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WO2022122004A1 - Cd73的抗原结合蛋白及其应用 - Google Patents

Cd73的抗原结合蛋白及其应用 Download PDF

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WO2022122004A1
WO2022122004A1 PCT/CN2021/136950 CN2021136950W WO2022122004A1 WO 2022122004 A1 WO2022122004 A1 WO 2022122004A1 CN 2021136950 W CN2021136950 W CN 2021136950W WO 2022122004 A1 WO2022122004 A1 WO 2022122004A1
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seq
binding protein
amino acid
acid sequence
isolated antigen
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PCT/CN2021/136950
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English (en)
French (fr)
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徐锦根
朱向阳
于海佳
韦小越
童玲
陈时
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上海华奥泰生物药业股份有限公司
华博生物医药技术（上海）有限公司
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Application filed by 上海华奥泰生物药业股份有限公司, 华博生物医药技术（上海）有限公司 filed Critical 上海华奥泰生物药业股份有限公司
Priority to US18/265,529 priority Critical patent/US20240026024A1/en
Priority to EP21902709.1A priority patent/EP4261224A1/en
Priority to CN202180079159.5A priority patent/CN116529265A/zh
Priority to JP2023535442A priority patent/JP2023554326A/ja
Priority to KR1020237023190A priority patent/KR20230119173A/ko
Priority to AU2021395129A priority patent/AU2021395129A1/en
Publication of WO2022122004A1 publication Critical patent/WO2022122004A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein of CD73 and its application.
  • CD73 also known as NT5E, is an extracellular 5-nuclease (NT5E) with a molecular weight of about 70Kd, which is anchored to the cell surface by glycophosphatidylinositol (GPI), mainly in the form of dimers. Under normal physiological conditions, CD73 can be expressed in a variety of tissues or organs, such as liver, large intestine, kidney, spleen, lung, ovary, lymph nodes and other tissues.
  • GPI glycophosphatidylinositol
  • CD73 is abnormally highly expressed on the surface of various tumor cells, including glioma, breast cancer, melanoma, non-small cell lung cancer, bladder cancer, ovarian cancer, colorectal cancer, etc., and clinical data show that The high expression of CD73 is closely related to the poor prognosis of tumor patients, so CD73 can be used as a potential target for clinical treatment and prognosis of various tumors.
  • the application provides an isolated antigen-binding protein that exhibits one or more desired functional properties, such as its ability to specifically bind CD73 or a functionally active fragment thereof; its ability to prevent or reduce activation of CD73; After the CD73 is combined, the isolated antigen-binding protein can bind to amino acids 143-157 of CD73 or their corresponding CD73 sequences, 178-191 amino acids or their corresponding CD73 sequences and 381-386 amino acids or their corresponding CD73 sequences. at least one amino acid residue in the corresponding CD73 sequence; capable of inhibiting the enzymatic activity of CD73; and capable of inducing the endocytosis of cell surface CD73.
  • the present application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells and methods of making the isolated antigen binding proteins.
  • the isolated antigen binding proteins described herein can be used to prevent and/or treat diseases and/or disorders, such as tumors.
  • the application provides an isolated antigen binding protein comprising HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1.
  • the isolated antigen binding protein comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.
  • the isolated antigen binding protein comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5.
  • the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4.
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1
  • the H-FR1 comprises SEQ ID NO: 7.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO: 8, SEQ ID The amino acid sequence shown in NO: 16 or SEQ ID NO: 21.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 9, SEQ ID The amino acid sequence shown in NO:17, SEQ ID NO:20 or SEQ ID NO:22.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 10 or SEQ ID NO: 10 or SEQ ID NO: 10 or SEQ ID NO: 10 The amino acid sequence shown in ID NO: 18.
  • the isolated antigen binding protein comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises SEQ ID NO: 11 or the amino acid sequence shown in SEQ ID NO:23.
  • the isolated antigen binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 12 or SEQ ID The amino acid sequence shown in NO:24.
  • the isolated antigen binding protein comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO: 13, SEQ ID The amino acid sequence shown in NO:25, SEQ ID NO:27 or SEQ ID NO:28.
  • the isolated antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3, and the L-FR4 comprises SEQ ID NO: 14 or SEQ ID NO: 14 The amino acid sequence shown in ID NO:26.
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35.
  • the isolated antigen binding protein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
  • the isolated antigen binding protein comprises any one of the groups of VH and VL selected from the group consisting of:
  • VH comprises the amino acid sequence shown in SEQ ID NO:29
  • VL comprises the amino acid sequence shown in SEQ ID NO:30
  • VH comprises the amino acid sequence shown in SEQ ID NO:31
  • VL comprises the amino acid sequence shown in SEQ ID NO:32
  • VH comprises the amino acid sequence shown in SEQ ID NO:33
  • VL comprises the amino acid sequence shown in SEQ ID NO:34
  • VH comprises the amino acid sequence shown in SEQ ID NO:35
  • VL comprises the amino acid sequence shown in SEQ ID NO:36
  • VH comprises the amino acid sequence shown in SEQ ID NO:33
  • VL comprises the amino acid sequence shown in SEQ ID NO:36
  • VH comprises the amino acid sequence shown in SEQ ID NO:35
  • VL comprises the amino acid sequence shown in SEQ ID NO:34.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human antibody heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgGl heavy chain constant region.
  • the isolated antigen binding protein comprises an antibody light chain constant region.
  • the antibody light chain constant region is derived from a human IgK constant region.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of specifically binding CD73 or a functionally active fragment thereof.
  • the CD73 comprises human CD73 and/or monkey CD73.
  • the isolated antigen binding protein is capable of preventing or reducing activation of CD73.
  • the isolated antigen-binding protein when binding to the CD73, binds to amino acids 143-157 of the amino acid sequence shown in SEQ ID NO: 40 or the corresponding CD73 sequence, SEQ ID NO: 40 : amino acids 178-191 of the amino acid sequence shown in 40 or the CD73 sequence corresponding thereto and amino acids 381-386 of the amino acid sequence shown in SEQ ID NO: 40 or at least one amino acid residue in the corresponding CD73 sequence base combined.
  • the amino acids 143-157 or their corresponding CD73 sequences are located in the N-terminal domain of the CD73, and the 178-191 amino acids or their corresponding CD73 sequences are located in the CD73 N-terminal domain, and the amino acids 381-386 or the CD73 sequence corresponding thereto are located in the C-terminal domain of CD73.
  • the isolated antigen binding protein is capable of inhibiting the enzymatic activity of CD73.
  • the isolated antigen binding protein is capable of inducing the endocytosis of cell surface CD73.
  • the present application also provides a polypeptide molecule comprising the isolated antigen-binding protein.
  • the polypeptide molecule comprises a fusion protein.
  • the present application also provides an isolated nucleic acid molecule encoding the isolated antigen binding protein or the polypeptide molecule.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the nucleic acid molecule and/or the vector.
  • the present application also provides an immunoconjugate comprising the isolated antigen-binding protein.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell and/or the described immunoconjugate, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the isolated antigen-binding protein, which comprises culturing the cells under conditions such that the isolated antigen-binding protein is expressed.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are in Use in the preparation of medicaments for the prevention and/or treatment of diseases and/or disorders.
  • the present application also provides a method for preventing and/or treating a disease and/or disorder, the method may comprise administering to a subject in need thereof the isolated antigen-binding protein, the polypeptide molecule, The nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.
  • the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition described in this application can be used with for the prevention and/or treatment of diseases and/or disorders.
  • the disease and/or disorder is a CD73-mediated disease and/or disorder.
  • the disease and/or disorder comprises a tumor.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the disease and/or disorder comprises breast cancer.
  • the present application also provides a method for detecting CD73 in a sample, comprising administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition.
  • the present application also provides a reagent or kit for detecting CD73 in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunization The conjugate and/or the pharmaceutical composition.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are in Use in the preparation of a kit for detecting the presence and/or content of CD73 in a sample.
  • Figure 1 shows the binding activity of the antigen-binding proteins 900581 and 900565 described in this application to CD73 on the cell surface.
  • Figure 2 shows the binding activity of the antigen binding proteins 900581 and 900694-900698 described in this application to CD73 on the cell surface.
  • Figure 3 shows the inhibition of the enzymatic activity of recombinant CD73 by the antigen binding proteins 900581 and 900694-900698 described in this application.
  • Figure 4 shows the inhibition of the enzymatic activity of cell surface CD73 by the antigen binding proteins 900581 and 900565 described in this application.
  • Figure 5 shows the inhibition of the enzymatic activity of cell surface CD73 by the antigen binding proteins 900581, 900697 and 900698 described in the present application.
  • Figure 6 shows the efficiency of the antigen binding protein 900581 described in the present application in inducing the endocytosis of CD73 on the cell surface.
  • Figure 7 shows a structural model of the antigen binding protein binding epitope of the present application.
  • FIGS 8A-8C show the competitive binding activity of the antigen binding proteins described in the present application with 900609.
  • Figure 9 shows the effect of the antigen binding protein described in the present application in treating tumors in vivo.
  • Figure 10 shows the effect of the antigen binding protein described in the present application in treating tumors in vivo.
  • CD73 is also referred to as “NT5E", “cluster of differentiation 73", “5'-nucleotidase (5'-NT)” or “extracellular 5'-nucleotidase”.
  • the term encompasses "full-length”, unprocessed CD73 and any form of CD73 produced by cellular processing.
  • CD73 includes full-length wild-type CD73 and mutants, fragments, variants, isoforms and homologues thereof.
  • the CD73 can comprise human CD73.
  • the sequence for human CD73 can be found under Uniprot accession number P21589.
  • the CD73 may comprise the amino acid sequence shown in SEQ ID NO:40.
  • isolated generally refers to artificial means obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated does not exclude the admixture of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the material.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding capacity that has been removed from its naturally occurring state.
  • isolated antigen-binding protein may comprise an antigen-binding moiety and, optionally, a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to the antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See, eg, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; trifunctional antibodies; tetrabodies; Fab, Fab', Fv fragments, F (ab') 2 , F(ab) 2 , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.
  • CDRs also referred to as “complementarity determining regions” generally refers to regions within the variable domains of antibodies, the sequences of which are highly variable and/or form structurally defined loops.
  • an antibody typically includes six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are also functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum.
  • the amino acid sequence numbering of the antigen binding protein can be according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http://imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res.
  • the CDRs of the antigen binding proteins can be determined according to the Kabat numbering system (see e.g. Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • variable domains of native heavy and light chains each comprise four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3 and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3 and L-FR4).
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable generally refers to the fact that certain segments of the variable domains may differ greatly in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are referred to as framework regions (FRs).
  • FRs framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs that form loops connecting, and in some cases forming part of, the ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide that includes an antigen-binding site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutant and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind human CD73). Typically, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen-binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form multivalent Combined basic 4-chain unit.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is connected to the H chain by one covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH and CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Particular amino acid residues are thought to form the interface between the light and heavy chain variable domains.
  • the VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Based on the amino acid sequence of the heavy chain (CH) constant domains, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, CD73).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • a Fab fragment may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) of the H chain ( VH ).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment can include all of the light chain, all of the variable regions of the heavy chain, and all or part of the first and second constant regions of the heavy chain.
  • Fab' fragments may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of whole antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen-binding activity and are capable of cross-linking antigens.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions.
  • Heavy chain variable regions and light chain variable regions include, for example, CDRs.
  • Fv fragments include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable
  • the regions are contiguous (eg, via synthetic linkers such as short flexible polypeptide linkers) and are capable of being expressed as single-chain polypeptides, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N- and C-termini of the polypeptide), and the scFv may include a VL-linker-VH or VH-linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or a VH domain or a VL domain, see eg Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term "monoclonal antibody” generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody comprising only human immunoglobulin protein sequences.
  • a fully human antibody may contain murine sugar chains if it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • mouse antibody or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Fully human antibodies can be generated in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.
  • polypeptide molecule and “polypeptide”, “peptide” are used interchangeably and generally refer to a polymer of amino acid residues.
  • fusion protein generally refers to a polypeptide having at least two moieties covalently linked together. Each of these moieties can be polypeptides with different properties.
  • the property may be a biological property, such as in vitro or in vivo activity.
  • the properties can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc.
  • the two moieties can be directly linked by a single peptide bond or by a peptide linker.
  • nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or analogs isolated from their natural environment or synthetically synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • a vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 Bacteriophages and animal viruses, etc.
  • Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses vesicle virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses vesicle virus
  • a vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
  • the carrier may also include components to assist its entry into
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject plasmid or vector, comprising a nucleic acid molecule described herein or a nucleic acid molecule described herein Carrier.
  • a cell can include the progeny of a single cell. Progeny may not necessarily be identical (in morphology or in genome) to the original parent cell due to natural, accidental or intentional mutation.
  • Cells can include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (eg, E.
  • the cells are mammalian cells. In certain embodiments, the mammalian cells are HEK293 cells.
  • the term “immunoconjugate” generally refers to the conjugation of the other agents (eg, chemotherapeutic agents, radioactive elements, cytostatic and cytotoxic agents) to the antibody or antigen-binding fragment thereof (eg, A conjugate formed by covalently linking a linker molecule) that can specifically bind to an antigen on a target cell via the antibody or antigen-binding fragment thereof, and deliver the other agent to the target cell (eg, tumor cells).
  • the other agents eg, chemotherapeutic agents, radioactive elements, cytostatic and cytotoxic agents
  • the term "pharmaceutical composition” generally refers to a composition for the prevention/treatment of a disease or disorder.
  • the pharmaceutical composition may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a carrier described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable preservatives preparation.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are free of the cells or mammals to which they are exposed at the dosages and concentrations employed. poisonous.
  • Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, Sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • the term “specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be found in a heterogeneous population of molecules, including biomolecules
  • the presence of the target determines the presence of the target.
  • an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets .
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • tumor generally refers to any new pathological tissue growth. Tumor cells can spread to other parts of the body locally or through the bloodstream and lymphatic system.
  • the tumors may include benign tumors and malignant tumors.
  • the tumor may include solid tumors and/or hematological tumors.
  • the tumor may include cancer.
  • examples of such tumors include, but are not limited to, glioma, breast cancer, melanoma, non-small cell lung cancer, bladder cancer, ovarian cancer, and colorectal cancer.
  • activation generally refers to the transformation from an inactive state to an active state.
  • preventing or reducing activation of CD73 can refer to preventing or reducing the conversion of CD73 from a catalytically inactive open conformation to a catalytically active closed conformation.
  • the term "reduction” generally refers to a reduction in quantity and/or degree compared to the original level.
  • said reducing activation of CD73 can refer to reducing activation of CD73 by at least about 0.5-fold, at least about 1-fold, at least about 1.5-fold, at least about 2-fold compared to the absence of said isolated antigen binding protein , at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times Or at least about 100 times.
  • the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, one or more substitutions, deletions, or additions to the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CD73 protein).
  • amino acid protein or polypeptide may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • the homologue may be at least about 85% (eg, having at least about 85%) the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CD73 protein). 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology Sexual proteins or polypeptides.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • FASTA and BLAST A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, “Rapid and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the CDRs of antibodies are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions.
  • the CDRs encompass CDR sequences that are divided according to any CDR division; variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the CDRs are determined by the IMGT numbering scheme.
  • the application provides an isolated antigen binding protein comprising HCDR3, which can comprise the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding protein may also comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the isolated antigen binding protein may also comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the isolated antigen binding protein may comprise HCDR3, HCDR2 and HCDR1.
  • HCDR3 of the isolated antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:3
  • HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2
  • HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:1 amino acid sequence.
  • the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the isolated antigen binding protein may also comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the isolated antigen binding protein can also comprise LCDR1, and the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:4.
  • the isolated antigen binding protein may comprise LCDR3, LCDR2 and LCDR1.
  • LCDR3 of the isolated antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:6
  • LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:5
  • LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4 amino acid sequence.
  • the isolated antigen binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1.
  • the isolated antigen-binding protein HCDR3 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:3, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the amino acid sequence of LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise as SEQ ID NO : 7, the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 19.
  • the isolated antigen binding protein may comprise H-FR2, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise, for example, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 8 The amino acid sequence shown in ID NO: 16 or SEQ ID NO: 21.
  • the isolated antigen-binding protein may comprise H-FR3, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise, for example, SEQ ID NO: 9, SEQ ID NO: 9, SEQ ID NO: 9, SEQ ID NO: 9, SEQ ID NO: 9
  • SEQ ID NO: 9 The amino acid sequence shown in ID NO: 17, SEQ ID NO: 20 or SEQ ID NO: 22.
  • the isolated antigen binding protein may comprise H-FR4, the N-terminus of the H-FR4 may be linked to the C-terminus of the HCDR3, and the H-FR4 may comprise as SEQ ID NO: 10 or The amino acid sequence shown in SEQ ID NO:18.
  • the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO, respectively, in order : the amino acid sequence shown in 10.
  • the H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO, respectively, in order : amino acid sequence shown in 18.
  • H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO, respectively, in order : amino acid sequence shown in 18.
  • H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO, respectively, in sequence : amino acid sequence shown in 18.
  • the isolated antigen binding protein may comprise L-FR1, the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 may comprise as SEQ ID NO : 11 or the amino acid sequence shown in SEQ ID NO: 23.
  • the isolated antigen binding protein may comprise L-FR2, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise, for example, SEQ ID NO: 12 or SEQ ID NO: 12 or SEQ ID NO: 12 or SEQ ID NO: 12.
  • SEQ ID NO: 12 amino acid sequence shown in ID NO:24.
  • the isolated antigen binding protein may comprise L-FR3, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise, for example, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 13 The amino acid sequence shown in ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the isolated antigen binding protein may comprise L-FR4, the N-terminus of the L-FR4 may be linked to the C-terminus of the LCDR3, and the L-FR4 may comprise as SEQ ID NO: 14 or The amino acid sequence shown in SEQ ID NO:26.
  • the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4.
  • L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO, respectively, in order : the amino acid sequence shown in 14.
  • L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO, respectively, in order : the amino acid sequence shown in 26.
  • L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO, respectively, in order : the amino acid sequence shown in 26.
  • L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO, respectively, in order : the amino acid sequence shown in 26.
  • the isolated antigen binding protein may comprise a VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35.
  • the isolated antigen binding protein may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
  • the isolated antigen binding protein may comprise the VH and VL.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:29, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:30.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:31, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:32.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:33, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:34.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:35, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:36. In certain embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:33, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:36. In certain embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:35, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:34.
  • the isolated antigen binding protein may comprise at least one CDR in the VH described herein.
  • the isolated antigen binding protein may comprise at least one CDR in the VL described herein.
  • the CDR can be obtained by dividing according to any dividing manner.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH described in the present application.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35.
  • the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3 in the VL described in the present application.
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgGl heavy chain constant region.
  • the isolated antigen binding protein may comprise an antibody light chain constant region.
  • the antibody light chain constant region may be derived from a human IgK constant region.
  • the isolated antigen-binding protein may comprise an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment may comprise a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies.
  • the VH of the chimeric antibody can comprise the amino acid sequence set forth in SEQ ID NO:29
  • the VL of the chimeric antibody can comprise the amino acid sequence set forth in SEQ ID NO:30.
  • the VH of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:31
  • the VL of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:32.
  • the VH of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:33
  • the VL of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:34.
  • the VH of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:35
  • the VL of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:36.
  • the VH of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:33
  • the VL of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:36.
  • the VH of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:35
  • the VL of the humanized antibody can comprise the amino acid sequence set forth in SEQ ID NO:34.
  • the isolated antigen-binding proteins described in the present application may comprise heavy chain and/or light chain sequences with one or more conservative sequence modifications.
  • conservative sequence modifications is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are replacement of amino acid residues with amino acid residues with similar side chains. Sets of amino acid residues with similar side chains are known in the art.
  • amino acid residue groups include those with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eglycine, as
  • one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described herein can be replaced with other amino acid residues of the same side chain group.
  • Those skilled in the art know that some conservative sequence modifications will not abolish antigen binding, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin.Can.Res.6:2835 -43.
  • CD73 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
  • the antigen binding proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like or the antigen-binding activity of the fusion protein.
  • the isolated antigen binding protein is capable of specifically binding CD73 or a functionally active fragment thereof.
  • the isolated antigen binding protein can be affinity screened by Biacore.
  • the isolated antigen-binding protein can bind to the CD73 protein with a KD of 5 ⁇ 10-9 M or less, for example, the antigen-binding protein described herein can be less than about 4 ⁇ 10-9 M, less than about 3 ⁇ 10-9M , less than about 2 ⁇ 10-9M , less than about 1 ⁇ 10-9M , less than about 7 ⁇ 10-10M , less than about 6 ⁇ 10-10M , less than about 5 ⁇ 10-10M , less than about 4 ⁇ 10-10 M, less than about 3 ⁇ 10-10 M, less than about 2 ⁇ 10-10 M, less than about 1 ⁇ 10-10 M, less than about 9 ⁇ 10-11 M, less than about 5 ⁇ 10-11 M, KD of less than about 2 x 10-11 M binds CD73.
  • the present application also includes the use of FACS methods to determine the binding activity of an antigen-binding protein to an antigen.
  • the antigen-binding proteins described herein can be present at less than or equal to about 0.5 ⁇ g/mL, less than or equal to about 0.4 ⁇ g/mL, less than or equal to about 0.3 ⁇ g/mL, less than or equal to about 0.2 ⁇ g/mL, less than or equal to about 0.2 ⁇ g/mL
  • An EC50 value of about 0.1 ⁇ g/mL binds to cell surface human CD73.
  • the antigen binding proteins described herein can be combined with an EC50 value of less than or equal to about 0.4 ⁇ g/mL, less than or equal to about 0.3 ⁇ g/mL, less than or equal to about 0.2 ⁇ g/mL, or less than or equal to about 0.1 ⁇ g/mL Cell surface human CD73 binding.
  • the isolated antigen binding protein is capable of binding CD73.
  • the antigen binding proteins described herein can also cross-react with monkey CD73.
  • cross-reactivity generally refers to the ability of an antibody to react with homologous proteins from other species.
  • the isolated antigen binding proteins described herein do not bind mouse and/or rat CD73.
  • titers can be detected by ELISA methods.
  • the isolated antigen binding protein can be tested for binding activity or functional activity using a FACS assay.
  • the isolated antigen binding protein can be subjected to affinity testing.
  • the isolated antigen binding protein is capable of preventing or reducing activation of CD73.
  • the isolated antigen binding protein can prevent or reduce the conversion of CD73 from a catalytically inactive open conformation to a catalytically active closed conformation.
  • the isolated antigen binding protein can block the catalytic center of CD73, thereby inhibiting the enzymatic activity of CD73.
  • the binding epitope of the isolated antigen binding protein can be determined by hydrogen deuterium exchange mass spectrometry (HDX-MS).
  • the isolated antigen binding protein has competitive binding activity with the CPI-006 antibody (Corvus).
  • the isolated antigen binding protein when combined with the CD73, is bound to the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37) of the N-terminal domain of CD73 or its corresponding CD73 sequence, 178-SKETPFLSNPGTNL -191 region (SEQ ID NO:38) or its corresponding CD73 sequence, and at least one amino acid residue in the 381-WNHVSM-386 region of the CD73 C-terminal domain (SEQ ID NO:39) or its corresponding CD73 sequence Combine.
  • the CD73 sequence corresponding thereto may refer to the amino acid sequence at the corresponding position in its homologue (or CD73 from other species).
  • the isolated antigen binding protein is capable of inhibiting the enzymatic activity of CD73.
  • the isolated antigen binding protein may have an enzymatic activity-inhibiting effect on recombinant CD73.
  • the isolated antigen-binding protein may have an enzymatic activity-inhibiting effect on human CD73.
  • the enzymatic activity described in this application can be detected by any method commonly used in the art. The detection of the enzymatic activity can include mixing the CD73 antigen-binding protein with the recombinant CD73 protein, adding AMP and ATP, Substrate addition Buffer, mix and equilibrate to room temperature for full-wavelength detection.
  • the isolated antigen-binding protein can be at less than or equal to about 0.8 ⁇ g/mL, less than or equal to about 0.7 ⁇ g/mL, less than or equal to about 0.6 ⁇ g/mL, less than or equal to about 0.5 ⁇ g/mL, less than or equal to about 0.5 ⁇ g/mL
  • An EC50 value of about 0.4 ⁇ g/mL, less than or equal to about 0.3 ⁇ g/mL, or less than or equal to about 0.2 ⁇ g/mL inhibits the enzymatic activity of the recombinant CD73 protein.
  • the isolated antigen-binding protein can be at less than or equal to about 6 ⁇ g/mL, less than or equal to about 5 ⁇ g/mL, less than or equal to about 4 ⁇ g/mL, less than or equal to about 3 ⁇ g/mL, less than or equal to about 2 ⁇ g/mL , an EC50 value of less than or equal to about 1 ⁇ g/mL, or less than or equal to about 0.5 ⁇ g/mL inhibits the enzymatic activity of CD73 protein on the cell membrane.
  • the isolated antigen binding protein is capable of inducing the endocytosis of cell surface CD73.
  • the endocytosis of proteins can be determined by fluorescent labeling.
  • the efficiency of the antigen binding protein in inducing the endocytosis of cell surface CD73 can be obtained by measuring the fluorescence intensity of the cells using flow cytometry.
  • the isolated antigen binding protein can induce CD73 endocytosis with an efficiency of about 50% or greater.
  • Polypeptide molecules Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions
  • the application provides polypeptide molecules that may comprise the isolated antigen binding proteins described herein.
  • the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule can be a fusion protein.
  • the application provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, eg, by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified (iv) synthetic, such as by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the carrier may also include components to assist its entry into cells, such as viral particles, liposomes or protein coats, but not only these materials.
  • the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the cells can be bacterial cells (eg, E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2/0 cells, NSO cells or myeloma cells.
  • CHO Chinese Hamster Ovary
  • LNCAP low-density lipoprotein
  • HeLa cells HeLa cells
  • 293T cells HeLa cells
  • COS-1 cells COS-1 cells
  • SP2/0 cells nuclear-derived cells
  • NSO cells myeloma cells.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application also provides immunoconjugates, which may comprise the isolated antigen binding proteins described herein.
  • an isolated antigen binding protein or fragment thereof described herein can be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, and the like.
  • the attachment can be through one or more covalent bonds, or non-covalent interactions, and can include chelation.
  • linkers which may be known in the art, can be used to form immunoconjugates.
  • immunoconjugates can be provided in the form of fusion proteins, which can be expressed from polynucleotides encoding the immunoconjugates.
  • the immunoconjugate may also comprise, for example, an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • Suitable drugs may include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerases Inhibitors of I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotics.
  • the antibody and therapeutic agent can be cross-linked by a linker that is cleavable, such as a peptidic, disulfide, or hydrazone-like linker.
  • the present application also provides a pharmaceutical composition, which can comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the The nucleic acid molecule, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, typically those active compounds with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as on the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with pharmaceutical administration, and is generally safe and nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application provides methods for preparing the antigen binding proteins.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application.
  • animals can be immunized with linked or naturally occurring CD73 or fragments thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • a hybridoma preparation method can be used to obtain spleen cells from immunized mice and fuse them with SP2/0 myeloma cells, and then select a hybridoma cell line through HAT.
  • CD73 can be used as an immunogen (antigen) for generating non-human antibodies specific for CD73 and screening the antibodies for biological activity.
  • the priming immunogen can be full-length mature human CD73, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • Chimeric human antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody may be an IgG antibody, and the IgG1 subtype may be used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods of the present application.
  • kappa chains or variants thereof can be used in the compounds and methods of the present application.
  • the present application provides that the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are prepared Use in a medicament for the prevention and/or treatment of a disease and/or disorder.
  • the present application also provides a method for preventing and/or treating a disease and/or disorder, the method may comprise administering to a subject in need thereof the isolated antigen-binding protein, the polypeptide molecule, The nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.
  • the use can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition described in this application can be used with for the prevention and/or treatment of diseases and/or disorders.
  • the disease and/or disorder may be a CD73 mediated disease and/or disorder.
  • the disease and/or disorder includes a tumor.
  • the tumors include solid tumors and/or hematological tumors.
  • the disease and/or disorder includes breast cancer.
  • the present application also provides a method for detecting CD73 in a sample, the method comprising administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunization The conjugate and/or the pharmaceutical composition.
  • the method of detecting CD73 in a sample is an in vitro method. In certain instances, the method of detecting CD73 in a sample is non-therapeutic. In certain instances, the method of detecting CD73 in a sample is not a diagnostic method.
  • the present application also provides a reagent or kit for detecting CD73 in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunization The conjugate and/or the pharmaceutical composition.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are in Use in the preparation of a kit for detecting the presence and/or content of CD73 in a sample.
  • the method for preparing murine monoclonal antigen-binding protein adopts the hybridoma preparation technology invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497).
  • human CD73 with His-tagged protein (Met 1-Lys 547) was used as an immunization antigen, and part of Freund's Adjuvant, Complete (Sigma cat no.F5881, alias: FCA) and Freund's Adjuvant, Incomplete (Sigma cat) were used.
  • no.F5506, alias: FICA was used for immunization, and the other part used Quick Antibody-Mouse 5w (Boaolong cat no.
  • KX0210041 adjuvant to immunize multiple BALB/c and CD1 mice subcutaneously at multiple points. After three immunizations, serum was taken to detect the titer by ELISA, and FACS was used to detect the binding activity and functional activity. Finally, the best mouse was selected to obtain splenocytes for fusion with SP2/0 myeloma cells.
  • the hybridoma cell line was screened by HAT, and the cell culture supernatant was used for FACS detection to screen out the monoclonal hybridoma cell line that specifically binds to human CD73 and monkey CD73, and then screened out the monoclonal cell line that inhibits the enzymatic activity of CD73. And the screened monoclonal cell lines were screened by affinity (Biacore), and finally a monoclonal hybridoma cell line expressing human CD73 antigen-binding protein was obtained for sequence analysis. The screening data are listed in Table 1.
  • the final monoclonal hybridoma cell line has high affinity and can bind to both human and monkey.
  • variable region gene-specific cDNAs for heavy and light chains were synthesized using the SMARTer 5'RACE Synthesis Kit (TAKARA, #634859) according to the manufacturer's instructions.
  • the 5' and 3' ends of the cDNA sequence were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, so that the resulting PCR products could be cloned into
  • the heavy chain carrier pHB-Fc and the light chain carrier pHB-CK are expressed on the existing recombinant antigen-binding protein.
  • the pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, in which the L234A and L235A (Eu numbering) mutations with the antigen-binding protein ADCC knockout (knock out, KO) effect on CH2; pHB-C ⁇ vector contains Human kappa light chain constant region gene sequence.
  • variable region sequence of the antigen-binding protein was obtained through analysis by selecting single clone colonies for Sanger and NGS sequencing.
  • the variable region sequence of the anti-CD73 antigen-binding protein expressed by 3A9 is as follows:
  • CDRs IMGT definition, each CDR list is as follows):
  • the expression vector obtained in Example 2.1 was amplified by Escherichia coli, and a sufficient amount of plasmid was prepared with an endotoxin-removing plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection and expression of antigen binding. protein.
  • the host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). By mixing the prepared two heavy chain carriers with the light chain carrier, respectively, and polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes, CHO-S cells were transfected and put into Incubate in the incubator for 5-7 days. The cell culture supernatant was collected by centrifugation, and purified by Protein A affinity chromatography column to obtain human mouse antigen-binding protein (the number of antigen-binding protein expressed by 3A9 is 900581).
  • the humanization of the antigen-binding protein adopts the 3D modeling method: first, the three-dimensional structure modeling of the mouse antigen-binding protein is carried out, and the optimal structural model is selected.
  • Antibody Modeling use homology modeling method to select 5-10 optimal structural solutions
  • Loop region is generally modeled by homology modeling method, if the CDR amino acid sequence alignment result shows less than 50% Identity, then use de novo modeling Methods
  • the structure model of CDR3 was constructed.
  • Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence (structural resolution higher than 2.5 angstroms), compare the automatic modeling models, and select the optimal structural model.
  • the variable region sequences of the antigen binding proteins were then compared with the available sequences in the NCBI IgBlast database, and through identification and analysis, human framework regions (FR regions) suitable for the construction of CDR-grafted heavy and light chains were finally determined.
  • the transformation site was designed, and the variable regions of the heavy and light chains of the antigen-binding protein 900581 of the present application were respectively subjected to humanization mutation design.
  • the designed humanized sequence should not affect the structural stability of the antibody, do not affect the binding of the antigen-binding protein to the antigen, do not introduce protein modification sites such as glycosylation and phosphorylation, and do not introduce sites that are easily oxidized and aminated. , enhance structural stability and other requirements.
  • humanized heavy chain sequences and 3 humanized light chain sequences were designed for the murine antigen-binding protein sequence of 900581, and the humanized point mutant antigen-binding protein expression plasmids were passed through CHO- S cell expression and purification to obtain a humanized antigen-binding protein.
  • detection methods such as ELISA, Biacore and flow cytometry, the receptor binding ability, functional inhibitory activity, non-specific binding characteristics, thermal stability and other indicators of the humanized antigen-binding protein were screened, and 5 excellent performance indicators were obtained.
  • Humanized anti-CD73 antigen binding protein The protein numbers of the obtained humanized anti-CD73 antigen binding proteins and the corresponding VH and VL sequences are shown below:
  • the underlined regions are CDRs (defined by IMGT), and 900694, 900695, 900696, 900697 and 900698 are the numbers of five anti-CD73 humanized antigen-binding proteins, respectively.
  • the chimeric antibody 900565 is derived from hybridoma cell line 115B10.
  • the heavy chain variable region (VH) amino acid sequence of 900565 is shown in SEQ ID NO: 41
  • the light chain variable region (VL) amino acid sequence of 900565 is shown in SEQ ID NO. :42.
  • All antigen-binding proteins were diluted to 30 ⁇ g/ml with 1% BSA-containing PBS solution (1% BSA/PBS), and then diluted 3-fold in 11 gradients, 20 ⁇ L per well was added to 96-well U-plate, and a negative control was set simultaneously. (Add only 1% BSA/PBS).
  • the 96-well U-shaped plate after the reaction was resuspended with 1% BSA/PBS, centrifuged (300g ⁇ 3min) to discard the supernatant, washed once in this way, and 1:200 diluted PE-goat anti-human-Fc (Jackson ImmunoResearch, #109-115-098), react at room temperature for 15 minutes in the dark; resuspend the reacted 96-well U-plate with 1% BSA/PBS, centrifuge (300g ⁇ 3min) to discard the supernatant, and wash 3 times in this way, and finally use each The wells were resuspended in 100 ⁇ L of 1% BSA/PBS, and the fluorescence intensity of the PE channel was detected by a flow cytometer (BD, #CantoII).
  • BD flow cytometer
  • antigen binding protein 900581 900565 EC50( ⁇ g/mL) 0.4258 2.141
  • antigen binding protein 900581 900694 900695 900696 900697 900698 EC50( ⁇ g/mL) 0.3466 0.6461 0.5942 0.6840 0.5176 0.6879
  • the recombinant CD73 protein was diluted to 0.3 ⁇ g/ml with TM Buffer (ROCKLAND, #MB-059), and 25 ⁇ L/well of each was added to a 96-well plate and mixed. Then, the diluted AMP and ATP were mixed 1:1, 50 ⁇ L/well was added to the above 96-well plate, and the reaction was carried out for 15 min. will finally Substrate addition Buffer, mix well and equilibrate to room temperature, then add to the well to be tested, 100 ⁇ L/well, react at room temperature for 10 min, and detect at full wavelength.
  • TM Buffer TM Buffer
  • antigen binding protein 900581 900694 900695 900696 900697 900698 EC50( ⁇ g/mL) 0.2621 0.6812 0.7460 0.7509 0.5854 0.5876
  • All antigen-binding proteins were diluted to 30 ⁇ g/ml in PBS and then diluted 5-fold in 6 gradients.
  • the percentage of enzymatic activity was assessed as follows: residual CD73 activity was: (CHOK1-huCD73-3F4 cells+Ab+ATP+AMP)-(ATP+AMP)/(CHOK1-huCD73-3F4 cells+ATP+AMP) -(ATP+AMP)*100.
  • antigen binding protein 900581 900565 EC50( ⁇ g/mL) 0.9437 1.625
  • antigen binding protein 900581 900697 900698 EC50( ⁇ g/mL) 1.853 1.972 1.431
  • Human CD73 protein was diluted with experimental buffer to a series of concentrations of 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM, 1.57nM as the analyte, and the experimental buffer was used as a 0 concentration control, at a flow rate of 30 ⁇ L/min, binding for 120s, Dissociate for 600s. 10mM Glycine pH 1.5 at a flow rate of 50 ⁇ L/min, regeneration 60s multi-cycle kinetic assay.
  • the Fit local model was used to fit the association rate constant (ka), the dissociation rate constant (kd) and the dissociation equilibrium rate constant (KD).
  • the fitting results are shown in Table 8, showing that all antigen-binding proteins were Binds to human CD73 protein, and most antigen-binding proteins have no significant difference in binding affinity with human CD73 protein.
  • Table 8 Binding affinity detection of 900581, 900694-900698 to human CD73 protein
  • 900581, 900694, 900695, 900696, 900697 and 900698 were respectively associated with human CD73 protein (manufacturer: ACRO Biosystems, product number: CD3-H52H7), mouse CD73/NT5E (manufacturer: ACRO Biosystems, product number: CD3-M52H9), rat CD73 (manufacturer: Novoprotein, product number: CB16) and monkey CD73 (manufacturer: Novoprotein, product number: CI21)
  • the binding kinetics experiments were detected by Biacore (GE, model number: T200), using protein A chip (manufacturer: GE, product number: 29- 1275-56), and the experimental buffer was HBS-EP+ (pH 7.4).
  • Human CD73 protein/monkey CD73/rat CD73/mouse CD73 were diluted with assay buffer to a series of concentrations of 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM, 1.57nM as analytes, and the assay buffer was used as 0 Concentration control, under the flow rate of 30 ⁇ L/min, binding for 120s and dissociation for 600s. 10mM Glycine pH 1.5 at a flow rate of 50 ⁇ L/min, regeneration 60s multi-cycle kinetic assay.
  • the Fit local model was used to fit the association rate constant (ka), the dissociation rate constant (kd) and the dissociation equilibrium rate constant (KD).
  • the fitting results are shown in Table 9, showing that the above antigen-binding proteins are all It can bind to human CD73 and monkey CD73 (manufacturer: Novoprotein, product number: CI21), and most of the antigen-binding proteins have no significant difference in binding affinity with human and monkey CD73 proteins. : ACRO Biosystems, product number: CD3-M52H9) and rat (manufacturer: Novoprotein, product number: CB16) CD73 protein binding.
  • Control protein 900222, chimeric protein 900581 and humanized protein 900697 and 900698 were diluted with PBS solution to 1 ⁇ g/ml, and each concentration was replicated in 2 wells.
  • the sequence of the control protein 900222 was derived from the mAb-CD73.4-IgG2 antibody sequence in the CD73 antibody patent US20190284293A1 of Bristol-Myers Squibb. Calu6 cells in the logarithmic growth phase were taken and prepared into 2 ⁇ 10 5 /ml cell suspension, 100 ⁇ L per well (about 20,000 cells) was added to a 96-well U-shaped plate, centrifuged at 300 g ⁇ 3 min, and the supernatant was removed.
  • antigen binding protein 900222 900581 900697 900698 Endocytosis efficiency (%) 44.18 78.75 98.17 96.35
  • 5-10 ⁇ M of antigen or antigen-binding protein or antigen-binding protein complex (1:1 molar ratio) (50 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM TCEP) were placed at 4 °C for 1 hour to form a complex. stable state.
  • 5 ⁇ L samples were then diluted into 20 ⁇ L D2O (deuterium) and placed into various HDX time points for mass spectrometry analysis (eg, 0, 10, 60, 300, 900 seconds). After a period of hydrogen deuterium exchange, the reaction was terminated by mixing with 25 ⁇ L of ice-cold 4M guanidine hydrochloride and 1% trifluoroacetic acid.
  • the sample tube was passed through a fixed pepsin column at a flow rate of 120 ⁇ L/min, and the enzymatically digested peptides were captured on a C18 capture column and desalted.
  • the desalted peptides were separated using a 2.1 mm x 5 cm C18 column (1.9 ⁇ m Hypersil Gold, Thermo Fisher) with a linear gradient of 4-40% acetonitrile and 0.3% formic acid over 8 minutes.
  • protein digestion and peptide separation were performed at 4°C.
  • Mass spectrometry data for hydrogen deuterium exchange was acquired using an Orbitrap mass spectrometer (Orbitrap Fusion TM Tribrid TM Mass Spectrometer, Thermo Fisher), measured at a resolution of 65,000 (m/z 400). Each sample had three HDX triplicates at each time point.
  • Orbitrap mass spectrometer Orbitrap Fusion TM Tribrid TM Mass Spectrometer, Thermo Fisher
  • Hydrogen deuterium exchange mass spectrometry (HDX-MS) assay results showed that the binding epitope of 900581 mainly included the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37) and the 178-SKETPFLSNPGTNL-191 region (SEQ ID NO: 38) of the N-terminal domain of CD73 ), and the 381-WNHVSM-386 region of the CD73 C-terminal domain (SEQ ID NO: 39), when the three discontinuous regions were positioned on the three-dimensional structural model of CD73, the epitope was found to be at the edge of the catalytically active center of CD73 , as shown in Figure 7.
  • CD73 After CD73 binds the substrate AMP, it will undergo a configuration change from the open conformation to the closed conformation. After replacing AMP with the non-hydrolyzed analog of AMP, APCP, because APCP cannot be hydrolyzed to generate adenosine, CD73 can be locked in the closed conformation. Intermediate state. As the concentration of APCP increases, the proportion of CD73 in the catalytic intermediate state in the closed conformation will increase accordingly. For the antigen-binding protein bound to the catalytic region, the proportion of the bound CD73 will decrease accordingly, while the proportion of the antigen-binding protein bound to the non-catalytic region will decrease accordingly. The combined ratio does not change.
  • cells are prepared into 1E6/ml cell suspension with 1% BSA, and 20 ⁇ L per well is added to a 96-well U-shaped plate.
  • the APCP was diluted with 1% BSA to 30 nM/30 ⁇ M, respectively, and 20 ⁇ L/well was added to the above-mentioned 96-well U-shaped plate, mixed well, and placed at room temperature for 30 min.
  • the antigen-binding protein to be tested was diluted to 90 ⁇ g/ml, and then diluted 3-fold for 10 gradients.
  • serially diluted antigen-binding protein was added to the above-mentioned 96-well U-shaped plate, 20 ⁇ L/well, mixed well, and placed at room temperature for 30 min. After the incubation, centrifuge at 300g ⁇ 3min, remove the supernatant, add 20 ⁇ L/well of secondary antibody PE goat anti-human IgG Fc (1:200), mix well, and place at room temperature for 15min; after the incubation, wash twice, 300g ⁇ 3min , flow detection PE-MFI.
  • CD73 humanized mice were used to establish an animal model of MDA-MB-231 triple-negative breast cancer and to test the efficacy of the tested antibodies.
  • 100 ⁇ l of MDA-MB-231 (ATCC bank) cells (2E6 cells) were inoculated into CD73 humanized mice to establish a CD73 humanized mouse MDA-MB-231 triple-negative breast cancer animal model, tumor-forming mice According to the tumor volume and body weight, the mice were evenly divided into 4 experimental groups, with 6 mice in each group. The specific dosing schedule is shown in the table below.
  • 900201 is a negative control antibody, which does not bind to CD73 antigen.
  • the heavy chain (HC) amino acid sequence of 900201 is shown in SEQ ID NO:43
  • the light chain (LC) amino acid sequence of 900201 is shown in SEQ ID NO:44.
  • the results show that 900581, 900697 and 900698 can significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer, and have no effect on the body weight growth indicators of mice.
  • the negative control antibody 900201 could not inhibit tumor growth.
  • the MDA-MB-231 triple-negative breast cancer animal model was established using NPG mice and the efficacy of the tested antibodies was tested.
  • the human breast cancer cells MDA-MB-231 used in this experiment were cultured in L-15 medium supplemented with 10% FBS in a CO2 -free 37°C incubator. Before the cells were continuously cultured for ten passages, about 5.0 ⁇ 10 6 MDA-MB-231 cells were suspended in 100 ⁇ L PBS, mixed with an equal volume of Matrigel, and then inoculated into the right side of the back of NPG humanized mice near the armpit by subcutaneous injection. Below, the inoculation volume is about 200 ⁇ L. Mice were anesthetized with 2-5% isoflurane prior to inoculation.
  • 900201 is a negative control antibody, which does not bind to CD73 antigen.
  • the heavy chain (HC) amino acid sequence of 900201 is shown in SEQ ID NO:43
  • the light chain (LC) amino acid sequence of 900201 is shown in SEQ ID NO:44.
  • Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca.
  • oleclumab and 900698 can significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer, while the negative control antibody 900201 cannot inhibit the growth of tumors.
  • NPG mice were used to establish a BxPC-3 pancreatic cancer animal model and to test the efficacy of the tested antibodies.
  • Human pancreatic cancer cells BxPC-3 used in this experiment were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator with 5% CO 2 . Before the cells were continuously cultured for ten passages, 100 ⁇ L of PBS containing about 1 ⁇ 10 7 BxPC-3 cells was mixed with an equal volume of Matrigel, and then inoculated into 18 NPG mice by subcutaneous injection on the right side of the back near the armpit, and the inoculation volume was about 200 ⁇ L. . Mice were anesthetized with 2-5% isoflurane prior to inoculation.
  • 900543 is a negative control antibody (900543 is the ADCC knockout type of 900201), which does not bind to CD73 antigen.
  • the heavy chain (HC) amino acid sequence of 900543 is shown in SEQ ID NO:47, and the light chain (LC) amino acid sequence of 900543 is shown in SEQ ID NO:44.
  • Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca.
  • oleclumab and 900698 can significantly inhibit the growth of BxPC-3 pancreatic cancer, while the negative control antibody 900543 cannot inhibit the growth of the tumor.

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Abstract

提供一种分离的抗原结合蛋白，其能够结合CD73。所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。还提供所述抗原结合蛋白的制备方法和应用。

Description

CD73的抗原结合蛋白及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种CD73的抗原结合蛋白及其应用。
背景技术
CD73，也称NT5E，是一种分子量约为70Kd的细胞外5-核酸酶(NT5E)，通过糖磷脂酰肌醇(GPI)锚定于细胞表面，主要以二聚体形式存在。在正常生理状态下，CD73可表达于多种组织或器官，如肝脏，大肠，肾脏，脾脏，肺，卵巢，淋巴结等组织。
大量临床前研究结果显示，CD73在多种肿瘤细胞表面异常高表达，包括脑胶质瘤，乳腺癌，黑色素瘤，非小细胞肺癌，膀胱癌，卵巢癌，结直肠癌等，且临床数据显示CD73的高表达与肿瘤病人的不良预后密切相关，因此CD73可作为多种肿瘤临床治疗及预后的潜在靶标。
但是，目前市面上尚未有针对人CD73的抗体或小分子药物问世，迫切需要开发更多的靶向肿瘤CD73的药物，为治疗CD73异常表达的癌症提供新的思路和前景。
发明内容
本申请提供了一种分离的抗原结合蛋白，其表现出一种或多种期望的功能特性，例如其能够特异性结合CD73或其功能活性片段；其能够阻止或减少CD73的活化；当与所述CD73结合后，所述分离的抗原结合蛋白能够与CD73的第143-157位氨基酸或与其对应的CD73序列、第178-191位氨基酸或与其对应的CD73序列和第381-386位氨基酸或与其对应的CD73序列中的至少一个氨基酸残基相结合；能够抑制CD73的酶活性；以及能够诱导细胞表面CD73的内吞。本申请还提供编码所述分离的抗原结合蛋白的核酸分子、表达载体、宿主细胞和制备所述分离的抗原结合蛋白的方法。本申请所述分离的抗原结合蛋白可以用于预防和/或治疗疾病和/或病症，例如肿瘤。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:7、SEQ ID NO:15或SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:8、SEQ ID NO:16或SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:11或SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:12或SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:13、SEQ ID NO:25、SEQ ID NO:27或SEQ ID NO:28所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR4，所述L-FR4的N末端与所述 LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:14或SEQ ID NO:26所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33或SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VL，所述VL包含SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含选自下述的任一组VH和VL：
1)所述VH包含SEQ ID NO:29所示的氨基酸序列，且所述VL包含SEQ ID NO:30所示的氨基酸序列；
2)所述VH包含SEQ ID NO:31所示的氨基酸序列，且所述VL包含SEQ ID NO:32所示的氨基酸序列；
3)所述VH包含SEQ ID NO:33所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列；
4)所述VH包含SEQ ID NO:35所示的氨基酸序列，且所述VL包含SEQ ID NO:36所示的氨基酸序列；
5)所述VH包含SEQ ID NO:33所示的氨基酸序列，且所述VL包含SEQ ID NO:36所示的氨基酸序列；以及
6)所述VH包含SEQ ID NO:35所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人抗体重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人IgG重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人IgG1重链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链恒定区。
在某些实施方式中，所述抗体轻链恒定区源自人Igκ恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合CD73或其功能活性片段。
在某些实施方式中，所述CD73包括人CD73和/或猴CD73。
在某些实施方式中，所述分离的抗原结合蛋白能够阻止或减少CD73的活化。
在某些实施方式中，当与所述CD73结合时，所述分离的抗原结合蛋白与SEQ ID NO:40所示的氨基酸序列的第143-157位氨基酸或与其对应的CD73序列、SEQ ID NO:40所示的氨基酸序列的第178-191位氨基酸或与其对应的CD73序列和SEQ ID NO:40所示的氨基酸序列的第381-386位氨基酸或与其对应的CD73序列中的至少一个氨基酸残基相结合。
在某些实施方式中，所述第143-157位氨基酸或与其对应的CD73序列位于所述CD73的N端结构域，所述第178-191位氨基酸或与其对应的CD73序列位于所述CD73的N端结构域，且所述第381-386位氨基酸或与其对应的CD73序列位于所述CD73的C端结构域。
在某些实施方式中，所述分离的抗原结合蛋白能够抑制CD73的酶活性。
在某些实施方式中，所述分离的抗原结合蛋白能够诱导细胞表面CD73的内吞。
另一方面，本申请还提供了一种多肽分子，其包含所述分离的抗原结合蛋白。
在某些实施方式中，所述多肽分子包含融合蛋白。
另一方面，本申请还提供了一种分离的核酸分子，其编码所述分离的额抗原结合蛋白或所述多肽分子。
另一方面，本申请还提供了一种载体，其包含所述核酸分子。
另一方面，本申请还提供了一种细胞，其包含所述核酸分子和/或所述载体。
另一方面，本申请还提供了一种免疫缀合物，其包含所述分离的抗原结合蛋白。
另一方面，本申请还提供了一种药物组合物，其包含所述分离的抗原结合蛋白，所述的多肽分子、所述的核酸分子、所述的载体、所述的细胞和/或所述的免疫缀合物，以及任选地药学上可接受的载剂。
另一方面，本申请还提供了一种制备所述分离的抗原结合蛋白的方法，其包括在使得所述分离的抗原结合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
另一方面，本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物，其可以用于预防和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症是CD73介导的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述疾病和/或病症包括乳腺癌。
另一方面，本申请还提供了检测样品中CD73的方法，其包括施用所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
另一方面，本申请还提供了检测样品中CD73的试剂或试剂盒，其包含所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中CD73的存在和/或含量。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述抗原结合蛋白900581、900565与细胞表面CD73的结合活性。
图2显示的是本申请所述抗原结合蛋白900581、900694-900698与细胞表面CD73的结合活性。
图3显示的是本申请所述抗原结合蛋白900581、900694-900698对重组CD73的酶活性的抑制。
图4显示的是本申请所述抗原结合蛋白900581、900565对细胞表面CD73的酶活性的抑制。
图5显示的是本申请所述抗原结合蛋白900581、900697和900698对细胞表面CD73的酶活性的抑制。
图6显示的是本申请所述抗原结合蛋白900581诱导细胞表面CD73内吞的效率。
图7显示的是本申请所述抗原结合蛋白结合表位的结构模型。
图8A-8C显示的是本申请所述抗原结合蛋白与900609的竞争结合活性。
图9显示的是本申请所述抗原结合蛋白的体内治疗肿瘤的效果。
图10显示的是本申请所述抗原结合蛋白的体内治疗肿瘤的效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“CD73”也称“NT5E”、“分化簇73”、“5’-核苷酸酶(5’-NT)”或“胞外5’-核苷酸酶”。所述术语涵盖“全长”、未加工的CD73以及由细胞加工所产生的任何形式的CD73。在本申请中，术语“CD73”包括全长野生型CD73及其突变体、片段、变体、同种型和同源物。在某些实施方式中，所述CD73可以包括人CD73。例如针对人CD73的序列可以见Uniprot登录号P21589下。在本申请中，所述CD73可以包含如SEQ ID NO:40所示的氨基酸序列。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的抗原结合蛋白”通常指脱离了其天然存在状态的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工 框架区。此类框架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。抗原结合蛋白的实例包括但不限于：人抗体、人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv，dAb，IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；或IgG4抗体以及其片段。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。例如，所述抗原结合蛋白的氨基酸序列编号可以按照IMGT编号方案(IMGT,the international ImMunoGeneTics information system@imgt.cines.fr；http：//imgt.cines.fr；Lefranc等,1999,Nucleic Acids Res.27:209-212；Ruiz等,2000 Nucleic Acids Res.28:219-221；Lefranc等,2001,Nucleic Acids Res.29:207-209；Lefranc等,2003,Nucleic Acids Res.31:307-310；Lefranc等,2005,DevComp Immunol 29:185-203)。例如，所述抗原结合蛋白的CDR可以根据Kabat编号系统确定(参见例如Kabat EA&Wu TT(1971)Ann NY AcadSci 190:382-391和Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)。
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。它通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合人CD73)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，CD73)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(V H)的第一恒定区(C H1)组成。
在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。
在本申请中，术语“单克隆抗体”通常指单分子组成的抗体分子制备物。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单 克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
术语“全人源抗体”通常指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术在美国专利：6,150,584、6,458,592、6,420,140中描述。其它技术，如使用文库，是本领域已知的。
在本申请中，术语“多肽分子”和“多肽”、“肽”可以互换使用，通常指氨基酸残基的聚合物。术语“融合蛋白”通常指具有至少两个共价连接在一起的部分的多肽。其中每个部分可以是具有不同特性的多肽。该特性可以是生物学性质，例如体外或体内活性。该性质也可以是简单的化学或物理性质，例如与靶分子的结合，反应的催化等。这两个部分可以通过单个肽键或通过肽接头直接连接。
在本申请中，术语“核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类可以包括逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293细胞。
在本申请中，术语“免疫缀合物”通常指所述其他试剂(例如，化疗剂、放射性元素、细胞生长抑制剂和细胞毒性剂)与所述抗体或其抗原结合片段缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述抗体或其抗原结合片段与靶细胞上的抗原特异性结合，将所述其他试剂递送至靶细胞(例如，肿瘤细胞)。
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载 体可包括例如缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，例如钠；和/或非离子表面活性剂。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
术语“肿瘤”通常指任何新的病理性的组织增生。肿瘤细胞可以局部地或通过血流和淋巴系统扩散到身体其他部分。在本申请中，所述肿瘤可以包括良性肿瘤和恶性肿瘤。在本申请中，所述肿瘤可以包括实体瘤和/或血液瘤。在本申请中，所述肿瘤可以包括癌症。在本申请中，所述肿瘤的实例包括但不限于：脑胶质瘤、乳腺癌、黑色素瘤、非小细胞肺癌、膀胱癌、卵巢癌和结直肠癌。
在本申请中，术语“活化”通常指从无活性状态向有活性状态的转化。在某些实施方式中，阻止或减少CD73的活化可以指阻止或减少CD73从无催化活性的开放构象向催化活性的封闭构象的转化。
在本申请中，术语“减少”通常指与原水平相比，在数量和/或程度上的降低。例如，所述减少CD73的活化可以指与所述分离的抗原结合蛋白不存在的情况下相比，将CD73的活化减少至少约0.5倍、至少约1倍、至少约1.5倍、至少约2倍、至少约5倍、至少约10倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约60倍、至少约70倍、至少约80倍、至少约90倍或至少约100倍。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合CD73蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋 白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合CD73蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
分离的抗原结合蛋白
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，所述CDR由IMGT编号方案来确定。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含HCDR3，所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白还可以包含HCDR2，所述HCDR2可以包含如SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白还可以包含HCDR1，所述HCDR1可以包含如SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，HCDR2和HCDR1。例如，所述分离的抗原结合蛋白的HCDR3可以包含SEQ ID NO:3所示的氨基酸序列，HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，且HCDR1可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白还可以包含LCDR2，所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白还可以包含LCDR1，所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，LCDR2和LCDR1。例如，所 述分离的抗原结合蛋白的LCDR3可以包含SEQ ID NO:6所示的氨基酸序列，LCDR2可以包含SEQ ID NO:5所示的氨基酸序列，且LCDR1可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1。例如，本申请所述的分离的抗原结合蛋白HCDR3可以包含SEQ ID NO:3所示的氨基酸序列，HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，HCDR1可以包含SEQ ID NO:1所示的氨基酸序列，LCDR3可以包含SEQ ID NO:6所示的氨基酸序列，LCDR2可以包含SEQ ID NO:5所示的氨基酸序列，且LCDR1可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端可以与所述HCDR1的N末端直接或间接相连，所述H-FR1可以包含如SEQ ID NO:7、SEQ ID NO:15或SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2可以位于所述HCDR1与所述HCDR2之间，所述H-FR2可以包含如SEQ ID NO:8、SEQ ID NO:16或SEQ ID NO:21所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3可以位于所述HCDR2与所述HCDR3之间，所述H-FR3可以包含如SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N末端可以与所述HCDR3的C末端相连，所述H-FR4可以包含如SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4。例如，所述分离的抗原结合蛋白的H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:19、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:18所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端可以与所 述LCDR1的N末端直接或间接相连，所述L-FR1可以包含如SEQ ID NO:11或SEQ ID NO:23所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR2，所述L-FR2可以位于所述LCDR1与所述LCDR2之间，所述L-FR2可以包含如SEQ ID NO:12或SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR3，所述L-FR3可以位于所述LCDR2与所述LCDR3之间，所述L-FR3可以包含如SEQ ID NO:13、SEQ ID NO:25、SEQ ID NO:27或SEQ ID NO:28所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR4，所述L-FR4的N末端可以与所述LCDR3的C末端相连，所述L-FR4可以包含如SEQ ID NO:14或SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，L-FR2，L-FR3和L-FR4。例如，所述分离的抗原结合蛋白的L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列。例如，所述分离的抗原结合蛋白的L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列。例如，所述分离的抗原结合蛋白的L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:27和SEQ ID NO:26所示的氨基酸序列。例如，所述分离的抗原结合蛋白的L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:28和SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VH，所述VH可以包含SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33或SEQ ID NO:35所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VL，所述VL可以包含SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含所述VH和VL。在某些实施方式中，所述VH可以包含SEQ ID NO:29所示的氨基酸序列，且所述VL可以包含SEQ ID NO:30所示的氨基酸序列。在某些实施方式中，所述VH可以包含SEQ ID NO:31所示的氨基酸序列，且所述VL可以包含SEQ ID NO:32所示的氨基酸序列。在某些实施方式中，所述VH可以包含SEQ ID NO:33所示的氨基酸序列，且所述VL可以包含SEQ ID NO:34所示的氨基酸序列。在某些实施方式中，所述VH可以包含SEQ ID NO:35所示的氨基酸序列，且所述VL 可以包含SEQ ID NO:36所示的氨基酸序列。在某些实施方式中，所述VH可以包含SEQ ID NO:33所示的氨基酸序列，且所述VL可以包含SEQ ID NO:36所示的氨基酸序列。在某些实施方式中，所述VH可以包含SEQ ID NO:35所示的氨基酸序列，且所述VL可以包含SEQ ID NO:34所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述的VH中的至少一个CDR。在本申请中，所述分离的抗原结合蛋白可以包含本申请所述的VL中的至少一个CDR。所述CDR可以根据任何划分方式划分得到。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述VH中的HCDR1，HCDR2和HCDR3。所述VH可以包含SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33或SEQ ID NO:35所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述VL中的LCDR1，LCDR2和LCDR3。所述VL可以包含SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区。所述抗体重链恒定区可以源自人IgG重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1重链恒定区。
在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区。所述抗体轻链恒定区可以源自人Igκ恒定区。
在本申请中，所述分离的抗原结合蛋白可以包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段可以包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体可以包括单克隆抗体、嵌合抗体、人源化抗体和/或全人源抗体。
例如，所述嵌合抗体的VH可以包含SEQ ID NO:29所示的氨基酸序列，且所述嵌合抗体的VL可以包含SEQ ID NO:30所示的氨基酸序列。
例如，所述人源化抗体的VH可以包含SEQ ID NO:31所示的氨基酸序列，且所述人源化抗体的VL可以包含SEQ ID NO:32所示的氨基酸序列。
例如，所述人源化抗体的VH可以包含SEQ ID NO:33所示的氨基酸序列，且所述人源化抗体的VL可以包含SEQ ID NO:34所示的氨基酸序列。
例如，所述人源化抗体的VH可以包含SEQ ID NO:35所示的氨基酸序列，且所述人源 化抗体的VL可以包含SEQ ID NO:36所示的氨基酸序列。
例如，所述人源化抗体的VH可以包含SEQ ID NO:33所示的氨基酸序列，且所述人源化抗体的VL可以包含SEQ ID NO:36所示的氨基酸序列。
例如，所述人源化抗体的VH可以包含SEQ ID NO:35所示的氨基酸序列，且所述人源化抗体的VL可以包含SEQ ID NO:34所示的氨基酸序列。
此外，需要说明的是，本申请所述分离的抗原结合蛋白可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述分离的抗原结合蛋白中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请所述分离的抗原结合蛋白的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改不会使抗原结合性消失，具体可以参见，例如，Brummell et al.,(1993)Biochem 32:1180-8；de Wildt et al.,(1997)Prot.Eng.10:835-41；Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870；Hall et al.,(1992)J.Immunol.149:1605-12；Kelley and O'Connell(1993)Biochem.32:6862-35；Adib-Conquy et al.,(1998)Int.Immunol.10:341-6and Beers et al.,(2000)Clin.Can.Res.6:2835-43。
可以通过本领域已知的各种测定鉴别、筛选或表征本申请所述的CD73抗原结合蛋白。
例如，可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白或融合蛋白的抗原结合活性。
在本申请中，所述分离的抗原结合蛋白能够特异性结合CD73或其功能活性片段。
在某些实施方式中，所述分离的抗原结合蛋白可以通过Biacore进行亲和力筛选。所述分离的抗原结合蛋白可以以5×10 -9M或更低的KD与CD73蛋白相结合，例如，本申请所述的抗原结合蛋白可以以小于约4×10 -9M、小于约3×10 -9M、小于约2×10 -9M、小于约1×10 -9M、 小于约7×10 -10M、小于约6×10 -10M、小于约5×10 -10M、小于约4×10 -10M、小于约3×10 -10M、小于约2×10 -10M、小于约1×10 -10M、小于约9×10 -11M、小于约5×10 -11M、小于约2×10 -11M的KD结合CD73。在某些实施方式中，本申请也包括使用FACS方法测定抗原结合蛋白与抗原的结合活性。例如，本申请所述抗原结合蛋白可以以小于或等于约0.5μg/mL、小于或等于约0.4μg/mL、小于或等于约0.3μg/mL、小于或等于约0.2μg/mL、小于或等于约0.1μg/mL的EC50值与细胞表面人CD73结合。例如，本申请所述抗原结合蛋白可以以小于或等于约0.4μg/mL、小于或等于约0.3μg/mL、小于或等于约0.2μg/mL、小于或等于约0.1μg/mL的EC50值与细胞表面人CD73结合。
在某些实施方式中，所述分离的抗原结合蛋白能够结合CD73。在某些情形中，本申请所述的抗原结合蛋白还可以与猴的CD73交叉反应。例如，通过FACS所检测的。在本申请中，“交叉反应”通常指抗体与来自其他物种的同源蛋白反应的能力。
在某些实施方式中，本申请所述分离的抗原结合蛋白不与小鼠和/或大鼠的CD73结合。
例如，可以通过ELISA方法检测效价。例如，可以使用FACS检测检测所述分离的抗原结合蛋白的结合活性或功能活性。例如，可以对所述分离的抗原结合蛋白进行亲和力检测。
在本申请中，所述分离的抗原结合蛋白能够阻止或减少CD73的活化。例如，所述分离的抗原结合蛋白能够阻止或减少CD73由无催化活性的开放构象向催化活性的封闭构象的转化。例如，所述分离的抗原结合蛋白能够将CD73催化中心封闭，从而抑制CD73的酶活。
在某些实施方式中，所述分离的抗原结合蛋白的结合表位可以通过氢氘交换质谱(HDX-MS)测定。在某些实施方式中，所述分离的抗原结合蛋白与CPI-006抗体(Corvus)具有竞争结合活性。
在本申请中，当与所述CD73相结合时，所述分离的抗原结合蛋白与CD73N端结构域的143-NIKAKGPLASQISGL-157区域(SEQ ID NO:37)或与其对应的CD73序列，178-SKETPFLSNPGTNL-191区域(SEQ ID NO:38)或与其对应的CD73序列，以及CD73C端结构域的381-WNHVSM-386的区域(SEQ ID NO:39)或与其对应的CD73序列中的至少一个氨基酸残基相结合。
在本申请中，“与其对应的CD73序列”可以指其同源物(或者来自其他物种的CD73)中相应位置处的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够抑制CD73的酶活性。例如，所述分离的抗原结合蛋白可以对重组CD73具有酶活性抑制的效果。例如，所述分离的抗原结合蛋白可以对人CD73具有酶活性抑制的效果。本申请所述酶活性可以通过本领域常用的任何方法检测 得到。所述酶活性的检测可以包括将CD73抗原结合蛋白与重组CD73蛋白混匀，加入AMP和ATP，将
Figure PCTCN2021136950-appb-000001
底物加入
Figure PCTCN2021136950-appb-000002
缓冲液，混匀并平衡至室温反应，进行全波长检测。例如，所述分离的抗原结合蛋白可以以小于或等于约0.8μg/mL、小于或等于约0.7μg/mL、小于或等于约0.6μg/mL、小于或等于约0.5μg/mL、小于或等于约0.4μg/mL、小于或等于约0.3μg/mL、小于或等于约0.2μg/mL的EC50值抑制重组CD73蛋白的酶活性。例如，所述分离的抗原结合蛋白可以以小于或等于约6μg/mL、小于或等于约5μg/mL、小于或等于约4μg/mL、小于或等于约3μg/mL、小于或等于约2μg/mL、小于或等于约1μg/mL、小于或等于约0.5μg/mL的EC50值抑制细胞膜上CD73蛋白的酶活性。
在本申请中，所述分离的抗原结合蛋白能够诱导细胞表面CD73的内吞。在某些实施方式中，可以通过荧光标记的方法判断蛋白的内吞情况。例如，可以使用流式细胞术检测细胞的荧光强度获得所述抗原结合蛋白诱导细胞表面CD73内吞的效率。在某些情形下，所述分离的抗原结合蛋白可以以约50％或更高的效率诱导CD73的内吞。
多肽分子、核酸分子、载体、细胞、免疫缀合物和药物组合物
另一方面，本申请提供了多肽分子，其可以包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，所述多肽分子可以包含融合蛋白。在某些实施方式中，所述多肽分子可以为融合蛋白。
另一方面，本申请提供了分离的核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(ii)通过克隆重组产生的；(iii)纯化的，例如通过酶切和凝胶电泳分级分离；或者(iv)合成的，例如通过化学合成。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的 载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、293T细胞、COS-1细胞、SP2/0细胞、NS0细胞或骨髓瘤细胞。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请还提供了免疫缀合物，其可以包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，可以将本申请所述的分离的抗原结合蛋白或其片段与另一试剂，如化学治疗剂、毒素、免疫治疗剂、成像探针、分光镜探针等的连接。该连接可以通过一个或多个共价键，或非共价相互作用，并且可以包括螯合作用。可以使用多种接头(所述接头可以为本领域所知)以形成免疫缀合物。此外，可以以融合蛋白质的形式提供免疫缀合物，其可以由编码免疫缀合物的多核苷酸表达。所述免疫缀合物还可以包含例如抗体-药物缀合物(ADC)。合适的药物可以包括细胞毒素、烷化剂、DNA小沟结合分子、DNA嵌入剂、DNA交联剂、组蛋白去乙酰化酶抑制剂、核输出抑制剂、蛋白酶体抑制剂、拓扑异构酶I或II的抑制剂、热激蛋白抑制剂、酪氨酸激酶抑制剂、抗生素和抗有丝分裂剂。在ADC中，抗体和治疗剂可以通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
制备方法
另一方面，本申请提供了制备所述的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，可以用连接或天然存在的CD73或其片段，免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。例如，可以使用杂交瘤制备方法，获取已经过免疫的小鼠的脾细胞与SP2/0骨髓瘤细胞进行融合，经过HAT筛选出杂交瘤细胞株。
任何合适形式的CD73都可以作为免疫原(抗原)，用于产生对CD73特异的非人抗体，筛选所述抗体的生物学活性。例如，激发免疫原可以是全长的成熟人CD73，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。
嵌合人源抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体可以是IgG抗体，可以使用IgG1亚型。可以通过用下文实施例中描述的生物学测定筛选抗体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本申请的化合物和方法中使用。例如，本申请的化合物和方法中可以使用κ链或其变体。
方法和用途
另一方面，本申请提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载 体、所述细胞、所述免疫缀合物和/或所述药物组合物。
在本申请中，所述使用可以通过不同方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物，其可以用于预防和/或治疗疾病和/或病症。
在本申请中，所述疾病和/或病症可以是CD73介导的疾病和/或病症。
在本申请中，所述疾病和/或病症包括肿瘤。
在本申请中，所述肿瘤包括实体瘤和/或血液瘤。
在本申请中，所述疾病和/或病症包括乳腺癌。
另一方面，本申请还提供了检测样品中CD73的方法，所述方法包括施用所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
在某些情形中，所述检测样品中CD73的方法是体外方法。在某些情形中，所述检测样品中CD73的方法为非治疗目的。在某些情形中，所述检测样品中CD73的方法不是诊断方法。
另一方面，本申请还提供了检测样品中CD73的试剂或试剂盒，其包含所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中CD73的存在和/或含量。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 抗人CD73的鼠源抗原结合蛋白的制备方法
1.1制备产生鼠源抗原结合蛋白的杂交瘤细胞
制备鼠源单克隆抗原结合蛋白的方法采用Kohler和Milstein 1975年发明的杂交瘤制备技术(Nature，1975，256：495-497)。首先将人CD73带有His标签蛋白(Met 1-Lys 547)作为免疫抗原，一部分使用弗氏佐剂：Freund’s Adjuvant,Complete(Sigma cat no.F5881，别名:FCA) 与Freund’s Adjuvant,Incomplete(Sigma cat no.F5506，别名：FICA)进行免疫，另一部分使用Quick Antibody-Mouse 5w(博奥龙cat no.KX0210041)佐剂，分别对多只BALB/c、CD1小鼠进行多点皮下免疫。经过三次免疫后取血清用ELISA法检测效价，FACS检测结合活性及功能活性。最终挑选最佳小鼠获取脾细胞与SP2/0骨髓瘤细胞进行融合。经过HAT筛选出杂交瘤细胞株，取细胞培养上清用FACS检测方法，筛选出特异性结合人CD73、猴CD73的单克隆杂交瘤细胞株，再次筛选出具有抑制CD73酶活性的单克隆细胞株，并对筛选出的单克隆细胞株进行亲和力(Biacore)筛选，最终得到表达人CD73抗原结合蛋白的单克隆杂交瘤细胞株进行序列分析，筛选数据列举见表1。
表1杂交瘤筛选
Figure PCTCN2021136950-appb-000003
经过高通量筛选，最终得到的单克隆杂交瘤细胞株具有高亲和力，可以与人和猴都具有结合活性。
实施例2 抗CD73抗原结合蛋白可变区基因测序以及各抗原结合蛋白的制备
2.1杂交瘤细胞中抗原结合蛋白的可变区基因测序及克隆
基于TAKARA的5’RACE技术原理，克隆出由杂交瘤细胞株3A9表达的小鼠抗原结合 蛋白可变区的cDNA序列。简言之，用SMARTer 5’RACE合成试剂盒(TAKARA，#634859)按说明书合成重链和轻链的可变区基因特异性cDNA。用PCR引物修饰cDNA序列的5’和3’端，所述引物设计成为分别在重链和轻链可变区cDNA上增加合适的前导序列，使所得PCR产物能够通过无缝克隆的方法克隆到现有重组抗原结合蛋白表达的重链载体pHB-Fc和轻链载体pHB-Cκ上。pHB-Fc表达载体上含有人IgG1重链恒定区基因序列，其中CH2上带有抗原结合蛋白ADCC敲除(knock out，KO)效应的L234A和L235A(Eu numbering)突变；pHB-Cκ载体上含有人κ轻链恒定区基因序列。将重链和轻链可变区PCR扩增产物通过In-fusion克隆试剂(TAKARA,#639650)克隆到表达载体上，并转化到E.coli DH5α大肠杆菌感受态细胞(益生生技，#FYE607-80VL)中。通过挑选单克隆菌落进行Sanger以及NGS测序，经分析获得抗原结合蛋白可变区序列。其中3A9表达的抗CD73抗原结合蛋白可变区序列如下：
3A9 VH SEQ ID NO：29
Figure PCTCN2021136950-appb-000004
3A9 VL SEQ ID NO：30
Figure PCTCN2021136950-appb-000005
其中，下划线区为CDRs(IMGT定义，各CDR列表如下)：
表2.鼠源CD73抗原结合蛋白CDR序列
Figure PCTCN2021136950-appb-000006
2.2抗原结合蛋白的表达
将实施例2.1中所得表达载体经过大肠杆菌扩增，用去内毒素质粒抽提试剂盒(天根生化科技(北京)有限公司，#DP117)制备足量质粒，用于瞬时转染表达抗原结合蛋白。表达 所用的宿主细胞为CHO-S细胞(赛默飞，#R80007)。通过将制备所得的两种重链载体分别和轻链载体一起，与聚醚酰亚胺(PEI，Polysciences，#24765-1)混合形成脂质体复合物后，转染CHO-S细胞，放入培养箱中培养5-7天。离心收集细胞培养液上清，通过Protein A亲和层析柱纯化得到人鼠抗原结合蛋白(3A9表达的抗原结合蛋白编号为900581)。
2.3抗原结合蛋白900694-900698的制备
抗原结合蛋白的人源化采用3D建模的方法：先对鼠源抗原结合蛋白进行三维结构建模，选出最优结构模型，具体为分别使用Discovery Studio和
Figure PCTCN2021136950-appb-000007
Antibody Modeling，采用同源建模方法选取5-10个最优结构解，Loop区域一般使用同源建模方法建模，如CDR氨基酸序列比对结果显示低于50％Identity，则使用从头建模方法搭建CDR3结构模型。使用PDB BLAST调取序列最接近的10个抗体晶体结构模型(结构分辨率高于2.5埃)，对比自动建模模型，选取最优的结构模型。然后将抗原结合蛋白的可变区序列与NCBI IgBlast数据库中的可用序列比较，通过鉴定和分析，最终确定了适合在其上构建CDR移植重链和轻链的人源构架区(FR区)。
改造时，根据人抗体FR区保守的氨基酸残基以及抗体FR区中重要的氨基酸残基，设计改造位点，对本申请抗原结合蛋白900581的重轻链的可变区分别进行人源化突变设计，设计的人源化序列应满足不影响抗体结构稳定性，不影响抗原结合蛋白与抗原结合，不引入糖基化、磷酸化等蛋白修饰位点，不引入易被氧化、氨基化等位点，增强结构稳定性等要求。经分析本次针对900581的鼠源抗原结合蛋白序列共设计了3条人源化重链序列以及3条人源化轻链序列，并将人源化点突变抗原结合蛋白表达质粒分别经CHO-S细胞表达，纯化后得到人源化抗原结合蛋白。利用ELISA，Biacore和流式细胞术等检测方法，对人源化抗原结合蛋白的受体结合能力，功能抑制活性和非特异结合特性，热稳定性等指标进行筛选，获得了5个性能优异的人源化抗CD73抗原结合蛋白。所获得人源化抗CD73抗原结合蛋白的蛋白号以及相应的VH和VL序列下所示：
900694 VH SEQ ID NO:31
Figure PCTCN2021136950-appb-000008
900694 VL SEQ ID NO:32
Figure PCTCN2021136950-appb-000009
900695以及900697 VH SEQ ID NO:33
Figure PCTCN2021136950-appb-000010
900695以及900698 VL SEQ ID NO:34
Figure PCTCN2021136950-appb-000011
900696以及900698 VH SEQ ID NO:35
Figure PCTCN2021136950-appb-000012
900696以及900697 VL SEQ ID NO:36
Figure PCTCN2021136950-appb-000013
其中，下划线区为CDRs(IMGT定义)，900694，900695，900696，900697以及900698分别为5个抗CD73人源化抗原结合蛋白编号。
实施例3 抗CD73抗原结合蛋白对细胞表面人CD73的结合活性检测
抗CD73抗原结合蛋白900581、900565以及900581抗原结合蛋白的人源化抗原结合蛋白900694，900695，900696，900697以及900698与细胞表面人CD73的细胞(CHOK1-huCD73-3F4，华博生物)的结合活性检测。其中嵌合抗体900565来源于杂交瘤细胞株115B10。900565的重链可变区(VH)氨基酸序列如SEQ ID NO:41所示，900565的轻链可变区(VL)氨基酸序列如SEQ ID NO:42所示。
所有抗原结合蛋白均用含1％BSA的PBS溶液(1％BSA/PBS)稀释至30μg/ml，再3倍稀释11个梯度，每孔20μL加入96孔U型板中，同步设定阴性对照(只加1％BSA/PBS)。取对数生长期内表达人CD73的细胞(CHOK1-huCD73-3F4，华博生物)悬液，离心(1000rpm×5min)弃培养液，用1％BSA/PBS重悬至活细胞密度为1×10 6/mL，每孔20μL(2×10 4个细胞)加入已有抗CD73抗原结合蛋白的96孔U型板中室温反应30min。将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤1遍，加入1:200稀释的PE-羊抗人-Fc(Jackson ImmunoResearch，#109-115-098)，室温避光反应15min；将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤3遍，最终用每孔100μL 1％BSA/PBS重悬，用流式细胞仪(BD，#CantoⅡ)检测PE通道的荧光强度。
900581抗原结合蛋白结合活力结果见图1及表3，900694-900698抗原结合蛋白结合活力 结果见图2及表4，结果表明900581抗原结合蛋白与细胞表面表达人CD73的细胞(CHOK1-huCD73-3F4，华博生物)有很好的亲和力，900694-900698与900581的结合活性相当。
表3.900581、900565对细胞表面人CD73结合活性
抗原结合蛋白 900581 900565
EC50(μg/mL) 0.4258 2.141
表4.900581、900694-900698对细胞表面人CD73结合活性
抗原结合蛋白 900581 900694 900695 900696 900697 900698
EC50(μg/mL) 0.3466 0.6461 0.5942 0.6840 0.5176 0.6879
实施例4 抗CD73抗原结合蛋白对重组CD73酶活性抑制的效果检测
900581以及900694，900695，900696，900697以及900698对重组CD73蛋白(ACRO，#CD3-H52H7-100μg)蛋白酶活性抑制的效果检测。具体操作如下：用TM Buffer(ROCKLAND，#MB-059)将系列CD73抗原结合蛋白稀释成120μg/mL，再5倍稀释10个梯度，25μL/well加入96孔板中。再用TM Buffer(ROCKLAND，#MB-059)将重组CD73蛋白稀释至0.3μg/ml，各25μL/well加入96孔板中，混匀。然后将稀释好的AMP和ATP进行1:1混合，50μL/well加入上述96孔板中，反应15min。最后将
Figure PCTCN2021136950-appb-000014
底物加入
Figure PCTCN2021136950-appb-000015
缓冲液，混匀并平衡至室温，然后加入待测孔，100μL/well，室温反应10min，全波长检测。
900581，900694，900695，900696，900697以及900698对重组CD73蛋白(ACRO，#CD3-H52H7-100μg)酶活性抑制的结果见图3以及表5，结果表明900694，900695，900696，900697和900698与900581抗原结合蛋白对重组CD73蛋白酶活性抑制的效果相当。
表5.900581、900694-900698对重组CD73蛋白酶活性抑制
抗原结合蛋白 900581 900694 900695 900696 900697 900698
EC50(μg/mL) 0.2621 0.6812 0.7460 0.7509 0.5854 0.5876
实施例5 抗CD73抗原结合蛋白对细胞膜上CD73酶活性抑制的效果检测
所有抗原结合蛋白均用PBS溶液稀释至30μg/ml，再以5倍比例稀释6个梯度。取对数 生长期内表达人CD73的细胞(CHOK1-huCD73-3F4，华博生物)悬液，用培养基制备成4X10 5/ml细胞悬液，每孔100ul(约40000个细胞)加入96孔U型板中,1500rpm×5min离心，去上清。然后用系列稀释的抗原结合蛋白重悬细胞，每孔加入100ul，混匀，37℃孵育20min。孵育结束后，1500rpm离心5min，去上清，200ul/well PBS洗涤1遍，然后用100μL/well 180μM AMP重悬，37℃孵育60min。孵育结束后，1500rpm×5min离心，取50μL/well上清，转移至96孔黑板中，再加入50ul/well配制好的ATP，混匀，37℃反应15min。最后将
Figure PCTCN2021136950-appb-000016
底物加入
Figure PCTCN2021136950-appb-000017
缓冲液，混匀并平衡至室温，然后加入待测孔，100μL/well，室温反应10min，全波长检测。酶活性的百分比按照如下所述进行评估：残余CD73的活性为：(CHOK1-huCD73-3F4细胞+Ab+ATP+AMP)-(ATP+AMP)/(CHOK1-huCD73-3F4细胞+ATP+AMP)-(ATP+AMP)*100。
抗CD73嵌合抗体900581，900565对CD73的细胞(CHOK1-huCD73-3F4，华博生物)酶活性抑制的效果见图4以及表6，900581，900697以及900698对CD73的细胞(CHOK1-huCD73-3F4，华博生物)酶活性抑制的效果见图5以及表7，结果表明900697，900698与900581对细胞膜上CD73酶活性抑制的效果基本一致。
表6.900581、900565对细胞膜上CD73酶活性抑制
抗原结合蛋白 900581 900565
EC50(μg/mL) 0.9437 1.625
表7.900581、900697、900698对细胞膜上CD73酶活性抑制
抗原结合蛋白 900581 900697 900698
EC50(μg/mL) 1.853 1.972 1.431
实施例6 抗CD73抗原结合蛋白与人CD73的结合亲和力检测
900581，900694，900695，900696，900697以及900698与人CD73蛋白(厂家：ACRO Biosystems，货号：CD3-H52H7)的结合动力学实验运用Biacore(GE，型号为T200)检测，实验缓冲液为HBS-EP+(pH7.4)，protein A芯片(厂家：GE,货号：29-1275-56)。
设置样品仓和流路温度为25℃，protein A芯片分别捕获抗原结合蛋白作为配体固定约100RU。人CD73蛋白用实验缓冲液稀释至50nM，25nM，12.5nM，6.25nM，3.12nM，1.57nM一系列浓度作为分析物，并以实验缓冲液作为0浓度对照，30μL/min流速下，结合120s，解 离600s。10mM Glycine pH 1.5在50μL/min流速下，再生60s多循环动力学检测。采用1:1结合模型，选用Fit local模式拟合出结合速率常数(ka)，解离速率常数(kd)和解离平衡速率常数(KD)，拟合结果见表8，显示所有抗原结合蛋白均与人CD73蛋白结合，且大部分抗原结合蛋白与人CD73蛋白的结合亲和力无显著差异。
表8：900581、900694-900698对人CD73蛋白的结合亲和力检测
Ligand Analyte ka(1/Ms) kd(1/s) KD(M)
900581 人CD73 2.67E+05 4.39E-04 1.65E-9
900694 人CD73 3.29E+05 4.93E-04 1.50E-9
900695 人CD73 2.95E+04 6.03E-05 2.04E-9
900696 人CD73 2.19E+05 9.28E-04 4.23E-9
900697 人CD73 3.80E+05 4.92E-04 1.29E-9
900698 人CD73 3.39E+05 1.06E-03 3.12E-9
实施例7 抗CD73抗原结合蛋白与不同种属CD73(人、小鼠、大鼠、猴)的结合亲和力检测
900581，900694，900695，900696，900697以及900698分别与人CD73蛋白(厂家：ACRO Biosystems，货号：CD3-H52H7)，小鼠CD73/NT5E(厂家：ACRO Biosystems，货号：CD3-M52H9)，大鼠CD73(厂家：Novoprotein，货号：CB16)和猴CD73(厂家：Novoprotein，货号：CI21)的结合动力学实验运用Biacore(GE，型号为T200)检测，使用protein A芯片(厂家：GE，货号：29-1275-56)，实验缓冲液为HBS-EP+(pH7.4)。
设置样品仓和流路温度为25℃，Protein A芯片分别捕获抗原结合蛋白作为配体固定为约100RU。人CD73蛋白/猴CD73/大鼠CD73/小鼠CD73分别用实验缓冲液稀释至50nM，25nM，12.5nM，6.25nM，3.12nM，1.57nM一系列浓度作为分析物，并以实验缓冲液作为0浓度对照，30μL/min流速下，结合120s，解离600s。10mM Glycine pH 1.5在50μL/min流速下，再生60s多循环动力学检测。采用1:1结合模型，选用Fit local模式拟合出结合速率常数(ka)， 解离速率常数(kd)和解离平衡速率常数(KD)，拟合结果见表9，显示上述抗原结合蛋白均与人CD73，猴CD73(厂家：Novoprotein，货号：CI21)都能结合，且大部分抗原结合蛋白与人、猴CD73蛋白的结合亲和力无显著差异，同时所有抗原结合蛋白均不与小鼠(厂家：ACRO Biosystems，货号：CD3-M52H9)和大鼠(厂家：Novoprotein，货号：CB16)的CD73蛋白结合。
表9：900581、900694-900698对猴CD73蛋白的结合亲和力检测
Ligand Analyte ka(1/Ms) kd(1/s) KD(M)
900581 猴CD73 3.49E+05 3.58E-04 1.05E-9
900694 猴CD73 5.78E+05 1.17E-04 2.03E-9
900695 猴CD73 1.80E+05 1.48E-04 8.22E-10
900696 猴CD73 3.54E+05 2.14E-04 6.04E-10
900697 猴CD73 6.41E+04 1.37E-04 2.13E-9
900698 猴CD73 1.08E+05 1.15E-04 1.41E-9
实施例8 抗CD73抗原结合蛋白诱导细胞表面CD73内吞能力的检测
将对照蛋白900222、嵌合蛋白900581以及人源化蛋白900697、900698蛋白用PBS溶液稀释至1μg/ml，每个浓度重复2孔。其中，对照蛋白900222序列来源于百时美施贵宝公司的CD73抗体专利US20190284293A1中的mAb-CD73.4-IgG2抗体序列。取对数生长期内的Calu6细胞，将细胞制备成2×10 5/ml细胞悬液，每孔100μL(约20000个细胞)加入96孔U型板中，300g×3min离心，去上清。再用稀释的抗原结合蛋白重悬细胞，100μL/well，混匀，4℃孵育30min。孵育结束后，300g×3min离心，去上清，1％BSA重悬，一半37℃孵育，另一半4℃孵育，孵育时间为5h。孵育结束后，取出对应时间的孔，300g×3min离心去上清，加入PE anti-huIgG Fc(1:200)，50μL/孔，混匀，4℃反应30min。孵育结束后，洗2遍，300g×3min，流式检测荧光强度。抗CD73抗原结合蛋白诱导的细胞表面CD73内吞检测结果如下表10以 及图6所示，结果表明嵌合蛋白900581以及人源化蛋白900697、900698蛋白诱导CD73内吞的效果显著优于对照蛋白900222，且人源化蛋白900697以及900698的诱导内吞效果又比嵌合体900581要好。
表10.抗CD73抗原结合蛋白诱导细胞表面CD73内吞效率
抗原结合蛋白 900222 900581 900697 900698
内吞效率(％) 44.18 78.75 98.17 96.35
实施例9 抗CD73抗原结合蛋白的结合表位鉴定
9.1氢氘交换质谱(HDX-MS)测定900581抗原结合蛋白的结合表位
将5～10μM抗原或抗原结合蛋白或抗原-结合蛋白复合物(1:1molar ratio)(50mM HEPES，pH 7.4,150mM NaCl，4mM TCEP)分别在4℃下放置1小时，使之形成复合物的稳定状态。在4℃，之后将5μL样品稀释到20μL D 2O(氘)中并放置到不同的HDX时间点进入质谱分析(例如，0,10,60,300,900秒)。氢氘交换一段时间后，通过与25μL冰冷的4M盐酸胍和1％三氟乙酸混合来终止反应。在停止反应后立即将样品管置于干冰上直至样品注入HDX LEAP PAL3.0平台。注射后到全自动氢氘交换平台后，样品以120μL/min流速通过固定的胃蛋白酶柱，并将酶解的肽段捕获在C18捕获柱上并脱盐。在8分钟内，用2.1mm×5cm C18柱(1.9μm Hypersil Gold，Thermo Fisher)以4-40％乙腈和0.3％甲酸的线性梯度分离脱盐的肽段。在样品处理过程中，蛋白质酶解和肽段分离均在4℃下进行。使用Orbitrap质谱仪(Orbitrap Fusion TM Tribrid TM Mass Spectrometer，Thermo Fisher)获得氢氘交换的质谱数据，测量的分辨率为65,000(m/z 400)。每一个样品在每一个时间点都有三次HDX测定(triplicates)。
氢氘交换质谱(HDX-MS)测定结果表明900581的结合表位主要包括CD73N端结构域的143-NIKAKGPLASQISGL-157区域(SEQ ID NO:37)，178-SKETPFLSNPGTNL-191区域(SEQ ID NO:38)，以及CD73C端结构域的381-WNHVSM-386的区域(SEQ ID NO:39)，将该三段非连续区域定位到CD73的三维结构模型上时，发现该表位处于CD73催化活性中心边缘，具体如图7所示。当900581结合在该表位时可以将CD73催化中心非常高效地封闭，从而最大效率地抑制CD73的酶活。查阅专利和文献表明目前本公司的900581是迄今为止首次报道的结合在CD73催化中心周围的并给出具体表位信息的治疗性抗原结合蛋白。
9.2非水解形式的AMP类似物APCP竞争结合实验验证900581抗原结合蛋白的结合表位
CD73结合底物AMP之后会发生从开放构象到封闭构象的构型改变，采用AMP非水解形式的类似物APCP替代AMP之后，由于APCP无法水解生成腺苷，从而可以将CD73锁定在封闭构象的催化中间状态。随着APCP浓度的增加，处于封闭构象的催化中间状态的CD73比例会相应增加，对于结合在催化区域的抗原结合蛋白，则结合上去的比例会相应减少，而结合在非催化区域的抗原结合蛋白结合上去的比例则不会变化。该方法具体操作如下：将细胞用1％BSA制备成1E6/ml细胞悬液，每孔20μL加入96孔U型板中。将APCP用1％BSA分别稀释成30nM/30μM，20μL/well加入上述96孔U型板中，混匀，室温放置30min。将待测抗原结合蛋白稀释至90μg/ml，再分别3倍稀释10个梯度。将系列稀释的抗原结合蛋白加入上述96孔U型板中，20μL/well，混匀，室温放置30min。孵育结束后，300g×3min离心，去上清，再20μL/well加入二抗PE羊抗人IgG Fc(1:200)，混匀，室温放置15min；孵育结束后，洗涤2遍，300g×3min，流式检测PE-MFI。
检测结果如图8A-8C所示，其中900609是Corvus公司的被证明结合在催化中心周围的CPI-006抗体，TNP是阴性对照抗体。结果表明900581抗原结合蛋白的表现和CPI-006抗体类似，随着APCP浓度的增加结合到CD73上的抗原结合蛋白比例会逐渐降低，从而进一步证明900581结合在CD73催化活性中心周围。
实施例10 抗CD73抗原结合蛋白的体内治疗肿瘤的效果检测
10.1
本实验利用CD73人源化小鼠建立MDA-MB-231三阴性乳腺癌动物模型并测试受试抗体的药效。首先，将MDA-MB-231(ATCC库)细胞(2E6个)100μl接种于CD73人源化小鼠中建立CD73人源化小鼠MDA-MB-231三阴性乳腺癌动物模型，成瘤小鼠根据小鼠肿瘤体积和体重平均分配到4个实验组中，每组6只，腹腔注射给药，每周给药2次共给药6次。具体给药方案见下表。其中，900201为阴性对照抗体，不与CD73抗原结合。900201的重链(HC)氨基酸序列如SEQ ID NO:43所示，900201的轻链(LC)氨基酸序列如SEQ ID NO:44所示。
结果显示900581、900697以及900698能够显著抑制MDA-MB-231三阴性乳腺癌的生长，同时对小鼠的体重生长指标没有影响。而阴性对照抗体900201则不能抑制肿瘤的生长。
表11给药方案
Figure PCTCN2021136950-appb-000018
Figure PCTCN2021136950-appb-000019
10.2
利用NPG小鼠建立MDA-MB-231三阴性乳腺癌动物模型并测试受试抗体的药效。用于本实验的人乳腺癌细胞MDA-MB-231以L-15培养基添加10％FBS培养于含无CO 2的37℃培养箱。细胞连续培养十代之前，将约含5.0×10 6MDA-MB-231细胞混悬于100μL PBS中，和等体积Matrigel混匀后，通过皮下注射接种于NPG人源化小鼠背部右边靠近腋下，接种体积为200μL左右。接种前用2-5％异氟烷将小鼠麻醉。在接种当天，经尾静脉注射1.0×10 7PBMC(100μL)，当肿瘤生长到平均约50-80mm 3左右时，18只荷瘤小鼠将根据肿瘤体积和体重被随机分成3组，每组6只。分组当天进行给药,具体给药方案见表12。其中，900201为阴性对照抗体，不与CD73抗原结合。900201的重链(HC)氨基酸序列如SEQ ID NO:43所示，900201的轻链(LC)氨基酸序列如SEQ ID NO:44所示。Oleclumab为阿斯利康开发的抗CD73单克隆抗体。
结果如下表12和图9所示，oleclumab以及900698能够显著抑制MDA-MB-231三阴性乳腺癌的生长，而阴性对照抗体900201则不能抑制肿瘤的生长。
表12三阴性乳腺癌模型给药方案
Figure PCTCN2021136950-appb-000020
实施例11 抗CD73抗原结合蛋白的体内治疗胰腺癌的效果检测
本实验利用NPG小鼠建立BxPC-3胰腺癌动物模型并测试受试抗体的药效。用于本实验的人胰腺癌细胞BxPC-3，以RPMI-1640培养基添加10％FBS培养于含5％CO 2的37℃培养箱。细胞连续培养十代之前，将约含1×10 7BxPC-3细胞的PBS 100μL和等体积Matrigel混 匀后，通过皮下注射接种于18只NPG小鼠背部右边靠近腋下，接种体积为200μL左右。接种前用2-5％异氟烷将小鼠麻醉。在接种当天，经尾静脉注射1.0×10 7PBMC(100μL)，当肿瘤生长到平均约50-80mm 3左右时，18只荷瘤小鼠将根据肿瘤体积和体重被随机分成3组，每组6只。分组当天进行给药,具体给药方案见表13。其中，900543为阴性对照抗体(900543是900201的ADCC敲除型)，不与CD73抗原结合。900543的重链(HC)氨基酸序列如SEQ ID NO:47所示，900543的轻链(LC)氨基酸序列如SEQ ID NO:44所示。Oleclumab为阿斯利康开发的抗CD73单克隆抗体。
结果如下表13和图10所示，oleclumab以及900698能够显著抑制BxPC-3胰腺癌的生长，而阴性对照抗体900543则不能抑制肿瘤的生长。
表13胰腺癌模型给药方案
Figure PCTCN2021136950-appb-000021
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (50)

  1. 分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含LCDR3，所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含LCDR2，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其包含LCDR1，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。
  7. 根据权利要求3-6中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:7、SEQ ID NO:15或SEQ ID NO:19所示的氨基酸序列。
  8. 根据权利要求2-7中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:8、SEQ ID NO:16或SEQ ID NO:21所示的氨基酸序列。
  9. 根据权利要求2-8中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
  11. 根据权利要求6-10中任一项所述的分离的抗原结合蛋白，其包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:11或SEQ ID NO:23所示的氨基酸序列。
  12. 根据权利要求6-11中任一项所述的分离的抗原结合蛋白，其包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:12或SEQ ID NO:24所示的氨基酸序列。
  13. 根据权利要求5-12中任一项所述的分离的抗原结合蛋白，其包含L-FR3，所述L-FR3位 于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:13、SEQ ID NO:25、SEQ ID NO:27或SEQ ID NO:28所示的氨基酸序列。
  14. 根据权利要求4-13中任一项所述的分离的抗原结合蛋白，其包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:14或SEQ ID NO:26所示的氨基酸序列。
  15. 根据权利要求6-14中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含VH，所述VH包含SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33或SEQ ID NO:35所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含VL，所述VL包含SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34或SEQ ID NO:36所示的氨基酸序列。
  18. 根据权利要求17所述的分离的抗原结合蛋白，其包含选自下述的任一组VH和VL：
    1)所述VH包含SEQ ID NO:29所示的氨基酸序列，且所述VL包含SEQ ID NO:30所示的氨基酸序列；
    2)所述VH包含SEQ ID NO:31所示的氨基酸序列，且所述VL包含SEQ ID NO:32所示的氨基酸序列；
    3)所述VH包含SEQ ID NO:33所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列；
    4)所述VH包含SEQ ID NO:35所示的氨基酸序列，且所述VL包含SEQ ID NO:36所示的氨基酸序列；
    5)所述VH包含SEQ ID NO:33所示的氨基酸序列，且所述VL包含SEQ ID NO:36所示的氨基酸序列；以及
    6)所述VH包含SEQ ID NO:35所示的氨基酸序列，且所述VL包含SEQ ID NO:34所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含抗体重链恒定区。
  20. 根据权利要求19所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人抗体重链恒定区。
  21. 根据权利要求19-20中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自 人IgG重链恒定区。
  22. 根据权利要求19-21中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG1重链恒定区。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区。
  24. 根据权利要求23所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区源自人Igκ恒定区。
  25. 根据权利要求1-24中任一项所述的分离的抗原结合蛋白，其包含抗体或其抗原结合片段。
  26. 根据权利要求25所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
  27. 根据权利要求25-26中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其能够特异性结合CD73或其功能活性片段。
  29. 根据权利要求28所述的分离的抗原结合蛋白，所述CD73包括人CD73和/或猴CD73。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其能够阻止或减少CD73的活化。
  31. 根据权利要求28-30中任一项所述的分离的抗原结合蛋白，当与所述CD73结合时，所述分离的抗原结合蛋白与SEQ ID NO:40所示的氨基酸序列的第143-157位氨基酸或与其对应的CD73序列、SEQ ID NO:40所示的氨基酸序列的第178-191位氨基酸或与其对应的CD73序列和SEQ ID NO:40所示的氨基酸序列的第381-386位氨基酸或与其对应的CD73序列中的至少一个氨基酸残基相结合。
  32. 根据权利要求31所述的分离的抗原结合蛋白，其中所述第143-157位氨基酸或与其对应的CD73序列位于所述CD73的N端结构域，所述第178-191位氨基酸或与其对应的CD73序列位于所述CD73的N端结构域，且其中所述第381-386位氨基酸或与其对应的CD73序列位于所述CD73的C端结构域。
  33. 根据权利要求1-32中任一项所述的分离的抗原结合蛋白，其能够抑制CD73的酶活性。
  34. 根据权利要求1-33中任一项所述的分离的抗原结合蛋白，其能够诱导细胞表面CD73的内吞。
  35. 多肽分子，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白。
  36. 根据权利要求35所述的多肽分子，其包含融合蛋白。
  37. 分离的核酸分子，其编码权利要求1-34中任一项所述的分离的抗原结合蛋白或权利要求 35-36中任一项所述的多肽分子。
  38. 载体，其包含权利要求37中所述的核酸分子。
  39. 细胞，其包含权利要求37中所述的核酸分子和/或权利要求38所述的载体。
  40. 免疫缀合物，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白。
  41. 药物组合物，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35-36中任一项所述的多肽分子、权利要求37所述的核酸分子、权利要求38所述的载体、权利要求39所述的细胞和/或权利要求40所述的免疫缀合物，以及任选地药学上可接受的载剂。
  42. 制备权利要求1-34中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述分离的抗原结合蛋白表达的条件下，培养权利要求39所述的细胞。
  43. 权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35-36中任一项所述的多肽分子、权利要求37所述的核酸分子、权利要求38所述的载体、权利要求39所述的细胞、权利要求40所述的免疫缀合物和/或权利要求41所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  44. 根据权利要求43所述的用途，其中所述疾病和/或病症是CD73介导的疾病和/或病症。
  45. 根据权利要求43-44中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  46. 根据权利要求45所述的用途，其中所述肿瘤包括实体瘤和/或血液瘤。
  47. 根据权利要求43-46中任一项所述的用途，其中所述疾病和/或病症包括乳腺癌。
  48. 检测样品中CD73的方法，所述方法包括施用权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35-36中任一项所述的多肽分子、权利要求37所述的核酸分子、权利要求38所述的载体、权利要求39所述的细胞、权利要求40所述的免疫缀合物和/或权利要求41所述的药物组合物。
  49. 检测样品中CD73的试剂或试剂盒，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35-36中任一项所述的多肽分子、权利要求37所述的核酸分子、权利要求38所述的载体、权利要求39所述的细胞、权利要求40所述的免疫缀合物和/或权利要求41所述的药物组合物。
  50. 权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35-36中任一项所述的多肽分子、权利要求37所述的核酸分子、权利要求38所述的载体、权利要求39所述的细胞、权利要求40所述的免疫缀合物和/或权利要求41所述的药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中CD73的存在和/或含量。
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