WO2020143836A1 - Cd73抗体及其制备方法和应用 - Google Patents

Cd73抗体及其制备方法和应用 Download PDF

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Publication number
WO2020143836A1
WO2020143836A1 PCT/CN2020/071838 CN2020071838W WO2020143836A1 WO 2020143836 A1 WO2020143836 A1 WO 2020143836A1 CN 2020071838 W CN2020071838 W CN 2020071838W WO 2020143836 A1 WO2020143836 A1 WO 2020143836A1
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Prior art keywords
antibody
variable region
chain variable
heavy chain
light chain
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Ceased
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PCT/CN2020/071838
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English (en)
French (fr)
Inventor
王冬旭
段清
刘礼乐
杨达志
刘虎
韩烨
谢荣荣
邵晓慧
王鹏
钟勤
黄亚珺
吴建
王美玲
王远东
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Shanghai Pharmaexplorer Co Ltd
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Shanghai Pharmaexplorer Co Ltd
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Priority to EP20738698.8A priority Critical patent/EP3909982A4/en
Priority to CN202080008682.4A priority patent/CN113316591B/zh
Priority to US17/422,060 priority patent/US20220098319A1/en
Publication of WO2020143836A1 publication Critical patent/WO2020143836A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a CD73 antibody and its preparation method and application.
  • tumor immunotherapy has become the focus of the field of tumor therapy, in which therapeutic monoclonal antibodies against immune checkpoints have shown anti-tumor activity in the treatment of some tumor types such as melanoma and non-small cell lung cancer.
  • Cytotoxic T lymphocyte-associated antigen 4 cytotoxic T lymphocyte-associated antigen-4, CTLA-4
  • programmed death molecule 1 and its ligands programmed cell death 1/programmed cell death ligand 1, PD-1/PD -L1
  • the low response rate of single medication is the main problem of existing tumor immunotherapy.
  • CTLA4 antibody carried out clinical trials, which showed the presence of toxicity (causing tissue-specific inflammation) and low response rate. The most obvious clinical effect occurred in the treatment of melanoma, but the objective response rate was only 15%.
  • PD-1 and PD-L1 Hodgkin's lymphoma, Merkel cell carcinoma, and connective tissue proliferative melanoma have the highest objective response rate, reaching 50-90%; the response rate of melanoma treatment 35-40%; the response rate of non-small cell lung cancer, head and neck cancer, bladder cancer, kidney cancer, and hepatocellular carcinoma is only 15-25%.
  • Tumor is a disease with multiple pathways and multiple targets.
  • the objective response rate of a single therapeutic drug is low. It is likely that tumor cells choose other compensatory pathways to meet growth when a certain signaling pathway is inhibited by drugs. In order to improve the existing therapeutic effect and reduce the amount of toxic antibodies, tumor immunotherapy will become an important development trend.
  • CD73 glycosylphosphatidylinositol
  • CD73 dephosphorylates extracellular nucleotide monophosphate (AMP) to produce adenosine.
  • Extracellular adenosine binds to a variety of cell-specific adenosine receptors (A1, A2A, A2B, and A3), activates the adenosine pathway, and plays an important role in immunosuppression and angiogenesis.
  • CD73 is highly expressed on the surface of various tumor cells, including bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer and breast cancer. Upregulation of CD73 expression is associated with cancer cell proliferation, metastasis, angiogenesis, and shorter patient survival. Therefore, CD73 can be used as a new drug target and biomarker for the treatment of cancer.
  • the present invention provides a CD73 antibody with high affinity and strong specificity, and a preparation method and application thereof.
  • a heavy chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 10n+1, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, Or 9.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1 or 101.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 11.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.21.
  • a heavy chain of an antibody having a heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain further includes a heavy chain constant region.
  • the heavy chain constant region is of human or murine origin.
  • the heavy chain constant region is a human antibody heavy chain IgG1 constant region.
  • the heavy chain constant region is a human antibody heavy chain IgG1-TM constant region.
  • the IgG1-TM constant region is IgG1 and contains mutations at three positions L234F, L235E and P331S.
  • a light chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO. 10n+6 or SEQ ID NO. 103, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO. 6 or 103.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO. 16.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.26.
  • a light chain of an antibody said light chain having a light chain variable region as described in the third aspect of the invention.
  • the light chain further includes a light chain constant region.
  • the light chain constant region is of human or murine origin.
  • the light chain constant region is a human antibody light chain kappa constant region.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention,
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the amino acid sequence of any of the above CDRs includes derived, deleted, modified, and/or substituted 1, 2 or 3 amino acid derived CDR sequences, and the VH and VL containing the derived CDR sequences
  • the derived antibody can retain the affinity of binding to CD73.
  • the ratio (F1/F0) of the affinity F1 of the derived antibody to CD73 and the affinity F0 of the corresponding non-derivatized antibody to CD73 is 0.5-2, preferably 0.7-1.5, And better still 0.8-1.2.
  • the number of added, deleted, modified and/or substituted amino acids is 1-5 (eg 1-3, preferably 1-2, more preferably 1).
  • the derived sequence that has been added, deleted, modified, and/or substituted for at least one amino acid and can retain the binding affinity of CD73 is an amino acid sequence with homology or sequence identity of at least 96%.
  • the antibody further includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the heavy chain constant region is a human antibody heavy chain IgG1 constant region
  • the light chain constant region is a human antibody light chain kappa constant region
  • the heavy chain constant region is a human antibody heavy chain IgG1-TM constant region
  • the light chain constant region is a human antibody light chain kappa constant region
  • the heavy chain variable region of the antibody further includes a framework region of human origin, and/or the light chain variable region of the antibody further includes a framework region of human origin.
  • the heavy chain variable region of the antibody further includes a murine framework region, and/or the light chain variable region of the antibody further includes a murine framework region.
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.
  • the ratio of the immunogenicity Z1 of the chimeric antibody in humans to the immunogenicity Z0 of non-chimeric antibodies (such as murine antibodies) in humans is 0 -0.5, preferably 0-0.2, more preferably 0-0.05 (e.g. 0.001-0.05).
  • the antibody is a partially or fully humanized, or fully human monoclonal antibody.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is an antibody full-length protein or an antigen-binding fragment.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the antibody is in the form of a drug conjugate.
  • the antibody has one or more characteristics selected from the group consisting of:
  • the antibody has a heavy chain variable region according to the first aspect of the present invention and a light chain variable region according to the third aspect of the present invention;
  • heavy chain variable region and the light chain variable region include CDRs selected from the group consisting of:
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the antibody has a heavy chain variable region according to the first aspect of the present invention and a light chain variable region according to the third aspect of the present invention; wherein,
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.3,
  • VH-CDR2 shown in SEQ ID NO. 4, and
  • VH-CDR3 shown in SEQ ID NO.5;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO. 9, and
  • VL-CDR3 shown in SEQ ID NO.10;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.14, and
  • VH-CDR3 shown in SEQ ID NO.15;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.19, and
  • VL-CDR3 shown in SEQ ID NO.20;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.24, and
  • VH-CDR3 shown in SEQ ID NO.25;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.29.
  • VL-CDR3 shown in SEQ ID NO.30.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101; and/ Or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 103.
  • the antibody heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.1; and the antibody light chain variable region contains the amino acid sequence shown in SEQ ID NO.6.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 101; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 103.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.11; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.16.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 21; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 26.
  • the antibody is selected from the following group:
  • the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID No. 1, 11, 21, 31, 41, 51, 61, 71, 81, 91 or 101 in the sequence listing
  • the amino acid sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity.
  • amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 103 in the sequence listing Has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • a recombinant protein in a sixth aspect of the present invention, includes:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or polymer.
  • the recombinant protein includes:
  • the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain variable region according to the third aspect of the present invention such as The light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention.
  • polynucleotide encoding the heavy chain variable region is shown in SEQ ID NO. 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, or 102; and /Or, the polynucleotide encoding the light chain variable region is shown in SEQ ID NO. 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, or 104.
  • polynucleotide encoding the heavy chain variable region sequence and the polynucleotide encoding the light chain variable region sequence are selected from the group consisting of:
  • the vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell containing the vector or genome of the eighth aspect of the present invention incorporating the polynucleoside of the seventh aspect of the present invention acid.
  • an antibody conjugate comprising:
  • a coupling portion coupled to the antibody portion is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody part and the coupling part are coupled by a chemical bond or a linker.
  • an immune cell which expresses or is exposed to the cell membrane the antibody of the fifth aspect of the present invention.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, and the third according to the invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition includes 0.01-99.99% of the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, and the tenth according to the invention
  • the antibody conjugate, the immune cell according to the eleventh aspect of the present invention, or a combination thereof and a pharmaceutical carrier of 0.01 to 99.99% the percentage is a mass percentage of the pharmaceutical composition.
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, and a second aspect of the present invention
  • the recombinant protein according to the sixth aspect of the invention, the antibody conjugate according to the tenth aspect of the invention, the immune cell according to the eleventh aspect of the invention, or a combination thereof, wherein the active ingredient is used In (a) preparation of diagnostic reagents or kits; and/or (b) preparation of drugs for preventing and/or treating diseases related to abnormalities of CD73 expression or function.
  • the diagnostic reagent is a test piece or a test plate.
  • the disease associated with abnormal CD73 expression or function is a tumor.
  • the tumor is selected from the group consisting of bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck cancer, prostate cancer, Pancreatic cancer.
  • the diagnostic reagent or kit is used for:
  • the drug is used to prevent and/or treat diseases related to abnormal expression or function of CD73, and the diseases related to abnormal expression or function of CD73 are tumors.
  • the tumor is selected from the group consisting of bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer, breast cancer lung cancer, head and neck cancer, prostate cancer, pancreas cancer.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the diagnostic reagent or kit is used to diagnose CD73-related diseases.
  • the diagnostic reagent or kit is used to detect CD73 protein in a sample.
  • a fourteenth aspect of the present invention a method for in vitro detection (including diagnostic or non-diagnostic) of CD73 protein in a sample is provided.
  • the method includes the steps of:
  • composition for in vitro detection of CD73 protein in a sample which comprises the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, The antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the eleventh aspect of the present invention, or a combination thereof is used as an active ingredient.
  • a detection board comprises: a substrate (supporting plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the invention 2.
  • a kit in a seventeenth aspect of the present invention, includes:
  • a first container containing the antibody of the present invention and/or
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • the recombinant polypeptide is isolated from the culture, and the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
  • a pharmaceutical combination including:
  • a first active ingredient comprising the antibody 1 according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the tenth aspect according to the invention
  • a second active ingredient which includes a second antibody or a chemotherapeutic agent.
  • the second antibody is selected from the group consisting of CTLA4 antibody, PD-1 antibody, and PD-L1 antibody.
  • the second antibody is a PD-1 antibody.
  • the second active ingredient is an A2AR inhibitor.
  • the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.
  • the antibody according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention Or the combination of the immune cell according to the eleventh aspect of the present invention, and/or the pharmaceutical composition according to the twelfth aspect of the present invention and a second antibody or chemotherapeutic agent is related to the preparation for the treatment of CD73 expression or functional abnormalities Use of medicines for diseases.
  • the second antibody is selected from the group consisting of CTLA4 antibody, PD-1 antibody, and PD-L1 antibody.
  • the second antibody is a PD-1 antibody.
  • the second active ingredient is an A2AR inhibitor.
  • an effective amount of the antibody according to the fifth aspect of the present invention is administered to a subject in need thereof, or as The recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention, or the immune cell according to the eleventh aspect of the invention, or the tenth according to the invention.
  • the pharmaceutical composition according to the second aspect, or a combination thereof is administered to a subject in need thereof, or as The recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention, or the immune cell according to the eleventh aspect of the invention, or the tenth according to the invention.
  • the disease associated with abnormal CD73 expression or function is a tumor.
  • the tumor is selected from the group consisting of bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck cancer, prostate cancer, Pancreatic cancer.
  • the method further includes: administering a safe and effective amount of the second antibody to the subject before, during, and/or after the first active ingredient is administered.
  • the second antibody is selected from the group consisting of CTLA4 antibody, PD-1 antibody, and PD-L1 antibody.
  • the second antibody is a PD-1 antibody.
  • FIG. 1 Flow cytometry (FACS) detection of the binding of murine antibodies to human CD73, cynomolgus monkey CD73 and murine CD73.
  • mIgG1 is the isotype control
  • MFI (mean fluorescence) intensity is the average fluorescence intensity.
  • Anti-CD73 murine antibody inhibits the enzyme activity of human CD73.
  • mIgG1 is the isotype control
  • Anti-CD73 murine antibody restores AMP-mediated inhibition of T cell proliferation.
  • Activated is the percentage of T cell proliferation without AMP and without antibody
  • Activated+AMP500uM is the percentage of T cell proliferation with AMP and without antibody
  • mIgG1 is the isotype control
  • FIG. 5 Flow cytometry (FACS) detection of chimeric antibody binding to human CD73, cynomolgus monkey CD73 and murine CD73.
  • HIgG1 is the control;
  • MFI (mean fluorescence) intensity is the average fluorescence intensity
  • Anti-CD73 chimeric antibody inhibits the enzyme activity of human CD73.
  • HIgG1 is the control;
  • Anti-CD73 chimeric antibody mediates CD73 endocytosis.
  • HIgG1 is the control;
  • Anti-CD73 chimeric antibody restores CD4+ T cell proliferation.
  • Activated is the percentage of T cell proliferation without AMP and without antibody
  • Activated+AMP 800uM is the percentage of T cell proliferation with AMP and without antibody
  • hIgG1 is the control
  • Tab2 is Innate Pharma's anti-human CD73 antibody 11E1.
  • the present inventors have obtained diverse antibody sequences using different immunization strategies (different mouse strains, multiple antigens, and different routes of administration), and selected various properties combined with human CD73 Specific anti-CD73 monoclonal antibodies are more excellent (such as excellent in vitro activity in all aspects).
  • the present invention uses techniques such as immunization of SJL mice, hybridomas, molecular biology (sequencing, vector construction), etc., to provide a set of human-mouse chimeric antibodies that bind to CD73, containing mouse-derived antibody heavy chains and light Chain variable region and human antibody constant region. All variable regions contain CDR1, CDR2, CRR3 three complementarity determining regions or hypervariable regions.
  • variable regions of such antibodies can be humanized and combined with human antibody constant regions into fully human antibody molecules.
  • the CD73 enzyme activity experiment, endocytosis experiment and T cell proliferation experiment prove that the obtained CD73 antibody has excellent biological activity: compared with MEDI9447, it can obviously inhibit CD73 enzyme activity and promote the endocytosis of CD73, compared with BMS-986179. Effectively restore the proliferation of T cells mediated by AMP.
  • the present invention also provides the use of the anti-CD73 monoclonal antibody, including improving the tumor microenvironment, activating tumor-specific immune response, inhibiting tumor growth, and applying it alone or in combination with other anti-tumor drugs for tumor immunotherapy.
  • the present invention also provides the use of the anti-CD73 monoclonal antibody in combination with a variety of immune checkpoint antibodies or chemotherapeutic agents to effectively inhibit tumor growth, thereby preparing a medicament for treating diseases related to abnormal expression of CD73 or dysfunction. On this basis, the present invention has been completed.
  • VH-CDR1 and “CDR-H1” are used interchangeably, both refer to the CDR1 of the heavy chain variable region;
  • VH-CDR2 and “CDR-H2” are used interchangeably, both refer to the heavy chain CDR2 of the variable region;
  • VH-CDR3 and “CDR-H3” are used interchangeably, and both refer to the CDR3 of the variable region of the heavy chain.
  • VL-CDR1 and CDR-L1 can be used interchangeably, both refer to CDR1 of the light chain variable region;
  • VL-CDR2 and CDR-L2 can be used interchangeably, both refer to the light chain variable region CDR2;
  • VL-CDR3 and CDR-L3 are used interchangeably and both refer to CDR3 of the light chain variable region.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain parts of the variable region of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669) (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
  • immunoglobulins can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of its constant region. According to the amino acid sequence of the constant region of its heavy chain, immunoglobulins can be divided into different types. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which divide this segment into 4 framework regions (FR)
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention includes not only whole antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies.
  • a chimeric antibody is a molecule in which different parts come from different animal species, such as a variable region with a monoclonal antibody from a mouse, and a chimeric antibody from a human immunoglobulin constant region (see, for example, U.S. Patent No. 4,816,567 and US Patent 4,816,397, hereby incorporated by reference in its entirety).
  • Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see US Patent 5,585,089, It is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • CDRs complementarity determining regions
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes conservative variants, which means that there are at most 10, preferably at most 8, more preferably at most 5, compared with the amino acid sequence of the antibody of the present invention, most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservatively variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the antibody is an anti-CD73 antibody.
  • the present invention provides a high specificity and high affinity antibody against CD73, which includes a heavy chain and a light chain, the heavy chain contains a heavy chain variable region (VH) amino acid sequence, and the light chain contains a light chain variable Region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable Region
  • the heavy chain variable region (VH) has a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
  • the light chain variable region (VL) has a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the heavy chain variable region (VH) includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • the light chain variable region (VL) includes the following three complementarity determining regions CDR:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2 or 3; preferably n is 0 or 1;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the antibodies described herein are full-length antibody antibodies, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single chain antibodies (single chain antibodies, scFv), single domain antibodies (single domain antibodies) , SdAb) and single-region antibodies (Signle-domain antibodies) one or more, and monoclonal antibodies or polyclonal antibodies prepared by the above antibodies.
  • the monoclonal antibody can be developed by various routes and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the antibody (anti-CD73 antibody) in the present invention may be a full-length protein (such as IgG1, IgG2a, IgG2b or IgG2c), or a protein fragment containing an antigen-antibody binding domain (such as Fab, F(ab'), sdAb, ScFv fragment).
  • a full-length protein such as IgG1, IgG2a, IgG2b or IgG2c
  • a protein fragment containing an antigen-antibody binding domain such as Fab, F(ab'), sdAb, ScFv fragment.
  • the antibody (anti-CD73 antibody) in the present invention may be a wild-type protein or a mutant protein that has achieved a certain effect through a specific mutation, for example, the mutation is used to eliminate the effector function of the antibody.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, more preferably fully human Source antibody.
  • the antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab') 2 or other known antibody derivatives in the art, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or other subtypes of any one or several.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the animal is preferably a mammal, such as a rat.
  • the antibody of the present invention may be a chimeric antibody, humanized antibody, CDR grafted and/or modified antibody targeting CD73 (eg, human CD73).
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the original amino acid sequence, more preferably not more than 35%, more preferably 1-33% , More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of added, deleted, modified and/or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably For 1-2.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101.
  • the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, or 103.
  • amino acid sequence of the heavy chain variable region (VH) and/or the light chain variable region (VL) of the antibody targeting CD73 is shown in Table 2 below:
  • the antibody targeting CD73 is Hu030-2, 42A5A7, 56F12H8, 66H6C12, 24D6B4, 60G1C8, 69C9E12, 71E10B3, 77B9A3, 80H7D6, or 125A4E10.
  • the antibodies targeting CD73 are Hu030-2, 42A5A7, 56F12H8, 66H6C12.
  • the antibody targeting CD73 is Hu030-2, or 42A5A7.
  • the present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of the CD73 antibody, and/or Or, one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the CD73 antibody,
  • the sequence of the heavy chain CDR1-3 is as follows:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4,
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • the sequence of the light chain CDR1-3 is as follows:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; preferably n is 0 or 1;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the recombinant protein of the present invention includes the heavy chain variable region of the CD73 antibody and/or the light chain variable region of the CD73 antibody.
  • the heavy chain variable region of the antibody contains SEQ ID NO.1 , 11, 21, 31, 41, 51, 61, 71, 81, 91, or 101; the light chain variable region of the antibody contains SEQ ID NO. 6, 16, 26, 36, 46 , 56, 66, 76, 86, 96, or 103.
  • the recombinant protein of the present invention includes the heavy chain variable region of the CD73 antibody and the light chain variable region of the CD73 antibody.
  • the heavy chain variable region of the antibody contains SEQ ID NO.1, 11 , 21, 31, 41, 51, 61, 71, 81, 91, or 101
  • the light chain variable region of the antibody contains SEQ ID NO. 6, 16, 26, 36, 46, The amino acid sequence shown in 56, 66, 76, 86, 96, or 103.
  • sequence numbers of the amino acid sequences of the recombinant protein and the heavy chain CDR1-3 and light chain CDR1-3 included in the recombinant protein are shown in Table 3:
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and/or substituted for at least one amino acid, and capable of retaining the binding affinity of CD73.
  • the recombinant protein further includes an antibody heavy chain constant region and/or an antibody light chain constant region.
  • the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region Or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a rat-derived light chain antibody constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.
  • the recombinant protein is a conventional protein in the art, preferably, it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody, fragment, scFv) ), single domain antibody (single domain antibody, sdAb) and single domain antibody (Signle-domain antibody) one or more, and the monoclonal antibody or polyclonal antibody prepared by the above antibody.
  • the monoclonal antibody can be developed in various ways and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which include light chain variable regions, light chain constant regions, and Fd segments of heavy chain constant regions.
  • the protein fragments of the antigen-antibody binding domain are Fab and F(ab').
  • the single domain antibody is a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
  • the single-region antibody is a conventional single-region antibody in the art, which includes only the heavy chain variable region.
  • the preparation method of the recombinant protein is a conventional preparation method in the art.
  • the preparation method is preferably isolated from expression transformants that recombinantly express the protein or obtained by artificially synthesizing protein sequences.
  • the method for separating and obtaining the recombinant transformant that expresses the protein is preferably obtained by cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain recombinant expression
  • the transformant can be isolated and purified by culturing the obtained recombinant expression transformant to obtain the recombinant protein.
  • the present invention also provides a nucleic acid encoding the heavy chain variable region or light chain variable region of the aforementioned antibody (eg, anti-CD73 antibody) or recombinant protein or anti-CD73 antibody.
  • a nucleic acid encoding the heavy chain variable region or light chain variable region of the aforementioned antibody (eg, anti-CD73 antibody) or recombinant protein or anti-CD73 antibody.
  • the preparation method of the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining the nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above protein by artificial full sequence synthesis .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately substituted, deleted, changed, inserted or added to provide a polynucleotide homologue.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting, or adding one or more bases of the gene encoding the protein sequence within the range of maintaining antibody activity.
  • the invention also provides a recombinant expression vector containing the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, it is constructed by connecting the nucleic acid molecule of the present invention to various expression vectors.
  • the expression vector is a variety of conventional vectors in the art, as long as it can contain the aforementioned nucleic acid molecule.
  • the vector preferably includes various plasmids, cosmids, bacteriophages or viral vectors.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: it is prepared by transforming the above-mentioned recombinant expression vector into a host cell.
  • the host cell is a variety of conventional host cells in the art, as long as it satisfies the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed.
  • the host cell is E. coli TG1 or E. coli BL21 cells (expressing single chain antibody or Fab antibody), or HEK293 or CHO cells (expressing full-length IgG antibody).
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electric conversion method.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the DNA sequence encoding the antibody (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, etc.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to the personnel.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding tests (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).
  • the binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed in a cell, on a cell membrane, or secreted out of a cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with protein precipitation agent (salting out method)
  • centrifugation osmotic disruption
  • ultrasonic treatment ultracentrifugation
  • molecular sieve chromatography gel filtration
  • adsorption layer Analysis ion exchange chromatography
  • ADC Antibody-drug conjugate
  • the invention also provides an antibody-drug conjugate (ADC) based on the antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-coupled drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a drug having therapeutic activity.
  • the effector molecule may be one or more of toxic protein, chemotherapeutic drug, small molecule drug or radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that makes the effector molecule and antibody form a covalent bond, such as glutaraldehyde and the like.
  • the coupling agent using a carboxyl group may be any one or several of cis-aconitic anhydride-based coupling agents (such as cis-aconitic anhydride) and acylhydrazone-based coupling agents (coupling sites are acylhydrazones).
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent can be directly or indirectly linked to the antibody via a linker.
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and antibody.
  • the joint may be degradable or non-degradable.
  • the degradable linker is typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degraded linkers, including peptide group-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that can degrade under reducing conditions (such as disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
  • the linker Before connecting to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Mercapto-specific reactive reactive groups are preferred and include: for example maleimide compounds, halogenated amides (eg iodine, bromine or chlorinated); halogenated esters (eg iodine, bromine or chlorinated) ); halogenated methyl ketone (such as iodine, bromine or chloro), benzyl halide (such as iodine, bromine or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker may include, for example, maleimide attached to the antibody through
  • the drug may be any drug that is cytotoxic, inhibits cell growth, or immunosuppresses.
  • the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker.
  • the drug may have an amino group, a carboxyl group, a mercapto group, a hydroxyl group, or a ketone group that can bond with the linker.
  • the drug is directly connected to the linker, the drug has a reactive active group before being connected to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors
  • typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes
  • benzodiazepines or benzodiazepine containing drugs such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines) and vinca alkaloids (vinca alkaloids).
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • vinca alkaloids vinca alkaloids
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two-step or multi-step method.
  • the cysteine residue reacts with the reactive part of the linker in the first step, and in the subsequent step, the functional group on the linker reacts with the drug, thereby forming an ADC.
  • the functional group on the linker is selected to facilitate specific reaction with the appropriate reactive group on the drug moiety.
  • the azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety.
  • the drug is covalently bonded to the linker through 1,3-dipolar cycloaddition between the azide and alkynyl groups.
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • React with alcohols and activated esters, such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • activated esters such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody and the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the present invention include: binding the antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the method of the invention further comprises: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • the present invention also provides the use of the antibody, antibody conjugate ADC, recombinant protein, and/or immune cell of the present invention, for example, for preparing a diagnostic preparation or preparing a medicine.
  • the medicine is a medicine for preventing and/or treating diseases related to abnormal expression or function of CD73.
  • the diseases related to abnormal expression or function of CD73 are conventional diseases related to abnormal expression or function of CD73 in the art.
  • the disease related to abnormal expression or function of CD73 is tumor/cancer.
  • the cancer is conventional cancer in the art, preferably bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck cancer, Prostate cancer, pancreatic cancer.
  • antibodies, ADCs, recombinant proteins, and/or immune cells of the present invention include (but are not limited to):
  • the tumors include (but are not limited to): bladder cancer, blood cancer, glioma, malignant glioma, melanoma, ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck cancer, prostate cancer, pancreatic cancer.
  • the antibody of the present invention or its ADC can be used in a detection application, for example, to detect a sample, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention shall include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or an puncture or needle biopsy of an organ.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the antibody of the present invention may be fixed to the detection plate.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, which may be an aqueous solution, a non-aqueous solution or a suspension, more preferably tablets, capsules, granules , Injections or infusions.
  • the antibody of the present invention may also be used for cell therapy by expressing a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating diseases related to abnormal expression or function of CD73.
  • the pharmaceutical composition of the present invention can be directly used to bind CD73 protein molecules, and thus can be used to prevent and treat diseases such as tumors.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the aforementioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutical Acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptide of the present invention can be used together with other therapeutic agents.
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier may be any suitable physiological or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the above protein and 0.01-99.99% of the pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
  • the administration amount of the pharmaceutical composition is an effective amount
  • the effective amount is an amount capable of relieving or delaying the progression of a disease, degenerative or traumatic condition.
  • the effective amount can be determined on an individual basis and will be based in part on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors such as individual basis and using no more than routine experimentation.
  • a safe and effective amount of an immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
  • the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of diseases related to abnormal expression or function of CD73.
  • the disease related to abnormal expression or function of CD73 is tumor/cancer.
  • the present invention also provides a method for detecting CD73 protein in a sample (for example, detecting overexpressing CD73 cells), which includes the steps of contacting the above-mentioned antibody with the sample to be tested in vitro and detecting whether the above-mentioned antibody binds to the sample to be tested It is sufficient to form an antigen-antibody complex.
  • overexpression is conventional in the art, and refers to the overexpression of RNA or protein of the CD73 protein in the sample to be detected (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation changes), and due to protein Changes in delivery mode (increased nuclear localization) result in local overexpression and increased functional activity (eg in the case of increased enzymatic hydrolysis of the substrate).
  • the detection method of whether the above forms an antigen-antibody complex is a conventional detection method in the art, preferably a flow cytometry (FACS) test.
  • FACS flow cytometry
  • the invention provides a composition for detecting CD73 protein in a sample, which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • a composition for detecting CD73 protein in a sample which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • it also includes a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.
  • the antibody obtained by the present invention can have both excellent ability to mediate CD73 endocytosis and restore T cell proliferation.
  • the room temperature described in the examples is conventional room temperature in the art, and is generally 10-30°C.
  • the PBS described in the examples is PBS phosphate buffer, pH 7.2.
  • the present invention uses an advanced antibody transgenic mouse technology platform to prepare fully human sequence monoclonal antibodies.
  • the anti-CD73 antibody obtained by the present invention can be prepared by various routes and methods, including:
  • Monoclonal antibodies prepared from conventional animals, such as mice, can clone the antibody heavy chain variable region and light chain variable region genes by conventional molecular biology methods, and the variable region genes can be grafted to human antibody constant region genes to form humans Mouse chimeric antibody (US Pat. No. 4,816,567, Cabilly et al), to greatly reduce the immunogenicity of the human body when used. Furthermore, the CDR domain of the variable region of the murine antibody can be grafted onto the structure of the human antibody, thereby reducing the composition of the murine antibody to less than 5%, greatly increasing the safety of the antibody in the human body.
  • the antibody obtained in this way is called humanized antibody, and is currently the main product in the antibody drug market (US Pat. No. 5,225,539 to 55, Winter, and US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen) .
  • a series of human and mouse chimeric monoclonal antibodies are prepared in the present invention.
  • These anti-human CD73 antibodies are prepared by immunizing Balb/c and SJL mice, optimized hybridoma technology preparation, molecular biology and antibody engineering technology, with mouse antibody heavy chain and light chain variable regions and human antibodies Constant area.
  • the amino acid sequence of the CD73 protein in the extracellular region was added with a His6 tag at the C-terminal of 27-547, and then cloned into the pTT5 vector to obtain pTT5-hCD73ECD-His6. It was transiently transfected into CHO cells. After 9 days, the cell culture fluid was collected, centrifuged to remove cell components, and the supernatant medium was filtered with a 0.22 micron filter head. Then, the culture supernatant containing the CD73 protein was loaded onto a nickel affinity chromatography column, and the change of the ultraviolet absorption value (A280nm) was monitored with an ultraviolet (UV) detector.
  • UV ultraviolet
  • mice 6-8 week old Balb/c and SJL mice (provided by SLAC) were used. The mice were reared under SPF conditions after receiving.
  • the first immunization of PD-1 protein was emulsified with Freund's complete adjuvant, and intraperitoneal injection of 0.25 ml, 100 ⁇ g protein per mouse.
  • the booster immune PD-1 protein was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml of 50 ⁇ g protein per mouse.
  • the interval between the first immunization and the first booster immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks.
  • Blood was collected 7 days after each booster immunization, and the antibody titer and specificity in the serum were detected by ELISA and FACS.
  • Immunogen 2 the full-length amino acid sequence of human CD73 was cloned into pLVX-IRES-puro vector. After plasmid transfection of HEK293 cell line, lentivirus infection of CHOK1 and BW5147 cell lines and selective cultivation in puromycin-containing medium for 2 weeks, subcloning in 96-well culture plate by limiting dilution method, about 2 weeks later Select a part of the monoclonal wells and expand into 6-well plates. The amplified clones were screened by flow cytometry with anti-CD73 specific antibody. Choose cell lines with better growth, higher fluorescence intensity, and monoclonal cells to continue to expand the culture and cryopreservation with liquid nitrogen.
  • CD73 cells were immunized with 6-8 week old female Balb/c and SJL/J (provided by Shanghai Slake Breeding). After receiving, the mice were reared under SPF conditions.
  • Human CD73-transfected HEK293/Renca stable cell line was expanded to 75-90% confluence in T-75 cell culture flasks, drained the medium, washed with DMEM/1640 basal medium 1-2 times, and then pancreas Enzyme treatment and collection of cells.
  • HEK293 cells were washed 1-2 times with DMEM basal medium, after cell counting, the cells were diluted with PBS to 1x10 7 cells per ml; Renca cells were treated with mitomycin for 4 hours, washed with PBS 2-3 times for cells After counting, the cells were diluted with PBS to 1 ⁇ 10 7 cells per ml.
  • Each mouse was injected intraperitoneally with 0.5 ml of cell suspension at each immunization. The interval between the first and second immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks. Blood was collected 7 days after each booster immunization, and antibody titer and specificity in serum were detected by FACS.
  • Gene gun immunization The plasmid was coated onto 1.0 ⁇ M gold colloidal bullets and immunized with Helios gene gun (Bio-rad). The detailed method was formulated according to the Helios gene gun instruction manual. 6-8 weeks old female Balb/c and SJL/J (provided by Shanghai Slake Breeding) were reared under SPF conditions. All mice were immunized 3-4 times with a gene gun through the abdomen, with 4 shots each time and 1.0 ⁇ g cDNA amount per shot. The interval between the initial immunization and the first booster immunization, and booster immunization were 2 weeks. Blood was collected 7 days after each booster immunization, and the antibody titer in serum was detected by ELISA or FACS. Usually the FACS titer of most mice can reach more than 1:1000 after 2-3 immunizations.
  • In vivo electroporation immunization 6-8 week old female Balb/c and SJL/J (provided by Shanghai Slake Breeding) were raised under SPF conditions after receiving. All mice received intradermal injections of CD73 full-length amino acid sequence cDNA 3-4 times on both sides of the base of the prepared tail, each injection of 50 ⁇ g/20 ⁇ l on each side. Immediately afterwards, the injection site was electroporated using AgileP ⁇ lse system. For the detailed method, refer to the instructions of AgileP ⁇ lse (BTX Harvard apparatus). The interval between the initial immunization and the first booster immunization, and booster immunization were 2 weeks. Blood was collected 7 days after each booster immunization, and the antibody titer in serum was detected by ELISA or FACS. Usually the FACS titer of most mice can reach more than 1:1000 after 3-4 immunizations.
  • mice with titers that meet the requirements can choose to perform cell fusion and hybridoma preparation.
  • the last immunization of protein-immunized and gene-immunized mice was injected intraperitoneally with 50 ⁇ g of purified hCD73ECD-His each, and the last immunization of cell-immunized mice was intraperitoneally injected with 0.5-1x10 7 cells each. After 3-5 days, the mice were sacrificed and spleen cells or lymphocytes were collected.
  • the desired clones select the desired clones and perform subcloning in 96-well plates by limiting dilution method. 7-10 days after subcloning, preliminary screening was performed with Acumen, and 3-4 positive monoclonals were selected and expanded into 24-well plates to continue cultivation. After 2-3 days, the antigen binding was positive by FACS and the biological activity was evaluated by CD73 enzyme activity test. Based on the test results of the 24-well plate samples, an optimal clone was selected for expansion culture, liquid nitrogen freezing, antibody production and purification.
  • the hybridoma cells were expanded into T-75 cell culture flasks and passaged for 2-3 passages with production medium (Hybridoma seruM free maximM, Invitrogen). After the hybridoma cells grow well, inoculate the cell culture spinner flask. Add 200-500 ml of production medium to each 2-liter culture spinner flask and inoculate the cell with a density of 0.5-1.0x10 5 /ml. Close the cap tightly and place the spinner bottle on the spinner in a 37°C incubator, and adjust the speed to 3 rpm.
  • the cell culture fluid was collected, the cells were removed by centrifugation or filtration, and clarified by filtration with a 0.22-0.45 micron filter membrane.
  • the treated cell culture supernatant can be purified immediately or frozen at -30°C.
  • the monoclonal antibody in the supernatant of hybridoma cell culture can be purified by Protein A (Protein A) affinity chromatography column. According to the size of the sample size, prepare a corresponding volume of chromatography column. For small volumes of 200-300ml purification, 1-2ml protein A column is required. The protein A column was first equilibrated with equilibration buffer (Tris-HCl, pH7.4), and then the culture supernatant was added to the chromatography column, and the flow rate was controlled at 3-4 ml/min. After loading the sample, wash the chromatography column with 3-5 column bed volume using equilibrium buffer.
  • equilibration buffer Tris-HCl, pH7.4
  • IgG1 was eluted with eluent (0.1M sodium citrate buffer, pH 4.5); other subclasses of IgG were eluted with eluent (0.1M sodium citrate buffer, pH 3.5).
  • the antibody bound to the column is monitored by an ultraviolet detector for elution. Collect the eluted antibody (ultraviolet absorption peak), add 10% volume of 1.0M Tris-HCl buffer to neutralize the pH, then immediately dialyze with PBS overnight, change the fluid once the next day and continue dialysis for 2-3 hours.
  • the antibody after dialysis was collected, sterile filtered with a 0.22 micron filter, and stored aseptically. Separate samples for detection and analysis of protein concentration, purity and endotoxin.
  • Flow cytometry detects the binding of antibodies to CD73 expressing cells of human, cynomolgus monkey, and mouse.
  • the CD73-transfected CHOK1 stable cell line was expanded to 75-90% confluence in T-75 cell culture flasks, the medium was drained, washed 1-2 times with PBS, and then trypsin (Tryple express: Life technology) Handle and collect cells. Wash the cells 1-2 times with PBS buffer, after cell counting, dilute the cells with PBS to 1-2x106 cells per ml, add 2% fetal bovine serum (FBS) blocking solution, incubate on ice for 20-30 minutes, then use The HBSS was centrifugally washed twice.
  • FBS fetal bovine serum
  • FACS buffer PBS + 2% FBS
  • FACS buffer PBS + 2% FBS
  • the antibody sample to be tested 100 ⁇ l per well, 4 Incubate for 1-2 hours.
  • Wash twice by centrifugation with FACS buffer add 100 ⁇ l of fluorescent (Alexa 488) labeled secondary antibody per well, and incubate at 4 degrees for 0.5-1.0 hours.
  • the cells were centrifugally washed with FACS buffer for 2-3 times, 100 ⁇ l of fixative solution (4% Paraformaldehyde) was added to suspend cells, and after 5-10 minutes, they were washed with FACS buffer for 1-2 times.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACSCalibur, BD).
  • CD73 enzyme activity experiment After digesting the CHOK1-hCD73 cells, they were diluted with TM buffer (25mM Tris, 5mM MgCl 2 , pH 7.5) to 2 ⁇ 10 4 cells per ml, and added to a 96-well reaction plate (Corning Cat#3799) at 100 ml per well. Centrifuge and remove supernatant. At the same time, the antibody to be tested was prepared as a 4 ⁇ solution with TM buffer, and the cells in the 96-well plate were resuspended in 50 ⁇ l per well, and incubated at 37 degrees for 30 minutes.
  • TM buffer 25mM Tris, 5mM MgCl 2 , pH 7.5
  • the AMP was prepared into a 4 ⁇ solution (800 ⁇ M) with TM buffer, added to a 96-well plate at 50 ⁇ l per well, mixed well, and incubated at 37 degrees for 30 minutes. Centrifuge the 96-well plate at 300 ⁇ g, remove 50 ⁇ l of the supernatant (cannot attract cells), and transfer to a 96-well detection plate (Corning Cat#3903). Add 50 ⁇ l of 2 ⁇ ATP solution (130 ⁇ M) per well and 100 ⁇ l of CellTiter Glo reaction solution per well, mix well. After 10 minutes in the dark, read the fluorescence value on the microplate reader.
  • CD4+ T cell isolation kit was used to isolate CD4 positive T cells from human peripheral blood cells (PBMC). Resuspend in PBS + 1% BSA to 2 ⁇ 10 6 cells per ml, add the same volume of 2 ⁇ CFSE solution (4 ⁇ M), mix and place at 37 degrees for 10 minutes. Add 40% volume of FBS, mix and place at 37 degrees for 10 minutes. Wash twice with a large volume of PBS solution. Resuspend with T-cell culture solution containing anti-CD2/CD3/CD28 magnetic beads (Miltenyi Biotec, 130-091-441) to 1.5 ⁇ 10 6 cells per ml, and add 100 ml per well to 96-well plates.
  • PBMC peripheral blood cells
  • RNA isolation After the supernatant of the subclonal culture was tested for antigen binding, 1-5 ⁇ 10 7 hybridoma cells were collected by centrifugation. Add 1mL Trizol to mix and transfer to a 1.5ml centrifuge tube, let stand at room temperature for 5min; add 0.2ml chloroform, shake for 15s, let stand for 2min, then centrifuge at 4°C, 12000g ⁇ 5min, take the supernatant and transfer to a new 1.5ml centrifuge In the tube; add 0.5ml of isopropanol, gently mix the liquid in the tube, let stand at room temperature for 10min, then centrifuge at 4°C, 12000g ⁇ 15min, discard the supernatant; add 1ml of 75% ethanol, and gently wash the precipitate. 4°C, 12000g ⁇ 5min, discard the supernatant and dry it, add an appropriate amount of DEPC H 2 O to dissolve (55°C water bath for
  • Reverse transcription and PCR Take 1 ⁇ g of tRNA, configure 20 ⁇ l system, add reverse transcriptase and react at 42°C for 60min, then at 70°C for 10min to terminate the reaction.
  • Configure 50 ⁇ l PCR system including 1 ⁇ l cDNA, 25pmol of each primer, 1 ⁇ l DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs; set PCR program, pre-denaturation 95°C3min, denaturation 95°C30s, annealing 55°C30s, extension 72°C 35s, after 35 cycles, it was extended at 72°C for 5min.
  • the extension temperature can be adjusted according to the actual situation.
  • Cloning and sequencing take 5 ⁇ l PCR products for agarose gel electrophoresis detection, and use the column recovery kit to purify positive test samples; perform ligation reaction: sample 50ng, T vector 50ng, ligase 0.5 ⁇ l, buffer 1 ⁇ l, reaction system 10 ⁇ l , Reaction at 16°C for half an hour; take 5 ⁇ l of ligation product into 100 ⁇ l of competent cells, ice bath for 5min, then heat shock at 42°C water bath for 1min, put it back on ice for 1min, then add 650 ⁇ l of antibiotic-free SOC medium at 37°C Resuscitate at 200 RPM for 30 min on a shaker, take out 200 ⁇ l coated on LB solid medium containing antibiotics and incubate in a 37°C incubator overnight; the next day, use primers M13F and M13R on the T carrier to configure 30 ⁇ l PCR system for colony PCR.
  • mice Different immunization strategies (protein immunization, cell immunization, gene immunization) are used to immunize mice. Fusion and screening were performed from mice, and these hybridoma cell supernatants were used for clonal screening. The clones of particular interest were isolated and purified to produce murine antibodies including 24D6, 37F8, 42A5, 56F12, 57G8, 60G1, 66H6, 69C9, 47F12, 71E10, 77B9, 78E6, 80H7 and 125A4.
  • CHOK1-hCD73 transfected with human CD73
  • CHOK1-cCD73 transfected with cynomolgus CD73
  • CHOK1-mCD73 transfected with murine CD73
  • CHOK1 negative for human CD73, cynomolgus CD73, murine CD73
  • Cells hybridoma cells expressed and purified CD73 antibody as the primary antibody
  • Alexa 488 Donkey anti-mouse IgG (H+L) Alexa 488 Donkey anti-mouse IgG (H+L) (Invitrogen, A21202) was used as the secondary antibody, and the following method was used to generate the titration binding curve:
  • the CD73-transfected CHOK1 stable cell line was expanded to 75-90% confluence in T-75 cell culture flasks, the medium was drained, washed 1-2 times with PBS, and then trypsin (Tryple express: Life technology) Handle and collect cells. Wash the cells 1-2 times with PBS buffer, after cell counting, dilute the cells with PBS to 1-2x10 6 cells per ml, add 2% fetal bovine serum (FBS) blocking solution, and incubate on ice for 20-30 minutes, then Wash twice with HBSS centrifugation.
  • FBS fetal bovine serum
  • the collected cells were suspended in FACS buffer (PBS+2% FBS) to 2 ⁇ 10 6 cells/ml, added to 96-well FACS reaction plate at 100 ⁇ l per well, and centrifuged at 300 g for 5 minutes to discard the supernatant.
  • the blocking solution of anti-CD73 antibody was set to an initial concentration of 10ug/ml and serially diluted at 8 points.
  • the cells were centrifugally washed with FACS buffer 2-3 times, 100 ⁇ l of fixing solution (4% Paraformaldehyde) was added to suspend cells, and after 5-10 minutes, the cells were centrifuged with FACS buffer 1-2 times. The cells were suspended with 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACSCalibur, BD), see Figure 1 and Table 4.
  • TM buffer 25mM Tris, 5mM MgCl 2 , pH 7.5
  • TM buffer 25mM Tris, 5mM MgCl 2 , pH 7.5
  • the antibody to be tested was prepared as a 4 ⁇ solution with TM buffer, serially diluted at 6 points, and the cells in the 96-well plate were resuspended with 50 ⁇ l per well, and incubated at 37 degrees for 30 minutes.
  • the AMP was prepared into a 4 ⁇ solution (800 ⁇ M) with TM buffer, added to a 96-well plate at 50 ⁇ l per well, mixed well, and incubated at 37 degrees for 30 minutes. Centrifuge a 96-well plate at 300 ⁇ g, remove 50 ⁇ l of supernatant, and transfer to a 96-well detection plate (Corning Cat#3903). Add 50 ⁇ l of 2 ⁇ ATP solution (130 ⁇ M) per well and 100 ⁇ l of CellTiter Glo reaction solution per well, mix well. After 10 minutes in the dark, read the fluorescence value on the microplate reader. The results are shown in Figure 2 and Table 5.
  • Figure 2 shows that mAb020, 024, 030, 032, 033, 034, 036, 038, 039, 041, 042, 043, 044, 065 antibodies can inhibit the enzymatic activity of human CD73 on the cell surface.
  • the maximum inhibition percentage and IC50 of each antibody are shown in Table 5.
  • the CD73 antibody expressed and purified by hybridoma cells was used to incubate CHOK1-hCD73 cells at 37°C, and antibody-mediated CD73 endocytosis was detected by FACS.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACSCalibur, BD).
  • FACS Fluorescence-Activated Cell Sorting
  • Figure 3 shows the time curves of mAb020, 024, 030, 032, 033, 034, 036, 038, 039, 041, 042, 043, 044, 065 antibody-mediated CD73 endocytosis.
  • the results show that most antibodies can effectively and significantly mediate CD73 endocytosis, such as mab020, 030, 033, 034, 042.
  • AMP undergoes CD73 dephosphorylation to form adenosine, which is bound by adenosine receptors on T cells to inhibit the proliferation of effector T cells.
  • the anti-CD73 antibody blocks the role of CD73, inhibits the formation of adenosine, and restores T cell proliferation.
  • CD4+ T cell isolation kit was used to isolate CD4 positive T cells from human peripheral blood cells (PBMC). Resuspend in PBS + 1% BSA to 2 ⁇ 10 6 cells per ml, add the same volume of 2 ⁇ CFSE solution (4 ⁇ M), mix and place at 37 degrees for 10 minutes. Add 40% volume of FBS, mix and place at 37 degrees for 10 minutes. Wash twice by centrifugation with PBS solution. Resuspend to 1.5 ⁇ 10 6 cells/ml with T cell culture solution (RMPI 1640+10%FBS+1%P/S) containing anti-CD2/CD3/CD28 magnetic beads (Miltenyi Biotec, 130-091-441) , Add 100ml per well to a 96-well plate.
  • T cell culture solution RMPI 1640+10%FBS+1%P/S
  • RNA isolation After the supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (that is, after verification and activity measurement of Examples 2 to 5), 5 ⁇ 10 7 hybridoma cells were collected by centrifugation and added 1mL Trizol was mixed and transferred to a 1.5mL centrifuge tube and allowed to stand at room temperature for 5 minutes. Add 0.2mL of chloroform and shake for 15 seconds. After standing for 2 minutes, centrifuge at 12,000g for 5 minutes at 4°C. Take the supernatant and transfer to a new 1.5mL centrifuge tube.
  • Reverse transcription and PCR Take 1 ⁇ g of total RNA and configure 20 ⁇ l system. After adding reverse transcriptase, react at 42°C for 60 minutes and at 7°C for 10 minutes to terminate the reaction. Configure 50 ⁇ l PCR system, including 1 ⁇ l cDNA, 25pmol of each primer, 1 ⁇ l DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs. The PCR program was set, pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 35 seconds, and extension at 72°C for 5 minutes after 35 cycles to obtain PCR products.
  • the kit used for reverse transcription is PrimeScript RT Master, purchased from Takara, catalog number RR036; the kit used for PCR is Q5 ultra-fidelity enzyme, purchased from NEB, catalog number M0492.
  • Cloning and sequencing take 5 ⁇ l PCR products for agarose gel electrophoresis detection, and use the column recovery kit to purify the positive test samples, of which the recovery kit is Gel & PCR Clean-up, purchased from MACHEREY-NAGEL, Catalog No. 740609.
  • Perform ligation reaction sample 50 ng, T vector 50 ng, ligase 0.5 ⁇ l, buffer 1 ⁇ l, reaction system 10 ⁇ l, ligation product at 16°C for half an hour.
  • the ligation kit is T4 DNA ligase, purchased from NEB, article number M0402; Take 5 ⁇ l of the ligation product and add it to 100 ⁇ l of competent cells (Ecos 101 competent cells, purchased from Yeastern, Catalog No.
  • Example 4 Preparation of murine human chimeric antibody.
  • Example 1 has obtained purified CD73 antibody from the culture supernatant of hybridoma cells, and the heavy chain variable region and light chain of CD73 antibody have been clarified according to the sequencing results of Example 6 Variable region sequence.
  • the heavy chain variable region sequence of the CD73 antibody was recombined into an expression vector containing a signal peptide and a human heavy chain antibody IgG1-TM constant region (IgG1 contains mutations at three positions of L234F, L235E and P331S to reduce the effects of ADCC and CDC) (Among them, the IgG1 expression vector was purchased from Invitrogen.
  • the point mutation modification and recombination steps are routine steps.
  • the light chain variable region sequence of the CD73 antibody is recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region.
  • Recombinant plasmid and verified by sequencing are the same as the sequencing method in Example 6).
  • Use alkaline lysis method kit (purchased from MACHEREY-NAGEL) to weigh the recombinant plasmid with increased purity, the quality is more than 500 ⁇ g, and filtered through 0.22 ⁇ m
  • the membrane purchased from Millopore was filtered for transfection.
  • 293E cells purchased from Invitrogen
  • Freestyle 293 expression medium purchased from Invitrogen.
  • the shaker was set at 37°C, 130 RPM, and 8% CO 2 (v/v) concentration.
  • Freestyle 293 expression medium added 10% (v/v) F68 (purchased from Invitrogen) to a final F68 concentration of 0.1% (v/v) at the time of transfection.
  • the antibody titer of the culture broth was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes) and filtered through a 0.22 ⁇ m filter membrane to obtain the filtered cell supernatant for purification.
  • mAb020,024,030,032,033,034,036,038,039,041,042,043,044,065 chimeric antibodies can inhibit the enzyme activity of human CD73 on the cell surface.
  • the maximum inhibition percentage and EC50 of each antibody are shown in Table 7.
  • Figure 7 shows the time curves of mAb020,024,030,032,033,034,036,038,039,041,042,043,044,065 chimeric antibody mediated CD73 endocytosis.
  • Anti-CD73 chimeric antibody restores T cell proliferation.
  • mAb020,024,030,032,033,034,036,038,041,042,044,065 chimeric antibodies can restore CD4+ T cell proliferation.
  • Octet Red96 is used as the test instrument, and AHC biosensor is used as the test sensor.
  • the AHC sensor has been fixed with Anti-human IgG Fc antibody, which can be used to directly capture the 15 antibodies in this experiment, and then the sensor is immersed in the analysis Sample (antigen).
  • the regeneration buffer (Glycine PH1.5) and the neutralization buffer (1*PBS buffer) are alternately soaked for 5 seconds, and the sensor is regenerated five times in total. The maximum number of sensor regenerations is 10.
  • the running buffer in this experiment is a sample dilution (1*PBS buffer containing 0.02% Tween20 and 0.1% BSA), namely the Baseline step, Dissociation step, and the buffer used in the blank analyte sample.
  • Four sensors are used in this experiment.
  • Sample processing use sample diluent (1*PBS buffer containing 0.02% Tween20 and 0.1% BSA) to dilute all antibodies to a working concentration of 10ug/mL, and gradually dilute the analyte sample (antigen) to three jobs Concentration: 200nM, 100nM, 50nM.
  • the candidate antibody mAb030 heavy chain variable region and light chain variable region have no important hotspot.
  • the germline gene sequence with the highest homology to the candidate antibody mAb030 heavy chain variable region and light chain variable region was selected as the variable region graft skeleton by sequence alignment (NCBI-Igblast): IGHV4-38-2*02 and IGKV1 -39*01.
  • sequence alignment NCBI-Igblast
  • Amplification primers were synthesized by Genewiz, and then the variable regions of light and heavy chains were amplified by PCR method, respectively.
  • Configure 50 ⁇ L reaction system including 50-100ng of heavy chain variable region, light chain variable region, forward and reverse primers 1ul, 1ul pfxD enzyme (purchased from invitrogen, 12344-012), 10*pfx buffer 5ul ( The supplier has the same enzyme as pfx) and water to make up to 50 ⁇ L.
  • the PCR program was set, pre-denatured at 95°C for 5 minutes, denatured at 95°C for 30 seconds, annealed at 56°C for 30 seconds, extended at 68°C for 30 seconds, and extended at an additional 68°C for 10 minutes after 25 cycles to obtain PCR products. Take 5 ⁇ L of PCR product for agarose gel electrophoresis detection, and use the recovery kit to purify the positive test sample.
  • the recovery kit is PureLink Quick Extraction kit, purchased from Qiagen, Catalog No. 28706.
  • ligation reaction insert 20-40ng, digested expression vector 60-100ng, recombinant enzyme Exnase (purchased from Vazyme, Catalog No. C112-01/02) 1 ⁇ L, buffer 2 ⁇ L, reaction system 10 ⁇ L, at 37 The reaction product was obtained after half an hour at °C to obtain the ligation product, namely the constructed recombinant vector.
  • Exnase purchased from Vazyme, Catalog No. C112-01/02
  • the buffer is the buffer used to purchase the recombinase; the heavy chain variable region is directionally cloned into an expression vector containing a signal peptide and a human antibody heavy chain IgG4 (S228P) constant region (where the expression vector is purchased from Invitrogen, recombination step) Is a conventional step), the light chain variable region is directionally cloned into an expression vector containing a signal peptide and a human antibody light chain kappa constant region (where the expression vector is purchased from Invitrogen, and the recombination step is a conventional step).
  • Add 10 ⁇ L of the ligation product to 100 ⁇ L of competent cells (Ecos 101 Competent Cells, purchased from Yeastern, Catalog No.
  • the recombinant vectors with the correct sequence of heavy and light chains were amplified, and then FreeStyleTM293-F cells (purchased from Invitrogen) were transiently transfected to produce antibodies.
  • the density of 293-F cells should be 1-1.2 ⁇ 106 cells/mL.
  • 100 ⁇ g of the recombinant vector constructed above and 200 ⁇ g of transfection reagent polyethyleneimine (PEI) are required.
  • Recombinant vector and PEI were added to 5 mL medium, and allowed to stand at room temperature for 5 minutes. After filtering with a 0.22 ⁇ m filter, the mixture of recombinant vector and PEI was allowed to stand at room temperature for 15 minutes.
  • the above mixture was slowly added to the cells, and cultured at 130 rpm in a 37°C, 8% (v/v) CO 2 incubator.
  • the culture supernatant and cell pellet were taken every day to detect antibody expression.
  • the cell culture solution was centrifuged at 3000 g for 30 minutes, and the supernatant was collected and filtered with a 0.22 ⁇ m filter.
  • the monoclonal antibody in 200 mL of clear supernatant was purified with 1 mL of MabSelectTM SuReTM column (purchased from GE Healthcare).
  • MabSelectTMSuReTMcolumn is first equilibrated with equilibration buffer (PBS phosphate buffer, pH7.2), MabSelectTMSuReTMcolumn. After loading the sample, wash the MabSelectTMSuReTMcolumn with equilibration buffer. The volume of the equilibration buffer is 5 times the volume of the protein A column bed. The monoclonal antibody bound to MabSelectTMSuReTMcolumn was eluted with an eluent (0.1M glycine hydrochloric acid buffer, pH 3.0). Collect the eluted antibody and add 10% (v/v) 1.0M Tris-HCl buffer to neutralize the pH.
  • equilibration buffer PBS phosphate buffer, pH7.2
  • Example 2 The same method as in Example 2. The results are shown in Table 11, showing that the affinity of the humanized antibody is comparable to that of the chimeric antibody.
  • the amino acid sequence and nucleotide sequence of V030 of Hu030-2 are shown in SEQ ID No.: 101 and 102, and the amino acid sequence and nucleotide sequence of VL of Hu030-2 are shown in SEQ ID No.: 103 and 104.
  • the three CDRs of VH and three CDRs of Hu030-2 antibody are the same as those of antibody 030 (ie clone 42A5A7), which are SEQ ID No.: 3, 4 and 5, and SEQ ID No.: 8, 9 and 10.
  • MedImmune's antibody MEDI9447 is in clinical phase I/II, the antibody is obtained by phage display technology; BMS company's antibody BMS-986179 is in clinical phase I/II, the antibody is obtained by immunizing humanized mice.
  • Experimental data in animals show that MEDI9447 alone has no significant effect on inhibiting the growth of CT26 tumors, and the anti-PD1 antibody can greatly increase the anti-tumor effect; and BMS-986179 can not recognize the mouse-derived CD73 protein, so there is no reference in vivo data .
  • both antibodies can inhibit the enzyme activity of CD73 to a certain extent, mediate the endocytosis of CD73, and restore the proliferation of T cells mediated by AMP.
  • MEDI9447 has a weaker effect on endocytosis
  • BMS-986179 has a weaker ability to restore AMP-mediated T cell proliferation.
  • these two antibodies can inhibit the enzyme activity of CD73 to a certain extent, mediate the endocytosis of CD73, and restore the proliferation of T cells mediated by AMP.
  • MEDI9447 has a weaker effect on endocytosis
  • BMS-986179 has a weaker ability to restore AMP-mediated T cell proliferation.
  • the anti-CD73 antibody obtained by the present invention such as mab030, can not only exhibit excellent endocytosis effect, but also provide the possibility of reducing CD73 on the surface of the cell membrane; it can also strongly restore the proliferation of T cells mediated by AMP and become a complete In terms of outstanding performance of anti-CD73 antibodies.

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Abstract

提供了一种靶向CD73的抗体、其制备方法和用途。提供的单克隆抗体能够高特异性地结合CD73抗原，其具有很高的亲和力并且具有显著抗肿瘤等活性。

Description

CD73抗体及其制备方法和应用 技术领域
本发明属于生物医药领域，具体地涉及一种CD73抗体及其制备方法和应用。
背景技术
近几年，肿瘤免疫治疗已成为肿瘤治疗领域的焦点，其中针对免疫检查点的治疗性单克隆抗体已在一些肿瘤类型如黑色素瘤和非小细胞肺癌等的治疗中显示了抗肿瘤活性，靶向细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)和程序性死亡分子1及其配体(programmed cell death 1/programmed cell death ligand 1,PD-1/PD-L1)的免疫检查点抗体已获得美国FDA的批准。
然而，单一用药响应率低是现有肿瘤免疫疗法的主要问题。2000年，CTLA4抗体开展临床实验，表现为存在毒性(引起组织特异的炎症反应)以及响应率低下。最明显的临床效果发生在黑色毒瘤的治疗中，但客观响应率仅为15％。在PD-1和PD-L1的临床实验中，霍奇金淋巴瘤、默克尔细胞癌、结缔组织增生性黑素瘤的客观响应率最高，达到50-90％；黑色素瘤治疗的响应率为35-40％；非小细胞肺癌、头颈癌、膀胱癌、肾癌、肝细胞癌的响应率仅有15-25％。肿瘤是一种多通路多靶点的疾病，单一的治疗药物客观响应率低，很可能是由于肿瘤细胞在某条信号通路被药物抑制时，选择其他代偿通路满足生长。为了提高现有的治疗效果、降低产生毒性的抗体用量，肿瘤免疫联合疗法将成为重要的发展趋势。
肿瘤使用多种手段来逃避免疫消除，因此更好地理解肿瘤微环境的免疫抑制是非常必要的。肿瘤微环境中氧含量低、缺乏营养物质、pH值常为酸性。肿瘤细胞有多种调节机制以适应恶劣的生存环境，其中最重要的途径之一是通过上调CD73(外-5'-核苷酸酶)表达量进而改变嘌呤代谢。CD73是一个70KD大小的蛋白，以非共价键形成二聚体，其C末端通过糖基磷脂酰肌醇(GPI)锚定在细胞膜上。CD73将细胞外的单磷酸核苷酸(AMP)脱磷酸化，产生腺苷。胞外的腺苷与多种细胞表面特异的腺苷受体(A1，A2A，A2B和A3)结合，激活腺苷通路，在免疫抑制、血管生成中有重要作用。
研究表明CD73在多种肿瘤细胞表面高表达，包括膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌和乳腺癌等。CD73表达量上调与癌细胞增殖、转移、血管形成、和较短患者存活期相关。因此，CD73可作为一个新的药物作用靶点和生物标记，用于治疗癌症。
发明内容
为了克服目前缺少安全且特异性高的CD73抗体的不足，本发明提供一种亲和力高、特异性强的CD73抗体及其制备方法和应用。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区具有选自 下组的互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.10n+1所示的氨基酸序列，其中，n为0、1、2、3、4、5、6、7、8、或9。
在另一优选例中，所述重链可变区具有SEQ ID NO.1或101所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.11所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.21所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明的第一方面所述的重链可变区。
在另一优选例中，所述重链还包括重链恒定区。
在另一优选例中，所述重链恒定区为人源或鼠源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1恒定区。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1-TM恒定区。
在另一优选例中，所述IgG1-TM恒定区为IgG1，且含L234F,L235E和P331S三个位点突变。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区具有选自下组的互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.10n+6或SEQ ID NO.103所示的氨基酸序列，其中，n为0、1、2、3、4、5、6、7、8、或9。
在另一优选例中，所述轻链可变区具有SEQ ID NO.6或103所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.16所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.26所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明的第三 方面所述的轻链可变区。
在另一优选例中，所述轻链还包括轻链恒定区。
在另一优选例中，所述轻链恒定区为人源或鼠源的。
在另一优选例中，所述轻链恒定区为人源抗体轻链kappa恒定区。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2或3个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的VH和VL所构成的衍生抗体能够保留与CD73结合的亲和力。
在另一优选例中，所述的衍生抗体与CD73结合的亲和力F1与相应非衍生的抗体与CD73结合的亲和力F0之比(F1/F0)为0.5-2，较佳地为0.7-1.5，和更佳地0.8-1.2。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列为同源性或序列相同性为至少96％的氨基酸序列。
在另一优选例中，所述的抗体还包括重链恒定区和/或轻链恒定区。
在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1恒定区，且所述轻链恒定区为人源抗体轻链kappa恒定区。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1-TM恒定区，且所述轻链恒定区为人源抗体轻链kappa恒定区。
在另一优选例中，所述抗体的重链可变区还包括人源的框架区，和/或所述抗体的轻链可变区还包括人源的框架区。
在另一优选例中，所述抗体的重链可变区还包括鼠源的框架区，和/或所述抗体的轻链可变区还包括鼠源的框架区。
在另一优选例中，所述抗体选自下组：动物源抗体、嵌合抗体、人源化抗体、全人抗体、或其组合。
在另一优选例中，所述的嵌合抗体在人中的免疫原性Z1与非嵌合的抗体(如鼠源抗体)在人中的免疫原性Z0之比(Z1/Z0)为0-0.5，较佳地0-0.2，更佳地0-0.05(如0.001-0.05)。
在另一优选例中，所述的抗体是部分或全人源化、或全人的单克隆抗体。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述抗体为抗体全长蛋白、或抗原结合片段。
在另一优选例中，所述抗体为双特异性抗体、或多特异性抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)抑制CD73的酶活；
(b)促进CD73的内吞；
(c)恢复AMP所介导T细胞的增殖；
(d)改善肿瘤微环境，激活肿瘤特异免疫反应
(e)抑制肿瘤细胞迁移或转移；
(b)抑制肿瘤生长。
在另一优选例中，所述的抗体具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区；
其中，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
Figure PCTCN2020071838-appb-000001
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述的抗体具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区；其中，
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.3所示的VH-CDR1，
SEQ ID NO.4所示的VH-CDR2，和
SEQ ID NO.5所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.8所示的VL-CDR1，
SEQ ID NO.9所示的VL-CDR2，和
SEQ ID NO.10所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.13所示的VH-CDR1，
SEQ ID NO.14所示的VH-CDR2，和
SEQ ID NO.15所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.18所示的VL-CDR1，
SEQ ID NO.19所示的VL-CDR2，和
SEQ ID NO.20所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.23所示的VH-CDR1，
SEQ ID NO.24所示的VH-CDR2，和
SEQ ID NO.25所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.28所示的VL-CDR1，
SEQ ID NO.29所示的VL-CDR2，和
SEQ ID NO.30所示的VL-CDR3。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51、61、71、81、91或101所示的氨基酸序列；和/或所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56、66、76、86、96、或103所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.6所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.101所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.103所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.11所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.16所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.21所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.26所示的氨基酸序列。
在另一优选例中，所述的抗体选自下组：
Figure PCTCN2020071838-appb-000002
Figure PCTCN2020071838-appb-000003
在另一优选例中，所述重链可变区的氨基酸序列与如序列表中SEQ ID NO.1、11、21、31、41、51、61、71、81、91或101所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO.6、16、26、36、46、56、66、76、86、96、或103所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白包括：
(i)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述重组蛋白包括：
(i)选自下组的抗体，
Figure PCTCN2020071838-appb-000004
以及
(ii)任选的协助表达和/或纯化的标签序列。
在本发明的第七方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：
(1)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(2)如本发明的第六方面所述的重组蛋白。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO.2、12、22、32、42、52、62、72、82、92、或102所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.7、17、27、37、47、57、67、77、87、97、或104所示。
在另一优选例中，编码所述重链可变区序列的多核苷酸和编码所述轻链可变区序列的多核苷酸选自下组：
Figure PCTCN2020071838-appb-000005
在本发明的第八方面，提供了一种载体，所述载体含有本发明本发明的第七方面中任一项所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第九方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有本发明的第八方面所述的载体或基因组中整合有本发明的第七方面所述的多核苷酸。
在本发明的第十方面，提供了一种抗体偶联物，该抗体偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四 方面所述的轻链、或如本发明的第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十一方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明的第五方面所述的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十二方面，提供了一种药物组合物，所述药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在本发明的第十三方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合，其中所述活性成分被用于(a)制备诊断试剂或试剂盒；和/或(b)制备预防和/或治疗与CD73表达或功能异常相关的疾病的药物。
在另一优选例中，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述CD73表达或功能异常相关的疾病为肿瘤。
在另一优选例中，所述肿瘤选自下组：膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌、肺癌、头颈癌、前列腺癌、胰腺癌。
在另一优选例中，所述诊断试剂或试剂盒用于：
(1)检测样品中CD73蛋白；和/或
(2)检测肿瘤细胞中内源性的CD73蛋白；和/或
(3)检测表达CD73蛋白的肿瘤细胞；
而所述药物用于预防和/或治疗与CD73表达或功能异常相关的疾病，所述与CD73表达或功能异常相关的疾病为肿瘤。
在另一优选例中，所述肿瘤选自下组：膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌肺癌、头颈癌、前列腺癌、胰腺癌。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的诊断试剂或试剂盒用于诊断CD73相关疾病。
在另一优选例中，所述的诊断试剂或试剂盒用于检测样品中CD73蛋白。
在本发明的第十四方面，提供了一种体外检测(包括诊断性或非诊断性)样品中CD73蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD73蛋白。
在本发明的第十五方面，提供了一种体外检测样品中CD73蛋白的组合物，其包括如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合作为活性成分。
在本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合。
在本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明抗体的二抗；
或者，
所述试剂盒含有如本发明的第十六方面所述的检测板。
在本发明的第十八方面，提供了一种重组多肽的制备方法，该方法包括：
(a)在适合表达的条件下，培养如本发明的第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明的第五方面所述的抗体或如本发明的第六方面所述的重组蛋白。
在本发明的第十九方面，提供了一种药物组合，包括：
(i)第一活性成分，所述第一活性成分包括如本发明的第五方面所述的抗体1、或如本 发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合；
(ii)第二活性成分，所述第二活性成分包括第二抗体、或化疗剂。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体、PD-L1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在另一优选例中，所述的第二活性成分为A2AR抑制剂。
在另一优选例中，所述化疗剂选自下组：多西他赛、卡铂、或其组合。
在本发明的第二十方面，提供了本发明的第五方面所述的抗体，或本发明的第六方面所述的重组蛋白、或本发明的第十方面所述的抗体偶联物、或本发明的第十一方面所述的免疫细胞、和/或本发明的第十二方面所述的药物组合物与第二抗体或化疗剂的组合在制备用于治疗CD73表达或功能异常相关的疾病的药物中的用途。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体、PD-L1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在另一优选例中，所述的第二活性成分为A2AR抑制剂。
在本发明的第二十一方面，提供了一种治疗与CD73表达或功能异常相关的疾病的方法，向有需要的对象施用有效量的如本发明的第五方面所述的抗体、或如本发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合。
在另一优选例中，所述与CD73表达或功能异常相关的疾病为肿瘤。
在另一优选例中，所述肿瘤选自下组：膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌、肺癌、头颈癌、前列腺癌、胰腺癌。
在另一优选例中，所述的方法还包括：在施用第一活性成分之前、之中和/或之后，向所述对象施用安全有效量的第二抗体。
在另一优选例中，所述的第二抗体选自下组：CTLA4抗体、PD-1抗体、PD-L1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1流式细胞实验(FACS)检测鼠源抗体与人CD73、食蟹猴CD73和鼠CD73的结合。其中mIgG1为同型对照；MFI(mean fluorescence intensity)为平均荧光强度。
图2抗CD73鼠源抗体抑制人CD73的酶活。其中mIgG1为同型对照；
图3抗CD73鼠源抗体介导CD73内吞。其中mIgG1为同型对照；
图4抗CD73鼠源抗体恢复AMP介导的抑制T细胞增殖。其中，Activated为无AMP和无抗体时T细胞的增殖百分比；Activated+AMP 500uM为有AMP、无抗体时T细胞的增殖百分比；mIgG1为同型对照；
图5流式细胞实验(FACS)检测嵌合抗体与人CD73、食蟹猴CD73和鼠CD73的结合。其中hIgG1为对照；MFI(mean fluorescence intensity)为平均荧光强度
图6抗CD73嵌合抗体抑制人CD73的酶活。其中hIgG1为对照；
图7抗CD73嵌合抗体介导CD73内吞。其中hIgG1为对照；
图8抗CD73嵌合抗体恢复CD4+T细胞增殖。其中，Activated为无AMP和无抗体时T细胞的增殖百分比；Activated+AMP 800uM为有AMP、无抗体时T细胞的增殖百分比；hIgG1为对照；Tab2为Innate Pharma的抗人CD73抗体11E1。
具体实施方式
本发明人通过广泛而深入的研究，采用不同免疫策略(不同小鼠品系、多种抗原、不同施用途径)获得了多样性的抗体序，从中挑选得到了与人CD73相结合的、各类性质都更为出色(如体外活性各方面都优异)的特异性抗CD73单克隆抗体。具体而言，本发明采用免疫SJL小鼠、杂交瘤、分子生物学(测序、构建载体)等技术，提供了一组与CD73结合的人鼠嵌合抗体，含有小鼠源抗体重链和轻链可变区和人源抗体恒定区。所有可变区均含有CDR1，CDR2，CRR3三个互补决定区或高变区。该类抗体可变区可进行人源化，与人源抗体恒定区组合为全人抗体分子。CD73酶活实验、内吞实验和T细胞增殖实验证明所得CD73抗体具有优异的生物活性：相比于MEDI9447能明显地抑制CD73的酶活、促进CD73的内吞，相比于BMS-986179能更有效地恢复AMP所介导T细胞的增殖。此外，本发明还提供了所述抗CD73单克隆抗体的用途，包括改善肿瘤微环境，激活肿瘤特异免疫反应，抑制肿瘤生长，单独或与其它抗肿瘤药物联合应用于用于肿瘤免疫治疗。本发明还提供了所述抗CD73单克隆抗体与多种免疫检查点抗体或化疗剂联用有效抑制肿瘤生长，从而用于制备用于治疗CD73表达或功能异常相关的疾病的药物的用途。在此基础上完成了本发明。
术语
本发明中，“VH-CDR1”与“CDR-H1”可互换使用，均指重链可变区的CDR1；“VH-CDR2”与“CDR-H2”可互换使用，均指重链可变区的CDR2；“VH-CDR3”与“CDR-H3”可互换使用，均指重链可变区的CDR3。“VL-CDR1”与“CDR-L1”可互换使用，均指轻链可变区的CDR1；“VL-CDR2”与“CDR-L2”可互换使用，均指轻链可变区的CDR2；“VL-CDR3”与“CDR-L3”可互换使用，均指轻链可变区的CDR3。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白 分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗CD73的抗体
本发明中，所述抗体为抗CD73的抗体。本发明提供一种针对CD73的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，
所述的重链可变区(VH)具有选自下组的互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
所述的轻链可变区(VL)具有选自下组的互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
优选地，重链可变区(VH)包括以下三个互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
轻链可变区(VL)包括以下三个互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
各n独立地为0、1、2或3；较佳地n为0或1；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
较佳地，本文所述抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和 技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
本发明中的抗体(抗CD73的抗体)可以是全长蛋白(如IgG1,IgG2a,IgG2b或者IgG2c)，也可以是包含抗原抗体结合域的蛋白片段(例如Fab，F(ab'),sdAb,ScFv片段)。
本发明中的抗体(抗CD73的抗体)可以是野生型蛋白，也可以是经过特定突变已达到某种特定效果的突变型蛋白，例如利用突变消除抗体的效应子功能。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向CD73(例如人CD73)的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51、61、71、81、91、或101所示的氨基酸序列。
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56、66、76、86、96、或103所示的氨基酸序列。
在另一优选例中，所述靶向CD73的抗体的重链可变区(VH)氨基酸序列，和/或，轻链可变区(VL)氨基酸序列如下表2所示：
表2
Figure PCTCN2020071838-appb-000006
Figure PCTCN2020071838-appb-000007
在另一优选例中，所述靶向CD73的抗体为Hu030-2、42A5A7、56F12H8、66H6C12、24D6B4、60G1C8、69C9E12、71E10B3、77B9A3、80H7D6、或125A4E10。
在另一优选例中，所述靶向CD73的抗体为Hu030-2、42A5A7、56F12H8、66H6C12。
在另一优选例中，所述靶向CD73的抗体为Hu030-2、或42A5A7。
重组蛋白
本发明还提供一种重组蛋白，其包括CD73抗体的重链CDR1(VH-CDR1)、重链CDR2(VH-CDR2)和重链CDR3(VH-CDR3)中的一种或多种，和/或，CD73抗体的轻链CDR1(VL-CDR1)、轻链CDR2(VL-CDR2)和轻链CDR3(VL-CDR3)中的一种或多种，
所述重链CDR1-3的序列如下：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，
SEQ ID NO.10n+5所示的VH-CDR3；
所述轻链CDR1-3的序列如下：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
各n独立地为0、1、2、3、4、5、6、7、8、或9；较佳地n为0或1；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括CD73抗体的重链可变区和/或CD73抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51、61、71、81、91、或101所示的氨基酸序列；所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56、66、76、86、96、或103所示的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括CD73抗体的重链可变区和CD73抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51、61、71、81、91、或101所示的氨基酸序列，且所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56、66、76、86、96、或103所示的氨基酸序列。
在另一优选例中，所述重组蛋白及其包括的重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号如表3所示：
表3重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号
Figure PCTCN2020071838-appb-000008
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
较佳地，所述的重组蛋白还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为大鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为大鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
所述的重组蛋白为本领域常规的蛋白质，较佳地，其为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab')。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述重组蛋白的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述重组蛋白。
核酸
本发明还提供一种核酸，其编码上述的抗体(例如抗CD73的抗体)或重组蛋白或抗CD73的抗体的重链可变区或轻链可变区。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
载体
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或 基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和 DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2020071838-appb-000009
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
应用
本发明还提供了本发明抗体、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。
较佳地，所述的药物是用于预防和/或治疗与CD73表达或功能异常相关的疾病的药物。
本发明中，所述与CD73表达或功能异常相关的疾病是本领域常规的与CD73表达或功能异常相关的疾病。较佳地，所述与CD73表达或功能异常相关的疾病为肿瘤/癌症。
本发明中，所述癌症为本领域常规的癌症，较佳地为膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌、肺癌、头颈癌、前列腺癌、胰腺癌。
本发明抗体、ADC、重组蛋白、和/或免疫细胞的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是CD73高表达的肿瘤。所述肿瘤包括(但并不限于)：膀胱癌、血癌、神经胶质瘤、恶性胶质瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌、肺癌、头颈癌、前列腺癌、胰腺癌。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如， 所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明所述的药物组合物是用于预防和/或治疗与CD73表达或功能异常相关的疾病的药物组合物。
本发明的药物组合物可直接用于结合CD73蛋白分子，因而可用于预防和治疗肿瘤等疾病。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供上述药物组合物在制备预防和/或治疗与CD73表达或功能异常相关的疾病的药物中的应用。较佳地，所述与CD73表达或功能异常相关的疾病为肿瘤/癌症。
检测样品中CD73蛋白的方法、组合物
本发明还提供一种检测样品中CD73蛋白(例如检测过表达CD73细胞)的方法，包括如下的步骤：上述的抗体与待检样品在体外接触，检测上述的抗体与所述待检样品是否结合形成抗原-抗体复合物即可。
所述的过表达的含义为本领域常规，指CD73蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在 底物的酶水解作用增加的情况下)。
本发明中，上述是否结合形成抗原-抗体复合物的检测方式是本领域常规的检测方式，较佳地为流式细胞实验(FACS)检测。
本发明提供一种检测样品中CD73蛋白的组合物，其包括上述的抗体、重组蛋白、抗体偶联物、免疫细胞、或其组合作为活性成分。较佳地，其还包括上述的抗体的功能片段组成的化合物作为活性成分。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的主要优点在于：
(1)本发明所获得的抗体与MEDI9447和BMS anti-CD73识别不同的抗原表位；
(2)；本发明所获得的抗体可同时具备优异的介导CD73内吞能力和恢复T细胞增殖的能力。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。
材料和方法
本发明采用先进的抗体转基因小鼠技术平台以制备全人源序列单克隆抗体。
本发明所得到的抗CD73抗体可以由多种途径和方法制备，包括：
(1)传统的杂交瘤制备技术
该技术由Kohler and Milstein在40年前建立(Kohler and Milstein 1975，Nature 256:495)，现在已广泛应用于科研、诊断、治疗等许多相关的单克隆抗体的制备和生产之中。其基本方法虽然沿用至今，但在许多方面都有所变化、改进和创新，包括不同品系动物如转基因动物的使用、电融合技术的引入、高效筛选技术设备的应用如ClonePix设备等，使杂交瘤技术的应用更多样化和更加高效。从常规动物如小鼠等制备的单抗，可以通过常规分子生物学方法克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体(U.S.Pat.No.4,816,567，Cabilly et al)，以大大降低人体使用时的免疫原性。更进一步，鼠源抗体可变区的CDR结构域可以嫁接到人源抗体架构上，从而使鼠源抗体成分降低到5％以下，大大增加了抗体在人体内使用的安全性。这一途径得到抗体称为人源化抗体，并 且是目前抗体药物市场的主要产品(U.S.Pat.No.5,225,539to 55,Winter,and U.S.Pat.Nos.5,530,101；5,585,089；5,693,762and 6,180,370to Queen et al)。
在本发明的一个优选实例中，本发明制备了一系列人鼠嵌合单克隆抗体。这些抗人CD73抗体的是通过免疫Balb/c和SJL小鼠、优化的杂交瘤技术制备、分子生物学和抗体工程技术制备，具有小鼠源抗体重链和轻链可变区和人源抗体恒定区。
(2)Balb/c和SJL小鼠免疫
制备包括胞外区CD73蛋白、CD73重组细胞株、CD73 DNA载体的表达质粒等免疫原。
免疫原1)，胞外区CD73蛋白氨基酸序列27-547C末端加了His6标签，然后克隆到pTT5载体中，得到pTT5-hCD73ECD-His6。将其瞬时转染CHO细胞，9天后收集细胞培养液，离心去除细胞成分，将上清培养基进行0.22微米滤头过滤。然后将含CD73蛋白的培养上清上样到镍亲和层析柱，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS(磷酸盐缓冲液，pH7.5)和PBS(含0.1％Triton X114和0.1％Triton X100，pH7.5)清洗层析柱到紫外吸收回到基线，然后用含适量咪唑的PBS(pH7.5)洗脱，收集从柱上洗脱下来的带His标签的CD73胞外区蛋白(hCD73ECD-His),用PBS在4度冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装-80度保存。
CD73蛋白免疫采用6-8周龄Balb/c和SJL小鼠(斯莱克(SLAC)提供)，小鼠接收后在SPF条件下饲养。初次免疫PD-1蛋白用福氏完全佐剂乳化后腹腔注射0.25毫升，100微克蛋白每只小鼠。加强免疫PD-1蛋白用福氏不完全佐剂乳化后腹腔注射0.25毫升，50微克蛋白每只小鼠。初次免疫与第一次加强免疫之间隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用ELISA和FACS检测血清中抗体效价和特异性。
免疫原2)，人CD73全长氨基酸序列被克隆到pLVX-IRES-puro载体。对HEK293细胞系进行质粒转染、CHOK1与BW5147细胞系进行慢病毒感染并在含puromycin的培养基中选择性培养2周后，用有限稀释法在96孔培养板中亚克隆，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用抗CD73特异性抗体经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存。
CD73细胞免疫采用6-8周龄雌性Balb/c和SJL/J(上海斯莱克繁殖提供)，小鼠接收后在SPF条件下饲养。人CD73转染的HEK293/Renca稳定细胞系在T-75细胞培养瓶中扩大培养至75-90％汇合度，吸尽培养基，用DMEM/1640基础培养基洗涤1-2次，然后用胰酶处理和收集细胞。HEK293细胞用DMEM基础培养基洗涤1-2次,进行细胞计数后将细胞用PBS稀释至1x10 7细胞每毫升；Renca细胞用丝裂霉素处理4小时，用PBS洗2-3次，进行细胞计数后将细胞用PBS稀释至1x10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用FACS检测血清中抗体效价和特异性。
免疫原3)，CD73全长氨基酸序列cDNA被克隆到pCP载体上，采用基因枪免疫或者体内电穿孔免疫方法。
基因枪免疫：质粒被包被到1.0μM金胶体子弹上，用Helios基因枪(Bio-rad)免疫。详细方法根据Helios基因枪说明书进行制定。6-8周龄雌性Balb/c和SJL/J(上海斯莱克繁殖提供)接收后在SPF条件下饲养。所有小鼠经腹部用基因枪免疫3-4次，每次4枪，每枪1.0微克cDNA量。初次免疫与第一次加强免疫，以及加强免疫之间均隔2周。每次加强免疫7天后采血，用ELISA或FACS检测血清中抗体效价。通常大部分小鼠经2-3次免疫后FACS效价可达到1:1000以上。
体内电穿孔免疫：6-8周龄雌性Balb/c和SJL/J(上海斯莱克繁殖提供)接收后在SPF条件下饲养。所有小鼠经备皮的尾巴根部两侧皮内注射3-4次CD73全长氨基酸序列cDNA，每次每侧注射50μg/20μl。随后立即使用AgilePμlse system给予注射部位电穿孔，详细方法参照AgilePμlse(BTX Harvard apparatus)说明书进行。初次免疫与第一次加强免疫，以及加强免疫之间均隔2周。每次加强免疫7天后采血，用ELISA或FACS检测血清中抗体效价。通常大部分小鼠经3-4次免疫后FACS效价可达到1:1000以上。
(3)杂交瘤细胞的制备和抗体筛选
效价符合要求的小鼠可选择进行细胞融合和杂交瘤制备。细胞融合前，蛋白免疫和基因免疫的小鼠最后一次免疫用每只50微克纯化的hCD73ECD-His腹腔注射，细胞免疫的小鼠最后一次免疫用每只0.5-1x10 7细胞腹腔注射。3-5天后处死小鼠，收集脾细胞或者淋巴细胞。加入NH 4OH至终浓度1％以细胞悬液中的红细胞，用DMEM基础培养基离心清洗细胞2-3次，然后按5:1比率与小鼠骨髓瘤细胞SP2/0混合，采用传统的PEG细胞融合方法或高效电融合方法进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1xHAT的DMEM选择性培养基中，按1x10 5/20微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中。10-14天后用Acumen(微孔板细胞检测法)筛选细胞融合板上清，阳性克隆扩增到24孔板扩大培养，2-3天后对24孔板上清进行抗体亚型分析，用FACS确定对CD73阳性细胞的结合活性，CD73酶活实验确定抗体样品对CD73代谢AMP的抑制作用。
根据24孔板筛选结果，选择所需克隆用有限稀释法在96孔板进行亚克隆。亚克隆后7-10天用Acumen进行初步筛选，挑选3-4个阳性单克隆扩增到24孔板继续培养。2-3天后用FACS确定抗原结合阳性并用CD73酶活实验评估生物活性。根据24孔板样品检测结果，挑选一个最优克隆进行扩大培养、液氮冻存、抗体生产和纯化。
(4)小鼠杂交瘤细胞单克隆抗体的生产和纯化
将杂交瘤细胞扩增到T-75细胞培养瓶并用生产培养基(Hybridoma seruM free mediuM,Invitrogen)顺化传代2-3代。待杂交瘤细胞生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶加入200-500毫升生产培养基，接种细胞密度为0.5-1.0x10 5/毫升。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，调到转速3转/分钟。连续旋转培养10-14天后，收集细胞培养液，用离心或过滤去除细胞，并用0.22-0.45微米的滤膜过滤澄清。处理后的细胞培养上清可马上进行纯化或-30℃冻存。
杂交瘤细胞培养上清中的单克隆抗体可用蛋白A(Protein A)亲和层析柱进行纯化。根据样品量的大小，准备相应的体积的层析柱。对于200-300ml的小体积纯化， 需要1-2ml蛋白A柱。蛋白A柱先用平衡缓冲液(Tris-HCl,pH7.4)平衡，然后将培养上清加到层析柱，控制流速在3-4ml/分钟。上样完毕，用平衡缓冲液清洗层析柱3-5柱床体积。用洗脱液(0.1M柠檬酸钠缓冲液，pH4.5)洗脱IgG1；用洗脱液(0.1M柠檬酸钠缓冲液，pH3.5)洗脱其他亚类的IgG。结合在柱上的抗体，用紫外检测器监测洗脱情况。收集洗脱的抗体(紫外吸收峰)，加入10％体积的1.0M Tris-HCl缓冲液中和pH，然后立即用PBS透析过夜，第二天换液一次并继续透析2-3小时。收集透析后的抗体，用0.22微米的滤器进行无菌过滤，无菌保存。分装小样进行蛋白浓度、纯度、内毒等检测分析。
(5)纯化后先导抗体的检定
a)抗原结合反应
流式细胞实验(FACS)检测抗体与人、食蟹猴、小鼠CD73表达细胞的结合。CD73转染的CHOK1稳定细胞株在T-75细胞培养瓶中扩大培养至75-90％汇合度，吸尽培养基，用PBS洗涤1-2次，然后用胰酶(Tryple express:Life technology)处理和收集细胞。用PBS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用PBS稀释至1-2x106细胞每毫升，加入2％胎牛血清(FBS)封闭液，冰上孵育20-30分钟，然后用HBSS离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+2％FBS)悬浮至2x10 6细胞/ml,按每孔100微升加入到96孔FACS反应板中，加入待测抗体样品每孔100微升，4度孵育1-2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，4度孵育0.5-1.0小时。用FACS缓冲液离心洗涤2-3次，加入每孔100微升固定液(4％Paraformaldehyde)悬浮细胞，5-10分钟后用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果。
b)生物学功能分析
CD73酶活实验。将CHOK1-hCD73细胞消化后，用TM缓冲液(25mM Tris,5mM MgCl 2,pH 7.5)稀释至2×10 4细胞每毫升，按每孔100毫升加入到96孔反应板(Corning Cat#3799)中，离心，去除上清。同时，用TM缓冲液将待测抗体配置成4×溶液，以50微升每孔重悬96孔板中的细胞，37度孵育30分钟。用TM缓冲液将AMP配置成4×溶液(800μM)，以50微升每孔加入96孔板，混匀，37度孵育30分钟。300×g离心96孔板，取出50微升上清(不能吸到细胞)，转移至96孔检测板(Corning Cat#3903)中。先后加入50微升每孔2×ATP溶液(130μM)和100微升每孔CellTiter Glo反应液，混匀。暗处放置10分钟后在酶标仪上读取荧光值。
内吞实验。将CHOK1-hCD73细胞消化后，用FACS缓冲液悬浮至2x10 6细胞/ml,按每孔100毫升加入到96孔反应板中，离心，去除上清。加入20ug/ml待测抗体每孔100微升，4度孵育1-2小时，用FACS缓冲液洗去未结合的抗体。在37度/4度放置0,1,2,4小时后取出，加入识别表位不同于待测抗体的检测抗体1ug/ml，4度孵育1小时后，用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果。
T细胞增殖实验。用CD4+T细胞分离试剂盒从人外周血细胞(PBMC)中分离CD4 阳性T细胞。用PBS+1％BSA重悬至2×10 6细胞每毫升，加入同体积的2×CFSE溶液(4μM)，混匀后放置37度10分钟。加入40％体积的FBS，混匀后放置37度10分钟。用大体积的PBS溶液离心洗涤2次。用含有anti-CD2/CD3/CD28磁珠(Miltenyi Biotec,130-091-441)的T细胞培养液重悬至1.5×10 6细胞每毫升，按照100毫升每孔加入至96孔板中。加入50微升每孔4×待测抗体溶液，混匀，37度孵育0.5小时。再加入50微升每孔4×AMP溶液(2mM)。CD4+T细胞放置于37度5％CO 2培养箱中3-5天，用FACS(FACSCalibur,BD)检测和分析结果。
(6)抗体重链和轻链可变区基因和氨基酸序列测定和CDR结构域分析
轻重链可变区氨基酸序列测定。总RNA分离：亚克隆培养的上清检验过抗原结合后，通过离心搜集1-5×10 7杂交瘤细胞。加入1mL Trizol混匀并转移到1.5ml离心管中，室温静置5min；加0.2ml氯仿，振荡15s，静置2min后于4℃离心，12000g×5min，取上清转移到新的1.5ml离心管中；加入0.5ml异丙醇，将管中液体轻轻混匀，室温静置10min后于4℃离心，12000g×15min，弃上清；加入1ml 75％乙醇，轻轻洗涤沉淀。4℃，12000g×5min，弃上清并晾干，加入适量的DEPC H 2O溶解(55℃水浴促溶10min)。
逆转录与PCR：取1μg tRNA，配置20μl体系，加入逆转录酶后于42℃反应60min，于70℃反应10min终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，预变性95℃3min，变性95℃30s，退火55℃30s，延伸72℃35s，35个循环后再额外于72℃延伸5min。注：延伸温度可根据实际情况有所调整。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化；进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时；取5μl连接产物加入100μl的感受态细胞中，冰浴5min，而后于42℃水浴热激1min，放回冰上1min后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30min，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养；次日，使用T载体上引物M13F和M13R配置30μl PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含抗生素的LB固体培养皿上以保存菌株；PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序。
实施例1：CD73特异性鼠源抗体的制备
制备包括胞外区CD73蛋白、CD73重组细胞株、CD73 DNA载体的表达质粒等免疫原。
用不同的免疫策略(蛋白免疫、细胞免疫、基因免疫)来免疫小鼠。自小鼠实施融合并筛选，并使用这些杂交瘤细胞上清进行克隆筛选。分离了尤其受关注的克隆并生产纯化，得到包括24D6、37F8、42A5、56F12、57G8、60G1、66H6、69C9、47F12、71E10、77B9、78E6、80H7及125A4的鼠源抗体。
实施例2：抗CD73鼠源抗体的性质鉴定
2.1流式细胞实验(FACS)检测鼠源抗体与人、食蟹猴、小鼠CD73表达细胞的结合
使用CHOK1-hCD73(经人CD73转染)，CHOK1-cCD73(经食蟹猴CD73转染)，CHOK1-mCD73(经鼠CD73转染)及CHOK1(人CD73、食蟹猴CD73、鼠CD73阴性)细胞，杂交瘤细胞表达并纯化的CD73抗体作为一抗，Alexa 
Figure PCTCN2020071838-appb-000010
488驴抗鼠IgG(H+L)(Invitrogen，A21202)作为二抗，进行以下方法产生滴定结合曲线：
CD73转染的CHOK1稳定细胞株在T-75细胞培养瓶中扩大培养至75-90％汇合度，吸尽培养基，用PBS洗涤1-2次，然后用胰酶(Tryple express:Life technology)处理和收集细胞。用PBS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用PBS稀释至1-2x10 6细胞每毫升，加入2％胎牛血清(FBS)封闭液，冰上孵育20-30分钟，然后用HBSS离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+2％FBS)悬浮至2x10 6细胞/ml，按每孔100微升加入到96孔FACS反应板中，300g离心5分钟弃掉上清。抗CD73抗体用封闭液配置成初始浓度10ug/ml，8点连续稀释。加入待测抗体样品每孔100微升，4度孵育1-2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，4度孵育0.5-1.0小时。用FACS缓冲液离心洗涤2-3次，加入每孔100微升固定液(4％Paraformaldehyde)悬浮细胞，5-10分钟后用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果，见图1和表4。
图1结果表明：mAb020、024、030、032、033、034、036、038、039、041、042、043、044、065抗体均能结合到细胞表面的人CD73、食蟹猴CD73上，但是不能结合到鼠CD73上。每一抗体获得的结合的EC50显示于表4中。
表4流式细胞实验(FACS)检测鼠源抗体与人CD73、食蟹猴CD73和鼠CD73的结合
Figure PCTCN2020071838-appb-000011
Figure PCTCN2020071838-appb-000012
2.2抗CD73鼠源抗体对酶活性的抑制实验
将CHOK1-hCD73细胞消化后，用TM缓冲液(25mM Tris,5mM MgCl 2,pH 7.5)稀释至2×10 4细胞每毫升，按每孔100毫升加入到96孔反应板(Corning Cat#3799)中，离心，去除上清。同时，用TM缓冲液将待测抗体配置成4×溶液，6点连续稀释，以50微升每孔重悬96孔板中的细胞，37度孵育30分钟。用TM缓冲液将AMP配置成4×溶液(800μM)，以50微升每孔加入96孔板，混匀，37度孵育30分钟。300×g离心96孔板，取出50微升上清，转移至96孔检测板(Corning Cat#3903)中。先后加入50微升每孔2×ATP溶液(130μM)和100微升每孔CellTiter Glo反应液，混匀。暗处放置10分钟后在酶标仪上读取荧光值。结果见图2和表5。
图2表示mAb020、024、030、032、033、034、036、038、039、041、042、043、044、065抗体均能抑制细胞表面人CD73的酶活性。每一抗体的最大抑制百分比和IC50显示于表5中。
表5抗CD73鼠源抗体抑制人CD73的酶活
Figure PCTCN2020071838-appb-000013
2.3抗CD73鼠源抗体介导的内吞实验
使用杂交瘤细胞表达并纯化的CD73抗体，在37度孵育CHOK1-hCD73细胞，用FACS检测抗体介导的CD73内吞。
将CHOK1-hCD73细胞消化后，用FACS缓冲液悬浮至2x10 6细胞/ml,按每孔100毫升加入到96孔反应板中，离心，去除上清。加入20ug/ml待测抗体每孔100微升，4度孵育1-2小时，用FACS缓冲液洗去未结合的抗体。在37度/4度放置0,1,2,4小时后 取出，加入识别表位不同于待测抗体的检测抗体(Alexa 488标记)1ug/ml，4度孵育1小时后，用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果。4度0h的数据MFI读值作为对照，所有计算值均为与该对照相比的百分比。结果见图3。
图3展示了mAb020、024、030、032、033、034、036、038、039、041、042、043、044、065抗体介导CD73内吞的时间曲线。结果表明：大部分抗体能有效并显著介导CD73内吞，例如mab020、030、033、034、042。
2.4抗CD73鼠源抗体恢复T细胞增殖
AMP经过CD73去磷酸化作用形成腺苷，通过被T细胞上的腺苷受体结合，抑制效应T细胞的增殖。本实施例通过抗CD73的抗体阻断CD73作用，抑制腺苷形成，恢复T细胞增殖。
用CD4+T细胞分离试剂盒从人外周血细胞(PBMC)中分离CD4阳性T细胞。用PBS+1％BSA重悬至2×10 6细胞每毫升，加入同体积的2×CFSE溶液(4μM)，混匀后放置37度10分钟。加入40％体积的FBS，混匀后放置37度10分钟。用PBS溶液离心洗涤2次。用含有anti-CD2/CD3/CD28磁珠(Miltenyi Biotec,130-091-441)的T细胞培养液(RMPI 1640+10％FBS+1％P/S)重悬至1.5×10 6细胞每毫升，按照100毫升每孔加入至96孔板中。加入50微升每孔4×待测抗体溶液，混匀，37度孵育0.5小时。再加入50微升每孔4×AMP溶液(2mM)。CD4+T细胞放置于37度5％CO 2培养箱中3-5天，用FACS(FACSCalibur,BD)检测和分析结果。结果见图4。
图4结果表明：mAb020、024、030、032、033、034、036、038、039、041、042、043、044、065抗体均能恢复CD4+T细胞增殖。
实施例3：轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例2～5的检定和活性测定后)，通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促溶10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自 Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2020071838-appb-000014
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4 DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μl PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health,Bethesda,Md.(1991)。
各抗体的测序结果如表A和表B所示。
表A抗体的VH、VH-CDR1、VH-CDR2、VH-CDR3，VL、VL-CDR1、VL-CDR2、VL-CDR3的序列编号(SEQ ID NO.)
Figure PCTCN2020071838-appb-000015
表B序列信息
Figure PCTCN2020071838-appb-000016
Figure PCTCN2020071838-appb-000017
Figure PCTCN2020071838-appb-000018
Figure PCTCN2020071838-appb-000019
Figure PCTCN2020071838-appb-000020
Figure PCTCN2020071838-appb-000021
实施例4：制备鼠人嵌合抗体。
(一)质粒构建与准备：实施例1已从杂交瘤细胞的培养上清液中获得了纯化的CD73抗体，并根据实施例6的测序结果明确了CD73抗体的重链可变区和轻链可变区序列。将CD73抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1-TM恒定区(IgG1含L234F,L235E和P331S三个位点突变，以减少ADCC和CDC作用)的表达载体(其中IgG1表达载体购买自Invitrogen，点突变改造和重组步骤均为常规步骤，将CD73抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例6中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
(二)细胞转染：
在培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM，8％CO 2(v/v)浓度。
Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68 终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。
取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293expression medium中至293E的细胞密度为1.5×10 6/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
(三)抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料(购自GE)，使用0.1M NaOH处理30分钟或者5个柱体积的0.5M NaOH冲洗。对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％(v/v)Triton×100对柱料清洗。使用5个柱体积的PBS(PBS磷酸缓冲液，pH7.2)进行平衡，将步骤(2)所得过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用0.5倍柱体积洗脱液的pH8.5的1M Tris-HCl(1.5M NaCl)中和，收获鼠人嵌合CD73抗体。上述所用溶液均需要新配置。收获鼠人嵌合CD73抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒检测抗体内毒素含量。
实施例5：嵌合抗体的性质鉴定
5.1流式细胞方法鉴定抗CD73嵌合抗体对人、食蟹猴、鼠CD73的结合
方法同实施例2。结果如图5所示。
结果表明：mAb020,024,030,032,033,034,036,038,039,041,042,043,044,065嵌合抗体均能结合到细胞表面的人CD73、食蟹猴CD73上，但是不能结合到鼠CD73上。每一抗体获得的结合的EC50显示于表6中。
表6流式细胞实验(FACS)检测嵌合抗体与人CD73、食蟹猴CD73和鼠CD73的结合
Figure PCTCN2020071838-appb-000022
Figure PCTCN2020071838-appb-000023
5.2抗CD73嵌合抗体对酶活性的抑制实验。
方法同实施例3。结果如图6所示。
结果表明：mAb020,024,030,032,033,034,036,038,039,041,042,043,044,065嵌合抗体均能抑制细胞表面人CD73的酶活性。每一抗体的最大抑制百分比和EC50显示于表7中。
表7抗CD73嵌合抗体抑制人CD73的酶活
Figure PCTCN2020071838-appb-000024
5.3抗CD73嵌合抗体介导的内吞实验。
方法同实施例4。图7展示了mAb020,024,030,032,033,034,036,038,039,041,042,043,044,065嵌合抗体介导CD73内吞的时间曲线。
结果表明：嵌合抗体mab020、030、034、042能有效并显著介导CD73内吞。
5.4抗CD73嵌合抗体恢复T细胞增殖。
方法同实施例5。结果如图8所示。
结果表明：mAb020,024,030,032,033,034,036,038,041,042,044,065嵌合抗体均能恢复CD4+T细胞增殖。
5.5 SPR鉴定抗CD73嵌合抗体亲和力
本实验选用Octet Red96为测试仪器，选用AHC生物传感器为测试传感器，AHC传感器上已固定有Anti-human IgG Fc抗体，可用其直接捕获本实验中的15个抗体，紧接着将该传感器浸于分析物样品(抗原)中。本实验共运行五个步骤：1、Baseline(120s)2、Loading(捕获抗体)(300s)3、Baseline(120s)4、Association(结合抗原)(180s)5、Dissociation(抗原解离)(1200s)。测试完成后使用再生缓冲液(Glycine PH1.5)与中和缓冲液(1*PBS buffer)交替浸润5秒，共循环五次对传感器进行再生。传感器再生次数最多为10次。本实验中的运行缓冲液为样品稀释液(含有0.02％Tween20及0.1％BSA的1*PBS缓冲液)，即Baseline步骤，Dissociation步骤，空白分析物样品中所使用的缓冲液。本实验一次运行使用4根传感器。
样品处理：使用样品稀释液(含有0.02％Tween20及0.1％BSA的1*PBS缓冲液)将所有的抗体稀释至10ug/mL的工作浓度，以及将分析物样品(抗原)梯度稀释为三个工作浓度：200nM,100nM,50nM。
数据分析使用Octet Data Analysis(version 7.0)计算响应信号值(偶联的分析物样品信号扣减掉空白的分析物样品信号)，并使用1:1结合模型拟合数据。结果见表8。
表8抗CD73嵌合抗体对hCD73 ECD-His亲和力
Figure PCTCN2020071838-appb-000025
结果表明：所测抗体的KD值均在纳摩水平，且与MEDI9447相当或更佳，这表明本发明的这些抗体对人CD73 ECD均有优异的亲和力。
实施例6人源化抗体的制备
经过序列分析，候选抗体mAb030重链可变区和轻链可变区均无重要的hotspot。 通过序列比对(NCBI-Igblast)选择与候选抗体mAb030重链可变区，轻链可变区同源性最高的胚系基因序列作为可变区移植骨架：IGHV4-38-2*02和IGKV1-39*01。在选定人抗体骨架后，通过同源建模，预测在鼠抗恒定区中可能决定结构的关键氨基酸，对嫁接的骨架区进行回复突变设计。
根据以上原则，设计10个重链可变区序列(mab030VH.g0，mab030VH.g1，mab030VH.g2，mab030VH.g3)和4个轻链可变区序列(mab030VL.g0,mab030VL.g1，mab030VL.g2,mab030VL.g3)。随后做交叉组合进行表达，共得到下列16个人源化抗体，见表9。
表9人源化抗体设计组合
  Hu030VH g0 Hu030VH g1 Hu030VH g2 Hu030VH g3
Hu030VL g0 Hu030-1 Hu030-2 Hu030-3 Hu030-4
Hu030VL g1 Hu030-5 Hu030-6 Hu030-7 Hu030-8
Hu030VL g2 Hu030-9 Hu030-10 Hu030-11 Hu030-12
Hu030VL g3 Hu030-13 Hu030-14 Hu030-15 Hu030-16
载体构建：扩增引物由Genewiz合成，随后通过PCR方法分别扩增轻链和重链的可变区。配置50μL反应体系，包括50-100ng的重链可变区，轻链可变区、正向反向引物各1ul、1ul pfxD酶(购自invitrogen，12344-012)、10*pfx缓冲液5ul(供应商同pfx同酶)以及加水补足至50μL。设置PCR程序，预变性95℃5分钟，变性95℃30秒，退火56℃30秒，延伸68℃30秒，25个循环后再额外68℃延伸10min，得到PCR产物。取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用回收试剂盒纯化，其中回收试剂盒为PureLink Quick Gel extraction kit，购自Qiagen，货号28706。
表达纯化：进行连接反应：插入片段20-40ng，酶切过的表达载体60-100ng，重组酶Exnase(购自Vazyme，货号C112-01/02)1μL，缓冲液2μL，反应体系10μL，于37℃反应半小时得到连接产物，即构建好的重组载体。缓冲液为该重组酶配套购买使用的缓冲液；将重链可变区定向克隆到包含信号肽和人源抗体重链IgG4(S228P)恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)，将轻链可变区定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)中。将10μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，42℃水浴热激60秒，放回冰上3分钟，取出80μL涂布于含氨苄青霉素的LB固体培养基上，于37℃孵箱过夜培养。次日，使用表达载体上引物pEF1A和pSV40，配置30μL PCR体系，进行菌落PCR。菌落PCR的体系为：引物各1μL，15μL的PCR预混液(购自Novoprotein)，补足至30μL。用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出4.5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序。
将序列正确的重组抗体重、轻链的表达载体进行扩增，随后瞬时转染FreeStyleTM293-F细胞(购自Invitrogen)以生产抗体。转染时，293-F细胞的密度应为1-1.2×106个/mL，100mL细胞需要100μg上述已构建好的重组载体和200μg的转染试剂聚乙烯亚 胺(PEI)。将重组载体和PEI分别加入到5mL培养基中，室温静置5分钟，0.22μm滤膜过滤后，将重组载体和PEI的混合物于室温静置15分钟。然后将上述混合物缓慢地加入到细胞中，在37℃、8％(v/v)CO2培养箱中以130rpm的转速培养。每天采取培养上清和细胞沉淀检测抗体的表达。5天后，3000g离心细胞培养液30分钟，收集上清液，0.22μm滤器过滤。用1mL MabSelectTMSuReTMcolumn(购自GE Healthcare)纯化200mL澄清上清液中的单克隆抗体。MabSelectTMSuReTMcolumn先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，MabSelectTMSuReTMcolumn。上样完毕后用平衡缓冲液清洗MabSelectTMSuReTMcolumn，平衡缓冲液的体积为蛋白A柱柱床体积的5倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH3.0)洗脱结合在MabSelectTMSuReTMcolumn上的单克隆抗体。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH。然后立即用PBS磷酸缓冲液透析过夜。收集透析后的单克隆抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的CD73人源化抗体。将所得抗体进行蛋白浓度、纯度检测分析。
结果如下表10所示，结果显示，人源化抗体的产量和纯度分析均表现正常。
表10抗CD73人源化抗体表达和纯化结果
Figure PCTCN2020071838-appb-000026
实施例7 SPR鉴定人源化抗CD73抗体的亲和力
方法同实施例2。结果如表11所示，显示人源化抗体亲和力与嵌合抗体相当。
表11抗CD73人源化抗体对hCD73ECD-His亲和力
Figure PCTCN2020071838-appb-000027
Figure PCTCN2020071838-appb-000028
从表中可以看出，以Hu030-2为代表的人源化抗体具有更为优异的性能。Hu030-2的VH的氨基酸序列和核苷酸序列如SEQ ID No.:101和102，Hu030-2的VL的氨基酸序列和核苷酸序列如SEQ ID No.:103和104所示。Hu030-2抗体的VH的3个CDR以及VL的3个CDR与抗体030(即克隆42A5A7)的相同，分别为SEQ ID No.:3、4和5，以及SEQ ID No.:8、9和10。
讨论
目前，MedImmune和BMS两家公司的抗CD73抗体处于临床阶段。其中，MedImmune公司的抗体MEDI9447处于临床一/二期，该抗体通过噬菌体展示技术得到；BMS公司的抗体BMS-986179处于临床一/二期，该抗体通过免疫人源化小鼠得到。动物体内实验数据表明，MEDI9447单独使用对于抑制CT26肿瘤生长无明显作用，与抗PD1抗体联用可大幅增加抗肿瘤效果；而BMS-986179由于不能识别鼠源CD73蛋白，故无可参考的体内数据。从体外实验数据可知，这两个抗体均能一定程度上抑制CD73的酶活，介导CD73的内吞，恢复AMP所介导T细胞的增殖。但相比之下，MEDI9447促内吞的效果较弱，BMS-986179恢复AMP所介导T细胞的增殖能力较弱。
目前，从MedImmune和BMS两家公司提供的体外实验数据可知，这两个抗体均能一定程度上抑制CD73的酶活，介导CD73的内吞，恢复AMP所介导T细胞的增殖。但相比之下，MEDI9447促内吞的效果较弱，BMS-986179恢复AMP所介导T细胞的增殖能力较弱。而本发明得到的抗CD73抗体，例如mab030，既能表现出优异的促内吞效果，为减少细胞膜表面的CD73提供可能；也能强有力地恢复AMP所介导T细胞的增殖，成为一个全方面的表现出色的抗CD73抗体。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO.10n+3所示的VH-CDR1，
    SEQ ID NO.10n+4所示的VH-CDR2，和
    SEQ ID NO.10n+5所示的VH-CDR3；
    其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO.10n+8所示的VL-CDR1，
    SEQ ID NO.10n+9所示的VL-CDR2，和
    SEQ ID NO.10n+10所示的VL-CDR3；
    其中，各n独立地为0、1、2、3、4、5、6、7、8、或9；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链，
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  6. 如权利要求5所述的抗体，其特征在于，所述的抗体具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区；
    其中，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
    Figure PCTCN2020071838-appb-100001
    Figure PCTCN2020071838-appb-100002
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  7. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区含有SEQ ID NO.101所示的氨基酸序列；和所述抗体的轻链可变区含有SEQ ID NO.103所示的氨基酸序列。
  8. 如权利要求6所述的抗体，其特征在于，所述的抗体选自下组：
    Figure PCTCN2020071838-appb-100003
  9. 一种重组蛋白，其特征在于，所述的重组蛋白包括：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  10. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (2)如权利要求9所述的重组蛋白。
  11. 如权利要求10所述的多核苷酸，其特征在于，编码所述重链可变区的多核苷酸如SEQ ID NO.2、12、22、32、42、52、62、72、82、92、或102所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.7、17、27、37、47、57、67、77、87、97、或104所示。
  12. 如权利要求11所述的多核苷酸，其特征在于，编码所述重链可变区序列的多核苷酸和编码所述轻链可变区序列的多核苷酸选自下组：
    Figure PCTCN2020071838-appb-100004
  13. 一种载体，其特征在于，所述载体含有本发明权利要求10-12中任一项所述的多核苷酸。
  14. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求13所述的载体或基因组中整合有权利要求10-12中任一项所述的多核苷酸。
  15. 一种抗体偶联物，其特征在于，该抗体偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  16. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有权利要求5-8中任一项所述的抗体。
  17. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、如权利要求9所述的重组蛋白、如权利要求15所述的抗体偶联物、权利要求16所述的免疫细胞、或其组合；以及
    (ii)药学上可接受的载体。
PCT/CN2020/071838 2019-01-11 2020-01-13 Cd73抗体及其制备方法和应用 Ceased WO2020143836A1 (zh)

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