WO2023246325A1 - 靶向cd73的纳米抗体及纳米抗体-药物偶联物、其制备方法和用途 - Google Patents

靶向cd73的纳米抗体及纳米抗体-药物偶联物、其制备方法和用途 Download PDF

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WO2023246325A1
WO2023246325A1 PCT/CN2023/092108 CN2023092108W WO2023246325A1 WO 2023246325 A1 WO2023246325 A1 WO 2023246325A1 CN 2023092108 W CN2023092108 W CN 2023092108W WO 2023246325 A1 WO2023246325 A1 WO 2023246325A1
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seq
antibody
nanobody
targeting
drug
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French (fr)
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余科
刘亮
孟韬
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Fudan University
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Fudan University
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Priority to JP2024575344A priority Critical patent/JP2025522547A/ja
Priority to PCT/CN2023/100479 priority patent/WO2023246623A1/zh
Priority to CN202380009587.XA priority patent/CN117616051A/zh
Priority to EP23826272.9A priority patent/EP4545561A1/en
Priority to US18/878,580 priority patent/US20250382376A1/en
Publication of WO2023246325A1 publication Critical patent/WO2023246325A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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Definitions

  • the present invention relates to the field of medicine, and in particular to CD73-targeted Nanobodies (CD73-Nb) and CD73 Nanobody-drug conjugates (Nanobody-drug conjugate, CD73-NDC), their preparation methods and uses.
  • CD73-Nb CD73-targeted Nanobodies
  • CD73 Nanobody-drug conjugates CD73-NDC
  • CD73 is abnormally expressed in a variety of tumors, including lung cancer, breast cancer, melanoma and glioma, and is closely related to tumor metastasis, recurrence, drug resistance and immune suppression.
  • CD73 is a 5 ⁇ -nucleotidase anchored to the cell membrane surface. It is the main rate-limiting enzyme that dephosphorylates adenosine monophosphate (AMP) to generate adenosine (ADO) through an enzymatic reaction.
  • AMP adenosine monophosphate
  • ADO adenosine
  • Adenosine participates in a variety of physiological and pathological processes in the body by binding to the corresponding adenosine receptors (A1AR, A2AR, A2BR, A3AR), including forming an immunosuppressive microenvironment, such as inhibiting the proliferation and function of T lymphocytes and inhibiting dendrites.
  • A1AR, A2AR, A2BR, A3AR adenosine receptors
  • the differentiation and maturation of dendritic cells (DC) affects the polarization of macrophages and promotes the release of anti-inflammatory cytokines such as TGF ⁇ , IL-10 etc.
  • Drug resistance is a major obstacle that affects the effectiveness of tumor treatment. Studies have shown that CD73 is involved in the resistance of tumor chemotherapy drugs, small molecule targeted drugs and immune checkpoint inhibitors. In addition, CD73 is also involved in the processes of epithelial-mesenchymal transition, angiogenesis, metastasis and recurrence of tumors. Therefore, by targeting CD73,
  • Nanobodies are alpaca naturally produced antibodies composed only of heavy chains.
  • the target recognition module consists of a single variable region (VHH) with a molecular weight of only 12-14kd. It is expected to overcome the solid tumor penetration of traditional monoclonal antibodies. The defect of low sex.
  • Nanobodies can be connected to functional domains such as Fc, other nanobodies, or peptide tags or toxins.
  • nanobodies Due to its small size, higher diffusion rate, vascular permeability, and tumor penetration, it has a more even tissue distribution than traditional monoclonal antibodies. These properties make them particularly suitable for specific and effective tumor-targeted therapy in vivo.
  • nanobodies also have the advantages of low immunogenicity, high stability, high resistance to degradation, low cost of prokaryotic expression and production, and easy product characterization.
  • the development of tumor treatment drugs based on nanobodies is still in the early exploratory research stage.
  • Antibody-drug conjugate uses monoclonal antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby accurately delivering anti-tumor drugs (such as small molecule chemotherapy drugs, etc.) to tumors Target cells are released to achieve precise tumor killing.
  • ADCs are currently used for targeted drug delivery to tumor tissues.
  • ADC has a long terminal half-life in the body, which can lead to unnecessary toxicity of loaded toxins to normal tissues or cells.
  • Nanobody-drug conjugates (NDC) prepared by nanobodies not only retain the advantages of traditional ADCs, but also have high vascular permeability, strong tumor penetration, and fast arrival at target cells, and can It can improve the accumulation of drugs in tumors, and at the same time moderately control the plasma exposure and half-life of drugs, which will help to further improve the therapeutic effect and overall treatment window of solid tumors, and is expected to become a new class of anti-tumor drugs with the most potential.
  • CD73-Nb and CD73-NDC can provide new treatments for tumor patients with abnormal expression of CD73 in clinical treatment alone or in combination.
  • the purpose of the present invention is to provide a CD73-targeting Nanobody and Nanobody-drug conjugate, their preparation methods and uses.
  • a Nanobody targeting CD73 is provided, and the complementarity determining region CDR of the VHH chain of the Nanobody is one or more selected from the following group:
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining CD73 binding affinity.
  • the CDR region of the Nanobody VHH chain contains at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably, any one of SEQ ID NO: 1-36. amino acid sequences with at least 99% sequence similarity.
  • the VHH chain includes CDR1, CDR2 and CDR3 selected from the following combinations:
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the VHH chain of the Nanobody further includes a framework region (FR).
  • FR framework region
  • the framework region FR is of human, mouse, rabbit or camel origin.
  • the framework region FR includes a human-derived FR region, a mouse-derived or camel-derived FR region.
  • an antibody targeting CD73 is provided, the antibody comprising one or more VHH chains of the Nanobody targeting CD73 as described in the first aspect of the present invention.
  • the VHH chain of the CD73-targeting Nanobody has the following characteristics: SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, The amino acid sequence shown in SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO.36.
  • the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).
  • CAR chimeric antigen receptor antibody
  • the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.
  • the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, more preferably 1.
  • the at least one amino acid sequence that has been added, deleted, modified and/or substituted is an amino acid sequence with at least 80% homology.
  • the derived sequence with addition, deletion, modification and/or substitution of at least one amino acid has the catalytic function of inhibiting cell surface CD73 or recombinant CD73 protease.
  • the antibody is in the form of a drug conjugate.
  • the Nanobody has a binding affinity EC 50 of ELISA for human CD73 protein extracellular domain (CD73-ECD) of 0.014 to 0.07 ⁇ g/mL (0.175 to 0.875 nM), or by ForteBio surface plasmon resonance (The binding constant KD value measured by SPR is 0.795 ⁇ 1.997nM.
  • the antibody has one or more properties selected from the following group:
  • CD73 that specifically binds to tumor cells and/or immune/stromal cells in the tumor microenvironment
  • a multispecific antibody in the third aspect of the present invention, includes: the CD73-targeting Nanobody described in the first aspect of the present invention or the target of the second aspect of the present invention. Antibodies to CD73.
  • the multispecific antibody further includes a second antigen-binding region targeting a target selected from the following group: EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22 , B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or its combination.
  • a target selected from the following group: EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22 , B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or its
  • the second antigen-binding region is a Nanobody.
  • the multispecific antibody includes one or more second antigen-binding regions.
  • the multispecific antibody further includes the Fc segment of the antibody.
  • the antigen-binding region is an antibody or an antibody fragment
  • the antibody fragment includes: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecule; (vi) dAb fragment.
  • the fourth aspect of the present invention provides a recombinant protein, said recombinant protein having:
  • the recombinant protein further includes: (iv) an optional tag sequence to assist expression and/or purification.
  • the recombinant protein is a monomer, dimer, or multimer.
  • polypeptide molecules or fragments with therapeutic functions include but are not limited to: targeting EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, Polypeptide molecules or fragments of GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, and TLR7.
  • the recombinant protein includes a fusion protein.
  • the fusion protein includes multispecific antibodies and chimeric antibodies.
  • the functional domain that improves protein physicochemical properties or druggability includes Fc segment, anti-albumin nanobody (HLE), and albumin binding domain (ABD).
  • the fusion protein has the following elements from the N-C terminus:
  • the VHH chain of the CD73-targeting Nanobody is selected from the following group: SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO .20, the amino acid sequence shown in SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO.36.
  • the Fc segment is a human IgG Fc segment.
  • amino acid sequence of the Fc segment is selected from the following group: SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ The amino acid sequence shown in ID NO.42.
  • a fifth aspect of the present invention provides a CAR construct, wherein the antigen-binding region of the CAR construct is the VHH chain of the Nanobody as described in the first aspect of the present invention.
  • the immune cells are from humans or non-human mammals (such as mice).
  • a coupling moiety coupled to the Nanobody moiety being selected from the group consisting of detectable markers, drugs, toxins, cytokines, enzymes, protein degraders, oligonucleotides, or the like; combination.
  • the immunoconjugate is a Nanobody drug conjugate.
  • Nanobody part and the coupling part are coupled through chemical bonds or linkers.
  • the chemical label is an isotope, an immunotoxin and/or a chemical drug.
  • the biomarker is biotin, avidin or enzyme label.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolite drugs, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • auristatins camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes (
  • the toxin is selected from the following group:
  • Diagnostic isotope the said diagnostic isotope is selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of the CD73-targeting Nanobody as described in the first aspect of the present invention or the present invention.
  • the CD73-targeting antibody of the second aspect is provided.
  • Tubulin inhibitors such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);
  • the pharmaceutical composition includes single drugs, compound drugs, or synergistic drugs.
  • the pharmaceutical composition also contains other biologically active substances, such as drugs for treating tumors.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the ninth aspect of the present invention provides the use of an active ingredient selected from the following group: the CD73-targeting Nanobody as described in the first aspect of the present invention, the second aspect of the present invention
  • the immunoconjugate according to the seventh aspect of the present invention, or a combination thereof, the active ingredient is used for (a) preparing detection reagents, detection plates or kits; and/or (b) preparing prevention and/or treatment Drugs for CD73-related diseases.
  • the detection reagent, detection plate or kit is used for:
  • the detection reagent, detection plate or kit is used to diagnose CD73-related diseases.
  • the drug is used to treat or prevent tumors with high CD73 expression, tumor migration, or tumor drug resistance.
  • the tumor resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.
  • the drug is used for a purpose selected from the following group:
  • CD73 that specifically binds to tumor cells and/or immune/stromal cells in the tumor microenvironment
  • the CD73-related disease is selected from the group consisting of cancer, autoimmune diseases, metabolic-related diseases, infectious diseases, or combinations thereof.
  • the CD73-related diseases include: tumor occurrence, growth and/or metastasis.
  • the cancer includes solid tumors and blood cancers.
  • the cancer is a tumor with high expression of CD73.
  • the cancer is a drug-resistant tumor.
  • the tumor with high expression of CD73 refers to the ratio of CD73 transcript and/or protein level L1 in tumor tissue to transcript and/or protein level L0 in normal tissue, L1/L0 ⁇ 2 , preferably ⁇ 3.
  • the sample is combined with the CD73-targeting Nanobody according to the first aspect of the present invention, the CD73-targeting antibody according to the second aspect of the present invention, or the CD73-targeting Nanobody according to the seventh aspect of the present invention.
  • the fourteenth aspect of the present invention provides a detection plate, which includes: a substrate (support plate) and a test strip, and the test strip contains the CD73-targeting gene described in the first aspect of the invention.
  • a detection plate which includes: a substrate (support plate) and a test strip, and the test strip contains the CD73-targeting gene described in the first aspect of the invention.
  • a first container containing the CD73-targeting Nanobody according to the first aspect of the present invention or the CD73-targeting antibody according to the second aspect of the present invention and/or
  • a sixteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, characterized in that the method includes:
  • the method further includes: administering other drugs or treatment methods to the subject in need for combined treatment.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.
  • nucleotide sequence of the alpaca-derived VHH sequence of the CD73-targeting Nanobody described in the first aspect of the present invention is cloned into an expression vector containing the nucleotide sequence of the human antibody constant region, it is expressed by transfecting animal cells. people -Alpaca chimeric antibodies.
  • a method for preparing humanized antibodies comprising the steps:
  • the nucleotide sequence of the CDR region of the VHH chain of the CD73-targeting Nanobody described in the first aspect of the present invention is implanted into the nucleotide sequence template containing the human antibody FR region, and then cloned into a nucleotide sequence template containing the human antibody constant After constructing the expression vector of the region, humanized antibodies are expressed by transfecting animal cells.
  • a method for inhibiting tumor growth in a model animal comprising the steps of: administering to a subject in need the CD73-targeting Nanobody described in the first aspect of the present invention, the present invention
  • the CD73-targeting antibody of the second aspect, the Nanobody-drug conjugate of the present invention, or the CAR-T cell of the Nanobody comprising the steps of: administering to a subject in need the CD73-targeting Nanobody described in the first aspect of the present invention, the present invention
  • the CD73-targeting antibody of the second aspect, the Nanobody-drug conjugate of the present invention, or the CAR-T cell of the Nanobody comprising the steps of: administering to a subject in need the CD73-targeting Nanobody described in the first aspect of the present invention, the present invention
  • the CD73-targeting antibody of the second aspect, the Nanobody-drug conjugate of the present invention, or the CAR-T cell of the Nanobody comprising the steps of: administering to a subject in need the CD73-targeting Nanobody
  • cross-linking reaction of the preparation method is as shown in Figure 24.
  • the reducing reagent in step (1) is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or disulfide threitol (DTT).
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • beta-mercaptoethanol beta-mercaptoethylamine hydrochloride
  • DTT disulfide threitol
  • the buffer is selected from the following group: potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH)/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA ) buffer, disodium hydrogen phosphate-citric acid/sodium chloride (NaCl)/diethyltriaminepentacetic acid (DTPA), boric acid-borax/sodium chloride (NaCl)/diethyltriaminepentacetic acid (DTPA) ), histidine-sodium hydroxide/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), and PBS/diethyltriaminepentaacetic acid (DTPA).
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • DTPA diethyltriaminepentaacetic acid
  • DTPA disodium
  • the volume proportion of the organic solvent in the reaction solution does not exceed 15%.
  • linker drug preliminarily dissolved in 10 mg/ml in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA)
  • ACN acetonitrile
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • DMA diethyl acetamide
  • the drug-antibody conjugation ratio (DAR) of the obtained antibody-drug conjugate is relatively uniform. There are certain differences for DAR NDC, if you need to obtain a sample with better homogeneity, you can further use but are not limited to the following methods for separation and purification: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC).
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • IEC ion exchange chromatography
  • Figure 1 shows the surface plasmon resonance (SPR) analysis to determine the binding affinity constants (KD) of four chimeric nanobodies 3-D7, 5-E11, 4-D04, and 4-B02 with human CD73-ECD.
  • SPR surface plasmon resonance
  • Figure 2 shows the ELISA binding affinity (Binding affinity EC 50 ) of 3-D7, 5-E11, 4-D04, and 4-B02 to CD73-ECD.
  • Figure 3 shows the inhibitory activity (IC50) of 3-D7, 5-E11, 4-D04, and 4-B02 on the catalytic function of recombinant human CD73 enzyme.
  • Figure 4 shows the binding affinity (Binding affinity EC 50 ) detection results of 3-D7, 5-E11, 4-D04, and 4-B02 to MDA-MB-231 ( Figure 4A) or NCI-H1299 ( Figure 4B) cell surface CD73. .
  • Figure 6 shows the binding affinity (EC 50 ) detection results of 3-D7, 4-D04, 5-E11, and 4-B02 to CD73 on the cell surface of SW1573 ( Figure 6A) and NCI-H1373 ( Figure 6B).
  • Figure 7 shows the detection results of the binding affinity (Binding affinity EC 50 ) of 3-D7, 5-E11, 4-D04, and 4-B02 to CD73 on the surface of HCC-44 cells.
  • Figure 8 shows the inhibitory activity of 3-D7, 5-E11, 4-D04, and 4-B02 on the catalytic function of CD73 enzyme on the surface of MDA-MB-231 cells, showing the inhibition curve and IC 50 value.
  • Figure 9 shows that 3-D7, 5-E11, 4-D04, and 4-B02 can effectively reverse the inhibitory effect of adenosine monophosphate (AMP) on the proliferation of human T lymphocytes.
  • the experiment used sorting to obtain CD3+ human T cells, and then cultured them for 5 days to calculate the cell proliferation rate and EC 50 value.
  • AMP adenosine monophosphate
  • Figure 10 shows that 3-D7, 5-E11, 4-D04, and 4-B02 can effectively reverse the inhibitory effect of AMP on the expression of INF-g in human T lymphocytes.
  • the test uses sorting to obtain CD3 + human T cells, and after culturing them for 5 days, the T cell culture supernatant is detected.
  • Figure 12 shows the binding activity of chimeric nanobodies 1-B11, 1-E7, 2-B10, 2-G9, 3-A3, 3-F1 and CD73 on the surface of MDA-MB-231 cells, using HEK293F cells for transient culture. 100 ⁇ L, 10 ⁇ L, and 1 ⁇ L of the supernatant after 2 days were tested.
  • Figure 20A is the SDS-PAGE after the expression and purification of the chimeric Nanobody 5-E11, corresponding to SEQ NO.40, SEQ NO.41, SEQ NO.42;
  • Figure 20B is the SDS-PAGE after the purification of the 5-E11 recombinant fusion protein. Corresponds to SEQ NO.45.
  • Figure 21 is the mass spectrum of the chimeric Nanobody 3-D7-vc-MMAE.
  • Figure 22 is the mass spectrum of the chimeric Nanobody 5-E11-vc-MMAE.
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • single domain antibody VHH
  • nanobody the terms “single domain antibody (VHH)” and “nanobody” have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody, constructing a single domain consisting of only one heavy chain variable region Antibody (VHH), which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and form The binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable 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 natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by 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, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules formed by combining with the antibodies of the present invention or fragments thereof of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the anti-CD73 protein antibody or fragment thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably, and all refer to polypeptides that specifically bind to the CD73 protein, such as proteins or polypeptides with heavy chain variable regions. . They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to the heavy chain of the antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), and the amino acid sequences of the four FRs. Relatively conservative and not directly involved in binding reactions. These CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • the present invention provides a variety of high specificity and high affinity Nanobodies targeting CD73, which only include heavy chains, and the heavy chains contain heavy chain variable region (VH) amino acid sequences (listed in Table-1 and Table-2). out).
  • VH heavy chain variable region
  • the heavy chain variable region (VH) amino acid sequence has CDR1, CDR2, and CDR3 of the following polypeptide sequences:
  • the antibody has the function of inhibiting cell surface and recombinant CD73 protease catalysis, and the antibody can be quickly endocytosed by cells and enter lysosomes.
  • the antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, human-animal chimeric antibodies, preferably humanized antibodies, and more preferably fully humanized antibodies.
  • the antibody derivatives of the present invention can be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and/or modified antibody targeting human CD73.
  • the sequence is located in the CDR region of the variable region (VH).
  • the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and 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 can be 1-7, more preferably 1-5, more preferably 1-3, even more preferably For 1-2 pieces.
  • amino acid sequence number of the heavy chain variable region (VH) of the chimeric antibody is listed in Table 1.
  • the amino acid sequence number of the complementarity determining region (CDR) of the antibody is listed in Table 2.
  • Recombinant protein or fusion protein
  • the recombinant protein (or fusion protein) containing the CD73 Nanobody of the present invention is also included.
  • One preferred fusion protein is a multispecific antibody that further includes a second antigen binding region targeting a target selected from the group consisting of: EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG- 3. FGL1, TLR7, or their combination.
  • the multispecific antibody includes one or more second antigen-binding regions, or further includes a third antigen-binding region.
  • the antibody of the present invention refers to a polypeptide that has CD73 protein binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above-described CDR regions that have the same function as the antibodies of the invention. These variant forms include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also encompasses fragments of the Nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed within the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, infiltration sterilization, sonication, 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.
  • the linker Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) and activated esters, such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • activated esters such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • the oligonucleotide drug is a nucleic acid drug targeting small molecules, including antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), and messenger RNA.
  • ASO antisense nucleic acid
  • siRNA small interfering RNA
  • miRNA microRNA
  • saRNA small activating RNA
  • messenger RNA messenger RNA.
  • mRNA messenger RNA
  • aptamer aptamer
  • ribozyme ribozyme
  • ARC antibody nucleic acid conjugate drug
  • LU is the joint/linker
  • Drugs that can be used to constitute the NDC of the present invention include, but are not limited to: cytotoxic agents (such as cytotoxic small molecule drugs).
  • the drug is the camptothecin derivative SN-38.
  • SN-38 is the biologically active metabolite of irinotecan hydrochloride (CPT-11) and is a type of topoisomerase inhibitor. SN-38 causes the strongest inhibition of DNA topoisomerase I, inhibits DNA synthesis in a dose-dependent and time-dependent manner, and causes frequent DNA single-strand breaks. The structure of SN-38 is shown in the figure below.
  • the drug is the camptothecin derivative Exatecan. It is a synthetic analog of the topoisomerase I inhibitor camptothecin. It is more active than SN-38 and can cause the strongest inhibition of DNA topoisomerase I. It inhibits DNA synthesis in a dose-dependent and time-dependent manner. And cause frequent DNA single-strand breaks.
  • the structure of Exatecan is shown in the following formula.
  • PBD pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD dimers pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD is a type of natural product produced by Streptomyces. Its unique property is its ability to form non-twisted covalent adducts at the minor groove of DNA, specifically at the purine-guanine-purine sequence.
  • the application of PBD as part of a small molecule strategy to target DNA sequences and as a novel anticancer and antibacterial drug has attracted increasing interest.
  • a flexible carbon chain is used to connect the C8/C8' hydroxyl groups of two PBD units, and the resulting dimer has enhanced biological activity.
  • PBD dimers are thought to produce sequence-selective DNA damage, such as inverted 5'-Pu-GATC-Py-3' interstrand cross-links, resulting in their biological activity. These compounds have been shown to be highly potent cytotoxic drugs and may serve as candidates for antibody-drug conjugates.
  • linkers or “linkers of antibody drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.
  • the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by cells, the antibody is enzymatically hydrolyzed in the lysosome, releasing the small molecule drug, connected molecule, an active molecule composed of antibody amino acid residues.
  • the resulting change in the structure of the drug molecule does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect).
  • Cleavable linkers can cleave within target cells and release the active drug (the small molecule drug itself).
  • Cleavable linkers can be divided into two main categories: chemically labile linkers and enzyme-labile linkers. son. Chemically unstable linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc. Linkers that are sensitive to pH are often called acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH7.3-7.5), but will be destroyed in weakly acidic endosomes (pH5.0-6.5) and lysosomes (pH4.5-5.0). hydrolysis.
  • the first generation of antibody drug conjugates mostly used this type of linkers, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers generally have short half-lives (2-3 days). This short half-life limits the application of pH-sensitive linkers in new generation antibody drug conjugates to a certain extent.
  • Glutathione-sensitive linkers are also called disulfide linkers. Drug release is caused by the difference between high intracellular glutathione concentrations (millimolar range) and relatively low glutathione concentrations in the blood (micromolar range). This is especially true for tumor cells, where low oxygen levels lead to increased activity of reductase enzymes and thus higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have good stability in plasma.
  • Self-releasing linkers are generally embedded between the cleavable linker and the active drug, or themselves are part of the cleavable linker.
  • the mechanism of action of the self-releasing linker is: when the cleavable linker is broken under appropriate conditions, the self-releasing linker can spontaneously undergo structural rearrangement, thereby releasing the active drug connected to it.
  • Common suicide linkers include para-aminobenzyl alcohols (PAB) and ⁇ -glucuronides ( ⁇ -Glucuronide).
  • the invention also provides the use of the antibody of the invention, for example, for the preparation of diagnostic preparations or the preparation of medicaments for the prevention and/or treatment of CD73-related diseases.
  • CD73-related diseases include tumor occurrence, growth and/or metastasis, tumor drug resistance-related diseases, inflammation, metabolism-related diseases, etc.
  • the uses of the antibodies, NDC or CAR-T of the present invention include (but are not limited to):
  • the tumors include (but are not limited to): breast cancer (such as triple negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, tuberculosis Rectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc.; especially triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, and malignant glioma, More preferred are triple negative breast cancer and/or non-small cell lung cancer.
  • autoimmune diseases include (but are not limited to): systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, and multiple sclerosis.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis inflammation, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.
  • the metabolism-related diseases include (but are not limited to): diabetes, food-borne obesity, and adipose inflammation.
  • the composition is a pharmaceutical composition, which contains the above The antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention can also be expressed in cells from a nucleotide sequence and used for cell therapy.
  • the antibody can be used for chimeric antigen receptor T cell immunotherapy (CAR-T).
  • 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 tumors and other diseases.
  • other therapeutic agents may be used simultaneously.
  • 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 above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation 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 conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. Additionally, the polypeptides of the invention may be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the Nanobody-drug conjugate provided by the present invention can target a specific cell population and bind to a specific protein (antigen) on the cell surface, thereby making the drug in an active form through the endocytosis of the conjugate or the penetration of the drug. Released into cells, therefore, the Nanobody-drug conjugate of the present invention can be used to treat target diseases.
  • the above-mentioned antibody-drug conjugate can be administered to a subject in a therapeutically effective amount through a suitable route (for example, people).
  • a subject in need of treatment may be a patient at risk for, or suspected of having, a condition related to the activity or expression of a particular antigen. Such patients can be identified through routine physical examination.
  • Nanobody-drug conjugates of the present invention delivery can be performed by conventional methods in the art. For example, it can be introduced into cells through the use of liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector may be delivered locally by direct injection or by use of an infusion pump.
  • the Nanobody of the present invention has excellent biological activity and specificity, has high affinity (its EC 50 is 0.014-0.070 ⁇ g/mL as measured by ELISA), and has inhibitory activity of CD73 enzyme function (its IC is measured by enzyme activity 50 is 4.66 ⁇ 38.18ng/mL). In addition, it has good binding affinity to CD73 of tumor cells (EC 50 measured by FACS is 0.037-0.227 ⁇ g/mL), and inhibits the function of tumor cell CD73 enzyme (IC 50 value is 0.083-0.48 ⁇ g/mL). It can be used Make therapeutic antibodies targeting CD73.
  • Nanobody and NDC of the present invention have significant anti-tumor activity without obvious toxic side effects on normal cells.
  • the Nanobody of the present invention has a significant protective effect on the proliferation of human T lymphocytes, can effectively reverse the proliferation inhibition of T lymphocytes by adenosine monophosphate (AMP), and promote the expression and secretion of INF ⁇ . Its EC 50 is 0.0008 ⁇ 0.0247 ⁇ g/mL.
  • Nanobody-drug conjugate (NDC) of the present invention has excellent CD73-dependent anti-tumor activity, that is, it has no obvious toxic side effects on cells with normal or low expression of CD73-, while it has no obvious toxic side effects on cells with medium or high expression of CD73- of tumor cells It has extremely high cell-killing activity, and its IC 50 is 0.0025 to 0.0658 ⁇ g/mL as determined by the cell proliferation inhibition test.
  • the NDC of the present invention has no obvious toxic or side effects on the proliferation of normal human T lymphocytes, and its IC 50 is >15 ⁇ g/mL as measured by a cell proliferation inhibition test.
  • the NDC of the present invention has a significant protective effect on the proliferation of human T lymphocytes, can effectively reverse the proliferation inhibition of T lymphocytes by adenosine monophosphate (AMP) and promote the expression and secretion of INF ⁇ , and its EC 50 is 0.0046 ⁇ 0.0379 ⁇ g/mL.
  • AMP adenosine monophosphate
  • CD73-ECD extracellular domain of human CD73 protein
  • Alpaca (Alpaca) was immunized using the CD73 extracellular domain protein prepared above. The immunization interval was 21 days. Part of the peripheral blood (PBMC) was collected 10 days after the last immunization. After PBMC isolation and VHH antibody fragment cloning, a phage display library was constructed. and yeast display libraries. Through repeated panning, monoclonal ELISA, FACS screening and other steps, 4 highly active and independent sequence Nanobodies (3-D7, 5-E11, 4-D04, 4-B02) were obtained from the phage display library. The display library obtained 5 active antibodies with independent sequences (1-E7, 2-B10, 2-G9, 3-A3, 3-F1).
  • SPR Surface plasmon resonance
  • CD73-ECD CD73 protein extracellular domain
  • coating solution Dilute the CD73 protein extracellular domain (CD73-ECD) to 1 ⁇ g/mL with coating solution, and coat the ELISA plate with 100 ⁇ L/well at 4°C overnight. Wash away excess antigen, block with 1% BSA for 2 hours at room temperature, and then add 5-fold gradient dilution of Each monoclonal antibody, 100 ⁇ L/well, was incubated at room temperature for 1 h; unbound antibodies were washed away, and horseradish peroxidase-labeled Staphylococcus protein A at an appropriate concentration was added, 100 ⁇ L/well, and incubated at room temperature for 0.5 h. Wash away the unbound secondary antibody, add TMB chromogenic solution and react for about 15 minutes. Add 1M HCL, 50 ⁇ L/well, to terminate the chromogenic reaction, then measure the absorbance at 450 nm and analyze the data.
  • 3-D7, 5-E11, 4-D04, and 4-B02 have strong affinity for CD73-ECD, with EC 50 of 0.070 ⁇ g/mL, 0.052 ⁇ g/mL, and 0.035 ⁇ g respectively. /mL, 0.014 ⁇ g/mL.
  • Nanobodies 1-E7, 2-B10, 2-G9, 3-A3, and 3-F1 all have strong CD73-ECD binding affinity.
  • CD73 extracellular domain Dilute the human recombinant CD73 enzyme (CD73 extracellular domain) to 0.1 ⁇ g/mL with antigen diluent, and spread it evenly into a 96-well low-adsorption culture plate, 25 ⁇ L/well.
  • test results are shown in Figure 3.
  • 3-D7, 5-E11, 4-D04, and 4-B02 all have the ability to significantly inhibit the hydrolysis of AMP by recombinant CD73 protease, and their IC 50 are 38.18ng/mL, 9.47ng/mL, respectively. 5.63ng/mL, 4.66ng/mL.
  • the above results show that the CD73 monoclonal antibody in this example has significant inhibitory activity on the catalytic function of recombinant human CD73 enzyme.
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to MDA-MB-231, with EC 50 of 0.120 ⁇ g/mL, 0.037 ⁇ g/mL, and 0.059 respectively. ⁇ g/mL, 0.037 ⁇ g/mL;
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to NCI-H1299, with EC 50 of 0.222 ⁇ g/mL, 0.068 ⁇ g/mL, and 0.080 ⁇ g/mL respectively.
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to PC9, with EC 50 of 0.133 ⁇ g/mL, 0.041 ⁇ g/mL, 0.057 ⁇ g/mL, respectively. 0.044 ⁇ g/mL;
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to NCI-H1975, with EC 50 of 0.106 ⁇ g/mL, 0.057 ⁇ g/mL, and 0.068 ⁇ g/mL respectively.
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to SW1573, with EC 50 of 0.116 ⁇ g/mL, 0.062 ⁇ g/mL, 0.067 ⁇ g/mL, respectively. 0.061 ⁇ g/mL;
  • 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinity to HC44, with EC 50 of 0.234 ⁇ g/mL, 0.096 ⁇ g/mL, 0.114 ⁇ g/mL, respectively. 0.105 ⁇ g/mL;
  • Nanobodies 1-E7, 2-B10, 2-G9, 3-A3, and 3-F1 all have strong CD73-ECD binding affinity.
  • the above results show that the CD73 Nanobody of this embodiment can target CD73 of human tumor cells and has good binding activity.
  • CD73-high-expressing triple-negative breast cancer cell MDA-MB-231 was used as the target cell.
  • 3-D7, 5-E11, 4-D04, and 4-B02 can all significantly inhibit the function of CD73 on the surface of MDA-MB-231 cells to catalyze the hydrolysis of AMP, with IC 50 of 0.483 ⁇ g/mL respectively. 0.088 ⁇ g/mL, 0.093 ⁇ g/mL, 0.083 ⁇ g/mL.
  • T lymphocyte proliferation test The T lymphocytes obtained by the above sorting were fluorescently labeled, and the pre-prepared CFSE (carboxyfluorescein succinimidyl ester) was added to the cell suspension (final concentration 2.5 ⁇ M), and then labeled at 37°C for 5 minutes. Wash 3 times with PBS.
  • CFSE carboxyfluorescein succinimidyl ester
  • FACS flow cell sorter
  • CD73 Nanobodies 3-D7, 5-E11, 4-D04, and 4-B02 have a significant protective effect on the proliferation of human T lymphocytes, and can effectively reverse the inhibition of T lymphocyte proliferation by AMP.
  • Their EC 50 are 0.0247 ⁇ 0.0052 ⁇ g/mL, 0.0008 ⁇ 0.0002 ⁇ g/mL, 0.0025 ⁇ 0.0007 ⁇ g/mL, and 0.0012 ⁇ 0.0004 ⁇ g/mL respectively.
  • CD73 nanobodies 3-D7, 5-E11, 4-D04, and 4-B02 can effectively reverse the inhibitory effect of AMP on T cell expression/secretion of INF- ⁇ .
  • the above results show that the CD73 Nanobody of this example can not only inhibit the CD73 enzyme activity on the surface of tumor cells, but also inhibit the enzyme activity of CD73 on the surface of T cells, reverse the impact of AMP on T cell proliferation and function, and protect T cells.
  • the 3-D07 protein stock solution was mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer. Dilute to 5 mg/mL, add 3-4 times excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stir the reaction solution at 25°C for 2.5 hours.
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriaminepentaacetic acid
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the coupling reaction mixture was purified by pH 6.0 histidine-acetic acid/sucrose gel filtration using a desalting column, and peak samples were collected based on the UV280 ultraviolet absorption value. Then it was sterilized through a filtration device with a pore size of 0.22 microns and stored at -80°C.
  • the 5-E11 protein stock solution was mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer. Dilute to 5 mg/mL, add 3-4 times excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stir the reaction solution at 25°C for 2.5 hours.
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriaminepentaacetic acid
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the coupling reaction mixture was purified by pH 6.0 histidine-acetic acid/sucrose gel filtration using a desalting column, and peak samples were collected based on the UV280 ultraviolet absorption value. Then it was sterilized through a filtration device with a pore size of 0.22 microns and stored at -80°C.
  • the 4-D04 protein stock solution was mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer. Dilute to 5 mg/mL, add 3-4 times excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stir the reaction solution at 25°C for 2.5 hours.
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriaminepentaacetic acid
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the coupling reaction mixture was purified by pH 6.0 histidine-acetic acid/sucrose gel filtration using a desalting column, and peak samples were collected based on the UV280 ultraviolet absorption value. Then it was sterilized through a filtration device with a pore size of 0.22 microns and stored at -80°C.
  • the cell lines used in the examples were purchased from the American Type Culture Collection (ATCC), the Cell Bank of the Chinese Academy of Sciences, and Nanjing Kebai Biotechnology Co., Ltd., and were cultured according to the corresponding instructions, including: MDA-MB-453, MDA-MB- 231, PC9, NCI-H1975, NCI-H441, SW1573.
  • the above cells in the logarithmic growth phase were seeded into a 96-well cell culture plate at a density of 200-2000 cells per well (depending on the growth rate of different cells), 150 ⁇ L/well, 37°C, 5% CO 2 After culturing for about 5 hours, add different concentrations of CD73-NDCs (15 ⁇ g/mL ⁇ 0.00019 ⁇ g/mL).
  • cytotoxicity (IC50 value) of CD73-NDC showed that the cytotoxic activity of CD73-NDC was directly related to the CD73 expression level of the test cells, and therefore was judged to be CD73 target-specific cytotoxicity.
  • Table-3 summarizes the IC50 values of some cell proliferation inhibition tests.
  • PBMC Human peripheral blood mononuclear cell cryopreservation tubes were provided by Jiangsu Xidier Biotechnology Co., Ltd.
  • PBMC peripheral blood mononuclear cell
  • a sorting kit (supplier: Stemcell, Cat#1795) to sort out CD3-positive T lymphocytes and then fluorescently label them.
  • the pre-prepared fluorescent dye CFSE (carboxyfluorescein succinimidyl ester) was added to the cell suspension (final concentration 2.5 ⁇ M), incubated at 37°C for 5 min, and washed three times with PBS before use in the experiment.
  • FACS flow cell sorter
  • CD73-NDC not only has no toxic effect on T lymphocytes, but also can effectively reverse the inhibitory effect of AMP/Ado on the proliferation of T lymphocytes.
  • CD73-NDC 3-D7-MMAE and 5-E11-MMAE both have good CD73-specific tumor cell killing activity, that is, they have a strong inhibitory effect on the proliferation of tumor cells with medium and high expression of CD73-, and have a strong inhibitory effect on CD73-low expression.
  • the expressed cells proliferate without obvious toxicity;
  • CD73-NDC 3-D7-MMAE and 5-E11-MMAE have no obvious toxicity against the cell proliferation of normal human T lymphocytes;
  • CD73-NDC 3-D7-MMAE and 5-E11-MMAE not only have no toxic effect on T lymphocytes, but can also effectively reverse the inhibitory effect of AMP/Ado on the proliferation of T lymphocytes;
  • chimeric Nanobodies and recombinant fusion proteins derived from 3-D7, 5-E11 can be expressed, produced and purified with high efficiency.

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Abstract

本发明公开了一种新的靶向CD73的纳米抗体(nanobody,Nb)及纳米抗体-药物偶联物(nanobody-drug conjugate,NDC)、其制备方法和用途。本发明的单克隆纳米抗体以及相应的NDC能够高效、高特异性地结合纯化的CD73蛋白、多种肿瘤细胞以及免疫细胞表面的CD73，阻断CD73酶的催化活性，其具有很高的亲和力及很低的免疫原性，并且具有显著抗肿瘤作用。

Description

靶向CD73的纳米抗体及纳米抗体-药物偶联物、其制备方法和用途 技术领域
本发明涉及医药领域，尤其涉及靶向CD73的纳米抗体(CD73-Nb)及CD73纳米抗体-药物偶联物(Nanobody-drug conjugate,CD73-NDC)、其制备方法和用途。
背景技术
近年来，针对肿瘤的治疗已经取得了显著进展，其中靶向治疗和免疫疗法为肿瘤治疗提供了新的思路，然而患者对药物的低响应率和快速耐药使得肿瘤的治疗依然面临大的挑战，因此寻找新的治疗靶点和药物成为治疗难治型肿瘤的方向。近期研究表明，肿瘤进展和耐药的发生往往与肿瘤的转移、复发和免疫抑制性的微环境有关，而CD73是与之密切相关的分子机制之一。
CD73异常表达于多种肿瘤，包括肺癌，乳腺癌，黑色素瘤和脑胶质瘤等，与肿瘤的转移、复发、耐药和免疫抑制密切相关。CD73是锚定于细胞膜表面的5`-核苷酸酶，是通过酶促反应将一磷酸腺苷脱磷酸(AMP)生成腺苷(ADO)的主要限速酶。腺苷通过结合相应的腺苷受体(A1AR、A2AR、A2BR、A3AR)参与体内多种生理和病理进程，包括形成免疫抑制性的微环境，如抑制T淋巴细胞的增殖和功能，抑制树突状细胞(dendritic cells，DC)的分化和成熟，影响巨噬细胞的极化，促进免疫抑制细胞如骨髓来源的抑制性细胞(Myeloid-derived suppressor cells，MDSC)释放抗炎细胞因子，如TGFβ、IL-10等。耐药是影响肿瘤治疗效果的极大障碍，研究表明CD73参与了肿瘤化疗药物、小分子靶向药物以及免疫检查点抑制剂的耐药。此外，CD73还参与了肿瘤的上皮间质转化、血管新生、转移和复发等进程。因此，通过靶向CD73，通过单药或联合用药，可为肿瘤治疗提供全新的策略。
单克隆抗体药物已经为癌症治疗带来了诸多改善，然而，传统单抗的分子量为150kd，使其实体瘤的穿透性和药物分布受限，治疗效果还有待进一步提高。纳米抗体(Nb)是羊驼自然产生的仅由重链组成的抗体，其中，目标识别模块由单个可变区(VHH)组成，分子量仅为12-14kd，有望克服传统单抗实体瘤穿透性低的缺陷。纳米抗体可与Fc等功能结构域、其他纳米体、或多肽标签或毒素进行连接。由于其体积小，具有更高的扩散率，血管通透性以及肿瘤穿透力等特点，比传统的单克隆抗体具有更均匀的组织分布。这些特性使它们特别适合于体内特定和有效的肿瘤靶向治疗。并且纳米抗体还具有免疫原性低、稳定性高、降解抗性高、原核生物表达生产成本低、产品表征容易等优势。不过，仅管众多潜在优势，基于纳米抗体的肿瘤治疗药物开发迄今尚处于早期探索研究阶段。
抗体-药物偶联物(Antibody-drug conjugate，ADC)，是利用单克隆抗体特异性识别肿瘤细胞表面特定抗原的特点，从而实现精准地将抗肿瘤药物(如小分子化疗药物等)递送到肿瘤靶细胞并释放，达到精准杀伤肿瘤的目的。ADC目前已用于靶向给药至肿瘤组织，但由于其分子量大，瘤内穿透力较弱等限制，导致在肿瘤内给药效果差异大，实体瘤应用不理想。并且ADC在体内的终末半衰期长，这可导致荷载毒素对正常组织或细胞产生不必要的毒性。而通过纳米抗体制备的纳米抗体-药物偶联物(nanobody-drug conjugate,NDC)不仅保留了传统ADC的优点，还具有血管通透性高、肿瘤穿透力强、到达靶细胞速度快，可以提高药物在肿瘤的累积，同时还可适度控制药物的血浆暴露量及半衰期，有助于进一步提高对实体瘤治疗效果和整体治疗窗，有望成为最具潜力的一类全新的抗肿瘤药物。
综上所述，基于CD73在肿瘤发展和复发耐药等机制中的重要作用，开发靶向CD73-Nb及CD73-NDC药物能够为临床上单独或联合治疗CD73异常表达的肿瘤患者提供新的治疗策略。
发明内容
本发明的目的在于提供一种靶向CD73的纳米抗体及纳米抗体-药物偶联物、其制备方法和用途。
在本发明的第一方面，提供了一种靶向CD73的纳米抗体，所述纳米抗体的VHH链的互补决定区CDR为选自下组的一种或多种：
(1)SEQ ID NO.1所示的CDR1，
SEQ ID NO.2所示的CDR2，和
SEQ ID NO.3所示的CDR3；
或者，
(2)SEQ ID NO.5所示的CDR1，
SEQ ID NO.6所示的CDR2，和
SEQ ID NO.7所示的CDR3；
或者，
(3)SEQ ID NO.9所示的CDR1，
SEQ ID NO.10所示的CDR2，和
SEQ ID NO.11所示的CDR3，
或者，
(4)SEQ ID NO.13所示的CDR1，
SEQ ID NO.14所示的CDR2，和
SEQ ID NO.15所示的CDR3，
或者，
(5)SEQ ID NO.17所示的CDR1，
SEQ ID NO.18所示的CDR2，和
SEQ ID NO.19所示的CDR3，
或者，
(6)SEQ ID NO.21所示的CDR1，
SEQ ID NO.22所示的CDR2，和
SEQ ID NO.23所示的CDR3，
或者，
(7)SEQ ID NO.25所示的CDR1，
SEQ ID NO.26所示的CDR2，和
SEQ ID NO.27所示的CDR3，
或者，
(8)SEQ ID NO.29所示的CDR1，
SEQ ID NO.30所示的CDR2，和
SEQ ID NO.31所示的CDR3，
或者，
(9)SEQ ID NO.33所示的CDR1，
SEQ ID NO.34所示的CDR2，和
SEQ ID NO.35所示的CDR3。
在另一优选例中，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，所述的纳米抗体VHH链的CDR区包含与SEQ ID NO:1-36中任一具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的纳米抗体VHH链CDR区的氨基酸序列与SEQ ID NO:1-36中任一相比包含一个或多个氨基酸取代，优选保守氨基酸取代。
在另一优选例中，所述VHH链包含选自以下组合的CDRl、CDR2和CDR3：
(1)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体3-D7的CDR)；或
(2)SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体5-E11的CDR)；或
(3)SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体4-D04的CDR)；或
(4)SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体4-B02的CDR)；或
(5)SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体1-E7的CDR)；或
(6)SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体2-B10的CDR)；或
(7)SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体2-G9的CDR)；或
(8)SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体3-A3的CDR)；或
(9)SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35所示的互补决定区CDR1、CDR2、CDR3(对应纳米抗体3-F1的CDR)。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述纳米抗体的VHH链还包括框架区(FR)。
在另一优选例中，所述的框架区FR为人源、鼠源、兔源或骆驼源。
在另一优选例中，所述的框架区FR包括人源的FR区、鼠源或骆驼源的FR区。
在另一优选例中，所述靶向CD73的纳米抗体的VHH链具有如SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.12、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.24、SEQ ID NO.28、SEQ ID NO.32或SEQ ID NO.36所示的氨基酸序列。
在本发明的第二方面，提供了一种靶向CD73的抗体，所述抗体包括一个或多个如本发明第一方面所述的靶向CD73的纳米抗体的VHH链。
在另一优选例中，所述靶向CD73的纳米抗体的VHH链具有如SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.12、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.24、SEQ ID NO.28、SEQ ID NO.32或SEQ ID NO.36所示的氨基酸序列。
在另一优选例中，所述抗体为单体、二价抗体、和/或多价抗体。
在另一优选例中，所述抗体为动物源抗体、人源化抗体、嵌合抗体或嵌合抗原受体抗体(CAR)。
在另一优选例中，所述人源化抗体的CDR区包含1、2、或3个氨基酸的变化。
在另一优选例中，所述的动物为非人哺乳动物，较佳地为鼠、羊、兔、骆驼。
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，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
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，不超过初始氨基酸序列总氨基酸数量的40％，较佳地为20％，更佳地为10％。
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1-7个，较佳地为1-3个，更佳地为1个。
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
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的衍生序列具有抑制细胞表面CD73或重组CD73蛋白酶催化功能。
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，所述纳米抗体对人CD73蛋白胞外区(CD73-ECD)ELISA结合亲和力EC50为0.014～0.07μg/mL(0.175～0.875nM)，或者经ForteBio表面等离子体共振(SPR)测定的结合常数KD值为0.795～1.997nM。
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催化腺苷一磷酸(AMP)水解生成腺苷的活性；
(b)特异结合肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的CD73；
(c)抑制肿瘤/肿瘤微环境CD73催化AMP水解的活性；
(d)抑制肿瘤细胞迁移或转移；
(e)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(f)促进免疫细胞的增殖存活及功能，从而提高肿瘤免疫的效果；
(g)结合肿瘤细胞后被内吞至细胞内溶酶体。
在本发明的第三方面，提供了一种多特异性抗体，所述的多特异性抗体包含：本发明第一方面所述的靶向CD73的纳米抗体或本发明第二方面所述的靶向CD73的抗体。
在另一优选例中，多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：EGFR、TGFβ、BCMA、B7H6、GUCY2C、DLL3、CD38、CD123、CD19、CD20、CD22、B7-H3、GPC3、HER2、PMSA、CD28、4-1BB、OX40、CD40、CD27、CD3、CTLA4、PD1、PDL1、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、TLR7，或其组合。
在另一优选例中，所述的第二抗原结合区为纳米抗体。
在另一优选例中，所述多特异性抗体包括一个或多个第二抗原结合区。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
在另一优选例中，所述抗原结合区为抗体或抗体片段，所述抗体片段包括：(i)Fab片段；(ii)F(ab')2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段。
本发明的第四方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的靶向CD73的纳米抗体、或如本发明第二方面所述的抗 CD73抗体；以及
(ii)任选的具有治疗功能的多肽分子或片段；和/或
(iii)任选的提升蛋白理化性质或成药性的功能域。
在另一优选例中，所述提升蛋白理化性质或成药性包括延长靶向CD73的纳米抗体的半衰期。
在另一优选例中，所述重组蛋白还包括：(iv)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列选自下组：6His标签、GGGS序列、FLAG标签。
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，所述具有治疗功能的多肽分子或片段包括但不限于：靶向EGFR、TGFβ、BCMA、B7H6、GUCY2C、DLL3、CD38、CD123、CD19、CD20、CD22、B7-H3、GPC3、HER2、PMSA、CD28、4-1BB、OX40、CD40、CD27、CD3、CTLA4、PD1、PDL1、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、TLR7的多肽分子或片段。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括但不限于：胰岛素、IL-2、干扰素、降钙素、GHRH肽、肠肽类似物、白蛋白、抗体片段、细胞因子。
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，所述的融合蛋白包括多特异性抗体、嵌合抗体。
在另一优选例中，所述提升蛋白理化性质或成药性的功能域包括Fc段、抗白蛋白纳米抗体(HLE)、白蛋白结合域(ABD)。
在另一优选例中，所述的融合蛋白从N-C端具有如下元件：
A-B；
其中，A元件为靶向CD73的纳米抗体；B元件为Fc段或白蛋白；
“-”代表肽键。
在另一优选例中，所述靶向CD73的纳米抗体的VHH链选自下组：SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.12、SEQ ID NO.16、SEQ ID NO.20、SEQ ID NO.24、SEQ ID NO.28、SEQ ID NO.32或SEQ ID NO.36所示的氨基酸序列。
在另一优选例中，所述Fc段为人IgG Fc段。
在另一优选例中，所述Fc段的氨基酸序列选自下组：SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42所示的氨基酸序列。
在另一优选例中，所述的融合蛋白选自下组：3-D7、5-E11、4-D04、4-B02、1-E7、2-B10、2-G9、3-A3、3-F1、3-D7-FC1、3-D7-FC2、3-D7-FC3、5-E11-FC1、5-E11-FC2、5-E11-FC3、3-D7-HLE、3-D7-ABD、5-E11-ABD。
本发明的第五方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域为如本发明第一方面所述的纳米抗体的VHH链。
本发明的第六方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明的第五方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：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)。
本发明的第七方面，提供了一种免疫偶联物，所述的免疫偶联物含有：
(a)抗体部分，所述抗体部分为本发明第一方面所述的靶向CD73的纳米抗体或本发明第二方面所述的靶向CD73的抗体；和
(b)与所述纳米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、酶、蛋白降解剂、寡核苷酸或其组合。
在另一优选例中，所述免疫偶联物为纳米抗体药物偶联物。
在另一优选例中，所述的纳米抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在另一优选例中，所述的偶联部分为化学标记和生物标记。
在另一优选例中，所述化学标记为同位素、免疫毒素和/或化学药物。
在另一优选例中，所述生物标记为生物素、亲和素或酶标记。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：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、或其组合。
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))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述可检测标记物包括放射性核素，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述蛋白降解剂为肿瘤相关蛋白的降解剂，所述肿瘤相关蛋白选自下组：EGFR、NF-κB、RIPK2、BCR-ABL、HER2、c-Met、TBK1、CDK、ALK、Akt、CK2、ERK1/2、FLT3、PI3K、BTK、TRK、Fak、BRD、AR、ER、MetAp-2、BCL-XL、Sirt2、HDAC6、Pirin、SMAD3、ARNT、PCAF/GCN5、Tau、EZH2、IRAK4、 STAT3FRS2、RAS(例如KRAS、HRAS和NRAS)。
在另一优选例中，所述KRAS包括KRAS-G12C、KRAS-G12D。
在另一优选例中，所述蛋白降解剂包括PROTAC(蛋白降解靶向嵌合体)。
在另一优选例中，所述PROTAC靶向EGFR、KRAS(包括KRAS-G12C、KRAS-G12D)。
在另一优选例中，所述寡核苷酸包括反义寡核苷酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)或RNA适配(Aptamer)。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明的第一方面所述的靶向CD73的纳米抗体或本发明的第二方面所述的靶向CD73的抗体。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明的第一方面所述的靶向CD73的纳米抗体或本发明的第二方面所述的靶向CD73的抗体。
在另一优选例中，所述的检测为体内检测或体外检测。
在另一优选例中，所述免疫偶联物用于诊断和/或治疗表达CD73蛋白的肿瘤。
在另一优选例中，所述抗体药物偶联物ADC如下分子式所示：
其中：
nAb是靶向CD73的纳米抗体，
LU是接头(又称连接子)；
D是药物；
而且下标p是选自1-8的值。
在另一优选例中，LU选自马来酰亚胺基己酰基(MC)、马来酰亚胺(MAL)、琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-甲酸酯)(SMCC)接头与抗体部分连接，并包含缬氨酸-瓜氨酸(VC)、缬氨酸-丙氨酸(VA)、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(GGFG)、丙氨酸-丙氨酸-丙氨酸(AAA)、对氨基苄氧基羰基(PAB)、聚乙二醇(PEG)的一种或多种的连接子。
在另一优选例中，其中所述抗体通过与选自下组的部分反应共价结合至所述接头：马来酰亚胺基己酰基(MC)、马来酰亚胺(MAL)、琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-甲酸酯)(SMCC)等。
在另一优选例中，其中D选自下组具有抗肿瘤活性的化合物：
(i)微管蛋白抑制剂，如美登素衍生物(DM1、DM4)，单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF)；
(ii)作用于DNA毒素，如倍癌霉素(duocarmycin)、吡咯并苯二氮卓(PBD)；
(iii)拓扑异构酶抑制剂，喜树碱、SN38、依喜替康、Dxd。
在另一优选例中，其中所述LU-D化合物选自下组：
本发明的第八方面，提供了一种药物组合物，所述的药物组合物包含：
(i)如本发明的第一方面所述的靶向CD73的纳米抗体、如本发明的第二方面所述的靶向CD73的抗体、如本发明的第三方面的多特异性抗体、如本发明的第四方面所述的重组蛋白、如本发明的第六方面所述的重组的免疫细胞或如本发明的第七方面所述的免疫偶联物；
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物还包含其他生物活性物质，如治疗肿瘤的药物。
在另一优选例中，所述的药物组合物的施用方式选自下组：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述的药物组合物的剂型选自下组：液态、固体、或凝胶态。
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。
本发明的第九方面，提供了一种活性成分的用途，所述的活性成分选自下组：如本发明的第一方面所述的靶向CD73的纳米抗体、如本发明的第二方面所述的靶向CD73的抗体、如本发明的第三方面的多特异性抗体、如本发明的第四方面所述的重组蛋白、如本发明的第六方面所述的重组的免疫细胞、如本发明的第七方面所述的免疫偶联物、或其组合，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗CD73相关疾病的药物。
在另一优选例中，所述检测试剂、检测板或试剂盒用于：
(1)检测样品中的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。
在另一优选例中，所述的检测类型，包括但不限于用于流式检测、细胞免疫荧光检测、酶联免疫吸附检测、免疫印迹检测等。
在另一优选例中，所述的检测试剂、检测板或试剂盒用于诊断CD73相关疾病。
在另一优选例中，所述的药物用于治疗或预防CD73高表达的肿瘤、肿瘤迁移、或肿瘤耐药。
在另一优选例中，所述的肿瘤耐药包括：肿瘤免疫治疗药物的耐药、肿瘤靶向治疗药物的耐药、常规肿瘤化疗的耐药，放射治疗的不敏感。
在另一优选例中，所述的药物用于选自下组的用途：
(a)抑制CD73催化腺苷一磷酸(AMP)水解生成腺苷的活性；
(b)特异结合肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的CD73；
(c)抑制肿瘤/肿瘤微环境CD73催化AMP水解的活性；
(d)抑制肿瘤细胞迁移或转移；
(e)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(f)促进免疫细胞的增殖、存活及功能，从而提高肿瘤免疫的效果。
在另一优选例中，所述CD73相关疾病选自下组：癌症、自身免疫疾病、代谢相关疾病、感染疾病、或其组合。
在另一优选例中，所述CD73相关的疾病包括：肿瘤的发生、生长和/或转移。
在另一优选例中，所述的癌症包括实体瘤、血液癌。
在另一优选例中，所述的癌症为CD73高表达的肿瘤。
在另一优选例中，所述的CD73高表达的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、卵巢癌、前列腺癌癌、直肠癌、脑胶质瘤、黑色素瘤、白血病、淋巴瘤、或其组合。
在另一优选例中，所述的癌症为耐药性肿瘤。
在另一优选例中，所述的CD73高表达的肿瘤指肿瘤组织中CD73转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
本发明的第十方面，提供了一种多核苷酸，其特征在于，所述的多核苷酸编码选自下组的多肽：
(1)本发明的第一方面所述的靶向CD73的纳米抗体、本发明的第二方面所述的靶向CD73的抗体、或如本发明的第三方面的多特异性抗体；
(2)如本发明的第四方面所述的重组蛋白；或
(3)如本发明的第五方面所述的CAR构建物。
在另一优选例中，所述多核苷酸包括RNA、DNA或cDNA。
本发明的第十一方面，提供了一种载体，其特征在于，所述的载体含有如本发明的第十方面所述的多核苷酸。
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。
本发明的第十二方面，提供了一种遗传工程化的宿主细胞，其特征在于，所述的宿主细胞含有本发明的第十一方面所述的载体或基因组中整合有本发明的第十方面 所述的多核苷酸。
本发明的第十三方面，提供了一种体外检测(包括诊断性或非诊断性)样品中CD73的方法，其特征在于，所述方法包括步骤：
(1)在体外，将所述样品与本发明的第一方面所述的靶向CD73的纳米抗体、本发明的第二方面所述的靶向CD73的抗体或如本发明的第七方面所述的免疫偶联物接触；
(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。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
本发明的第十四方面，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，所述的测试条含有本发明第一方面所述的靶向CD73的纳米抗体或本发明第二方面所述的靶向CD73的抗体或本发明第七方面所述的免疫偶联物。
本发明的第十五方面，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第一方面所述的靶向CD73的纳米抗体或本发明第二方面所述的靶向CD73的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明第一方面所述的靶向CD73的纳米抗体或本发明第二方面所述的靶向CD73的抗体的二抗；
或者，所述试剂盒含有本发明第十四方面所述的检测板。
本发明的第十六方面，提供了一种重组多肽的制备方法，其特征在于，所述方法包括：
(a)在适合表达的条件下，培养本发明的第十二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一方面所述的靶向CD73的纳米抗体、如本发明的第二方面所述的靶向CD73的抗体、如本发明的第三方面的多特异性抗体、如本发明的第四方面所述的重组蛋白。
本发明的第十七方面，提供了一种治疗CD73相关疾病的方法，其特征在于，所述方法包括：给需要的对象施用如本发明的第一方面所述的靶向CD73的纳米抗体、如本发明的第二方面所述的靶向CD73的抗体、如本发明的第三方面的多特异性抗体、如本发明的第四方面所述的重组蛋白、如本发明的第六方面所述的重组的免疫细胞、如本发明的第七方面所述的免疫偶联物、如本发明的第八方面所述的药物组合物、或其组合。
在另一优选例中，所述的方法还包括：给需要的对象施用其他药物或治疗方法进行联合治疗。
在另一优选例中，所述的其他药物或治疗方法包括：抗肿瘤免疫治疗药物、肿瘤靶向药物、肿瘤化疗药物、肿瘤放射治疗。
在另一优选例中，所述的抗肿瘤免疫治疗药物包括PD-1、PD-L1单抗。
在本发明的第十八方面，提供了一种制备嵌合抗体的方法，包括步骤：
将本发明第一方面所述的靶向CD73的纳米抗体的羊驼来源的VHH序列的核苷酸序列克隆入含有人抗体恒定区的核苷酸序列的表达载体后，通过转染动物细胞表达人 -羊驼嵌合抗体。
在本发明的第十九方面，提供了一种制备人源化抗体的方法，包括步骤：
将本发明第一方面所述的靶向CD73的纳米抗体的VHH链的CDR区的核苷酸序列植入含人源抗体FR区的核苷酸序列模板，再将其克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。
在本发明的第二十方面，提供了一种抑制肿瘤细胞生长和迁移的方法，包括步骤：给需要的对象施用本发明的第一方面所述的靶向CD73的纳米抗体、本发明的第二方面所述的靶向CD73的抗体或本发明所述的纳米抗体-药物偶联物、或所述纳米抗体的CAR-T细胞、或其组合。
在本发明的第二十一方面，提供了一种抑制肿瘤在模型动物体内生长的方法，包括步骤：给需要的对象施用本发明的第一方面所述的靶向CD73的纳米抗体、本发明的第二方面所述的靶向CD73的抗体、本发明所述的纳米抗体-药物偶联物、或所述纳米抗体的CAR-T细胞。
在另一优选例中，所述药物可以实施单独给药，或组合用药包括肿瘤免疫疗法、肿瘤靶向药物、细胞毒药物、放射治疗。
在本发明的第二十二方面，提供了一种本发明的第七方面所述的纳米抗体-药物偶联物的制备方法，包括步骤：
(1)用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
(2)用式DL连接子-药物缀合物与步骤(1)中得到的经还原后的抗体在缓冲液与有机溶剂混合液中进行交联(偶联)，得到具有不同DAR值的抗体-药物偶联物1a、1b、1c或1d。
在另一优选例中，所述制备方法的交联反应如图24所示。
在另一优选例中，所述步骤(1)中所述抗体经还原试剂还原，从而使抗体链间二硫键被还原，产生巯基基团。
在另一优选例中，所述步骤(1)中还原试剂为三(2-羧乙基)膦盐酸盐(TCEP)、beta-巯基乙醇、beta-巯基乙胺盐酸盐、或二硫苏糖醇(DTT)。
在另一优选例中，所述的缓冲液选自下组：磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)缓冲液、磷酸氢二钠-柠檬酸/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)、硼酸-硼砂/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)、组氨酸-氢氧化钠/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)，和PBS/二乙基三胺五乙酸(DTPA)。
在另一优选例中，所述的步骤(2)中，有机溶剂在反应液中的体积占比不超过15％。
在另一优选例中，所述的步骤(2)中的有机溶剂选自下组：乙腈(ACN)、二甲基甲酰胺(DMF)、二甲基乙酰胺(DMA)、二甲基亚砜(DMSO)。
再加入连接子药物(预先10mg/ml溶在乙腈(ACN)、二甲亚砜(DMSO)、二甲基甲酰胺(DMF)或二乙基乙酰胺(DMA)中)，并保证反应液中有机溶剂的体积占比不超过15％，偶联反应于0-37℃搅动2-4小时。若采用TCEP还原，也可不需除去剩余TCEP，直接加入取代马来酰亚胺类化合物进行偶联。
采用脱盐柱将偶联反应混合物用琥珀酸钠/NaCl缓冲液或组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。或超滤数遍。然后过滤除菌，所得产物低温保存。
所得抗体药物偶联物的药物抗体偶联比(DAR)较为均一。对于DAR有一定差别的 NDC，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
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为表面等离子体共振(SPR)分析测定4个嵌合纳米抗体3-D7，5-E11，4-D04，4-B02与人CD73-ECD的结合亲和力常数(KD)。
图2为3-D7，5-E11，4-D04，4-B02对CD73-ECD的ELISA结合亲和力(Binding affinity EC50)。
图3为3-D7，5-E11，4-D04，4-B02对重组人CD73酶催化功能的抑制活性(IC50)。
图4为3-D7，5-E11，4-D04，4-B02对MDA-MB-231(图4A)或NCI-H1299(图4B)细胞表面CD73的结合亲和力(Binding affinity EC50)检测结果。
图5为3-D7，5-E11，4-D04，4-B02对PC9(图5A)和NCI-H1975(图5B)细胞表面CD73的结合亲和力(Binding affinity EC50)检测结果。
图6为3-D7，4-D04，5-E11，4-B02对SW1573(图6A)和NCI-H1373(图6B)细胞表面CD73的结合亲和力(Binding affinity EC50)检测结果。
图7为3-D7，5-E11，4-D04，4-B02对HCC-44细胞表面CD73的结合亲和力(Binding affinity EC50)检测结果。
图8为3-D7，5-E11，4-D04，4-B02对MDA-MB-231细胞表面CD73酶催化功能的抑制活性，展示抑制曲线和IC50值。
图9为3-D7，5-E11，4-D04，4-B02能有效逆转腺苷单磷酸(AMP)对人T淋巴细胞的增殖抑制作用。试验采用分选获得CD3+人T细胞，将其培养5天后统计细胞增殖率和EC50值。
图10为3-D7，5-E11，4-D04，4-B02能有效逆转AMP对人T淋巴细胞INF-g表达的抑制作用。试验采用分选获得CD3+人T细胞，将其培养5天后检测T细胞培养上清。
图11为嵌合纳米抗体1-B11、1-E7、2-B10、2-G9、3-A3、3-F1与人CD73-ECD的ELISA结合活性，采用HEK293F细胞瞬转培养2天后的上清100μL、10μL、1μL进行检测。
图12为为嵌合纳米抗体1-B11、1-E7、2-B10、2-G9、3-A3、3-F1与MDA-MB-231细胞表面CD73的结合活性，采用HEK293F细胞瞬转培养2天后的上清100μL、10μL、1μL进行检测。
图13为嵌合纳米抗体3-D7，5-E11，4-D04，4-B02与MDA-MB-231细胞结合导致内吞(Internalization)至细胞内溶酶体。所述抗体(5μg/mL)和细胞孵育4℃1小时，或37℃4小时后置于荧光显微镜显微镜观察结果。
图14为嵌合纳米抗体-药物偶联物(NDC)3-D7-MMAE，5-E11-MMAE以及对照物Hu001-MMAE(CD73-ADC)对乳腺癌细胞MDA-MB-453(图14A)和MDA-MB-231(图14B)的增殖抑制活性的检测结果。
图15为3-D7-MMAE，5-E11-MMAE以及对照物Hu001-MMAE对肺癌细胞PC9(图15A)和NCI-H1975(图15B)的增殖抑制活性的检测结果。
图16为3-D7-MMAE，5-E11-MMAE以及对照物Hu001-MMAE对肺癌细胞NCI-H441(图16A)和SW1573(图16B)的增殖抑制活性的检测结果。
图17A为人T淋巴细胞与不同浓度的3-D7-MMAE，5-E11-MMAE孵育5天后读取固定体积的活细胞数，以增殖率(相对于溶媒/缓冲液)绘制抑制曲线图；图17B为3-D7-MMAE，5-E11-MMAE能有效逆转腺苷单磷酸(AMP)对人T淋巴细胞的增殖抑制作用。试验采用分选获得CD3+人T淋巴细胞，将其培养5天后统计细胞增殖率。
图18为3-D7-MMAE和5-E11-MMAE能有效逆转AMP对人T淋巴细胞INF-g表达的抑制作用。试验采用分选获得CD3+人T细胞，将其培养5天后检测T细胞培养上清。
图19A为嵌合纳米抗体3-D7表达纯化后的SDS-PAGE，对应SEQ NO.37,SEQ NO.38,SEQ NO.39；图19B为3-D7重组融合蛋白纯化后的SDS-PAGE，对应SEQ NO.43,SEQ NO.44。
图20A为嵌合纳米抗体5-E11表达纯化后的SDS-PAGE，对应SEQ NO.40,SEQ NO.41,SEQ NO.42；图20B为5-E11重组融合蛋白纯化后的SDS-PAGE，对应SEQ NO.45。
图21为嵌合纳米抗体3-D7-vc-MMAE的质谱图。
图22为嵌合纳米抗体5-E11-vc-MMAE的质谱图。
图23为嵌合纳米抗体4-D04-vc-MMAE的质谱图。
图24为本发明的纳米抗体-药物偶联物的制备方法的交联反应式，其中，具有不同DAR值的抗体-药物偶联物分别如1a、1b、1c或1d所示。
具体实施方式
本发明人通过广泛而深入的研究，经对羊驼(Alpaca)进行免疫和建库，大量筛选噬菌体展示库及酵母展示库，获得了3-D7、5-E11、4-D04、4-B02等多个纳米抗体。所述抗体能高特异性的结合人CD73蛋白，ELISA测定其EC50分别为0.070μg/mL，0.052μg/mL，0.035μg/mL，0.014μg/mL；所述抗体对重组表达或细胞来源的人CD73酶均具有高效的抑制活性；能有效逆转AMP对人T淋巴细胞的增殖抑制作用。此外，基于3-D7，5-E11，4-D04设计的纳米抗体-药物偶联物(CD73-NDC)，不仅保留了与抗体上述一致的作用，还能够特异性的杀死CD73中高表达的肿瘤，从多方面均表现出了良好的抑瘤活性及调控免疫微环境的潜力。在此基础上完成了本发明。
术语
如本文所用，术语“本发明纳米抗体”、“本发明的抗CD73纳米抗体”、“本发明CD73纳米抗体”可互换使用，均指特异性识别和结合于CD73(包括人CD73)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO.:1-40所示的纳米抗体。
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“单域抗体(VHH)”、“纳米抗体”(nanobody)具有相同的含义，指单克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
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。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗CD73蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合CD73蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定区域来描述，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。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
抗CD73的纳米抗体
本发明提供多款靶向CD73的高特异性和高亲和力的纳米抗体，其仅包括重链，所述重链含有重链可变区(VH)氨基酸序列(由表-1、表-2列出)。
优选地，重链可变区(VH)氨基酸序列具有以下多肽序列的CDR1、CDR2、CDR3：
a1)CDR1为SEQ ID NO.1：GFLLDYYV，SEQ ID NO.5:GFTLDYYN，SEQ ID NO.9：GFTLDYYN，SEQ ID NO.13：GFPLDYYS，SEQ ID NO.17：GFTLDYYN，SEQ ID NO.21：GFTLDDNA，SEQ ID NO.25：GFLLDYYT，SEQ ID NO.29：GFPLDYYT，SEQ ID NO.33：GFTLDYYT；
a2)CDR2为SEQ ID NO.2:ISGSDRST，SEQ ID NO.6:SSNSGGST，SEQ ID NO.10: ISSSEGST，SEQ ID NO.14:IGSSDGST，SEQ ID NO.18:ISNSDGST，SEQ ID NO.22:MRTSDGDT，SEQ ID NO.26:ISSSDSSP，SEQ ID NO.30:ISSSDSSP，SEQ ID NO.34；IRSSDSSP；
a3)HCDR3为SEQ ID NO.3:AADPSPVTVQAMCLPRFPVPY，SEQ ID NO.7:AAYRGWHPSLDARVYDY，SEQ ID NO.11:AADRYYYCSLHVSQYDY,SEQ ID NO.15:AALKYYYCSGHEAIYDY,SEQ ID NO.19:AAYRGWHPSLDARVYDY,SEQ ID NO.23:AAALGTYYSGFYYLAGDGMDY,SEQ ID NO.27:ALRANVYDY,SEQ ID NO.31:SLRANVYDY,SEQ ID NO.35:SLTANVYDY；
a4)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列。
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。
优选地，所述的抗体具有抑制细胞表面及重组CD73蛋白酶催化功能，所述抗体能快速被细胞内吞进入溶酶体。
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-动物嵌合抗体、优选为人源化抗体，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。
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人CD73的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID NO.1～3、SEQ ID NO.5～7、SEQ ID NO.9～11、NO.13～15、NO.17～19、NO.21～23、NO.25～27、NO.29～31、NO.33～35中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列，位于可变区(VH)的CDR区。
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，所述的原始抗体为人-羊驼嵌合抗体3-D7，5-E11，4-D04、4-B02、1-E7、2-B10、2-G9、3-A3、3-F1。
在另一优选例中，所述嵌合抗体的重链可变区(VH)的氨基酸序列编号由表1列出。
在另一优选例中，所述抗体的互补决定区(CDR)的氨基酸序列编号由表2列出。
本发明的抗体可以联合应用，用于构建CAR构建物、包含CAR构建物的重组的免疫细胞、抗体药物偶联物等用途，还可以用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗CD73相关疾病的药物。
表-1：本发明序列表中各序列的代表含义

表-2：本发明纳米抗体的CDR氨基酸序列
重组蛋白(或融合蛋白)
在本发明中，还包括含有本发明的CD73纳米抗体的重组蛋白(或融合蛋白)。一种优选的融合蛋白为多特异性抗体，所述多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：EGFR、TGFβ、BCMA、B7H6、GUCY2C、DLL3、CD38、CD123、CD19、CD20、CD22、B7-H3、GPC3、HER2、PMSA、CD28、4-1BB、OX40、CD40、CD27、CD3、CTLA4、PD1、PDL1、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、TLR7，或其组合。
较佳地，所述多特异性抗体包括一个或多个第二抗原结合区，或进一步地包含第三抗原结合区。
此外，本发明的重组蛋白(或融合蛋白)除了含有本发明的CD73纳米抗体外，还包括任选的协助表达和/或纯化的标签序列(例如6His标签、GGGS序列、FLAG标签)；或包括任选的具有治疗功能的多肽分子或片段；或任选的协助理化或者药学的蛋白功能域(例如能够延长纳米抗体体内半衰期的分子，如HLE、ABD)。
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。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有CD73蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”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A进行氨基酸替换而产生。
表A

本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是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。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
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。
纳米抗体-药物偶联物(Nanobody-drug conjugate，NDC)
本发明还提供了基于本发明抗体的免疫偶联物，优选地为纳米抗体偶联药物(nanobody-drug conjugate，NDC)。
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。
纳米抗体可以偶联药物从而形成抗体药物偶联物(NDCs)。典型地，NDC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NDC。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NDC。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NDC。
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。
较佳地，本发明中的纳米抗体药物偶联物NDC中的与所述纳米抗体部分偶联的偶联部分包括蛋白降解剂或寡核苷酸药物。
较佳地，所述寡核苷酸药物为靶向小分子的核酸药物，包括反义核酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)、适配体(aptamer)、核酶(ribozyme)、抗体核酸偶联药物(ARC)等。
如本文所用，所述蛋白降解剂为肿瘤相关蛋白的降解剂，所述肿瘤相关蛋白选自下组：
(1)激酶类，如RIPK2、BCR-ABL、EGFR、HER2、c-Met、TBK1、CDK2/4/6/9、ALK、Akt、CK2、ERK1/2、FLT3、PI3K、BTK、Fak等；
(2)BET蛋白，如BRD2/4/6/9；
(3)核受体，如AR、ER等；
(4)其他蛋白，如MetAp-2、Bcl-xL、Sirt2、HDAC6、Pirin、SMAD3、ARNT、PCAF/GCN5、Tau、FRS2、RAS(KRAS、HRAS和NRAS)等。
较佳地，所述蛋白降解剂包括PROTAC(蛋白降解靶向嵌合体)，更佳地，所述PROTAC靶向EGFR、KRAS(例如KRAS-G12C、KRAS-G12D)。
本发明还提供了制备NDC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(NDC)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，纳米抗体药物偶联物NDC如下分子式所示：
其中：
nAb是纳米抗体，
LU是接头/连接子；
D是药物；
而且下标p是选自1到8的值。
药物(Drug)
如本文所用，“药物”泛指任何具有期望的生物活性，并具有反应性官能团以便制备本发明所述偶联物的化合物。期望的生物活性包括，诊断，治愈，缓解，治疗，预防人或其它动物的疾病。因此，只要具有必需的反应性官能团，术语“药物”涉及的化合物包括正式国家药典，以及例如美国正式同种疗法药典，正式全国处方集，或者其任何增补本等确认的药物。典型的药物列于医师案头用药参考(PDR)和美国食品药品监督管理局(FDA)的橙皮书。应理解，随着新型药物不断被发现和发展，这些药物也应纳入本发明所述偶联药物的中的“药物”。
可用于构成本发明NDC的药物包括但并不限于：细胞毒剂(例如细胞毒类小分子药物)。
术语“细胞毒剂”是指抑制或阻止细胞表达活性、细胞功能和/或造成细胞破坏的物质。该术语包括放射性同位素、化学治疗剂以及毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。细胞毒剂的例子包括但不限于：耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂以及糖皮质激素和其它化学治疗剂，以及放射性同位素，如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212或213、P32和包括Lu177在内的Lu的放射性同位素。抗体也可与能够将前药转化成其活性形式的抗癌前药活化酶偶联。
一种优选的本发明药物是美登素或类美登素。美登素化合物通过抑制微管蛋白的微管形成来抑制细胞增殖。类美登素是美登素的衍生物。美登素和类美登素都具有高效的细胞毒性，但是它们在癌症治疗的临床应用上具有很大的局限性，这主要是源于此类分子对肿瘤的低选择性。但是，这种高细胞毒性促使它们成为抗体药物偶联物的首选药物部分。以下列出了去乙酰基美登素的结构。
另一种优选的本发明药物是耳抑素肽类药物。耳抑素肽类药物是海兔毒素10(Dolastatin10)的类似物，而后者是从海洋软体动物海兔体内分离出来的具有生物活性的多肽。海兔毒素10通过结合微管蛋白(与长春新碱同样的结合区域)而抑制微管蛋白聚合。海兔毒素10，耳抑素肽PE，耳抑素肽E都是线性多肽，含有四个氨基酸(其中三个氨基酸是海兔毒素类化合物所独有的)和C-端酰胺基团。两个代表性的耳抑素肽类化合物，单甲基耳抑素肽E(MMAE)和单甲基耳抑素肽F(MMAF)，都是抗体药物偶联物的首选药物。
另一种优选的本发明药物是微管溶素(Tubulysin)。微管溶素首先由研究小组从粘细菌培养物中分离，是非常有效的细胞生长抑制剂，其通过抑制微管蛋白聚合并从而诱导细胞凋亡而起作用。微管溶素中的微管溶素D是最有效的，具有超过大多数其他微管蛋白调节剂(包括埃博霉素、长春碱和紫杉醇)10至100倍的活性。紫杉醇和长春碱目前用于多种癌症的治疗，而埃博霉素衍生物正在临床试验中进行活性评价。微管溶素D的合成衍生物将提供与抑制和关键结合相互作用有关的必要信息，并且作为抗癌剂可以具有优越的性质，所述抗癌剂作为分离的实体或作为靶向抗体或配体上的化学弹头。微管溶素D是复杂的四肽，其可以分为四个区域，Mep(D-N-甲基哌啶甲酸)、Ile(异亮氨酸)、Tuv(管式缬氨酸，tubuvaline)和Tup(管式苯丙氨酸，tubuphenylalanine)，如下式所示：
另一种优选的本发明药物是可抑制微管聚合的微生物来源的cryptophycin衍生物。Cryptophycin是从蓝藻的培养物中分离得到的一种能抑制微管生成的新型抗肿瘤活性物质，对多种肿瘤有活性。Cryptophycin是一个脂溶性化合物，含有2个肽键及2个酯键，有5个光学活性中心及1个环氧基团。双肽双酯键都在一个大环结构中。Cryptophycin衍生物CP1和CP2结构如下式所示：
另一种优选的本发明药物是新型抗微管剂Taltobulin(HTI-286、SPA-110)。Taltobulin抑制纯化微管的多聚化，干扰细胞内微管组织、诱导有丝分裂阻断以及诱导细胞凋亡。Taltobulin是细胞增殖强效抑制剂，对18种人肿瘤细胞系的平均IC50为2.5nM。和目前应用的抗微管剂相比，Taltobulin并不是p-糖蛋白的合适的底物，其中Taltobulin结构如下图所示。
一方面，药物是喜树碱类药物衍生物SN-38。SN-38是伊立替康盐酸盐(CPT-11)的生物活性代谢物，为一类拓扑异构酶抑制剂。SN-38引起DNA拓扑异构酶I的抑制最强，剂量依赖性和时间依赖性地抑制DNA的合成，并造成频繁的DNA单链断裂。其中SN-38结构如下图所示。
一方面，药物是喜树碱类药物衍生物Exatecan。是拓扑异构酶I抑制剂喜树碱的合成类似物，活性较SN-38更强，可引起DNA拓扑异构酶I的抑制最强，剂量依赖性和时间依赖性地抑制DNA的合成，并造成频繁的DNA单链断裂。其中Exatecan结构如下式所示。
另一种优选的本发明药物是鹅膏覃碱类药物(alpha-Amanitin)，结构如下图所示。alpha-Amanitin是一种来自毒蘑菇鬼笔鹅蕈(Amanita phalloides)的真菌毒素，二环八肽，能抑制真核RNA聚合酶Ⅱ与RNA聚合酶Ⅲ转录。
另一种优选的本发明药物是苯并二吡咯类抗生素(duocarmycins,CC-1065等)和其它的环丙基吡咯吲哚-4-酮(cyclopropapyrroloind-4-one,CPI)衍生物。这类化合物是有效的DNA小沟结合-烷基化试剂。环丙苯并吲哚-4-酮(cyclopropabenzindol-4-one，CBI)类似物的化学结构更稳定，生物活性更高，而且与它们含有天然CPI烷基化亚基的父系化合物相比更容易合成。一个代表性的CBI衍生物是酚羟基保护衍生物CBI，具有弱化的前药毒性和增强的水溶性(其中CBI-seco类结构通式如下图所示)：
另一种优选的本发明药物是吡咯并苯二氮卓类(pyrrolo[2,1-c][1,4]benzodi-azepines，PBDs)或者PBD二聚体类(PBD dimers)。PBD是一类由链霉菌产生的天然产物，其独特特性在于能够在DNA小沟，确切是在嘌呤-鸟嘌呤-嘌呤序列处，形成非扭曲的共价加和物。应用PBD作为部分小分子策略靶向锁定DNA序列以及作为新型的抗癌和抗菌药物引起了越来越多的兴趣。应用一个柔性碳链连接两个PBD单元的C8/C8’的羟基基团，所得的二聚体具有增强的生物活性。PBD二聚体被认为是可以产成序列选择性的DNA损伤，例如倒序的5’-Pu-GATC-Py-3’链间交联，从而导致其生物活性。这些化合物已被证明是高效的细胞毒性药物，可作为抗体药物偶联物的备选药物。
另一种优选的本发明药物是PNU-159682衍生物，PNU-159682是Nemorubicin在人肝微粒体中的主要活性代谢产物，与MMDX和阿霉素相比，活性提高3000倍。
另一方面，药物并不仅仅局限于上述提到的类别，还包括所有可用于抗体药物偶联物的药物。并且尤其是那些能够通过与接头的酰胺键来配位，如通过具有碱性胺基(一级胺或二级胺)来配位的细胞毒素，例如上文中所示的细胞毒素D1-D12的结构。
连接子
按照在细胞内药物释放的机制，“连接子”或“抗体药物偶联物的连接子”可被分为两类：不可断裂连接子和可断裂连接子。
对于含有不可断裂连接子的抗体药物偶联物，其药物释放机制为：偶联物与抗原结合并被细胞内吞后，抗体在溶酶体中被酶解，释放出由小分子药物，连接子，和抗体氨基酸残基共同组成的活性分子。由此带来的药物分子结构改变并不减弱其细胞毒性，但由于活性分子是带电荷的(氨基酸残基)，从而导致其不能渗入邻近细胞。因此，此类活性药物不能杀死邻近不表达靶向抗原(抗原阴性细胞)的肿瘤细胞(旁观者效应,bystander effect)。
可断裂连接子，顾名思义，可以在目标细胞内断裂并释放出活性药物(小分子药物本身)。可断裂连接子可分为两个主要的类别：化学不稳定连接子和酶不稳定连接 子。化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值，谷胱甘肽浓度等。对pH值敏感的连接子，通常又称为酸断裂连接子。这样的连接子在血液的中性环境下相对稳定(pH7.3-7.5)，但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)内将会被水解。第一代的抗体药物偶联物大多应用这类连接子，例如腙，碳酸酯，缩醛，缩酮类。由于酸断裂连接子有限的血浆稳定性，基于此类连接子的抗体药物偶联物通常具有较短的半衰期(2-3天)。这种较短的半衰期在一定程度上限制了pH敏感连接子在新一代抗体药物偶联物中的应用。
对于谷胱甘肽敏感的连接子，又称二硫键连接子。药物释放是基于细胞内谷胱甘肽的高浓度(毫摩尔范围)与血液中相对较低的谷胱甘肽浓度(微摩尔范围)差异引起的。对于肿瘤细胞而言尤其如此，其低含氧量导致还原酶的活性增强，因而导致更高的谷胱甘肽浓度。二硫键具有热力学稳定性，因此在血浆中具有较好的稳定性。
酶不稳定连接子，如肽连接子，能够更好地控制药物释放。肽连接子能够被溶酶体内蛋白酶，如组织蛋白酶(CathepsinB)或纤溶酶(在一些肿瘤组织中此类酶含量增加)，有效地切断。这种肽连接被认为在血浆循环中非常稳定，这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常在不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性，酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。典型的酶不稳定连接子包括Val-Cit(VC)，Val-Ala(VA),Gly-Gly-Phe-Gly(GGFG)等。
自释放连接子一般嵌合在可断裂连接子与活性药物之间，或者本身就是可断裂连接子的一部分。自释放连接子的作用机制是：当可断裂连接子在合宜的条件下断裂后，自释放连接子能够自发地进行结构重排，进而释放与之连接的活性药物。常见的自杀式连接子包括对氨基苄醇类(PAB)和β-葡萄糖醛酸苷类(β-Glucuronide)等。
应用
本发明还提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗CD73相关的疾病的药物。所述CD73相关的疾病包括肿瘤发生、生长和/或转移、肿瘤耐药相关疾病、炎症、代谢相关疾病等。
本发明抗体、NDC或CAR-T等的用途，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)：乳腺癌(如三阴性乳腺癌)、肺癌(如非小细胞肺癌)、胰腺癌、恶性脑胶质瘤、胃癌、肝癌、食道癌、肾癌、结直肠癌、膀胱癌、前列腺癌、子宫内膜癌、卵巢癌、宫颈癌、白血病、骨髓癌、血管肉瘤等；尤其是三阴性乳腺癌、非小细胞肺癌、胰腺癌、恶性脑胶质瘤，更优选为三阴性乳腺癌和/或非小细胞肺癌。
(ii)诊断、预防和/或治疗自身免疫疾病。所述自身免疫疾病包括(但并不限于)：系统性红斑狼疮、类风湿关节炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症。
(iii)诊断、预防和/或治疗炎症。所述炎症包括(但并不限于)：风湿性关节炎、骨关节炎、强直性脊柱炎、痛风、莱特尔综合征、牛皮癣性关节病、感染性关节炎、结核性关节炎、病毒性关节炎、真菌性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病、特发性肺纤维化。
(iv)诊断、预防和/或治代谢相关疾病。所述代谢相关疾病包括(但并不限于)：糖尿病、食源性肥胖和脂肪炎症。
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NDC或相应的CAR-T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。
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。此外，还可同时使用其他治疗剂。
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。
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。
对于NDC而言，由于本发明提供的纳米抗体-药物偶联物可以靶向瞄准特殊的细胞群体，与细胞表面特异蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内，因此，本发明的纳米抗体-药物偶联物可以用于治疗目标疾病，上面提到的抗体-药物偶联物可以以治疗有效量，通过合适的途径给予受试者(例如人)。需要治疗的受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。
当用本发明的纳米抗体-药物偶联物治疗时，可以通过本领域常规的方法进行递送。例如，它可以通过使用脂质体，水凝胶，环糊精，生物可降解的纳米胶囊，或生物粘附性微球被引入到细胞中。或者，所述核酸或载体可在本地通过直接注射或通过使用输注泵递送。
本发明的主要优点包括：
(a)本发明纳米抗体具有优异的生物活性和特异性，并具有很高的亲和力(ELISA测定其EC50为0.014～0.070μg/mL)，具有CD73酶功能的抑制活性(酶活性测定其IC50为4.66～38.18ng/mL)。此外，对肿瘤细胞的CD73具有良好的结合亲合力(FACS测定其EC50为0.037～0.227μg/mL)，并抑制肿瘤细胞CD73酶的功能(IC50值为0.083～0.48μg/mL),可用做靶向CD73的治疗抗体。
(b)本发明的纳米抗体和NDC具有显著的抗肿瘤活性，而对于正常的细胞无明显毒副作用。
(c)本发明的纳米抗体对人T淋巴细胞具有显著的增殖保护作用，能有效逆转腺苷单磷酸(AMP)对T淋巴细胞的增殖抑制,并促进INFγ的表达和分泌，其EC50为0.0008～0.0247μg/mL。
(d)本发明所述的纳米抗体-药物偶联物(NDC)具有优异的CD73-依赖的抗肿瘤活性，即对CD73-正常或低表达的细胞无明显毒副作用，而对CD73-中高表达的肿瘤细 胞具有极高的杀伤活性，细胞增殖抑制试验测定其IC50为0.0025～0.0658μg/mL。
(e)本发明所述的NDC对正常人T淋巴细胞的增值没有明显的毒副作用，细胞增殖抑制试验测定其IC50为>15μg/mL。
(f)本发明的NDC对人T淋巴细胞具有显著的增殖保护作用，能有效逆转腺苷单磷酸(AMP)对T淋巴细胞的增殖抑制并促进INFγ的表达和分泌，其EC50为0.0046～0.0379μg/mL。
下面结合具体实施例，进一步陈述本发明。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Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)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。
实施例1靶向人CD73纳米抗体的发现和制备
首先制备人CD73蛋白的胞外区(CD73-ECD)作为抗原。参照NCBI:NP_002517.1氨基酸的第27位到第547位，采用基因克隆技术和哺乳动物载体表达体系获得碳末端多组氨酸标记(C-terminus polyhistidine-tagged)的抗原：
利用上述制备的CD73胞外区蛋白对羊驼(Alpaca)进行免疫，免疫间隔时间21天，最后一次免疫10天后采集部分外周血(PBMC)；经PBMC分离及VHH抗体片段克隆，构建噬菌体展示库和酵母展示库。通过反复淘选，单克隆ELISA，FACS筛选等步骤，从噬菌体展示库获得4款高活性且具独立序列的纳米抗体(3-D7，5-E11，4-D04，4-B02)，从酵母展示库获得5款活性抗体且具独立序列(1-E7、2-B10、2-G9、3-A3、3-F1)。
实施例2 CD73纳米抗体对人CD73抗原亲和力SPR的测定
表面等离子体共振(SPR)测定：使用Protein A传感器固化抗体，固化高度为1.5nM左右。缓冲液为PBST(PBS+0.02％tween20)，CD73-ECD蛋白样品稀释至500，250，125，62.5，31.3，0nM。亲和力检测：平衡60s，结合180s，解离180s，检测温度25℃，使用ForteBio OCTET R2系统进行动力学表征分析。
检测结果如图1所示，3-D7，5-E11，4-D04，4-B02对CD73-ECD有很强的亲和性,结合常数(KD)分别为1.997nM,0.826nM,0.919nM,0.795nM。
实施例3 CD73纳米抗体对人CD73抗原亲和力ELISA的测定
用包被液将CD73蛋白胞外区(CD73-ECD)稀释成1μg/mL，包被ELISA板，100μL/孔，4℃，过夜。洗去多余抗原，用1％BSA于室温封闭2h，然后加入5倍梯度稀释的 各单克隆抗体，100μL/孔，室温孵育1h；洗去未结合的抗体，加入合适浓度辣根过氧化物酶标记的葡萄球菌A蛋白，100μL/孔，室温孵育0.5h。洗去未结合的二抗，加入TMB显色液反应大约15min，加入1M HCL，50μL/孔，终止显色反应，然后在450nm处测定吸光度，并分析数据。
检测结果如图2所示，3-D7，5-E11，4-D04，4-B02对CD73-ECD有很强的亲和性，EC50分别0.070μg/mL，0.052μg/mL，0.035μg/mL，0.014μg/mL。
针对酵母来源的5款抗体，经HEK293F细胞瞬转表达后继续培养2天后取上清，采用100μL、10μL、1μL进行ELISA测定。
检测结果如图11所示，纳米抗体1-E7、2-B10、2-G9、3-A3、3-F1均有较强的CD73-ECD结合亲合力。
综上结果说明，本实施例CD73纳米抗体与人CD73抗原具有良好的结合活性。
实施例4 CD73纳米抗体对重组人CD73酶催化功能的抑制活性的测定
用抗原稀释液将人重组CD73酶(CD73胞外区)稀释成0.1μg/mL，均匀铺到96孔低吸附培养板中，25μL/孔。将50μL按照3倍梯度从300ng/mL稀释到0.137ng/mL的CD73抗体加入培养板中，混匀(终浓度为1350ng/mL～0.069ng/mL)，37℃孵育1h后，加入25μL含有1.2mM AMP和0.4mM ATP的混合液，37℃孵育1h。取出上述反应液50μL加入另一96孔白板中，向每孔加入50μL CellTiter-Glo试剂，混匀避光反应3-5min，采用酶标仪检测荧光信号强度。
检测结果如图3所示，3-D7，5-E11，4-D04，4-B02均具有显著抑制重组CD73蛋白酶水解AMP的活性，其IC50分别为38.18ng/mL、9.47ng/mL、5.63ng/mL，4.66ng/mL。
综上结果说明，本实施例CD73单克隆抗体对重组人CD73酶催化功能具有显著的抑制活性。
实施例5 CD73纳米抗体对肿瘤细胞表面CD73的结合亲和力的测定
采用CD73-中高表达的三阴性乳腺癌细胞MDA-MB-231、非小细胞肺癌细胞NCI-H1299、PC9、SW1573、NCI-H1975和NCI-H1373作为靶细胞，将100μL按照5倍梯度从10μg/mL稀释到0.016μg/mL的受试抗体作为一抗，分别与悬浮于100μL RPMI-1640无血清培养基中的1x105个肿瘤细胞混匀，使其终浓度为5μg/mL稀释到0.008μg/mL，然后于4℃孵育1h，PBS洗涤细胞两次以去除未结合的一抗，再将靶细胞与200μL，2μg/mL，PE标记的二抗4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μL PBS中，通过流式细胞仪测定受试抗体对相应细胞表面CD73的结合亲和力(Binding affinity)。
检测结果如图4A所示，3-D7，5-E11，4-D04，4-B02对MDA-MB-231具有优异的结合亲和力，EC50分别为0.120μg/mL、0.037μg/mL、0.059μg/mL、0.037μg/mL；
检测结果如图4B所示，3-D7，5-E11，4-D04，4-B02对NCI-H1299具有优异的结合亲和力，EC50分别为0.222μg/mL、0.068μg/mL、0.080μg/mL、0.078μg/mL；
检测结果如图5A所示，3-D7，5-E11，4-D04，4-B02对PC9具有优异的结合亲和力，EC50分别为0.133μg/mL、0.041μg/mL、0.057μg/mL、0.044μg/mL；
检测结果如图5B所示，3-D7，5-E11，4-D04，4-B02对NCI-H1975具有优异的结合亲和力，EC50分别为0.106μg/mL、0.057μg/mL、0.068μg/mL、0.055μg/mL；
检测结果如图6A所示，3-D7，5-E11，4-D04，4-B02对SW1573具有优异的结合亲和力，EC50分别为0.116μg/mL、0.062μg/mL、0.067μg/mL、0.061μg/mL；
检测结果如图6B所示，3-D7，5-E11，4-D04，4-B02对NCI-H1373具有优异的结 合亲和力，EC50分别为0.227μg/mL、0.135μg/mL、0.163μg/mL、0.112μg/mL；
检测结果如图7所示，3-D7，5-E11，4-D04，4-B02对HC44具有优异的结合亲和力，EC50分别为0.234μg/mL、0.096μg/mL、0.114μg/mL、0.105μg/mL；
针对酵母来源的6款抗体，经HEK293F细胞瞬转表达后继续培养2天后取上清，采用100μL、10μL、1μL与MDA-MB-231细胞孵育后进行流式细胞仪测定其结合活性。
检测结果如图12所示，纳米抗体1-E7、2-B10、2-G9、3-A3、3-F1均有较强的CD73-ECD结合亲合力。
综上结果说明，本实施例CD73纳米抗体能够以人源肿瘤细胞的CD73为作用靶点并具有良好的结合活性。
实施例6 CD73纳米抗体对肿瘤细胞表面CD73酶催化功能的影响
采用CD73-高表达的三阴性乳腺癌细胞MDA-MB-231作为靶细胞。铺合适数量的肿瘤细胞(经预实验确认)于96孔板中，37℃培养16小时之后，用无血清RPMI-1640培养基洗涤细胞3次，将50μL按照3倍梯度从10μg/mL稀释到0.0045μg/mL的受试抗体加入96孔板中，37℃孵育30min后，加入25μL 0.9mM的AMP，置37℃，5％CO2培养3h。取出上述培养上清25μL加入另一个96孔白板中，加入25μL0.1mM ATP，混匀。向每孔加入50μL CellTiter-Glo试剂，混匀避光反应3～5min，采用酶标仪检测荧光信号强度。
检测结果如图8所示，3-D7，5-E11，4-D04，4-B02均能显著抑制MDA-MB-231细胞表面CD73催化水解AMP的功能，IC50分别为0.483μg/mL，0.088μg/mL，0.093μg/mL，0.083μg/mL。
实施例7 CD73纳米抗体对T淋巴细胞增殖和表达IFN-γ的保护作用
PBMC的复苏、扩增与分选：首先采用含有500ng/mL CD3/CD28抗体和100IU/mL IL-2的培养基将PBMC复苏培养3-4天，然后采用分选试剂盒(Stemcell,Cat#1795)分选PBMC后获得CD3阳性的T淋巴细胞。
T淋巴细胞增殖试验：将上述分选得到的T淋巴细胞进行荧光标记，将预先配好的CFSE(carboxyfluorescein succinimidyl ester)加入细胞悬液(终浓度2.5μM)，使其在37℃标记5min后以PBS洗涤3次。然后，将CFSE-标记的T细胞铺至96孔板中(2x104个细胞/孔)，每孔加入50μL按照梯度稀释的纳米抗体(终浓度15μg/mL～0.00019μg/mL，n＝4)，并加入50μL腺苷单磷酸(AMP，终浓度为0.25mM)，混匀，培养4-5天后收集培养上清，采用流式细胞分选仪(FACS)读取并统计固定体积的细胞数，使用Flowjo软件绘制细胞增殖曲线并计算EC50值。摄取50μL/孔T细胞培养上清用于检测IFN-γ蛋白浓度，采用ELISA试剂盒，并参照试剂盒所提供的技术操作步骤。
结果如图9显示，CD73纳米抗体3-D7，5-E11，4-D04，4-B02对人T淋巴细胞具有显著的增殖保护作用，能有效逆转AMP对T淋巴细胞的增殖抑制，其EC50分别为0.0247±0.0052μg/mL，0.0008±0.0002μg/mL，0.0025±0.0007μg/mL，0.0012±0.0004μg/mL。
结果如图10显示，CD73纳米抗体3-D7，5-E11，4-D04，4-B02能有效逆转AMP对T细胞表达/分泌INF-γ的抑制作用。
综上结果说明，本实施例CD73纳米抗体不仅能够抑制肿瘤细胞表面CD73酶活性，还能够对T细胞表面CD73的酶活功能抑制，逆转AMP对T细胞增殖和功能的影响，保护T细胞。
实施例8 CD73抗体与肿瘤细胞结合导致内吞至细胞内溶酶体
将50％密度MDA-MB-231细胞铺于26孔板中，于37℃培养16h后加入5μg/mL的CD73纳米抗体，分别于37℃孵育4h或4℃孵育1h，PBS洗涤三次以去除未与细胞结合的抗体，采用4％的多聚甲醛于室温固定30min。PBS洗涤三次，采用0.4％Triton X-100透化10min。PBS洗涤三次后，37℃条件下孵育Lamp-2(兔抗人)抗体1h，以标记细胞溶酶体的位置。采用PBS洗去未结合的抗体，37℃孵育R-PE标记的羊抗人和Alexa Fluor488标记的驴抗兔二抗30min。洗去未结合的二抗，用DAPI染色10min以标记细胞核位置，之后用荧光显微镜(200×)观察抗体内吞情况。
结果如图13所示，3-D7，5-E11，4-D04，4-B02均能快速并大幅度的被MDA-MB-231细胞内吞至溶酶体。该结果表明，本发明的抗体适合用于制备NDC，提示CD73-NDC将具有良好的NDC药物特性，可用于广谱且高特异性靶向CD73阳性肿瘤治疗药物的前景。
实施例9 3-D7-vc-MMAE、5-E11-vc-MMAE、4-D01-vc-MMAE的制备
(一)3-D7-vc-MMAE偶联物的制备
将3-D07蛋白原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入3-4倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动2.5小时。
将上述反应液冷至0-10℃，未经纯化加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的mc-vc-MMAE(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于10-25℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.2 2微米孔径的过滤装置除菌，-80℃保存。
通过质谱分析抗体偶联物3-D7-vc-MMAE的质量数与理论分子量差值(图21)，计算获得精确载药量约为4。
(二)5-E11-vc-MMAE偶联物的制备
将5-E11蛋白原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入3-4倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动2.5小时。
将上述反应液冷至0-10℃，未经纯化加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的mc-vc-MMAE(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于10-25℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.2 2微米孔径的过滤装置除菌，-80℃保存。
通过质谱分析抗体偶联物5-E11-vc-MMAE的质量数与理论分子量差值(图22)，计算获得精确载药量为4(图22)。
(三)4-D04-vc-MMAE偶联物的制备
将4-D04蛋白原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入3-4倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动2.5小时。
将上述反应液冷至0-10℃，未经纯化加入适量的二乙基乙酰胺(DMA)，再加入6 倍过量摩尔比的mc-vc-MMAE(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于10-25℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.2 2微米孔径的过滤装置除菌，-80℃保存。
通过质谱分析抗体偶联物4-D04-vc-MMAE的质量数与理论分子量差值(图22)，计算获得精确载药量约为4(图23)。
实施例10 CD73-NDC针对CD73高表达的肿瘤细胞的体外抗肿瘤活性
实例所使用细胞系购自于美国典型培养物保藏中心(ATCC)、中国科学院细胞库、南京科佰生物有限公司，并按照相应的说明进行培养，包括：MDA-MB-453、MDA-MB-231、PC9、NCI-H1975、NCI-H441、SW1573。将上述处于对数生长期的细胞，分别以每孔200-2000个细胞的密度(依不同细胞的生长速率而定)接种至96孔细胞培养板中，150μL/孔，37℃，5％CO2培养约5h后，分别加入不同浓度的CD73-NDCs(15μg/mL～0.00019μg/mL)，每个药物浓度设置2-4个复孔，及相应的溶媒对照和空白对照孔，作用5-9天后(根据细胞生长速度，保证细胞分裂足够次数)，倾去培养液，加入MTS反应液(购自Promega,cat#G3581)，100μL/孔，于37℃反应至预期颜色深浅，测定每组的细胞活力(OD490nm)，并按照以下公式计算细胞存活率：存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。通过GraphPad Prism 8软件分析上述数据,并分别计算上述CD73抗体-药物偶联物在不同细胞株上的IC50值。
两种优选的CD73-NDC：3-D7-MMAE、5-E11-MMAE以及作为对照的CD73-ADC Hu001-MMAE的体外抗肿瘤活性结果分别如图14至图16所示。
如图14A所示，CD73-NDCs对CD73低表达的细胞MDA-MB-453的增殖抑制作用不明显，而对CD73中高表达的MDA-MB-231(图14B)、PC9(图15A)、NCI-H1975(图15B)、NCI-H441(图16A)、SW1573(图16B)均显示很强的细胞增殖抑制作用。
总体而言，CD73-NDC的细胞毒性(IC50值)表明CD73-NDC的细胞毒活性与受试细胞的CD73表达水平直接相关，因此判断为CD73靶标特异性细胞毒性。表-3汇总了部分细胞增殖抑制测试的IC50值。
表-3：CD73纳米抗体-药物偶联物的体外抗肿瘤活性
实施例11 CD73-NDC对T淋巴细胞增殖和表达IFN-γ的保护作用
人外周血单核细胞(peripheral blood mononuclear cell，PBMC)冻存管均由江苏西迪尔生物技术有限公司提供。首先采用含有500ng/mL CD3/CD28抗体和100IU/mL IL-2的培养基将PBMC复苏培养3-4天，然后采用分选试剂盒(供应商：Stemcell, Cat#1795)分选出CD3阳性的T淋巴细胞，再对其进行荧光标记。将预先配好的荧光染料CFSE(carboxyfluorescein succinimidyl ester)加入细胞悬液(终浓度2.5μM)，使其在37℃孵育5min后以PBS洗涤3次后用于试验。
先是观察不同浓度CD73-NDC对T细胞增殖曲线的影响，将CFSE-标记的T细胞铺至96孔板中(2x104个细胞/孔),每孔加入按照梯度稀释的CD73-NDC(终浓度15μg/mL～0.000192μg/mL，n＝2)或加入对照溶媒，培养5天后采用FACS读取活细胞数，作剂量曲线图计算IC50值。如图17A所示，3-D7-MMAE和5-E11-MMAE在受试浓度区间未显示明显的毒副作用(IC50>15μg/mL)。
在另一组试验中，将CFSE-标记的T细胞铺至96孔板中(2x104个细胞/孔)，每孔加入50μL按照梯度稀释的NDC(终浓度3μg/mL～0.00019μg/mL，n＝2)，并加入50μL AMP(终浓度为0.25mM)，混匀，培养4-5天后收集培养上清，采用流式细胞分选仪(FACS)读取并统计固定体积的细胞数，使用Flowjo软件绘制细胞增殖曲线并计算EC50值。结果如图17B显示，3-D7-MMAE和5-E11-MMAE对人T淋巴细胞具有显著的增殖保护作用，能有效逆转AMP对T淋巴细胞的增殖抑制，其EC50分别为0.0379±0.0066μg/mL，0.0046±0.0012μg/mL。
摄取50μL/孔T细胞培养上清用于检测IFN-γ蛋白浓度，采用ELISA试剂盒(Sinobiological，Cat#KIT11725A)，并参照试剂盒所提供的技术操作步骤。结果如图18显示，3-D7-MMAE和5-E11-MMAE能有效逆转AMP对T细胞表达/分泌INF-g的抑制作用
本实施例的结果说明CD73-NDC对T淋巴细胞不仅无毒性作用，并且能有效逆转AMP/Ado对T淋巴细胞的增殖的抑制作用。
综上，CD73-NDC相关实施例的研究明确表明：
1、CD73-NDC 3-D7-MMAE和5-E11-MMAE均具有良好的CD73特异的肿瘤细胞杀伤活性，即针对CD73-中高表达的肿瘤细胞增殖具有极强的抑制作用，而对CD73-低表达的细胞增殖无明显毒性；
2、CD73-NDC 3-D7-MMAE和5-E11-MMAE针对正常人的T淋巴细胞的细胞增殖无明显毒性；
3、CD73-NDC 3-D7-MMAE和5-E11-MMAE对T淋巴细胞不仅无毒性作用，并且能有效逆转AMP/Ado对T淋巴细胞的增殖的抑制作用；
实施例12 CD73纳米抗体及融合蛋白的批量表达生产和纯化
将3-D7，5-E11纳米抗体可变区序列克隆至hIgG1表达载体(3-D7-FC1，3-D7-FC2，3-D7-FC3，5-E11-FC1，5-E11-FC2，5-E11-FC3)，或重组/融合蛋白表达载体(3-D7-HLE，3-D7-ABD，5-E11-ABD)，经瞬转HEK293后置无血清培养基悬浮培养6天。收集培养上清开展纯化，蛋白定量和SDS-PAGE检测。
如图19，图20所示，来源于3-D7，5-E11的嵌合纳米抗体和重组融合蛋白均能高效率地表达，生产和纯化。
本发明纳米抗体序列
SEQ NO.1：3-D7 CDR1 GFLLDYYV
SEQ NO.2：3-D7 CDR2 ISGSDRST
SEQ NO.3：3-D7 CDR3 AADPSPVTVQAMCLPRFPVPY
SEQ NO.4：3-D7 VH
SEQ NO.5:5-E11 CDR1 GFTLDYYN
SEQ NO.6:5-E11 CDR2 SSNSGGST
SEQ NO.7:5-E11 CDR3 AAYRGWHPSLDARVYDY
SEQ NO.8:5-E11 VH
SEQ NO.9:4-D04 CDR1 GFTLDYYN
SEQ NO.10:4-D04 CDR2 ISSSEGST
SEQ NO.11:4-D04 CDR3 AADRYYYCSLHVSQYDY
SEQ NO.12:4-D04 VH
SEQ NO.13:4-B02 CDR1 GFPLDYYS
SEQ NO.14:4-B02 CDR2 IGSSDGST
SEQ NO.15:4-B02 CDR3 AALKYYYCSGHEAIYDY
SEQ NO.16:4-B02 VH
SEQ NO.17:1-E7 CDR1 GFTLDYYN
SEQ NO.18:1-E7 CDR2 ISNSDGST
SEQ NO.19:1-E7 CDR3 AAYRGWHPSLDARVYDY
SEQ NO.20:1-E7 VH
SEQ NO.21:2-B10 CDR1 GFTLDDNA
SEQ NO.22:2-B10 CDR2 MRTSDGDT
SEQ NO.23:2-B10 CDR3 AAALGTYYSGFYYLAGDGMDY
SEQ NO.24:2-B10 VH
SEQ NO.25:2-G9 CDR1 GFLLDYYT
SEQ NO.26:2-G9 CDR2 ISSSDSSP
SEQ NO.27:2-G9 CDR3 ALRANVYDY
SEQ NO.28:2-G9 VH
SEQ NO.29:3-A3 CDR1 GFPLDYYT
SEQ NO.30:3-A3 CDR2 ISSSDSSP
SEQ NO.31:3-A3 CDR3 SLRANVYDY
SEQ NO.32:3-A3 VH
SEQ NO.33:3-F1 CDR1 GFTLDYYT
SEQ NO.34:3-F1 CDR2 IRSSDSSP
SEQ NO.35:3-F1 CDR3 SLTANVYDY
SEQ NO.36:3-F1 VH
SEQ NO.37:3-D7-FC1
SEQ NO.38:3-D7-FC2
SEQ NO.39:3-D7-FC3
SEQ NO.40:5E11-FC1
SEQ NO.41:5E11-FC2
SEQ NO.42:5E11-FC3
SEQ NO.43:3-D7-HLE
SEQ NO.44:3-D7-ABD
SEQ NO.45:5-E11-ABD
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 (10)

  1. 一种靶向CD73的纳米抗体，其特征在于，所述纳米抗体的VHH链的互补决定区CDR为选自下组的一种或多种：
    (1)SEQ ID NO.1所示的CDR1，
    SEQ ID NO.2所示的CDR2，和
    SEQ ID NO.3所示的CDR3；
    或者，
    (2)SEQ ID NO.5所示的CDR1，
    SEQ ID NO.6所示的CDR2，和
    SEQ ID NO.7所示的CDR3；
    或者，
    (3)SEQ ID NO.9所示的CDR1，
    SEQ ID NO.10所示的CDR2，和
    SEQ ID NO.11所示的CDR3，
    或者，
    (4)SEQ ID NO.13所示的CDR1，
    SEQ ID NO.14所示的CDR2，和
    SEQ ID NO.15所示的CDR3，
    或者，
    (5)SEQ ID NO.17所示的CDR1，
    SEQ ID NO.18所示的CDR2，和
    SEQ ID NO.19所示的CDR3，
    或者，
    (6)SEQ ID NO.21所示的CDR1，
    SEQ ID NO.22所示的CDR2，和
    SEQ ID NO.23所示的CDR3，
    或者，
    (7)SEQ ID NO.25所示的CDR1，
    SEQ ID NO.26所示的CDR2，和
    SEQ ID NO.27所示的CDR3，
    或者，
    (8)SEQ ID NO.29所示的CDR1，
    SEQ ID NO.30所示的CDR2，和
    SEQ ID NO.31所示的CDR3，
    或者，
    (9)SEQ ID NO.33所示的CDR1，
    SEQ ID NO.34所示的CDR2，和
    SEQ ID NO.35所示的CDR3。
  2. 如权利要求1所述的纳米抗体，其特征在于，所述纳米抗体的VHH链还包括框架区(FR)。
  3. 一种靶向CD73的抗体，其特征在于，所述抗体包括一个或多个如权利要求2所述的靶向CD73的纳米抗体的VHH链。
  4. 一种多特异性抗体，其特征在于，所述的多特异性抗体包含：权利要求1所述的靶向CD73的纳米抗体或权利要求3所述的靶向CD73的抗体。
  5. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的靶向CD73的纳米抗体、或如权利要求3所述的抗CD73抗体；以及
    (ii)任选的具有治疗功能的多肽分子或片段；和/或
    (iii)任选的提升蛋白理化性质或成药性的功能域。
  6. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合区域为如权利要求1所述的纳米抗体的VHH链。
  7. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求6所述的CAR构建物。
  8. 一种免疫偶联物，其特征在于，所述的免疫偶联物含有：
    (a)抗体部分，所述抗体部分为权利要求1所述的靶向CD73的纳米抗体或权利要求3所述的靶向CD73的抗体；和
    (b)与所述纳米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、酶、蛋白降解剂、寡核苷酸或其组合。
  9. 如权利要求8所述的免疫偶联物，其特征在于，所述免疫偶联物为如下分子式所示的抗体药物偶联物ADC：
    其中：
    nAb是靶向CD73的纳米抗体，
    LU是接头(又称连接子)；
    D是药物；
    而且下标p是选自1-8的值。
  10. 一种药物组合物，其特征在于，所述的药物组合物包含：
    (i)如权利要求1所述的靶向CD73的纳米抗体、如权利要求3所述的靶向CD73的抗体、如权利要求4的多特异性抗体、如权利要求5所述的重组蛋白、如权利要求7所述的重组的免疫细胞或如权利要求8所述的免疫偶联物；
    (ii)药学上可接受的载体。
PCT/CN2023/092108 2022-06-22 2023-05-04 靶向cd73的纳米抗体及纳米抗体-药物偶联物、其制备方法和用途 Ceased WO2023246325A1 (zh)

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