WO2023138643A1 - Cd39抗原结合蛋白及其应用 - Google Patents

Cd39抗原结合蛋白及其应用 Download PDF

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WO2023138643A1
WO2023138643A1 PCT/CN2023/073060 CN2023073060W WO2023138643A1 WO 2023138643 A1 WO2023138643 A1 WO 2023138643A1 CN 2023073060 W CN2023073060 W CN 2023073060W WO 2023138643 A1 WO2023138643 A1 WO 2023138643A1
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amino acid
seq
acid sequence
sequence shown
binding protein
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PCT/CN2023/073060
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English (en)
French (fr)
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徐锦根
于海佳
任晓琛
李雪
占一帆
朱向阳
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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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Publication of WO2023138643A1 publication Critical patent/WO2023138643A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • This application relates to the field of biomedicine, in particular to a CD39 antigen-binding protein and its application.
  • CD39 also known as external nucleoside triphosphate diphosphate hydrolase 1 (ENTPDase1), is an integral membrane protein that phosphorylates ATP to generate ADP and AMP.
  • CD39 is expressed in many solid tumor cells. CD39 can participate in tumor immune escape by inhibiting the activation, clonal expansion and homing of tumor-specific T cells, impairing the killing of tumor cells by effector T lymphocytes. CD39 can directly cause the regulation of cancer cell growth, differentiation, invasion, migration, metastasis and angiogenesis. CD39 is important for both the initiation of angiogenesis and the progression of angiogenesis.
  • the application provides an isolated antigen-binding protein, wherein the antigen-binding protein has one or more of the following characteristics: a) as determined by FACS detection, with an EC of less than about 0.5 ⁇ g/mL 50 Value combined with human CD39; b) determined by FACS detection, with an EC of less than about 0.7 ⁇ g/mL 50 Values combined with cynomolgus monkey (cyno) CD39; c) measured by Biacore assay to less than about 1.3 ⁇ 10 -8 M of K D. Value combined with human CD39; d) determined by Biacore detection method to be less than about 1.1 ⁇ 10 -7 M of K D.
  • cynomolgus monkey (cyno) CD39 binds to cynomolgus monkey (cyno) CD39; e) determined by ATPase activity assay with an EC of less than about 0.8 ⁇ g/mL 50 value inhibits ATPase activity in cells expressing CD39; and/or, f) inhibits CD39 function in tumor cells.
  • the isolated antigen binding protein contains at least one CDR in the VH of the heavy chain variable region, and the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-8, 11-12.
  • said isolated antigen binding protein comprises HCDR3, and said HCDR3 comprises SEQ ID NO: 29 (AX 1 x 2 x 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 x 12 x 13 x 14 DX 15 x 16 x 17 , where X 1 for A or W, X 2 for A or G, X 3 for E or G, X 4 for A or D, X 5 for D or G, X 6 for S or V, X 7 for S or V, X 8 for G or W, X 9 for F or G, X 10 for G or T, X 11 for P or T, X 12 is D, P or T, X 13 for L or W, X 14 is A, C, G or M, X 15 for W or Y, X 16 for G or W, X 17 is the amino acid sequence shown in G or S).
  • the isolated antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 or 17.
  • the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 6, 9 and 10.
  • the isolated antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • the isolated antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or 4.
  • the isolated antigen-binding protein comprises HCDR1-HCDR3, wherein, a) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2; and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; b) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5; DR3 comprises the amino acid sequence shown in SEQ ID NO:6; c) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2; and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9; or, d) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1; The amino acid sequence shown in NO:10.
  • the isolated antigen-binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18 or 22.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:19, 23 and 27.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:20, 24, 26 and 28.
  • the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 21 or 25.
  • the antigen binding protein of described separation comprises H-FR1 ⁇ H-FR4, wherein: a) described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:18; Described H-FR2 comprises the aminoacid sequence shown in SEQ ID NO:19; Described H-FR3 comprises the aminoacid sequence shown in SEQ ID NO:20; Described H-FR4 comprises the aminoacid sequence shown in SEQ ID NO:21; Amino acid sequence; the H-FR2 The H-FR3 comprises the amino acid sequence shown in SEQ ID NO:24; the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:25; c) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:18; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:19; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:26; The amino acid sequence shown in NO:21; or, d) the H-FR1 includes the amino acid sequence shown in SEQ ID NO:
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence shown in any one of SEQ ID NO:7, 8, 11 and 12.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises a dAb.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies and affinity matured antibodies.
  • the present application provides a polypeptide molecule comprising the isolated antigen-binding protein described herein.
  • the polypeptide molecule comprises a fusion protein.
  • the present application provides a nucleic acid molecule encoding the isolated antigen-binding protein described herein or the polypeptide molecule described herein.
  • the present application provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein.
  • the present application provides a cell comprising the nucleic acid molecule described in the present application, the vector described in the present application and/or the immunoconjugate described in the present application.
  • the present application provides a pharmaceutical composition, which comprises the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the nucleic acid molecule described herein, the carrier described herein, the immunoconjugate described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a method for preparing the isolated antigen-binding protein described herein, the method comprising culturing the cell described herein under conditions such that the isolated antigen-binding protein is expressed.
  • the present application provides an isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the immunoconjugate described in the present application, the cell described in the present application and/or the use of the pharmaceutical composition described in the present application in the preparation of a medicament, and the medicament is used to prevent and/or treat diseases and/or conditions.
  • the disease and/or condition is mediated by CD39.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the disease and/or condition comprises pancreatic cancer and/or colorectal cancer.
  • the present application provides a method for detecting CD39 in a sample, the method comprising administering the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the nucleic acid molecule described herein, the carrier described herein, the immunoconjugate described herein, the cell described herein and/or the pharmaceutical composition described herein.
  • the present application provides a reagent or kit for detecting CD39 in a sample, which comprises the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the immunoconjugate described in the present application, the cell described in the present application and/or the pharmaceutical composition described in the present application.
  • the present application provides the use of the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the immunoconjugate described in the present application, the cell described in the present application and/or the pharmaceutical composition described in the present application in the preparation of a kit for detecting the presence and/or content of CD39 in a sample.
  • Figure 1 shows the results of nested PCR amplification of candidate VHH antibodies of the present application.
  • Figure 2 shows the observation results of the screening and enrichment of the immune library by the Drop method plated plate versus the Drop method plated plate.
  • Figure 3 shows the alignment results of the Unique sequences of the candidate VHH antibodies of the present application.
  • FIG. 4 shows the SDS-PAGE results of the antigen-binding proteins described in this application.
  • Figure 5 shows the FACS detection results of the affinity of the antigen-binding protein described in the present application to the human CD39 antigen on the cell surface.
  • Figure 6 shows the FACS detection results of the affinity of the antigen-binding protein described in this application to the monkey CD39 antigen on the cell surface.
  • Figure 7 shows the detection results of the antigen binding protein described in the present application for its ability to inhibit the activity of CD39 antigenase on the cell surface.
  • Figure 8 shows the results of monoclonal sequencing analysis of the candidate affinity-matured antigen-binding proteins of the present application.
  • 9A-9B show the results of FACS detection of the affinity of the affinity-matured antigen-binding protein of the present application to the CD39 antigen on the cell surface.
  • 10A-10B show the detection results of the ability of the affinity-matured antigen-binding protein of the present application to inhibit the activity of CD39 antigenase on the cell surface.
  • Figure 11 shows the detection results of the antigen-binding protein described in this application in treating pancreatic cancer in vivo.
  • Figure 12 shows the detection results of the antigen-binding protein described in this application in treating colorectal cancer in vivo.
  • CD39 generally refers to the extracellular nucleoside triphosphate diphosphate hydrolase 1 polypeptide encoded by the ENTPD1 gene in humans.
  • Other names for CD39 include ENTPD1, ATPDase, NTPDase-1 and SPG64.
  • CD39 catalyzes the hydrolysis of ⁇ - and ⁇ -phosphate residues of extracellular nucleoside triphosphates (NTP; eg, adenosine triphosphate or ATP) and nucleoside diphosphates (NDP; eg, adenosine diphosphate or ADP), converting these molecules to nucleoside monophosphate (NMP; eg, adenosine monophosphate or AMP) derivatives.
  • NMP nucleoside monophosphate
  • AMP nucleoside monophosphate
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • the "isolated antigen binding protein” may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to 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.
  • FR antibody-derived protein framework regions
  • Such frameworks include, but are not limited to, those containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein.
  • Antibody-derived framework regions as well as fully synthetic framework regions comprising, for example, 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; and its fragments.
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop.
  • antibodies comprise 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 capable of functioning and stabilizing 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 various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum.
  • the amino acid sequence numbering of the antigen-binding protein can be numbered 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.28:2 19-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203).
  • IMGT the international ImMunoGeneTics information system@imgt.cines.fr
  • http://imgt.cines.fr http://imgt.cines.fr
  • Lefranc et al. 1999, Nucle
  • the CDRs of the antigen binding protein 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, 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 can be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class), and comprise the antigen-binding site.
  • variable generally refers to certain segments of the variable domain between antibodies that may differ in sequence. big difference.
  • 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 called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs, which form a circular link and, in some cases, form part of the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (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, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated and grafted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically bind human CD39). Typically, such fragments will include the 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 antibodies are composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 basic 4-chain units that can be combined with J chains to form multivalent combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • 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 the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to 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, CD39).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, see for example Ward et al. WO 06/003388 and other published patent applications of Domantis Ltd.
  • monoclonal antibody generally refers to a preparation of antibody molecules of single molecular composition.
  • Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants.
  • monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • the modifier "monoclonal” denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • an affinity matured antibody generally refers to a binding molecule (such as an antigen-binding protein) that has undergone affinity maturation, which is a process of increasing the affinity of a binding molecule (such as an antigen-binding protein) to a target antigen.
  • an affinity matured antibody can include an antibody having one or more changes in one or more CDRs (e.g., a change in one or more amino acid residues in one or more CDRs) that results in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have those changes.
  • the affinity matured antibody may retain the same epitope as the parental antibody.
  • polypeptide molecule and “polypeptide” and “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 a polypeptide with different properties.
  • the property may be a biological property, such as in vitro or in vivo activity.
  • the property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. These two moieties can be linked directly by a single peptide bond or by a peptide linker.
  • nucleic acid molecule generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially 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 can be expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; Chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but not
  • 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's plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • Cells may include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (e.g., E.
  • the cells are mammalian cells. In certain embodiments, the mammalian cells are HEK293 cells.
  • the term "immunoconjugate” generally refers to a conjugate formed by conjugating the other agent (e.g., chemotherapeutic agent, radioactive element, cytostatic agent, and cytotoxic agent) to the antibody or its antigen-binding fragment (e.g., covalently linked through a linker molecule), and the conjugate can deliver the other agent to the target cell (e.g., tumor cell) through the specific binding of the antibody or its antigen-binding fragment to the antigen on the target cell.
  • the other agent e.g., chemotherapeutic agent, radioactive element, cytostatic agent, and cytotoxic agent
  • the conjugate can deliver the other agent to the target cell (e.g., tumor cell) through the specific binding of the antibody or its antigen-binding fragment to the antigen on the target cell.
  • the term "pharmaceutical composition” generally refers to a composition for preventing/treating a disease or condition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or 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.
  • pharmaceutically acceptable carrier generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dosages and concentrations employed.
  • 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 the binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets.
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding includes, but does not require exclusive binding.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • tumor generally refers to any new pathological growth of tissue. Tumor cells can spread to other parts of the body locally or through the bloodstream and lymphatic system.
  • the tumor may include benign tumors and malignant tumors.
  • the tumor may include solid tumor and/or hematological tumor.
  • the tumor may include cancer.
  • examples of the tumor may include, but not limited to: breast cancer.
  • the application provides an isolated antigen-binding protein, wherein the antigen-binding protein has one or more of the following characteristics:
  • a) binds to human CD39 with an EC50 value of less than about 0.5 ⁇ g/mL as determined by FACS detection;
  • the EC50 value can be less than about 0.4 ⁇ g/mL, less than about 0.3 ⁇ g/mL, less than about 0.2 ⁇ g/mL, less than about 0.1 ⁇ g/mL, less than about 0.09 ⁇ g/mL, less than about 0.08 ⁇ g/mL, less than about 0.07 ⁇ g/mL, less than about 0.06 ⁇ g/mL, less than about 0.05 ⁇ g/mL, less than about 0.04 ⁇ g/mL, less than about 0.03 ⁇ g/mL g/mL, less than about 0.02 ⁇ g/mL, less than about 0.01 ⁇ g/mL or lower.
  • b) binds to cynomolgus monkey (cyno) CD39 with an EC50 value of less than about 0.7 ⁇ g/mL as determined by FACS assay;
  • the EC50 value can be less than about 0.6 ⁇ g/mL, less than about 0.5 ⁇ g/mL, less than about 0.4 ⁇ g/mL, less than about 0.3 ⁇ g/mL, less than about 0.2 ⁇ g/mL, less than about 0.1 ⁇ g/mL, less than about 0.09 ⁇ g/mL, less than about 0.08 ⁇ g/mL, less than about 0.07 ⁇ g/mL, less than about 0.06 ⁇ g/mL, less than about 0.05 ⁇ g/mL mL, less than about 0.04 ⁇ g/mL, less than about 0.03 ⁇ g/mL, less than about 0.02 ⁇ g/mL, less than about 0.01 ⁇ g/mL or lower.
  • c) binds to human CD39 with a KD value of less than about 1.3 ⁇ 10 ⁇ 8 M as determined by a Biacore assay
  • the KD value may be less than about 1 ⁇ 10 -8 M, less than about 9 ⁇ 10 -9 M, less than about 8 ⁇ 10 -9 M, less than about 7 ⁇ 10 -9 M, less than about 6 ⁇ 10 -9 M, less than about 5 ⁇ 10 -9 M, less than about 4 ⁇ 10 -9 M, less than about 3 ⁇ 10 -9 M , less than about 2 ⁇ 10 -9 M 9 M, less than about 1 ⁇ 10-9 M, less than about 8 ⁇ 10-10 M, less than about 6 ⁇ 10-10 M, less than about 4 ⁇ 10-10 M, less than about 2 ⁇ 10-10 M , less than about 1 ⁇ 10-10 M or lower.
  • d) binds to cynomolgus monkey (cyno) CD39 with a KD value of less than about 1.1 ⁇ 10 ⁇ 7 M as determined by a Biacore assay;
  • the KD value may be less than about 1 ⁇ 10 -7 M, less than about 9 ⁇ 10 -8 M, less than about 8 ⁇ 10 -8 M, less than about 7 ⁇ 10 -8 M , less than about 6 ⁇ 10 -8 M, less than about 5 ⁇ 10 -8 M, less than about 4 ⁇ 10 -8 M, less than about 3 ⁇ 10 -8 M, less than about 2 ⁇ 10 -8 M, less than about 1 ⁇ 10 -8 M, less than about 8 ⁇ 10 -9 M, less than about 6 ⁇ 10 -9 M, less than about 4 x 10 -9 M , less than about 2 x 10 -9 M, less than about 1 x 10 -9 M or less.
  • e inhibits ATPase activity in cells expressing CD39 with an EC50 value of less than about 0.8 ⁇ g/mL as determined by an ATPase activity assay; and/or,
  • the EC 50 The value can be less than about 0.7 ⁇ g/mL, less than about 0.6 ⁇ g/mL, less than about 0.5 ⁇ g/mL, less than about 0.4 ⁇ g/mL, less than about 0.3 ⁇ g/mL, less than about 0.2 ⁇ g/mL, less than about 0.1 ⁇ g/mL, less than about 0.09 ⁇ g/mL, less than about 0.08 ⁇ g/mL, less than about 0.07 ⁇ g/mL, less than about 0.06 ⁇ g/mL, less than about 0.0 5 ⁇ g/mL, less than about 0.04 ⁇ g/mL, less than about 0.03 ⁇ g/mL, less than about 0.02 ⁇ g/mL, less than about 0.01 ⁇ g/mL or lower.
  • the isolated antigen-binding protein contains at least one CDR in the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 7-8, 11-12.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.
  • SEQ ID NO: 29 AX 1 x 2 x 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 x 12 x 13 x 14 DX 15 x 16 x 17 , where X 1 for A or W, X 2 for A or G, X 3 for E or G, X 4 for A or D, X 5 for D or G, X 6 for S or V, X 7 for S or V, X 8 for G or W, X 9 for F or G, X 10 for G or T, X 11 for P or T, X 12 is D, P or T, X 13 for L or W, X 14 is A, C, G or M, X 15 for W or Y, X 16 for G or W, X 17 for G or S.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6 or 17.
  • SEQ ID NO: 17 AWGEDDSSWFGTX 1 LX 2 DYWG, wherein X 1 is D or T, and X 2 is C, G or M.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, 6, 9 and 10.
  • said isolated antigen binding protein may comprise HCDR2, and said HCDR2 may comprise The amino acid sequence shown in SEQ ID NO:2 or 5.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or 4.
  • the isolated antigen-binding protein may comprise HCDR1-HCDR3, wherein,
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR may cover the CDR sequence divided according to any CDR division method; it may also cover its variants, the variants include substitution, deletion and/or addition of one or more amino acids in the amino acid sequence of the CDR.
  • the variants include substitution, deletion and/or addition of one or more amino acids in the amino acid sequence of the CDR.
  • the CDRs are determined by the IMGT numbering scheme.
  • the isolated antigen-binding protein described in the present application may comprise heavy chain and/or light chain sequences with one or more conservative sequence modifications.
  • conservative sequence modification refers to the amino acid modification that will not significantly affect or change 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 mutations and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues with similar side chains. amino acids with similar side chains Residue sets are known in the art.
  • amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in the present application may 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 cause antigen binding to disappear, for example, 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.
  • CD39 antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.
  • the antigen-binding activity of the antigen-binding protein or fusion protein of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting e.g., Western blot
  • flow cytometry e.g., FACS
  • immunohistochemistry e.g., immunofluorescence, etc.
  • the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 18 or 22.
  • the isolated antigen-binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:19, 23 and 27.
  • the isolated antigen-binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:20, 24, 26 and 28.
  • the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 21 or 25.
  • the isolated antigen-binding protein may comprise H-FR1 ⁇ H-FR4, wherein:
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:24;
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:25;
  • the isolated antigen binding protein may comprise HCDR1-3 and H-FR1 ⁇ H-FR4, wherein:
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the isolated antigen-binding protein may be referred to as 17-13;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:18;
  • H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:19;
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:26;
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:21;
  • the isolated antigen-binding protein can be referred to as 17-22;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10;
  • H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:18;
  • H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:27;
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:28;
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:21;
  • the isolated antigen-binding protein can be referred to as 17-35; or,
  • H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:22;
  • H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:24;
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:25;
  • the isolated antigen binding protein may be referred to as 17-17.
  • the isolated antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:7,8,11,12.
  • the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.
  • said antigen-binding fragments include dAbs.
  • the antigen-binding fragment may be VHH.
  • the antibody is selected from one or more of the following groups: monoclonal antibody and affinity matured antibody.
  • the isolated antigen binding protein may further comprise a heavy chain constant region.
  • the isolated antigen binding protein may comprise an Fc region.
  • the Fc region can be derived from the Fc region of human IgG.
  • Affinity maturation usually means that in humoral immunity, the average affinity of antibodies produced in second response is higher than that in primary immune response.
  • those skilled in the art can use well-known techniques to modify the antibody to obtain the affinity matured antibody. For example, engineering can be performed by means of error-prone PCR, strand displacement, site-directed mutagenesis, and/or DNA shuffling.
  • the isolated antigen-binding protein may also have one or more of the following characteristics:
  • eATP extracellular adenosine triphosphate
  • eADP extracellular adenosine diphosphate
  • eAMP extracellular adenosine monophosphate
  • Polypeptide molecules Polypeptide molecules, nucleic acid molecules, vectors, immunoconjugates, cells and pharmaceutical compositions
  • polypeptide molecules which may comprise an isolated antigen binding protein described herein.
  • the polypeptide molecule comprises a fusion protein.
  • the application provides an isolated nucleic acid molecule that may encode an isolated antigen binding protein described herein or a polypeptide molecule described herein.
  • an isolated nucleic acid molecule may encode an isolated antigen binding protein described herein or a polypeptide molecule described herein.
  • it may be produced or synthesized by (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification; (ii) recombinant production by cloning; (iii) purified, such as by enzymatic cleavage and fractionation by gel electrophoresis; or (iv) synthetic, such as by chemical synthesis.
  • in vitro amplification such as by polymerase chain reaction (PCR) amplification
  • recombinant production by cloning recombinant production by cloning
  • purified such as by enzymatic cleavage and fractionation by gel electrophoresis
  • synthetic such as by chemical synthesis.
  • the present application provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • 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 permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled 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 the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.
  • 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 linkage 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.
  • the immunoconjugates can be provided in the form of fusion proteins, which can be expressed from a polynucleotide encoding the immunoconjugates.
  • the immunoconjugates may also comprise, for example, antibody-drug conjugates (ADCs).
  • Suitable drugs may include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.
  • ADCs antibodies and therapeutic agents can be cross-linked by a linker that is cleavable, such as a peptide, disulfide, or hydrazone linker.
  • the present application provides a cell, which may comprise the nucleic acid molecule described in the present application, the vector described in the present application and/or the immunoconjugate described in the present application.
  • each or each host cell comprises one or more nucleic acid molecules or vectors described herein.
  • each or each host cell comprises a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like.
  • the cells are bacterial cells (e.g., 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 SP2/0 cells
  • NSO cells myeloma cells.
  • myeloma cells myeloma cells.
  • the vectors described in this application 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 a pharmaceutical composition, which may 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 nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application A carrier and/or a cell as described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also contain suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives.
  • suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives 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 composition may also contain more than one active compound, generally those active compounds with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with drug administration, and is generally safe and non-toxic.
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, 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 different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. Such 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 a method for preparing the antigen-binding protein.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.
  • 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 CD39 or fragments thereof.
  • Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, one or more routes may be used.
  • hybridoma preparation methods can be used.
  • CD39 can be used as an immunogen (antigen) for producing non-human antibodies specific to CD39 and screening the biological activity of the antibodies.
  • the priming immunogen can be full-length mature human CD39, including native homodimers, or single/multiple epitope-containing peptides.
  • Immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • the application provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the immunoconjugate, the cell and/or the pharmaceutical composition in the preparation of a medicament. for the prevention and/or treatment of diseases and/or conditions.
  • the present application also provides a method for preventing and/or treating a disease and/or disorder, the method may comprise administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the immunoconjugate, the cell and/or the pharmaceutical composition described in the present application to a subject in need.
  • 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 immunoconjugate, the cell and/or the pharmaceutical composition described herein can be used to prevent and/or treat diseases and/or conditions.
  • the disease and/or disorder may be a CD39-mediated disease and/or disorder.
  • the diseases and/or conditions may include tumors.
  • the tumor may include solid tumor and/or hematological tumor.
  • the diseases and/or conditions may include pancreatic cancer and/or colorectal cancer.
  • the present application also provides a method for detecting CD39 in a sample, the method 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 method for detecting CD39 in a sample may be an in vitro method.
  • the method for detecting CD39 in a sample may be for non-therapeutic purposes.
  • the method for detecting CD39 in a sample is not a diagnostic method.
  • the present application also provides a reagent or kit for detecting CD39 in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the immunoconjugate, the cell and/or the pharmaceutical composition.
  • the present application also provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the immunoconjugate, the cell and/or the pharmaceutical composition in the preparation of a kit for detecting the presence and/or content of CD39 in a sample.
  • the CD39 antigen was mixed with Freund's adjuvant in equal volumes, emulsified and injected subcutaneously at multiple points to immunize an alpaca alpaca, Once every 3 weeks or so, a total of 3 times of immunization to stimulate B cells to express antigen-specific nanobodies.
  • 50ml of peripheral blood was collected, and plasma and lymphocytes were collected for antibody titer titration and nanobody library construction.
  • the immune effect is excellent, the plasma antibody titer reaches 1:128000, and the OD450 value of the plasma antibody diluted 32000 times is ⁇ 0.5 (1.37), which fully meets the requirements for library construction.
  • the construction method of the alpaca immune library is as follows: use the PBMC obtained from alpaca blood collection after three immunizations, use a special kit to extract total RNA, reverse transcribe it into cDNA, and use nested PCR to amplify VHH.
  • the PCR results are shown in Figure 1.
  • the VHH fragment was inserted into the pShort phagemid and electrotransferred into the supercompetent cell SU320+H ⁇ , and the storage capacity and GA recombination efficiency were measured.
  • the storage capacity was 7.8E9CFM, and the GA recombination efficiency reached more than 80% (the results are shown in Table 1), which fully met the relevant indicators of high-quality immune library.
  • the PEG8000/NaCl precipitation method was used to separate and purify the phage to obtain the antibody library. Adjust the phage concentration, aliquot and freeze at -80°C for later use.
  • CD39 antigen at 5 ⁇ g/ml, 100 ⁇ l/well into a 96-well plate for coating, and overnight at 4°C. The next day, 1% PVA solution was added and incubated at room temperature for 1 h to block. After blocking, the antibody library (1E13pfu/ml, 100 ⁇ l/well) was added and incubated at room temperature for 1 h. Then add 200ul of PT buffer to each well to wash 8-10 times. After washing, add 100mM HCl to each well, incubate at room temperature for 5min, then add 1M Tris-HCl to neutralize and elute. Infect NEB alpha 5F' cells with the phage-containing eluate, add helper phage, and culture overnight.
  • the Drop method was used to coat the plate, and the enrichment degree was counted the next day.
  • the overnight culture product was added to PEG8000/NaCl for precipitation, the phages were isolated and purified, and entered into the next round of elution and the enrichment and purity were counted.
  • the results of elution by the Drop method are shown in Figure 2.
  • After enrichment, single clones were picked randomly, and positive clones were identified by Phage-ELISA. Finally, multiple single clones were selected for sequencing according to the OD value of the ELISA positive wells and the difference with the control wells. Further, sequences with abnormal sequencing were eliminated, and clones with identical sequences were merged.
  • 900690 positive control antibody, derived from Surface Oncology’s anti-CD39 monoclonal antibody
  • the heavy chain (HC) amino acid sequence of 900690 is shown in SEQ ID NO:30
  • the light chain (LC) amino acid sequence of 900690 is shown in SEQ ID NO:31
  • the results of the binding activity of 17-13,17-17 to human CD39 on the cell surface are shown in Figure 5 and Table 3
  • the results of binding activity to monkey CD39 on the cell surface As shown in Figure 6 and Table 4, the results show that the 17-13 and 17-17 antigen binding proteins have good affinity to the human CD39 and monkey CD39 antigen proteins expressed on the cell surface.
  • All anti-CD39 antigen-binding proteins were diluted to 30 ⁇ g/mL with TM buffer (Rockland, #MB-059), and then 3 ⁇ 9 ⁇ , ⁇ 10 ⁇ ,TM buffer ⁇ , ⁇ ; ⁇ CD39 ⁇ (CHOK1-huCD39-10B6, ⁇ ) ⁇ , ⁇ (1000rpm,5min) ⁇ , ⁇ TM buffer ⁇ 1 ⁇ 10 5 /mL, ⁇ 50 ⁇ L(5 ⁇ 10 3 ⁇ ) ⁇ 96 ⁇ , ⁇ 50 ⁇ L/ ⁇ ,37°C ⁇ 1h; ⁇ TM buffer ⁇ 60 ⁇ M ⁇ ATP ⁇ 50 ⁇ L/ ⁇ , ⁇ 1h; ⁇ 96 ⁇ (300g,3min), ⁇ 50 ⁇ L ⁇ (Corning,#3916) ⁇ ; ⁇ (Promega, #G7572) was added to the wells to be tested at 50 ⁇ L/well, reacted at room temperature for 10 minutes, and detected at full wavelengths.
  • TM buffer Rockland, #MB-059
  • CD39 antigen-binding proteins 17-13, 17-17 and 900690 to human CD39 protein was detected using Biacore (GE, model 8K), the experimental buffer was HBS-EP+ (pH7.4), Protein A chip (manufacturer: GE, product number: 29-1275-56).
  • 1% BSA/PBS is used as a negative control for use; a certain amount of human CD39-expressing cells (CHOK1-huCD39 , Huabo Biology) suspension, centrifuged (1000rpm, 5min), discarded the supernatant, resuspended the cells with 1% BSA/PBS to a viable cell density of 1 ⁇ 106/mL, 20 ⁇ L per well (2 ⁇ 10 4 1) into a 96-well U-shaped plate, then add the diluted antibody and negative control into the corresponding wells at 20 ⁇ L/well, and incubate at room temperature for 30 minutes; then resuspend the 96-well plate with 1% BSA/PBS, centrifuge (300g, 3min), discard the supernatant, and repeat the washing again, then add PE Goat Anti-human IgG, Fc (Jackson ImmunoRe) diluted with 1% BSA/PBS 1:200 search, #109-115-098), incubate at room temperature in the dark for 15 min;
  • Affinity matured CD39 antigen binding protein 17-22, 17-35 and parental antibody 17-13 pair before affinity maturation The results of the inhibition of human CD39 enzyme activity on the cell surface are shown in Figures 10A-10B and Table 11. The results show that the affinity-matured CD39 antigen-binding proteins 17-22 and 17-35 have improved ability to inhibit the enzyme activity of human CD39 expressed on the cell surface compared with the parent antibody 17-13 before affinity maturation.
  • Table 11 17-13, 17-35 and 17-22 inhibit human CD39 enzyme activity on cell surface
  • CD39 antigen-binding proteins 17-22 and 17-13 The binding kinetics of CD39 antigen-binding proteins 17-22 and 17-13 to human CD39 protein (manufacturer: ACRO Biosystems, product number: CD9-H52H4) and monkey CD39 protein (manufacturer: Sino Biological Inc, product number 90310-C08B) was detected using Biacore (GE, model 8K), the experimental buffer was HBS-EP+ (pH7.4), Protein A chip (manufacturer: GE , Item No.: 29-1275-56).
  • NPG mice were used to establish BxPC-3 pancreatic cancer animal models and test the efficacy of the tested antibodies.
  • Human pancreas used in this experiment Adenocarcinoma cells BxPC-3 were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO 2 . Before the cells were continuously cultured for ten generations, 100 ⁇ L of PBS containing about 1 ⁇ 10 7 BxPC-3 cells and an equal volume of Matrigel were mixed and inoculated into 18 NPG mice by subcutaneous injection on the right side of the back near the armpit with a volume of about 200 ⁇ L. Mice were anesthetized with 2-5% isoflurane prior to inoculation.
  • PBMCs On the day of inoculation, 1.0X10 7 PBMCs (100 ⁇ L) were injected via the tail vein. When the tumor grew to an average of about 50-80 mm 3 , 18 tumor-bearing mice were randomly divided into 3 groups according to tumor volume and body weight, with 6 mice in each group. Dosing was carried out on the day of grouping, and the specific dosing regimen was shown in Table 13 below.
  • 900543 is a negative control antibody that does not bind to CD39 antigen.
  • the amino acid sequence of the heavy chain (HC) of 900543 is shown in SEQ ID NO:13
  • the amino acid sequence of the light chain (LC) of 900543 is shown in SEQ ID NO:14.
  • 900689 is an anti-CD39 monoclonal antibody developed by Innate pharma, which is currently in phase 1 clinical trials.
  • the amino acid sequence of the heavy chain (HC) of 900689 is shown in SEQ ID NO:15
  • the amino acid sequence of the light chain (LC) of 900689 is shown in SEQ ID NO:16.
  • Example 11 In vivo detection of the effect of CD39 antigen-binding protein in treating colorectal cancer
  • CD39/CD73 double humanized mice (B-hCD73/hCD39(v3) mice) to establish MC38 colorectal cancer animal model and test the efficacy of the tested antibodies.
  • MC38 colorectal cancer cells used in this experiment were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO 2 . Before the cells were continuously cultured for ten generations, 100 ⁇ L of PBS containing about 5 ⁇ 10 5 MC38 cells and an equal volume of Matrigel were mixed and inoculated into 12 CD39/CD73 double humanized mice by subcutaneous injection on the right side of the back near the axilla, with an inoculation volume of about 100 ⁇ L. Mice were anesthetized with 2-5% isoflurane prior to inoculation.
  • 900543 is a negative control antibody that does not bind to CD39 antigen.
  • the results are shown in Table 14 and Figure 12 below. The results indicated that 17-22 could significantly inhibit the growth of MC38 colorectal cells, while the negative control antibody 900543 could not inhibit the growth of tumors.
  • the heavy chain (HC) amino acid sequence of 900543 is shown in SEQ ID NO: 13
  • the light chain (LC) amino acid sequence of 900543 is shown in SEQ ID NO: 13 ID NO:14.

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Abstract

本申请涉及一种分离的抗原结合蛋白及其应用，其中所述抗原结合蛋白质具有下列特征中的一种或多种：通过FACS检测法测定，以小于0.5μg/mL的EC50值与人CD39结合；通过FACS检测法测定，以小于0.7μg/mL的EC50值与食蟹猕猴CD39结合；通过Biacore检测法测定，以小于1.3×10-8M的KD值与人CD39结合；通过Biacore检测法测定，以小于1.1×10-7M的KD值与食蟹猕猴(cyno)CD39结合；通过ATP酶活性分析测定，以小于0.8μg/mL的EC50值抑制在表达CD39的细胞中的ATP酶活性；和/或，抑制肿瘤细胞的CD39功能。

Description

CD39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CD39抗原结合蛋白及其应用。
背景技术
CD39，也称为外核苷三磷酸二磷酸水解酶1(ENTPDase1)，是将ATP磷酸水解以生成ADP及AMP的完整膜蛋白质。CD39表达在许多实体瘤细胞中，CD39可通过抑制肿瘤特异性T细胞的激活、克隆扩增以及归巢，从而参与肿瘤免疫逃逸，损害由效应T淋巴细胞进行的肿瘤细胞杀灭。CD39可直接造成癌细胞生长、分化、入侵、迁移、转移及血管生成的调节。CD39对于血管生成的起始以及血管新生的进展均是重要的。
目前亟需靶向CD39的调节剂。
发明内容
一方面，本申请提供了一种分离的抗原结合蛋白，其中所述抗原结合蛋白质具有下列特征中的一种或多种：a)通过FACS检测法测定，以小于约0.5μg/mL的EC50值与人CD39结合；b)通过FACS检测法测定，以小于约0.7μg/mL的EC50值与食蟹猕猴(cyno)CD39结合；c)通过Biacore检测法测定，以小于约1.3×10-8M的KD值与人CD39结合；d)通过Biacore检测法测定，以小于约1.1×10-7M的KD值与食蟹猕猴(cyno)CD39结合；e)通过ATP酶活性分析测定，以小于约0.8μg/mL的EC50值抑制在表达CD39的细胞中的ATP酶活性；和/或，f)抑制肿瘤细胞的CD39功能。
在某些实施方式中，所述的分离的抗原结合蛋白含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:7-8、11-12中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO:29(AX1X2X3X4X5X6X7X8X9X10X11X12X13X14DX15X16X17，其中X1为A或W，X2为A或G，X3为E或G，X4为A或D，X5为D或G，X6为S或V，X7为S或V，X8为G或W，X9为F或G，X10为G或T，X11为P或T，X12为D、P或T，X13为L或W，X14为A、C、G或M，X15为W或Y，X16为G或W，X17为G或S)所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO:6或17所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO:3、6、9和10中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:2或5所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO:1或4所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR1～HCDR3，其中，a)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；b)所述HCDR1包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2包含SEQ ID NO:5所示的氨基酸序列；且所述HCDR3包含SEQ ID NO:6所示的氨基酸序列；c)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:9所示的氨基酸序列；或者，d)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述的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:18或22所示的氨基酸序列。
在某些实施方式中，所述的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:19、23和27中任一项所示的氨基酸序列。
在某些实施方式中，所述的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:20、24、26和28中任一项所示的氨基酸序列。
在某些实施方式中，所述的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:21或25所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR1～H-FR4，其中:a)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3包含SEQ ID NO:20所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列；b)所述H-FR1包含SEQ ID NO:22所示的氨基酸序列；所述H-FR2 包含SEQ ID NO:23所示的氨基酸序列；所述H-FR3包含SEQ ID NO:24所示的氨基酸序列；所述H-FR4包含SEQ ID NO:25所示的氨基酸序列；c)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3包含SEQ ID NO:26所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列；或者，d)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:27所示的氨基酸序列；所述H-FR3包含SEQ ID NO:28所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:7、8、11和12中任一项所示的氨基酸序列。
在某些实施方式中，所述的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：单克隆抗体和亲和力成熟抗体。
另一方面，本申请提供了一种多肽分子，其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，所述的多肽分子包含融合蛋白。
另一方面，本申请提供了一种核酸分子，其编码本申请所述的分离的抗原结合蛋白或本申请所述的多肽分子。
另一方面，本申请提供了一种载体，其包含本申请所述的核酸分子。
另一方面，本申请提供了一种免疫缀合物，其包含本申请所述的分离的抗原结合蛋白。
另一方面，本申请提供了一种细胞，其包含本申请所述的核酸分子、本申请所述的载体和/或本申请所述的免疫缀合物。
另一方面，本申请提供了一种药物组合物，其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、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫缀合物和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
另一方面，本申请提供了一种制备本申请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，培养本申请所述的细胞。
另一方面，本申请提供了一种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、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫缀合物、本申请所述的的细胞和/或本申请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。
在某些实施方式中，所述疾病和/或病症由CD39介导。
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/或病症包括胰腺癌和/或结直肠癌。
另一方面，本申请提供了一种检测样品中CD39的方法，所述方法包括施用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、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫缀合物、本申请所述的细胞和/或本申请所述的药物组合物。
另一方面，本申请提供了一种检测样品中CD39的试剂或试剂盒，其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、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫缀合物、本申请所述的细胞和/或本申请所述的药物组合物。
另一方面，本申请提供了一种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、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫缀合物、本申请所述的细胞和/或本申请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CD39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。对附图简要说明如下：
图1显示的是本申请候选VHH抗体的巢式PCR扩增结果。
图2显示的是Drop法涂板对Drop法涂板对免疫库进行筛选以及富集现象的观察结果。
图3显示的是本申请候选VHH抗体的Unique序列比对结果。
图4显示的是本申请所述抗原结合蛋白的SDS-PAGE结果。
图5显示的是本申请所述抗原结合蛋白对细胞表面人CD39抗原的亲和力的FACS检测结果。
图6显示的是本申请所述抗原结合蛋白对细胞表面猴CD39抗原的亲和力的FACS检测结果。
图7显示的是本申请所述抗原结合蛋白对细胞表面CD39抗原酶活性抑制能力的检测结果。
图8显示的是本申请候选亲和力成熟抗原结合蛋白的单克隆测序分析结果。
图9A-9B显示的是亲和力成熟的本申请所述抗原结合蛋白对细胞表面CD39抗原的亲和力的FACS检测结果。
图10A-10B显示的是亲和力成熟的本申请所述抗原结合蛋白对细胞表面CD39抗原酶活性抑制能力的检测结果。
图11显示的是本申请所述抗原结合蛋白在体内治疗胰腺癌的检测结果。
图12显示的是本申请所述抗原结合蛋白在体内治疗结直肠癌的检测结果。
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“CD39”通常是指由ENTPD1基因在人体内编码的胞外核苷三磷酸二磷酸水解酶1多肽。CD39的其它名称包括ENTPD1、ATPD酶、NTPD酶-1和SPG64。CD39催化细胞外三磷酸核苷(NTP；例如三磷酸腺苷或ATP)和二磷酸核苷(NDP；例如，二磷酸腺苷或ADP)的γ-和β-磷酸残基水解，将这些分子转化为一磷酸核苷(NMP；例如，一磷酸腺苷或AMP)衍生物。CD39的示例性氨基酸序列示于NCBI参考序列:NP_001767.3中。
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,2000Nucleic 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“VH”和“VL”(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CD39)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，CD39)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“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“亲和力成熟”通常指结合分子(例如抗原结合蛋白)已经历亲和力成熟，亲和力成熟为使结合分子(例如抗原结合蛋白)对靶抗原的亲和力增加的过程。因此，亲和力成熟抗体可以包括在一个或多个CDR中具有一种或多种变化(例如一个或多个CDR中的一个或多个氨基酸残基的变化)的抗体，与不具有那些变化的亲本抗体相比，所述一种或多种变化使所述抗体对抗原的亲和力改善。在某些情况下，所述亲和力成熟抗体可以与亲本抗体保留相同的表位。
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，所述肿瘤的实例可以包括但不限于：乳腺癌。
发明详述
分离的抗原结合蛋白
一方面，本申请提供了一种分离的抗原结合蛋白，其中所述抗原结合蛋白质具有下列特征中的一种或多种：
a)通过FACS检测法测定，以小于约0.5μg/mL的EC50值与人CD39结合；
例如，所述EC50值可以小于约0.4μg/mL、小于约0.3μg/mL、小于约0.2μg/mL、小于约0.1μg/mL、小于约0.09μg/mL、小于约0.08μg/mL、小于约0.07μg/mL、小于约0.06μg/mL、小于约0.05μg/mL、小于约0.04μg/mL、小于约0.03μg/mL、小于约0.02μg/mL、小于约0.01μg/mL或更低。
b)通过FACS检测法测定，以小于约0.7μg/mL的EC50值与食蟹猕猴(cyno)CD39结合；
例如，所述EC50值可以小于约0.6μg/mL、小于约0.5μg/mL、小于约0.4μg/mL、小于约0.3μg/mL、小于约0.2μg/mL、小于约0.1μg/mL、小于约0.09μg/mL、小于约0.08μg/mL、小于约0.07μg/mL、小于约0.06μg/mL、小于约0.05μg/mL、小于约0.04μg/mL、小于约0.03μg/mL、小于约0.02μg/mL、小于约0.01μg/mL或更低。
c)通过Biacore检测法测定，以小于约1.3×10-8M的KD值与人CD39结合；
例如，可以所述KD值可以小于约1×10-8M、小于约9×10-9M、小于约8×10-9M、小于约7×10-9M、小于约6×10-9M、小于约5×10-9M、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8×10-10M、小于约6×10-10M、小于约4×10-10M、小于约2×10- 10M、小于约1×10-10M或更低。
d)通过Biacore检测法测定，以小于约1.1×10-7M的KD值与食蟹猕猴(cyno)CD39结合；
例如，所述KD值可以小于约1×10-7M、小于约9×10-8M、小于约8×10-8M、小于约7×10- 8M、小于约6×10-8M、小于约5×10-8M、小于约4×10-8M、小于约3×10-8M、小于约2×10-8M、小于约1×10-8M、小于约8×10-9M、小于约6×10-9M、小于约4×10-9M、小于约2×10-9M、小于约1×10-9M或更低。
e)通过ATP酶活性分析测定，以小于约0.8μg/mL的EC50值抑制在表达CD39的细胞中的ATP酶活性；和/或，
例如，所述EC50值可以小于约0.7μg/mL、小于约0.6μg/mL、小于约0.5μg/mL、小于约0.4μg/mL、小于约0.3μg/mL、小于约0.2μg/mL、小于约0.1μg/mL、小于约0.09μg/mL、小于约0.08μg/mL、小于约0.07μg/mL、小于约0.06μg/mL、小于约0.05μg/mL、小于约0.04μg/mL、小于约0.03μg/mL、小于约0.02μg/mL、小于约0.01μg/mL或更低。
f)抑制肿瘤细胞的CD39功能。
在本申请中，所述的分离的抗原结合蛋白含重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:7-8、11-12中任一项所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR3，且所述HCDR3可以包含SEQ ID NO:29所示的氨基酸序列。
其中，SEQ ID NO:29：AX1X2X3X4X5X6X7X8X9X10X11X12X13X14DX15X16X17，其中X1为A或W，X2为A或G，X3为E或G，X4为A或D，X5为D或G，X6为S或V，X7为S或V，X8为G或W，X9为F或G，X10为G或T，X11为P或T，X12为D、P或T，X13为L或W，X14为A、C、G或M，X15为W或Y，X16为G或W，X17为G或S。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR3，且所述HCDR3可以包含SEQ ID NO:6或17所示的氨基酸序列。
其中，SEQ ID NO:17：AWGEDDSSWFGTX1LX2DYWG，其中X1为D或T，X2为C、G或M。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR3，且所述HCDR3可以包含SEQ ID NO:3、6、9和10中任一项所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR2，且所述HCDR2可以包含 SEQ ID NO:2或5所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，且所述HCDR1可以包含SEQ ID NO:1或4所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1～HCDR3，其中，
a)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:3所示的氨基酸序列；
b)所述HCDR1可以包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:5所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:6所示的氨基酸序列；
c)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:9所示的氨基酸序列；或者，
d)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列。
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可以涵盖根据任何CDR划分方式划分得到的CDR序列；也可以涵盖其变体，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。在本申请中，所述CDR由IMGT编号方案来确定。
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。在本申请中，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CD39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，所述的分离的抗原结合蛋白可以包含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可以包含SEQ ID NO:18或22所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:19、23和27中任一项所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:20、24、26和28中任一项所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含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可以包含SEQ ID NO:21或25所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR1～H-FR4，其中:
a)所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:20所示的氨基酸序列；所 述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列；
b)所述H-FR1可以包含SEQ ID NO:22所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:23所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:24所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:25所示的氨基酸序列；
c)所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:26所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列；或者，
d)所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:27所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:28所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列。
例如，在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1-3和H-FR1～H-FR4，其中:
a)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:3所示的氨基酸序列；
且所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:20所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列，
例如，在本申请中，所述的分离的抗原结合蛋白可以称为17-13；
b)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:9所示的氨基酸序列；
且所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:26所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列；
例如，在本申请中，所述的分离的抗原结合蛋白可以称为17-22；
c)所述HCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:10所示的氨基酸序列；
且所述H-FR1可以包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:27所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:28所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:21所示的氨基酸序列；
例如，在本申请中，所述的分离的抗原结合蛋白可以称为17-35；或者，
d)所述HCDR1可以包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:5所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:6所示的氨基酸序列；
且所述H-FR1可以包含SEQ ID NO:22所示的氨基酸序列；所述H-FR2可以包含SEQ ID NO:23所示的氨基酸序列；所述H-FR3可以包含SEQ ID NO:24所示的氨基酸序列；所述H-FR4可以包含SEQ ID NO:25所示的氨基酸序列；
例如，在本申请中，所述的分离的抗原结合蛋白可以称为17-17。
在本申请中，所述的分离的抗原结合蛋白可以包含VH，所述VH可以包含SEQ ID NO:7、8、11、12中任一项所示的氨基酸序列。
在本申请中，所述的分离的抗原结合蛋白可以包含抗体或其抗原结合片段。
在本申请中，所述抗原结合片段包括dAb。在本申请中，所述抗原结合片段可以为VHH。
在本申请中，所述抗体选自下组中的一种或多种：单克隆抗体和亲和力成熟抗体。
在本申请中，所述分离的抗原结合蛋白还可以包含重链恒定区。例如，所述分离的抗原结合蛋白可以包含Fc区。例如，所述Fc区可以源自人IgG的Fc区。
亲和力成熟(affinity maturation)通常是指在体液免疫中，再次应答所产生抗体的平均亲和力高于初次免疫应答。目前本领域技术人员可以使用熟知的技术对抗体进行改造，从而获得所述亲和力成熟抗体。例如，可以通过易错PCR、链置换、定点突变和/或DNA改组的手段进行改造。
在本申请中，所述的分离的抗原结合蛋白还可以具有下列特征中的一种或多种：
抑制或减少人CD39将细胞外三磷酸腺苷(eATP)或细胞外二磷酸腺苷(eADP)转化为细胞外一磷酸腺苷(eAMP)；增加或提高eATP水平；降低或减少细胞外腺苷；维持、增加或提高eATP的免疫刺激水平；增加或提高淋巴细胞的增殖；增加或提高一种或多种树突细胞活化标志物的表达；增加或提高一种或多种细胞因子从树突细胞的分泌；增加或提高肿瘤中的巨噬细胞浸润；和/或，增加或提高巨噬细胞吸引趋化因子的分泌。
多肽分子、核酸分子、载体、免疫缀合物、细胞和药物组合物
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，所述多肽分子包含融合蛋白。
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或本申请所述的多肽分子。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。
例如，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，本申请提供了一种细胞，其可以包含本申请所述的核酸分子、本申请所述的载体和/或本申请所述的免疫缀合物。在某些实施方式中，每种或每个宿主细胞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。
在本申请中，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。
在本申请中，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。
在本申请中，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。
在本申请中，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CD39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。
任何合适形式的CD39都可以作为免疫原(抗原)，用于产生对CD39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CD39，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。
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、所述免疫缀合物、所述细胞和/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、所述免疫缀合物、所述细胞和/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、所述免疫缀合物、所述细胞和/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CD39介导的疾病和/或病症。
在本申请中，所述疾病和/或病症可以包括肿瘤。
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，所述疾病和/或病症可以包括胰腺癌和/或结直肠癌。
另一方面，本申请还提供了检测样品中CD39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。
在本申请中，所述检测样品中CD39的方法可以是体外方法。在本申请中，所述检测样品中CD39的方法可以为非治疗目的。在本申请中，所述检测样品中CD39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CD39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、所述免疫缀合物、所述细胞和/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、所述免疫缀合物、所述细胞和/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CD39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抗原结合蛋白、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 CD39抗原结合蛋白文库的构建
将CD39抗原与弗氏佐剂等体积混合，乳化后于皮下多点注射，免疫一只alpaca羊驼， 每隔3周左右一次，共免疫3次，刺激B细胞表达抗原特异性的纳米抗体。免疫结束后，采集50ml外周血，分别收集血浆及淋巴细胞用于抗体效价滴定及纳米抗体库构建。经ELISA测定免疫效果优良，血浆抗体效价达到1：128000，且血浆抗体稀释32000倍后其OD450值≥0.5(1.37)，已完全满足建库要求。
羊驼免疫库构建方法具体如下：使用羊驼三免后采血分离所得PBMC，使用专用试剂盒提取总RNA，再反转录为cDNA，并利用巢式PCR扩增VHH，PCR结果见图1。然后将VHH片段插入到pShort噬菌粒并电转到超级感受态细胞SU320+Hφ中并测定库容和GA重组效率，库容大小为7.8E9CFM，GA重组效率达到80％以上(结果如表1所示)，完全满足高质量免疫库的相关指标。待噬菌体扩增到一定数量后采用PEG8000/NaCl沉淀法，分离纯化噬菌体，获得抗体库。调整噬菌体浓度，分装冻存于-80℃备用。
表1羊驼免疫库相关指标统计
实施例2 CD39抗原结合蛋白的筛选及鉴定
将CD39抗原以5μg/ml，100μl/孔加入到96孔板中包被，4℃过夜。第二天加入1％的PVA溶液室温孵育1h进行封闭。封闭完成后加入抗体库(1E13pfu/ml，100μl/孔)，室温孵育1h。然后每孔加入200ul的PT缓冲液清洗8～10次。清洗完成后每孔加入100mM HCl，室温孵育5min，再加入1M Tris-HCl中和洗脱。将含噬菌体的洗脱液感染NEB alpha 5F’细胞，加入辅助噬菌体后，过夜培养。同时采用Drop法涂板，第二天统计富集程度。将过夜培养的产物加入PEG8000/NaCl进行沉淀，分离纯化噬菌体，进入下一轮淘筛并统计富集纯度，Drop法涂板淘筛结果如图2所示。出现富集后，随机挑取单克隆，通过Phage-ELISA鉴定阳性克隆。最后根据ELISA阳性孔OD值的高低以及与对照孔的差异，共选择多个单克隆进行测序。进一步，剔除其中测序异常的序列、合并序列相同的克隆。
为了获得更多的特异性单克隆，本项目先后进行了多次阳性单克隆鉴定。最终，共获得13条特异性单克隆序列，根据CDR(特别是HCDR3)的差异，可分为3组，结果如图3。其中17-13和17-17的序列如下表2所示，将纳米抗体序列构建到含有人Fc蛋白序列的表达载体上并采用293F细胞进行表达，17-13-hFc和17-17-hFc蛋白表达和纯化如图4所示。
表2 17-13和17-17抗体的序列

实施例3 CD39抗原结合蛋白对细胞表面人和猴CD39的结合活性检测
将所有抗CD39的抗原结合蛋白用含1％BSA的PBS溶液(1％BSA/PBS)稀释至60μg/mL,再3倍稀释10次，共11个梯度浓度，1％BSA/PBS为阴性对照，待用；取一定量的处于对数生长期内的表达人CD39的细胞(CHOK1-huCD39，华博生物)和猴CD39的细胞(CHOK1-rhCD39，华博生物)悬液，离心(1000rpm，5min)后弃上清，用1％BSA/PBS重悬细胞至活细胞密度为1×106/mL,每孔20μL(2×104个)加入96孔U型板中，再将已稀释好的抗原结合蛋白和阴性对照以20μL/孔加入相应的孔中，室温孵育30min；而后将96孔板用1％BSA/PBS重悬，离心(300g，3min)，弃上清，如此重复洗涤一遍，加入用1％BSA/PBS 1：200稀释的PE Goat Anti-human IgG,Fc(Jackson Immuno Research，#109-115-098)，室温避光孵育15min；将反应后的96孔板用1％BSA/PBS重悬，离心(300g，3min)并弃上清，重复洗涤2遍，最终用100μL/孔的1％BSA/PBS重悬，用流式细胞仪(BD，#CantoII)检测PE通道的荧光强度。
900690(阳性对照抗体，来源于Surface Oncology公司的抗CD39单克隆抗体，900690的重链(HC)氨基酸序列如SEQ ID NO:30所示，900690的轻链(LC)氨基酸序列如SEQ ID NO:31所示)，17-13,17-17与细胞表面人CD39结合活力结果见图5及表3，与细胞表面猴CD39结合活力结果见图6及表4，结果表明17-13，17-17抗原结合蛋白与细胞表面表达人CD39以及猴CD39抗原蛋白都有很好的亲和力。
表3 900690、17-13、17-17与细胞表面人CD39结合活力
表4 900690、17-13、17-17与细胞表面猴CD39结合活力
实施例4 CD39抗原结合蛋白对细胞表面人CD39酶活性抑制效果检测
将所有抗CD39的抗原结合蛋白用TM buffer(Rockland，#MB-059)稀释至30μg/mL,再 3倍稀释9次，共10个梯度浓度，TM buffer作为对照，待用；取一定量的处于对数生长期内的表达人CD39的细胞(CHOK1-huCD39-10B6，华博生物)悬液，离心(1000rpm，5min)后弃上清，用TM buffer重悬细胞至活细胞密度为1×105/mL,每孔50μL(5×103个)加入96孔板中，再将已稀释好的抗原结合蛋白和对照以50μL/孔加入相应的孔中，37℃孵育1h；而后将用TM buffer稀释至60μM的ATP以50μL/孔加入相应的孔中，孵育1h；而后将96孔板离心(300g，3min)，并取出50μL加入黑板(Corning，#3916)中；将(Promega，#G7572)以50μL/孔加入待测孔，室温反应10min，全波长检测。
900690、17-13、17-17对细胞表面人CD39酶活性抑制的结果见图7以及表5，结果表明只有17-13对细胞表面人CD39酶活性有很明显的抑制效果，阳性对照900690只有微弱的抑制效果，而17-17几乎没有抑制效果。
表5 900690、17-13、17-17对细胞表面人CD39酶活性抑制
实施例5 CD39抗原结合蛋白与人CD39的结合亲和力检测
CD39抗原结合蛋白17-13,17-17以及900690与人CD39蛋白(厂家：ACRO Biosystems，货号：CD9-H52H4)的结合动力学实验使用Biacore(GE，型号为8K)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150RU。人CD39蛋白用实验缓冲液稀释至50nM，25nM，12.5nM，6.25nM，3.13nM，1.56nM，0.78nM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300s。10mM Glycine pH 1.5在50μL/min流速下，再生60s，使用多循环动力学检测。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6，显示所有抗原结合蛋白均与人CD39蛋白结合，且17-13抗体与人CD39蛋白的结合亲和力要优于对照抗体900690。
表6 900690、17-13、17-17对人CD39蛋白的结合亲和力检测
实施例6 CD39抗原结合蛋白17-13的亲和力成熟
为了进一步增强具有功能活性的17-13纳米抗体对CD39抗原的亲和力和生物活性，我们对17-13进行了亲和力成熟改造。首先通过对17-13纳米抗体的3个CDR区序列进行分析，选择出亲和力成熟突变的CDR关键位点。并基于CDR区序列构建3个突变文库(小库)Lib 17-13H1、Lib 17-13H2、和Lib 17-13H3，进行生物淘筛。建库数据(表7)显示：多样性＝库容*突变效率大于理论多样性，可以用于后续筛选。生物掏筛并富集成功后，每个小库随机挑取40个克隆，进行单克隆测序和突变分析，确定与亲和力相关的热点(Hot spot)区域；然后对热点区域进行组合突变，构建组合突变文库(1个大库，Lib CL)，建库数据(表8)显示：多样性＝库容*突变效率大于理论多样性；随机挑取单克隆，进行测序，结果(图8)显示：热点区域突变成功，可以用于后续筛选。生物淘筛并富集成功后，挑取96个克隆进行phage ELISA；然后对phage ELISA阳性、且OD值高于母本抗体的克隆进行测序和亲和力排序，并最终挑选亲和力最高的两个序列17-22以及17-35进行亲和力结合验证，17-22以及17-35的序列如表9所示。
表7深度突变抗体库的建库数据
表8组合突变抗体库的建库数据
表9 17-22和17-35抗体的序列
实施例7亲和力成熟的CD39抗原结合蛋白与细胞表面人CD39的亲和力
将亲和力成熟的CD39抗原结合蛋白17-22,17-35以及亲和力成熟前的母本抗体17-13用含1％BSA的PBS溶液(1％BSA/PBS)稀释至60μg/mL,再3倍稀释10次，共11个梯度浓度，1％BSA/PBS为阴性对照，待用；取一定量的处于对数生长期内的表达人CD39的细胞(CHOK1-huCD39，华博生物)悬液，离心(1000rpm，5min)后弃上清，用1％BSA/PBS重悬细胞至活细胞密度为1×106/mL,每孔20μL(2×104个)加入96孔U型板中，再将已稀释好的抗体和阴性对照以20μL/孔加入相应的孔中，室温孵育30min；而后将96孔板用1％BSA/PBS重悬，离心(300g，3min)，弃上清，如此重复洗涤一遍，加入用1％BSA/PBS 1：200稀释的PE Goat Anti-human IgG,Fc(Jackson ImmunoResearch，#109-115-098)，室温避光孵育15min；将反应后的96孔板用1％BSA/PBS重悬，离心(300g，3min)并弃上清，重复洗涤2遍，最终用100μL/孔的1％BSA/PBS重悬，用流式细胞仪(BD，#CantoII)检测PE通道的荧光强度。
亲和力成熟的CD39抗原结合蛋白17-22、17-35以及亲和力成熟前的母本抗体17-13与细胞表面人CD39结合活力结果见图9A-9B及表10，结果表明亲和力成熟的CD39抗原结合蛋白17-22、17-35与细胞表面表达人CD39的亲和力相较于亲和力成熟前的母本抗体17-13都有改善。
表10 17-13、17-35以及17-22与细胞表面人CD39结合活力
实施例8亲和力成熟的CD39抗原结合蛋白对细胞表面人CD39酶活性抑制效果检测
将亲和力成熟的CD39抗原结合蛋白17-22、17-35以及亲和力成熟前的母本抗体17-13用TM buffer(Rockland，#MB-059)稀释至30μg/mL,再3倍稀释9次，共10个梯度浓度，TM buffer作为对照，待用；取一定量的处于对数生长期内的表达人CD39的细胞(CHOK1-huCD39-10B6，华博生物)悬液，离心(1000rpm，5min)后弃上清，用TM buffer重悬细胞至活细胞密度为1×105/mL,每孔50μL(5×103个)加入96孔板中，再将已稀释好的抗原结合蛋白和对照以50μL/孔加入相应的孔中，37℃孵育1h；而后将用TM buffer稀释至60μM的ATP以50μL/孔加入相应的孔中，孵育1h；而后将96孔板离心(300g，3min)，并取出50μL加入黑板(Corning，#3916)中；将(Promega，#G7572)以50μL/孔加入待测孔，室温反应10min，全波长检测。
亲和力成熟的CD39抗原结合蛋白17-22、17-35以及亲和力成熟前的母本抗体17-13对 细胞表面人CD39酶活性抑制的结果见图10A-10B以及表11，结果表明亲和力成熟的CD39抗原结合蛋白17-22、17-35对细胞表面表达人CD39的酶活抑制能力相较于亲和力成熟前的母本抗体17-13都有改善。
表11 17-13、17-35以及17-22对细胞表面人CD39酶活性抑制
实施例9亲和力成熟的CD39抗原结合蛋白与人和猴CD39的结合亲和力检测
CD39抗原结合蛋白17-22以及17-13与人CD39蛋白(厂家：ACRO Biosystems，货号：CD9-H52H4)以及猴CD39蛋白(厂家：Sino Biological Inc,货号90310-C08B)的结合动力学实验使用Biacore(GE，型号为8K)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150RU。人和猴CD39蛋白分别用实验缓冲液稀释至50nM，25nM，12.5nM，6.25nM，3.13nM，1.56nM，0.78nM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300s。10mM Glycine pH 1.5在50μL/min流速下，再生60s，使用多循环动力学检测。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12，显示亲和力成熟的CD39抗原结合蛋白17-22对人和猴CD39抗原的结合亲和力相较于亲和力成熟前的母本抗体17-13都有改善。
表12 17-13、17-22对人和猴CD39蛋白的结合亲和力检测
实施例10 CD39抗原结合蛋白的体内治疗胰腺癌的效果检测
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％CO2的37℃培养箱。细胞连续培养十代之前，将约含1×107BxPC-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1.0X107PBMC(100μL)，当肿瘤生长到平均约50-80mm3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，具体给药方案见下表13。
其中，900543为阴性对照抗体，不与CD39抗原结合。900543的重链(HC)氨基酸序列如SEQ ID NO:13所示，900543的轻链(LC)氨基酸序列如SEQ ID NO:14所示。900689为Innate pharma开发的抗CD39单克隆抗体，目前处于临床1期。900689的重链(HC)氨基酸序列如SEQ ID NO:15所示，900689的轻链(LC)氨基酸序列如SEQ ID NO:16所示。
结果如下表13和图11所示，结果说明17-22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
实施例11 CD39抗原结合蛋白的体内治疗结直肠癌的效果检测
利用CD39/CD73双人源化小鼠(B-hCD73/hCD39(v3)mice)建立MC38结直肠癌动物模型并测试受试抗体的药效。用于本实验的MC38结直肠癌细胞，以RPMI-1640培养基添加10％FBS培养于含5％CO2的37℃培养箱。细胞连续培养十代之前，将约含5×105MC38细胞的PBS 100μL和等体积Matrigel混匀后，通过皮下注射接种于12只CD39/CD73双人源化小鼠背部右边靠近腋下，接种体积为100μL左右。接种前用2-5％异氟烷将小鼠麻醉。
在接种当天，当肿瘤生长到平均约100-150mm3左右时，12只荷瘤小鼠将根据肿瘤体积和体重被随机分成2组，每组6只。分组当天进行给药，具体给药方案见下表。其中，900543为阴性对照抗体，不与CD39抗原结合。结果如下表14和图12所示，结果说明17-22能够显著抑制MC38结直肠细胞的生长，而阴性对照抗体900543则不能抑制肿瘤的生长。900543的重链(HC)氨基酸序列如SEQ ID NO:13所示，900543的轻链(LC)氨基酸序列如SEQ  ID NO:14所示。
表14
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 (33)

  1. 分离的抗原结合蛋白，其中所述抗原结合蛋白质具有下列特征中的一种或多种：
    a)通过FACS检测法测定，以小于约0.5μg/mL的EC50值与人CD39结合；
    b)通过FACS检测法测定，以小于约0.7μg/mL的EC50值与食蟹猕猴(cyno)CD39结合；
    c)通过Biacore检测法测定，以小于约1.3×10-8M的KD值与人CD39结合；
    d)通过Biacore检测法测定，以小于约1.1×10-7M的KD值与食蟹猕猴(cyno)CD39结合；
    e)通过ATP酶活性分析测定，以小于约0.8μg/mL的EC50值抑制在表达CD39的细胞中的ATP酶活性；和/或，
    f)抑制肿瘤细胞的CD39功能。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:7-8、11-12中任一项所示的氨基酸序列。
  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HCDR3，且所述HCDR3包含SEQ ID NO:29(AX1X2X3X4X5X6X7X8X9X10X11X12X13X14DX15X16X17，其中X1为A或W，X2为A或G，X3为E或G，X4为A或D，X5为D或G，X6为S或V，X7为S或V，X8为G或W，X9为F或G，X10为G或T，X11为P或T，X12为D、P或T，X13为L或W，X14为A、C、G或M，X15为W或Y，X16为G或W，X17为G或S)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HCDR3，且所述HCDR3包含SEQ ID NO:6或17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HCDR3，且所述HCDR3包含SEQ ID NO:3、6、9和10中任一项所示的氨基酸序列。
  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HCDR2，且所述HCDR2包含SEQ ID NO:2或5所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:1或4所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其包含HCDR1～HCDR3，其中，
    a)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列；
    b)所述HCDR1包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2包含SEQ ID NO:5所示的氨基酸序列；且所述HCDR3包含SEQ ID NO:6所示的氨基酸序列；
    c)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:9所示的氨基酸序列；或者，
    d)所述HCDR1包含SEQ ID NO:1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；且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
  9. 根据权利要求1-8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:18或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-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:19、23和27中任一项所示的氨基酸序列。
  11. 根据权利要求1-10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:20、24、26和28中任一项所示的氨基酸序列。
  12. 根据权利要求1-11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:21或25所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其包含H-FR1～H-FR4，其中:
    e)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3包含SEQ ID NO:20所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列；
    f)所述H-FR1包含SEQ ID NO:22所示的氨基酸序列；所述H-FR2包含SEQ ID NO:23所示的氨基酸序列；所述H-FR3包含SEQ ID NO:24所示的氨基酸序列；所述H-FR4包含SEQ ID NO:25所示的氨基酸序列；
    g)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:19所示的氨基酸序列；所述H-FR3包含SEQ ID NO:26所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列；或者，
    h)所述H-FR1包含SEQ ID NO:18所示的氨基酸序列；所述H-FR2包含SEQ ID NO:27所示的氨基酸序列；所述H-FR3包含SEQ ID NO:28所示的氨基酸序列；所述H-FR4包含SEQ ID NO:21所示的氨基酸序列。
  14. 根据权利要求1-13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:7、8、11和12中任一项所示的氨基酸序列。
  15. 根据权利要求1-14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。
  16. 根据权利要求15所述的分离的抗原结合蛋白，其中所述抗原结合片段包括dAb。
  17. 根据权利要求15-16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：单克隆抗体和亲和力成熟抗体。
  18. 多肽分子，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白。
  19. 根据权利要求18所述的多肽分子，其包含融合蛋白。
  20. 核酸分子，其编码权利要求1-17中任一项所述的分离的抗原结合蛋白或权利要求18-19中任一项所述的多肽分子。
  21. 载体，其包含权利要求20所述的核酸分子。
  22. 免疫缀合物，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白。
  23. 细胞，其包含权利要求20所述的核酸分子、权利要求21所述的载体和/或权利要求22所述的免疫缀合物。
  24. 药物组合物，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-19中任一项所述的多肽分子、权利要求20所述的核酸分子、权利要求21所述的载体、权利要求22所述的免疫缀合物和/或权利要求23所述的细胞，以及任选地药学上可接受的载剂。
  25. 制备权利要求1-17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23所述的细胞。
  26. 权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-19中任一项所述的多肽分子、权利要求20所述的核酸分子、权利要求21所述的载体、权利要求22所述的免疫缀合物、权利要求23所述的细胞和/或权利要求24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。
  27. 根据权利要求26所述的用途，其中所述疾病和/或病症由CD39介导。
  28. 根据权利要求26-27中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  29. 根据权利要求28所述的用途，其中所述肿瘤包括实体瘤和/或血液瘤。
  30. 根据权利要求26-29中任一项所述的用途，其中所述疾病和/或病症包括胰腺癌和/或结直肠癌。
  31. 检测样品中CD39的方法，所述方法包括施用权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-19中任一项所述的多肽分子、权利要求20所述的核酸分子、权利要求21所述的载体、权利要求22所述的免疫缀合物、权利要求23所述的细胞和/或权利要求24所述的药物组合物。
  32. 检测样品中CD39的试剂或试剂盒，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-19中任一项所述的多肽分子、权利要求20所述的核酸分子、权利要求21所述的载体、权利要求22所述的免疫缀合物、权利要求23所述的细胞和/或权利要求24所述的药物组合物。
  33. 权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-19中任一项所述的多肽分子、权利要求20所述的核酸分子、权利要求21所述的载体、权利要求22所述的免疫缀合物、权利要求23所述的细胞和/或权利要求24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CD39的存在和/或含量。
PCT/CN2023/073060 2022-01-24 2023-01-19 Cd39抗原结合蛋白及其应用 WO2023138643A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111448211A (zh) * 2017-07-31 2020-07-24 提圣纳医疗公司 抗cd39抗体、包含抗cd39抗体的组合物和使用抗cd39抗体的方法
CN112714768A (zh) * 2019-08-27 2021-04-27 科望(苏州)生物医药科技有限公司 新型抗cd39抗体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111448211A (zh) * 2017-07-31 2020-07-24 提圣纳医疗公司 抗cd39抗体、包含抗cd39抗体的组合物和使用抗cd39抗体的方法
CN112714768A (zh) * 2019-08-27 2021-04-27 科望(苏州)生物医药科技有限公司 新型抗cd39抗体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DUAN YINGHUI: "Generation and characterization of nanobodies against the ecto-enzymes CD39/CD73", DISSERTATION, 1 August 2021 (2021-08-01), pages 1 - 75, XP093079643 *

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