WO2024032777A1 - Anti-cd73 antibody or antigen fragment thereof and use thereof - Google Patents

Anti-cd73 antibody or antigen fragment thereof and use thereof Download PDF

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WO2024032777A1
WO2024032777A1 PCT/CN2023/112604 CN2023112604W WO2024032777A1 WO 2024032777 A1 WO2024032777 A1 WO 2024032777A1 CN 2023112604 W CN2023112604 W CN 2023112604W WO 2024032777 A1 WO2024032777 A1 WO 2024032777A1
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seq
amino acid
antibody
acid sequence
antigen
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PCT/CN2023/112604
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French (fr)
Chinese (zh)
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邱奕凯
胡桐桐
周艺
刘�东
连凯
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南京蓬勃生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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 relates to antibodies or antigen-binding fragments thereof capable of specifically binding to the CD73 protein and the use of such preparations.
  • the antibody or antigen-binding fragment thereof can be used to treat diseases related to CD73 activity.
  • the adenosine pathway can downregulate inflammation and suppress immune responses to maintain immune homeostasis.
  • TME tumor microenvironment
  • ATP ATP will be released into the tumor TME, and ATP is subsequently broken down into adenosine by CD39 and CD73 proteins.
  • Abundant extracellular adenosine has an immunosuppressive effect. It inhibits the expansion and cytotoxicity of T cells and NK cells through A2a receptors and A2b receptors, hinders antigen presentation by dendritic cells, and is beneficial to regulatory T cells ( regulatory T cells) and myeloid-derived suppressor cells (MDSC) (Ref. 1).
  • CD39/CD73 tandem reaction The breakdown of extracellular ATP is mediated through the CD39/CD73 tandem reaction, with CD39 catalyzing the conversion of ATP into AMP and CD73 catalyzing the conversion of AMP into adenosine.
  • Adenosine is detected at higher concentrations in solid tumors than in surrounding tissue, suggesting its role in suppressing immune responses in the TME and tumor progression.
  • Cell surface CD73 protein and soluble CD73 directly decompose AMP into adenosine, making CD73 a hot target for tumor treatment.
  • CD73 expression is induced in response to hypoxia (HIFa), inflammation (TGF ⁇ ), and cell death caused by chemotherapy. It responds directly to HIF ⁇ signaling, which may explain why CD73 expression is increased in tumor cells under hypoxic conditions (Ref. 2).
  • TGFb signaling stabilizes H1F ⁇ protein and indirectly enhances CD73 expression.
  • CD73 is also expressed on Tregs and CD4+ memory T cells, especially the surface protein used to mark Th17.
  • Olecumab (MEDI9447) is an anti-CD73 monoclonal antibody and is the first anti-CD73 antibody drug in clinical trial 2. Olecumab alleviates adenosine-mediated immunosuppression in vitro and in vivo in CT26 syngeneic animal models. Olecumab 10 mg/kg inhibited tumor growth by 50% and showed a large increase in CD8+ cytotoxic T lymphocytes in TIL (tumor-infiltrating lymphocytes).
  • the combined administration of Olecumab and PD-1 antibody has an additive effect on tumor rejection in mice and can increase the secretion of IFN ⁇ and TNF ⁇ by T cells. Therefore, it may be possible to combine anti-CD73 antibodies with other immune checkpoint blockers in cancer treatment.
  • Other CD73 antibody drugs in phase 1 clinical trials include Bristol-Myers Squibb’s BMS-986179, Corvus Pharmaceuticals Inc.’s CPI-006 (Reference 4), Gilead Sciences’ bispecific antibody GS-1423 and Akeso's AK119, most of which The drug is in clinical trials combined with other immune checkpoint blockers, such as PD-1, PD-L1, or CTLA-4 (Reference 1).
  • antibodies or antigen-binding fragments thereof including:
  • VH Heavy chain variable domain
  • Heavy chain CDR1 which includes an amino acid sequence selected from one of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74 and 82;
  • Heavy chain CDR2 (HCDR2), which includes an amino acid sequence selected from one of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75 and 83; and
  • Heavy chain CDR3 (HCDR3), which includes an amino acid sequence selected from one of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84 and 105; and
  • VL Light chain variable domain
  • LCDR1 Light chain CDR1 (LCDR1), which includes an amino acid sequence selected from one of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78 and 86;
  • LCDR2 Light chain CDR2
  • LCDR2 which includes an amino acid sequence selected from one of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79 and 87;
  • LCDR3 Light chain CDR3 (LCDR3), which includes an amino acid sequence selected from one of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80 and 88,
  • the antibody or antigen-binding fragment thereof can specifically bind to CD73, preferably human CD73.
  • the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 18, 19 and 20, respectively
  • the VL includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 22, 23 and 24 respectively.
  • the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 2, 3 and 4 respectively, and the VL includes SEQ ID NO: 6 and 7 respectively. and LCDR1, LCDR2 and LCDR3 with the sequences shown in 8; 4)
  • the VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 10, 11 and 12 respectively, and the VL includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 10, 11 and 12 respectively, and the VL includes SEQ ID NO: 14 respectively.
  • the VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 26, 27 and 28 respectively, and the VL includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 26, 27 and 28 respectively, and the VL includes SEQ ID NO. : LCDR1, LCDR2 and LCDR3 of the sequences shown in 30, 31 and 32; 6)
  • the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 42, 43 and 44 respectively or having SEQ ID NO: 42 respectively.
  • the VL includes LCDR1, LCDR2 and LCDR3 with the sequences shown in SEQ ID NO:46, 47 and 48 respectively; 7)
  • the VH includes the LCDR1, HCDR2 and LCDR3 with the sequences shown in SEQ ID NO:46, 47 and 48 respectively; 7)
  • the VH includes SEQ ID NO. : HCDR1, HCDR2 and HCDR3 of the sequences shown in 50, 51 and 52, the VL includes SEQ ID NO: 54, respectively.
  • the VH includes HCDR1, HCDR2 and HCDR3 respectively having the sequences shown in SEQ ID NO: 58, 59 and 60, and the VL includes respectively having SEQ ID NO: 58, 59 and 60.
  • the VH includes HCDR1, HCDR2 and HCDR3 respectively having the sequences shown in SEQ ID NO: 66, 67 and 68, and the VL includes respectively having the sequences of SEQ ID NO: LCDR1, LCDR2 and LCDR3 with the sequences shown in NO:70, 71 and 72; 10)
  • the VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO:74, 75 and 76 respectively, and the VL includes respectively the LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NO:78, 79 and 80; or 11) the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO:82, 83 and 84 respectively, the VL Included are LCDR1, LCDR2 and LCDR3 having the sequences shown in SEQ ID NO:86, 87 and 88 respectively.
  • the VH includes an amino acid that is at least 80% identical to a sequence selected from one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, and 81 Sequence, the VL includes an amino acid sequence that is at least 80% identical to a sequence selected from one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85.
  • the VH includes an amino acid sequence selected from one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81 or relative to SEQ ID NO: 1
  • the amino acid sequence of one of , 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81 includes variants with up to 3 amino acid substitutions
  • the VL includes a variant selected from SEQ ID NO: 5, 13, 21 , the amino acid sequence of one of 29, 37, 45, 53, 61, 69, 77 and 85 or relative to one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85
  • An amino acid sequence includes variants with up to 3 amino acid substitutions.
  • the VH includes the amino acid sequence shown in SEQ ID NO: 17, and the VL includes the amino acid sequence shown in SEQ ID NO: 21; 2) the VH includes the amino acid sequence shown in SEQ ID NO: 33 Sequence, the VL includes the amino acid sequence shown in SEQ ID NO: 37; 3) the VH includes the amino acid sequence shown in SEQ ID NO: 1, the VL includes the amino acid sequence shown in SEQ ID NO: 5; 4) the VH includes the amino acid sequence shown in SEQ ID NO: 9, and the VL includes the amino acid sequence shown in SEQ ID NO: 13; 5) The VH includes the amino acid sequence shown in SEQ ID NO: 25, and the VL includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 29; 6) the VH includes the amino acid sequence shown in SEQ ID NO: 41, the VL includes the amino acid sequence shown in SEQ ID NO: 45; 7) the VH includes the amino acid sequence shown in SEQ ID NO: 49 Sequence, the VL includes the amino acid sequence
  • the enzymatic activity of the antibody or antigen-binding fragment thereof against the CD73 expressed on the cell surface has an IC50 value of 10 ⁇ 1 ⁇ g/mL to 10 ⁇ 3 ⁇ g/mL.
  • the antibody or antigen-binding fragment thereof has an EC50 value for binding to cells expressing the CD73 of 1 ⁇ g/mL to 10 ⁇ 2 ⁇ g/mL.
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody, scFv, Fab, Fab', (Fab') 2 , or Fv.
  • the VH of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 91, 93, 89, 95, 97, 99 or 101
  • the VL of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 91, 93, 89, 95, 97, 99 or 101.
  • the VH includes the amino acid sequence shown in SEQ ID NO:91 and the VL includes the amino acid sequence shown in SEQ ID NO:92; 2) the VH includes the amino acid sequence shown in SEQ ID NO:93 The sequence and the VL include the amino acid sequence shown in SEQ ID NO: 94; 3) the VH includes the amino acid sequence shown in SEQ ID NO: 89 and the VL includes the amino acid sequence shown in SEQ ID NO: 90; 4) the VH includes the amino acid sequence shown in SEQ ID NO:95 and the amino acid sequence shown in the VLSEQ ID NO:96; 5) the VH includes the amino acid sequence shown in SEQ ID NO:97 and the VL includes the amino acid sequence shown in SEQ ID NO:98 The amino acid sequence is shown; 6) the VH includes the amino acid sequence shown in SEQ ID NO: 99 and the VL includes the amino acid sequence shown in SEQ ID NO: 100; or 7) the VH includes the amino acid sequence shown in SEQ ID NO: 101 And the VL includes the amino acid sequence
  • the enzymatic activity of the humanized antibody against the CD73 expressing the cell surface expression has an IC50 value of 10 ⁇ 1 ⁇ g/mL to 10 ⁇ 2 ⁇ g/mL.
  • the KD value for binding between the antibody or antigen-binding fragment thereof and the CD73 is 10 ⁇ 8 M to 10 ⁇ 11 M.
  • the VH and/or VL are linked to the constant region of an immunoglobulin; preferably, the VH is linked to the IgGl constant region.
  • bispecific antibodies the aforementioned antibodies or antigen-binding fragments thereof and a second antibody portion.
  • the second antibody portion is capable of binding an antigen other than CD73.
  • the antigen other than CD73 is CD39, CTLA-4, PD-L1, TIM-3, LAG-3, or A2aR.
  • the second antibody moiety is Fab, Fab', (Fab') 2 , Fv, scFv, or sdAb.
  • polynucleotides encoding the above-claimed antibodies or antigen-binding fragments thereof or bispecific antibodies.
  • vectors for the above polynucleotides are provided herein.
  • cells comprising the polynucleotides or vectors described above.
  • this article provides a pharmaceutical composition for cancer treatment, including: the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned bispecific antibody; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes one or more other anti-cancer agents.
  • the cancer is a solid cancer.
  • the cancer is selected from non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), or pancreatic cancer.
  • provided herein is the use of the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, polynucleotide, vector or cell in the preparation of a medicament for treating cancer.
  • the cancer is a solid cancer.
  • the cancer is selected from non-small cell lung cancer, triple negative breast cancer, or pancreatic cancer.
  • a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody or antigen-binding fragment thereof, a bispecific antibody or the above-mentioned pharmaceutical composition.
  • the subject is a human.
  • the disease is cancer.
  • the disease is solid cancer.
  • the solid cancer is selected from non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), or pancreatic cancer.
  • the present invention blocks CD73 enzyme activity through anti-CD73 antibodies, which can alleviate the immunosuppression of the tumor microenvironment by the adenosine pathway and is beneficial to T cells. and the specific killing effect of NK cells on tumors.
  • the antibody we identified has better antagonistic ability to CD73 extracellular-5'-nucleotidase than MEDI9447 and is highly diverse in multi-epitope targeting. These antibody leads may provide good candidates for antibody therapy, bispecific antibody design, and combination therapy with chemotherapy and radiotherapy.
  • Figure 1 Inhibition of AMP hydrolysis by 52A6B3 and 100C5A3.
  • Figure 2 Inhibition of AMP hydrolysis by 52A8B3, 96A5A5 and 134D2H6.
  • Figure 3 Inhibition of AMP hydrolysis by 141G7E9, 143H3C5 and 143E5D2.
  • Figure 4 Inhibition of AMP hydrolysis by 9E12E3, 22E1B4 and 35D8G8.
  • Figure 5 FACS data of anti-CD73 mouse monoclonal antibody binding to the human CD73-CHO-K1 cell line.
  • Figure 6 The inhibitory effect of 7 humanized antibodies on CD73-mediated AMP hydrolysis.
  • A shows the inhibitory effect of 5 humanized antibodies on CD73-mediated AMP hydrolysis;
  • B shows the other 2 humanized antibodies.
  • Antibodies inhibit CD73-mediated AMP hydrolysis.
  • Figure 7 FACS data of anti-CD73 antibodies binding to CD73 in some embodiments: Panel A) shows the binding ability of the antibody to human CD73; Panel B) shows the binding ability of the antibody to cynomolgus monkey CD73.
  • Figure 8 Epitope identification of antibodies and MEDI19447 in some examples.
  • the present disclosure provides anti-CD73 monoclonal antibodies and uses thereof.
  • This disclosure relates to heavy chain variable domains (VH) and light chain variable structures of mouse anti-CD73 monoclonal antibodies (9E12E3, 22E1E4, 35D8G8, 52A8B3, 52A6B3, 96A5A5, 100C5A3, 134D2H6, 141G7E9, 143E5D2 and 143H3C5 clones)
  • Amino acid sequence of domain (VL) It also relates to the amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) that have been humanized or post-translationally modified on these mouse anti-CD73 monoclonal antibody clones.
  • the present disclosure also relates to methods of producing anti-CD73 monoclonal antibodies.
  • the present disclosure provides anti-human CD73 chimeric antibodies by fusing the variable regions of the heavy and light chains of the disclosed mouse anti-CD73 monoclonal antibodies with the constant regions of human IgG.
  • the present invention provides humanized versions of the heavy chain variable domain (VH) and light chain variable domain (VL) of mouse anti-CD73 monoclonal antibodies.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the CDRs of mouse anti-CD73 monoclonal antibodies are grafted onto human IgG framework sequences, and the resulting humanized variable domains can be further fused to the constant regions of human IgG.
  • the term "about” means a value within ⁇ 10% of a given numerical value.
  • the terms “comprises,” “contains,” “having,” and similar expressions mean that non-recited elements are not excluded. These terms also include instances that consist solely of the recited elements.
  • Antibodies refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen.
  • the basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. According to the conservative differences in amino acid sequences, heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. The variable regions of a heavy chain and a light chain interact to form the antigen-binding site (Fv).
  • variable region the composition and order of amino acid residues in certain regions are more variable than other regions (backbone regions, FR) within the variable region, which are called hypervariable regions (HVR).
  • the hypervariable regions are actually antibodies. and Key site for antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both heavy and light chains have three complementarity determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively.
  • the amino acid sequences of the CDRs can be determined using art-recognized numbering schemes, such as the Kabat, Chothia, IMGT, AbM, or Contact numbering schemes.
  • the CDR sequences of the antibodies described herein have been determined according to the Kabat numbering scheme.
  • Antibodies can be divided into five main different types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. These antibody types can be further divided into subclasses based on the size of the hinge region, the position of the interchain disulfide bond, and the molecular weight, such as IgGl, IgG2a, IgG2b, and IgG3.
  • the light chain can be divided into two types: kappa and lambda.
  • the subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.
  • the "antigen-binding fragment" of an antibody molecule refers to the amino acid fragment in the antibody molecule that participates in specific antigen binding, such as Fab, Fab', (Fab') 2 , scFv, sdAb, etc.
  • Fab fragment-specific antigen binding
  • Fab' fragment-specific antigen binding
  • Fab' fragment-specific antigen binding
  • F(ab') 2 fragment-specific antigen binding
  • Single chain fragment variable is composed of an antibody heavy chain variable region and a light chain variable region connected through a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and light chain form Fv segments through non-covalent interactions, so scFv can better retain its affinity activity for antigens.
  • Single domain antibody also known as “VHH antibody” refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region but no light chain. From a structural point of view, single domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids. Subsequently, researchers discovered more single-domain antibodies with antigen-binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, and when used in the human body, they can easily reach tissues that are difficult for ordinary antibody molecules to reach. or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; they are more stable and can withstand changes in temperature and pH, as well as the effects of denaturants and proteases.
  • Fc fragment refers to the handle region of a Y"-shaped antibody molecule, that is, the crystallizable fragment (fragment crystallizable, Fc) includes the second and third constant domains (CH2 and CH3 domains) of the heavy chain. It can be passed through proteolytic enzymes (such as papain) hydrolyzes the antibody molecule to obtain the antibody Fc region.
  • the Fc region can include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides.
  • the Fc fragment can be from IgG, IgM, IgD, IgE, or IgA.
  • the Fc region is from IgG1, IgG2, IgG3, or IgG4.
  • Fc fragment also includes fragments from native Fc fragments that have been altered but still retain their effector function.
  • a "variant Fc fragment” includes an amino acid sequence that has at least one amino acid change in the amino acid sequence of a native Fc fragment.
  • a variant Fc fragment has At least one amino acid substitution, for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions.
  • the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least About 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity.
  • Effector functions of "Fc fragments” may include binding to Fc receptors, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, etc.
  • the Fc fragment may be modified to enhance, weaken, or eliminate its effector function.
  • Mouse antibody refers to an antibody in which the variable and constant regions, if any, are derived from mouse or rat immunoglobulin sequences.
  • Mouse antibodies can be conveniently obtained by immunizing mice or rats with corresponding antigens and isolating the antibodies of interest therefrom. Alternatively, it can be obtained by immunizing mice or rats with the corresponding antigen and then isolating and culturing cells (such as B cells) expressing the antibody of interest. Or, after immunizing mice or rats with the corresponding antigen, isolate and culture the cells expressing the target antibody, and fuse them with immortalized cells such as myeloma cells to obtain hybridoma cells. The hybridoma cells can be cultured for a long time and in large quantities. Obtain the target antibody (such as monoclonal antibody).
  • the "murine antibody” is a mouse antibody.
  • Humanized antibody refers to a non-human antibody, that is, an antibody in which the variable and constant regions (if any) are not derived from human immunoglobulins, have been artificially modified so that they contain the amino acid sequence of a human antibody, thereby Obtained chimeric antibodies.
  • Humanized antibodies may comprise the constant regions and/or framework regions of human antibodies.
  • Humanized antibodies can be obtained by genetic engineering means, for example, replacing the constant region of a murine antibody with a human antibody's constant region and/or replacing the mouse antibody's framework region with a human antibody's framework region. This humanization transformation usually does not affect the binding specificity of the original antibody to the corresponding antigen, so such antigens are also included in the scope of the present invention.
  • Human antibody or “humanized antibody” refers to an antibody in which both the variable and constant regions (if any) are derived from human germline immunoglobulin sequences.
  • Human antibodies can be obtained through a variety of techniques, including phage antibody library technology, single B cell cloning technology, and transgenic mouse technology (for example, using the introduction of human germline immunoglobulin genes and the removal of the mouse's own germline immunoglobulin genes). transgenic mice), etc.
  • animal-derived antibodies such as mouse-derived antibodies
  • human antibodies have the advantages of low immunogenicity and high safety when used in human patients.
  • the term "monoclonal antibody” as used herein refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single epitope on the antigen.
  • the modifier "monoclonal” indicates the uniform character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • the monoclonal antibodies of the invention can be produced by hybridoma methods or recombinant DNA methods well known in the art, or obtained by screening methods described elsewhere herein.
  • an “isolated” antibody is an antibody that has been isolated and/or recovered from its natural or production environment (eg, cell culture). Contaminating components in the production environment, such as components from recombinantly transfected cells (e.g. enzymes, hormones and other protein or non-protein components, nucleic acids), often interfere with the research, diagnostic or therapeutic use of antibodies. Therefore, preferably, the isolated antibody is substantially free of association with other components of its natural or production environment.
  • epitope refers to the portion of a molecule (eg, an antigen) to which an antibody binds.
  • An epitope may comprise a non-contiguous portion of the molecule (e.g., amino acids in a polypeptide that are not contiguous in the main sequence of the polypeptide but are sufficiently close to each other in the trivalent and tetravalent structure of the polypeptide to be bound by an antibody Residues).
  • Bispecific antibodies refer to antibody molecules that have two different binding sites and can recognize and bind to two different antigens respectively.
  • one binding site of the bispecific antibody can be used to bind immune cells (e.g. T cells), another binding site is used to bind tumor cells, thereby enhancing the killing effect of immune cells on tumor cells while reducing side effects such as off-target toxicity.
  • the two binding sites of the bispecific antibody respectively bind to proteins or antigens related to the tumor microenvironment and immunosuppression (such as CD73 and PD-L1).
  • This bifunctional antibody usually has higher efficacy than monoclonal antibody drugs as a tumor treatment drug.
  • antibody molecules can be engineered to include multiple different binding sites, creating "trispecific antibodies,”"tetraspecificantibodies,” etc.
  • the "multispecific antibody” used in this article covers these bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.
  • Fusion protein refers to a protein molecule composed of at least two different peptide segments that is artificially produced (for example, through genetic engineering technology). These peptides do not exist in nature or do not exist in the same protein molecule. Common examples of fusion proteins including antibody fragments include chimeric antibodies, antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also known as immunotoxins), enzyme-labeled antibodies for immunoassays, and chimeric antigen receptors (CAR) etc.
  • Targeting or “specific binding” means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher affinity for another molecule (e.g., a tumor cell surface antigen) relative to other molecules also present in the environment. Binding affinity. "Targeting” or “specific binding” does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.
  • EC 50 concentration for 50% of maximal effect refers to the concentration that causes 50% of the maximum effect.
  • concentration of the antibody molecule that produces half of the maximum fluorescence intensity. The lower the EC50 value, the greater the binding affinity to the antigen on the cell.
  • KD values can also be used to measure the binding affinity between an antibody and its antigen.
  • the KD value is the equilibrium dissociation constant between the antibody and its antigen, that is, the ratio of k off /k on . Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.
  • the inhibitory effect of an inhibitor on the biological activity of an active molecule can be measured by the IC 50 value (half of the maximum inhibitory concentration).
  • IC 50 indicates the concentration of inhibitor required to inhibit the activity of the active molecule (such as CD73) by half (such as catalyzing the conversion of AMP to adenosine). The smaller the IC 50 value, the stronger its ability to inhibit the active molecule.
  • Variant refers to a product sequence (eg, CDR sequence, VH or VL sequence) resulting from the introduction of differences in amino acid sequence relative to a reference sequence (eg, CDR sequence, VH or VL sequence disclosed herein).
  • a reference sequence eg, CDR sequence, VH or VL sequence disclosed herein.
  • a few amino acids can be replaced, deleted, added, and the resulting product can be verified or screened for its binding ability to the corresponding antigen CD73 or its ability to inhibit it, thereby obtaining
  • Corresponding variants of the CD73-targeting antibodies or antigen-binding fragments thereof provided herein are also included in the scope of the present invention.
  • the antibodies or antigen-binding fragments thereof disclosed herein may have at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid changes in the full length or CDR sequence.
  • Purification tag refers to the amino acid sequence expressed together with the target protein or polypeptide in the form of a fusion protein for purifying the target protein or polypeptide, including but not limited to His6 tag, Flag tag, MBP (maltose binding protein) tag and GST (gluten thione sulfhydryl transferase) tag, SUMO (small ubiquitin related modifier), etc. These tags can be removed by enzymatic digestion after purification, or they can be used with tags (such as His6 tags) without affecting the normal function of the target protein or peptide.
  • Detectable label refers to an amino acid sequence or other chemical group linked to a protein or polypeptide, used to indicate the presence or content of the protein or polypeptide in a sample, or used to track the protein or polypeptide in a subject's body or cells location information.
  • detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP); fluorescent groups (such as FAM, FITC) or fluorescent proteins (such as GFP ); radioactive isotopes (such as 3 H, 14 C, 35 S).
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • fluorescent groups such as FAM, FITC
  • fluorescent proteins such as GFP
  • radioactive isotopes such as 3 H, 14 C, 35 S.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably to refer to polymers of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence” refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.
  • vector refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (eg, a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (eg, nucleic acid, plasmid, virus, etc.).
  • the vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and/or enhancer), and/or one or more Selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, such as beta-galactose).
  • expression vector refers to a vector used to express a polypeptide of interest in a host cell.
  • “Host cell” refers to a cell that is or has been the recipient of a vector or isolated polynucleotide.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells.
  • Host cells include the progeny of a single host cell, and the progeny may not necessarily be identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations.
  • Host cells also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.
  • Treatment refers to the treatment of a subject to obtain a beneficial or desired clinical result.
  • Treatment encompasses a variety of treatments, including administration of any possible drug to the subject, surgery, radiation, etc.
  • beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g. metastasis, e.g. metastasize to the lungs or lymph nodes), prevent or delay disease recurrence, delay or slow down disease progression, improve disease conditions, inhibit disease or disease progression, block its development and remission (whether partial or complete remission).
  • the methods provided herein encompass any one or more of these aspects of treatment. In accordance with the above, “treatment” does not require complete removal of all symptoms of a condition or disease or complete alleviation.
  • terapéuticaally effective amount refers to an amount of active compound sufficient to elicit the biological or medical response desired by the clinician in a subject.
  • the "therapeutically effective dose” of the fusion protein of the present invention can be determined by those skilled in the art based on the route of administration, the subject's weight, age, condition and other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.
  • Solid cancer refers to a tangible tumor that can be detected by imaging examination methods such as CT, magnetic resonance, etc.
  • solid tumors include gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, etc.
  • pharmaceutically acceptable carrier refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be administered safely and are suitable for use by humans and/or Administration to the animal without undue adverse side effects while being suitable for maintaining the viability of the drug or active agent therein.
  • various carriers well known in the art may be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols , alginic acid, phosphate buffer, emulsifier, isotonic saline and/or pyrogen-free water, etc.
  • the pharmaceutical compositions provided herein can be made into clinically acceptable dosage forms such as powders and injections.
  • composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, etc. route of administration.
  • Subject refers to an animal, such as a mammal, including (but not limited to) humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, goats, mammals Laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets.
  • the subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects.
  • a subject refers to an individual in need of treatment of a disease or condition.
  • a subject receiving treatment can be a patient who has a condition associated with the treatment, or is at risk of developing the condition.
  • the subject is a healthy individual or an individual suffering from a disease other than that of concern.
  • the subject is a human, such as a human patient.
  • the term is often used interchangeably with "patient,” “subject,” “subject,” etc.
  • sequence identity refers to the identity between two amino acid or nucleotide sequences (such as a query sequence and a reference sequence) A quantity of degree, usually expressed as a percentage. Usually, before calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matching at that position; if the amino acid residues or bases in the two sequences are different, the two sequences are considered to be inconsistent or matching at that position. mismatch.
  • the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity.
  • the number of gaps and/or the gap length is also taken into account.
  • the publicly available alignment software BLAST available on the web page ncbi.nlm.nih.gov
  • BLAST can be used to obtain an optimal sequence alignment and calculate two amino acids or nucleotides by using default settings Sequence identity between sequences.
  • CD73 also known as extracellular-5'-nucleotidase (ecto-5'-NT or EC 3.1.3.5), is a cell surface phosphatase that catalyzes the dephosphorylation of extracellular AMP to produce adenosine. glycosides.
  • Physiologically, CD73 is induced by hypoxia to control inflammation at the site of injury.
  • Pathologically, CD73 is often found to be overexpressed on regulatory T cells (Tregs) and tumor cells. Increased CD73 activity leads to the accumulation of adenosine in the tumor microenvironment (TME).
  • TME tumor microenvironment
  • adenosine By binding to adenosine receptors A2a and A2b, adenosine suppresses innate and adaptive immunity by regulating many immune cells, such as macrophages, dendritic cells, natural killer cells, and T effector cells. Therefore, it is speculated that the immunosuppression of adenosine may be alleviated by inhibiting the activity of CD73 in the TME. In fact, in vivo animal studies have shown that inhibiting CD73 activity can suppress tumor tumor formation and growth, suggesting that CD73 is a promising target for cancer treatment.
  • the term "human CD73” refers to CD73 of human origin. An exemplary amino acid sequence of human CD73 is represented in GenBank accession number P21589.1.
  • anti-CD73 monoclonal antibodies of the present disclosure may cross-react with CD73 from species other than human or with other proteins structurally related to human CD73.
  • the anti-CD73 monoclonal antibodies provided herein are fully specific for human CD73 and do not exhibit species or other types of cross-reactivity.
  • anti-CD73 antibodies or antigen-binding fragments thereof that specifically bind CD73.
  • the anti-CD73 antibody or antigen-binding fragment thereof binds to CD73 molecules with relatively high binding affinity.
  • the ability of an anti-CD73 antibody or antigen-binding fragment thereof to bind to CD73 can be measured by assay methods such as enzyme-linked immunosorbent assay (ELISA) and flow cytometric fluorescence sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometric fluorescence sorting
  • SPR surface plasmon resonance technology
  • BLI biolayer interference
  • Anti-CD73 antibodies or antigen-binding fragments thereof bind to CD73 and inhibit its activity, such as the catalytic activity of converting AMP to adenosine.
  • anti-CD73 monoclonal antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH comprising a variable domain selected from SEQ ID NO.
  • HCDR1 of SEQ ID NO:50 (SYGIN), SEQ ID NO:58 (GYYMH), SEQ ID NO:66 (SYAMS), SEQ ID NO:74 (RYGIH) or SEQ ID NO:82 (GYYMH) or including up to
  • the HCDR1 variant with 3 (such as 1, 2 or 3) amino acid substitutions is selected from SEQ ID NO: 3 (YINPGDDYIKYSQNFKG), SEQ ID NO: 11 (YINPSDAYTTYSQNFKG), SEQ ID NO: 19 (NIYPGNSDTNNNEKFRN), SEQ ID NO:27(EIYPGSGNTYYNEKFKD), SEQ ID NO:35(NIYPGNSNTNYNEKFKS), SEQ ID NO:
  • the anti-CD73 antibody or antigen-binding fragment thereof is an inhibitor of CD73 and has an enzymatic activity against said CD73 expressed on the cell surface of about 10 -1 ⁇ g/mL to about 10 -3 ⁇ g /mL.
  • IC 50 value In some embodiments, the IC50 value of the enzymatic activity of the anti-CD73 antibody or antigen-binding fragment thereof against CD73 expressed on the cell surface is less than the IC50 value of the reference antibody MEDI9447 against CD73.
  • the anti-CD73 antibody, or antigen-binding fragment thereof binds CD73-expressing cells with an EC50 value of about 1 ⁇ g/mL to about 10 ⁇ 2 ⁇ g/mL (as determined by FASC). In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof binds to cells expressing CD73 with an EC 50 value that is less than the EC 50 value of the reference antibody MEDI9447 binding to cells expressing CD73.
  • the anti-CD73 antibody or antigen-binding fragment thereof includes an HCDR3 selected from SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, or 105 and LCDR3 of SEQ ID NO:8, 16, 24, 32, 40, 48, 56, 64, 72, 80 or 88, while amino acid substitutions occur in the CDR1 and CDR2 domains of VH and VL.
  • anti-CD73 antibodies or antigen-binding fragments thereof, whose VH includes a VH selected from the group consisting of SEQ ID NO: 2 (NYWIH), SEQ ID NO: 10 (NYWMH), SEQ ID NO: 18 (SYWIN), SEQ ID NO:26(NYGIS), SEQ ID NO:34(SYWIN), SEQ ID NO:42(NYGIT), SEQ ID NO:50(SYGIN), SEQ ID NO:58(GYYMH), SEQ The HCDR1 of ID NO:66 (SYAMS), SEQ ID NO:74 (RYGIH) or SEQ ID NO:82 (GYYMH) or a variant of this HCDR1 including up to 3 (eg 1, 2 or 3) amino acid substitutions, preferably From SEQ ID NO:3(YINPGDDYIKYSQNFKG), SEQ ID NO:11(YINPSDAYTTYSQNFKG), SEQ ID NO:19(NIYPGNSDTNN
  • amino acid substitutions 1, 2 or 3) amino acid substitutions, and selected from SEQ ID NO:8(LQYGDTTYT), SEQ ID NO:16(LQYGENTYT), SEQ ID NO:24(QHSRELPWT), SEQ ID NO:32(QQRYSTPYT), SEQ ID NO:40(QQGYILPFT), SEQ ID NO:48( QQHSSPPFT), SEQ ID NO:56 (LQHGESPFT), SEQ ID NO:64 (LQYDEFPNT), SEQ ID NO:72 (QQDYSSPWT), SEQ ID NO:80 (LQYDELYT) or SEQ ID NO:88 (LQYDEFPYT).
  • the anti-CD73 antibody or antigen-binding fragment thereof is an inhibitor of CD73 and has an enzymatic activity against said CD73 expressed on the cell surface of about 10 -1 ⁇ g/mL to about 10 -3 ⁇ g /mL. IC 50 value. In some embodiments, the IC50 value of the enzymatic activity of the anti-CD73 monoclonal antibody against CD73 expressed on the cell surface is less than the IC50 value of the reference antibody MEDI9447 against CD73. In some embodiments, the anti-CD73 monoclonal antibody binds CD73-expressing cells with an EC50 value of about 1 ⁇ g/mL to about 10 ⁇ 2 ⁇ g/mL (as determined by FASC). In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, binds cells expressing CD73 with an EC50 value that is less than the EC50 value of the reference antibody MEDI9447 binding cells expressing CD73.
  • amino acid substitutions mentioned may be conservative amino acid substitutions.
  • Conservative amino acid substitutions can generally be described as the replacement of one amino acid residue by another amino acid residue of similar chemical structure and having little or essentially no effect on the function, activity, or other biological properties of the polypeptide.
  • Conservative amino acid substitutions are well known in the art.
  • Conservative substitutions may, for example, be the substitution of one amino acid in the following groups (a)-(e) by another amino acid in the same group: (a) Small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
  • anti-CD73 antibodies or antigen-binding fragments thereof e.g., anti-CD73 monoclonal antibodies
  • their VHs include HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 3, and 4, respectively
  • Its VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:6, 7 and 8 respectively
  • Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:10, 11 and 12 respectively
  • its VL includes SEQ ID NO:10, 11 and 12 respectively.
  • VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 34, 35 and 36, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 38, 39 and 40; 6) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:42, 43 and 44 respectively or HCDR1, HCDR2 and HCDR3 of SEQ ID NO:42, 43 and 105 respectively and its VL includes SEQ ID NO:46 and 47 respectively and 48 LCDR1, LCDR2 and LCDR3; 7) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:50, 51 and 52 respectively, and its VL includes LCDR1, LCDR2 of SEQ ID NO:54, 55 and 56 respectively.
  • VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 58, 59 and 60, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 62, 63 and 64; 9) Its VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 66, 67 and 68, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 70, 71 and 72; 10) Its VH includes SEQ ID NO: 70, 71 and 72 respectively.
  • HCDR1, HCDR2 and HCDR3 of NO:74, 75 and 76 whose VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:78, 79 and 80 respectively; or 11) whose VH includes SEQ ID NO:82 and 83 respectively and HCDR1, HCDR2 and HCDR3 of 84, whose VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:86, 87 and 88 respectively.
  • anti-CD73 antibodies or antigen-binding fragments thereof (such as anti-CD73 monoclonal antibodies), which comprise: 1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VH comprising SEQ ID NO: 5 VL (9E12E3) with the amino acid sequence shown; 2) VH including the amino acid sequence shown in SEQ ID NO: 9 and VL (22E1E4) including the amino acid sequence shown in SEQ ID NO: 13; 3) VL (22E1E4) including the amino acid sequence shown in SEQ ID NO: 17 VH showing the amino acid sequence and VL including the amino acid sequence shown in SEQ ID NO: 21 (35D8G8); 4) VH including the amino acid sequence shown in SEQ ID NO: 25 and VL including the amino acid sequence shown in SEQ ID NO: 29 ( 52A8B3); 5) VH including the amino acid sequence shown in SEQ ID NO: 33 and VL (52A6B3) including the amino acid sequence shown in SEQ ID NO
  • an anti-CD73 antibody or an antigen-binding fragment thereof (such as an anti-CD73 monoclonal antibody) is provided, comprising: 1) having at least 80% (e.g., at least 85%) the same amino acid sequence as shown in SEQ ID NO: 1 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity of the VH of the amino acid sequence and includes the amino acid sequence shown in SEQ ID NO: 5 A VL whose sequence has an amino acid sequence of at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; 2) Includes an amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or VH and include amino acid sequences having at least 80% (e.g., at least 85%,
  • Sequence VL includes the same amino acid as shown in SEQ ID NO: 65 VH and amino acid sequences whose sequences have at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity Includes an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 69 ) VL with an amino acid sequence having sequence identity; 10) including a VL having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) with the amino acid sequence shown in SEQ ID NO:73 , 96%, 97%, 98% or 99%) and include VHs having an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 9
  • the anti-CD73 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.
  • the CDRs described above can be used in various pairwise combinations to generate some humanized anti-CD73 antibodies.
  • Humanizing substitutions are known to those skilled in the art.
  • potentially useful humanizing replacement amino acids can be identified by comparing the FR sequences of VH or VL derived from other species with the corresponding FR sequences of one or more closely related human VH or VL, and then using known methods in the art. Methods (also described herein) introduce one or more such potentially useful humanized replacement amino acids into the VH or VL.
  • a humanized antibody can be obtained by replacing all CDR sequences of a human antibody VH or VL (ie, all CDR sequences between the four FR sequences of a human antibody) with murine CDR sequences.
  • the heavy and light chains of an antibody are humanized simultaneously.
  • the resulting humanized antibodies can be tested for binding affinity to CD73, binding stability, and/or other desired properties, such as the ability to inhibit the activity of CD73.
  • the anti-CD73 antibodies or antigen-binding fragments thereof provided herein may be partially or fully humanized.
  • the resulting humanized antibody such as a humanized antibody or antigen-binding fragment thereof, binds to CD73 with a KD, EC50 or IC50 value as described herein.
  • anti-CD73 humanized antibodies, or antigen-binding fragments thereof whose VH includes the amino acid sequence of any one of SEQ ID NO: 89, 91, 93, 95, 97, 99, and 101 or is identical to
  • the amino acid sequence of any one of SEQ ID NO: 89, 91, 93, 95, 97, 99 and 101 has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99%) sequence identity of the amino acid sequence, the VL of which includes the amino acid sequence of any one of SEQ ID NO: 90, 92, 94, 96, 98, 100 and 102 or with
  • the amino acid sequence of any one of SEQ ID NO: 90, 92, 94, 96, 98, 100 and 102 has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %,
  • the anti-CD73 humanized antibody or antigen-binding fragment thereof has an IC50 value for enzymatic activity against said CD73 expressed on the cell surface of about 10 "1 ⁇ g/mL to about 10" 2 ⁇ g/mL. In some embodiments, the anti-CD73 humanized antibody or antigen-binding fragment thereof binds to CD73 with a KD value of about 10 "8 M to about 10" 11 M.
  • anti-CD73 humanized antibodies or antigen-binding fragments thereof which include: 1) VH of the sequence set forth in SEQ ID NO:89 and VL (human origin) of the sequence set forth in SEQ ID NO:90 9E12E3); 2) VH of the sequence shown in SEQ ID NO: 91 and VL of the sequence shown in SEQ ID NO: 92 (humanized 35D8G8); 3) VH and SEQ of the sequence shown in SEQ ID NO: 93 VL of the sequence shown in ID NO:94 (humanized 52A6B3); 4) VH of the sequence shown in SEQ ID NO:95 and VL of the sequence shown in SEQ ID NO:96 (humanized 96A5A5); 5) VH of the sequence shown in SEQ ID NO:97 and VL of the sequence shown in SEQ ID NO:98 (humanized 134D2H6); 6) VH of the sequence shown in SEQ ID NO:99 and the sequence shown in SEQ ID NO:100
  • the VL (human origin) of the sequence set
  • the anti-CD73 humanized antibodies, or antigen-binding fragments thereof, provided herein further comprise immunoglobulin constant region sequences.
  • the immunoglobulin constant region may be selected from IgG1, IgG2 or IgG4.
  • the anti-CD73 humanized antibody or antigen-binding fragment thereof comprises human IgG1 constant region sequence.
  • the heavy chain of the CD73 humanized antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region sequence
  • the light chain of the CD73 humanized antibody or antigen-binding fragment thereof comprises a human Kappa chain constant region. district.
  • the human IgG1 heavy chain constant region sequence can be the amino acid sequence shown in SEQ ID NO: 103
  • the human Kappa chain constant region can be the amino acid sequence shown in SEQ ID NO: 104.
  • anti-CD73 antibodies that compete for binding to CD73 with any of the anti-CD73 antibodies or antigen-binding fragments provided above (i.e., "competitive anti-CD73 antibodies”).
  • the competing anti-CD73 antibody competes for binding to the same epitope on CD73 with any of the anti-CD73 antibodies or antigen-binding fragments provided above.
  • Such competitive binding can be detected, for example, by an ELISA assay.
  • Anti-CD73 antibodies eg, anti-CD73 monoclonal antibodies
  • Anti-CD73 antibodies described herein can be prepared using any method known in the art or as described herein.
  • Rodent monoclonal antibodies can be obtained using methods known in the art, such as by immunizing rodents (such as mice or rats) and obtaining hybridomas therefrom, or by cloning Fab fragments using molecular biology techniques known in the art or single-chain Fc (scFv) libraries, followed by selection by ELISA screening or using phage display.
  • rodents such as mice or rats
  • scFv single-chain Fc
  • DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the antibody heavy and light chains).
  • Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, the host cell can be a prokaryotic cell or a eukaryotic cell (eg, a mammalian cell).
  • nucleic acid molecules of the present application may be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or produced semi-synthetically, for example, by combined chemical synthesis and recombinant methods.
  • Linkage of coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences Ligation can be performed using established methods such as restriction digestion, ligation, and molecular cloning. According to the well-known knowledge of those skilled in the art, changing (eg, replacing, deleting, etc.) the nucleotide sequence encoding the protein will not change the amino acids of the protein, such as codon optimization and other methods.
  • an expression vector comprising the above polynucleotide.
  • Vectors well known to those skilled in the art, such as plasmids, phage vectors or viral vectors.
  • the vector is a recombinant expression vector, such as a plasmid.
  • These vectors include any elements to support the functions of conventional expression vectors, including, for example, promoters, ribosome binding elements, terminators, enhancers, selectable markers, and origins of replication.
  • the promoter can be a conventional promoter, an inducible promoter or a repressible promoter.
  • expression vectors are known in the art that are capable of delivering nucleic acids into cells and can be used to produce antibodies or antigen-binding fragments thereof within cells. According to the methods in the embodiments of the present invention, conventional cloning technology or artificial gene synthesis can be used to generate recombinant expression vectors.
  • a host cell or cell-free expression system comprising the above-mentioned expression vector.
  • any host cell conventional in the art can be used for the expression of antibodies or antigen-binding fragments thereof.
  • the host cell is E. coli TG1 or BL21 (for expression of, for example, scFv or Fab antibodies), CHO-DG44, CHO-3E7, CHO-K1, or HEK293.
  • the recombinant expression vector is transfected into the host cell through conventional methods (such as chemical transfection, thermal transfection, or electrotransfection) and is stably integrated into the host cell genome, so that the recombinant nucleic acid can be effectively expressed.
  • the cell-free expression system described in this application is a new protein preparation system that is different from traditional protein expression systems. It uses DNA or mRNA as a template and utilizes organelles, protein folding factors and other related enzymes in cell extracts, by adding Amino acids, nucleotides, polymerases and energy substances are used to achieve protein expression in vitro.
  • the antigen-binding fragment of an anti-CD73 antibody can be linked to an Fc fragment to form a fusion protein.
  • the Fc fragment can be located at the C-terminus and N-terminus of the antigen-binding fragment of the anti-CD73 antibody.
  • the Fc fragment may be located at the C-terminus of the antigen-binding fragment of the anti-CD73 antibody.
  • the fusion protein formed by the antigen-binding fragment of the anti-CD73 antibody and the Fc fragment has the ability to specifically bind to CD73 and also has the effector function of the Fc fragment, such as mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cell death. Toxicity (ADCC), mediating phagocytosis, etc.
  • the Fc fragment can be derived from the constant region of an immunoglobulin, such as IgGl, IgG2 or IgG4.
  • the antigen-binding fragment of an anti-CD73 antibody can be linked to a protein tag to form a fusion protein.
  • Protein tags can include purification tags and detectable tags. Purification tags include but are not limited to His6 tag, Flag tag, MBP tag, GST tag, SUMO) tag, etc.
  • the detectable label can be used to indicate the presence or content of the anti-CD73 antibody or antigen-binding fragment thereof in a sample, or to provide information about the location of the anti-CD73 antibody or antigen-binding fragment thereof in the body or cells of a subject.
  • detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; fluorescent proteins, such as GFP. Due to anti-CD73 antibodies or its antigens The specific binding ability of the binding fragment to CD73 can be determined by the amount of the detectable tag connected to the anti-CD73 antibody or its antigen-binding fragment, and thereby the content of CD73 in the sample.
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • GFP fluorescent proteins
  • anti-CD73 antibodies or antigen-binding fragments thereof can be linked to cytokines or therapeutic proteins to form fusion proteins.
  • the specific binding ability of anti-CD73 antibodies or antigen-binding fragments thereof to CD73 can be used to deliver cytokines or therapeutic proteins to specific tissues or cells in a targeted manner to achieve treatment with cytokines or therapeutic proteins. effect.
  • multispecific binding peptides that include at least one domain (or functional unit) that binds CD73 and one or more additional binding domains.
  • the one or more additional binding domains may bind to a second antigen or protein other than CD73.
  • the multispecific antibody is a bispecific antibody.
  • the first antigen-binding domain of the bispecific antibody includes an anti-CD73 antibody or an antigen-binding fragment thereof provided herein and is capable of specifically binding to CD73; the second antigen-binding domain is capable of specifically binding to suppressive immunity.
  • Checkpoint molecules such as those that specifically bind CTLA-4, PD-L1, TIM-3 or LAG-3.
  • the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding CTLA-4.
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the first antigen-binding domain of the bispecific antibody is capable of binding CD73 and the second antigen-binding domain is capable of binding PD-L1 or PD-1.
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding TIM-3.
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding LAG-3.
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the first antigen-binding domain of the bispecific antibody includes an anti-CD73 antibody or an antigen-binding fragment thereof provided herein and is capable of specifically binding to CD73; the second antigen-binding domain is capable of specifically binding to CD39-CD73 -Proteins (enzymes or receptors) in the adenosine pathway.
  • the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding CD39.
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding adenosine 2A receptor (A2AR).
  • A2AR adenosine 2A receptor
  • the first antigen-binding domain of the bispecific antibody is in the form of scFv
  • the second antigen-binding domain is in the form of scFv or sdAb.
  • the bispecific antibody further includes an Fc fragment.
  • the presence of the Fc fragment facilitates multimerization of binding domains and may provide associated effector functions.
  • compositions and methods of treatment are provided.
  • the specific binding ability of the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein to CD73 is similar to that of the control antibody MEDI9447, and the binding ability of some CD73 antibodies or antigen-binding fragments thereof is better than that of the control antibody MEDI9447.
  • the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein have similar inhibitory abilities to CD73 as the control antibody MEDI9447, and some anti-CD73 antibodies or antigen-binding fragments thereof have better inhibitory abilities than the control antibody MEDI9447.
  • anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein can be formulated into pharmaceutical compositions together with pharmaceutically acceptable carriers, and administered to subjects. Administered for tumor prevention or treatment.
  • the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein, as well as fusion proteins or multispecific antibody molecules including the anti-CD73 antibodies or antigen-binding fragments thereof can be combined with one or more other drugs (e.g., anti-CD73 antibodies or antigen-binding fragments thereof). tumor agent) combined administration.
  • the anti-tumor agent can be a small molecule compound, such as a CD39 inhibitor, a CD73 inhibitor, or an A2AR inhibitor.
  • the disease or condition treated is a tumor, preferably a solid tumor.
  • the solid tumor may be metastatic colorectal cancer, hormone-refractory prostate cancer, melanoma, metastatic head and neck cancer, metastatic ovarian cancer, squamous cell carcinoma, etc., especially non-small cell lung cancer (NSCLC), triple-negative breast cancer cancer (TNBC) and pancreatic cancer, etc.
  • NSCLC non-small cell lung cancer
  • TNBC triple-negative breast cancer cancer
  • pancreatic cancer etc.
  • the anti-CD73 antibodies or antigen-binding fragments thereof (and fusion proteins or multispecific antibody molecules comprising the anti-CD73 antibodies or antigen-binding fragments thereof) disclosed herein may be administered using any suitable method or route, and optionally in combination. Other antineoplastic agents. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.
  • Nucleic acid molecules encoding anti-CD73 antibodies or antigen-binding fragments thereof, expression vectors including the nucleic acid molecules, and host cells transfected with the nucleic acid molecules or expression vectors can also be used for the above therapeutic purposes in various ways.
  • the expression vector can be introduced into the subject through gene therapy methods known in the art to express the protein or polypeptide of interest (anti-CD73 antibody or antigen-binding fragment thereof and a fusion protein including anti-CD73 antibody or antigen-binding fragment thereof or multispecific antibody molecules) to achieve therapeutic purposes.
  • the effective dosage for this purpose may depend on the severity of the disease and the overall state of the patient's own immune system. Dosage regimens will also vary with the disease state and subject status, and will generally range from a single bolus dose or continuous infusion to multiple doses per day (eg, every 4-6 hours). Clinicians skilled in the art can readily determine whether a subject is a candidate for such treatment, for example, by utilizing clinical trials, physical examinations, and the subject's family history.
  • anti-CD73 antibodies or antigen-binding fragments thereof provided herein, as well as other forms of molecules including anti-CD73 antibodies or antigen-binding fragments thereof (eg, fusion proteins or bispecific antibody molecules), can specifically bind to CD73 in a sample.
  • detecting the amount of the anti-CD73 antibody or its antigen-binding fragment-CD73 complex formed, or the amount of the anti-CD73 antibody or its antigen-binding fragment in the formed anti-CD73 antibody or its antigen-binding fragment-CD73 complex it is convenient to Determine the amount (or presence) of CD73 in the sample.
  • the anti-CD73 antibodies or antigen-binding fragments thereof provided herein can be linked to various detection tags to facilitate detection by various means, including but not limited to bioluminescence, fluorescence, radioactive labeling , the product production volume of enzymatic reaction, etc.
  • detection tags including but not limited to bioluminescence, fluorescence, radioactive labeling , the product production volume of enzymatic reaction, etc.
  • the anti-CD73 antibodies or antigen-binding fragments thereof provided herein, as well as other forms of molecules including the anti-CD73 antibodies or antigen-binding fragments thereof (e.g., fusion proteins or bispecific antibody molecules), can be placed in a container to form a assay or therapy.
  • Reagent test kit These containers may be in the form of boxes, ampoules, vials, tubes, bags or other suitable containers known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for preserving medications. If required, instructions for use are also provided with the container.
  • Instructions may generally include information on how to use an anti-CD73 antibody, or antigen-binding fragment thereof, in conjunction with a peptide, or a composition comprising an anti-CD73 antibody, or antigen-binding fragment thereof, for the treatment or prevention of tumors (eg, solid tumors), and may include, for example, guidance on therapeutic agents (e.g., anti-CD73 antibodies or antigen-binding fragments thereof); dosage regimens for the treatment or prevention of neoplasia (e.g., multiple myeloma); precautions; warnings; indications; contraindications; adverse reactions; animal pharmacology; Clinical studies; and/or reference materials. Instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate paper, booklet, card or folded print provided in or with the container.
  • therapeutic agents e.g., anti-CD73 antibodies or antigen-binding fragments thereof
  • mice were immunized with human CD73-HIS protein (R&D SYSTEMS, Cat#5795-EN). Dissolve the antigen in PBS solution, or form an emulsion with CFA (complete Freund's adjuvant; primary immunization) or IFA (incomplete Freund's adjuvant; enhanced immunity) for immunization.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • the antigen is administered intraperitoneally or subcutaneously in the back.
  • Each animal received 4 doses (the first immunization was 50 ⁇ g per mouse, then the next three doses were 25 ⁇ g per mouse).
  • spleen cells from selected mice were extracted in a sterile environment and fused with sp2/0 cells following standard hybridoma generation protocols. Fusion cells were cultured in DMEM medium containing 1 ⁇ HAT (hypoxanthine, aminopterin, thymidine) and 10% fetal calf serum for 6 days. In the present invention, a total of 160 96-well plate hybridomas were screened. All hybridoma supernatants were analyzed for their binding ability to human CD73 by ELISA. Anti-human CD73 positive clones were screened based on ELISA signal (OD450).
  • 408 anti-hCD73 hybridoma strains generated from A-J mice (ELISA OD>2.0) and 62 anti-hCD73 hybridoma strains generated from Balb/c mice (ELISA OD>0.3) were selected by ELISA.
  • Flow cytometry was used to detect their binding ability to the CHO-K1 stable cell line overexpressing human CD73.
  • Antibody isotypes were detected (Clonotyping System-HRP, Southern Biotech; Birmingham, AL), antibodies were purified with Protein-A magnetic beads (GenScript, Cat# L00695), eluted with 0.5 M sodium citrate solution (pH 3.5), and eluted with Neutralized with 0.5M Tris-HCl (pH9.0). Change the storage buffer to PBS and determine the concentration using nanodrops (Thermofisher, Cat# ND-ONE-W).
  • Table 1 Binding signals of antibodies and CD73 in 124 monoclonal supernatants measured by ELISA
  • the functional assay of the anti-CD73 mouse antibody is based on the activity of the Calu-6 cell surface CD73 enzyme, which reflects true post-translational modifications in non-small cell lung cancer (NSCLC) cells.
  • NSCLC non-small cell lung cancer
  • Promega kit CellTiter Kit, Catalog #: G7571
  • CD73 on Calu-6 cells can decompose AMP into adenosine and inorganic phosphate, which can enhance luciferase activity and emit light. Therefore, antibodies that block CD73 enzymatic activity will reduce light emission.
  • a total of 124 antibodies at 1 ⁇ g/ml were tested for their ability to antagonize CD73 protein activity.
  • Calu-6 cells ATCC, classification number: HTB-56 TM
  • the original medium was replaced with fresh medium containing anti-CD73 antibodies at different concentrations and added to the corresponding culture plate wells. Incubate the assay plate for 30 minutes at room temperature.
  • AMP solution was added to cells and then incubated at 37°C for 2 hours.
  • CD73 activity ((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100
  • Inhibition rate % (anti-CD73 antibody activity) (1-((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100
  • CHO cells ATCC, classification number: CCL-61 TM ).
  • IC 50 is equivalent to MEDI-9447.
  • the above 11 kinds of mouse antibodies including 9E12E3, 22E1E4, 35D8G8, 52A6B3, 52A8B3, 96A5A5, 100C5A3, 134D2H6, 141G7E9, 143E5D2 and 143H3C5) were sequenced.
  • Table 3 lists the VH, VL and CDR amino acid sequences of these anti-CD73 antibodies.
  • the EC 50 of antibodies 52A6B3, 52A8B3, 96A5A5, 100C5A3 and 134D2H6 is lower than MEDI9447, indicating that their binding ability is better than MEDI9447.
  • the EC 50 of 141G7E9, 143E5D2 and 143H3C5 is higher than MEDI9447.
  • Table 4 lists the EC50 for all antibodies.
  • CDRs, HV loops and FRs were analyzed, and homology modeling was performed to obtain the model structure of the mouse antibody.
  • the solvent-accessible surface area of the backbone residues was calculated, and based on the results, the buried backbone amino acid residues (fast solvent-accessible surface area ⁇ 15%) were determined.
  • a human receptor for VH and VL was chosen that had the same top sequence as the mouse counterpart.
  • the CDRs of the mouse antibody are directly grafted onto the human acceptor framework (Human-IgG1) to obtain the sequence of the grafted antibody without any reverse mutation, in which certain amino acids are reverted to the mouse framework sequence.
  • DNA sequences encoding humanized light and heavy chains were synthesized, introduced into pcDNA3.4, and transfected into Expi293F cells (Genscript Probio, catalog number: SC1975) to produce antibody proteins.
  • Humanized antibodies were compared to syngeneic mouse antibodies by surface plasmon resonance (SPR) using Biacore 8K (Cytiva, Cat#29125379). Briefly, mouse and human antibodies were affinity captured by binding to Protein A via the sensor chip Protein A (Cytiva, Cat#: 29127555). Human CD73CED protein (R&D SYSTEMS, Cat#5795-EN) is in the mobile phase, using a 1:1 binding model for association and dissociation. The association contact time was 120 seconds, the separation contact time was 360 seconds, and the flow rate was 30 ⁇ l/min. The antibody-antigen binding affinity results are shown in Table 6. The binding affinity of the antibody to CD73 after humanization is comparable to that of the mouse antibody.
  • IC 50 half the maximum inhibitory concentration of CD73 protein on Calu-6 cells.
  • the specific method was the same as the in vitro functional test method of anti-CD73 mouse antibody in Example 2, and the measurement results were As shown in Figures 6A and 6B, 35D5G8 and 52A6B3 showed better inhibitory effects on CD73 enzyme activity than MEDI-9447.
  • Table 7 and Table 8 (two batches of experimental results) list the IC 50 values of all humanized anti-CD73 antibodies.
  • humanized 96A5A5 and 52A6B3 performed better than MEDI-9447.
  • IC50 value humanized 143E5D2 lost its ability to inhibit CD73, although its binding affinity to human CD73ECD protein was not reduced.
  • FACS FACS was used to detect the binding ability of the antibody on the CHO-K1 cell line overexpressing human/cynomolgus monkey.
  • the specific method was the same as the FACS detection of mouse antibody and human CD73+/CHO-K1 in Example 2.
  • the secondary antibody was goat anti-human IgG (H+L) labeled with 1 ⁇ g/mL fluorophore (iFluor 647).
  • Figures 7A and 7B show that the detection antibodies are both able to bind to CHO-K1/Human CD73 cells and CHO-K1/Cyno CD73 cells, and exhibit binding abilities that are better than or equivalent to MEDI-9447.

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Abstract

Provided is an anti-CD73 antibody specifically binding to CD73 or an antigen-binding fragment thereof. Also provided is use of the anti-CD73 antibody or the antigen-binding fragment thereof in tumor treatment.

Description

抗CD73抗体或其抗原片段及其应用Anti-CD73 antibodies or antigen fragments thereof and their applications
交叉引用cross reference
本申请要求于2022年08月12日提交中国专利局的申请号为202210965534.2、名称为“抗CD73抗体或其抗原片段及其应用”的中国专利申请的优先权，并将其全部内容通过引用结合在本申请中。This application claims priority to the Chinese patent application with application number 202210965534.2 and titled "Anti-CD73 Antibody or Antigen Fragment and Applications thereof" submitted to the China Patent Office on August 12, 2022, and the entire content of which is incorporated by reference. in this application.
技术领域Technical field
本申请涉及能够与CD73蛋白特异性结合的抗体或其抗原结合片段以及此类制剂的用途。该抗体或其抗原结合片段可用于治疗与CD73活性有关的疾病。The present application relates to antibodies or antigen-binding fragments thereof capable of specifically binding to the CD73 protein and the use of such preparations. The antibody or antigen-binding fragment thereof can be used to treat diseases related to CD73 activity.
背景技术Background technique
腺苷途径能下调炎症并抑制免疫反应以维持免疫稳态。在肿瘤微环境(TME)中，当免疫原性肿瘤细胞死亡时，ATP将被释放到肿瘤TME中，随后ATP通过CD39和CD73蛋白分解成腺苷。丰富的细胞外腺苷具有免疫抑制作用，它通过A2a受体和A2b受体抑制T细胞和NK细胞的扩张和细胞毒性，阻碍树突状细胞的抗原递呈，同时有利于调节性T细胞(调节性T细胞)和骨髓源性抑制细胞(MDSC)的发育(参考文献1)。The adenosine pathway can downregulate inflammation and suppress immune responses to maintain immune homeostasis. In the tumor microenvironment (TME), when immunogenic tumor cells die, ATP will be released into the tumor TME, and ATP is subsequently broken down into adenosine by CD39 and CD73 proteins. Abundant extracellular adenosine has an immunosuppressive effect. It inhibits the expansion and cytotoxicity of T cells and NK cells through A2a receptors and A2b receptors, hinders antigen presentation by dendritic cells, and is beneficial to regulatory T cells ( regulatory T cells) and myeloid-derived suppressor cells (MDSC) (Ref. 1).
细胞外ATP的分解是通过CD39/CD73串联反应介导的，CD39催化ATP转化为AMP，CD73催化AMP转化为腺苷。在实体瘤中检测到的腺苷浓度比周围组织高，表明其在TME和肿瘤进展中抑制免疫反应的作用。细胞表面CD73蛋白和可溶性CD73直接将AMP分解为腺苷，CD73因此成为肿瘤治疗的热点靶点。CD73在缺氧(HIFa)、炎症(TGFβ)和化疗导致的细胞死亡时被诱导表达。它直接响应HIFα信号转导，这可以解释为什么肿瘤细胞在缺氧状态下CD73表达升高(参考文献2)。TGFb信号稳定H1Fα蛋白并间接增强CD73表达。CD73也在Treg、CD4+记忆性T细胞上表达，尤其是被用来标记Th17的表面蛋白。The breakdown of extracellular ATP is mediated through the CD39/CD73 tandem reaction, with CD39 catalyzing the conversion of ATP into AMP and CD73 catalyzing the conversion of AMP into adenosine. Adenosine is detected at higher concentrations in solid tumors than in surrounding tissue, suggesting its role in suppressing immune responses in the TME and tumor progression. Cell surface CD73 protein and soluble CD73 directly decompose AMP into adenosine, making CD73 a hot target for tumor treatment. CD73 expression is induced in response to hypoxia (HIFa), inflammation (TGFβ), and cell death caused by chemotherapy. It responds directly to HIFα signaling, which may explain why CD73 expression is increased in tumor cells under hypoxic conditions (Ref. 2). TGFb signaling stabilizes H1Fα protein and indirectly enhances CD73 expression. CD73 is also expressed on Tregs and CD4+ memory T cells, especially the surface protein used to mark Th17.
在过去的10年中，TME中CD73腺苷途径的免疫抑制受到高度关注。CD73抗体的治疗潜力已在临床前研究(参考文献3)中得到证实，并正在进行临床试验。Olecumab(MEDI9447)是抗CD73单克隆抗体，是临床试验2中的第一个抗CD73抗体药物。在CT26同系动物模型中，Olecumab在体外和体内缓解腺苷介导的免疫抑制。10mg/kg Olecumab可抑制50％的肿瘤生长，并在TIL(肿瘤浸润淋巴细胞)中显示出CD8+细胞毒性T淋巴细胞的大量增加。结合PD-1抗体治疗，Olecumab与PD-1抗体联合给药在小鼠肿瘤排斥反应具有相加效应，能增加T细胞分泌IFNγ以及TNFα。因此在癌症治疗中，将抗CD73抗体与其他免疫检查点阻断剂相结合是可能的。临床一期试验中的其他CD73抗体药物包括百时美施贵宝的BMS-986179、Corvus药物公司(Corvus Pharmaceuticals Inc.)的CPI-006(参考文献4)、Gilead Sciences的双特异型抗体GS-1423和Akeso的AK119，其中大多数 药物在临床试验中结合了其他免疫检查点阻断剂，例如PD-1、PD-L1或CTLA-4(参考文献1)。Over the past 10 years, immunosuppression of the CD73 adenosine pathway in the TME has received great attention. The therapeutic potential of CD73 antibodies has been demonstrated in preclinical studies (Ref. 3) and clinical trials are ongoing. Olecumab (MEDI9447) is an anti-CD73 monoclonal antibody and is the first anti-CD73 antibody drug in clinical trial 2. Olecumab alleviates adenosine-mediated immunosuppression in vitro and in vivo in CT26 syngeneic animal models. Olecumab 10 mg/kg inhibited tumor growth by 50% and showed a large increase in CD8+ cytotoxic T lymphocytes in TIL (tumor-infiltrating lymphocytes). Combined with PD-1 antibody treatment, the combined administration of Olecumab and PD-1 antibody has an additive effect on tumor rejection in mice and can increase the secretion of IFNγ and TNFα by T cells. Therefore, it may be possible to combine anti-CD73 antibodies with other immune checkpoint blockers in cancer treatment. Other CD73 antibody drugs in phase 1 clinical trials include Bristol-Myers Squibb’s BMS-986179, Corvus Pharmaceuticals Inc.’s CPI-006 (Reference 4), Gilead Sciences’ bispecific antibody GS-1423 and Akeso's AK119, most of which The drug is in clinical trials combined with other immune checkpoint blockers, such as PD-1, PD-L1, or CTLA-4 (Reference 1).
发明内容Contents of the invention
在一方面，本文提供了分离的抗体或其抗原结合片段，包括：In one aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, including:
I)重链可变结构域(VH)，包括：I) Heavy chain variable domain (VH), including:
1)重链CDR1(HCDR1)，其包括选自SEQ ID NO:2、10、18、26、34、42、50、58、66、74和82之一的氨基酸序列；1) Heavy chain CDR1 (HCDR1), which includes an amino acid sequence selected from one of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74 and 82;
2)重链CDR2(HCDR2)，其包括选自SEQ ID NO:3、11、19、27、35、43、51、59、67、75和83之一的氨基酸序列；以及2) Heavy chain CDR2 (HCDR2), which includes an amino acid sequence selected from one of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75 and 83; and
3)重链CDR3(HCDR3)，其包括选自SEQ ID NO:4、12、20、28、36、44、52、60、68、76、84和105之一的氨基酸序列；以及3) Heavy chain CDR3 (HCDR3), which includes an amino acid sequence selected from one of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84 and 105; and
II)轻链可变结构域(VL)，包括：II) Light chain variable domain (VL), including:
1)轻链CDR1(LCDR1)，其包括选自SEQ ID NO:6、14、22、30、38、46、54、62、70、78和86之一的氨基酸序列；1) Light chain CDR1 (LCDR1), which includes an amino acid sequence selected from one of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78 and 86;
2)轻链CDR2(LCDR2)，其包括选自SEQ ID NO:7、15、23、31、39、47、55、63、71、79和87之一的氨基酸序列；以及2) Light chain CDR2 (LCDR2), which includes an amino acid sequence selected from one of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79 and 87; and
3)轻链CDR3(LCDR3)，其包括选自SEQ ID NO:8、16、24、32、40、48、56、64、72、80和88之一的氨基酸序列，3) Light chain CDR3 (LCDR3), which includes an amino acid sequence selected from one of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80 and 88,
其中所述抗体或其抗原结合片段能够特异性结合CD73，优选人CD73。Wherein the antibody or antigen-binding fragment thereof can specifically bind to CD73, preferably human CD73.
在一些实施方案中，1)所述VH包括分别具有SEQ ID NO:18、19和20所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:22、23和24所示序列的LCDR1、LCDR2和LCDR3；2)所述VH包括分别具有SEQ ID NO:34、35和36所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:38、39和40所示序列的LCDR1、LCDR2和LCDR3；3)所述VH包括分别具有SEQ ID NO:2、3和4所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:6、7和8所示序列的LCDR1、LCDR2和LCDR3；4)所述VH包括分别具有SEQ ID NO:10、11和12所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:14、15和16所示序列的LCDR1、LCDR2和LCDR3；5)所述VH包括分别具有SEQ ID NO:26、27和28所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:30、31和32所示序列的LCDR1、LCDR2和LCDR3；6)所述VH包括分别具有SEQ ID NO:42、43和44所示序列的HCDR1、HCDR2和HCDR3或分别具有SEQ ID NO:42、43和105所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:46、47和48所示序列的LCDR1、LCDR2和LCDR3；7)所述VH包括分别具有SEQ ID NO:50、51和52所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:54、 55和56所示序列的LCDR1、LCDR2和LCDR3；8)所述VH包括分别具有SEQ ID NO:58、59和60所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:62、63和64所示序列的LCDR1、LCDR2和LCDR3；9)所述VH包括分别具有SEQ ID NO:66、67和68所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:70、71和72所示序列的LCDR1、LCDR2和LCDR3；10)所述VH包括分别具有SEQ ID NO:74、75和76所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:78、79和80所示序列的LCDR1、LCDR2和LCDR3；或11)所述VH包括分别具有SEQ ID NO:82、83和84所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:86、87和88所示序列的LCDR1、LCDR2和LCDR3。In some embodiments, 1) the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 18, 19 and 20, respectively, and the VL includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 22, 23 and 24 respectively. The sequences of LCDR1, LCDR2 and LCDR3; 2) the VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO:34, 35 and 36 respectively, and the VL includes the sequences shown in SEQ ID NO:38, 39 and 40 respectively. LCDR1, LCDR2 and LCDR3 of the sequences shown; 3) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 2, 3 and 4 respectively, and the VL includes SEQ ID NO: 6 and 7 respectively. and LCDR1, LCDR2 and LCDR3 with the sequences shown in 8; 4) The VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 10, 11 and 12 respectively, and the VL includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 10, 11 and 12 respectively, and the VL includes SEQ ID NO: 14 respectively. , LCDR1, LCDR2 and LCDR3 with the sequences shown in 15 and 16; 5) The VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 26, 27 and 28 respectively, and the VL includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO: 26, 27 and 28 respectively, and the VL includes SEQ ID NO. : LCDR1, LCDR2 and LCDR3 of the sequences shown in 30, 31 and 32; 6) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 42, 43 and 44 respectively or having SEQ ID NO: 42 respectively. , HCDR1, HCDR2 and HCDR3 with the sequences shown in 43 and 105. The VL includes LCDR1, LCDR2 and LCDR3 with the sequences shown in SEQ ID NO:46, 47 and 48 respectively; 7) The VH includes the LCDR1, HCDR2 and LCDR3 with the sequences shown in SEQ ID NO:46, 47 and 48 respectively; 7) The VH includes SEQ ID NO. : HCDR1, HCDR2 and HCDR3 of the sequences shown in 50, 51 and 52, the VL includes SEQ ID NO: 54, respectively. LCDR1, LCDR2 and LCDR3 of the sequences shown in 55 and 56; 8) The VH includes HCDR1, HCDR2 and HCDR3 respectively having the sequences shown in SEQ ID NO: 58, 59 and 60, and the VL includes respectively having SEQ ID NO: 58, 59 and 60. LCDR1, LCDR2 and LCDR3 of the sequences shown in 62, 63 and 64; 9) The VH includes HCDR1, HCDR2 and HCDR3 respectively having the sequences shown in SEQ ID NO: 66, 67 and 68, and the VL includes respectively having the sequences of SEQ ID NO: LCDR1, LCDR2 and LCDR3 with the sequences shown in NO:70, 71 and 72; 10) The VH includes HCDR1, HCDR2 and HCDR3 with the sequences shown in SEQ ID NO:74, 75 and 76 respectively, and the VL includes respectively the LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NO:78, 79 and 80; or 11) the VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO:82, 83 and 84 respectively, the VL Included are LCDR1, LCDR2 and LCDR3 having the sequences shown in SEQ ID NO:86, 87 and 88 respectively.
在一些实施方案中，所述VH包括与选自SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的序列有至少80％一致性的氨基酸序列，所述VL包括与选自SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的序列有至少80％一致性的氨基酸序列。In some embodiments, the VH includes an amino acid that is at least 80% identical to a sequence selected from one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, and 81 Sequence, the VL includes an amino acid sequence that is at least 80% identical to a sequence selected from one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85.
在一些实施方案中，所述VH包括选自SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的氨基酸序列或相对于SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的氨基酸序列包括至多3个氨基酸替换的变体，所述VL包括选自SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的氨基酸序列或相对于SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的氨基酸序列包括至多3个氨基酸替换的变体。In some embodiments, the VH includes an amino acid sequence selected from one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81 or relative to SEQ ID NO: 1 The amino acid sequence of one of , 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81 includes variants with up to 3 amino acid substitutions, and the VL includes a variant selected from SEQ ID NO: 5, 13, 21 , the amino acid sequence of one of 29, 37, 45, 53, 61, 69, 77 and 85 or relative to one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85 An amino acid sequence includes variants with up to 3 amino acid substitutions.
在一些实施方案中，1)所述VH包括SEQ ID NO:17所示氨基酸序列，所述VL包括SEQ ID NO:21所示氨基酸序列；2)所述VH包括SEQ ID NO:33所示氨基酸序列，所述VL包括SEQ ID NO:37所示氨基酸序列；3)所述VH包括SEQ ID NO:1所示氨基酸序列，所述VL包括SEQ ID NO:5所示氨基酸序列；4)所述VH包括SEQ ID NO:9所示氨基酸序列，所述VL包括SEQ ID NO:13所示氨基酸序列；5)所述VH包括SEQ ID NO:25所示氨基酸序列，所述VL包括SEQ ID NO:29所示氨基酸序列；6)所述VH包括SEQ ID NO:41所示氨基酸序列，所述VL包括SEQ ID NO:45所示氨基酸序列；7)所述VH包括SEQ ID NO:49所示氨基酸序列，所述VL包括SEQ ID NO:53所示氨基酸序列；8)所述VH包括SEQ ID NO:57所示氨基酸序列，所述VL包括SEQ ID NO:61所示氨基酸序列；9)所述VH包括SEQ ID NO:65所示氨基酸序列，所述VL包括SEQ ID NO:69所示氨基酸序列；10)所述VH包括SEQ ID NO:73所示氨基酸序列，所述VL包括SEQ ID NO:77所示氨基酸序列；或11)所述VH包括SEQ ID NO:81所示氨基酸序列，所述VL包括SEQ ID NO:85所示氨基酸序列。In some embodiments, 1) the VH includes the amino acid sequence shown in SEQ ID NO: 17, and the VL includes the amino acid sequence shown in SEQ ID NO: 21; 2) the VH includes the amino acid sequence shown in SEQ ID NO: 33 Sequence, the VL includes the amino acid sequence shown in SEQ ID NO: 37; 3) the VH includes the amino acid sequence shown in SEQ ID NO: 1, the VL includes the amino acid sequence shown in SEQ ID NO: 5; 4) the VH includes the amino acid sequence shown in SEQ ID NO: 9, and the VL includes the amino acid sequence shown in SEQ ID NO: 13; 5) The VH includes the amino acid sequence shown in SEQ ID NO: 25, and the VL includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 29; 6) the VH includes the amino acid sequence shown in SEQ ID NO: 41, the VL includes the amino acid sequence shown in SEQ ID NO: 45; 7) the VH includes the amino acid sequence shown in SEQ ID NO: 49 Sequence, the VL includes the amino acid sequence shown in SEQ ID NO: 53; 8) the VH includes the amino acid sequence shown in SEQ ID NO: 57, the VL includes the amino acid sequence shown in SEQ ID NO: 61; 9) the VH includes the amino acid sequence shown in SEQ ID NO:65, and the VL includes the amino acid sequence shown in SEQ ID NO:69; 10) The VH includes the amino acid sequence shown in SEQ ID NO:73, and the VL includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 77; or 11) the VH includes the amino acid sequence shown in SEQ ID NO:81, and the VL includes the amino acid sequence shown in SEQ ID NO:85.
在一些实施方案中，所述抗体或其抗原结合片段针对细胞表面表达的所述CD73的酶活性具有10-1μg/mL至10-3μg/mL的IC50值。 In some embodiments, the enzymatic activity of the antibody or antigen-binding fragment thereof against the CD73 expressed on the cell surface has an IC50 value of 10 −1 μg/mL to 10 −3 μg/mL.
在一些实施方案中，所述抗体或其抗原结合片段与表达所述CD73的细胞结合的EC50值为1μg/mL至10-2μg/mL。In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 value for binding to cells expressing the CD73 of 1 μg/mL to 10 −2 μg/mL.
在一些实施方案中，所述抗体为鼠抗体、嵌合抗体、人源化抗体或人抗体。In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
在一些实施方案中，所述抗体或其抗原结合片段为单克隆抗体、scFv、Fab、Fab’、(Fab’)2或Fv。In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, scFv, Fab, Fab', (Fab') 2 , or Fv.
在一些实施方案中，所述人源化抗体的VH包括选自SEQ ID NO:91、93、89、95、97、99或101的氨基酸序列，所述人源化抗体的VL包括选自SEQ ID NO:92、94、90、96、98、100或102的氨基酸序列。In some embodiments, the VH of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 91, 93, 89, 95, 97, 99 or 101, and the VL of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 91, 93, 89, 95, 97, 99 or 101. ID NO: 92, 94, 90, 96, 98, 100 or 102 amino acid sequence.
在一些实施方案中，1)所述VH包括SEQ ID NO:91所示氨基酸序列和所述VL包括SEQ ID NO:92所示氨基酸序列；2)所述VH包括SEQ ID NO:93所示氨基酸序列和所述VL包括SEQ ID NO:94所示氨基酸序列；3)所述VH包括SEQ ID NO:89所示氨基酸序列和所述VL包括SEQ ID NO:90所示氨基酸序列；4)所述VH包括SEQ ID NO:95所示氨基酸序列和所述VLSEQ ID NO:96所示氨基酸序列；5)所述VH包括SEQ ID NO:97所示氨基酸序列和所述VL包括SEQ ID NO:98所示氨基酸序列；6)所述VH包括SEQ ID NO:99所示氨基酸序列和所述VL包括SEQ ID NO:100所示氨基酸序列；或7)所述VH包括SEQ ID NO:101所示氨基酸序列和所述VL包括SEQ ID NO:102所示氨基酸序列。In some embodiments, 1) the VH includes the amino acid sequence shown in SEQ ID NO:91 and the VL includes the amino acid sequence shown in SEQ ID NO:92; 2) the VH includes the amino acid sequence shown in SEQ ID NO:93 The sequence and the VL include the amino acid sequence shown in SEQ ID NO: 94; 3) the VH includes the amino acid sequence shown in SEQ ID NO: 89 and the VL includes the amino acid sequence shown in SEQ ID NO: 90; 4) the VH includes the amino acid sequence shown in SEQ ID NO:95 and the amino acid sequence shown in the VLSEQ ID NO:96; 5) the VH includes the amino acid sequence shown in SEQ ID NO:97 and the VL includes the amino acid sequence shown in SEQ ID NO:98 The amino acid sequence is shown; 6) the VH includes the amino acid sequence shown in SEQ ID NO: 99 and the VL includes the amino acid sequence shown in SEQ ID NO: 100; or 7) the VH includes the amino acid sequence shown in SEQ ID NO: 101 And the VL includes the amino acid sequence shown in SEQ ID NO:102.
在一些实施方案中，所述人源化抗体针对表达所述细胞表面表达的所述CD73的酶活性具有10-1μg/mL至10-2μg/mL的IC50值。In some embodiments, the enzymatic activity of the humanized antibody against the CD73 expressing the cell surface expression has an IC50 value of 10 −1 μg/mL to 10 −2 μg/mL.
在一些实施方案中，所述抗体或其抗原结合片段与所述CD73之间结合的KD值为10- 8M至10-11M。In some embodiments, the KD value for binding between the antibody or antigen-binding fragment thereof and the CD73 is 10 −8 M to 10 −11 M.
在一些实施方案中，所述VH和/或VL连接至免疫球蛋白的恒定区；优选地，所述VH连接至IgG1恒定区。In some embodiments, the VH and/or VL are linked to the constant region of an immunoglobulin; preferably, the VH is linked to the IgGl constant region.
另一方面，本文提供了双特异性抗体，其上述抗体或其抗原结合片段和第二抗体部分。In another aspect, provided herein are bispecific antibodies, the aforementioned antibodies or antigen-binding fragments thereof and a second antibody portion.
在一些实施方案中，所述第二抗体部分能够结合不同于CD73的抗原。In some embodiments, the second antibody portion is capable of binding an antigen other than CD73.
在一些实施方案中，所述不同于CD73的抗原为CD39、CTLA-4、PD-L1、TIM-3、LAG-3或A2aR。In some embodiments, the antigen other than CD73 is CD39, CTLA-4, PD-L1, TIM-3, LAG-3, or A2aR.
在一些实施方案中，所述第二抗体部分为Fab、Fab’、(Fab’)2、Fv、scFv或sdAb。In some embodiments, the second antibody moiety is Fab, Fab', (Fab') 2 , Fv, scFv, or sdAb.
另一方面，本文提供了编码权利要求上述抗体或其抗原结合片段或双特异性抗体的多核苷酸。In another aspect, provided herein are polynucleotides encoding the above-claimed antibodies or antigen-binding fragments thereof or bispecific antibodies.
另一方面，本文提供了上述多核苷酸的载体。In another aspect, provided herein are vectors for the above polynucleotides.
另一方面，本文提供了包含上述多核苷酸或载体的细胞。In another aspect, provided herein are cells comprising the polynucleotides or vectors described above.
另一方面，本文提供了用于癌症治疗的药物组合物，包括：上述抗体或其抗原结合片段，或上述双特异性抗体；以及药学上可接受的载体。On the other hand, this article provides a pharmaceutical composition for cancer treatment, including: the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned bispecific antibody; and a pharmaceutically acceptable carrier.
在一些实施方案中，所述药物组合物还包括一种或更多种其他抗癌剂。 In some embodiments, the pharmaceutical composition further includes one or more other anti-cancer agents.
在一些实施方案中，所述癌症为实体癌。In some embodiments, the cancer is a solid cancer.
在一些实施方案中，所述癌症选自非小细胞肺癌(NSCLC)、三阴性乳腺癌(TNBC)或胰腺癌。In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), or pancreatic cancer.
另一方面，本文提供了包含上述抗体或其抗原结合片段、双特异性抗体、多核苷酸、载体或细胞在制备用于治疗癌症的药物中的用途。In another aspect, provided herein is the use of the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, polynucleotide, vector or cell in the preparation of a medicament for treating cancer.
在一些实施方案中，所述癌症为实体癌。In some embodiments, the cancer is a solid cancer.
在一些实施方案中，所述癌症选自非小细胞肺癌、三阴性乳腺癌或胰腺癌。In some embodiments, the cancer is selected from non-small cell lung cancer, triple negative breast cancer, or pancreatic cancer.
再一方面，本文提供一种治疗疾病的方法，包括向有需要的受试者施用治疗有效量的上文所述的抗体或其抗原结合片段、双特异性抗体或上述药物组合物。In yet another aspect, provided herein is a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody or antigen-binding fragment thereof, a bispecific antibody or the above-mentioned pharmaceutical composition.
在一些实施方案中，所述受试者为人。In some embodiments, the subject is a human.
在一些实施方案中，所述疾病为癌症。在一些优选实施方案中，所述疾病为实体癌。在更有选的实施方案中，所述实体癌选自非小细胞肺癌(NSCLC)、三阴性乳腺癌(TNBC)或胰腺癌。In some embodiments, the disease is cancer. In some preferred embodiments, the disease is solid cancer. In a more preferred embodiment, the solid cancer is selected from non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), or pancreatic cancer.
在本发明中，我们鉴定了11种抗体，8种抗体对CD73胞外-5'-核苷酸酶的拮抗能力优于MEDI9447，并且在多表位靶向方面具有高度多样性。根据CD73在多种肿瘤组织中的高表达水平(参考文献5，参考文献6)，这些抗体先导物在肿瘤治疗中具有很高的潜力，具有广泛的适应症，包括非小细胞肺癌(NSCLC)、三阴性乳腺癌(TNBC)和胰腺癌等。同时，抗CD73抗体在联合治疗中具有高度的灵活性，包括PD-1、PD-L1、化疗、A2AR拮抗剂等。本发明的人源化抗体与人和食蟹猴的CD73都可以结合，方便了临床及临床前的研究。In the present invention, we identified 11 antibodies, 8 of which had better antagonistic abilities against CD73 extracellular-5'-nucleotidase than MEDI9447 and were highly diverse in multi-epitope targeting. Based on the high expression levels of CD73 in various tumor tissues (Ref. 5, Ref. 6), these antibody leads have high potential in tumor therapy with a wide range of indications, including non-small cell lung cancer (NSCLC). , triple negative breast cancer (TNBC) and pancreatic cancer, etc. At the same time, anti-CD73 antibodies are highly flexible in combination treatments, including PD-1, PD-L1, chemotherapy, A2AR antagonists, etc. The humanized antibody of the present invention can bind to both human and cynomolgus CD73, which facilitates clinical and preclinical research.
本发明根据CD73在免疫调节中的功能和肿瘤细胞中CD73的高表达以及高腺苷浓度，通过抗CD73抗体阻断CD73酶活性可减轻腺苷途径对肿瘤微环境的免疫抑制，有利于T细胞和NK细胞对肿瘤的特异性杀伤作用。Based on the function of CD73 in immune regulation and the high expression of CD73 and high adenosine concentration in tumor cells, the present invention blocks CD73 enzyme activity through anti-CD73 antibodies, which can alleviate the immunosuppression of the tumor microenvironment by the adenosine pathway and is beneficial to T cells. and the specific killing effect of NK cells on tumors.
在本发明中，我们鉴定出的抗体对CD73胞外-5'-核苷酸酶的拮抗能力优于MEDI9447，并且在多表位靶向方面具有高度多样性。这些抗体先导物可以为抗体治疗、双特异性抗体设计以及与化疗和放疗的联合治疗提供良好的候选物。In the present invention, the antibody we identified has better antagonistic ability to CD73 extracellular-5'-nucleotidase than MEDI9447 and is highly diverse in multi-epitope targeting. These antibody leads may provide good candidates for antibody therapy, bispecific antibody design, and combination therapy with chemotherapy and radiotherapy.
附图说明Description of drawings
图1：52A6B3和100C5A3对AMP水解的抑制作用。Figure 1: Inhibition of AMP hydrolysis by 52A6B3 and 100C5A3.
图2：52A8B3、96A5A5和134D2H6对AMP水解的抑制作用。Figure 2: Inhibition of AMP hydrolysis by 52A8B3, 96A5A5 and 134D2H6.
图3：141G7E9、143H3C5和143E5D2对AMP水解的抑制作用。Figure 3: Inhibition of AMP hydrolysis by 141G7E9, 143H3C5 and 143E5D2.
图4：9E12E3、22E1B4和35D8G8对AMP水解的抑制作用。Figure 4: Inhibition of AMP hydrolysis by 9E12E3, 22E1B4 and 35D8G8.
图5：抗CD73小鼠单克隆抗体结合人CD73-CHO-K1细胞系的FACS数据。 Figure 5: FACS data of anti-CD73 mouse monoclonal antibody binding to the human CD73-CHO-K1 cell line.
图6：7种人源化抗体对CD73介导的AMP水解的抑制作用，(A)为5种人源化抗体对CD73介导的AMP水解的抑制作用；(B)为另2种人源化抗体对CD73介导的AMP水解的抑制作用。Figure 6: The inhibitory effect of 7 humanized antibodies on CD73-mediated AMP hydrolysis. (A) shows the inhibitory effect of 5 humanized antibodies on CD73-mediated AMP hydrolysis; (B) shows the other 2 humanized antibodies. Antibodies inhibit CD73-mediated AMP hydrolysis.
图7：一些实施例中抗CD73抗体结合CD73的FACS数据：图A)显示了抗体对人CD73的结合能力；图B)显示了抗体对食蟹猴CD73的结合能力。Figure 7: FACS data of anti-CD73 antibodies binding to CD73 in some embodiments: Panel A) shows the binding ability of the antibody to human CD73; Panel B) shows the binding ability of the antibody to cynomolgus monkey CD73.
图8：一些实施例中抗体与MEDI19447的表位鉴定。Figure 8: Epitope identification of antibodies and MEDI19447 in some examples.
具体实施方式Detailed ways
本公开提供了抗CD73单克隆抗体及其应用。本公开涉及小鼠抗CD73单克隆抗体(9E12E3、22E1E4、35D8G8、52A8B3、52A6B3、96A5A5、100C5A3、134D2H6、141G7E9、143E5D2和143H3C5克隆)的重链可变结构域(VH)和轻链可变结构域(VL)的氨基酸序列。它还涉及到在这些小鼠抗CD73单克隆抗体克隆上进行人源化或翻译后修饰后的重链可变结构域(VH)和轻链可变结构域(VL)的氨基酸序列。本公开还涉及到抗CD73单克隆抗体的产生方法。The present disclosure provides anti-CD73 monoclonal antibodies and uses thereof. This disclosure relates to heavy chain variable domains (VH) and light chain variable structures of mouse anti-CD73 monoclonal antibodies (9E12E3, 22E1E4, 35D8G8, 52A8B3, 52A6B3, 96A5A5, 100C5A3, 134D2H6, 141G7E9, 143E5D2 and 143H3C5 clones) Amino acid sequence of domain (VL). It also relates to the amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) that have been humanized or post-translationally modified on these mouse anti-CD73 monoclonal antibody clones. The present disclosure also relates to methods of producing anti-CD73 monoclonal antibodies.
本公开通过将所披露的小鼠抗CD73单克隆抗体的重链和轻链的可变区与人IgG的恒定区融合，提供抗人CD73嵌合抗体。本发明提供了小鼠抗CD73单克隆抗体的重链可变结构域(VH)和轻链可变结构域(VL)的人源化形式。在一些例子中，小鼠抗CD73单克隆抗体的CDR被嫁接到人IgG框架序列上，所获得的人源化可变结构域还可以进一步与人IgG的恒定区域融合。The present disclosure provides anti-human CD73 chimeric antibodies by fusing the variable regions of the heavy and light chains of the disclosed mouse anti-CD73 monoclonal antibodies with the constant regions of human IgG. The present invention provides humanized versions of the heavy chain variable domain (VH) and light chain variable domain (VL) of mouse anti-CD73 monoclonal antibodies. In some examples, the CDRs of mouse anti-CD73 monoclonal antibodies are grafted onto human IgG framework sequences, and the resulting humanized variable domains can be further fused to the constant regions of human IgG.
定义definition
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
术语“或”是指列举的可选择要素中的单个要素，除非上下文明确地另外指出。术语“和/或”是指所列举的可选择要素中的任意一个、任意两个、任意三个、任意更多个或其全部。The term "or" refers to a single element of a listed alternative element, unless the context clearly dictates otherwise. The term "and/or" refers to any one, any two, any three, any more or all of the listed optional elements.
本文所用术语“约”表示给定数值的±10％范围内的值。As used herein, the term "about" means a value within ±10% of a given numerical value.
本文所用术语“包含”、“含有”、“具有”以及类似的表述表示不排除未列举的要素。这些术语也包括仅由所列举的要素组成的情形。As used herein, the terms "comprises," "contains," "having," and similar expressions mean that non-recited elements are not excluded. These terms also include instances that consist solely of the recited elements.
“抗体”指由浆细胞(效应B细胞)分泌、被机体免疫***用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。经典抗体分子的基本结构是由2个相同重链和2个相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。在可变区中，某些区域氨基酸残基的组成和排列次序比可变区内的其它区域(骨架区，FR)更易变化，称为高变区(HVR)，高变区实际上是抗体与 抗原结合的关键部位。由于这些高变区序列与抗原决定簇互补，故又称为互补决定区(complementarity-determining region、CDR)。重链和轻链均具有三个互补决定区，分别称为HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3。CDR的氨基酸序列可以使用本领域公认的编号方案来确定，例如Kabat、Chothia、IMGT、AbM或Contact编号方案。在一个具体实施方案中，已经根据Kabat编号方案确定了本文所述的抗体的CDR序列。根据抗体重链恒定区的氨基酸序列，可将抗体分为五种主要的不同类型：IgA、IgD、IgE、IgG和IgM。这些抗体类型根据铰链区的大小，链间二硫键的位置和分子量的不同可进一步分为亚类，例如，IgGl、IgG2a、IgG2b和IgG3等。根据抗体轻链恒定区氨基酸组成和排列的不同，可将轻链分为κ和λ两种类型。不同类别的免疫球蛋白的亚单位结构和三维构象在本领域内是已知的。"Antibodies" refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen. The basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. According to the conservative differences in amino acid sequences, heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. The variable regions of a heavy chain and a light chain interact to form the antigen-binding site (Fv). In the variable region, the composition and order of amino acid residues in certain regions are more variable than other regions (backbone regions, FR) within the variable region, which are called hypervariable regions (HVR). The hypervariable regions are actually antibodies. and Key site for antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both heavy and light chains have three complementarity determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively. The amino acid sequences of the CDRs can be determined using art-recognized numbering schemes, such as the Kabat, Chothia, IMGT, AbM, or Contact numbering schemes. In a specific embodiment, the CDR sequences of the antibodies described herein have been determined according to the Kabat numbering scheme. Antibodies can be divided into five main different types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. These antibody types can be further divided into subclasses based on the size of the hinge region, the position of the interchain disulfide bond, and the molecular weight, such as IgGl, IgG2a, IgG2b, and IgG3. According to the different amino acid composition and arrangement of the constant region of the antibody light chain, the light chain can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.
抗体分子的“抗原结合片段”指抗体分子中参与抗原特异性结合的氨基酸片段，例如，Fab、Fab’、(Fab’)2、scFv和sdAb等。本领域技术人员已知如何获得这些抗原结合片段。例如，经典抗体分子可经木瓜蛋白酶消化而得到Fab片段，经胃蛋白酶消化得到F(ab’)2，通过以还原剂处理断开F(ab’)2铰链区之间的二硫键而形成Fab’片段。The "antigen-binding fragment" of an antibody molecule refers to the amino acid fragment in the antibody molecule that participates in specific antigen binding, such as Fab, Fab', (Fab') 2 , scFv, sdAb, etc. The person skilled in the art knows how to obtain these antigen-binding fragments. For example, a classical antibody molecule can be digested with papain to obtain the Fab fragment, and pepsin digested to obtain F(ab') 2 , which is formed by treatment with a reducing agent to break the disulfide bond between the hinge regions of F(ab') 2 Fab' fragment.
单链抗体(single chain fragment variable、scFv)，是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。Single chain fragment variable (scFv) is composed of an antibody heavy chain variable region and a light chain variable region connected through a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and light chain form Fv segments through non-covalent interactions, so scFv can better retain its affinity activity for antigens.
“单域抗体(single domain antibody，sdAb)”，或者也称为“VHH抗体”，指具有抗原结合能力，包括重链可变区而无轻链的抗体分子。从结构上看，单域抗体也可以认为是抗体分子的一种抗原结合片段。其首先在骆驼科动物中被发现，随后，研究人员通过抗体库(例如噬菌体展示文库)筛选发现了更多的具有抗原结合能力的单域抗体。单域抗体相对于普通抗体分子(例如，经典四聚体抗体分子)或其抗原结合片段具有一些优势，例如包括但不限于：分子量更小，使用于人体时易于到达普通抗体分子难以到达的组织或部位，或者，能够接触到蛋白或多肽中普通抗体分子难以接触到的抗原表位；更加稳定，能够耐受例如温度和pH的变化以及变性剂和蛋白酶的作用。"Single domain antibody (sdAb)", also known as "VHH antibody", refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region but no light chain. From a structural point of view, single domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids. Subsequently, researchers discovered more single-domain antibodies with antigen-binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, and when used in the human body, they can easily reach tissues that are difficult for ordinary antibody molecules to reach. or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; they are more stable and can withstand changes in temperature and pH, as well as the effects of denaturants and proteases.
“Fc片段”指Y”形抗体分子的柄部区域，即可结晶片段(fragment crystallizable，Fc)包括重链的第二和第三恒定结构域(CH2和CH3结构域)。可通过蛋白水解酶(如木瓜蛋白酶)水解抗体分子得到抗体Fc区。在一些实例中，Fc区可包含铰链、CH2和CH3。当Fc区包含铰链时可介导两个含Fc的多肽之间的二聚化。Fc片段可来自IgG、IgM、IgD、IgE或IgA。在一些实例中，Fc区来自IgG1、IgG2、IgG3或IgG4。“Fc片段”还包括来自天然Fc片段，经改动但仍保持其效应功能的变体Fc片段。“变体Fc片段”包含在天然Fc片段的氨基酸序列上具有至少一个氨基酸变动的氨基酸序列。在一些实例中，变体Fc片段相比于亲本Fc片段(天然Fc片段)具有至少一个氨基酸取代，例如在亲本Fc片段中约1至约10个氨基酸被取代，且优选约1至约5个氨基酸取代。在一些实例中，变体Fc片段Fc区与亲本Fc片段具有至少约80％序列一致性、至少约90％序列一致性、至少约95％、至少 约96％、至少约97％、至少约98％或至少约99％序列一致性。“Fc片段”的效应功能可包括与Fc受体的结合、Clq结合和补体依赖性细胞毒性(CDC)、抗体依赖性细胞介导的细胞毒性(ADCC)、介导吞噬作用等。在一些情形下，可视情况下对Fc片段进行改造，一以增强、减弱或消除其效应功能。"Fc fragment" refers to the handle region of a Y"-shaped antibody molecule, that is, the crystallizable fragment (fragment crystallizable, Fc) includes the second and third constant domains (CH2 and CH3 domains) of the heavy chain. It can be passed through proteolytic enzymes (such as papain) hydrolyzes the antibody molecule to obtain the antibody Fc region. In some examples, the Fc region can include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment can be from IgG, IgM, IgD, IgE, or IgA. In some examples, the Fc region is from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes fragments from native Fc fragments that have been altered but still retain their effector function. Variant Fc fragment. A "variant Fc fragment" includes an amino acid sequence that has at least one amino acid change in the amino acid sequence of a native Fc fragment. In some examples, a variant Fc fragment has At least one amino acid substitution, for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some examples, the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least About 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. Effector functions of "Fc fragments" may include binding to Fc receptors, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, etc. In some cases, the Fc fragment may be modified to enhance, weaken, or eliminate its effector function.
“鼠抗体”指可变区和恒定区(如果有的话)衍生自小鼠或大鼠免疫球蛋白序列的抗体。鼠抗体可方便地以相应抗原免疫小鼠或大鼠并从其分离目的抗体而获得。或者，在以相应抗原免疫小鼠或大鼠后，分离并培养表达目的抗体的细胞(如B细胞)而获得。又或者，在以相应抗原免疫小鼠或大鼠后，分离并培养表达目的抗体的细胞，将其与永生化细胞如骨髓瘤细胞融合而获得杂交瘤细胞，培养杂交瘤细胞则可长期和大量获得目的抗体(如单克隆抗体)。在一些实施方案中，所述“鼠抗体”是小鼠抗体。"Murine antibody" refers to an antibody in which the variable and constant regions, if any, are derived from mouse or rat immunoglobulin sequences. Mouse antibodies can be conveniently obtained by immunizing mice or rats with corresponding antigens and isolating the antibodies of interest therefrom. Alternatively, it can be obtained by immunizing mice or rats with the corresponding antigen and then isolating and culturing cells (such as B cells) expressing the antibody of interest. Or, after immunizing mice or rats with the corresponding antigen, isolate and culture the cells expressing the target antibody, and fuse them with immortalized cells such as myeloma cells to obtain hybridoma cells. The hybridoma cells can be cultured for a long time and in large quantities. Obtain the target antibody (such as monoclonal antibody). In some embodiments, the "murine antibody" is a mouse antibody.
“人源化抗体”是指对非人抗体，即可变区和恒定区(如果有的话)非衍生自人免疫球蛋白的抗体进行人为改造以使其含有人抗体的氨基酸序列，由此获得的嵌合抗体。人源化抗体可以包含人抗体的恒定区和/或骨架区。人源化抗体可以通过基因工程手段获得，例如以人抗体的恒定区替换鼠抗体的恒定区和/或以人抗体的骨架区替换鼠抗体的骨架区。这种人源化改造通常不影响原抗体与对应抗原的结合特异性，因此这样的抗原也包括在本发明的范围内。"Humanized antibody" refers to a non-human antibody, that is, an antibody in which the variable and constant regions (if any) are not derived from human immunoglobulins, have been artificially modified so that they contain the amino acid sequence of a human antibody, thereby Obtained chimeric antibodies. Humanized antibodies may comprise the constant regions and/or framework regions of human antibodies. Humanized antibodies can be obtained by genetic engineering means, for example, replacing the constant region of a murine antibody with a human antibody's constant region and/or replacing the mouse antibody's framework region with a human antibody's framework region. This humanization transformation usually does not affect the binding specificity of the original antibody to the corresponding antigen, so such antigens are also included in the scope of the present invention.
“人抗体”或“人源抗体”指可变区和恒定区(如果有的话)均衍生自人生殖系免疫球蛋白序列的抗体。人抗体可通过多种技术获得，包括噬菌体抗体库技术、单个B细胞克隆技术、转基因小鼠技术(例如，使用引入了人生殖系免疫球蛋白基因并去除了小鼠自身生殖系免疫球蛋白基因的转基因小鼠)等。相对于动物源抗体(例如鼠源抗体)，人抗体在用于人患者时具有免疫原性小、安全性高的优势。"Human antibody" or "humanized antibody" refers to an antibody in which both the variable and constant regions (if any) are derived from human germline immunoglobulin sequences. Human antibodies can be obtained through a variety of techniques, including phage antibody library technology, single B cell cloning technology, and transgenic mouse technology (for example, using the introduction of human germline immunoglobulin genes and the removal of the mouse's own germline immunoglobulin genes). transgenic mice), etc. Compared with animal-derived antibodies (such as mouse-derived antibodies), human antibodies have the advantages of low immunogenicity and high safety when used in human patients.
本文使用的术语“单克隆抗体”指均一的、仅针对某一特定抗原表位的抗体。与典型地包括针对不同抗原决定簇(表位)的不同抗体的多克隆抗体相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体可通过本领域公知的杂交瘤方法或重组DNA方法产生，或通过本文其它地方描述的筛选方法获得。The term "monoclonal antibody" as used herein refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" indicates the uniform character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method. The monoclonal antibodies of the invention can be produced by hybridoma methods or recombinant DNA methods well known in the art, or obtained by screening methods described elsewhere herein.
“分离的”抗体是已经从其自然环境或生产环境(例如细胞培养物)中被分离和/或回收的抗体。生产环境中的污染成分，如来自重组转染细胞的成分(例如酶、激素和其他蛋白或非蛋白组分、核酸)通常会干扰抗体的研究、诊断或治疗用途。因此，优选地，该分离的抗体基本上与其自然环境或生产环境中的其他成分没有关联。An "isolated" antibody is an antibody that has been isolated and/or recovered from its natural or production environment (eg, cell culture). Contaminating components in the production environment, such as components from recombinantly transfected cells (e.g. enzymes, hormones and other protein or non-protein components, nucleic acids), often interfere with the research, diagnostic or therapeutic use of antibodies. Therefore, preferably, the isolated antibody is substantially free of association with other components of its natural or production environment.
“表位”指分子(如抗原)中与抗体结合的分子部分。表位可以包含该分子的非相邻的部分(例如，多肽中，在该多肽的主要序列上不相邻、但在该多肽的三价和四价结构中彼此足够靠近以被抗体结合的氨基酸残基)。An "epitope" refers to the portion of a molecule (eg, an antigen) to which an antibody binds. An epitope may comprise a non-contiguous portion of the molecule (e.g., amino acids in a polypeptide that are not contiguous in the main sequence of the polypeptide but are sufficiently close to each other in the trivalent and tetravalent structure of the polypeptide to be bound by an antibody Residues).
“双特异性抗体”指具有两个不同的结合位点、能分别识别和结合两种不同的抗原的抗体分子。在一个实施方案中，双特异性抗体的一个结合位点可用于结合免疫细胞(例如 T细胞)，另一个结合位点用于结合肿瘤细胞，进而增强免疫细胞对肿瘤细胞的杀伤作用，同时减少脱靶毒性等副作用。在另一实施方案中，双特异性抗体的两个结合位点分别结合与肿瘤微环境与免疫抑制有关的蛋白或抗原(如CD73和PD-L1)。这种具有双功能的抗体作为***的药物通常比单抗药物有更高的疗效。类似地，可对抗体分子进行改造以使其包括多个不同的结合位点，生成“三特异性抗体”、“四特异性抗体”等。本文使用的“多特异性抗体”涵盖这些双特异性抗体、三特异性抗体、四特异性抗体等。"Bispecific antibodies" refer to antibody molecules that have two different binding sites and can recognize and bind to two different antigens respectively. In one embodiment, one binding site of the bispecific antibody can be used to bind immune cells (e.g. T cells), another binding site is used to bind tumor cells, thereby enhancing the killing effect of immune cells on tumor cells while reducing side effects such as off-target toxicity. In another embodiment, the two binding sites of the bispecific antibody respectively bind to proteins or antigens related to the tumor microenvironment and immunosuppression (such as CD73 and PD-L1). This bifunctional antibody usually has higher efficacy than monoclonal antibody drugs as a tumor treatment drug. Similarly, antibody molecules can be engineered to include multiple different binding sites, creating "trispecific antibodies,""tetraspecificantibodies," etc. The "multispecific antibody" used in this article covers these bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.
“融合蛋白”指人为生成(例如通过基因工程技术)的由至少两个不同肽段构成的蛋白分子。这些肽段在自然界中不存在，或者不存在于同一个蛋白分子中。常见的包括抗体片段的融合蛋白的实例包括嵌合抗体、抗体-细胞因子融合蛋白、抗体-细胞毒素融合蛋白(也称为免疫毒素)、用于免疫检测的酶标抗体、嵌合抗原受体(CAR)等。"Fusion protein" refers to a protein molecule composed of at least two different peptide segments that is artificially produced (for example, through genetic engineering technology). These peptides do not exist in nature or do not exist in the same protein molecule. Common examples of fusion proteins including antibody fragments include chimeric antibodies, antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also known as immunotoxins), enzyme-labeled antibodies for immunoassays, and chimeric antigen receptors (CAR) etc.
“靶向”或“特异性结合”指，相对于环境中同时存在的其他分子，一种分子(例如抗体或其抗原结合片段)对另一种分子(如肿瘤细胞表面抗原)具有更高的结合亲和力。“靶向”或“特异性结合”并不排除该分子可以对一种以上的分子具有结合亲和力，例如双特异性抗体可以对两种不同抗原具有高亲和力。"Targeting" or "specific binding" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher affinity for another molecule (e.g., a tumor cell surface antigen) relative to other molecules also present in the environment. Binding affinity. "Targeting" or "specific binding" does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.
EC50(concentration for 50％of maximal effect)指引起50％最大效应的浓度。在FACS中用于表示抗体分子与细胞上对应抗原的结合能力时，可指产生最大荧光强度一半时的抗体分子浓度。EC50值越低，则与细胞上抗原的结合亲和力越大。EC 50 (concentration for 50% of maximal effect) refers to the concentration that causes 50% of the maximum effect. When used in FACS to express the binding ability of an antibody molecule to the corresponding antigen on a cell, it can refer to the concentration of the antibody molecule that produces half of the maximum fluorescence intensity. The lower the EC50 value, the greater the binding affinity to the antigen on the cell.
KD值也可以用于衡量抗体与其抗原之间的结合亲和力。KD值是抗体与其抗原之间的平衡解离常数，即koff/kon的比值。因此KD值越低(浓度越低)，抗体的亲和力越高。KD values can also be used to measure the binding affinity between an antibody and its antigen. The KD value is the equilibrium dissociation constant between the antibody and its antigen, that is, the ratio of k off /k on . Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.
对于一种抑制剂对活性分子的生物活性的抑制效果，可通过IC50值(半数最大抑制浓度)来衡量。IC50表示需要何种抑制剂浓度才能将该活性分子(例如CD73)的活性(如催化AMP转化为腺苷)抑制一半。IC50值越小，表明其对该活性分子的抑制能力越强。The inhibitory effect of an inhibitor on the biological activity of an active molecule can be measured by the IC 50 value (half of the maximum inhibitory concentration). IC 50 indicates the concentration of inhibitor required to inhibit the activity of the active molecule (such as CD73) by half (such as catalyzing the conversion of AMP to adenosine). The smaller the IC 50 value, the stronger its ability to inhibit the active molecule.
“变体”指相对于参照序列(例如本文公开的CDR序列、VH或VL序列)在氨基酸序列上引入了差异而产生的产物序列(例如CDR序列、VH或VL序列)。本领域技术人员可以理解的是，在本文提供的具体序列基础上，可以通过对少数氨基酸进行替换、删除、添加并验证或筛选所得产物与相应抗原CD73的结合能力或对其抑制能力，从而获得本文提供的靶向CD73的抗体或其抗原结合片段的相应变体，这些变体也应包括在本发明的范围内。例如，在本文公开的抗体或其抗原结合片段在全长或CDR序列上，可以有至少1个且不超过10，或不超过5、4、3、2或1个氨基酸的改变。"Variant" refers to a product sequence (eg, CDR sequence, VH or VL sequence) resulting from the introduction of differences in amino acid sequence relative to a reference sequence (eg, CDR sequence, VH or VL sequence disclosed herein). Those skilled in the art can understand that, based on the specific sequences provided herein, a few amino acids can be replaced, deleted, added, and the resulting product can be verified or screened for its binding ability to the corresponding antigen CD73 or its ability to inhibit it, thereby obtaining Corresponding variants of the CD73-targeting antibodies or antigen-binding fragments thereof provided herein are also included in the scope of the present invention. For example, the antibodies or antigen-binding fragments thereof disclosed herein may have at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid changes in the full length or CDR sequence.
“纯化标签”指与目的蛋白或多肽以融合蛋白形式一起表达的用于纯化该目的蛋白或多肽的氨基酸序列，包括但不限于His6标签、Flag标签、MBP(麦芽糖结合蛋白)标签和GST(谷胱甘肽巯基转移酶)标签、SUMO(小泛素相关修饰物(small ubiquitin related modifier))等。这些标签可在纯化后通过酶切去除，或者在不影响目的蛋白或多肽正常功能情况下可带标签使用(例如His6标签)。 "Purification tag" refers to the amino acid sequence expressed together with the target protein or polypeptide in the form of a fusion protein for purifying the target protein or polypeptide, including but not limited to His6 tag, Flag tag, MBP (maltose binding protein) tag and GST (gluten thione sulfhydryl transferase) tag, SUMO (small ubiquitin related modifier), etc. These tags can be removed by enzymatic digestion after purification, or they can be used with tags (such as His6 tags) without affecting the normal function of the target protein or peptide.
“可检测标签”指与蛋白或多肽连接的氨基酸序列或其他化学基团，用于指示样品中该蛋白或多肽的存在或含量，或者用于跟踪该蛋白或多肽在受试者体内或细胞内的位置信息。可检测标签的实例包括免疫检测中可使用的各种酶，例如辣根过氧化物酶(HRP)、碱性磷酸酶(ALP)；荧光基团(如FAM、FITC)或荧光蛋白(如GFP)；放射性同位素(例如3H、14C、35S)。当可检测标签为酶时，可通过酶的酶学活性来确定与酶连接的蛋白或多肽的存在或含量。"Detectable label" refers to an amino acid sequence or other chemical group linked to a protein or polypeptide, used to indicate the presence or content of the protein or polypeptide in a sample, or used to track the protein or polypeptide in a subject's body or cells location information. Examples of detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP); fluorescent groups (such as FAM, FITC) or fluorescent proteins (such as GFP ); radioactive isotopes (such as 3 H, 14 C, 35 S). When the detectable label is an enzyme, the presence or content of the protein or polypeptide linked to the enzyme can be determined by the enzymatic activity of the enzyme.
本文中，术语“核酸分子”、“核酸”和“多核苷酸”可互换使用，指核苷酸聚合物。此类核苷酸聚合物可含有天然和/或非天然核苷酸且包括(但不限于)DNA、RNA和PNA。“核酸序列”指包含于核酸分子或多核苷酸中的核苷酸线性序列。As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to polymers of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.
术语“载体”指可经工程改造以含有目的多核苷酸(例如目的多肽的编码序列)的核酸分子或可在宿主细胞中复制的核酸分子(例如，核酸、质粒或病毒等)。载体可包括以下组件中的一个或更多个：复制起点、一个或更多个调控目的多核苷酸的表达的调控序列(诸如启动子和/或增强子子)和/或一个或更多个可选择标记物基因(诸如抗生素抗性基因和可用于比色分析中的基因，例如β-半乳糖)。术语“表达载体”指用于在宿主细胞中表达目的多肽的载体。The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (eg, a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (eg, nucleic acid, plasmid, virus, etc.). The vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and/or enhancer), and/or one or more Selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, such as beta-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.
“宿主细胞”指可为或已为载体或经分离多核苷酸的接受体的细胞。宿主细胞可为原核细胞或真核细胞。示例性真核细胞包括哺乳动物细胞，诸如灵长类动物或非灵长类动物细胞；真菌细胞，诸如酵母；植物细胞；以及昆虫细胞。非限制性示例性哺乳动物细胞包括(但不限于)NSO细胞、293以及CHO细胞，以及其衍生细胞，诸如293-6E、CHO-DG44、CHO-K1、CHO-S和CHO-DS细胞。宿主细胞包括单个宿主细胞的后代，且后代可能由于自然、偶然或故意突变而不一定与原始母细胞完全一致(在形态或基因组DNA互补方面)。宿主细胞也包括在活体内经本文提供的核酸分子或表达载体转染的细胞。"Host cell" refers to a cell that is or has been the recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include the progeny of a single host cell, and the progeny may not necessarily be identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. Host cells also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.
“治疗”指对受试者进行处理以获得有益的或所期望的临床结果。本文所用的“治疗”涵盖各种处理手段，包括以任何可能的药物向受试者给药、手术、辐射等。出于本发明的目的，有益或所期望的临床结果包括但不限于以下的任一种或多种：减轻一种或更多种症状、减弱疾病程度、预防或延迟疾病扩散(例如转移，例如转移至肺或***)、预防或延迟疾病复发、延迟或减缓疾病进展、改善疾病病况、抑制疾病或疾病进展、阻滞其发展和缓解(无论部分抑或完全缓解)。本文所提供的方法涵盖这些治疗方面中的任一种或多种。按照以上内容，“治疗”不需要完全去除病症或疾病的所有症状或完全缓解。"Treatment" refers to the treatment of a subject to obtain a beneficial or desired clinical result. "Treatment" as used herein encompasses a variety of treatments, including administration of any possible drug to the subject, surgery, radiation, etc. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g. metastasis, e.g. metastasize to the lungs or lymph nodes), prevent or delay disease recurrence, delay or slow down disease progression, improve disease conditions, inhibit disease or disease progression, block its development and remission (whether partial or complete remission). The methods provided herein encompass any one or more of these aspects of treatment. In accordance with the above, "treatment" does not require complete removal of all symptoms of a condition or disease or complete alleviation.
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明融合蛋白的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg或更多活性成分。The term "therapeutically effective amount" refers to an amount of active compound sufficient to elicit the biological or medical response desired by the clinician in a subject. The "therapeutically effective dose" of the fusion protein of the present invention can be determined by those skilled in the art based on the route of administration, the subject's weight, age, condition and other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.
“实体癌(或实体瘤)”指有形肿瘤，可通过诸如CT、磁共振等的影像学检查方法检测到。实体瘤的实例包括胃癌、肺癌、结直肠、胰腺癌、乳腺癌及肝癌等。 "Solid cancer (or solid tumor)" refers to a tangible tumor that can be detected by imaging examination methods such as CT, magnetic resonance, etc. Examples of solid tumors include gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, etc.
提及药物组合物，所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水和/或无热原水等。本文所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。The term "pharmaceutically acceptable carrier" as used with reference to pharmaceutical compositions refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be administered safely and are suitable for use by humans and/or Administration to the animal without undue adverse side effects while being suitable for maintaining the viability of the drug or active agent therein. Depending on the route of administration, various carriers well known in the art may be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols , alginic acid, phosphate buffer, emulsifier, isotonic saline and/or pyrogen-free water, etc. The pharmaceutical compositions provided herein can be made into clinically acceptable dosage forms such as powders and injections. The pharmaceutical composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, etc. route of administration.
“受试者”指动物，例如哺乳动物，包括(但不限于)人类、啮齿动物、猿猴、猫科动物、犬科动物、马科动物、牛科动物、猪科动物、绵羊、山羊、哺乳类实验动物、哺乳类农畜、哺乳类运动动物和哺乳类宠物。受试者可为雄性或雌性且可为任何适龄受试者，包括婴儿、幼年、青年、成年和老年受试者。在一些实例中，受试者指需要治疗疾病或病症的个体。在一些实例中，接受治疗的受试者可为患者，其患有与该治疗有关联的病症，或有风险患上该病症。在另一些实例中，受试者为健康个体或者为患有非所关注疾病的个体。在特定实例中，受试者为人类，诸如人类患者。该术语通常可与“患者”、“检测对象”、“治疗对象”等互换使用。"Subject" refers to an animal, such as a mammal, including (but not limited to) humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, goats, mammals Laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets. The subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects. In some examples, a subject refers to an individual in need of treatment of a disease or condition. In some examples, a subject receiving treatment can be a patient who has a condition associated with the treatment, or is at risk of developing the condition. In other examples, the subject is a healthy individual or an individual suffering from a disease other than that of concern. In certain examples, the subject is a human, such as a human patient. The term is often used interchangeably with "patient," "subject," "subject," etc.
提及氨基酸或核苷酸序列时，术语“序列一致性(sequence identity)”(也称为“序列同一性”)指两氨基酸或核苷酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两氨基酸或核苷酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的氨基酸残基或碱基相同，则认为两序列在该位置一致或匹配；两序列中的氨基酸残基或碱基不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两氨基酸或核苷酸序列之间的序列一致性。When referring to an amino acid or nucleotide sequence, the term "sequence identity" (also called "sequence identity") refers to the identity between two amino acid or nucleotide sequences (such as a query sequence and a reference sequence) A quantity of degree, usually expressed as a percentage. Usually, before calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matching at that position; if the amino acid residues or bases in the two sequences are different, the two sequences are considered to be inconsistent or matching at that position. mismatch. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the gap length is also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the web page ncbi.nlm.nih.gov) can be used to obtain an optimal sequence alignment and calculate two amino acids or nucleotides by using default settings Sequence identity between sequences.
“CD73”也被称为胞外-5'-核苷酸酶(ecto-5'-NT或EC 3.1.3.5)，是一种细胞表面磷酸酶，催化细胞外AMP的去磷酸化以产生腺苷。在生理上，CD73被缺氧诱导以控制损伤部位的炎症。在病理学上，CD73经常被发现在调节性T细胞(Tregs)和肿瘤细胞上过度表达。CD73活性的提高导致了腺苷在肿瘤微环境(TME)中的积累。通过与腺苷受体A2a和A2b结合，腺苷通过调节许多免疫细胞，如巨噬细胞、树突状细胞、自然杀伤细胞和T效应细胞，抑制先天和适应性免疫。因此，有推测，腺苷的免疫抑制可能通过抑制TME中CD73的活性而得到缓解。事实上，体内的动物研究表明，抑制CD73的活性可以抑制肿 瘤的形成和生长，这表明CD73是一个有希望的癌症治疗目标。术语“人CD73”是指源自人类的CD73。人CD73的一个示例性氨基酸序列在GenBank登录号P21589.1中表示。"CD73", also known as extracellular-5'-nucleotidase (ecto-5'-NT or EC 3.1.3.5), is a cell surface phosphatase that catalyzes the dephosphorylation of extracellular AMP to produce adenosine. glycosides. Physiologically, CD73 is induced by hypoxia to control inflammation at the site of injury. Pathologically, CD73 is often found to be overexpressed on regulatory T cells (Tregs) and tumor cells. Increased CD73 activity leads to the accumulation of adenosine in the tumor microenvironment (TME). By binding to adenosine receptors A2a and A2b, adenosine suppresses innate and adaptive immunity by regulating many immune cells, such as macrophages, dendritic cells, natural killer cells, and T effector cells. Therefore, it is speculated that the immunosuppression of adenosine may be alleviated by inhibiting the activity of CD73 in the TME. In fact, in vivo animal studies have shown that inhibiting CD73 activity can suppress tumor tumor formation and growth, suggesting that CD73 is a promising target for cancer treatment. The term "human CD73" refers to CD73 of human origin. An exemplary amino acid sequence of human CD73 is represented in GenBank accession number P21589.1.
在一些实施方案中，本公开的抗CD73单克隆抗体可与人类以外的物种的CD73或与人类CD73结构相关的其他蛋白质发生交叉反应。在一些实施方案中，本文提供的抗CD73单克隆抗体对人CD73具备完全的特异性，不表现出物种或其他类型的交叉反应性。In some embodiments, anti-CD73 monoclonal antibodies of the present disclosure may cross-react with CD73 from species other than human or with other proteins structurally related to human CD73. In some embodiments, the anti-CD73 monoclonal antibodies provided herein are fully specific for human CD73 and do not exhibit species or other types of cross-reactivity.
抗CD73抗体或其抗原结合片段Anti-CD73 antibody or antigen-binding fragment thereof
本文提供了特异性结合CD73的抗CD73抗体或其抗原结合片段。该抗CD73抗体或其抗原结合片段以相对较高的结合亲和力结合CD73分子。例如下文实施例中所阐述的，抗CD73抗体或其抗原结合片段与CD73结合能力可通过诸如酶联免疫吸附测定(ELISA)和流式细胞荧光分选技术(FACS)的测定方法来进行测量。另外，也可通过本领域已知的其他蛋白相互作用测定方法来测定，例如，表面等离子体共振技术(SPR)、生物层干涉(BLI)技术。抗CD73抗体或其抗原结合片段与CD73结合后可抑制其活性，例如将AMP转化为腺苷的催化活性。Provided herein are anti-CD73 antibodies or antigen-binding fragments thereof that specifically bind CD73. The anti-CD73 antibody or antigen-binding fragment thereof binds to CD73 molecules with relatively high binding affinity. For example, as described in the Examples below, the ability of an anti-CD73 antibody or antigen-binding fragment thereof to bind to CD73 can be measured by assay methods such as enzyme-linked immunosorbent assay (ELISA) and flow cytometric fluorescence sorting (FACS). In addition, it can also be measured by other protein interaction measurement methods known in the art, such as surface plasmon resonance technology (SPR) and biolayer interference (BLI) technology. Anti-CD73 antibodies or antigen-binding fragments thereof bind to CD73 and inhibit its activity, such as the catalytic activity of converting AMP to adenosine.
在一些实施方案中，本文提供了抗CD73单克隆抗体或其抗原结合片段，其包括重链可变结构域(VH)和轻链可变结构域(VL)，该VH包括选自SEQ ID NO:2(NYWIH)、SEQ ID NO:10(NYWMH)、SEQ ID NO:18(SYWIN)、SEQ ID NO:26(NYGIS)、SEQ ID NO:34(SYWIN)、SEQ ID NO:42(NYGIT)、SEQ ID NO:50(SYGIN)、SEQ ID NO:58(GYYMH)、SEQ ID NO:66(SYAMS)、SEQ ID NO:74(RYGIH)或SEQ ID NO:82(GYYMH)的HCDR1或包括至多3个(如1、2或3个)氨基酸替换的该HCDR1变体，选自SEQ ID NO:3(YINPGDDYIKYSQNFKG)、SEQ ID NO:11(YINPSDAYTTYSQNFKG)、SEQ ID NO:19(NIYPGNSDTNNNEKFRN)、SEQ ID NO:27(EIYPGSGNTYYNEKFKD)、SEQ ID NO:35(NIYPGNSNTNYNEKFKS)、SEQ ID NO:43(EIYPGSGNAYYKENFKG)、SEQ ID NO:51(EIYPGSGNTYYNEKFKV)、SEQ ID NO:59(RINPYNGATTYNQNFKD)、SEQ ID NO:67(TISSGGSSTYYPDSVKG)、SEQ ID NO:75(VIWVGGSTNYNSTLMS)或SEQ ID NO:83(RINPYNGATNYNQNFKD)的HCDR2或包括至多3个(如1、2或3个)氨基酸替换的该HCDR2变体，以及选自SEQ ID NO:4(SPIYYNDFDY)、SEQ ID NO:12(SPIYYNNFDY)、SEQ ID NO:20(ENGNNDWYFDV)、SEQ ID NO:28(FDYSGYYYALDY)、SEQ ID NO:36(EGDTYSWYFDV)、SEQ ID NO:44或105(FDYIYYNGLEY或FDYIYYNALEY)、SEQ ID NO:52(NWDYYFDY)、SEQ ID NO:60(SIGSSPFDY)、SEQ ID NO:68(RGTTEVPHFDY)、SEQ ID NO:76(DGGITTVVADY)或SEQ ID NO:84(SHYGSFDY)的HCDR3或包括至多3个(如1、2或3个)氨基酸替换的该HCDR3变体；该VL包括选自SEQ ID NO:6(KASQDIKSYLS)、SEQ ID NO:14(KASQDIKSYLS)、SEQ ID NO:22(RASQSVSTSGYSYMH)、SEQ ID NO:30 (KASQDLSTAVA)、SEQ ID NO:38(SASSSISSNYLH)、SEQ ID NO:46(KASQDVTTAVA)、SEQ ID NO:54(KASQDIKGYLG)、SEQ ID NO:62(KASQDINSYLS)、SEQ ID NO:70(KASQSVSNEAA)、SEQ ID NO:78(KASQDINSFLS)或SEQ ID NO:86(KASQDINSYLS)的LCDR1或包括至多3个(如1、2或3个)氨基酸替换的该LCDR1变体，选自SEQ ID NO:7(YATSLAD)、SEQ ID NO:15(YATDLAD)、SEQ ID NO:23(LASNLKS)、SEQ ID NO:31(PASFLYT)、SEQ ID NO:39(GTSNLAS)、SEQ ID NO:47(SASHRYT)、SEQ ID NO:55(YTTNLAD)、SEQ ID NO:63(RGNWLVD)、SEQ ID NO:71(YASNRYS)、SEQ ID NO:79(RAKRLVD)或SEQ ID NO:87(RANILID)的LCDR2或包括至多3个(如1、2或3个)氨基酸替换的该LCDR2变体，和选自SEQ ID NO:8(LQYGDTTYT)、SEQ ID NO:16(LQYGENTYT)、SEQ ID NO:24(QHSRELPWT)、SEQ ID NO:32(QQRYSTPYT)、SEQ ID NO:40(QQGYILPFT)、SEQ ID NO:48(QQHSSPPFT)、SEQ ID NO:56(LQHGESPFT)、SEQ ID NO:64(LQYDEFPNT)、SEQ ID NO:72(QQDYSSPWT)、SEQ ID NO:80(LQYDELYT)或SEQ ID NO:88(LQYDEFPYT)的LCDR3或包括至多3个(如1、2或3个)氨基酸替换的该LCDR3变体。在一些实施方案中，该抗CD73抗体或其抗原结合片段为CD73的抑制剂，并且针对细胞表面表达的所述CD73的酶活性具有约10-1μg/mL至约10- 3μg/mL的IC50值。在一些实施方案中，该抗CD73抗体或其抗原结合片段针对细胞表面表达的所述CD73的酶活性的IC50值小于参照抗体MEDI9447针对CD73的IC50值。在一些实施方案中，该抗CD73抗体或其抗原结合片段结合表达CD73的细胞的EC50值为约1μg/mL至约10-2μg/mL(如通过FASC检测的)。在一些实施方案中，该抗CD73抗体或其抗原结合片段结合表达CD73的细胞的EC50值小于参照抗体MEDI9447结合表达CD73的细胞的EC50值。In some embodiments, provided herein are anti-CD73 monoclonal antibodies, or antigen-binding fragments thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH comprising a variable domain selected from SEQ ID NO. :2(NYWIH), SEQ ID NO:10(NYWMH), SEQ ID NO:18(SYWIN), SEQ ID NO:26(NYGIS), SEQ ID NO:34(SYWIN), SEQ ID NO:42(NYGIT) , the HCDR1 of SEQ ID NO:50 (SYGIN), SEQ ID NO:58 (GYYMH), SEQ ID NO:66 (SYAMS), SEQ ID NO:74 (RYGIH) or SEQ ID NO:82 (GYYMH) or including up to The HCDR1 variant with 3 (such as 1, 2 or 3) amino acid substitutions is selected from SEQ ID NO: 3 (YINPGDDYIKYSQNFKG), SEQ ID NO: 11 (YINPSDAYTTYSQNFKG), SEQ ID NO: 19 (NIYPGNSDTNNNEKFRN), SEQ ID NO:27(EIYPGSGNTYYNEKFKD), SEQ ID NO:35(NIYPGNSNTNYNEKFKS), SEQ ID NO:43(EIYPGSGNAYYKENFKG), SEQ ID NO:51(EIYPGSGNTYYNEKFKV), SEQ ID NO:59(RINPYNGATTYNQNFKD), SEQ ID NO:67(TISSGGSSTYYPDSVKG ), the HCDR2 of SEQ ID NO:75 (VIWVGGSTNYNSTLMS) or SEQ ID NO:83 (RINPYNGATNYNQNFKD) or a variant thereof comprising up to 3 (eg 1, 2 or 3) amino acid substitutions, and selected from SEQ ID NO: 4(SPIYYNDFDY), SEQ ID NO:12(SPIYYNNFDY), SEQ ID NO:20(ENGNNDWYFDV), SEQ ID NO:28(FDYSGYYYALDY), SEQ ID NO:36(EGDTYSWYFDV), SEQ ID NO:44 or 105(FDYIYYNGLEY or FDYIYYNALEY), SEQ ID NO:52(NWDYYFDY), SEQ ID NO:60(SIGSSPFDY), SEQ ID NO:68(RGTTEVPHFDY), SEQ ID NO:76(DGGITTVVADY) or SEQ ID NO:84(SHYGSFDY) HCDR3 Or the HCDR3 variant including at most 3 (such as 1, 2 or 3) amino acid substitutions; the VL includes selected from SEQ ID NO: 6 (KASQDIKSYLS), SEQ ID NO: 14 (KASQDIKSYLS), SEQ ID NO: 22 (RASQSVSTSGYSYMH), SEQ ID NO:30 (KASQDLSTAVA), SEQ ID NO: 38 (SASSSISSNYLH), SEQ ID NO: 46 (KASQDVTTAVA), SEQ ID NO: 54 (KASQDIKGYLG), SEQ ID NO: 62 (KASQDINSYLS), SEQ ID NO: 70 (KASQSVSNEAA), SEQ LCDR1 of ID NO:78 (KASQDINSFLS) or SEQ ID NO:86 (KASQDINSYLS) or a variant thereof comprising up to 3 (eg 1, 2 or 3) amino acid substitutions, selected from SEQ ID NO:7 (YATSLAD) , SEQ ID NO:15(YATDLAD), SEQ ID NO:23(LASNLKS), SEQ ID NO:31(PASFLYT), SEQ ID NO:39(GTSNLAS), SEQ ID NO:47(SASHRYT), SEQ ID NO: 55 (YTTNLAD), SEQ ID NO:63 (RGNWLVD), SEQ ID NO:71 (YASNRYS), SEQ ID NO:79 (RAKRLVD) or SEQ ID NO:87 (RANILID) or include up to 3 (such as 1 , 2 or 3) of the LCDR2 variant with amino acid substitutions, and is selected from the group consisting of SEQ ID NO: 8 (LQYGDTTYT), SEQ ID NO: 16 (LQYGENTYT), SEQ ID NO: 24 (QHSRELPWT), SEQ ID NO: 32 ( QQRYSTPYT), SEQ ID NO: 40 (QQGYILPFT), SEQ ID NO: 48 (QQHSSPPFT), SEQ ID NO: 56 (LQHGESPFT), SEQ ID NO: 64 (LQYDEFPNT), SEQ ID NO: 72 (QQDYSSPWT), SEQ ID The LCDR3 of NO:80 (LQYDELYT) or SEQ ID NO:88 (LQYDEFPYT) or a variant thereof including up to 3 (eg 1, 2 or 3) amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof is an inhibitor of CD73 and has an enzymatic activity against said CD73 expressed on the cell surface of about 10 -1 μg/mL to about 10 -3 μg /mL. IC 50 value. In some embodiments, the IC50 value of the enzymatic activity of the anti-CD73 antibody or antigen-binding fragment thereof against CD73 expressed on the cell surface is less than the IC50 value of the reference antibody MEDI9447 against CD73. In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, binds CD73-expressing cells with an EC50 value of about 1 μg/mL to about 10 −2 μg/mL (as determined by FASC). In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof binds to cells expressing CD73 with an EC 50 value that is less than the EC 50 value of the reference antibody MEDI9447 binding to cells expressing CD73.
在一些实施方案中，该抗CD73抗体或其抗原结合片段包括选自SEQ ID NO:4、12、20、28、36、44、52、60、68、76、84或105的HCDR3和选自SEQ ID NO:8、16、24、32、40、48、56、64、72、80或88的LCDR3，而氨基酸替换出现在VH和VL的CDR1和CDR2结构域中。In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof includes an HCDR3 selected from SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, or 105 and LCDR3 of SEQ ID NO:8, 16, 24, 32, 40, 48, 56, 64, 72, 80 or 88, while amino acid substitutions occur in the CDR1 and CDR2 domains of VH and VL.
因此，在一些实施方案中，本文提供了这样的抗CD73抗体或其抗原结合片段，其VH包括选自SEQ ID NO:2(NYWIH)、SEQ ID NO:10(NYWMH)、SEQ ID NO:18(SYWIN)、SEQ ID NO:26(NYGIS)、SEQ ID NO:34(SYWIN)、SEQ ID NO:42(NYGIT)、SEQ ID NO:50(SYGIN)、SEQ ID NO:58(GYYMH)、SEQ ID NO:66(SYAMS)、SEQ ID NO:74(RYGIH)或SEQ ID NO:82(GYYMH)的HCDR1或包括至多3个(如1、2或3个)氨基酸替换的该HCDR1变体，选自SEQ ID NO:3(YINPGDDYIKYSQNFKG)、SEQ ID NO:11(YINPSDAYTTYSQNFKG)、SEQ ID NO:19(NIYPGNSDTNNNEKFRN)、SEQ ID NO:27(EIYPGSGNTYYNEKFKD)、SEQ ID NO:35(NIYPGNSNTNYNEKFKS)、SEQ ID NO:43(EIYPGSGNAYYKENFKG)、SEQ ID NO:51(EIYPGSGNTYYNEKFKV)、SEQ ID NO:59(RINPYNGATTYNQNFKD)、SEQ ID NO:67(TISSGGSSTYYPDSVKG)、SEQ ID NO: 75(VIWVGGSTNYNSTLMS)或SEQ ID NO:83(RINPYNGATNYNQNFKD)的HCDR2或包括至多3个(如1、2或3个)氨基酸替换的该HCDR2变体，以及选自SEQ ID NO:4(SPIYYNDFDY)、SEQ ID NO:12(SPIYYNNFDY)、SEQ ID NO:20(ENGNNDWYFDV)、SEQ ID NO:28(FDYSGYYYALDY)、SEQ ID NO:36(EGDTYSWYFDV)、SEQ ID NO:44或105(FDYIYYNGLEY或FDYIYYNALEY)、SEQ ID NO:52(NWDYYFDY)、SEQ ID NO:60(SIGSSPFDY)、SEQ ID NO:68(RGTTEVPHFDY)、SEQ ID NO:76(DGGITTVVADY)或SEQ ID NO:84(SHYGSFDY)的HCDR3；其VL包括选自SEQ ID NO:6(KASQDIKSYLS)、SEQ ID NO:14(KASQDIKSYLS)、SEQ ID NO:22(RASQSVSTSGYSYMH)、SEQ ID NO:30(KASQDLSTAVA)、SEQ ID NO:38(SASSSISSNYLH)、SEQ ID NO:46(KASQDVTTAVA)、SEQ ID NO:54(KASQDIKGYLG)、SEQ ID NO:62(KASQDINSYLS)、SEQ ID NO:70(KASQSVSNEAA)、SEQ ID NO:78(KASQDINSFLS)或SEQ ID NO:86(KASQDINSYLS)的LCDR1或包括至多3个(如1、2或3个)氨基酸替换的该LCDR1变体，选自SEQ ID NO:7(YATSLAD)、SEQ ID NO:15(YATDLAD)、SEQ ID NO:23(LASNLKS)、SEQ ID NO:31(PASFLYT)、SEQ ID NO:39(GTSNLAS)、SEQ ID NO:47(SASHRYT)、SEQ ID NO:55(YTTNLAD)、SEQ ID NO:63(RGNWLVD)、SEQ ID NO:71(YASNRYS)、SEQ ID NO:79(RAKRLVD)或SEQ ID NO:87(RANILID)的LCDR2或包括至多3个(如1、2或3个)氨基酸替换的该LCDR2变体，和选自SEQ ID NO:8(LQYGDTTYT)、SEQ ID NO:16(LQYGENTYT)、SEQ ID NO:24(QHSRELPWT)、SEQ ID NO:32(QQRYSTPYT)、SEQ ID NO:40(QQGYILPFT)、SEQ ID NO:48(QQHSSPPFT)、SEQ ID NO:56(LQHGESPFT)、SEQ ID NO:64(LQYDEFPNT)、SEQ ID NO:72(QQDYSSPWT)、SEQ ID NO:80(LQYDELYT)或SEQ ID NO:88(LQYDEFPYT)的LCDR3。在一些实施方案中，该抗CD73抗体或其抗原结合片段为CD73的抑制剂，并且针对细胞表面表达的所述CD73的酶活性具有约10-1μg/mL至约10- 3μg/mL的IC50值。在一些实施方案中，该抗CD73单克隆抗体针对细胞表面表达的所述CD73的酶活性的IC50值小于参照抗体MEDI9447针对CD73的IC50值。在一些实施方案中，该抗CD73单克隆抗体结合表达CD73的细胞的EC50值为约1μg/mL至约10-2μg/mL(如通过FASC检测的)。在一些实施方案中，该抗CD73抗体或其抗原结合片段结合表达CD73的细胞的EC50值小于参照抗体MEDI9447结合表达CD73的细胞的EC50值。Accordingly, in some embodiments, provided herein are anti-CD73 antibodies, or antigen-binding fragments thereof, whose VH includes a VH selected from the group consisting of SEQ ID NO: 2 (NYWIH), SEQ ID NO: 10 (NYWMH), SEQ ID NO: 18 (SYWIN), SEQ ID NO:26(NYGIS), SEQ ID NO:34(SYWIN), SEQ ID NO:42(NYGIT), SEQ ID NO:50(SYGIN), SEQ ID NO:58(GYYMH), SEQ The HCDR1 of ID NO:66 (SYAMS), SEQ ID NO:74 (RYGIH) or SEQ ID NO:82 (GYYMH) or a variant of this HCDR1 including up to 3 (eg 1, 2 or 3) amino acid substitutions, preferably From SEQ ID NO:3(YINPGDDYIKYSQNFKG), SEQ ID NO:11(YINPSDAYTTYSQNFKG), SEQ ID NO:19(NIYPGNSDTNNNEKFRN), SEQ ID NO:27(EIYPGSGNTYYNEKFKD), SEQ ID NO:35(NIYPGNSNTNYNEKFKS), SEQ ID NO: 43 (EIYPGSGNAYYKENFKG), SEQ ID NO: 51 (EIYPGSGNTYYNEKFKV), SEQ ID NO: 59 (RINPYNGATTYNQNFKD), SEQ ID NO: 67 (TISSGGSSTYYPDSVKG), SEQ ID NO: 75 (VIWVGGSTNYNSTLMS) or the HCDR2 of SEQ ID NO:83 (RINPYNGATNYNQNFKD) or the HCDR2 variant including up to 3 (such as 1, 2 or 3) amino acid substitutions, and selected from the group consisting of SEQ ID NO:4 (SPIYYNDFDY), SEQ ID NO: 12 (SPIYYNNFDY), SEQ ID NO: 20 (ENGNNDWYFDV), SEQ ID NO: 28 (FDYSGYYYALDY), SEQ ID NO: 36 (EGDTYSWYFDV), SEQ ID NO: 44 or 105 (FDYIYYNGLEY or FDYIYYNALEY), SEQ ID HCDR3 of NO: 52 (NWDYYFDY), SEQ ID NO: 60 (SIGSSPFDY), SEQ ID NO: 68 (RGTTEVPHFDY), SEQ ID NO: 76 (DGGITTVVADY) or SEQ ID NO: 84 (SHYGSFDY); its VL includes selected from SEQ ID NO: 6 (KASQDIKSYLS), SEQ ID NO: 14 (KASQDIKSYLS), SEQ ID NO: 22 (RASQSVSTSGYSYMH), SEQ ID NO: 30 (KASQDLSTAVA), SEQ ID NO: 38 (SASSSISSNYLH), SEQ ID NO: 46 (KASQDVTTAVA), SEQ ID NO:54(KASQDIKGYLG), SEQ ID NO:62(KASQDINSYLS), SEQ ID NO:70(KASQSVSNEAA), SEQ ID NO:78(KASQDINSFLS) or SEQ ID NO:86(KASQDINSYLS) Or the LCDR1 variant comprising up to 3 (such as 1, 2 or 3) amino acid substitutions, selected from SEQ ID NO: 7 (YATSLAD), SEQ ID NO: 15 (YATDLAD), SEQ ID NO: 23 (LASNLKS) , SEQ ID NO:31(PASFLYT), SEQ ID NO:39(GTSNLAS), SEQ ID NO:47(SASHRYT), SEQ ID NO:55(YTTNLAD), SEQ ID NO:63(RGNWLVD), SEQ ID NO: 71 (YASNRYS), the LCDR2 of SEQ ID NO:79 (RAKRLVD) or SEQ ID NO:87 (RANILID) or a variant thereof comprising up to 3 (e.g. 1, 2 or 3) amino acid substitutions, and selected from SEQ ID NO:8(LQYGDTTYT), SEQ ID NO:16(LQYGENTYT), SEQ ID NO:24(QHSRELPWT), SEQ ID NO:32(QQRYSTPYT), SEQ ID NO:40(QQGYILPFT), SEQ ID NO:48( QQHSSPPFT), SEQ ID NO:56 (LQHGESPFT), SEQ ID NO:64 (LQYDEFPNT), SEQ ID NO:72 (QQDYSSPWT), SEQ ID NO:80 (LQYDELYT) or SEQ ID NO:88 (LQYDEFPYT). In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof is an inhibitor of CD73 and has an enzymatic activity against said CD73 expressed on the cell surface of about 10 -1 μg/mL to about 10 -3 μg /mL. IC 50 value. In some embodiments, the IC50 value of the enzymatic activity of the anti-CD73 monoclonal antibody against CD73 expressed on the cell surface is less than the IC50 value of the reference antibody MEDI9447 against CD73. In some embodiments, the anti-CD73 monoclonal antibody binds CD73-expressing cells with an EC50 value of about 1 μg/mL to about 10 −2 μg/mL (as determined by FASC). In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, binds cells expressing CD73 with an EC50 value that is less than the EC50 value of the reference antibody MEDI9447 binding cells expressing CD73.
在一些实施方案中，所提及的氨基酸替换可以为保守氨基酸取代。保守氨基酸取代通常可被描述为一种氨基酸残基被类似化学结构的另一种氨基酸残基取代，并且对多肽的功能、活性或其他生物学性质几乎没有或基本上没有影响。保守氨基酸取代是本领域众所周知的。保守性取代可例如是下列(a)-(e)组中的一个氨基酸被同组内的另一个氨基酸取代：(a)小的脂肪族非极性或弱极性残基：Ala、Ser、Thr、Pro和Gly；(b)极性带负电荷的残基及其(不带电荷的)酰胺：Asp、Asn、Glu和Gln；(c)极性带正电荷的残基：His、Arg 和Lys；(d)大的脂肪族非极性残基：Met、Leu、Ile、Val和Cys；和(e)芳族残基：Phe、Tyr和Trp。In some embodiments, the amino acid substitutions mentioned may be conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as the replacement of one amino acid residue by another amino acid residue of similar chemical structure and having little or essentially no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may, for example, be the substitution of one amino acid in the following groups (a)-(e) by another amino acid in the same group: (a) Small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
在一些实施方案中，本文提供了抗CD73抗体或其抗原结合片段(如抗CD73单克隆抗体)，其中1)其VH包括分别为SEQ ID NO:2、3和4的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:6、7和8的LCDR1、LCDR2和LCDR3；2)其VH包括分别为SEQ ID NO:10、11和12的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:14、15和16的LCDR1、LCDR2和LCDR3；3)其VH包括分别为SEQ ID NO:18、19和20的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:22、23和24的LCDR1、LCDR2和LCDR3；4)其VH包括分别为SEQ ID NO:26、27和28的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:30、31和32的LCDR1、LCDR2和LCDR3；5)其VH包括分别为SEQ ID NO:34、35和36的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:38、39和40的LCDR1、LCDR2和LCDR3；6)其VH包括分别为SEQ ID NO:42、43和44的HCDR1、HCDR2和HCDR3或分别为SEQ ID NO:42、43和105的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:46、47和48的LCDR1、LCDR2和LCDR3；7)其VH包括分别为SEQ ID NO:50、51和52的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:54、55和56的LCDR1、LCDR2和LCDR3；8)其VH包括分别为SEQ ID NO:58、59和60的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:62、63和64的LCDR1、LCDR2和LCDR3；9)其VH包括分别为SEQ ID NO:66、67和68的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:70、71和72的LCDR1、LCDR2和LCDR3；10)其VH包括分别为SEQ ID NO:74、75和76的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:78、79和80的LCDR1、LCDR2和LCDR3；或11)其VH包括分别为SEQ ID NO:82、83和84的HCDR1、HCDR2和HCDR3，其VL包括分别为SEQ ID NO:86、87和88的LCDR1、LCDR2和LCDR3。In some embodiments, provided herein are anti-CD73 antibodies or antigen-binding fragments thereof (e.g., anti-CD73 monoclonal antibodies), wherein 1) their VHs include HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 3, and 4, respectively, Its VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:6, 7 and 8 respectively; 2) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:10, 11 and 12 respectively, and its VL includes SEQ ID NO:10, 11 and 12 respectively. LCDR1, LCDR2 and LCDR3 of ID NO:14, 15 and 16; 3) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:18, 19 and 20 respectively, and its VL includes SEQ ID NO:22 and 23 respectively. and 24 LCDR1, LCDR2 and LCDR3; 4) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:26, 27 and 28 respectively, and its VL includes LCDR1, LCDR2 of SEQ ID NO:30, 31 and 32 respectively. and LCDR3; 5) Its VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 34, 35 and 36, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 38, 39 and 40; 6) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:42, 43 and 44 respectively or HCDR1, HCDR2 and HCDR3 of SEQ ID NO:42, 43 and 105 respectively and its VL includes SEQ ID NO:46 and 47 respectively and 48 LCDR1, LCDR2 and LCDR3; 7) Its VH includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO:50, 51 and 52 respectively, and its VL includes LCDR1, LCDR2 of SEQ ID NO:54, 55 and 56 respectively. and LCDR3; 8) Its VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 58, 59 and 60, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 62, 63 and 64; 9) Its VH includes HCDR1, HCDR2 and HCDR3 respectively as SEQ ID NO: 66, 67 and 68, and its VL includes LCDR1, LCDR2 and LCDR3 respectively as SEQ ID NO: 70, 71 and 72; 10) Its VH includes SEQ ID NO: 70, 71 and 72 respectively. HCDR1, HCDR2 and HCDR3 of NO:74, 75 and 76, whose VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:78, 79 and 80 respectively; or 11) whose VH includes SEQ ID NO:82 and 83 respectively and HCDR1, HCDR2 and HCDR3 of 84, whose VL includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO:86, 87 and 88 respectively.
在一些实施方案中，本文提供了抗CD73抗体或其抗原结合片段(如抗CD73单克隆抗体)，其包括：1)包括SEQ ID NO:1所示氨基酸序列的VH和包括SEQ ID NO:5所示氨基酸序列的VL(9E12E3)；2)包括SEQ ID NO:9所示氨基酸序列的VH和包括SEQ ID NO:13所示氨基酸序列的VL(22E1E4)；3)包括SEQ ID NO:17所示氨基酸序列的VH和包括SEQ ID NO:21所示氨基酸序列的VL(35D8G8)；4)包括SEQ ID NO:25所示氨基酸序列的VH和包括SEQ ID NO:29所示氨基酸序列的VL(52A8B3)；5)包括SEQ ID NO:33所示氨基酸序列的VH和包括SEQ ID NO:37所示氨基酸序列的VL(52A6B3)；6)包括SEQ ID NO:41所示氨基酸序列的VH和包括SEQ ID NO:45所示氨基酸序列的VL(96A5A5)；7)包括SEQ ID NO:49所示氨基酸序列的VH和包括SEQ ID NO:53所示氨基酸序列的VL(100C5A3)；8)包括SEQ ID NO:57所示氨基酸序列的VH和包括SEQ ID NO:61所示氨基酸序列的VL(134D2H6)；9)包括SEQ ID NO:65所示氨基酸序列的VH和包括SEQ ID NO: 69所示氨基酸序列的VL(141G7E9)；10)包括SEQ ID NO:73所示氨基酸序列的VH和包括SEQ ID NO:77所示氨基酸序列的VL(143E5D2)；或11)包括SEQ ID NO:81所示氨基酸序列的VH和包括SEQ ID NO:85所示氨基酸序列的VL(143H3C5)。In some embodiments, provided herein are anti-CD73 antibodies or antigen-binding fragments thereof (such as anti-CD73 monoclonal antibodies), which comprise: 1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VH comprising SEQ ID NO: 5 VL (9E12E3) with the amino acid sequence shown; 2) VH including the amino acid sequence shown in SEQ ID NO: 9 and VL (22E1E4) including the amino acid sequence shown in SEQ ID NO: 13; 3) VL (22E1E4) including the amino acid sequence shown in SEQ ID NO: 17 VH showing the amino acid sequence and VL including the amino acid sequence shown in SEQ ID NO: 21 (35D8G8); 4) VH including the amino acid sequence shown in SEQ ID NO: 25 and VL including the amino acid sequence shown in SEQ ID NO: 29 ( 52A8B3); 5) VH including the amino acid sequence shown in SEQ ID NO: 33 and VL (52A6B3) including the amino acid sequence shown in SEQ ID NO: 37; 6) VH including the amino acid sequence shown in SEQ ID NO: 41 and including VL (96A5A5) with the amino acid sequence shown in SEQ ID NO: 45; 7) VL (100C5A3) including the amino acid sequence shown in SEQ ID NO: 49 and VL (100C5A3) including the amino acid sequence shown in SEQ ID NO: 53; 8) including SEQ VH including the amino acid sequence shown in ID NO: 57 and VL (134D2H6) including the amino acid sequence shown in SEQ ID NO: 61; 9) VH including the amino acid sequence shown in SEQ ID NO: 65 and including SEQ ID NO: VL (141G7E9) with the amino acid sequence shown in 69; 10) VH including the amino acid sequence shown in SEQ ID NO: 73 and VL (143E5D2) including the amino acid sequence shown in SEQ ID NO: 77; or 11) including SEQ ID NO: VH having the amino acid sequence shown in SEQ ID NO: 81 and VL (143H3C5) including the amino acid sequence shown in SEQ ID NO: 85.
在一些实施方案中，提供了抗CD73抗体或其抗原结合片段(如抗CD73单克隆抗体)，其包括：1)包括与SEQ ID NO:1所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:5所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；2)包括与SEQ ID NO:9所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:13所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；3)包括与SEQ ID NO:17所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:21所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；4)包括与SEQ ID NO:25所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:29所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；5)包括与SEQ ID NO:33所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:37所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；6)包括与SEQ ID NO:41所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:45所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；7)包括与SEQ ID NO:49所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:53所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；8)包括与SEQ ID NO:57所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:61所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；9)包括与SEQ ID NO:65所示氨基酸 序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:69所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；10)包括与SEQ ID NO:73所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括与SEQ ID NO:77所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL；或11)包括与SEQ ID NO:81所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VH和包括SEQ ID NO:85所示氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列的VL。In some embodiments, an anti-CD73 antibody or an antigen-binding fragment thereof (such as an anti-CD73 monoclonal antibody) is provided, comprising: 1) having at least 80% (e.g., at least 85%) the same amino acid sequence as shown in SEQ ID NO: 1 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity of the VH of the amino acid sequence and includes the amino acid sequence shown in SEQ ID NO: 5 A VL whose sequence has an amino acid sequence of at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; 2) Includes an amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or VH and include amino acid sequences having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) sequence identity to the amino acid sequence shown in SEQ ID NO: 13 , 96%, 97%, 98% or 99%) VL of an amino acid sequence having sequence identity; 3) including an amino acid sequence having at least 80% (such as at least 85%, 90%, 91%) with the amino acid sequence shown in SEQ ID NO: 17 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) VH of an amino acid sequence having a sequence identity of at least 80% ( For example, a VL having an amino acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; 4) including a VL with SEQ ID The amino acid sequence shown in NO:25 has at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity The VH of the amino acid sequence includes at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%) with the amino acid sequence shown in SEQ ID NO:29 , 98% or 99%) VL with an amino acid sequence having sequence identity; 5) including a VL having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%) with the amino acid sequence shown in SEQ ID NO: 33 , 94%, 95%, 96%, 97%, 98% or 99%) sequence identity and includes a VH having an amino acid sequence having at least 80% (e.g., at least 85%, 90%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 37 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) VL of an amino acid sequence with sequence identity; 6) including the amino acid sequence shown in SEQ ID NO: 41 VH and amino acid sequences whose sequences have at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity Includes an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 45 ) VL with an amino acid sequence having sequence identity; 7) including a VL having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) with the amino acid sequence shown in SEQ ID NO: 49 , 96%, 97%, 98% or 99%) and include VHs having an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92) sequence identity to the amino acid sequence set forth in SEQ ID NO: 53 %, 93%, 94%, 95%, 96%, 97%, 98% or 99%) VL of an amino acid sequence having sequence identity; 8) including an amino acid sequence having at least 80% ( For example, VH and amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity include VH and SEQ ID NO: The amino acid sequence shown in 61 has at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity. Sequence VL; 9) includes the same amino acid as shown in SEQ ID NO: 65 VH and amino acid sequences whose sequences have at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity Includes an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 69 ) VL with an amino acid sequence having sequence identity; 10) including a VL having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) with the amino acid sequence shown in SEQ ID NO:73 , 96%, 97%, 98% or 99%) and include VHs having an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92) sequence identity to the amino acid sequence set forth in SEQ ID NO: 77 %, 93%, 94%, 95%, 96%, 97%, 98% or 99%) VL of an amino acid sequence having sequence identity; or 11) comprising at least 80% of the amino acid sequence shown in SEQ ID NO:81 (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a VH amino acid sequence including SEQ ID NO: The amino acid sequence shown in 85 has at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity. Sequential VL.
在一些实施方案中，该抗CD73抗体或其抗原结合片段(如抗CD73单克隆抗体)为鼠抗体、嵌合抗体、人抗体或人源化抗体。In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof (eg, anti-CD73 monoclonal antibody) is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.
上文描述的CDR可以以各种成对的组合来产生一些人源化的抗CD73抗体。人源化替换对于本领域的技术人员来说是已知的。例如，可以通过将其他物种来源的VH或VL的FR序列与一个或多个密切相关的人类VH或VL的相应FR序列进行比较来确定潜在有用的人源化替代氨基酸，然后使用本领域已知的方法(也如本文所述)将一个或更多个此类潜在有用的人源化替代氨基酸引入所述VH或VL中。在一些实施方案中，可通过以鼠CDR序列替换人抗体VH或VL的全部CDR序列(即人抗体的四个FR序列之间所有CDR序列)来获得人源化抗体。通常，同时对抗体的重链和轻链进行人源化处理。可以测试所得到的人源化抗体对CD73的结合亲和力、结合稳定性和/或其他需要的特性，如对CD73的活性的抑制能力。本文提供的抗CD73抗体或其抗原结合片段可以部分或完全人源化。优选的是，所得的人源化抗体，如人源化抗体或其抗原结合片段，与CD73结合，具有本文所述的KD、EC50或IC50值。The CDRs described above can be used in various pairwise combinations to generate some humanized anti-CD73 antibodies. Humanizing substitutions are known to those skilled in the art. For example, potentially useful humanizing replacement amino acids can be identified by comparing the FR sequences of VH or VL derived from other species with the corresponding FR sequences of one or more closely related human VH or VL, and then using known methods in the art. Methods (also described herein) introduce one or more such potentially useful humanized replacement amino acids into the VH or VL. In some embodiments, a humanized antibody can be obtained by replacing all CDR sequences of a human antibody VH or VL (ie, all CDR sequences between the four FR sequences of a human antibody) with murine CDR sequences. Typically, the heavy and light chains of an antibody are humanized simultaneously. The resulting humanized antibodies can be tested for binding affinity to CD73, binding stability, and/or other desired properties, such as the ability to inhibit the activity of CD73. The anti-CD73 antibodies or antigen-binding fragments thereof provided herein may be partially or fully humanized. Preferably, the resulting humanized antibody, such as a humanized antibody or antigen-binding fragment thereof, binds to CD73 with a KD, EC50 or IC50 value as described herein.
在一些实施方案中，本文提供了抗CD73人源化抗体或其抗原结合片段，其VH包括SEQ ID NO:89、91、93、95、97、99和101中任一项的氨基酸序列或者与SEQ ID NO:89、91、93、95、97、99和101中任一项的氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列，其VL包括SEQ ID NO:90、92、94、96、98、100和102中任一项的氨基酸序列或者与SEQ ID NO:90、92、94、96、98、100和102中任一项的氨基酸序列具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列一致性的氨基酸序列。在一些实施方案中，该抗CD73人源化抗体或其抗原结合片段针对细胞表面表达的所述CD73的酶活性具有约10-1μg/mL至约10-2μg/mL的IC50值。在一些实施方案中，该抗CD73人源化抗体或其抗原结合片段与CD73结合的KD值为约10-8M至约10-11M。 In some embodiments, provided herein are anti-CD73 humanized antibodies, or antigen-binding fragments thereof, whose VH includes the amino acid sequence of any one of SEQ ID NO: 89, 91, 93, 95, 97, 99, and 101 or is identical to The amino acid sequence of any one of SEQ ID NO: 89, 91, 93, 95, 97, 99 and 101 has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99%) sequence identity of the amino acid sequence, the VL of which includes the amino acid sequence of any one of SEQ ID NO: 90, 92, 94, 96, 98, 100 and 102 or with The amino acid sequence of any one of SEQ ID NO: 90, 92, 94, 96, 98, 100 and 102 has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99%) sequence identity of the amino acid sequence. In some embodiments, the anti-CD73 humanized antibody or antigen-binding fragment thereof has an IC50 value for enzymatic activity against said CD73 expressed on the cell surface of about 10 "1 μg/mL to about 10" 2 μg/mL. In some embodiments, the anti-CD73 humanized antibody or antigen-binding fragment thereof binds to CD73 with a KD value of about 10 "8 M to about 10" 11 M.
在一些实施方案中，本文提供了抗CD73人源化抗体或其抗原结合片段，其包括：1)SEQ ID NO:89所示序列的VH和SEQ ID NO:90所示序列的VL(人源化的9E12E3)；2)SEQ ID NO:91所示序列的VH和SEQ ID NO:92所示序列的VL(人源化的35D8G8)；3)SEQ ID NO:93所示序列的VH和SEQ ID NO:94所示序列的VL(人源化的52A6B3)；4)SEQ ID NO:95所示序列的VH和SEQ ID NO:96所示序列的VL(人源化的96A5A5)；5)SEQ ID NO:97所示序列的VH和SEQ ID NO:98所示序列的VL(人源化的134D2H6)；6)SEQ ID NO:99所示序列的VH和SEQ ID NO:100所示序列的VL(人源化的143E5D2)；或7)SEQ ID NO:101所示序列的VH和SEQ ID NO:102所示序列的VL(人源化的22E1B4VL)。In some embodiments, provided herein are anti-CD73 humanized antibodies or antigen-binding fragments thereof, which include: 1) VH of the sequence set forth in SEQ ID NO:89 and VL (human origin) of the sequence set forth in SEQ ID NO:90 9E12E3); 2) VH of the sequence shown in SEQ ID NO: 91 and VL of the sequence shown in SEQ ID NO: 92 (humanized 35D8G8); 3) VH and SEQ of the sequence shown in SEQ ID NO: 93 VL of the sequence shown in ID NO:94 (humanized 52A6B3); 4) VH of the sequence shown in SEQ ID NO:95 and VL of the sequence shown in SEQ ID NO:96 (humanized 96A5A5); 5) VH of the sequence shown in SEQ ID NO:97 and VL of the sequence shown in SEQ ID NO:98 (humanized 134D2H6); 6) VH of the sequence shown in SEQ ID NO:99 and the sequence shown in SEQ ID NO:100 The VL (humanized 143E5D2); or 7) the VH of the sequence shown in SEQ ID NO:101 and the VL of the sequence shown in SEQ ID NO:102 (humanized 22E1B4VL).
在一些实施方案中，本文提供的抗CD73人源化抗体或其抗原结合片段，其进一步包括免疫球蛋白恒定区序列。所述免疫球蛋白恒定区可选自IgG1，IgG2或IgG4。在一些具体实施方案中，所述抗CD73人源化抗体或其抗原结合片段包含人IgG1恒定区序列。在一些实施方案中，所述CD73人源化抗体或其抗原结合片段的重链包含人IgG1重链恒定区序列，所述CD73人源化抗体或其抗原结合片段的轻链包含人Kappa链恒定区。所述人IgG1重链恒定区序列可以是SEQ ID NO:103所示的氨基酸序列，所述人Kappa链恒定区可以是SEQ ID NO:104所示的氨基酸序列。In some embodiments, the anti-CD73 humanized antibodies, or antigen-binding fragments thereof, provided herein further comprise immunoglobulin constant region sequences. The immunoglobulin constant region may be selected from IgG1, IgG2 or IgG4. In some specific embodiments, the anti-CD73 humanized antibody or antigen-binding fragment thereof comprises human IgG1 constant region sequence. In some embodiments, the heavy chain of the CD73 humanized antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region sequence, and the light chain of the CD73 humanized antibody or antigen-binding fragment thereof comprises a human Kappa chain constant region. district. The human IgG1 heavy chain constant region sequence can be the amino acid sequence shown in SEQ ID NO: 103, and the human Kappa chain constant region can be the amino acid sequence shown in SEQ ID NO: 104.
在一些实施方案中，本文提供了抗CD73抗体，其与上文提供的任一抗CD73抗体或其抗原结合片段竞争性结合CD73(即“竞争性抗CD73抗体”)。在一些实施方案中，竞争性抗CD73抗体与上文提供的任一抗CD73抗体或其抗原结合片段竞争性结合CD73上的同一表位。这种竞争性结合可例如通过ELISA测定来进行检测。In some embodiments, provided herein are anti-CD73 antibodies that compete for binding to CD73 with any of the anti-CD73 antibodies or antigen-binding fragments provided above (i.e., "competitive anti-CD73 antibodies"). In some embodiments, the competing anti-CD73 antibody competes for binding to the same epitope on CD73 with any of the anti-CD73 antibodies or antigen-binding fragments provided above. Such competitive binding can be detected, for example, by an ELISA assay.
本文所述的抗CD73抗体(如抗CD73单克隆抗体)可以使用本领域已知的任何方法或如本文所述的方法制备。Anti-CD73 antibodies (eg, anti-CD73 monoclonal antibodies) described herein can be prepared using any method known in the art or as described herein.
啮齿类单克隆抗体可以用本领域已知的方法获得，如通过免疫啮齿类动物(如小鼠或大鼠)并从中获得杂交瘤，或通过使用本领域已知的分子生物学技术克隆Fab片段或单链Fc(scFv)库，随后通过ELISA筛选或使用噬菌体展示进行选择。对于单克隆抗体的重组生产，编码单克隆抗体的核酸被分离或合成，并***到可复制的载体中以进一步克隆(DNA的扩增)或表达。编码单克隆抗体的DNA很容易被分离出来，并使用常规程序进行测序(例如，通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针)。许多载体是可用的。载体的选择部分取决于要使用的宿主细胞。一般来说，宿主细胞可以是原核细胞或真核细胞(例如哺乳动物细胞)。Rodent monoclonal antibodies can be obtained using methods known in the art, such as by immunizing rodents (such as mice or rats) and obtaining hybridomas therefrom, or by cloning Fab fragments using molecular biology techniques known in the art or single-chain Fc (scFv) libraries, followed by selection by ELISA screening or using phage display. For recombinant production of monoclonal antibodies, the nucleic acid encoding the monoclonal antibody is isolated or synthesized and inserted into a replicable vector for further cloning (amplification of DNA) or expression. DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the antibody heavy and light chains). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, the host cell can be a prokaryotic cell or a eukaryotic cell (eg, a mammalian cell).
抗CD73抗体构建体Anti-CD73 antibody construct
本申请另一方面提供编码上述抗体或其抗原结合片段的分离的多核苷酸。本申请的核酸分子可以例如通过标准化学合成方法和/或重组方法合成，或半合成地产生，例如通过组合化学合成和重组方法。编码序列与转录调控元件和/或与其他氨基酸编码序列的连 接可以使用已确立的方法进行，例如限制酶切消化、连接和分子克隆。根据本领域技术人员公知的，改变(如，替换、删除等)编码蛋白的核苷酸序列不会改变蛋白的氨基酸，如密码子优化等方法。Another aspect of the application provides isolated polynucleotides encoding the above-described antibodies or antigen-binding fragments thereof. Nucleic acid molecules of the present application may be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or produced semi-synthetically, for example, by combined chemical synthesis and recombinant methods. Linkage of coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences Ligation can be performed using established methods such as restriction digestion, ligation, and molecular cloning. According to the well-known knowledge of those skilled in the art, changing (eg, replacing, deleting, etc.) the nucleotide sequence encoding the protein will not change the amino acids of the protein, such as codon optimization and other methods.
本申请再一方面提供包含上述多核苷酸的表达载体。本领域技术人员公知的载体，如质粒、噬菌体载体或病毒载体。在一些具体实施方案中，所述载体是重组表达载体，如质粒。这些载体包括任意元件以支撑其常规表达载体的功能，如，包括启动子、核糖体结合元件、终止子、增强子、选择性标记以及复制起点。其中启动子可以是常规启动子、诱导启动子或可抑制启动子。本领域公知许多表达载体能够将核酸传递至细胞内，并且能用于在细胞内产生抗体或其抗原结合片段。根据本发明实施例中的方法，常规的克隆技术或人工基因合成均可用于产生重组表达载体。Another aspect of the present application provides an expression vector comprising the above polynucleotide. Vectors well known to those skilled in the art, such as plasmids, phage vectors or viral vectors. In some specific embodiments, the vector is a recombinant expression vector, such as a plasmid. These vectors include any elements to support the functions of conventional expression vectors, including, for example, promoters, ribosome binding elements, terminators, enhancers, selectable markers, and origins of replication. The promoter can be a conventional promoter, an inducible promoter or a repressible promoter. Many expression vectors are known in the art that are capable of delivering nucleic acids into cells and can be used to produce antibodies or antigen-binding fragments thereof within cells. According to the methods in the embodiments of the present invention, conventional cloning technology or artificial gene synthesis can be used to generate recombinant expression vectors.
本申请又一方面提供包含上述表达载体的宿主细胞或无细胞表达***。在本发明从任何本领域常规的宿主细胞均可用于抗体或其抗原结合片段的表达。在一些实施方案中，所述宿主细胞是E.coli TG1或BL21(用于表达如scFv或Fab抗体)，CHO-DG44、CHO-3E7、CHO-K1或者HEK293。按照特定实施例，通过常规方法(如化学转染、热转染或电转染等方法)将重组表达载体转染入宿主细胞，稳定整合到宿主细胞基因组，因此能有效表达重组的核酸。本申请所述无细胞表达***是一种区别于传统蛋白表达***的新型蛋白制备***，以DNA或mRNA为模板，利用细胞抽提物中的细胞器，蛋白折叠因子及其他相关酶系，通过添加氨基酸、核苷酸、聚合酶和能量物质来实现蛋白表达的体外体统。Another aspect of the present application provides a host cell or cell-free expression system comprising the above-mentioned expression vector. In the present invention, any host cell conventional in the art can be used for the expression of antibodies or antigen-binding fragments thereof. In some embodiments, the host cell is E. coli TG1 or BL21 (for expression of, for example, scFv or Fab antibodies), CHO-DG44, CHO-3E7, CHO-K1, or HEK293. According to specific embodiments, the recombinant expression vector is transfected into the host cell through conventional methods (such as chemical transfection, thermal transfection, or electrotransfection) and is stably integrated into the host cell genome, so that the recombinant nucleic acid can be effectively expressed. The cell-free expression system described in this application is a new protein preparation system that is different from traditional protein expression systems. It uses DNA or mRNA as a template and utilizes organelles, protein folding factors and other related enzymes in cell extracts, by adding Amino acids, nucleotides, polymerases and energy substances are used to achieve protein expression in vitro.
融合蛋白fusion protein
在一些实施方案中，抗CD73抗体的抗原结合片段可以与Fc片段连接形成融合蛋白。Fc片段可位于抗CD73抗体的抗原结合片段的C末端和N末端。优选地，Fc片段可位于抗CD73抗体的抗原结合片段的C末端。抗CD73抗体的抗原结合片段与Fc片段形成的融合蛋白具有特异性结合CD73的能力，同时具有Fc片段的效应功能，例如介导补体依赖性细胞毒性(CDC)、抗体依赖性细胞介导的细胞毒性(ADCC)、介导吞噬作用等。另外，与Fc片段的融合可增加抗CD73抗体的抗原结合片段在体内的半衰期，以便在将抗CD73抗体的抗原结合片段用作治疗药物时增加其给药间隔时间。在一些实施方案中，所述Fc片段可来源于免疫球蛋白的恒定区，如IgG1，IgG2或IgG4。In some embodiments, the antigen-binding fragment of an anti-CD73 antibody can be linked to an Fc fragment to form a fusion protein. The Fc fragment can be located at the C-terminus and N-terminus of the antigen-binding fragment of the anti-CD73 antibody. Preferably, the Fc fragment may be located at the C-terminus of the antigen-binding fragment of the anti-CD73 antibody. The fusion protein formed by the antigen-binding fragment of the anti-CD73 antibody and the Fc fragment has the ability to specifically bind to CD73 and also has the effector function of the Fc fragment, such as mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cell death. Toxicity (ADCC), mediating phagocytosis, etc. In addition, fusion to the Fc fragment can increase the half-life of the antigen-binding fragment of the anti-CD73 antibody in vivo, thereby increasing the dosing interval when the antigen-binding fragment of the anti-CD73 antibody is used as a therapeutic drug. In some embodiments, the Fc fragment can be derived from the constant region of an immunoglobulin, such as IgGl, IgG2 or IgG4.
在一些实施方案中，抗CD73抗体的抗原结合片段可以与蛋白标签连接形成融合蛋白。蛋白标签可包括纯化标签和可检测标签。纯化标签包括但不限于His6标签、Flag标签、MBP标签、GST标签、SUMO)标签等。可检测标签可用于指示样品中抗CD73抗体或其抗原结合片段的存在或含量，或者用于抗CD73抗体或其抗原结合片段在受试者体内或细胞内的位置信息。可检测标签的实例包括免疫检测中可使用的各种酶，例如辣根过氧化物酶(HRP)、碱性磷酸酶(ALP)等；荧光蛋白，例如GFP。由于抗CD73抗体或其抗原 结合片段与CD73的特异性结合能力，可通过与抗CD73抗体或其抗原结合片段连接的可检测标签的量来确定抗CD73抗体或其抗原结合片段量，并进而确定样品中CD73的含量。In some embodiments, the antigen-binding fragment of an anti-CD73 antibody can be linked to a protein tag to form a fusion protein. Protein tags can include purification tags and detectable tags. Purification tags include but are not limited to His6 tag, Flag tag, MBP tag, GST tag, SUMO) tag, etc. The detectable label can be used to indicate the presence or content of the anti-CD73 antibody or antigen-binding fragment thereof in a sample, or to provide information about the location of the anti-CD73 antibody or antigen-binding fragment thereof in the body or cells of a subject. Examples of detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; fluorescent proteins, such as GFP. Due to anti-CD73 antibodies or its antigens The specific binding ability of the binding fragment to CD73 can be determined by the amount of the detectable tag connected to the anti-CD73 antibody or its antigen-binding fragment, and thereby the content of CD73 in the sample.
在另一些实施方案中，抗CD73抗体或其抗原结合片段可以与细胞因子或治疗性蛋白连接形成融合蛋白。这种情况下，可以借助抗CD73抗体或其抗原结合片段与CD73的特异性结合能力，将细胞因子或治疗性蛋白有目的地输送至特定的组织或细胞，实现细胞因子或治疗性蛋白的治疗作用。In other embodiments, anti-CD73 antibodies or antigen-binding fragments thereof can be linked to cytokines or therapeutic proteins to form fusion proteins. In this case, the specific binding ability of anti-CD73 antibodies or antigen-binding fragments thereof to CD73 can be used to deliver cytokines or therapeutic proteins to specific tissues or cells in a targeted manner to achieve treatment with cytokines or therapeutic proteins. effect.
多特异性抗体multispecific antibodies
在一些实施方案中，本文提供了多特异性的结合肽，其至少包括一个结合CD73的结构域(或功能单元)和一个或更多个额外的结合结构域。该一个或更多个额外的结合结构域可结合至除CD73外的第二抗原或蛋白质。In some embodiments, provided herein are multispecific binding peptides that include at least one domain (or functional unit) that binds CD73 and one or more additional binding domains. The one or more additional binding domains may bind to a second antigen or protein other than CD73.
在一些实施方案中，该多特异性抗体为双特异性抗体。In some embodiments, the multispecific antibody is a bispecific antibody.
在一些实施方案中，该双特异性抗体的第一抗原结合结构域包括本文提供的抗CD73抗体或其抗原结合片段，能够特异性结合CD73；第二抗原结合结构域能够特异性结合抑制性免疫检查点分子，例如特异性结合CTLA-4、PD-L1、TIM-3或LAG-3。In some embodiments, the first antigen-binding domain of the bispecific antibody includes an anti-CD73 antibody or an antigen-binding fragment thereof provided herein and is capable of specifically binding to CD73; the second antigen-binding domain is capable of specifically binding to suppressive immunity. Checkpoint molecules, such as those that specifically bind CTLA-4, PD-L1, TIM-3 or LAG-3.
因此，在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合CTLA-4。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。Thus, in one embodiment, the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding CTLA-4. Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合PD-L1或PD-1。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。In one embodiment, the first antigen-binding domain of the bispecific antibody is capable of binding CD73 and the second antigen-binding domain is capable of binding PD-L1 or PD-1. Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合TIM-3。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。In one embodiment, the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding TIM-3. Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合LAG-3。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。In one embodiment, the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding LAG-3. Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一些实施方案中，该双特异性抗体的第一抗原结合结构域包括本文提供的抗CD73抗体或其抗原结合片段，能够特异性结合CD73；第二抗原结合结构域能够特异性结合CD39-CD73-腺苷通路上的蛋白(酶或受体)。In some embodiments, the first antigen-binding domain of the bispecific antibody includes an anti-CD73 antibody or an antigen-binding fragment thereof provided herein and is capable of specifically binding to CD73; the second antigen-binding domain is capable of specifically binding to CD39-CD73 -Proteins (enzymes or receptors) in the adenosine pathway.
在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合CD39。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。 In one embodiment, the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding CD39. Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一个实施方案中，该双特异性抗体的第一抗原结合结构域能够结合CD73，第二抗原结合结构域能够结合腺苷2A受体(A2AR)。优选地，该双特异性抗体的第一抗原结合结构域为scFv形式，第二抗原结合结构域为scFv形式或sdAb形式。In one embodiment, the first antigen binding domain of the bispecific antibody is capable of binding CD73 and the second antigen binding domain is capable of binding adenosine 2A receptor (A2AR). Preferably, the first antigen-binding domain of the bispecific antibody is in the form of scFv, and the second antigen-binding domain is in the form of scFv or sdAb.
在一些实施方案中，双特异性抗体还包括Fc片段。Fc片段的存在可方便地形成结合结构域的多聚化，并且可提供相关的效应功能。In some embodiments, the bispecific antibody further includes an Fc fragment. The presence of the Fc fragment facilitates multimerization of binding domains and may provide associated effector functions.
药物组合物和治疗方法Pharmaceutical compositions and methods of treatment
本文公开的抗CD73抗体或其抗原结合片段与CD73的特异性结合能力与对照抗体MEDI9447类似，部分CD73抗体或其抗原结合片段的结合能力优于对照抗体MEDI9447。本文公开的抗CD73抗体或其抗原结合片段对CD73抑制能力与对照抗体MEDI9447类似，部分抗CD73抗体或其抗原结合片段的抑制能力优于对照抗体MEDI9447。The specific binding ability of the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein to CD73 is similar to that of the control antibody MEDI9447, and the binding ability of some CD73 antibodies or antigen-binding fragments thereof is better than that of the control antibody MEDI9447. The anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein have similar inhibitory abilities to CD73 as the control antibody MEDI9447, and some anti-CD73 antibodies or antigen-binding fragments thereof have better inhibitory abilities than the control antibody MEDI9447.
本文公开的抗CD73抗体或其抗原结合片段以及包括该抗CD73抗体或其抗原结合片段的融合蛋白或多特异性抗体分子可以与药学上可接受的载体一起配制为药物组合物，对受试者给药以进行肿瘤预防或治疗。The anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein, as well as fusion proteins or multispecific antibody molecules including the anti-CD73 antibodies or antigen-binding fragments thereof, can be formulated into pharmaceutical compositions together with pharmaceutically acceptable carriers, and administered to subjects. Administered for tumor prevention or treatment.
在一些实施方式中，本文公开的抗CD73抗体或其抗原结合片段以及包括该抗CD73抗体或其抗原结合片段的融合蛋白或多特异性抗体分子可以与一种或更多种其他药物(例如抗肿瘤剂)联合给药。在一些实施方案中，抗肿瘤剂可以为小分子化合物，例如CD39抑制剂、CD73抑制剂或A2AR抑制剂。In some embodiments, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein, as well as fusion proteins or multispecific antibody molecules including the anti-CD73 antibodies or antigen-binding fragments thereof, can be combined with one or more other drugs (e.g., anti-CD73 antibodies or antigen-binding fragments thereof). tumor agent) combined administration. In some embodiments, the anti-tumor agent can be a small molecule compound, such as a CD39 inhibitor, a CD73 inhibitor, or an A2AR inhibitor.
在一些实施方案中，所治疗的疾病或病症为肿瘤，优选实体瘤。所述实体瘤可以是转移性结直肠癌、激素难治性***癌、黑色素瘤、转移性头颈癌、转移性卵巢癌、鳞状细胞癌等，尤其是非小细胞肺癌(NSCLC)、三阴性乳腺癌(TNBC)和胰腺癌等。可以使用任何合适的方法或途径以本文公开的抗CD73抗体或其抗原结合片段(以及包括抗CD73抗体或其抗原结合片段的融合蛋白或多特异性抗体分子)给药，并任选地联用其他抗肿瘤剂。给药途径包括，例如，口服、静脉内、腹膜内、皮下或者肌内给药。In some embodiments, the disease or condition treated is a tumor, preferably a solid tumor. The solid tumor may be metastatic colorectal cancer, hormone-refractory prostate cancer, melanoma, metastatic head and neck cancer, metastatic ovarian cancer, squamous cell carcinoma, etc., especially non-small cell lung cancer (NSCLC), triple-negative breast cancer cancer (TNBC) and pancreatic cancer, etc. The anti-CD73 antibodies or antigen-binding fragments thereof (and fusion proteins or multispecific antibody molecules comprising the anti-CD73 antibodies or antigen-binding fragments thereof) disclosed herein may be administered using any suitable method or route, and optionally in combination. Other antineoplastic agents. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.
编码抗CD73抗体或其抗原结合片段的核酸分子、包括该核酸分子的表达载体以及经所述核酸分子或表达载体转染的宿主细胞也可通过各种方式用于上述治疗目的。例如，对于表达载体，可通过本领域已知的基因治疗手段引入受试者体内，表达目的蛋白或多肽(抗CD73抗体或其抗原结合片段以及包括抗CD73抗体或其抗原结合片段的融合蛋白或多特异性抗体分子)，从而达到治疗目的。Nucleic acid molecules encoding anti-CD73 antibodies or antigen-binding fragments thereof, expression vectors including the nucleic acid molecules, and host cells transfected with the nucleic acid molecules or expression vectors can also be used for the above therapeutic purposes in various ways. For example, the expression vector can be introduced into the subject through gene therapy methods known in the art to express the protein or polypeptide of interest (anti-CD73 antibody or antigen-binding fragment thereof and a fusion protein including anti-CD73 antibody or antigen-binding fragment thereof or multispecific antibody molecules) to achieve therapeutic purposes.
该用途的有效用量可取决于疾病的严重性和患者自身免疫***的总体状态。给药方案将也随疾病状态和受试者状态而变动，并且一般范围将从单次大量(bolus)给药或连续输注至每天多次给药(例如每隔4-6小时)。本领域熟练的临床医生可以例如通过利用临床试验、身体检查和受试者家族史，容易地确定某受试者是否为这种治疗的候选者。 The effective dosage for this purpose may depend on the severity of the disease and the overall state of the patient's own immune system. Dosage regimens will also vary with the disease state and subject status, and will generally range from a single bolus dose or continuous infusion to multiple doses per day (eg, every 4-6 hours). Clinicians skilled in the art can readily determine whether a subject is a candidate for such treatment, for example, by utilizing clinical trials, physical examinations, and the subject's family history.
包括抗CD73抗体或其抗原结合片段检测或治疗试剂盒Including anti-CD73 antibody or its antigen-binding fragment detection or treatment kit
本文提供的抗CD73抗体或其抗原结合片段以及包括抗CD73抗体或其抗原结合片段的其他形式分子(例如融合蛋白或双特异性抗体分子)可与样品中的CD73特异性结合。通过检测所形成的抗CD73抗体或其抗原结合片段-CD73复合物的量，或者所形成的抗CD73抗体或其抗原结合片段-CD73复合物中抗CD73抗体或其抗原结合片段的量，可方便的确定样品中的CD73的含量(或是否存在)。The anti-CD73 antibodies or antigen-binding fragments thereof provided herein, as well as other forms of molecules including anti-CD73 antibodies or antigen-binding fragments thereof (eg, fusion proteins or bispecific antibody molecules), can specifically bind to CD73 in a sample. By detecting the amount of the anti-CD73 antibody or its antigen-binding fragment-CD73 complex formed, or the amount of the anti-CD73 antibody or its antigen-binding fragment in the formed anti-CD73 antibody or its antigen-binding fragment-CD73 complex, it is convenient to Determine the amount (or presence) of CD73 in the sample.
如上文所描述的，用于该目的，本文提供的抗CD73抗体或其抗原结合片段可与各种检测标签连接，方便通过各种手段进行检测，这包括但不限于生物发光、荧光、放射性标记、酶促反应的产物生产量等。检测样品中CD73含量后，与正常人群中正常CD73含量进行比较，可用于确定提供该样品的受试者的疾病状况或严重程度。通过在受试者治疗过程随时间反复检测CD73含量变化，还可用于确定治疗手段是否有效，从而为治疗方案的改动提供依据。As described above, for this purpose, the anti-CD73 antibodies or antigen-binding fragments thereof provided herein can be linked to various detection tags to facilitate detection by various means, including but not limited to bioluminescence, fluorescence, radioactive labeling , the product production volume of enzymatic reaction, etc. After detecting the CD73 content in the sample, comparison with the normal CD73 content in the normal population can be used to determine the disease status or severity of the subject who provided the sample. By repeatedly detecting changes in CD73 levels over time during a subject's treatment, it can also be used to determine whether the treatment is effective, thereby providing a basis for changes in the treatment plan.
本文提供的抗CD73抗体或其抗原结合片段以及包括该抗CD73抗体或其抗原结合片段的其他形式分子(例如融合蛋白或双特异性抗体分子)可被置于容器中，以形成检测断或治疗试剂盒。这些容器可以是盒、安瓿瓶、小瓶、管子、袋子或本领域已知的合适容器形式。这些容器可以由塑料、玻璃、层压纸、金属箔或适用于保存药物的其他材料制成。如果需要，与该容器一起提供的还包括使用说明书。说明书通常可包括关于如何使用抗CD73抗体或其抗原结合片段结合肽或包括抗CD73抗体或其抗原结合片段的组合物用于治疗或预防肿瘤(例如实体瘤)的信息，例如可包括对治疗剂(如抗CD73抗体或其抗原结合片段)的描述；用于治疗或预防瘤形成(例如多发性骨髓瘤)的剂量方案；注意事项；警告；适应症；禁忌症；不良反应；动物药理学；临床研究；和/或参考资料。说明书可直接打印在容器(如果存在的话)上，或作为贴于容器的标签，或者作为提供于容器中或与容器在一起的单独的纸、册、卡或折叠印刷品。The anti-CD73 antibodies or antigen-binding fragments thereof provided herein, as well as other forms of molecules including the anti-CD73 antibodies or antigen-binding fragments thereof (e.g., fusion proteins or bispecific antibody molecules), can be placed in a container to form a assay or therapy. Reagent test kit. These containers may be in the form of boxes, ampoules, vials, tubes, bags or other suitable containers known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for preserving medications. If required, instructions for use are also provided with the container. Instructions may generally include information on how to use an anti-CD73 antibody, or antigen-binding fragment thereof, in conjunction with a peptide, or a composition comprising an anti-CD73 antibody, or antigen-binding fragment thereof, for the treatment or prevention of tumors (eg, solid tumors), and may include, for example, guidance on therapeutic agents (e.g., anti-CD73 antibodies or antigen-binding fragments thereof); dosage regimens for the treatment or prevention of neoplasia (e.g., multiple myeloma); precautions; warnings; indications; contraindications; adverse reactions; animal pharmacology; Clinical studies; and/or reference materials. Instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate paper, booklet, card or folded print provided in or with the container.
实施例1:抗CD73单克隆抗体的制备Example 1: Preparation of anti-CD73 monoclonal antibodies
免疫接种Immunization
根据现行动物福利法规，用人类CD73-HIS蛋白(R&D SYSTEMS,Cat#5795-EN)免疫3只Balb/c和3只A/J小鼠。将抗原溶在PBS溶液中，或与CFA(弗氏完全佐剂；初次免疫)或IFA(弗氏不完全佐剂；增强免疫)形成乳剂进行免疫。在一系列免疫方法中，抗原通过腹腔注射或背部皮下注射免疫。每只动物接受4剂(第一针免疫为每只鼠50μg，然后在接下来的三针是每只鼠25μg)。在免疫过程中，在每个免疫时间点后7天，从动物身上采集20μL血样，通过包被hCD73蛋白(R&D SYSTEMS，Cat#5795-EN)的ELISA方法检测血清中抗血清滴度，直到满足融合标准。在本发明中，5只小鼠(2只Balb/c小鼠和3只A-J小鼠)血清效价高，用于融合。In accordance with current animal welfare regulations, 3 Balb/c and 3 A/J mice were immunized with human CD73-HIS protein (R&D SYSTEMS, Cat#5795-EN). Dissolve the antigen in PBS solution, or form an emulsion with CFA (complete Freund's adjuvant; primary immunization) or IFA (incomplete Freund's adjuvant; enhanced immunity) for immunization. In a range of immunization methods, the antigen is administered intraperitoneally or subcutaneously in the back. Each animal received 4 doses (the first immunization was 50 μg per mouse, then the next three doses were 25 μg per mouse). During the immunization process, 20 μL blood samples were collected from the animals 7 days after each immunization time point, and the antiserum titer in the serum was detected by ELISA coated with hCD73 protein (R&D SYSTEMS, Cat#5795-EN) until the Fusion standards. In the present invention, 5 mice (2 Balb/c mice and 3 A-J mice) with high serum titers were used for fusion.
分泌抗hCD73抗体杂交瘤的筛选 Screening of hybridomas secreting anti-hCD73 antibodies
最后一次免疫后三天，按照标准杂交瘤生成方案，在无菌环境中提取选定小鼠的脾细胞并与sp2/0细胞融合。融合细胞在含有1×HAT(次黄嘌呤、氨基蝶呤、胸腺嘧啶核苷)以及10％的胎牛血清的DMEM培养基中培养6天。在本发明中，总共筛选了160个96孔板杂交瘤。通过ELISA分析所有杂交瘤上清液与人CD73的结合能力。根据ELISA信号(OD450)筛选抗人CD73的阳性克隆。在本发明中，通过ELISA选择了408株从A-J小鼠产生的抗hCD73杂交瘤(ELISA OD>2.0)和62株从Balb/c小鼠产生的抗hCD73杂交瘤(ELISA OD>0.3)，之后用流式细胞仪检测它们与过表达人CD73的CHO-K1稳定细胞系的结合能力，共有159株杂交瘤与细胞表面hCD73结合，这些亲代杂交瘤通过有限稀释进行亚克隆。将100μl细胞悬液梯度稀释至1-3个细胞/孔，在含有1×HAT(次黄嘌呤、氨基蝶呤、胸腺嘧啶核苷)以及10％的胎牛血清的DMEM培养基中培养。细胞培养1周后，使用ELISA和FACS进行新一轮筛选，直到获得阳性单克隆。选定的每个独特的克隆用于生产0.5mg杂交瘤纯化抗体，该抗体用于进一步鉴定。总共选择了124个阳性单克隆进行抗体生产和纯化，并通过ELISA进行抗体亲和力测定(表1)。检测抗体同种型(Clonotyping System-HRP,SouthernBiotech；Birmingham,AL)，用Protein-A磁珠(GenScript，Cat#L00695)纯化抗体，用0.5M柠檬酸钠溶液(pH3.5)洗脱，并用0.5M Tris-HCl(pH9.0)中和。将储存缓冲液改为PBS，用nanodrops(Thermofisher,Cat#ND-ONE-W)测定浓度。Three days after the last immunization, spleen cells from selected mice were extracted in a sterile environment and fused with sp2/0 cells following standard hybridoma generation protocols. Fusion cells were cultured in DMEM medium containing 1×HAT (hypoxanthine, aminopterin, thymidine) and 10% fetal calf serum for 6 days. In the present invention, a total of 160 96-well plate hybridomas were screened. All hybridoma supernatants were analyzed for their binding ability to human CD73 by ELISA. Anti-human CD73 positive clones were screened based on ELISA signal (OD450). In the present invention, 408 anti-hCD73 hybridoma strains generated from A-J mice (ELISA OD>2.0) and 62 anti-hCD73 hybridoma strains generated from Balb/c mice (ELISA OD>0.3) were selected by ELISA. Flow cytometry was used to detect their binding ability to the CHO-K1 stable cell line overexpressing human CD73. A total of 159 hybridoma strains bound to cell surface hCD73. These parental hybridomas were subcloned through limiting dilution. 100 μl of cell suspension was serially diluted to 1-3 cells/well and cultured in DMEM medium containing 1×HAT (hypoxanthine, aminopterin, thymidine) and 10% fetal calf serum. After 1 week of cell culture, a new round of screening was performed using ELISA and FACS until positive single clones were obtained. Each unique clone selected was used to produce 0.5 mg of hybridoma-purified antibody, which was used for further characterization. A total of 124 positive monoclones were selected for antibody production and purification, and antibody affinity determination was performed by ELISA (Table 1). Antibody isotypes were detected (Clonotyping System-HRP, Southern Biotech; Birmingham, AL), antibodies were purified with Protein-A magnetic beads (GenScript, Cat# L00695), eluted with 0.5 M sodium citrate solution (pH 3.5), and eluted with Neutralized with 0.5M Tris-HCl (pH9.0). Change the storage buffer to PBS and determine the concentration using nanodrops (Thermofisher, Cat# ND-ONE-W).
表1:ELISA测定的124个单克隆上清液中抗体与CD73的结合信号

Table 1: Binding signals of antibodies and CD73 in 124 monoclonal supernatants measured by ELISA

实施例2:抗hCD73小鼠抗体的特性Example 2: Characteristics of anti-hCD73 mouse antibodies
抗CD73小鼠抗体体外功能测定In vitro functional assay of anti-CD73 mouse antibodies
抗CD73小鼠抗体的功能测定基于Calu-6细胞表面CD73酶的活性，该酶的活性可以反应在非小细胞肺癌(NSCLC)细胞中真正的翻译后修饰。使用Promega试剂盒(CellTiter试剂盒，目录号#：G7571)进行抗CD73小鼠抗体功能测定，以抑制Calu-6细胞表面CD73酶活性。已知过量的AMP可阻断ATP依赖的荧光素酶活性，Calu-6细胞上的CD73能将AMP分解为腺苷和无机磷酸盐，可以增强荧光素酶活性并发光。因此，阻断CD73酶活性的抗体将减少光发射。共检测了124种1μg/ml的抗体对CD73蛋白活性的拮抗能力。选择单剂量抑制率大于50％的抗体进行多剂量抑制试验和IC50测定。简而言之，Calu-6细胞(ATCC，分类号：HTB-56TM)接种到96孔培养皿中，37℃培养过夜。然后，用含有不同浓度的抗CD73抗体的新鲜培养基替换原培养基，加至相应的培养板孔。在室温下培养分析板30分钟。为了检测CD73活性，将AMP溶液添加到细胞中，然后在37℃下培养2小时。为了将AMP(SIGMA)定量，将培养上清液转移到另一个96孔分析板上，并向相应的孔中添加100μM ATP(SIGMA)。在室温下培养5～10分钟后，将CellTiter Glo溶液添加到96孔分析板中，与Phrastar(BMG LABTECH，GR18010766)温和混合并读取分析板数据。抗体的抑制作用取决于AMP的量。ATP+AMP表示最大荧光素酶抑制(100％)，ATP+AMP+CD73表示无荧光素酶抑制(0％)。剩余CD73活性的公式如下所示：The functional assay of the anti-CD73 mouse antibody is based on the activity of the Calu-6 cell surface CD73 enzyme, which reflects true post-translational modifications in non-small cell lung cancer (NSCLC) cells. Promega kit (CellTiter Kit, Catalog #: G7571) to perform anti-CD73 mouse antibody functional assay to inhibit the activity of CD73 enzyme on the surface of Calu-6 cells. It is known that excess AMP can block ATP-dependent luciferase activity. CD73 on Calu-6 cells can decompose AMP into adenosine and inorganic phosphate, which can enhance luciferase activity and emit light. Therefore, antibodies that block CD73 enzymatic activity will reduce light emission. A total of 124 antibodies at 1 μg/ml were tested for their ability to antagonize CD73 protein activity. Select antibodies with single-dose inhibition rates greater than 50% for multi-dose inhibition tests and IC50 determination. Briefly, Calu-6 cells (ATCC, classification number: HTB-56 TM ) were seeded into 96-well culture dishes and cultured at 37°C overnight. Then, the original medium was replaced with fresh medium containing anti-CD73 antibodies at different concentrations and added to the corresponding culture plate wells. Incubate the assay plate for 30 minutes at room temperature. To detect CD73 activity, AMP solution was added to cells and then incubated at 37°C for 2 hours. To quantify AMP(SIGMA), transfer the culture supernatant to another 96-well assay plate and add 100 μM ATP(SIGMA) to the corresponding wells. After incubating at room temperature for 5 to 10 minutes, add the CellTiter Glo solution to the 96-well assay plate, mix gently with Phrastar (BMG LABTECH, GR18010766) and read the assay plate data. The inhibitory effect of antibodies depends on the amount of AMP. ATP+AMP indicates maximum luciferase inhibition (100%), ATP+AMP+CD73 indicates no luciferase inhibition (0%). The formula for remaining CD73 activity is as follows:
剩余CD73活性＝((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100Remaining CD73 activity = ((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100
抗CD73抗体抑制活性的公式如下：The formula for the inhibitory activity of anti-CD73 antibodies is as follows:
抑制率％(抗CD73抗体活性)＝(1-((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100Inhibition rate % (anti-CD73 antibody activity) = (1-((ATP+AMP+CD73+Abs)-(ATP+AMP)/(ATP+AMP+CD73)-(ATP+AMP))*100
通过Calu-6细胞共检测了20种抗CD73抗体对CD73蛋白的IC50(最大抑制浓度的一半)，8种抗体显示出与MEDI9447相当或优于MEDI9447的抑制能力，包括52A6B3、 52A8B3、96A5A5、100C5A3、134D2H6、141G7E9、143E5D2以及143H3C5(见图1-3)。为了确定抗体对交叉物种CD73的结合，通过流式细胞术检测20种抗CD73抗体对表达人(Probio，货号：RD00783)、食蟹猴(Probio，货号：RD00783)和小鼠CD73(Probio，货号：RD00783)CHO细胞(ATCC，分类号：CCL-61TM)的结合。三种抗体先导物9E12E3、22E1B4和35D8G8可结合小鼠源和食蟹猴源的CD73，根据图4和表2列出的Calu-6细胞表面CD73活性的IC50值，三种抗体结合CD73活性的IC50和MEDI-9447相当。将上述11种小鼠抗体(包括9E12E3、22E1E4、35D8G8、52A6B3、52A8B3、96A5A5、100C5A3、134D2H6、141G7E9、143E5D2和143H3C5)进行测序。表3列出了这些抗CD73抗体的VH、VL和CDR氨基酸序列。A total of 20 anti-CD73 antibodies were tested for IC 50 (half the maximum inhibitory concentration) of CD73 protein through Calu-6 cells, and 8 antibodies showed inhibitory capabilities equivalent to or better than MEDI9447, including 52A6B3, 52A8B3, 96A5A5, 100C5A3, 134D2H6, 141G7E9, 143E5D2 and 143H3C5 (see Figure 1-3). To determine antibody binding to cross-species CD73, 20 anti-CD73 antibodies expressing human (Probio, Catalog No. RD00783), cynomolgus monkey (Probio, Catalog No. RD00783), and mouse CD73 (Probio, Catalog No. : RD00783) CHO cells (ATCC, classification number: CCL-61 TM ). Three antibody leads, 9E12E3, 22E1B4 and 35D8G8, can bind to CD73 from mouse and cynomolgus monkey sources. According to the IC 50 values of CD73 activity on the surface of Calu-6 cells listed in Figure 4 and Table 2, the three antibodies bind to CD73 activity. IC 50 is equivalent to MEDI-9447. The above 11 kinds of mouse antibodies (including 9E12E3, 22E1E4, 35D8G8, 52A6B3, 52A8B3, 96A5A5, 100C5A3, 134D2H6, 141G7E9, 143E5D2 and 143H3C5) were sequenced. Table 3 lists the VH, VL and CDR amino acid sequences of these anti-CD73 antibodies.
FACS检测鼠抗体与人CD73+/CHO-K1过表达细胞的结合FACS detection of binding of mouse antibodies to human CD73+/CHO-K1 overexpressing cells
为了验证抗体与细胞表面抗原结合，大约1×105个过表达人的CD73(Probio,Cat No:RD00783)的CHO-K1细胞(ATCC,Cat No:CCL-61TM)进行反应，之后以3倍连续稀释抗CD73单克隆抗体(45至7.6×10-4μg/ml)与细胞孵育，然后用1μg/mL荧光团(iFluor 647)标记的山羊抗小鼠IgG(H+L)二抗染色。以平均荧光强度表示结合亲和力，如图5所示。抗体52A6B3、52A8B3、96A5A5、100C5A3和134D2H6的EC50低于MEDI9447，显示其结合能力比MEDI9447好。而141G7E9、143E5D2和143H3C5的EC50高于MEDI9447。表4列出了所有抗体的EC50。To verify the binding of the antibody to the cell surface antigen, approximately 1×10 5 CHO-K1 cells (ATCC, Cat No: CCL-61 TM ) overexpressing human CD73 (Probio, Cat No: RD00783) were reacted, followed by 3 Serially diluted anti-CD73 monoclonal antibody (45 to 7.6×10 -4 μg/ml) was incubated with cells, and then stained with 1 μg/mL fluorophore (iFluor 647)-labeled goat anti-mouse IgG (H+L) secondary antibody. . The binding affinity was expressed as the average fluorescence intensity, as shown in Figure 5. The EC 50 of antibodies 52A6B3, 52A8B3, 96A5A5, 100C5A3 and 134D2H6 is lower than MEDI9447, indicating that their binding ability is better than MEDI9447. The EC 50 of 141G7E9, 143E5D2 and 143H3C5 is higher than MEDI9447. Table 4 lists the EC50 for all antibodies.
表2:抗CD73小鼠抗体对Calu-6细胞表面的人CD73的IC50
Table 2: IC50 of anti-CD73 mouse antibodies against human CD73 on the surface of Calu-6 cells
表3:11种抗CD73小鼠抗体的VH、VL和CDR氨基酸序列


Table 3: VH, VL and CDR amino acid sequences of 11 anti-CD73 mouse antibodies


表4：抗CD73小鼠单克隆抗体FACS结合数据的EC50

Table 4: EC 50 for anti-CD73 mouse monoclonal antibody FACS binding data

实施例3人源化抗体的制备与分析Example 3 Preparation and analysis of humanized antibodies
候选抗体的人源化设计Humanized design of candidate antibodies
基于抗体可变结构域序列，分析CDRs、HV环和FRs，并进行同源性建模，以获得小鼠抗体的模型结构。计算了骨架残留物的溶剂可及表面积，根据结果，确定了掩埋的骨架氨基酸残基(速溶剂可及表面积<15％)。选择了一个VH和VL的人类受体，该受体与小鼠对应物具有相同的顶部序列。将小鼠抗体的CDR直接移植到人类受体框架上(Human-IgG1)，以获得移植抗体的序列，而无任何反向突变，其中某些氨基酸被回复成小鼠框架序列。七个克隆包括9E12E3、35D8G8、52A6B3、96A5A5、134D2H6、143E5D2、22E1B4，均已成功人源化。氨基酸序列如表5所示(所采用的恒定区序列如SEQ ID NO：103和104所示)。Based on the antibody variable domain sequence, CDRs, HV loops and FRs were analyzed, and homology modeling was performed to obtain the model structure of the mouse antibody. The solvent-accessible surface area of the backbone residues was calculated, and based on the results, the buried backbone amino acid residues (fast solvent-accessible surface area <15%) were determined. A human receptor for VH and VL was chosen that had the same top sequence as the mouse counterpart. The CDRs of the mouse antibody are directly grafted onto the human acceptor framework (Human-IgG1) to obtain the sequence of the grafted antibody without any reverse mutation, in which certain amino acids are reverted to the mouse framework sequence. Seven clones including 9E12E3, 35D8G8, 52A6B3, 96A5A5, 134D2H6, 143E5D2, and 22E1B4 have been successfully humanized. The amino acid sequence is shown in Table 5 (the constant region sequences used are shown in SEQ ID NO: 103 and 104).
合成了编码人源化轻链和重链的DNA序列，导入pcDNA3.4，并转染Expi293F细胞(Genscript Probio，目录号：SC1975)以产生抗体蛋白。DNA sequences encoding humanized light and heavy chains were synthesized, introduced into pcDNA3.4, and transfected into Expi293F cells (Genscript Probio, catalog number: SC1975) to produce antibody proteins.
将抗体特性与MEDI-9447进行比较Comparing antibody properties to MEDI-9447
使用Biacore 8K(Cytiva，Cat#29125379)通过表面等离子体共振(SPR)将人源化抗体与同源小鼠抗体进行比较。简单地说，小鼠和人类抗体通过传感器芯片蛋白A(Cytiva，Cat#：29127555)与蛋白A结合进行亲和捕获。人类CD73CED蛋白(R&D SYSTEMS，Cat#5795-EN)处于流动相，采用结合和解离用1:1的结合模型。关联接触时间为120秒，分离接触时间为360秒，流速为30μl/min。抗体-抗原结合亲和性结果如表6所示，抗体在人源化后与CD73的结合亲和力与鼠源抗体相当。Humanized antibodies were compared to syngeneic mouse antibodies by surface plasmon resonance (SPR) using Biacore 8K (Cytiva, Cat#29125379). Briefly, mouse and human antibodies were affinity captured by binding to Protein A via the sensor chip Protein A (Cytiva, Cat#: 29127555). Human CD73CED protein (R&D SYSTEMS, Cat#5795-EN) is in the mobile phase, using a 1:1 binding model for association and dissociation. The association contact time was 120 seconds, the separation contact time was 360 seconds, and the flow rate was 30 μl/min. The antibody-antigen binding affinity results are shown in Table 6. The binding affinity of the antibody to CD73 after humanization is comparable to that of the mouse antibody.
共检测了七种人源化抗CD73抗体对Calu-6细胞上CD73蛋白的IC50(最大抑制浓度的一半)，具体方法同实施例2的抗CD73小鼠抗体的体外功能测试方法，测定结果见图6A和6B,35D5G8和52A6B3显示出优于MEDI-9447的CD73酶活抑制效果。表7和表8(两批实验结果)列出了所有的人源化抗CD73抗体的IC50值，在七种人源化抗体中，人源化96A5A5和52A6B3表现出比MEDI-9447更好的IC50值，人源化143E5D2失去了对CD73的抑制能力，尽管其与人CD73ECD蛋白的结合亲和力没有降低。A total of seven humanized anti-CD73 antibodies were tested for IC 50 (half the maximum inhibitory concentration) of CD73 protein on Calu-6 cells. The specific method was the same as the in vitro functional test method of anti-CD73 mouse antibody in Example 2, and the measurement results were As shown in Figures 6A and 6B, 35D5G8 and 52A6B3 showed better inhibitory effects on CD73 enzyme activity than MEDI-9447. Table 7 and Table 8 (two batches of experimental results) list the IC 50 values of all humanized anti-CD73 antibodies. Among the seven humanized antibodies, humanized 96A5A5 and 52A6B3 performed better than MEDI-9447. IC50 value, humanized 143E5D2 lost its ability to inhibit CD73, although its binding affinity to human CD73ECD protein was not reduced.
为了进一步表征人源化抗体，FACS检测了抗体在过表达人/食蟹猴的CHO-K1细胞系上的结合能力，具体方法同实施例2的FACS检测鼠抗体与人CD73+/CHO-K1过表达细胞的结合，二抗用1μg/mL荧光团(iFluor 647)标记的山羊抗人IgG(H+L)。图7A、7B显示检测抗体均能够结合CHO-K1/Human CD73细胞和CHO-K1/Cyno CD73细胞，并且表现出优于MEDI-9447或与之相当的结合能力。 In order to further characterize the humanized antibody, FACS was used to detect the binding ability of the antibody on the CHO-K1 cell line overexpressing human/cynomolgus monkey. The specific method was the same as the FACS detection of mouse antibody and human CD73+/CHO-K1 in Example 2. For binding of expressing cells, the secondary antibody was goat anti-human IgG (H+L) labeled with 1 μg/mL fluorophore (iFluor 647). Figures 7A and 7B show that the detection antibodies are both able to bind to CHO-K1/Human CD73 cells and CHO-K1/Cyno CD73 cells, and exhibit binding abilities that are better than or equivalent to MEDI-9447.
为了验证人源化抗体的表位特异性，进行了针对MEDI9447抗体的ELISA表位竞争实验。96孔板包被100μl人CD73蛋白，0.5μg/ml，检测抗体和竞争抗体分别为人源化抗体和生物素标记的MEDI9447，通过辣根过氧化物酶标记的链霉亲和素检测OD450nm值。图8显示：人源化抗体35D5G8、52A6B3、22E1B4和MEDI9447有着类似的结合表位，而人源化抗体96A5A5的结合表位和MEDI9447不一样。To verify the epitope specificity of the humanized antibody, an ELISA epitope competition experiment against the MEDI9447 antibody was performed. The 96-well plate was coated with 100 μl human CD73 protein, 0.5 μg/ml. The detection antibody and competitive antibody were humanized antibody and biotin-labeled MEDI9447 respectively. The OD450nm value was detected by horseradish peroxidase-labeled streptavidin. Figure 8 shows that humanized antibodies 35D5G8, 52A6B3, 22E1B4 and MEDI9447 have similar binding epitopes, while the binding epitope of humanized antibody 96A5A5 is different from MEDI9447.
表5:七种人源化抗CD73抗体的VH、VL和CDR氨基酸序列

Table 5: VH, VL and CDR amino acid sequences of seven humanized anti-CD73 antibodies

表6:人源化抗体与CD73-ECD结合的亲和力

Table 6: Binding affinity of humanized antibodies to CD73-ECD

表7.人源化抗CD73抗体的IC50
Table 7. IC50 of humanized anti-CD73 antibodies
表8.抗CD73人源化抗体的IC50
Table 8. IC50 of anti-CD73 humanized antibodies
本发明的实施方式并不限于上述实施例所述，在不偏离本发明的精神和范围的情况下，本领域普通技术人员可以在形式和细节上对本发明做出各种改变和改进，而这些均被认为落入了本发明的保护范围。The implementation of the present invention is not limited to the above-described embodiments. Without departing from the spirit and scope of the present invention, those of ordinary skill in the art can make various changes and improvements in the form and details of the present invention, and these All are considered to fall within the protection scope of the present invention.
参考文献：references:
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Claims (27)

  1. 分离的抗体或其抗原结合片段，包括重链可变结构域(VH)和轻链可变结构域(VL)，其中：An isolated antibody or antigen-binding fragment thereof, including a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein:
    1)所述VH包括分别具有SEQ ID NO:18、19和20所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:22、23和24所示序列的LCDR1、LCDR2和LCDR3；1) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 18, 19 and 20 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 22, 23 and 24 respectively. LCDR3;
    2)所述VH包括分别具有SEQ ID NO:34、35和36所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:38、39和40所示序列的LCDR1、LCDR2和LCDR3；2) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 34, 35 and 36 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 38, 39 and 40 respectively. LCDR3;
    3)所述VH包括分别具有SEQ ID NO:2、3和4所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:6、7和8所示序列的LCDR1、LCDR2和LCDR3；3) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 2, 3 and 4 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 6, 7 and 8 respectively. LCDR3;
    4)所述VH包括分别具有SEQ ID NO:10、11和12所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:14、15和16所示序列的LCDR1、LCDR2和LCDR3；4) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 10, 11 and 12 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 14, 15 and 16 respectively. LCDR3;
    5)所述VH包括分别具有SEQ ID NO:26、27和28所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:30、31和32所示序列的LCDR1、LCDR2和LCDR3；5) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 26, 27 and 28 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 30, 31 and 32 respectively. LCDR3;
    6)所述VH包括分别具有SEQ ID NO:42、43和44所示序列的HCDR1、HCDR2和HCDR3或分别具有SEQ ID NO:42、43和105所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:46、47和48所示序列的LCDR1、LCDR2和LCDR3；6) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 42, 43 and 44 respectively or HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 42, 43 and 105 respectively, the VL includes LCDR1, LCDR2 and LCDR3 having the sequences shown in SEQ ID NO: 46, 47 and 48 respectively;
    7)所述VH包括分别具有SEQ ID NO:50、51和52所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:54、55和56所示序列的LCDR1、LCDR2和LCDR3；7) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 50, 51 and 52 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 54, 55 and 56 respectively. LCDR3;
    8)所述VH包括分别具有SEQ ID NO:58、59和60所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:62、63和64所示序列的LCDR1、LCDR2和LCDR3；8) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 58, 59 and 60 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 62, 63 and 64 respectively. LCDR3;
    9)所述VH包括分别具有SEQ ID NO:66、67和68所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:70、71和72所示序列的LCDR1、LCDR2和LCDR3；9) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 66, 67 and 68 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 70, 71 and 72 respectively. LCDR3;
    10)所述VH包括分别具有SEQ ID NO:74、75和76所示序列的HCDR1、HCDR2和HCDR3，所述VL包括分别具有SEQ ID NO:78、79和80所示序列的LCDR1、LCDR2和LCDR3；或 10) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 74, 75 and 76 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 78, 79 and 80 respectively. LCDR3; or
    11)所述VH包括分别具有SEQ ID NO:82、83和84所示序列的HCDR1、HCDR2和HCDR3、所述VL包括分别具有SEQ ID NO:86、87和88所示序列的LCDR1、LCDR2和LCDR3，11) The VH includes HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO:82, 83 and 84 respectively, and the VL includes LCDR1, LCDR2 and HCDR3 having the sequences shown in SEQ ID NO:86, 87 and 88 respectively. LCDR3,
    其中所述抗体或其抗原结合片段能够特异性结合CD73。Wherein the antibody or antigen-binding fragment thereof can specifically bind to CD73.
  2. 如权利要求1所述的抗体或其抗原结合片段，其中所述VH包括与选自SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的序列有至少80％一致性的氨基酸序列，所述VL包括与选自SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的序列有至少80％一致性的氨基酸序列。The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH includes one of An amino acid sequence having at least 80% identity to the sequence, the VL including an amino acid sequence having at least 80% identity to one of the sequences selected from SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85 % identity of the amino acid sequence.
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述VH包括选自SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的氨基酸序列或相对于SEQ ID NO:1、9、17、25、33、41、49、57、65、73和81之一的氨基酸序列包括至多3个氨基酸替换的变体，所述VL包括选自SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的氨基酸序列或相对于SEQ ID NO:5、13、21、29、37、45、53、61、69、77和85之一的氨基酸序列包括至多3个氨基酸替换的变体。The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VH includes one selected from SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81 The amino acid sequence or a variant including up to 3 amino acid substitutions relative to the amino acid sequence of one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73 and 81, the VL includes An amino acid sequence selected from one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 and 85 or relative to SEQ ID NO: 5, 13, 21, 29, 37, 45 The amino acid sequence of one of , 53, 61, 69, 77 and 85 includes variants with up to 3 amino acid substitutions.
  4. 如权利要求1或2所述的抗体或其抗原结合片段，其中：The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein:
    1)所述VH包括SEQ ID NO:17所示氨基酸序列，所述VL包括SEQ ID NO:21所示氨基酸序列；1) The VH includes the amino acid sequence shown in SEQ ID NO: 17, and the VL includes the amino acid sequence shown in SEQ ID NO: 21;
    2)所述VH包括SEQ ID NO:33所示氨基酸序列，所述VL包括SEQ ID NO:37所示氨基酸序列；2) The VH includes the amino acid sequence shown in SEQ ID NO: 33, and the VL includes the amino acid sequence shown in SEQ ID NO: 37;
    3)所述VH包括SEQ ID NO:1所示氨基酸序列，所述VL包括SEQ ID NO:5所示氨基酸序列；3) The VH includes the amino acid sequence shown in SEQ ID NO: 1, and the VL includes the amino acid sequence shown in SEQ ID NO: 5;
    4)所述VH包括SEQ ID NO:9所示氨基酸序列，所述VL包括SEQ ID NO:13所示氨基酸序列；4) The VH includes the amino acid sequence shown in SEQ ID NO: 9, and the VL includes the amino acid sequence shown in SEQ ID NO: 13;
    5)所述VH包括SEQ ID NO:25所示氨基酸序列，所述VL包括SEQ ID NO:29所示氨基酸序列；5) The VH includes the amino acid sequence shown in SEQ ID NO:25, and the VL includes the amino acid sequence shown in SEQ ID NO:29;
    6)所述VH包括SEQ ID NO:41所示氨基酸序列，所述VL包括SEQ ID NO:45所示氨基酸序列；6) The VH includes the amino acid sequence shown in SEQ ID NO:41, and the VL includes the amino acid sequence shown in SEQ ID NO:45;
    7)所述VH包括SEQ ID NO:49所示氨基酸序列，所述VL包括SEQ ID NO:53所示氨基酸序列；7) The VH includes the amino acid sequence shown in SEQ ID NO:49, and the VL includes the amino acid sequence shown in SEQ ID NO:53;
    8)所述VH包括SEQ ID NO:57所示氨基酸序列，所述VL包括SEQ ID NO:61所示氨基酸序列；8) The VH includes the amino acid sequence shown in SEQ ID NO: 57, and the VL includes the amino acid sequence shown in SEQ ID NO: 61;
    9)所述VH包括SEQ ID NO:65所示氨基酸序列，所述VL包括SEQ ID NO:69所示氨基酸序列；9) The VH includes the amino acid sequence shown in SEQ ID NO: 65, and the VL includes the amino acid sequence shown in SEQ ID NO: 69;
    10)所述VH包括SEQ ID NO:73所示氨基酸序列，所述VL包括SEQ ID NO:77所示氨基酸序列；或 10) The VH includes the amino acid sequence shown in SEQ ID NO:73, and the VL includes the amino acid sequence shown in SEQ ID NO:77; or
    11)所述VH包括SEQ ID NO:81所示氨基酸序列，所述VL包括SEQ ID NO:85所示氨基酸序列。11) The VH includes the amino acid sequence shown in SEQ ID NO:81, and the VL includes the amino acid sequence shown in SEQ ID NO:85.
  5. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段针对细胞表面表达的所述CD73的酶活性具有10-1μg/mL至10-3μg/mL的IC50值。The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the enzymatic activity of the antibody or antigen-binding fragment thereof against the CD73 expressed on the cell surface is 10 -1 μg/mL to 10 -3 μg/mL. IC 50 value.
  6. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与表达所述CD73的细胞结合的EC50值为1μg/mL至10-2μg/mL。The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the EC 50 value of the antibody or antigen-binding fragment thereof binding to cells expressing the CD73 is 1 μg/mL to 10 −2 μg/mL.
  7. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体为鼠抗体、嵌合抗体、人源化抗体或人抗体。The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.
  8. 如权利要求1或2所述的抗体或其抗原结合片段，其为单克隆抗体、scFv、Fab、Fab’、(Fab’)2或Fv。The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a monoclonal antibody, scFv, Fab, Fab', (Fab') 2 or Fv.
  9. 如权利要求7所述的抗体或其抗原结合片段，其中所述人源化抗体的VH包括选自SEQ ID NO:91、93、89、95、97、99或101的氨基酸序列，所述人源化抗体的VL包括选自SEQ ID NO:92、94、90、96、98、100或102的氨基酸序列。The antibody or antigen-binding fragment thereof according to claim 7, wherein the VH of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 91, 93, 89, 95, 97, 99 or 101, and the human The VL of the humanized antibody includes an amino acid sequence selected from SEQ ID NO: 92, 94, 90, 96, 98, 100 or 102.
  10. 如权利要求9所述的抗体或其抗原结合片段，其中：The antibody or antigen-binding fragment thereof according to claim 9, wherein:
    1)所述VH包括SEQ ID NO:91所示氨基酸序列和所述VL包括SEQ ID NO:92所示氨基酸序列；1) The VH includes the amino acid sequence shown in SEQ ID NO:91 and the VL includes the amino acid sequence shown in SEQ ID NO:92;
    2)所述VH包括SEQ ID NO:93所示氨基酸序列和所述VL包括SEQ ID NO:94所示氨基酸序列；2) The VH includes the amino acid sequence shown in SEQ ID NO:93 and the VL includes the amino acid sequence shown in SEQ ID NO:94;
    3)所述VH包括SEQ ID NO:89所示氨基酸序列和所述VL包括SEQ ID NO:90所示氨基酸序列；3) The VH includes the amino acid sequence shown in SEQ ID NO:89 and the VL includes the amino acid sequence shown in SEQ ID NO:90;
    4)所述VH包括SEQ ID NO:95所示氨基酸序列和所述VLSEQ ID NO:96所示氨基酸序列；4) The VH includes the amino acid sequence shown in SEQ ID NO: 95 and the amino acid sequence shown in the VLSEQ ID NO: 96;
    5)所述VH包括SEQ ID NO:97所示氨基酸序列和所述VL包括SEQ ID NO:98所示氨基酸序列；5) The VH includes the amino acid sequence shown in SEQ ID NO: 97 and the VL includes the amino acid sequence shown in SEQ ID NO: 98;
    6)所述VH包括SEQ ID NO:99所示氨基酸序列和所述VL包括SEQ ID NO:100所示氨基酸序列；或6) The VH includes the amino acid sequence shown in SEQ ID NO:99 and the VL includes the amino acid sequence shown in SEQ ID NO:100; or
    7)所述VH包括SEQ ID NO:101所示氨基酸序列和所述VL包括SEQ ID NO:102所示氨基酸序列。7) The VH includes the amino acid sequence shown in SEQ ID NO:101 and the VL includes the amino acid sequence shown in SEQ ID NO:102.
  11. 如权利要求10所述的抗体或其抗原结合片段，其中所述人源化抗体针对表达所述CD73的细胞酶抑制活性具有10-1μg/mL至10-2μg/mL的IC50值。The antibody or antigen-binding fragment thereof of claim 10, wherein the humanized antibody has an IC 50 value of 10 −1 μg/mL to 10 −2 μg/mL against cellular enzyme inhibitory activity expressing the CD73.
  12. 如权利要求10所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与所述CD73之间结合的KD值为10-8M至10-11M。The antibody or antigen-binding fragment thereof according to claim 10, wherein the KD value of the binding between the antibody or antigen-binding fragment thereof and CD73 is 10 -8 M to 10 -11 M.
  13. 如权利要求1所述的抗体或其抗原结合片段，其中所述VH和/或VL连接至免疫球蛋白的恒定区。The antibody or antigen-binding fragment thereof according to claim 1, wherein said VH and/or VL are linked to the constant region of an immunoglobulin.
  14. 如权利要求13所述的抗体或其抗原结合片段，其中所述VH连接至IgG1恒定区。 The antibody or antigen-binding fragment thereof of claim 13, wherein the VH is linked to an IgGl constant region.
  15. 双特异性抗体，其包括权利要求1-14中任一项所述的抗体或其抗原结合片段和第二抗体部分。A bispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-14 and a second antibody portion.
  16. 如权利要求15所述的双特异性抗体，其中所述第二抗体部分能够结合不同于CD73的抗原。The bispecific antibody of claim 15, wherein the second antibody portion is capable of binding an antigen different from CD73.
  17. 如权利要求15或16所述的双特异性抗体，其中所述不同于CD73的抗原为CD39、CTLA-4、PD-L1、TIM-3、LAG-3或A2aR。The bispecific antibody of claim 15 or 16, wherein the antigen different from CD73 is CD39, CTLA-4, PD-L1, TIM-3, LAG-3 or A2aR.
  18. 如权利要求15或16所述的双特异性抗体，其中所述第二抗体部分为Fab、Fab’、(Fab’)2、Fv、scFv或sdAb。The bispecific antibody of claim 15 or 16, wherein the second antibody portion is Fab, Fab', (Fab') 2 , Fv, scFv or sdAb.
  19. 编码权利要求1-14中任一项所述的抗体或其抗原结合片段或权利要求15-18中任一项所述的双特异性抗体的多核苷酸。A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-14 or the bispecific antibody according to any one of claims 15-18.
  20. 包含权利要求19所述多核苷酸的载体。A vector comprising the polynucleotide of claim 19.
  21. 包含权利要求19所述多核苷酸或权利要求20所述载体的细胞。A cell comprising the polynucleotide of claim 19 or the vector of claim 20.
  22. 用于癌症治疗的药物组合物，包括：Pharmaceutical compositions for cancer treatment, including:
    权利要求1-14中任一项所述的抗体或其抗原结合片段，或权利要求15-18中任一项所述的双特异性抗体；以及The antibody or antigen-binding fragment thereof according to any one of claims 1-14, or the bispecific antibody according to any one of claims 15-18; and
    药学上可接受的载体。Pharmaceutically acceptable carrier.
  23. 如权利要求22所述的药物组合物，还包括一种或更多种其他抗癌剂。The pharmaceutical composition of claim 22, further comprising one or more other anti-cancer agents.
  24. 如权利要求22或23所述的药物组合物，其中所述癌症为实体癌。The pharmaceutical composition of claim 22 or 23, wherein the cancer is a solid cancer.
  25. 权利要求1-14中任一项所述的抗体或其抗原结合片段、权利要求15-18中任一项所述的双特异性抗体、权利要求19所述的多核苷酸、权利要求20所述的载体或权利要求21所述的细胞在制备用于治疗癌症的药物中的用途。The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the bispecific antibody according to any one of claims 15 to 18, the polynucleotide according to claim 19, the polynucleotide according to claim 20 The use of the vector described in claim 21 or the cell described in claim 21 in the preparation of a drug for treating cancer.
  26. 如权利要求25所述的用途，其中所述癌症为实体癌。The use of claim 25, wherein the cancer is a solid cancer.
  27. 如权利要求25或26所述的用途，其中所述癌症选自非小细胞肺癌、三阴性乳腺癌或胰腺癌。 The use of claim 25 or 26, wherein the cancer is selected from non-small cell lung cancer, triple negative breast cancer or pancreatic cancer.
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