hypoxia, radiotherapy and chemotherapy lead to the upregulation of PDL1, CD73 and CD47 expression.
  • CD73 has the potential to be applied clinically as monotherapy or combination therapy.
  • Medimmune s oleclumab
  • I-Mab uliledlimab.
  • the fastest-growing CD73 antibody is in the third phase of clinical trials, and there is no marketed product. Therefore, the development and application of CD73 antibodies are urgently needed in this field.
  • the application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), the light chain variable region includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), where:
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.2
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.17
  • the sequence shown may have at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.17
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR3 is SEQ ID The sequence shown in NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1, and the sequence of HCDR2 is SEQ ID NO.17
  • the sequence shown is at least 80%, 85%, 90%, 95% or 99% identical to the sequence shown in SEQ ID NO.17
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or the same as SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.2
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.21
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.21
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.23
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1, and the sequence of HCDR2 is SEQ ID NO.23 The sequence shown or with the SEQ ID The sequence shown in NO.23 has at least 80%, 85%, 90%, 95% or 99% identity, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or has at least 80% identity with the sequence shown in SEQ ID NO.3 %, 85%, 90%, 95% or 99% identity, the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% with the sequence shown in SEQ ID NO.5 % or 99% identity, the sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.6, the sequence of LCDR3 The sequence is or is
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.10
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.10
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.27
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.27
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.30
  • the sequence shown may have at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.30
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.35
  • the sequence of LCDR2 is SEQ ID
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or with SEQ ID NO.15
  • the sequence shown in ID NO. 15 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.30
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15;
  • HCDR and LCDR sequences are defined according to Kabat.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region has an amino acid sequence shown in SEQ ID NO.4, 18, 19, 20, 22, 24, 12, 25, 26, 28, 29 or 31 or the same as SEQ ID NO.4, 18, 19 , 20, 22, 24, 12, 25, 26, 28, 29 or 31 have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence; and/ or
  • the light chain variable region has an amino acid sequence shown in SEQ ID NO.8, 33, 34, 16, 36, 37 or 38 or the same as shown in SEQ ID NO.8, 33, 34, 16, 36, 37 or 38 Amino acid sequences are amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein:
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.3
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.3
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity one sex; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.10
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.11
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with SEQ ID NO.11
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity;
  • HCDR sequence is defined according to Kabat.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a light chain variable region, the light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), wherein:
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.5, and the sequence of LCDR2 is SEQ ID The sequence shown in NO.6 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.6, the sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or with SEQ ID NO. The sequence shown in ID NO.7 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.5, and the sequence of LCDR2 is SEQ ID The sequence shown in NO.32 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.32, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or with SEQ ID NO.7 The sequence shown in ID NO.7 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.13
  • the sequence of LCDR2 is SEQ ID
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or with SEQ ID NO.14
  • the sequence shown in ID NO.15 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.35
  • the sequence of LCDR2 is SEQ ID
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or with SEQ ID NO.14
  • the sequence shown in ID NO.15 has at least 80%, 85%, 90%, 95% or 99% identity;
  • LCDR sequence is defined according to Kabat.
  • the present application provides an isolated nucleic acid molecule encoding the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects.
  • the present application provides a vector containing the nucleic acid molecule described in the fifth aspect.
  • the present application provides a host cell containing the nucleic acid molecule of the fifth aspect or the vector of the sixth aspect.
  • the present application provides an antibody-drug conjugate, which comprises the anti-CD73 antibody or the antigen-binding part thereof described in the first to fourth aspects coupled with a therapeutic agent.
  • the present application provides a pharmaceutical composition, which comprises the anti-CD73 antibody or its antigen-binding portion described in the first aspect to the fourth aspect or the antibody-drug conjugate described in the eighth aspect, and a pharmaceutically acceptable carrier.
  • the present application provides the anti-CD73 antibody or its antigen-binding portion described in the first to fourth aspects, the nucleic acid molecule described in the fifth aspect, the carrier described in the sixth aspect, and the anti-CD73 antibody described in the seventh aspect.
  • the present application provides a method for treating a tumor in an individual, especially a tumor expressing CD73 (CD73+) on the surface of tumor cells, the method comprising administering to the individual a therapeutically effective amount of the first aspect to the fourth aspect.
  • the present application provides a conjugate, which comprises the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects and a detectable label.
  • the present application provides a fusion protein comprising the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects.
  • Figure 1 shows the test results of the binding ability of exemplary antibodies of the present application to soluble CD73.
  • Figure 2 shows the test results of the binding ability of exemplary humanized antibodies of the present application to soluble CD73.
  • Figure 3 shows the test results of the binding ability of the exemplary humanized antibody of the present application to CD73 on the surface of human breast cancer MDA-MB-231 cells.
  • Figure 4 shows the test results of the binding ability of the exemplary humanized antibody of the present application to CD73 on the surface of human ovarian cancer SKOV3 cells.
  • Figure 5 shows the test results of the binding ability of the exemplary humanized antibody of the present application to cynomolgus CD73 on the cell surface.
  • Fig. 6 shows the results of the enzyme activity inhibition experiment of the exemplary humanized antibody of the present application on CD73 protein.
  • Fig. 7 shows the results of the enzyme activity inhibition experiment of the exemplary humanized antibody of the present application on the CD73 protein on the cell surface.
  • Figure 8 shows the experimental results of CD73 internalization induced by exemplary humanized antibodies of the present application.
  • Figure 9A shows the detection results of the exemplary humanized antibody of the present application recognizing the N-terminus of CD73 protein.
  • Figure 9B shows the detection results of the exemplary humanized antibody of the present application recognizing the C-terminus of CD73 protein.
  • Figure 10A shows the results of the volume change of xenograft tumors in A375 tumor-bearing mice resulting from the administration of exemplary antibodies of the present application and other test substances.
  • Fig. 10B shows the results of changes in the volume of xenograft tumors in individual A375 tumor-bearing mice resulting from the administration of exemplary antibodies of the present application and other test substances.
  • Figure 10C shows the results of the administration of exemplary antibodies of the present application and other test substances on the body weight of A375 tumor-bearing mice.
  • Figure 11A shows the results of the volume change of xenograft tumors in CT26 tumor-bearing mice produced by administration of exemplary humanized antibodies of the present application and other test substances.
  • Fig. 11B shows the results of the change of xenograft tumor volume in individual CT26 tumor-bearing mice produced by administration of exemplary humanized antibodies of the present application and other test substances.
  • Figure 11C shows the results of administration of exemplary humanized antibodies of the present application and other test substances on the body weight of CT26 tumor-bearing mice.
  • the term "subject” or “individual” refers to mammals, such as humans, but can also be other animals, such as wild animals, domestic animals or experimental animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, etc.) , marmots, ground squirrels, etc.).
  • antigen is a predetermined target to which an antibody can selectively bind.
  • antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.
  • an “antibody” may refer to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, thus encompassing intact/full-length antibodies, antibody monoclonal chain or any antigen-binding fragment of an antibody (also referred to as an "antigen-binding portion").
  • antibody and “antigen-binding fragment/antigen-binding part” appear in the same context, “antibody” can be understood as a complete body relative to "antigen-binding fragment/antigen-binding part", and the two together correspond to the generalized Antibody concept.
  • anti-CD73 antibody or "antibody that binds CD73” includes antibodies that are capable of binding CD73 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent when targeted to CD73.
  • the anti-CD73 antibody binds to an irrelevant non-CD73 protein to an extent that is less than about 10% of the binding of the antibody to CD73, such as by fluorescence activated cell sorting (FACS) analysis or an immunoassay (e.g., radioimmunoassay (RIA)) assay.
  • FACS fluorescence activated cell sorting
  • an immunoassay e.g., radioimmunoassay (RIA)
  • the antibody that binds CD73 has a dissociation constant (KD) of less than or equal to 500 nM, 100 nM, 10 nM, 9 nM, 8 nM , 7nM, 6nM, 5nM, 4nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM or 0.1nM.
  • KD dissociation constant
  • the anti-CD73 antibody binds to an epitope of the CD73 protein that is conserved between CD73 from different species (eg, between human and cynomolgus CD73).
  • agonist antibody is an antibody that elicits a response, eg, an antibody that mimics at least one functional activity of a polypeptide of interest.
  • Agonist antibodies include antibodies that are ligand mimics, e.g., wherein the ligand binds to a cell surface receptor, the binding induces cell signaling or activity through an intracellular cell signaling pathway, and wherein the antibody induces similar cellular Signal transduction or activation.
  • Full-length antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • These variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.
  • a full-length antibody may be of any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses of the above), but the antibody need not belong to any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • the subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also encompassed by antibodies according to the present application.
  • complementarity determining regions are regions in the variable region that have the greatest influence on the affinity and specificity of antibodies.
  • CDR amino acid sequence of VH or VL There are many common ways to define the CDR amino acid sequence of VH or VL, such as Kabat definition, IMGT definition, Chothia definition, etc.
  • the CDR amino acid sequences in the VH and VL amino acid sequences can usually be determined according to different definition methods. In embodiments of the present application, Kabat sense CDR amino acid sequences are utilized.
  • the amino acid sequences of the CDRs in the variable region amino acid sequence can be analyzed in a number of ways.
  • mouse antibody refers to an antibody that is derived from the fusion of B cells and myeloma cells of immunized mice, and the mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies are screened, and then screened, The antibodies obtained by preparation and purification, murine antibodies are generally immunogenic, so they need to be humanized later.
  • humanized antibody means an antibody obtained by grafting CDR sequences derived from the germline of another mammalian species, such as a mouse, onto human framework sequences. To preserve binding affinity, some residues of the backbone (termed FR) segments can be modified. Humanized antibodies or fragments thereof according to the present application can be prepared by techniques known to those skilled in the art;
  • chimeric antibody refers to an antibody in which the variable region sequences are from one species and the constant region sequences are from another species, eg, an antibody in which the variable region sequences are from a mouse antibody and the constant region sequences are from a human antibody.
  • the chimeric antibody or fragment thereof according to the present application can be produced by using gene recombination technology.
  • said chimeric antibodies can be produced by cloning recombinant DNA comprising a promoter and sequences encoding the variable regions of non-human, especially murine monoclonal antibodies according to the present application, and sequences encoding the constant regions of human antibodies .
  • the chimeric antibody of the present application encoded by such a recombinant gene will be, for example, a mouse-human chimera whose specificity is determined by a variable region derived from mouse DNA and whose isotype is determined by a variable region derived from human DNA. to determine the constant region.
  • partially humanized antibody refers to an antibody that contains constant regions from a human and variable regions (including CDRs) from a non-human (eg, murine).
  • si-humanized antibody is a type of humanized antibody, and refers to an antibody in which one antibody chain contains a murine variable region and the other antibody chain contains a humanized variable region, that is, a semi-humanized antibody .
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.
  • antigen-binding fragment or “antigen-binding portion” or “antigen-binding region” is used interchangeably and refers to a protein comprising amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen.
  • an antibody fragment such as Fv, Fab, F(ab') 2 or Fab', or any fragment that should be able to increase the half-life by chemical modification or by incorporation into liposomes
  • said chemical Modifications such as addition of poly(alkylene) glycols such as polyethylene glycol (“pegylated, PEGylated”) (known as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2 - PEG or a pegylated fragment of Fab'-PEG) (“PEG” is polyethylene glycol), which fragment has CD73 binding activity.
  • the functional fragment will consist of or comprise a partial sequence of the variable chain of the heavy or light chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.
  • Fragments will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which they are derived.
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; ( 2 ) F(ab') fragments, which may be monovalent fragments having two A bivalent fragment of a Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (ie a dimer of Fab'); (3) an Fv fragment with the VL and VH domains of a single arm of an antibody.
  • single-chain antibody refers to a single polypeptide chain that is connected by a VH domain and a VL domain via a peptide linker.
  • (scFv) 2 comprises two VH domains and two VL domains connected by a peptide linker, the two The VL domain is combined with the two VH domains via a disulfide bridge.
  • Fc fragment refers to a portion of the constant region of an antibody heavy chain, including the hinge, CH2 and CH3 fragments of the constant region .
  • the Fc region of an anti-CD73 antibody can be engineered or modified, including effector function-related modifications, such as to reduce or eliminate antibody-dependent cellular Toxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions/mutations in the Fc region of the antibody.
  • ADCC antibody-dependent cellular Toxicity
  • CDC complement-dependent cytotoxicity
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.
  • polyantibody also known as “multispecific antibody” is a molecule with binding specificities for at least two different antigens, among which, such molecules that only bind to two antigens are also called double antibodies (ie, bispecific antibodies).
  • Antibodies, BsAb are also known as “multispecific antibody”.
  • bispecific antibody refers to an antibody that has the ability to bind two epitopes at the same time.
  • the two epitopes can be on different antigens or on the same antigen.
  • Bispecific antibodies can have a variety of structural configurations.
  • a bispecific antibody can be composed of two Fc fragments and two antigen-binding parts fused to them respectively (similar to natural antibodies, except that the two arms bind different antigenic targets or epitopes), and the antigen-binding parts can be single Chain antibody (scfv) format or Fab fragment format.
  • a human antibody that specifically binds CD73 can include at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and specifically binds a CD73 polypeptide.
  • conservative substitutions also includes the use of substituted amino acids in place of the unsubstituted parent amino acid, so long as the antibody specifically binds CD73. Non-conservative substitutions are those that reduce activity or bind to CD73.
  • XaaaY such as L234A, L235A, etc.
  • aaa represents the sequence position of an amino acid or base
  • aaa represents the residue sequence position in the reference sequence; or it can also follow the position numbering method prevailing in the field, such as the EU numbering system, etc.
  • X represents the original amino acid or base at "aaa”
  • Y represents the changed amino acid or base at "aaa”.
  • L234A mutation of the human heavy chain constant region it means that the 234th position leucine (L) is mutated to alanine (A) according to the EU numbering system of the human heavy chain constant region.
  • drugs A and B can be administered separately in sequence, administered separately at the same time, formulated in the same drug and administered together.
  • drugs A and B can be prepared in the same composition, or in the same pharmaceutical kit, at the time of manufacture.
  • Drugs A and B can be physically separate, or joined together.
  • the administration of drugs A and B may be separate, simultaneous or overlapping in time.
  • isolated refers to a biological component (such as a nucleic acid, protein (including antibodies) or organelle) that has been separated from other biological components (ie, other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) are substantially isolated or purified.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified using standard purification methods.
  • the term also includes nucleic acids and proteins produced by recombinant expression in host cells as well as chemically synthesized nucleic acids.
  • derivative sequence refers to a sequence that has at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity to a related sequence and still has the same or similar function.
  • pharmaceutical composition refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant combined together to achieve a specific purpose.
  • the pharmaceutical composition includes combinations that are separated in time and/or space, as long as they can work together to achieve the purpose of the present application.
  • the components contained in the pharmaceutical composition eg, antibodies, nucleic acid molecules, nucleic acid molecule combinations and/or conjugates according to the present application
  • the ingredients contained in the pharmaceutical composition are administered to the individual separately, the ingredients may be administered to the individual simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic Saline solutions such as PBS (phosphate buffered saline), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene diacetates Alcohols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the present application is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • compositions according to the present application may comprise wetting agents, emulsifiers or buffer substances as additives.
  • the pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any suitable route, for example, it can be administered orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • the term "therapeutically effective amount” or “effective amount” refers to a dose sufficient to demonstrate benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend upon the individual condition and severity of the individual being treated.
  • the prescribing of treatment e.g. decisions on dosage etc.
  • the prescribing of treatment is ultimately the responsibility of and relies on the general practitioner and other medical practitioners to make decisions, usually taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration and what is known to the physician. other known factors.
  • the EC 50 value mainly refers to the concentration of a drug, antibody or toxin that can achieve 50% of the maximum biological effect after a specific exposure time.
  • EC 50 is also used to characterize the potency (including agonism and antagonism) of a compound at the cellular level, and the EC 50 value can be determined by methods such as ELISA.
  • fusion protein is a protein composed of at least two structural domains, each domain is not naturally associated, encoded by a separate gene, each gene is linked, and is recognized as a whole. Transcription and translation produce a single protein.
  • a "fusion protein” comprising an antibody or an antigen-binding part refers to a product obtained by fusing an antibody or an antigen-binding part with another biologically active protein using genetic engineering techniques. Antigen binding ability and unique biological properties of bioactive proteins fused to antibodies.
  • identity/homology/identity with respect to amino acid or nucleic acid sequences is defined as the percentage of residues in amino acid or nucleotide sequence variants that are identical after alignment of the sequences and introduction of gaps, if desired, to Maximum percent identity. Methods and computer programs for alignment are well known in the art.
  • tumor refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant.
  • malignancy refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • exemplary malignancies include: carcinomas, solid tumors, melanoma sarcomas, hematological tumors, germ cell tumors, and blastomas.
  • malignancies include: multiple myeloma, renal cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, including Gastrointestinal cancer of stomach, prostate, pancreas, peritoneum, hepatocellular carcinoma, glioblastoma, ovary, liver, urinary tract, hepatoma, rectum, colorectum, endometrium or uterus Carcinoma, salivary gland cancer, squamous cell carcinoma (eg, squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma and B-cell lymphoma, brain and head and neck cancer and associated metastases.
  • lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma
  • bladder cancer breast cancer, cervical cancer, colon cancer
  • blood cancer refers to the uncontrolled growth and proliferation of abnormal cells, which in most cases originate from the bone marrow, which is where blood cells are produced.
  • exemplary hematological tumors include various types of leukemia, multiple myeloma, and malignant lymphoma.
  • blood cancers include: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin Lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma
  • ALL
  • solid tumor refers to a tangible mass that can be palpated by clinical examination such as X-ray film, CT scan, B-ultrasound or palpation.
  • Clinically diagnosed and treated solid tumors are divided into malignant and benign.
  • Malignant solid tumors include: Pediatric Hodgkin's lymphoma: lymphocyte predominant, nodular sclerosis, mixed cellularity, lymphocytic depletion; Pediatric non-Hodgkin's lymphoma: prolymphoblastic lymphoma, small noncleaved Cell lymphoma (Burkitt/non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; renal tumors in children: Wilms tumor (Wilms tumor), clear cell renal cell carcinoma, Renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.; neuroblastoma in children: neuroblastoma, ganglion
  • the application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), the light chain variable region includes light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), where:
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.2
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1, and the sequence of HCDR2 is SEQ ID NO.17
  • the sequence shown is at least 80%, 85%, 90%, 95% or 99% identical to the sequence shown in SEQ ID NO.17
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or the same as SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.17
  • the sequence shown may have at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.17
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or with SEQ ID NO.6
  • the sequence shown has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85% identity with the sequence shown in SEQ ID NO.7 %, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.2
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.21
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.21
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.23
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.32 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.32 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID NO.23
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with the sequence shown in SEQ ID NO.
  • sequence shown in 3 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.6 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.6 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.7; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.10
  • the sequence shown or with the SEQ ID has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or has at least 80% identity with the sequence shown in SEQ ID NO.11 %, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% with the sequence shown in SEQ ID NO.13 % or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.14
  • the sequence of LCDR3 The sequence is or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.10
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.27
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.27
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity
  • sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.30
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.
  • the sequence shown in 11 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least 80%, 85%, 90%, 95% or the sequence shown in SEQ ID NO.14 99% identity
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.15; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID NO.30
  • the sequence of HCDR3 is SEQ ID
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or with SEQ ID NO.13
  • the sequence shown in ID NO.13 has at least 80%, 85%, 90%, 95% or 99% identity
  • the sequence of LCDR2 is the sequence shown in SEQ ID NO.14 or has at least the same sequence with the sequence shown in SEQ ID NO.14 80%, 85%, 90%, 95% or 99% identity
  • HCDR and LCDR sequences are defined according to Kabat.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.4, 18, 19, 20, 22, 24, 12, 25, 26, 28, 29 or 31 or the same , 18, 19, 20, 22, 24, 12, 25, 26, 28, 29 or 31 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity sequence.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.8, 33, 34, 16, 36, 37 or 38 or the or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence shown in 38.
  • the heavy chain variable region sequence is the sequence shown in SEQ ID NO.4, and the light chain variable region sequence is the sequence shown in SEQ ID NO.8; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.18, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.33; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.18, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.34; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.19, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.33; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.19, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.34; or
  • the heavy chain variable region sequence is the sequence shown in SEQ ID NO.20, and the light chain variable region sequence is the sequence shown in SEQ ID NO.33; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.20, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.34; or
  • the heavy chain variable region sequence is the sequence shown in SEQ ID NO.22, and the light chain variable region sequence is the sequence shown in SEQ ID NO.33; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.22, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.34; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.24, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.33; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.24, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.34; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.12, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.16; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.25, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.36; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.25, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.37; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.25, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.38; or
  • the heavy chain variable region sequence is the sequence shown in SEQ ID NO.26, and the light chain variable region sequence is the sequence shown in SEQ ID NO.36; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.26, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.37; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.26, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.38; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.28, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.36; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.28, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.37; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.28, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.38; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.29, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.36; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.29, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.37; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.29, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.38; or
  • the sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.31, and the sequence of the light chain variable region is the sequence shown in SEQ ID NO.36; or
  • sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.31, and the sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.37; or
  • the sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.31, and the sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.38.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region has an amino acid sequence shown in SEQ ID NO.4, 18, 19, 20, 22, 24, 12, 25, 26, 28, 29 or 31 or the same as SEQ ID NO.4, 18, 19 , 20, 22, 24, 12, 25, 26, 28, 29 or 31 have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence; and/ or
  • the light chain variable region has an amino acid sequence shown in SEQ ID NO.8, 33, 34, 16, 36, 37 or 38 or the same as shown in SEQ ID NO.8, 33, 34, 16, 36, 37 or 38 Amino acid sequences are amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a heavy chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein:
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.3
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1, and the sequence of HCDR2 is SEQ ID The sequence shown in NO.21 or with SEQ The sequence shown in ID NO.21 has at least 80%, 85%, 90%, 95% or 99% identity, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or has at least the same sequence with the sequence shown in SEQ ID NO.3 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.1
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or with SEQ ID NO.3
  • the sequence shown in ID NO.3 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.10
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with the sequence shown in SEQ ID NO.11
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of HCDR1 is the sequence shown in SEQ ID NO.9 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.9
  • the sequence of HCDR2 is SEQ ID
  • the sequence of HCDR3 is the sequence shown in SEQ ID NO.11 or with SEQ ID NO.11
  • the sequence shown in ID NO.11 has at least 80%, 85%, 90%, 95% or 99% identity;
  • HCDR sequence is defined according to Kabat.
  • the present application provides an anti-CD73 antibody or an antigen-binding portion thereof, the anti-CD73 antibody or an antigen-binding portion thereof comprising a light chain variable region, the light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), wherein:
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.5, and the sequence of LCDR2 is SEQ ID The sequence shown in NO.6 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.6, the sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or with SEQ ID NO. The sequence shown in ID NO.7 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.5 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.5, and the sequence of LCDR2 is SEQ ID The sequence shown in NO.32 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.32, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.7 or with SEQ ID NO.7 The sequence shown in ID NO.7 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.13 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.13
  • the sequence of LCDR2 is SEQ ID
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or with SEQ ID NO.14
  • the sequence shown in ID NO.15 has at least 80%, 85%, 90%, 95% or 99% identity; or
  • the sequence of LCDR1 is the sequence shown in SEQ ID NO.35 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO.35
  • the sequence of LCDR2 is SEQ ID
  • the sequence of LCDR3 is the sequence shown in SEQ ID NO.15 or with SEQ ID NO.15
  • the sequence shown in ID NO.15 has at least 80%, 85%, 90%, 95% or 99% identity;
  • LCDR sequence is defined according to Kabat.
  • the anti-CD73 antibody, or antigen-binding portion thereof binds human CD73. In some embodiments, the anti-CD73 antibody or antigen-binding portion thereof binds to an epitope of the CD73 protein that is conserved between CD73 from different species (eg, between human and cynomolgus CD73).
  • the anti-CD73 antibody, or antigen-binding portion thereof has ADCC activity.
  • the anti-CD73 antibody, or antigen-binding portion thereof has CDC activity.
  • the anti-CD73 antibody is a whole antibody, a single chain antibody (scFv), scFv-Fc, a bispecific antibody or a multispecific antibody.
  • the antigen binding portion of the anti-CD73 antibody is Fab, Fab', Fv or F(ab') 2 .
  • the anti-CD73 antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the anti-CD73 antibody is a monoclonal antibody.
  • the anti-CD73 antibody is an antibody of the IgM, IgD, IgG, IgA or IgE type. In some embodiments, the anti-CD73 antibody is an IgG antibody. In some more specific embodiments, the anti-CD73 antibody is an IgG1 antibody.
  • the anti-CD73 antibody is of the IgGl, IgG2 or IgG4 isotype.
  • the anti-CD73 antibody comprises a light chain constant region of the kappa subtype or the lambda subtype.
  • the anti-CD73 antibody comprises a human IgGl heavy chain constant region and a human kappa light chain constant region.
  • the anti-CD73 antibody or antigen-binding portion thereof is used in medical therapy.
  • the anti-CD73 antibody comprises a wild-type Fc region.
  • the anti-CD73 antibody, or antigen-binding portion thereof is engineered to have enhanced ADCC or CDC activity.
  • an anti-CD73 antibody comprises an engineered Fc region that renders the antibody having enhanced ADCC and/or CDC effects.
  • the anti-CD73 antibody, or antigen-binding portion thereof is engineered to have reduced ADCC or CDC activity.
  • the anti-CD73 antibody comprises an engineered Fc region such that the antibody has reduced ADCC and/or CDC effects.
  • antibodies with weakened or even eliminated ADCC or CDC activity may be beneficial, because too strong Fc effect may affect or even weaken the therapeutic effect of antibodies. Ways to weaken or eliminate Fc effects are known in the art, for example, 1) amino acid residue mutation modification (mainly weakening the binding to related receptors), 2) glycosylation modification, 3) IgG4 antibody modification, etc.
  • the antibody has a human IgG1 heavy chain constant region and contains mutations at one or more of L234, L235, P329, P331, wherein residue numbering follows the EU numbering system, e.g., the mutation is L234A One or more mutations in , L235A, P329A, P331S.
  • the antibody has a heavy chain variable region shown in SEQ ID NO.20, a light chain variable region shown in SEQ ID NO.33, and a light chain variable region shown in SEQ ID NO.39 The heavy chain constant region shown and the light chain constant region shown in SEQ ID NO.40.
  • the antibody has a heavy chain variable region shown in SEQ ID NO.31, a light chain variable region shown in SEQ ID NO.38, and a light chain variable region shown in SEQ ID NO.39 Shown heavy chain constant region and SEQ ID NO.40 Light chain constant region indicated.
  • the present application provides an isolated nucleic acid molecule encoding the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects.
  • the application provides a combination of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody of the application or an antigen-binding portion thereof and a heavy chain encoding an antibody of the application or an antigen-binding portion thereof chain of polynucleotides.
  • the polynucleotide is operably linked to a regulatory sequence that is recognized by a host cell transformed with the vector.
  • the present application provides a vector (such as an expression vector), which contains the nucleic acid molecule or polynucleotide combination described in the fifth aspect.
  • the expression vector of the present application comprises the nucleic acid molecule described in the present application or the combination of the polynucleotide described in the present application, and described polynucleotide and the polypeptide that allow its coding express in host cell or cell-free
  • the regulatory sequences expressed in the system are operably linked.
  • the choice of expression vector depends on the choice of the host cell and can be selected so as to have the desired expression and regulatory characteristics in the host cell of choice.
  • An "expression vector” is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and/or translation of another DNA sequence.
  • the nucleic acid in the vector may be operably linked to one or more expression control sequences.
  • "operably linked” means incorporated into a genetic construct such that expression control sequences effectively control the expression of a coding sequence of interest.
  • expression control sequences include promoters, enhancers, and transcription termination regions.
  • a promoter is an expression control sequence consisting of a region of a DNA molecule usually within 100 nucleotides upstream of the start of transcription (usually near the initiation site for RNA polymerase II).
  • the translation initiation site of the translational reading frame of the polypeptide must be positioned between 1 and about 50 nucleotides downstream of the promoter.
  • Enhancers provide expression specificity in terms of time, location and level. Unlike promoters, enhancers can function when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription initiation site.
  • a coding sequence is "operably linked to” and “under the control of” an expression control sequence in a cell when RNA polymerase is capable of transcribing the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence ".
  • Suitable expression vectors include, but are not limited to, plasmid and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus.
  • Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).
  • Expression vectors can include tag sequences.
  • Tag sequences are usually expressed as fusions to the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, FlagTM tags (Kodak, New Haven, CT), maltose E-binding protein and protein A.
  • a nucleic acid molecule encoding a CD73 fusion polypeptide is present in a vector containing nucleic acid encoding one or more domains of an Ig heavy chain constant region, e.g., corresponding to the C ⁇ 1 chain of a human immunoglobulin. Amino acid sequences of the hinge region, CH2 region and CH3 region (Fc fragment).
  • the present application provides a host cell containing the nucleic acid molecule of the fifth aspect or the vector of the sixth aspect.
  • the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a phage.
  • the prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
  • Eukaryotic host cells can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, such as BHK cells, CHO cells, COS cells, Mammalian cells such as myeloma cells.
  • the host cells are preferably mammalian cells, more preferably BHK cells, CHO cells, NSO cells or COS cells.
  • the present application provides an antibody-drug conjugate, also known as an antibody-drug conjugate, which comprises the anti-CD73 antibody or its antigen-binding portion described in the first to fourth aspects coupled to a therapeutic agent .
  • the therapeutic agent is an antineoplastic drug, eg, a cytotoxic drug, an immunopotentiator, or a radioisotope.
  • the type of cytotoxic drug includes tubulin inhibitors (eg, alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.
  • tubulin inhibitors eg, alkaloids
  • DNA topoisomerase inhibitors e.g., DNA damaging agents, antimetabolites, or antitumor antibiotics.
  • tubulin inhibitors include, but are not limited to, auristatin derivatives (such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (such as DM1, DM4, Ansamitocin, Mertansine or dolastatin and its derivatives).
  • auristatin derivatives such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)
  • maytansine alkaloid derivatives such as DM1, DM4, Ansamitocin, Mertansine or dolastatin and its derivatives.
  • the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and derivatives thereof, e.g., DXD, SN38, irinotecan, irinotecan hydrochloride, Camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, le Totecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)benzene Base]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2 -Cyano-3-(3,4-dihydroxyphenyl)
  • DNA damaging agents include, but are not limited to, calicheamicins, duocarmycins, antramycin derivatives PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine ).
  • antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, or 5-fluorouracil.
  • antitumor antibiotics include, but are not limited to, polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinones (such as doxorubicin or mitoxantrone hydrochloride).
  • radioisotopes include, but are not limited to, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P , 60 Co, or 177 Lu.
  • immune potentiating agents include, but are not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosine, or polyinosine glycosides.
  • an anti-CD73 antibody of the present application is covalently linked to a drug moiety via a linker.
  • the linker is a cleavable linker.
  • linkers are cleavable under intracellular conditions.
  • the linker is hydrolyzable at a pH of less than 5.5.
  • linkers are cleavable by intracellular proteases.
  • the linker is a cathepsin cleavable linker.
  • the linker comprises a dipeptide.
  • the dipeptide is valine (Val)-citrulline (Cit).
  • the antibody is attached to the linker through a cysteine sulfhydryl group of the antibody.
  • the antibody is linked to the linker via an amino group of the antibody, particularly an amino group of a glutamine residue.
  • linkers include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG)m-Val-Cit, NH2- (PEG)m-Val- Cit-pAB, wherein m is an integer from 1 to 8.
  • the linker is a non-cleavable linker, including without limitation, an acid-labile linker (e.g., a hydrazone linker), a disulfide bond-containing linker, a peptidase-sensitive linker (e.g., a peptide linker comprising an amino acid, such as valline acid and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or those designed to avoid multidrug transporter-mediated Resistant hydrophilic linkers, etc.
  • an acid-labile linker e.g., a hydrazone linker
  • a disulfide bond-containing linker e.g., a disulfide bond-containing linker
  • a peptidase-sensitive linker e.g., a peptide linker comprising an amino acid, such as valline acid and/or citrulline such
  • the present application provides a pharmaceutical composition, which comprises the anti-CD73 antibody or its antigen-binding portion described in the first aspect to the fourth aspect or the antibody-drug conjugate described in the eighth aspect, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is used to treat a tumor in an individual.
  • the tumor is a tumor that expresses CD73 (CD73+) on the surface of tumor cells.
  • the tumor is a tumor that highly expresses CD73 (CD73 + ) on the surface of tumor cells.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 60% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 70% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 80% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 90% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 means that at least 95% of the tumor cells in the tumor cell population express CD73. in some implementations In the scheme, a tumor highly expressing CD73 (CD73 + ) means that at least 98% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) means that at least 99% of the tumor cells in the tumor cell population express CD73.
  • the tumor is a solid tumor.
  • the tumor is a hematological tumor.
  • the tumor is a malignant tumor.
  • the tumor is cancer
  • the tumor is selected from the group consisting of ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, kidney cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer , colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer , brain cancer, lymphoma (eg, B-cell lymphoma), leukemia, and metastases of the above tumors.
  • lymphoma eg, B-cell lymphoma
  • the pharmaceutical composition may also contain one or more of: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives, Such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents etc.
  • the pharmaceutical compositions of the present application can be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules and the like.
  • the pharmaceutical composition of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration Drugs, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • compositions for therapeutic use may be formulated in the form of lyophilized formulations or aqueous solutions by mixing reagents of desired purity with, as appropriate, pharmaceutically acceptable carriers, excipients, etc. storage.
  • the present application provides the anti-CD73 antibody or its antigen-binding portion described in the first to fourth aspects, the nucleic acid molecule described in the fifth aspect, the carrier described in the sixth aspect, and the anti-CD73 antibody described in the seventh aspect.
  • the tumor is a tumor that expresses CD73 (CD73+) on the surface of tumor cells.
  • the tumor is a tumor that highly expresses CD73 (CD73 + ) on the surface of tumor cells.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 60% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 70% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 80% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 90% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 means that at least 95% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) means that at least 98% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) means that at least 99% of the tumor cells in the tumor cell population express CD73.
  • the tumor is a solid tumor.
  • the tumor is a hematological tumor.
  • the tumor is a malignant tumor.
  • the tumor is cancer
  • the tumor is selected from the group consisting of ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, kidney cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer , colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer , brain cancer, lymphoma (eg, B-cell lymphoma), leukemia, and metastases of the above tumors.
  • lymphoma eg, B-cell lymphoma
  • the present application provides the treatment of tumors in individuals, especially tumor cells expressing CD73 on the surface (CD73+) tumors, the method comprising administering to the individual a therapeutically effective amount of the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects, or the antibody drug conjugate described in the eighth aspect Or the pharmaceutical composition described in the ninth aspect.
  • the methods of treating or preventing a disease, disorder or condition comprise exposing cells to an anti-CD73 antibody in vitro.
  • the tumor is a tumor that expresses CD73 (CD73+) on the surface of tumor cells.
  • the tumor is a tumor that highly expresses CD73 (CD73 + ) on the surface of tumor cells.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 60% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 70% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 80% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 (CD73 + ) means that at least 90% of the tumor cells in the tumor cell population express CD73.
  • a tumor that highly expresses CD73 means that at least 95% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) means that at least 98% of the tumor cells in the tumor cell population express CD73. In some embodiments, a tumor that highly expresses CD73 (CD73 + ) means that at least 99% of the tumor cells in the tumor cell population express CD73.
  • the tumor is a solid tumor.
  • the tumor is a hematological tumor.
  • the tumor is a malignant tumor.
  • the tumor is cancer
  • the tumor is selected from the group consisting of ovarian cancer, breast cancer, melanoma, colorectal cancer, gastric cancer, kidney cancer, prostate cancer, pancreatic cancer, esophageal cancer, head and neck cancer, thyroid cancer, lung cancer, bladder cancer, cervical cancer , colon cancer, liver cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, squamous cell carcinoma, vulvar cancer, anal cancer, penile cancer , brain cancer, lymphoma (eg, B-cell lymphoma), leukemia, and metastases of the above tumors.
  • lymphoma eg, B-cell lymphoma
  • the anti-CD73 antibody or antigen-binding portion thereof of the present application can be used alone or in combination with other compositions in anti-tumor therapy.
  • an anti-CD73 antibody can be co-administered with at least one additional therapeutic agent and/or adjuvant.
  • the additional compound is a therapeutic antibody other than an anti-CD73 antibody.
  • the additional therapeutic agent is a therapeutic agent that targets the cancer immune cycle (e.g., immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody), A2AR antagonists, STAT-3 inhibitors), combination therapy with such therapeutic agents can be used to reduce tumor-mediated immunosuppression.
  • immune checkpoint inhibitors e.g., anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody
  • A2AR antagonists e.g., anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody
  • STAT-3 inhibitors e.g., anti-CTLA4 antibody
  • combination therapy with such therapeutic agents can be used to reduce tumor-mediated immunosuppression.
  • the present application provides a conjugate, which comprises the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects and a detectable label.
  • Detectable labels are, for example, detectable fluorescent labels, chemiluminescent labels, isotopic labels, and the like.
  • the present application provides a fusion protein comprising the anti-CD73 antibody or antigen-binding portion thereof described in the first to fourth aspects.
  • antibody fusion proteins include Fab fusion proteins, Fc fusion proteins, and single-chain antibody (scFv) fusion proteins, etc., which are named according to the different sites to which effector proteins (such as cytokines) are fused.
  • a conjugate may be formed by synthesizing an anti-CD73 antibody of the present application to which a linker is covalently attached to one or more non-antibody-acting agents.
  • an antibody of the application is conjugated or recombinantly fused to a diagnostic, detection, or therapeutic agent, or any other molecule. Conjugated or recombinantly fused antibodies can be used, for example, to monitor or predict the onset, occurrence, progression and/or severity of CD73-mediated diseases as part of a clinical trial approach, eg, to determine the efficacy of a particular therapy. Such diagnosis and detection can be accomplished, for example, by conjugating the antibody to a detectable label.
  • Detectable labels include, but are not limited to, enzymes, prosthetic groups, fluorescent labels, chemiluminescent labels, isotopic labels, and the like.
  • an antibody of the present application may be conjugated or recombinantly fused to a therapeutic moiety or drug moiety that modifies a given biological response (here also an antibody drug conjugate as described in the eighth aspect).
  • Therapeutic or drug moieties are not limited to typical chemotherapeutic agents.
  • the drug moiety can be a protein, peptide, polypeptide, small molecule toxin, etc. that has the desired biological activity.
  • mice Healthy BALB/c mice were immunized with recombinant human CD73 protein (Arco, Cat. No. CD3-H52H7). After the initial immunization, booster immunization was performed three times every 14 days. The peripheral serum of the mice was collected, and the titer of the mouse serum was detected by enzyme-linked immunosorbent assay (ELISA), and mice with high plasma antibody titers were selected for cell fusion. Pulse immunizations were performed 3 days before fusion via tail vein or intraperitoneally. On the day of fusion, the mouse spleen was taken, prepared into a single cell suspension, mixed with SP2/0 cells and splenocytes at a ratio of 1:2, and fused by electrofusion.
  • ELISA enzyme-linked immunosorbent assay
  • the hybridoma cells Place them in a CO 2 incubator at 37°C and culture them. When the hybridoma cells grow to a certain amount, the hybridoma supernatants are screened by ELISA. Positive clones were selected and screened for subclones by limiting dilution. Finally, the obtained monoclonal positive cells are extracted from RNA, reverse transcription and PCR amplification to obtain the heavy chain and light chain variable region fragments of the mouse immunoglobulin for sequencing. Finally, the heavy chain and light chain CDR region sequences and the amino acid sequences of the variable regions of 7C3C2 and 1F2F9 clones were obtained as follows:
  • Heavy chain CDR1 SYDIN (SEQ ID NO.1)
  • Heavy chain CDR2 WIYPRDGFTKYNEKFKG (SEQ ID NO.2)
  • Heavy chain CDR3 KEGYRGDWYFDV (SEQ ID NO.3)
  • Light chain CDR1 RASGNVHNYLA (SEQ ID NO.5)
  • Heavy chain CDR1 SYWMH (SEQ ID NO.9)
  • Heavy chain CDR2 NINPSNGGTKYNEKFKS (SEQ ID NO.10)
  • Heavy chain CDR3 RDYGSPSY (SEQ ID NO.11)
  • Light chain CDR2 RTNILLD (SEQ ID NO.14)
  • Light chain CDR3 LQYHEFPVT (SEQ ID NO.15)
  • Embodiment 2 ELISA method detects the affinity of chimeric antibody
  • a human-mouse chimeric antibody expression vector was constructed.
  • the VH and VL were respectively constructed on eukaryotic expression vectors containing the hIgG1-kappa constant region, which contained L234A, L235A, P329A, and P331S mutations (referred to as hIgG1 in this application, and the sequence of the heavy chain constant region is shown in SEQ ID NO.39. shown, the light chain constant region sequence is shown in SEQ ID NO.40, the following examples are the same).
  • the obtained eukaryotic expression vector was transiently transformed into HEK293 cells and cultured for 6 days.
  • the culture supernatant was harvested and purified through a ProteinA column to obtain the target antibodies, named 7C3C2-hIgG1 and 1F2F9-hIgG1 respectively, and the affinity of the antibodies to recombinant human CD73 protein (same as in Example 1) was determined by ELISA.
  • the EC 50 of 7C3C2-hIgG1 and 1F2F9-hIgG1 binding to CD73 protein were 0.017nM and 0.028nM, respectively. It can be seen that the EC 50 values of these two antibodies meet the requirements of further experiments.
  • the humanized light chain template for the mouse 7C3C2 is IGKV1-NL1; for the heavy chain, the closest human matching sequence is the IGHV1-8 gene; the humanized light chain template for the mouse 1F2F9 is the IGKV1-16 gene, The closest human heavy chain match is the IGHV1-3 gene.
  • the CDRs of the light and heavy chains were respectively grafted onto the framework sequences of the matching light chain and heavy chain genes to obtain humanized antibodies Hab7C3C2 and Hab1F2F9.
  • Discovery Studio and Antibody Modeling constructs a 3D model to analyze whether there are any framework positions that replace mouse amino acids with human amino acids that affect binding and/or CDR conformation. See Table 2 and Table 3 for the reverse mutation sequence. See Table 4 and Table 5 for relevant humanized antibodies.
  • Table 4 Schematic diagram of 7C3C2 humanized antibody combination
  • Humanized antibody VH and VL were respectively constructed on eukaryotic expression vectors containing hIgG1-kappa constant region, which contained L234A, L235A, P329A, P331S mutations (abbreviated as hIgG1 in this application).
  • the affinity of the derivatized antibody was Using the HIS1K biosensor, human CD73 protein was immobilized on the biosensor to a binding response threshold of 0.3 nm. After the 120s baseline step, the sensors were respectively immersed in the anti-CD73 antibody analyte diluted with 0.02% PBST buffer, the initial antibody concentration was 25nM, 2-fold serial dilution, and 7 concentration gradients were set.
  • the binding and dissociation times of ligand and analyte were 120s and 300s, respectively. According to the binding curve, the affinity data of the antigen and antibody are calculated by regression analysis software. Among all the detected antibodies, Hab7C3C2.5, Huab7C3C.9, Huab1F2F9.12 and Huab1F2F9.15 showed the highest affinity and fewer back mutations, the results are shown in Table 6.
  • Table 6 Affinity of humanized antibody to human CD73 protein detected by Fortebio method
  • Test object EC50 value (nM) Hab7C3C2.5 0.013 Hab1F2F9.15 0.011
  • the EC 50 of Hab7C3C2.5 and Huab1F2F9.15 binding to CD73 protein were 0.013nM and 0.011nM, respectively. It can be seen that the affinity of the humanized antibody is not affected compared with the mouse antibody.
  • This example compares exemplary antibodies of the present application to anti-CD73 antibodies described in other patent applications.
  • the MEDI9447 and Hu101-28 (CDR region sequences are from PCT patent applications WO 2016/075099 and WO2018/137598, respectively) antibodies were cloned into eukaryotic expression vectors containing hIgG1-kappa constant regions.
  • the tumor cell lines MDA-MB-231 and SKOV3 endogenously expressing human CD73 on the cell surface were used to evaluate the binding ability of humanized antibodies Hab7C3C2.5 and Hab1F2F9.15 to CD73 on the cell surface.
  • the cells were seeded in a 96-well plate at a density of 2 ⁇ 10 6 /ml, 100 ⁇ l/well.
  • the cross-binding ability of the exemplary humanized anti-CD73 antibody of the present application and cynomolgus monkey CD73 was detected by Fortebio assay.
  • the specific implementation steps include immobilizing the CD73 protein of cynomolgus monkey on the HIS1K biosensor. After the 120s baseline step, the sensors were respectively immersed in the anti-CD73 antibody analyte diluted with 0.02% PBST buffer. The initial concentration of the antibody was 25nM, and 7 concentration gradients were set respectively for affinity analysis experiments. The binding and dissociation times of ligand and analyte were 120s and 300s, respectively. According to the binding curve, the affinity data of the antigen and antibody were calculated by regression analysis software. The results are shown in Table 9, the exemplary antibody of the present application can cross-recognize the CD73 protein of cynomolgus monkey.
  • Table 9 Affinity of humanized antibody to cynomolgus monkey CD73 protein detected by Fortebio method
  • CHO-K1 cells that can stably express the monkey-derived full-length CD73 were constructed.
  • the cells were seeded in a 96-well plate at a density of 2 ⁇ 10 6 /ml, 100 ⁇ l/well. Wash three times with PBS buffer, discard the supernatant.
  • Antibody diluted in PBS buffer was added and incubated at 4°C for 1 h.
  • the cells were washed 3 times with PBS buffer, and then labeled with Alexa Fluor 488-labeled goat anti-human IgG antibody at 4°C in the dark for 50 min.
  • the cells were washed 3 times with PBS buffer, and the fluorescence intensity of the cells was read by flow cytometry.
  • the prepared humanized antibody can dose-dependently bind to the monkey expressed on the cell CD73 protein.
  • Table 10 The exemplary humanized antibody of the present application and the binding experiment of cynomolgus monkey CD73 on the cell surface
  • CD73-expressing MDA-MB-231 cells were diluted at 2 ⁇ 10 5 /ml, 100 ⁇ l/well was inoculated into a 96-well plate. The plate was incubated at 37° C. for 1 h, then AMP (1800 ⁇ M) was added to each well, 50 ⁇ l/well, and incubated at 37° C. for 2 hours.
  • the cells were centrifuged at 1000 rpm for 5 min, and 50 ⁇ l of the supernatant was transferred to a new 96-well plate, and 50 ⁇ l of ATP was added to make the final concentration 75 ⁇ M.
  • CellTiter-Glo reagent Promega was added at a ratio of 1:1 to measure the cellular CD73 enzyme activity. The results are shown in Figure 7 and Table 12: Hab7C3C.5 and Hab1F2F9.15 can inhibit the CD73 enzyme activity on MDA-MB-231 cells.
  • antibody-mediated internalization of CD73 was assessed by flow cytometry.
  • the cells were suspended in serum-free DMEM medium, and incubated with the tested antibodies for 1 h on ice at a concentration of 10 ⁇ g/ml.
  • the cells were washed three times with serum-free DMEM medium; the cells incubated with different antibodies were suspended in serum-free DMEM medium, and each was divided into two parts and spread in two 24-well plates.
  • One culture plate was incubated in a 37°C incubator; the other culture plate was placed in a 4°C refrigerator as a control group; cultured for 24 hours. Collect and wash the cells, add Alexa Fluor 488-labeled goat anti-human secondary antibody, and stain at 4°C for 1 h in the dark. Washed 3 times in PBS buffer and detected by flow cytometry.
  • the exemplary antibody of the present application can induce about 60% of CD73 receptor internalization, and maintain about 40% of the initial surface staining. All calculated values are percent internalization compared to the control (set as 100%).
  • the C-terminal part (SEQ ID NO.41) and the N-terminal part (SEQ ID NO.42) of the CD73 protein were respectively constructed, and the antibody recognition epitope was detected by ELISA method.
  • the CD73 N-terminal and C-terminal proteins were coated on the microplate at a concentration of 1 ⁇ g/ml, and the antibodies to be tested were serially diluted. The resulting affinity is shown in Figure 9A (N-terminal) and 9B (C-terminal).
  • the experimental results show that: compared with the control antibodies (MEDI9447 and Hu101-28), the binding epitopes of Hab7C3C.5 and Hab1F2F9.15 are different; Bind to the N-terminus of CD73 protein, and Hu101-28 binds to the C-terminus of CD73 protein.
  • the binding epitopes of Hab7C3C.5 and Hab1F2F9.15 of the present application are obviously different from those of MEDI9447 and Hu101-28.
  • the monotherapy efficacy of the 1F2F9 chimeric antibody was evaluated in a tumor xenograft model.
  • 4 ⁇ 106 A375 melanoma cells and 4 ⁇ 105 human PBMCs were mixed with Matrigel 1:1 and inoculated into NCG immunodeficient mice.
  • 30 mg/kg of 1F2F9 chimeric antibody or PBS control was injected intraperitoneally and every other day.
  • 24 days after treatment several mice had tumors that reached an upper limit (2000 mm 3 ). Day 24 after treatment therefore became the data endpoint for the analysis.
  • the mean tumor volume was 1448 ⁇ 176.76 mm 3 (mean ⁇ standard error) at 24 days of treatment.
  • human CD73/PD1/PDL1 knock-in BALB/c mice and CT26 cells (colon cancer cell line) overexpressing human CD73/PDL1 were used to evaluate Huab1F2F9.15, anti-PD-1 antibody and their combination efficacy.
  • 1 ⁇ 10 6 CT26-hCD73/hPDL1 cells were subcutaneously inoculated into genetic mice.
  • the day of grouping was defined as day D0, and administration began on the day of grouping.
  • the indicated doses of antibodies or PBS control were injected intraperitoneally twice a week.
  • the present application has obtained an antibody with high affinity that recognizes CD73 protein and CD73 at the cellular level.
  • the representative antibodies tested can potently inhibit the enzymatic activity of CD73, and at the same time induce the internalization of CD73 protein on tumor cells, and further reduce the enzymatic activity of CD73 by reducing the CD73 molecules on the cell surface, thereby achieving inhibition through multiple pathways Effect of CD73 enzymatic activity.
  • Epitope analysis shows that the exemplary antibody of the present application does not bind to either the N-terminal or the C-terminal protein of the CD73 protein alone, and has differential epitope binding characteristics from prior art antibodies such as MEDI9447 and Hu101-28.

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WO2025125545A1 (en) * 2023-12-15 2025-06-19 Sanofi Effectorless igg1 fc variants

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