WO2021043229A1 - 靶向肿瘤的重组双功能融合蛋白及其应用 - Google Patents

靶向肿瘤的重组双功能融合蛋白及其应用 Download PDF

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WO2021043229A1
WO2021043229A1 PCT/CN2020/113357 CN2020113357W WO2021043229A1 WO 2021043229 A1 WO2021043229 A1 WO 2021043229A1 CN 2020113357 W CN2020113357 W CN 2020113357W WO 2021043229 A1 WO2021043229 A1 WO 2021043229A1
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tumor
antibody
fusion protein
tgfβr
cells
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French (fr)
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余科
任志强
刘亮
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复旦大学
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    • A61K38/18—Growth factors; Growth regulators
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of medicine, in particular to a bifunctional fusion protein of CD73 or Tissue factor (TF)-targeting antibody and TGF ⁇ receptor (TGF ⁇ R), its preparation method, application and anti-tumor mechanism.
  • TF Tissue factor
  • TGF ⁇ R TGF ⁇ receptor
  • the research on tumors is not simply limited to the tumor cells themselves, the tumor microenvironment has also been proved to be an important basis for tumor occurrence and development. Studies have found that some highly expressed proteins such as CD73, TF, and TGF ⁇ can affect and reshape the tumor microenvironment, making it play an important role in the occurrence, development, metastasis and drug resistance of tumors.
  • CD73 is an ecto-5'-nuclease (NT5E) with a molecular weight of 70kD, which is anchored on the cell surface by glycophosphatidylinositol (GPI).
  • CD73 is abnormally expressed in a variety of tumors, including most refractory tumors such as non-small cell lung cancer, triple-negative breast cancer, and pancreatic cancer.
  • ATP/ADP generates a large amount of adenosine (ADO) under the catalysis of CD39/CD73 and is exposed around the cells.
  • ADO induced by tumor cells can produce immunosuppression and promote tumor immune escape (Immunological Reviews 2017; 276:121-144).
  • the main mechanism is as follows: 1) After ADO binds to the CD4+/CD8+ effector T cell surface receptor A2AR, it inhibits its proliferation and expansion through the cAMP signaling pathway, thereby reducing the cytotoxicity of T cells; 2) ADO can interfere with NK cells and The degree of adhesion between tumor cells reduces the cytotoxicity of NK cells; 3) ADO promotes its proliferation by activating a series of immunosuppressive cell subgroups such as Tregs, MDSC, etc., thereby enhancing the immunosuppressive effect and anti-inflammatory function around tumor cells ; 4) ADO can inhibit the differentiation of M1 type macrophages and activate M2 type macrophages.
  • CD73 is related to the growth and metastasis of tumor cells, as well as angiogenesis in the tumor environment.
  • Tissue factor is a transmembrane glycoprotein with a molecular weight of 47kD, which activates the exogenous coagulation reaction after the blood vessel is traumatized.
  • TF is abnormally activated and expressed in many tumor tissues, among which breast cancer, pancreatic cancer, lung cancer, and esophageal cancer have higher abnormal expression rates. Recent studies have shown that the abnormal expression of TF in tumors is an important reason for tumor resistance, immune invasion suppression and metastasis.
  • TF can drive the increase of thrombin levels in the tumor microenvironment, causing the deposition of fibrin and the formation of coagulation, thereby affecting the tumor microenvironment, causing tumor fibrosis and tumor stroma changes (CancerRes 2019; 79:3417-3430 ). These tumor-specific changes have blocked the invasion of many immune cells, affecting the resistance of tumors to chemotherapeutic drugs and immunotherapeutic drugs (Journal of Clinical Investigation 2019; 129: 1785-1800). Changes in the tumor microenvironment also lead to increased levels of MDSC and TAM (tumor-associated macrophages), which reduces the tumor-killing activity of immune effector cells. Abnormal expression of TF can also cause the tumor-killing effect of the complement system to be inhibited. In addition, TF can help tumor cell metastasis by promoting angiogenesis.
  • TGF ⁇ is a key inducer of epithelial-mesenchymal-transition (EMT).
  • EMT epithelial-mesenchymal-transition
  • TGF ⁇ has a strong immunosuppressive effect in the tumor microenvironment, and thus has an important regulatory effect on tumor occurrence, development, metastasis and drug resistance.
  • TGF ⁇ has an inhibitory effect on some immune cells that inhibit tumor cells in the tumor microenvironment.
  • TGF ⁇ can promote the transformation of anti-tumor MI macrophages into MII macrophages, and can also inhibit the recruitment and recruitment of MI macrophages. Secretion of anti-tumor cytokines.
  • TGF ⁇ can also inhibit the maturation of dendritic cells and the secretion of related cytokines, as well as promote the apoptosis of dendritic cells.
  • TGF ⁇ can also inhibit the differentiation of CD8 + T cells, the secretion of IFN- ⁇ , and promote the apoptosis of CD8 + T cells.
  • TGF ⁇ promotes immunosuppressive cells such as Treg cells in the tumor microenvironment.
  • TGF ⁇ can also promote fibrosis of tumor matrix, collagen deposition, and block immune infiltration.
  • the purpose of the present invention is to provide an antibody fusion protein targeting tumor CD73-TGF ⁇ and TF-TGF ⁇ .
  • the present invention provides antibodies against the tumor microenvironment and fusion proteins invented on the basis thereof, including antibodies targeting CD73, dual targeting CD73 and TGF ⁇ fusion proteins (anti-CD73-TGF ⁇ R fusion protein), and dual Targets the fusion protein of TF and TGF ⁇ (anti-TF-TGF ⁇ R fusion protein). They can improve the tumor microenvironment, increase the killing effect of the body's own immune system on tumors, and inhibit tumor proliferation, metastasis and drug resistance.
  • a recombinant bifunctional fusion protein comprising:
  • the first binding domain (D1) The first binding domain (D1);
  • the second binding domain (D2) is the second binding domain (D2);
  • the first binding domain specifically binds the target molecule CD73 or TF protein
  • the second binding domain specifically binds the target molecule TGF ⁇ protein.
  • the D1 is an antibody or antibody fragment that specifically binds to CD73 or TF protein.
  • the antibodies include: animal-derived antibodies (such as murine antibodies), chimeric antibodies, and humanized antibodies.
  • the antibody fragment comprises a heavy chain variable region and a light chain variable region.
  • the antibody fragments include single-chain variable region fragments (scFv) and double-chain variable region fragments (dcFv).
  • the D2 is a polypeptide fragment that specifically binds to the TGF ⁇ protein, and the polypeptide fragment is derived from the TGF ⁇ receptor.
  • the D2 is the extracellular region of TGF ⁇ receptor II.
  • D2 is shown in SEQ ID NO.:33.
  • the D1 and the D2 are connected by a connecting peptide.
  • D1 is an antibody fragment
  • the connecting peptide is an antibody constant region sequence
  • D1 is an antibody
  • D1 is an anti-CD73 monoclonal antibody or an anti-TF monoclonal antibody
  • D2 is connected to a region of D1 selected from the group consisting of a heavy chain variable region, a heavy chain constant region, and a light chain through a connecting peptide. Variable region, or a combination thereof.
  • D1 is an anti-CD73 monoclonal antibody or an anti-TF monoclonal antibody
  • D2 is connected to the end of the heavy chain constant region of D1 through a connecting peptide.
  • the bifunctional fusion protein is a homodimer.
  • the bifunctional fusion protein (monomer) has the structure shown in formula I from the N-terminus to the C-terminus:
  • T1, T2, and T3 are each independently none or the extracellular region of TGF ⁇ receptor II, and at least one is not none;
  • L1, L2, and L3 are each independently a key or joint element
  • VL stands for the light chain variable region of an anti-CD73 or TF antibody
  • CL stands for the light chain constant region of an anti-CD73 or TF antibody
  • VH stands for the variable region of the heavy chain of an anti-CD73 or TF antibody
  • CH represents the heavy chain constant region of an anti-CD73 or TF antibody
  • ⁇ represents a disulfide bond or a covalent bond
  • the bifunctional fusion protein has the activity of simultaneously binding to CD73 or TF and binding to TGF ⁇ .
  • the L1, L2, and L3 are each independently (G4S) 4 G.
  • the T2, T3, L2 and L3 are none.
  • T1 is the extracellular region of TGF ⁇ receptor II.
  • the CH includes CH1, CH2, and CH3.
  • the two monomers of the bifunctional fusion protein form a dimer through disulfide bonds on CH2 and CH3.
  • the anti-CD73 antibody is as described in the second aspect of the present invention.
  • the anti-TF antibody contains:
  • variable region of the antibody heavy chain as shown in SEQ ID NO.: 37;
  • an anti-CD73 antibody said antibody containing:
  • the antibody further contains a heavy chain constant region, and the heavy chain constant region is of human, murine or rabbit origin.
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:2.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 3, SEQ ID NO.: 9, SEQ ID NO.: 10.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 4, SEQ ID NO.: 12, and SEQ ID NO.: 13.
  • the heavy chain variable region has SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, The amino acid sequence shown in SEQ ID NO.: 19 and SEQ ID NO.: 20.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27.
  • the antibody also contains a light chain constant region, and the light chain constant region is of human, murine or rabbit origin.
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 5 and SEQ ID NO.: 8.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 6 and SEQ ID NO.: 11.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.:7.
  • the light chain variable region has an amino acid sequence shown in SEQ ID NO.: 21, SEQ ID NO.: 22, and SEQ ID NO.: 23.
  • the light chain variable region has an amino acid sequence shown in SEQ ID NO.: 28, SEQ ID NO.: 29, and SEQ ID NO.: 30.
  • the affinity EC 50 of the antibody to CD73 (such as the extracellular domain of human CD73 protein, CD73-ECD) is 0.083-0.131 nM.
  • the third aspect of the present invention provides a use of the recombinant bifunctional fusion protein of the first aspect of the present invention for (a) preparing detection reagents or kits; and/or (b) preparing prevention and/or Drugs for the treatment of CD73 or TF, and/or TGF ⁇ -related diseases.
  • D1 is an antibody or antibody fragment that specifically binds to CD73, and the detection reagent or kit is used for:
  • the detection reagent, detection plate or kit is used to diagnose CD73 and/or TGF ⁇ related diseases.
  • the drug is used to treat or prevent tumors with high expression of CD73 and/or TGF ⁇ , tumor migration, or tumor drug resistance.
  • the tumor drug resistance includes: tumor immunotherapy drug drug resistance, tumor targeted therapy drug drug resistance, conventional tumor chemotherapy drug resistance, and radiotherapy insensitivity.
  • the drug is used for use selected from the following group:
  • CD73 that specifically binds to tumor cells and/or immune/stromal cells in the tumor microenvironment
  • the CD73 and/or TGF ⁇ -related diseases are selected from the following group: cancer, autoimmune diseases, metabolism-related diseases, fibrosis-related diseases, infectious diseases, or combinations thereof.
  • the CD73 and/or TGF ⁇ -related diseases include: tumor occurrence, growth, drug resistance and/or metastasis.
  • the cancer includes solid tumors and blood cancers.
  • the cancer is a tumor with high expression of CD73 and/or TGF ⁇ .
  • the tumors with high expression of CD73 and/or TGF ⁇ are selected from the following group: breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, brain glioma, melanoma, Leukemia, lymphoma, or a combination thereof.
  • the cancer is a drug-resistant tumor.
  • the tumor with high expression of CD73 and/or TGF ⁇ refers to the level of CD73 and/or TGF ⁇ transcript and/or protein in tumor tissue L1 and the level of transcript and/or protein in normal tissue L0
  • the ratio is L1/L0 ⁇ 2, preferably ⁇ 3.
  • the metabolic-related diseases include diabetes, food-borne obesity, and fatty inflammation.
  • the fibrosis-related diseases include: pulmonary fibrosis, renal fibrosis, liver fibrosis, cardiovascular fibrosis, splenic fibrosis, bone marrow fibrosis, and nervous system fibrosis.
  • infectious diseases include: bacterial and viral infections.
  • D1 is an antibody or antibody fragment that specifically binds to TF protein, and the detection reagent or kit is used for:
  • the detection reagent, detection plate or kit is used to diagnose TF and/or TGF ⁇ related diseases.
  • the drug is used to treat or prevent tumors with high expression of TF and/or TGF ⁇ , tumor migration, or tumor drug resistance.
  • the tumor drug resistance includes: tumor immunotherapy drug drug resistance, tumor targeted therapy drug drug resistance, conventional tumor chemotherapy drug resistance, and radiotherapy insensitivity.
  • the drug is used for use selected from the following group:
  • the TF and/or TGF ⁇ -related diseases are selected from the following group: cancer, thrombotic diseases, inflammatory diseases, autoimmune diseases, metabolic related diseases, fibrosis related diseases, or a combination thereof.
  • the TF and/or TGF ⁇ -related diseases include: tumor occurrence, growth, drug resistance and/or metastasis.
  • the cancer is a tumor with high expression of TF and/or TGF ⁇ .
  • the tumor with high expression of TF and/or TGF ⁇ is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, brain glioma, melanoma, Leukemia, lymphoma, or a combination thereof.
  • the tumor with high expression of TF and/or TGF ⁇ refers to the level of TF and/or TGF ⁇ transcript and/or protein in tumor tissue L1 and the level of transcript and/or protein in normal tissue L0
  • the ratio is L1/L0 ⁇ 2, preferably ⁇ 3.
  • the fourth aspect of the present invention provides a pharmaceutical composition, which contains:
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the fifth aspect of the present invention provides a method for treating diseases related to CD73 and/or TGF ⁇ , which method administers the recombinant bifunctional fusion protein according to the first aspect of the present invention or the second aspect of the present invention to a subject in need.
  • the antibody of the aspect, or the pharmaceutical composition of the fourth aspect of the present invention is not limited to CD73 and/or TGF ⁇ .
  • the method further includes: administering other drugs or treatment methods to the subject in need for combined therapy.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapeutics, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.
  • the sixth aspect of the present invention provides a method for preparing an anti-CD73-TGF ⁇ R fusion protein, which includes the steps:
  • the seventh aspect of the present invention provides a polynucleotide which encodes the recombinant bifunctional fusion protein according to the first aspect of the present invention or the antibody according to the second aspect of the present invention.
  • the eighth aspect of the present invention provides a vector containing the polynucleotide of the seventh aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the ninth aspect of the present invention provides a genetically engineered host cell, which contains the vector according to the eighth aspect of the present invention or the genome integrated with the polynucleotide according to the seventh aspect of the present invention.
  • the tenth aspect of the present invention provides an immunoconjugate, which contains:
  • a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the eleventh aspect of the present invention provides a recombinant protein, which contains:
  • the twelfth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • the thirteenth aspect of the present invention provides a method for inhibiting the growth and migration of tumor cells, comprising the steps of: administering the recombinant bifunctional fusion protein as described in the first aspect of the present invention or the second aspect of the present invention to a subject in need The antibody.
  • the fourteenth aspect of the present invention provides a method for protecting the proliferation of T lymphocytes, including the steps of: administering the recombinant bifunctional fusion protein as described in the first aspect of the present invention or the second aspect of the present invention to a subject in need The antibody described in the aspect.
  • a method for inhibiting tumor growth in a model animal comprising the steps of: administering the recombinant bifunctional fusion protein as described in the first aspect of the present invention or the first aspect of the present invention to a subject in need The antibody of the two aspects.
  • the drugs can be administered alone or in combination, including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiotherapy.
  • the improvement effect and mechanism of the fusion protein of the present invention on the tumor immune microenvironment in vivo are provided.
  • the anti-CD73/TF-TGF ⁇ R fusion protein can increase the infiltration level of MI (tumor suppressor) macrophages in tumors in vivo, and enhance the anti-tumor effect.
  • MI tumor suppressor
  • the anti-CD73/TF-TGF ⁇ R fusion protein can reduce the level of tumor MII (tumor promoting) macrophages in vivo and reduce the level of immunosuppressive cells, thereby further improving the anti-tumor effect.
  • the anti-CD73-TGF ⁇ R fusion protein can increase the infiltration level of mature dendritic cells in the tumor microenvironment in vivo, and enhance the antigen presentation ability and tumor killing level.
  • the anti-CD73-TGF ⁇ R fusion protein in humanized immune reconstitution NSG mice, can increase the infiltration of CD45 + immune cells in tumors and enhance the anti-tumor effect of immune cells.
  • the anti-CD73-TGF ⁇ R fusion protein in humanized immune reconstitution NSG mice, can increase the infiltration of CD8 + T cells in tumors, and directly or indirectly increase the killing effect of effector T cells on tumors.
  • the anti-CD73/TF-TGF ⁇ R fusion protein of the present invention can show stronger and more excellent tumor microenvironment improvement activity than the CD73/TF monoclonal antibody, resulting in a more excellent improvement of anti-tumor T
  • effector cells such as cells and NK cells
  • Tregs regulatory T cells
  • MII tumor-promoting macrophages Wait.
  • the anti-CD73/TF-TGF ⁇ R fusion protein can more strongly improve the permeability of the tumor matrix than the CD73/TF monoclonal antibody, and increase the efficiency of drug entry into the tumor.
  • Figure 1 shows the selected 5 mouse monoclonal antibodies targeting human CD73 against human CD73-highly expressed MDA-MB-231 (CD73-P) and CD73-lowly expressed MDA-MB-453 (CD73-N).
  • CD73-P human CD73-highly expressed MDA-MB-231
  • CD73-N CD73-lowly expressed MDA-MB-453
  • Figure 2 shows the results of the ELISA measurement of the affinity of three chimeric antibodies mAb001c, mAb002c, and mAb004c to the human CD73-ECD antigen.
  • Figure 3 shows the determination of the inhibitory activity of three chimeric antibodies mAb001c, mAb002c, and mAb004c on the catalytic function of recombinant human CD73.
  • Figure 4 shows the specific binding activity of three chimeric antibodies mAb001c, mAb002c, and mAb004c to the CD73 antigen on the surface of breast tumor cell MDA-MB-231.
  • Figure 5 shows the specific binding activity of three chimeric antibodies mAb001c, mAb002c, and mAb004c to the CD73 antigen on the surface of lung tumor cell NCI-H1299.
  • Figure 6 shows the determination of the inhibitory activity of three chimeric antibodies mAb001c, mAb002c, and mAb004c on the enzymatic hydrolysis of AMP by CD73 on the surface of breast tumor cell MDA-MB-231.
  • Figure 7 shows the determination of the inhibitory activity of three chimeric antibodies mAb001c, mAb002c, and mAb004c on the AMP-catalyzed hydrolysis of CD73 on the surface of lung tumor cell NCI-H1299.
  • Figure 8 is a schematic diagram of the structure of the CD73(TF) antibody-TGF ⁇ R fusion protein of the present invention, wherein the CD73 antibody and TF antibody and the extracellular region of TGF ⁇ receptor II respectively pass through a (Gly 4 Ser) 4 Gly linker (Linker) Fused together.
  • Figure 9 is a schematic diagram of the construction process of the expression vector of the heavy chain of the CD73 antibody-TGF ⁇ R and TF antibody-TGF ⁇ R fusion protein respectively.
  • the DNA fragments of the extracellular region of Linker and TGF ⁇ R are inserted between the BspQI and BamHI restriction sites.
  • FIG. 10 The electrophoresis diagram of the expression vector plasmids against CD73-TGF ⁇ R fusion protein Hu001-14-TGF ⁇ R heavy chain with BspQI, BamHI single digestion, and BspQI/BamHI double digestion (A); SDS-PAGE gel analysis diagram (B ) Shows the electrophoresis of the fusion protein in reduced and non-reduced states.
  • FIG 11 The electrophoresis diagram of the expression vector plasmids against the heavy chain of the CD73-TGF ⁇ R fusion protein Hu001-32-TGF ⁇ R with BspQI, BamHI single digestion, and BspQI/BamHI double digestion (A); SDS-PAGE gel analysis (B) ) Shows the electrophoresis of the fusion protein in reduced and non-reduced states.
  • Figure 12 The electrophoresis diagram of the expression vector plasmids against the heavy chain of the CD73-TGF ⁇ R fusion protein Hu002-3-TGF ⁇ R with BspQI, BamHI single digestion, and BspQI/BamHI double digestion (A); SDS-PAGE gel analysis (B) ) Shows the electrophoresis of the fusion protein in reduced and non-reduced states.
  • Figure 13 shows the ELISA detection of the affinity of the anti-CD73-TGF ⁇ R fusion protein to the extracellular domain of human CD73.
  • Figure 14 is an assay to detect the inhibitory activity of the anti-CD73-TGF ⁇ R fusion protein on the catalytic function of recombinant human CD73 enzyme.
  • Figure 15 shows the affinity of anti-CD73-TGF ⁇ R fusion protein to TGF ⁇ 1 detected by ELISA.
  • Figure 16 shows the FACS detection of anti-CD73-TGF ⁇ R fusion protein on the CD73 antigen binding activity on the surface of lung tumor cell NCI-H1299.
  • Figure 17 shows the FACS detection of the anti-CD73-TGF ⁇ R fusion protein on the CD73 antigen binding activity on the surface of breast tumor cells MDA-MB-231.
  • Figure 18 shows that the anti-CD73-TGF ⁇ R fusion protein can effectively reverse the inhibitory effect of adenosine (AMP) on the proliferation of human T lymphocytes.
  • the experiment uses sorting to obtain CD3 + human T cells, and counts the cell proliferation rate after culturing them for 5 days.
  • Figure 19 shows the anti-tumor activity test of anti-CD73-TGF ⁇ R fusion protein Hu001-14-TGF ⁇ R in vivo.
  • NCI-H1299 non-small cell lung cancer cells were mixed with 50 ⁇ g antibody and then inoculated into the back of nude mice subcutaneously, and observed 2-3 times a week. The results show the tumor growth curve (top) and the tumor weight at the end of the experiment (bottom).
  • Figure 20 shows the IHC detection of the inhibitory activity of anti-CD73-TGF ⁇ R fusion protein Hu001-14-TGF ⁇ R on TGF ⁇ 1 in NCI-H1299 tumors.
  • Figure 21 shows the IHC detection of the anti-CD73-TGF ⁇ R fusion protein Hu001-14-TGF ⁇ R's inhibitory activity on macrophages (TAM) in the H1299 tumor microenvironment.
  • Figure 22 shows the in vivo anti-tumor activity of CD73 antibody Hu001-14 and anti-CD73-TGF ⁇ R fusion protein Hu001-14-TGF ⁇ R.
  • the doses were Hu001-14 (10mg/kg) and Hu001-14-TGF ⁇ R. (12.5mg/kg).
  • the results show the tumor growth curve (top) and the tumor weight at the end of the test on day 26 (bottom).
  • Figure 23 shows the detection of MI-type macrophage markers CD86 and F4/80 in NCI-H441 tumor by immunofluorescence (IF) double staining.
  • IF immunofluorescence
  • Figure 24 shows the immunohistochemistry (IHC) detection of MII-type macrophage markers CD206 and F4/80 in NCI-H441 tumor.
  • IHC immunohistochemistry
  • Figure 25 shows the detection of MII-type macrophage markers CD206 and F4/80 in NCI-H441 tumor by immunofluorescence (IF) double staining.
  • IF immunofluorescence
  • Figure 26 shows the detection of mature dendritic cell markers CD86 and CD11c in NCI-H441 tumor by immunofluorescence (IF) double staining.
  • IF immunofluorescence
  • Figure 27 shows the anti-tumor effect of Hu001-14-TGF ⁇ R on the human immune reconstitution mouse (NSG mouse) NCI-H1299 lung cancer xenograft model.
  • the count of CD45 + cells and CD8 + cells by flow cytometry confirmed the successful immune reconstitution in NSG mice (A); Hu001-14 and Hu001-14-TGF ⁇ R were evaluated in the NCI-H1299 tumor model (B ).
  • Figure 28 shows the infiltration of CD45 + immune cells and CD8 + cells in the NCI-H1299 tumor of human immune reconstitution mice (NSG mice) detected by immunohistochemistry (IHC).
  • Hu001-14-TGF ⁇ R can significantly increase the infiltration of CD45 + immune cells and CD8 + T cells in tumor tissues.
  • Figure 29 is the electrophoresis image (A) of the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R expression vector plasmid with BspQI, BamHI single enzyme digestion, and BspQI/BamHI double enzyme digestion (A); SDS-PAGE gel analysis image (B) shows The electrophoresis of the fusion protein in the reduced and non-reduced state.
  • Figure 30 shows the ELISA detection of the affinity of the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R to the extracellular domain of human TF (TF-ECD).
  • Figure 31 shows the affinity of the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R to TGF ⁇ 1 detected by ELISA.
  • Figure 32 shows the FACS detection of anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R on the surface TF antigen binding activity of pancreatic tumor cell BxPC3.
  • Figure 33 shows the FACS detection of anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R on the surface TF antigen binding activity of breast tumor cell MDA-MB-231.
  • Figure 34 shows the anti-tumor activity of TF antibody HuSC1-39 and anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R in vivo.
  • HCC1806 breast cancer cells were mixed with 20 ⁇ g hIgG1, 20 ⁇ g HuSC1-39 and 25 ⁇ g HuSC1-39-TGF ⁇ R, respectively, and then inoculated into NSG mouse breast pads, and observed 2 to 3 times a week. The results show the tumor weight at the end of the test (16 days of tumor growth).
  • Figure 35 shows the detection of MI-type macrophage markers CD86 and F4/80 in HCC1806 tumor by immunofluorescence double staining.
  • the results show that compared with the TF antibody HuSC1-39, the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R can more effectively reduce the infiltration level of MII-type macrophages and achieve a more effective anti-tumor therapeutic effect.
  • Figure 36 shows the immunohistochemistry (IHC) detection of MII-type macrophage markers CD206 and F4/80 in HCC1806 tumors.
  • IHC immunohistochemistry
  • Figure 37 shows the immunofluorescence double staining detection of MII-type macrophage markers CD206 and F4/80 in HCC1806 tumors. The results further confirmed that compared with HuSC1-39, the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R can more effectively inhibit the infiltration level of MII-type macrophages in tumors and improve the effect of anti-tumor treatment.
  • human-mouse chimeric antibodies mAb001c, mAb002c, and mAb004c can bind to CD73 antigen with high specificity, measured by ELISA Its EC 50 is 0.024nM, 0.016nM, 0.038nM, respectively.
  • Humanized antibodies designed based on mAb001c and mAb002c also have excellent characteristics.
  • two novel antibody fusion proteins have been obtained through the design, preparation, and in vitro and in vivo verification of the present invention.
  • Both the anti-CD73-TGF ⁇ R fusion protein and the anti-TF-TGF ⁇ R fusion protein have the advantages of high dual target binding affinity and strong specificity, and can be used to further enhance the anti-tumor immune function.
  • the present invention has been completed on this basis.
  • CD73-ECD human CD73 protein
  • CD73 extracellular domain protein to immunize Balb/c mice, and the dosage of CD73 extracellular domain protein is 50 ⁇ g/mouse to prepare immune spleen cells; timely prepare mouse myeloma cells (SP2/0) and feeder cells to Prepared for the needs of integration.
  • PEG-mediated fusion of spleen cells and SP2/0 cells is used to prepare hybridoma cells, and the cells with high titer, good morphology, and monoclonal growth are screened for subclonal screening. , Until the positive clone rate of three consecutive screenings is 100%, the cell line is expanded and cultivated and the library is built.
  • the cell culture supernatant is collected and purified, and the purified product is quantified and tested.
  • the biological activity and target specificity of the selected 5 hybridoma monoclonal antibodies were determined.
  • the supernatant of the monoclonal cell culture medium was detected by flow cytometry (FACS). All 5 antibodies can specifically bind to human CD73-highly expressed MDA-MB-231 cells ( CD73-P), but has no obvious binding activity to MDA-MB-453 cells (CD73-N) with low expression of CD73-.
  • the purified antibody samples were used for serial dilution and FACS detection.
  • mAb001, mAb002, mAb003, mAb004, and mAb005 have excellent binding affinity to MDA-MB-231 cells.
  • the EC 50 of FACS detection is 1.24nM, 0.65nM, 10.7nM, 4.69nM, 26.07nM.
  • mAb001, mAb002, and mAb004 are preferentially selected for antibody sequencing and identification.
  • VH heavy chain variable region
  • VL light chain variable region
  • CDR complementarity determining region
  • variable region sequences Three groups of variable region sequences (see SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7) Clone into a vector containing human IgG1 heavy chain constant region and Kappa chain constant region. After sequencing, the constructed chimeric antibody is expressed using transfection technology and mammalian expression system (FreeStyle TM 293T cells) And purified, the obtained human-mouse chimeric antibodies were numbered mAb001c, mAb002c, and mAb004c, respectively.
  • variable region sequence of the antibody contains several unfavorable amino acids, which have been modified by point mutations.
  • the point mutation (PTM) was cloned into the hIgG1 vector by matching the above-mentioned point mutation template to obtain the corresponding chimeric antibody mutant of the point mutation.
  • Step 2 ELISA determination of the affinity of chimeric antibody to human CD73 antigen
  • CD73-ECD CD73 protein extracellular domain
  • the coating solution Dilute the CD73 protein extracellular domain (CD73-ECD) to 1 ⁇ g/mL with the coating solution, and coat the ELISA plate with 100 ⁇ L/well, 4°C, overnight. Wash off the excess antigen, block with 1% BSA at room temperature for 2h, then add each monoclonal antibody of 3-fold dilution, 100 ⁇ L/well, incubate at room temperature for 1h; wash off the unbound antibody, add appropriate concentration of horseradish peroxidase Labeled anti-mouse secondary antibody, 100 ⁇ L/well, incubate at room temperature for 0.5h. Wash off the unbound secondary antibody, add TMB color developing solution to react for about 15 minutes, add 1N HCL, 50 ⁇ L/well, stop the color reaction, then measure its absorbance at 450nm and analyze the data.
  • mAb001c, mAb002c, and mAb004c have strong affinity for CD73-ECD, with EC 50 of 0.024nM, 0.016nM, and 0.038nM, respectively.
  • Step 3 Determination of the inhibitory activity of the chimeric antibody on the catalytic function of recombinant human CD73
  • CD73 extracellular region Dilute the human recombinant CD73 enzyme (CD73 extracellular region) to 0.1 ⁇ g/mL with antigen diluent, and spread it evenly on a 96-well low-adsorption culture plate at 25 ⁇ L/well.
  • CD73 antibody diluted in a 3-fold gradient from 2nM to 0.0009nM to the culture plate, mix well (final concentration is 1nM ⁇ 0.00045nM), incubate at 37°C for 1h, add 25 ⁇ L of a mixture containing 1.2mM AMP and 0.4mM ATP Incubate at 37°C for 1h.
  • mAb001c, mAb002c, and mAb004c all have the activity of significantly inhibiting the hydrolysis of AMP by recombinant CD73 protease, with IC 50 of 0.025 nM, 0.031 nM, and 0.039 nM, respectively.
  • Step 4 The binding affinity of the chimeric antibody to CD73 on the surface of tumor cells
  • the cells were washed twice with PBS to remove unbound primary antibody, and then the target cells were mixed with 200 ⁇ L, 2 ⁇ g/mL, PE Incubate the labeled secondary antibody at 4°C for 30 minutes, wash the cells twice with PBS to remove unbound secondary antibody, and finally resuspend the cells in 200 ⁇ L PBS. Determine the binding affinity of the tested antibody to CD73 on the surface of the corresponding cell by flow cytometry ( Binding affinity).
  • mAb001c, mAb002c, and mAb004c have excellent binding affinity to MDA-MB-231, with EC 50 of 0.71nM, 0.36nM, 2.5nM, respectively;
  • mAb001c, mAb002c, and mAb004c have the same excellent binding affinity to NCI-H1299, with EC 50 of 1.0 nM, 0.39 nM, and 2.2 nM, respectively;
  • the above results indicate that the monoclonal antibody of this example can target CD73 of human tumor cells.
  • Step 5 Chimeric antibody inhibits the catalytic function of CD73 enzyme on the surface of tumor cells
  • CD73-highly expressed triple-negative breast cancer cell MDA-MB-231 and non-small cell lung cancer cell NCI-H1299 were used as target cells.
  • the tested antibody was added to a 96-well plate, after incubating at 37°C for 30min, 25 ⁇ L of 0.9mM AMP was added and incubated at 37°C with 5% CO 2 for 3h (the final antibody concentration was 133.3nM ⁇ 0.06nM).
  • mAb001c, mAb002c, and mAb004c 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 1.86 nM, 0.79 nM and 4.16 nM, respectively.
  • mAb001c, mAb002c, and mAb004c inhibit the function of CD73 on the surface of NCI-H1299 cells to catalyze the hydrolysis of AMP, with IC 50 of 0.24 nM, 0.19 nM, and 0.39 nM, respectively.
  • the humanized template that best matches the non-CDR regions of mAb001c and mAb002c, and then transplant the CDR region of the antibody to the selected humanized template, replace the CDR region of the human template, and then The IgG1 constant region is recombined, and based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are backmutated.
  • the humanization of mAb001c obtained 7 humanized heavy chain variable regions (SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20), and 3 humanized light chain variable regions (SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO .:twenty three).
  • humanization of mAb002c obtains four humanized heavy chain variable regions (SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27) and 3 The variable region of the humanized light chain (SEQ ID NO.: 28, SEQ ID NO.: 29, SEQ ID NO.: 30).
  • the designed humanized variable region sequence was cloned into a vector containing human IgG1 heavy chain constant region and Kappa chain constant region through gene recombination technology. After sequencing, the transfection technology and mammalian expression system (FreeStyle TM 293 Cell) will be constructed humanized antibody expression vector. Combine and express these humanized heavy and light chains respectively, and finally mAb001c-series obtained 11 humanized antibodies, mAb002c-series obtained 12 humanized antibodies, and the corresponding heavy and light chain combinations of each antibody are shown in the table -2 shown.
  • the humanized antibodies in Table 2 were serially diluted, and their affinity for CD73 protein was determined by ELISA.
  • the experimental method was referred to Example 2.
  • the experimental results are shown in Table-3.
  • the two groups of humanized antibodies all have strong binding affinity to the CD73 protein, and the EC 50 value is 0.02 nM to 0.13 nM.
  • the humanized antibodies in Table-2 were serially diluted, and the effect of the antibodies on the enzyme activity of recombinant CD73 was determined with reference to Example 2.
  • Table-4 CD73 enzyme inhibitory activity of humanized antibodies
  • Step 4 The binding activity of humanized antibody to tumor cell CD73
  • the test results are shown in Table-5.
  • the two groups of humanized antibodies have high affinity for CD73 on the surface of NCI-H1299 cells, with EC 50 values ranging from 0.53 nM to 1.65 nM.
  • the test results are shown in Table-6.
  • the two groups of humanized antibodies have high affinity for CD73 on the surface of MDA-MB-231 cells, with EC 50 values ranging from 0.21 nM to 0.74 nM.
  • the function of binding TGF ⁇ is added, and the fusion protein of the antibody and the extracellular domain of TGF ⁇ receptor II (abbreviated as TGF ⁇ R) is designed.
  • TGF ⁇ R TGF ⁇ receptor II
  • the fusion protein is composed of two main parts: a light chain and a heavy chain; the light chain part is exactly the same as the light chain of the corresponding antibody, and the heavy chain part is composed of the heavy chain of the antibody and the TGF ⁇ R is composed of Linker. Connected.
  • the constant region of the antibody heavy chain (SEQ ID NO.: 31; in order to prevent the cleavage of the Linker binding site in the cell, the end lysine is mutated to alanine) and the end of the Linker (SEQ ID NO.: 32) N
  • the C-end of Linker is connected to TGF ⁇ R (SEQ ID NO.: 33) (Science Translational Medicine 2018; 16: 1-15).
  • Step 2 Construction of fusion protein heavy chain expression plasmid
  • the heavy chain part of the fusion protein was constructed on the basis of the original humanized antibody heavy chain expression vector (the original expression vector was from Shanghai Ruizhi Chemical, codenamed pCP), and was added to the end of the DNA sequence of the antibody Fc.
  • the expression vector of the antibody heavy chain was recovered by double digestion with BspQI and BamHI, and the synthesized DNA fragment containing Linker and TGF ⁇ R was also digested with BspQI and BamHI.
  • the target fragments are recovered, then ligated with T4 DNA ligase, transformed into competent cells of DH5 ⁇ , coated with ampicillin resistant plates, and single colonies are picked. Perform plasmid extraction and restriction enzyme digestion verification on a single colony, and perform sequencing verification to obtain a vector with the correct sequence.
  • the heavy chain (SEQ ID NO.: 34) of Hu001c-14-TGF ⁇ R (Hu001-14-TGF ⁇ R for short) and the heavy chain of Hu001c-32-TGF ⁇ R (Hu001-32-TGF ⁇ R for short) were constructed ( SEQ ID NO.: 35) and the heavy chain (SEQ ID NO.: 36) of Hu002c-3-TGF ⁇ R (Hu002-3-TGF ⁇ R for short).
  • the anti-CD73-TGF ⁇ R fusion protein consists of a heavy chain and a light chain.
  • the light chain number of Hu001-14-TGF ⁇ R and Hu001-32-TGF ⁇ R is SEQ ID NO.: 21, and the light chain number of Hu002-3-TGF ⁇ R is SEQ ID NO.: 29, as shown in Table-7.
  • SEQ ID NO.: 36 Amino acid sequence of Hu002c-3-TGF ⁇ R fusion protein heavy chain
  • the heavy chain and light chain expression vector plasmids were transfected into 293T cells by liposome for protein expression. After culturing in a petri dish for 5 days, the expression supernatant was collected, and then purified with protein A, and the purified protein was analyzed by SDS-PAGE.
  • Step 2 ELISA determination of the affinity of the fusion protein to human CD73 antigen
  • CD73-ECD CD73 protein extracellular domain
  • Hu001-14-TGF ⁇ R, Hu001-32-TGF ⁇ R, Hu002-3-TGF ⁇ R have strong affinity for CD73-ECD, with EC 50 of 0.09nM, 0.11nM, 0.06nM, respectively;
  • Step 3 Determination of the inhibitory activity of the fusion protein on the catalytic function of recombinant human CD73
  • step 3 in Example 2 to determine the inhibitory effect of the anti-CD73-TGF ⁇ R fusion protein on the catalytic function of recombinant human CD73 enzyme.
  • Hu001-14-TGF ⁇ R, Hu001-32-TGF ⁇ R, and Hu002-3-TGF ⁇ R all have significant inhibitory activity to hydrolyze AMP by recombinant CD73 protease, with IC 50 of 0.052nM, 0.083nM, 0.032nM, respectively ;
  • Step 4 Detect the binding affinity of the fusion protein to TGF ⁇ by ELISA
  • Human TGF ⁇ 1 (Sino biological, Cat#10804-HNAC) was used as an antigen to coat the microtiter plate, and the concentration of TGF ⁇ 1 was 0.5 ⁇ g/mL.
  • the experimental method refers to step 2, in which the final concentration of the primary antibody is 27 ⁇ g/mL, and it is diluted by 3 times.
  • Hu001-14-TGF ⁇ R, Hu001-32-TGF ⁇ R, Hu002-3-TGF ⁇ R have a strong affinity for TGF ⁇ , with EC 50 of 0.4nM, 0.26nM, 0.55nM, respectively.
  • Step 5 The specific binding of the fusion protein to CD73 on the surface of tumor cells
  • the triple-negative breast cancer cell MDA-MB-231 and non-small cell lung cancer cell NCI-H1299 with high expression of CD73 were used to determine the binding of the fusion protein to CD73 on the cell surface.
  • FACS flow cytometry
  • Hu001-14-TGF ⁇ R, Hu001-32-TGF ⁇ R, Hu002-3-TGF ⁇ R have a strong affinity for CD73 on the surface of tumor cells, and the EC 50 for H1299 is 1.6 respectively.
  • EC 50 for MDA-MB-231 is 1.5nM, 1.4nM;
  • Example 6 The protective effect of anti-CD73-TGF ⁇ R fusion protein on the proliferation of T lymphocytes
  • PBMC recovery, expansion and sorting of PBMC: first use a medium containing 500ng/mL CD3/CD28 antibody and 100IU/mL IL-2 to resuscitate PBMC for 3 to 4 days, and then use a sorting kit (Stemcell, Cat# 1795) CD3-positive T lymphocytes were obtained after sorting PBMC.
  • T cell proliferation test the T cells obtained by the above sorting are fluorescently labeled, and the pre-prepared CFSE (carboxyfluorescein succinimidyl ester) is added to the cell suspension (final concentration 2.5 ⁇ M), and the cells are labeled at 37°C for 5 minutes and then washed with PBS 3 times.
  • CFSE carboxyfluorescein succinimidyl ester
  • FACS flow cytometer
  • Step 1 Anti-tumor activity of anti-CD73-TGF ⁇ R in nude mice transplanted NCI-H299 tumor model
  • Step 2 The inhibitory activity of anti-CD73-TGF ⁇ R on the production of TGF ⁇ 1 in tumor tissues
  • the tumor samples obtained in step 1 were embedded in paraffin and sectioned.
  • the tumors in the hIgG1 group and the Hu001-14-TGF ⁇ R administration group were placed on the same paraffin section, and the obtained sections were stained by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the concentration of the primary antibody is 1:50
  • the secondary antibody Jackson Immuno, Cat#111-035-003
  • DAB color developing solution to color
  • hematoxylin to counterstain, dehydration and mount.
  • the obtained IHC imaging pictures are analyzed by ImageJ software, and the gray value of about 10 representative fields is calculated, and the final statistical average value ⁇ S.E.
  • the accumulation level of TGF ⁇ 1 expression in the hIgG1 group tumor was significantly higher than that in the Hu001-14-TGF ⁇ R administration group.
  • Step 3 Anti-CD73-TGF ⁇ R inhibits MII type macrophages in tumor microenvironment
  • the concentration of the primary antibody of CD206 is 1:200 dilution
  • F4/80 macrophage
  • the concentration of the primary antibody of the cell co-marker is 1:200 dilution
  • the concentration of the secondary antibody Jackson Immuno, Cat#111-035-003 is 1:200 dilution.
  • the ratio of F480/CD206 in the hIgG1 tumor group was significantly lower than that in the Hu001-14-TGF ⁇ R administration group, indicating that Hu001-14-TGF ⁇ R can reduce the level of MII-type macrophages in the tumor microenvironment.
  • Example 8 Anti-CD73-TGF ⁇ R administered by intravenous injection can significantly inhibit the growth of NCI-H441 tumor and improve the tumor immune microenvironment
  • Step 1 The inhibitory activity of intravenous injection of anti-CD73-TGF ⁇ R on tumor growth
  • the Hu001-14-TGF ⁇ R group can more significantly inhibit the growth of NCI-H441 tumors in nude mice.
  • Step 2 Intravenous injection of anti-CD73-TGF ⁇ R increased the infiltration level of MI macrophages in the tumor microenvironment
  • the tumor samples obtained in step 1 were embedded in paraffin and sectioned.
  • the tumors in the hIgG1 administration group, the Hu001-14 administration group and the Hu001-14-TGF ⁇ R administration group were placed on the same paraffin section, and then the paraffin section was performed Immunofluorescence (IF) staining. Dewaxing and hydration are performed first, then citric acid is used for antigen retrieval, and then blocking is performed.
  • Anti-CD86 PE-labeled primary antibody (Biolegend) and anti-F4/80 488-labeled primary antibody (Biolegend) are incubated overnight. The concentration of the primary antibody was 1:100 dilution, and the nucleus was stained with DAPI and then mounted with anti-quenching agent.
  • the results are shown in Figure 23.
  • the overall number of MI-type (CD86 + F4/80 + double positive) macrophages in the IgG1 group of tumors is very low, and the CD73 antibody Hu001-14 treatment group has a certain increase.
  • the Hu001-14-TGF ⁇ R treatment group can significantly and greatly increase the infiltration/expansion number of MI-type macrophages in the tumor microenvironment, thereby achieving anti-tumor effects.
  • Step 3 Intravenous injection of anti-CD73-TGF ⁇ R reduced the infiltration level of MII macrophages in the tumor microenvironment
  • step 2 of this embodiment IHC and IF staining were performed on the same paraffin sections used in step 2 of this embodiment respectively, and the methods were respectively referred to step 2 of embodiment 7 and step 2 of this embodiment.
  • concentration of anti-CD206 PE-labeled primary antibody (Biolegend) and anti-F4/80 488-labeled primary antibody (Biolegend) was 1:100 dilution.
  • Step 4 Intravenous injection of anti-CD73-TGF ⁇ R increased the level of mature dendritic cells in the tumor microenvironment
  • Immunofluorescence (IF) staining was performed on the same paraffin sections used in step 2 of this example.
  • the method refers to step 2, in which anti-CD86 PE-labeled primary antibody (Biolegend) and anti-CD11c FITC-labeled primary antibody (Biolegend) The concentration is 1:100 dilution.
  • Anti-CD73-TGF ⁇ R can promote the infiltration of human CD45 + immune cells and CD8 + T lymphocytes in transplanted tumors of NSG mice with immune reconstitution
  • Step 1 NSG mouse humanized immune reconstitution and anti-CD73-TGF ⁇ R inhibit NCI-H1299 tumor
  • the frozen human PBMC was resuscitated, the supernatant was removed by centrifugation, the cells were resuspended in PBMC medium containing 100 IU/mL IL-2, and placed in a cell incubator for 6 hours to recover. The cells were centrifuged, the culture medium was discarded, and then washed with PBS and resuspended to a cell concentration of 2.5x10 7 /mL.
  • Each NSG mouse was vaccinated with 5x10 6 PBMC through the tail vein, and a group without PBMC was set up at the same time.
  • Step 2 The effect of anti-CD73-TGF ⁇ R on the infiltration of CD45 + cells and CD8 + T cells in NCI-H1299 tumor
  • NCI-H1299 tumors were collected when the test was terminated on the 28th day, fixed with 4% neutral paraformaldehyde, dehydrated, paraffin embedded, sectioned and immunohistochemically stained to observe the effects of hIgG1, Hu001-14 and Hu001-14-TGF ⁇ R on tumors The impact of human CD45 + immune cells and CD8 + T cell infiltration.
  • Hu001-14 and Hu001-14-TGF ⁇ R both have clear tumor immunotherapy effects in the humanized NSG immune reconstruction mouse NCI-H1299 xenograft tumor model; compared with Hu001-14, Hu001- 14-TGF ⁇ R further improves the infiltration of immune effector cells into tumor tissues, improves the tumor immune microenvironment, and presents a more significant anti-tumor therapeutic effect.
  • the anti-TF-TGF ⁇ R fusion protein HuSC1-39-TGF ⁇ R consists of a heavy chain and a light chain.
  • the heavy chain part is composed of the humanized anti-TF antibody HuSC1-39 and the extracellular region of TGF ⁇ RII through Linker.
  • the heavy chain variable region of HuSC1-39 is SEQ ID NO.: 37.
  • the light chain variable region of HuSC1-39-TGF ⁇ R is the same as the light chain variable region of HuSC1-39 (SEQ ID NO.: 38).
  • the heavy chain variable region and light chain variable region of HuSC1-39 are quoted from CN201610705557.4.
  • the fusion protein HuSC1-39-TGF ⁇ R was constructed.
  • SEQ ID NO.: 37 Amino acid sequence of heavy chain variable region of anti-TF antibody HuSC1-39
  • SEQ ID NO.: 38 Amino acid sequence of light chain variable region of anti-TF antibody HuSC1-39
  • SEQ ID NO.: 39 Amino acid sequence of HuSC1-39-TGF ⁇ R fusion protein heavy chain
  • the result of the construction of the fusion protein heavy chain vector is shown in Figure 29A.
  • the obtained vector was digested with BspQI, BamHI single enzyme and BspQI/BamHI double digestion to confirm that the fragment after digestion was consistent with the expected result.
  • the expression vector plasmid fused with heavy chain and light chain was transfected into 293T cells by liposome for protein expression. After culturing in a petri dish for 5 days, the expression supernatant was collected, and then purified with protein A. The purified protein was analyzed by SDS-PAGE.
  • Step 2 ELISA determination of the affinity of HuSC1-39-TGF ⁇ R to human TF antigen
  • TF protein extracellular domain (TF-ECD) was diluted to 2 ⁇ g/mL with the coating solution, and the ELISA plate was coated. Refer to Example 5 for the specific method of ELISA determination.
  • HuSC1-39-TGF ⁇ R have a strong affinity for the TF-ECD, EC 50 of 0.095nM.
  • TGF ⁇ 1 was used as an antigen to coat the ELISA plate, and the concentration of TGF ⁇ 1 was 0.5 ⁇ g/mL.
  • concentration of TGF ⁇ 1 was 0.5 ⁇ g/mL.
  • the final concentration of the primary antibody was 27 ⁇ g/mL, with a 3-fold dilution.
  • HuSC1-39-TGF ⁇ R have a strong affinity for TGF ⁇ 1, EC 50 of 0.61nM.
  • Step 4 The specific binding of the fusion protein to TF on the surface of tumor cells
  • triple-negative breast cancer cells MDA-MB-231 and pancreatic cancer cells BxPC3 with high TF expression were used to determine the binding affinity of the fusion protein to cell surface TF.
  • HuSC1-39-TGF ⁇ R has a strong specific binding to TF on the surface of tumor cells.
  • the EC 50 for BxPC3 cells is 5.6 nM; the EC for MDA-MB-231 cells is binding. 50 is 4.7 nM.
  • Example 11 Anti-TF-TGF ⁇ R can more effectively inhibit the growth of HCC1806 tumor and improve the tumor immune microenvironment
  • Step 1 Anti-tumor activity of anti-TF-TGF ⁇ R in NSG mouse xenograft tumor model
  • HuSC1-39-TGF ⁇ R can significantly inhibit the growth of NCI-H1806 tumors in NSG mice, and its effect is better than that of TF antibody HuSC1-39.
  • Step 2 Anti-TF-TGF ⁇ R increased the level of MI macrophages in the tumor microenvironment
  • the tumor samples obtained in step 1 were embedded in paraffin and sectioned, and the tumor paraffin sections of the hIgG1 group, HuSC1-39 group and HuSC1-39-TGF ⁇ R group were made on the same slide, and immunofluorescence (IF) staining was performed. Dewaxing and hydration are performed first, then citric acid is used for antigen retrieval, and then blocking is performed.
  • IF immunofluorescence
  • CD86 and F4/80 dual fluorescence staining uses anti-CD86 PE-labeled primary antibody (Biolegend) and anti-F4/80 488-labeled primary antibody (Biolegend) at a concentration of 1:100 dilution, incubate overnight, DAPI stains the nucleus Afterwards, the slides were mounted with anti-quenching agent. Use a fluorescence microscope for imaging and recording. The obtained IF imaging pictures were analyzed by ImageJ software, and the representative visual fields were counted, and the final statistical average value ⁇ S.E.
  • the results are shown in Figure 35.
  • the expression level of CD86 + F4/80 + in the hIgG1 group was lower, indicating that there were fewer MI-type macrophages in the hIgG1 group, and the TF antibody HuSC1-39 administration group significantly increased MI-type macrophages.
  • the level of MI-macrophages in the tumor microenvironment is the highest in the HuSC1-39-TGF ⁇ R group, indicating that HuSC1-39-TGF ⁇ R may reduce the inhibition of MI-type macrophages by TGF ⁇ , thereby further increasing
  • the degree of MI-type macrophage infiltration in the tumor was determined, and the tumor killing effect was achieved.
  • Step 3 Anti-TF-TGF ⁇ R reduces the level of MII type macrophages in the tumor microenvironment
  • IHC staining and immunofluorescence (IF) were performed on the same paraffin sections used in step 2 of this example. Refer to step 2 of embodiment 7 and step 2 of this embodiment respectively.
  • HuSC1-39 and HuSC1-39-TGF ⁇ R both have a clear tumor suppression effect and an effect of improving the tumor microenvironment in the NSG mouse HCC1806 breast cancer xenograft model; compared with HuSC1-39, HuSC1 -39-TGF ⁇ R further improves the infiltration of anti-tumor immune effector cells into tumor tissues, down-regulates the level of immunosuppressive cells, thereby improving the tumor immune microenvironment.

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Abstract

一种靶向肿瘤的重组双功能融合蛋白及其应用，具体提供了一种重组双功能融合蛋白，其包含特异性结合靶分子CD73或TF蛋白的第一结合结构域和特异性结合靶分子TGFβ蛋白的第二结合结构域；以及所述抗体融合蛋白的制备方法及其应用。CD73/TF-TGFβR融合蛋白通过高特异性地阻抑肿瘤微环境中的CD73-TGFβ或TF-TGFβ表达水平，改善肿瘤免疫细胞环境，增强肿瘤治疗的效果。该融合蛋白具有很高的靶标亲和力及显著的抗肿瘤活性。

Description

靶向肿瘤的重组双功能融合蛋白及其应用 技术领域
本发明涉及医药领域，尤其涉及靶向CD73或靶向组织因子(Tissue factor,TF)的抗体与TGFβ受体(TGFβR)的双功能融合蛋白，其制备方法、用途及抗肿瘤机制。
背景技术
目前，对于肿瘤的研究不单纯的局限于肿瘤细胞本身，肿瘤的微环境也被证明是肿瘤发生和发展的重要基础。研究发现，一些肿瘤细胞高表达的蛋白如CD73、TF、TGFβ能够影响和重塑肿瘤微环境，使之对肿瘤的发生、发展、转移及耐药等具有很重要的作用。
CD73是一种分子量为70kD的ecto-5’-核酸酶(NT5E)，被糖磷脂酰肌醇(GPI)锚定于细胞表面。CD73在多种肿瘤中异常表达，包括非小细胞肺癌、三阴性乳腺癌、胰腺癌等大多数难治型肿瘤。在肿瘤微环境中，ATP/ADP在CD39/CD73的催化作用下生成大量的腺苷(Adenosine，ADO)暴露在细胞周围。肿瘤细胞诱导产生的ADO能够产生免疫抑制，促进肿瘤发生免疫逃逸(Immunological Reviews 2017；276:121-144)。主要机制表现为：1)ADO与CD4+/CD8+效应T细胞表面受体A2AR结合以后，通过cAMP信号通路抑制其增殖，扩增，从而降低了T细胞的细胞毒性；2)ADO能够干扰NK细胞与肿瘤细胞之间的黏附程度，降低NK细胞的细胞毒性；3)ADO通过激活一系列免疫抑制细胞亚群如Tregs、MDSC等促进其增殖，从而增强了肿瘤细胞周围的免疫抑制作用和抗炎功能；4)ADO能够抑制M1型巨噬细胞的分化，激活M2型巨噬细胞。此外，CD73与肿瘤细胞的生长和转移相关，也与肿瘤环境中的血管新生相关。
组织因子(Tissue factor,TF)是分子量47kD的跨膜糖蛋白，在血管受到创伤后激活外源性凝血反应。然而TF在许多肿瘤组织中异常激活表达，其中乳腺癌、胰腺癌、肺癌、食道癌中都有较高的异常表达率。近期研究表明，TF在肿瘤中的异常表达是肿瘤具有耐药、免疫侵润抑制和转移的重要原因。TF在肿瘤微环境中能够驱动凝血酶水平的提高，造成纤维蛋白的沉积以及凝结的形成，从而影响肿瘤的微环境，造成肿瘤纤维化作用和肿瘤基质改变(Cancer Res 2019；79:3417-3430)。这些肿瘤特异性的改变导致许多免疫细胞侵润受阻，影响肿瘤对化疗药物以及免疫治疗药物的耐药(Journal of Clinical Investigation 2019；129:1785-1800)。肿瘤微环境的改变还导致了诸如MDSC和TAM(肿瘤相关巨噬细胞)水平的提高，降低免疫效应细胞对肿瘤的杀伤活性。TF异常表达也能导致补体***的肿瘤杀伤作用被抑制。此外，TF通过对血管新生的促进作用能帮助肿瘤细胞转移。
TGFβ是上皮间质转化(Epithelial-mesenchymal-transition,EMT)的一个关键诱导因子。同时，TGFβ在肿瘤微环境中具有很强的免疫抑制的作用，进而对肿瘤发生发展、转移和耐药有重要的调控作用。一方面，TGFβ在肿瘤微环境对于一些抑制肿瘤细胞的免疫细胞产生抑制作用，TGFβ能够促进抗肿瘤的MI型巨噬细胞转变成MII型巨噬细胞，也能够抑制MI型巨噬细胞的招募和抗肿瘤的细胞因子的分泌。TGFβ也能够抑制树突状细胞的成熟和相关细胞因子的分泌，同时还能促进树突状细胞的凋亡。TGFβ还能够抑制CD8 +T细胞的分化，IFN-γ的分泌，促进CD8 +T细胞的凋亡。另一方面，TGFβ对肿瘤微环境中的Treg细胞等免疫抑制细胞起到了促进作用。此外，TGFβ还能够促进肿瘤基质的纤维化，胶原 沉积，造成免疫浸润受阻。
目前，本领域关于肿瘤微环境的研究还有一些不足，迫切需要开发新的改善肿瘤微环境的治疗药物，从而降低微环境对肿瘤细胞生长、转移及耐药等方面的支撑，提高肿瘤的治疗效果。
发明内容
本发明的目的在于提供一种靶向肿瘤CD73-TGFβ及TF-TGFβ的抗体融合蛋白。
具体地，本发明提供了针对肿瘤微环境的抗体及在其基础上发明的融合蛋白，包括靶向CD73的抗体，双靶向CD73与TGFβ的融合蛋白(抗CD73-TGFβR融合蛋白)，以及双靶向TF与TGFβ的融合蛋白(抗TF-TGFβR融合蛋白)。它们具有改善肿瘤微环境，增加人体自身免疫***对肿瘤的杀伤作用，抑制肿瘤增殖、转移和耐药等作用。
在本发明的第一方面，提供了一种重组双功能融合蛋白，所述重组双功能融合蛋白包含：
第一结合结构域(D1)；和
第二结合结构域(D2)；
其中，所述第一结合结构域特异性结合靶分子CD73或TF蛋白；
所述第二结合结构域特异性结合靶分子TGFβ蛋白。
在另一优选例中，所述D1为特异性结合CD73或TF蛋白的抗体或抗体片段。
在另一优选例中，所述的抗体包括：动物源抗体(如鼠源抗体)、嵌合抗体、人源化抗体。
在另一优选例中，所述的抗体片段包含重链可变区和轻链可变区。
在另一优选例中，所述的抗体片段包含单链可变区片段(scFv)、双链可变区片段(dcFv)。
在另一优选例中，所述D2为特异性结合TGFβ蛋白的多肽片段，并且所述多肽片段衍生自TGFβ受体。
在另一优选例中，所述D2为TGFβ受体II的胞外区，较佳地，D2如SEQ ID NO.:33所示。
在另一优选例中，所述D1和所述D2通过连接肽相连。
在另一优选例中，D1为抗体片段，且所述连接肽为抗体恒定区序列。
在另一优选例中，D1为抗体，且所述的连接肽为(G4S) n，较佳地，(G4S) nG，其中，n为正整数(例如1、2、3、4、5或6)，较佳地，n＝4，更佳地，所述的连接肽如SEQ ID NO.:32所示。
在另一优选例中，D1为抗CD73单克隆抗体或抗TF单克隆抗体，并且D2通过连接肽连接在D1的选自下组的区域：重链可变区、重链恒定区、轻链可变区、或其组合。
在另一优选例中，D1为抗CD73单克隆抗体或抗TF单克隆抗体，并且D2通过连接肽连接在D1的重链恒定区末端。
在另一优选例中，所述双功能融合蛋白为同源二聚体。
在另一优选例中，所述双功能融合蛋白(单体)从N端到C端具有式I所示的结构：
Figure PCTCN2020113357-appb-000001
其中，
T1、T2、T3各自独立地为无或TGFβ受体II的胞外区，且至少一个不为无；
L1、L2、L3各自独立地为键或接头元件；
VL代表抗CD73或TF抗体的轻链可变区；
CL代表抗CD73或TF抗体的轻链恒定区；
VH代表抗CD73或TF抗体的重链可变区；
CH代表抗CD73或TF抗体的重链恒定区；
“～”代表二硫键或共价键；
“-”代表肽键；
其中，所述双功能融合蛋白具有同时结合CD73或TF以及结合TGFβ的活性。
在另一优选例中，所述的L1、L2、L3各自独立地为(G4S) 4G。
在另一优选例中，所述的T2、T3、L2和L3为无。
在另一优选例中，T1为TGFβ受体II的胞外区。
在另一优选例中，所述的CH包含CH1、CH2和CH3。
在另一优选例中，所述的双功能融合蛋白的两个单体通过CH2和CH3上的二硫键形成二聚体。
在另一优选例中，所述的抗CD73抗体如本发明第二方面所述。
在另一优选例中，所述的抗TF的抗体含有：
(a)如SEQ ID NO.:37所示的抗体重链可变区；和
(b)如SEQ ID NO.:38所示的抗体轻链可变区。
在本发明的第二方面，提供了抗CD73的抗体，所述的抗体含有：
(c)抗体重链可变区；和
(d)抗体轻链可变区。
在另一优选例中，所述的抗体还含有重链恒定区，所述的重链恒定区为人源、鼠源或兔源的。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区具有SEQ ID NO.:2所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:3、SEQ ID NO.:9、SEQ ID NO.:10所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:4、SEQ ID NO.:12、SEQ ID NO.:13所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:14、SEQ ID NO.:15、SEQ ID NO.:16、SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:19、SEQ ID NO.:20所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26、SEQ ID NO.:27所示的氨基酸序列。
在另一优选例中，所述的抗体还含有轻链恒定区，所述的轻链恒定区为人源、鼠源或兔源的。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:5、SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:6、SEQ ID NO.:11所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:21、SEQ ID NO.:22、SEQ ID NO.:23所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30所示的氨基酸序列。
在另一优选例中，所述抗体对CD73(如人CD73蛋白胞外区,CD73-ECD)的亲和力EC 50为0.083-0.131nM。
本发明的第三方面，提供了一种本发明第一方面所述的重组双功能融合蛋白的用途，用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗CD73或TF、和/或TGFβ相关疾病的药物。
在另一优选例中，D1为特异性结合CD73的抗体或抗体片段，且所述检测试剂或试剂盒用于：
(1)检测样品中的CD73蛋白和/或TGFβ蛋白；和/或
(2)检测肿瘤细胞中内源性的CD73蛋白和/或肿瘤细胞分泌的TGFβ蛋白；和/或
(3)检测表达CD73蛋白和/或分泌TGFβ蛋白的肿瘤细胞。
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和/或TGFβ相关疾病。
在另一优选例中，所述的药物用于治疗或预防CD73和/或TGFβ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)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(e)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；
(f)抑制TGFβ诱导的能够促进肿瘤的免疫细胞的功能；
(g)抑制TGFβ诱导产生的肿瘤微环境的免疫侵润抑制作用和纤维化等；
(h)抑制肿瘤的耐药；
(i)抑制肿瘤细胞迁移或转移。
在另一优选例中，所述CD73和/或TGFβ相关疾病选自下组：癌症、自身免疫疾病、代谢相关疾病、纤维化相关疾病、感染疾病、或其组合。
在另一优选例中，所述CD73和/或TGFβ相关的疾病包括：肿瘤的发生、生长、耐药和/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和/或TGFβ高表达的肿瘤。
在另一优选例中，所述的CD73和/或TGFβ高表达的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、卵巢癌、***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和/或TGFβ高表达的肿瘤指肿瘤组织中CD73和/或TGFβ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。
在另一优选例中，所述代谢相关疾病包括：糖尿病、食源性肥胖和脂肪炎症。
在另一优选例中，所述纤维化相关疾病包括：肺纤维化、肾纤维化、肝纤维化、心血管纤维化、脾纤维化、骨髓纤维化和神经***纤维化。
在另一优选例中，所述感染疾病包括：细菌和病毒感染。
在另一优选例中，D1为特异性结合TF蛋白的抗体或抗体片段，且所述检测试剂或试剂盒用于：
(1)检测样品中的TF蛋白和/或TGFβ蛋白；和/或
(2)检测肿瘤细胞中内源性的TF蛋白和/或肿瘤细胞分泌的TGFβ蛋白；和/或
(3)检测表达TF蛋白和/或分泌TGFβ蛋白的肿瘤细胞。
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TF和/或TGFβ相关疾病。
在另一优选例中，所述的药物用于治疗或预防TF和/或TGFβ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TF诱导的凝血酶的形成以及纤维蛋白的产生；
(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TF；
(c)抑制肿瘤细胞表达的TF诱导的凝血酶的形成和下游的信号通路的改变，以及造
成的凝结和血栓形成；
(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)抑制TGFβ诱导的能够促进肿瘤的免疫细胞的功能；
(h)抑制肿瘤微环境的免疫侵润抑制作用和纤维化等；
(i)抑制肿瘤的耐药。
在另一优选例中，所述TF和/或TGFβ相关疾病选自下组：癌症、血栓类疾病、炎症性疾病、自身免疫疾病、代谢相关疾病、纤维化相关疾病、或其组合。
在另一优选例中，所述TF和/或TGFβ相关的疾病包括：肿瘤的发生、生长、耐药和/或转移。
在另一优选例中，所述的癌症为TF和/或TGFβ高表达的肿瘤。
在另一优选例中，所述的TF和/或TGFβ高表达的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、卵巢癌、***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TF和/或TGFβ高表达的肿瘤指肿瘤组织中TF和/或TGFβ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。
本发明的第四方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
本发明的第五方面，提供了一种治疗与CD73和/或TGFβ相关疾病的方法，所述方法给需要的对象施用本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述 的抗体、或本发明第四方面所述的药物组合物。
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。
本发明的第六方面，提供了一种制备抗CD73-TGFβR融合蛋白的方法，包括步骤：
(a)将本发明第二方面所述抗体或TF抗体的重链的表达载体进行双酶切，获取线性载体，再将带有相同酶切位点的Linker和TGFβRII胞外区的DNA片段***到线性载体中，获得融合蛋白重链的表达载体；
(b)将融合蛋白重链的表达载体和本发明第二方面抗体或TF抗体的轻链的表达载体一同转染动物细胞表达融合蛋白。
本发明的第七方面，提供了一种多核苷酸，它编码本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体。
本发明的第八方面，提供了一种载体，它含有本发明第七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。
本发明的第九方面，提供了一种遗传工程化的宿主细胞，它含有本发明第八方面所述的载体或基因组中整合有本发明第七方面所述的多核苷酸。
本发明第十方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
本发明第十一方面，提供了一种重组蛋白，该重组蛋白含有：
(a)如本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体；和
(b)任选的协助表达和/或纯化的标签序列。
本发明的第十二方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养本发明第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体。
本发明的第十三方面，提供了一种抑制肿瘤细胞生长和迁移的方法，包括步骤：给需要的对象施用如本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体。
本发明的第十四方面，提供了一种对T淋巴细胞增殖保护作用的方法，包括步骤： 给需要的对象施用如本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体。
在本发明的第十五方面，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：给需要的对象施用如本发明第一方面所述的重组双功能融合蛋白、或本发明第二方面所述的抗体。
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。
在本发明的第十六方面，提供了本发明的融合蛋白对体内肿瘤免疫微环境的改善作用及机制。
在另一优选例中，所述抗CD73/TF-TGFβR融合蛋白能够提高体内肿瘤内MI型(抑瘤)巨噬细胞的浸润水平，增强抗肿瘤的作用。
在另一优选例中，所述抗CD73/TF-TGFβR融合蛋白能够降低体内肿瘤MII型(促瘤)巨噬细胞的水平，降低免疫抑制细胞的水平，从而进一步提高抗肿瘤作用。
在另一优选例中，抗CD73-TGFβR融合蛋白能够提高成熟的树突状细胞在体内肿瘤微环境中的浸润水平，提升抗原呈递能力和肿瘤杀伤水平。
在另一优选例中，在人源化免疫重建NSG小鼠中，抗CD73-TGFβR融合蛋白能够提高CD45 +免疫细胞在肿瘤中的浸润，提升免疫细胞抗肿瘤的作用。
在另一优选例中，在人源化免疫重建NSG小鼠中，抗CD73-TGFβR融合蛋白能够提高CD8 +T细胞在肿瘤中的浸润，直接或间接提高效应T细胞对肿瘤的杀伤作用。
在另一优选例中，本发明所述的抗CD73/TF-TGFβR融合蛋白相比CD73/TF单克隆抗体能够显示更强、更优异的肿瘤微环境改善活性，导致更优异的提高抗肿瘤T细胞、NK细胞等效应细胞的浸润，抗肿瘤细胞因子IFN-γ的增多，同时能够更有效的降低免疫负调控细胞如MDSC、调节型T细胞(Tregs)、促瘤型(MII)巨噬细胞等。
在另一优选例中，所述抗CD73/TF-TGFβR融合蛋白相比CD73/TF单克隆抗体能够更强地改善肿瘤基质的通透性，提高药物进入肿瘤的效率。
应理解，在本发明范围内，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是选定的5个靶向人CD73鼠源单克隆抗体对人源CD73-高表达的MDA-MB-231(CD73-P)、CD73-低表达的MDA-MB-453(CD73-N)乳腺癌细胞的的结合活性(A)，以及纯化后5个抗体针对MDA-MB-231细胞的结合亲和力(B)。
图2为3个嵌合抗体mAb001c、mAb002c、mAb004c对人CD73-ECD抗原亲和力的ELISA测定的结果。
图3为3个嵌合抗体mAb001c、mAb002c、mAb004c对重组人CD73酶催化功能的抑制活性的测定。
图4为3个嵌合抗体mAb001c、mAb002c、mAb004c对乳腺肿瘤细胞MDA-MB-231表面CD73抗原特异性结合活性。
图5为3个嵌合抗体mAb001c、mAb002c、mAb004c对肺肿瘤细胞NCI-H1299表面 CD73抗原特异性结合活性。
图6为3个嵌合抗体mAb001c、mAb002c、mAb004c对乳腺肿瘤细胞MDA-MB-231表面CD73酶催化水解AMP功能的抑制活性的测定。
图7为3个嵌合抗体mAb001c、mAb002c、mAb004c对肺肿瘤细胞NCI-H1299表面CD73酶催化水解AMP功能的抑制活性的测定。
图8是本发明的CD73(TF)抗体-TGFβR融合蛋白的结构示意图，其中，CD73抗体、TF抗体分别与TGFβ受体II胞外区通过一个(Gly 4Ser) 4Gly的链接子(Linker)融合而成。
图9是分别由CD73抗体-TGFβR、TF抗体-TGFβR融合蛋白重链的表达载体的构建过程示意图，Linker和TGFβR胞外区DNA片段在BspQI和BamHI酶切位点之间***。
图10对抗CD73-TGFβR融合蛋白Hu001-14-TGFβR重链的表达载体质粒分别进行BspQI、BamHI单酶切，BspQI/BamHI双酶切的电泳图(A)；SDS-PAGE凝胶分析图(B)显示融合蛋白在还原和非还原状态的电泳情况。
图11对抗CD73-TGFβR融合蛋白Hu001-32-TGFβR重链的表达载体质粒分别进行BspQI、BamHI单酶切，BspQI/BamHI双酶切的电泳图(A)；SDS-PAGE凝胶分析图(B)显示融合蛋白在还原和非还原状态的电泳情况。
图12对抗CD73-TGFβR融合蛋白Hu002-3-TGFβR重链的表达载体质粒分别进行BspQI、BamHI单酶切，BspQI/BamHI双酶切的电泳图(A)；SDS-PAGE凝胶分析图(B)显示融合蛋白在还原和非还原状态的电泳情况。
图13为ELISA检测抗CD73-TGFβR融合蛋白对人CD73胞外区的亲和力。
图14为检测抗CD73-TGFβR融合蛋白对重组人CD73酶催化功能的抑制活性的测定。
图15为ELISA检测抗CD73-TGFβR融合蛋白对TGFβ1的亲和力。
图16为FACS检测抗CD73-TGFβR融合蛋白对肺肿瘤细胞NCI-H1299表面CD73抗原结合活性。
图17为FACS检测抗CD73-TGFβR融合蛋白对乳腺肿瘤细胞MDA-MB-231表面CD73抗原结合活性。
图18为检测抗CD73-TGFβR融合蛋白能有效逆转腺苷(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。
图19为抗CD73-TGFβR融合蛋白Hu001-14-TGFβR在体内抗肿瘤活性测试。体内试验采用NCI-H1299非小细胞肺癌细胞与50μg抗体混匀后接种至裸鼠背部皮下，每周观察2～3次。结果显示肿瘤生长曲线(上图)与试验终止时的肿瘤重量(下)。
图20为IHC检测抗CD73-TGFβR融合蛋白Hu001-14-TGFβR对NCI-H1299肿瘤中TGFβ1的抑制活性。
图21为IHC检测抗CD73-TGFβR融合蛋白Hu001-14-TGFβR对H1299肿瘤微环境中巨噬细胞(TAM)的抑制活性。
图22为CD73抗体Hu001-14、抗CD73-TGFβR融合蛋白Hu001-14-TGFβR的体内抗肿瘤活性。NCI-H441细胞接种后第一天随机分组(n＝6～8)，静脉给药每周2次，第一天开始给药，剂量分别Hu001-14(10mg/kg)，Hu001-14-TGFβR(12.5mg/kg)。结果显示肿瘤生长曲线(上)与第26天试验终止时的肿瘤重量(下)。
图23为免疫荧光(IF)双染检测NCI-H441肿瘤中MI-型巨噬细胞标志物CD86和F4/80。结果显示相比Hu001-14，Hu001-14-TGFβR能大幅度增加MI-型巨噬细胞的浸润肿瘤组织。
图24为免疫组化(IHC)检测NCI-H441肿瘤中MII-型巨噬细胞标志物CD206和F4/80。结果显示相比Hu001-14，Hu001-14-TGFβR能更有效的降低肿瘤组织中浸润的MII-型巨噬细胞。
图25为免疫荧光(IF)双染检测NCI-H441肿瘤中MII-型巨噬细胞标志物CD206和 F4/80。IF双染结果进一步确认了相比Hu001-14，Hu001-14-TGFβR能更有效的降低肿瘤组织中浸润的MII-型巨噬细胞。
图26为免疫荧光(IF)双染检测NCI-H441肿瘤中成熟树突状细胞标志物CD86和CD11c。结果显示相比Hu001-14，Hu001-14-TGFβR能大幅度增加肿瘤组织浸润成熟树突状细胞的水平。
图27为Hu001-14-TGFβR对人的免疫重建鼠(NSG鼠)NCI-H1299肺癌移植瘤模型的抑瘤作用。通过流式细胞术对CD45 +细胞和CD8 +细胞的计数确认了NSG鼠免疫重建成功(A)；对Hu001-14和Hu001-14-TGFβR在NCI-H1299肿瘤模型中进行了药效评估(B)。
图28为免疫组化(IHC)检测人的免疫重建鼠(NSG鼠)的NCI-H1299肿瘤中CD45 +免疫细胞和CD8 +细胞的浸润情况。相比Hu001-14，Hu001-14-TGFβR能更显著提高肿瘤组织中CD45 +免疫细胞和CD8 +T细胞的浸润程度。
图29为抗TF-TGFβR融合蛋白HuSC1-39-TGFβR表达载体质粒分别进行BspQI、BamHI单酶切，BspQI/BamHI双酶切的电泳图(A)；SDS-PAGE凝胶分析图(B)显示融合蛋白在还原和非还原状态的电泳情况。
图30为ELISA检测抗TF-TGFβR融合蛋白HuSC1-39-TGFβR对人TF胞外区(TF-ECD)的亲和力。
图31为ELISA检测抗TF-TGFβR融合蛋白HuSC1-39-TGFβR对TGFβ1的亲和力。
图32为FACS检测抗TF-TGFβR融合蛋白HuSC1-39-TGFβR对胰腺肿瘤细胞BxPC3表面TF抗原结合活性。
图33为FACS检测抗TF-TGFβR融合蛋白HuSC1-39-TGFβR对乳腺肿瘤细胞MDA-MB-231表面TF抗原结合活性。
图34为TF抗体HuSC1-39、抗TF-TGFβR融合蛋白HuSC1-39-TGFβR在体内抗肿瘤活性。体内试验采用HCC1806乳腺癌细胞分别与20μg hIgG1、20μg HuSC1-39和25μg HuSC1-39-TGFβR混匀后接种至NSG鼠乳垫处，每周观察2～3次。结果显示试验终止时(肿瘤生长16天)的肿瘤重量。
图35为免疫荧光双染检测HCC1806肿瘤中MI-型巨噬细胞标志物CD86和F4/80。结果显示相比TF抗体HuSC1-39，抗TF-TGFβR融合蛋白HuSC1-39-TGFβR能够更有效的降低MII-型巨噬细胞的浸润水平，实现更有效的抗肿瘤治疗效果。
图36为免疫组化(IHC)检测HCC1806肿瘤中MII-型巨噬细胞标志物CD206和F4/80。结果显示相比HuSC1-39，抗TF-TGFβR融合蛋白HuSC1-39-TGFβR能更有效的抑制瘤内MII-型巨噬细胞的浸润水平。
图37为免疫荧光双染检测HCC1806肿瘤中MII-型巨噬细胞标志物CD206和F4/80。结果进一步确认了相比HuSC1-39，抗TF-TGFβR融合蛋白HuSC1-39-TGFβR能更有效的抑制瘤内MII-型巨噬细胞的浸润水平，提高抗肿瘤治疗效果。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得了数个抗CD73单克隆抗体，其中，人-鼠嵌合抗体mAb001c、mAb002c、mAb004c能够高特异性地结合CD73抗原，ELISA测定其EC 50分别为0.024nM、0.016nM、0.038nM。基于mAb001c和mAb002c设计的人源化抗体也具有优异的特性。此外，通过本发明的设计、制备，以及体外和体内验证所获得了2种新型抗体融合蛋白。抗CD73-TGFβR融合蛋白、抗TF-TGFβR融合蛋白均具有双靶标结合亲和力高、特异性强的优势，能用于进一步增强抗肿瘤免疫功能。在此基础上完成了本发明。
实施例1靶向人CD73的鼠源单克隆抗体的制备和鉴定
步骤①杂交瘤细胞的制备
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，具体氨基酸序列如下(SEQ ID NO.:1)：
SEQ ID NO.:1人CD73蛋白的胞外区氨基酸序列
Figure PCTCN2020113357-appb-000002
利用上述制备的CD73胞外区蛋白免疫Balb/c小鼠，CD73胞外区蛋白的用量为50μg/只，以制备免疫脾细胞；适时的制备鼠骨髓瘤细胞(SP2/0)和饲养细胞以备融合之需。
待上述三种细胞准备完毕，通过PEG介导融合免疫脾细胞和SP2/0细胞，进行杂交瘤细胞的制备，并且从中筛选效价高、形态好、呈单克隆生长的细胞继续进行亚克隆筛选，直到连续三次筛选阳性克隆率全为100％，对该细胞株进行扩大培养和建库。
将筛选出来的杂交瘤细胞扩大培养后，收集细胞培养上清并进行纯化，对纯化产物定量并进行检测。
步骤②抗体测序、互补决定区(CDR)的鉴定
经过反复筛选，对选定的5个杂交瘤单克隆抗体进行生物活性和靶向特异性的测定。如图1A所示，采用流式细胞荧光分选仪(FACS)检测单克隆细胞培液的上清，5个抗体均可以特异性的结合人源CD73-高表达的MDA-MB-231细胞(CD73-P)，而对CD73-低表达的MDA-MB-453细胞(CD73-N)无明显结合活性。随后，采用纯化后的抗体样品进行梯度稀释、FACS检测，如图1B所示，mAb001、mAb002、mAb003、mAb004、mAb005针对MDA-MB-231细胞具有优异的结合亲和力，FACS检测其EC 50分别为1.24nM、0.65nM、10.7nM、4.69nM、26.07nM。
基于优异的特异性及亲和力，优先选取mAb001、mAb002、mAb004进行抗体测序鉴定。设计引物通过常规PCR技术扩增重链(VH)、轻链(VL)可变区片段，克隆入载体，测序。采用常规测序并通过Kabat数据库分析，得到以下重链可变区(VH)、轻链可变区(VL)氨基酸序列、互补决定区(CDR)信息(下划线“_”所示为CDR-1/2/3氨基酸序列)。
SEQ ID NO.:2 mAb001重链可变区(VH)氨基酸序列
Figure PCTCN2020113357-appb-000003
SEQ ID NO.:3 mAb002重链可变区(VH)氨基酸序列
Figure PCTCN2020113357-appb-000004
Figure PCTCN2020113357-appb-000005
SEQ ID NO.:4 mAb004重链可变区(VH)氨基酸序列
Figure PCTCN2020113357-appb-000006
SEQ ID NO.:5 mAb001轻链可变区(VL)氨基酸序列
Figure PCTCN2020113357-appb-000007
SEQ ID NO.:6 mAb002轻链可变区(VL)氨基酸序列
Figure PCTCN2020113357-appb-000008
SEQ ID NO.:7 mAb004轻链可变区(VL)氨基酸序列
Figure PCTCN2020113357-appb-000009
实施例2人-鼠嵌合CD73抗体的制备和检测
步骤①人-鼠嵌合抗体的制备、嵌合抗体的点突变
通过基因重组技术将3组可变区序列(参见SEQ ID NO.:2、SEQ ID NO.:3、SEQ ID NO.:4、SEQ ID NO.:5、SEQ ID NO.:6、SEQ ID NO.:7)克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后，利用转染技术和哺乳动物表达***(FreeStyle TM293T细胞)将构建的嵌合型抗体表达和纯化，所获得的人-鼠嵌合型抗体，分别编号为mAb001c、mAb002c、mAb004c。
所述抗体的可变区序列含有数个不利氨基酸，对其进行了点突变改造。以下列出经点突变后的重链可变区(VH)、轻链可变区(VL)的氨基酸序列(“_”所示为CDR氨基酸序列)。
SEQ ID NO.:8 mAb001-VL-SGS
Figure PCTCN2020113357-appb-000010
SEQ ID NO.:9 mAb002-VH-QG
Figure PCTCN2020113357-appb-000011
SEQ ID NO.:10 mAb002-VH-NA
Figure PCTCN2020113357-appb-000012
SEQ ID NO.:11 mAb002-VL-SG
Figure PCTCN2020113357-appb-000013
SEQ ID NO.:12 mAb004-VH-QG
Figure PCTCN2020113357-appb-000014
SEQ ID NO.:13 mAb004-VH-NA
Figure PCTCN2020113357-appb-000015
以上述点突变模板匹配获得点突变(PTM)克隆到hIgG1载体获得点突变的相应嵌合抗体突变体。
综上所述的人-鼠嵌合抗体和所述抗体突变体的编号、所述抗体的重链和轻链编号由表-1汇总列出。
表-1：人-鼠嵌合抗体及其突变体
Figure PCTCN2020113357-appb-000016
步骤②嵌合抗体对人CD73抗原亲和力的ELISA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3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抗鼠的二抗，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1N HCL，50μL/孔，终止显色反应，然后在450nm处测定其吸光度，并分析数据。
检测结果如图2所示，mAb001c、mAb002c、mAb004c对CD73-ECD有很强的亲和性，EC 50分别0.024nM、0.016nM、0.038nM。
步骤③嵌合抗体对重组人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2nM稀释到0.0009nM的CD73抗体加入培养板中，混匀(终浓度为1nM～0.00045nM),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，mAb001c、mAb002c、mAb004c均具有显著抑制重组CD73蛋白酶水解AMP的活性，其IC 50分别为0.025nM、0.031nM、0.039nM。
步骤④嵌合抗体对肿瘤细胞表面CD73的结合亲和力
采用CD73-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作为靶细胞，将100μL按照3倍梯度从200nM稀释到0.091nM的受试抗体作为一抗，分别与悬浮于100μLRPMI-1640无血清培养基中的1x10 5个MDA-MB-231混匀，或将100μL按照3倍梯度从100nM稀释到0.046nM的mAb001c、mAb002c、mAb004c作为一抗与悬浮于100μL RPMI-1640无血清培养基中的1x10 5个NCI-H1299细胞混匀，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4所示，mAb001c、mAb002c、mAb004c对MDA-MB-231具有优异的结合亲和力，EC 50分别为0.71nM、0.36nM、2.5nM；
检测结果如图5所示，mAb001c、mAb002c、mAb004c对NCI-H1299具有同样优异的结合亲和力，EC 50分别为1.0nM、0.39nM、2.2nM；
综上结果说明，本实施例单克隆抗体能够以人源肿瘤细胞的CD73为作用靶点。
步骤⑤嵌合抗体对肿瘤细胞表面CD73酶催化功能的抑制
采用CD73-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200nM稀释到0.091nM的受试抗体加入96孔板中，37℃孵育30min后，加入25μL的0.9mM的AMP，置37℃，5％CO 2培养3h(抗体终浓度133.3nM～0.06nM)。取出上述培养上清25μL加入另一个96孔白板中，加入25μL的0.1mM 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6所示，mAb001c、mAb002c、mAb004c均能显著抑制MDA-MB-231细胞表面CD73催化水解AMP的功能，IC 50分别为1.86nM、0.79nM和4.16nM。
检测结果如图7所示，mAb001c、mAb002c、mAb004c抑制NCI-H1299细胞表面CD73催化水解AMP的功能，IC 50分别为0.24nM、0.19nM、0.39nM。
实施例3人源化CD73抗体的制备和检测
步骤①人源化CD73抗体的制备
在Germline数据库中收索选取与mAb001c、mAb002c非CDR区匹配最好的人源化模板，然后将抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与IgG1恒定区重组，同时以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变。
具体地，mAb001c的人源化实施获得7个人源化重链的可变区(SEQ ID NO.:14、SEQ ID NO.:15、SEQ ID NO.:16、SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:19、SEQ ID NO.:20)，以及3个人源化轻链的可变区(SEQ ID NO.:21、SEQ ID NO.:22、SEQ ID NO.:23)。
SEQ ID NO.:14 mAb001-VH_HuG.3
Figure PCTCN2020113357-appb-000017
Figure PCTCN2020113357-appb-000018
SEQ ID NO.:15 mAb001-VH_HuG.5
Figure PCTCN2020113357-appb-000019
SEQ ID NO.:16 mAb001-VH_HuG.6
Figure PCTCN2020113357-appb-000020
SEQ ID NO.:17 mAb001-VH_HuG.7
Figure PCTCN2020113357-appb-000021
SEQ ID NO.:18 mAb001-VH_HuG.8
Figure PCTCN2020113357-appb-000022
SEQ ID NO.:19 mAb001-VH_HuG.9
Figure PCTCN2020113357-appb-000023
SEQ ID NO.:20 mAb001-VH_HuG.10
Figure PCTCN2020113357-appb-000024
SEQ ID NO.:21 mAb001-VK_HuG.1
Figure PCTCN2020113357-appb-000025
SEQ ID NO.:22 mAb001-VK_HuG.2
Figure PCTCN2020113357-appb-000026
SEQ ID NO.:23 mAb001-VK_HuG.0
Figure PCTCN2020113357-appb-000027
具体地，mAb002c的人源化实施获得4个人源化重链的可变区(SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26、SEQ ID NO.:27)以及3个人源化轻链的可变区(SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30)。
SEQ ID NO.:24 mAb002-VH_HuG0
Figure PCTCN2020113357-appb-000028
SEQ ID NO.:25 mAb002-VH_HuG1
Figure PCTCN2020113357-appb-000029
Figure PCTCN2020113357-appb-000030
SEQ ID NO.:26 mAb002-VH_HuG2
Figure PCTCN2020113357-appb-000031
SEQ ID NO.:27 mAb002-VH_HuG3
Figure PCTCN2020113357-appb-000032
SEQ ID NO.:28 mAb002-VK_HuG1
Figure PCTCN2020113357-appb-000033
SEQ ID NO.:29 mAb002-VK_HuG2
Figure PCTCN2020113357-appb-000034
SEQ ID NO.:30 mAb002-VK_HuG3
Figure PCTCN2020113357-appb-000035
通过基因重组技术将所设计的人源化可变区序列克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后，利用转染技术和哺乳动物表达***(FreeStyle TM293细胞)将构建的人源化抗体表达载体。分别组合表达这些人源化的重链及轻链，最终mAb001c-系列获得了了11个人源化抗体，mAb002c-系列获得了12个人源化抗体，各抗体相应的重链和轻链组合如表-2所示。
表-2：人源化抗体
Figure PCTCN2020113357-appb-000036
步骤②人源化抗体对CD73的亲和力
将表2中的人源化抗体梯度稀释，采用ELISA法测定其对CD73蛋白的亲和力，实验方法参照实施例2。
实验结果如表-3所示,所述2组人源化抗体均对CD73蛋白具有很强的结合亲和力，EC 50值为0.02nM～0.13nM。
表-3：人源化抗体对CD73亲和力的ELISA结果
Figure PCTCN2020113357-appb-000037
Figure PCTCN2020113357-appb-000038
步骤③人源化抗体对人CD73酶功能的抑制作用
将表-2中的人源化抗体进行梯度稀释，参照实施例2测定抗体对重组CD73酶活性的影响。
实验结果如表-4所示，所述2组人源化抗体均对CD73酶具有极强的抑制效果，其IC 50值为0.02nM～0.3nM。
表-4：人源化抗体的CD73酶抑制活性
Figure PCTCN2020113357-appb-000039
步骤④人源化抗体对肿瘤细胞CD73的结合活性
通过流式细胞仪测定将表2中的人源化抗体对NCI-H1299、MDA-MB-231肺癌细胞表面CD73的亲和力，实验方法参照实施例2。
试验结果如表-5所示，所述2组人源化抗体对NCI-H1299细胞表面CD73具有很高亲和活性，EC 50值为0.53nM～1.65nM。
试验结果如表-6所示，所述2组人源化抗体对MDA-MB-231细胞表面CD73具有很高亲和活性，EC 50值为0.21nM～0.74nM。
表-5人源化抗体对NCI-H299细胞CD73的结合活性
Figure PCTCN2020113357-appb-000040
表-6人源化抗体对MDA-MB-231细胞CD73的结合活性
Figure PCTCN2020113357-appb-000041
Figure PCTCN2020113357-appb-000042
实施例4抗体-TGFβR融合蛋白的设计和构建
步骤①融合蛋白的设计
在抗体功能的基础上添加了结合TGFβ的功能，设计抗体与TGFβ受体II胞外区(简称TGFβR)的融合蛋白。如图8所示，融合蛋白由两个主要的部分组成：轻链和重链；其中轻链部分与所对应的抗体的轻链完全相同，重链部分是由抗体的重链和TGFβR由Linker连接而成。抗体重链恒定区(SEQ ID NO.:31；为了防止细胞内对Linker结合部位的剪切，末端的赖氨酸突变成丙氨酸)末端与Linker(SEQ ID NO.:32)的N端相连，然后Linker的C端连接TGFβR(SEQ ID NO.：33)(Science Translational Medicine 2018；16:1-15)。
SEQ ID NO.:31抗体重链恒定区的氨基酸序列
Figure PCTCN2020113357-appb-000043
SEQ ID NO.:32 Linker的氨基酸序列
Figure PCTCN2020113357-appb-000044
SEQ ID NO.:33 TGFβ受体II胞外区的氨基酸序列
Figure PCTCN2020113357-appb-000045
步骤②融合蛋白重链表达质粒的构建
如图9所示，融合蛋白重链部分的构建在原有的人源化抗体重链表达载体(原始表达载体来自于上海睿智化学，代号为pCP)的基础上，通过在抗体Fc的DNA序列末端的BspQI酶切位点( GCTCTTC/N)和载体上原有的BamHI酶切位点( G/GATCC)，将Linker和TGFβRII胞外区的DNA序列引入到载体中。
将抗体重链的表达载体通过BspQI和BamHI进行双酶切回收，并且对合成的含有Linker和TGFβR的DNA片段利用BspQI和BamHI也进行双酶切。回收目的片段，然后 利用T4DNA连接酶进行连接，转化到DH5α的感受态细胞中，涂布氨苄抗性的平板，挑取单菌落。对单菌落进行质粒提取和酶切验证，并且进行测序验证，获得序列正确的载体。
实施例5抗CD73-TGFβR融合蛋白的制备和检测
步骤①融合蛋白的制备
按照实施例4的设计，构建Hu001c-14-TGFβR(简称Hu001-14-TGFβR)的重链(SEQ ID NO.:34)、Hu001c-32-TGFβR(简称Hu001-32-TGFβR)的重链(SEQ ID NO.:35)和Hu002c-3-TGFβR(简称Hu002-3-TGFβR)的重链(SEQ ID NO.:36)。抗CD73-TGFβR融合蛋白由重链和轻链组成，其中Hu001-14-TGFβR和Hu001-32-TGFβR的轻链序号为SEQ ID NO.:21，Hu002-3-TGFβR的轻链序号为SEQ ID NO.:29，如表-7所示。
表-7：抗CD73-TGFβR融合蛋白
Figure PCTCN2020113357-appb-000046
SEQ ID NO.:34 Hu001c-14-TGFβR融合蛋白重链的氨基酸序列
Figure PCTCN2020113357-appb-000047
SEQ ID NO.:35 Hu001c-32-TGFβR融合蛋白重链的氨基酸序列
Figure PCTCN2020113357-appb-000048
Figure PCTCN2020113357-appb-000049
SEQ ID NO.:36 Hu002c-3-TGFβR融合蛋白重链的氨基酸序列
Figure PCTCN2020113357-appb-000050
融合蛋白重链载体构建结果如图10A、图11A、图12A所示，通过对获取的载体分别进行BspQI、BamHI单酶切以及BspQI/BamHI双酶切，确认了酶切后片段与预期的结果一致。
重链和轻链的表达载体质粒通过脂质体转染到293T细胞中进行蛋白表达。在培养皿中培养5天之后收集表达上清，然后利用蛋白A进行纯化，纯化的蛋白通过SDS-PAGE进行分析。
蛋白纯化的结果如图10A、图11A、图12B所示，对还原和非还原状态的蛋白进行SDS-PAGE分析之后发现，蛋白的电泳分子量与理论分子量基本一致，说明纯化出的蛋白是目的蛋白；利用SDS-PAGE结果分析，Hu001-14-TGFβR、Hu001-32-TGFβR和Hu002-3-TGFβR的蛋白纯度分别为94.5％、96.3％、95.4％。
步骤②融合蛋白对人CD73抗原亲和力的ELISA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3倍梯度稀释的单克隆抗体，100μL/孔，室温孵育2h；洗去未结合的抗体，加入合适浓度辣根过氧化物酶标记的抗鼠的二抗，100μL/孔，室温孵育1h。洗去未结合的二抗，加入TMB显色液反应大约10min，加入2M H 2SO 4，50μL/孔，终止显色反应，然后在450nm处测定其吸光度，并分析数据。
检测结果如图13所示，Hu001-14-TGFβR、Hu001-32-TGFβR、Hu002-3-TGFβR对CD73-ECD有很强的亲和性，EC 50分别0.09nM、0.11nM、0.06nM；
步骤③融合蛋白对重组人CD73酶催化功能的抑制活性的测定
参照实施例2的步骤③进行测定抗CD73-TGFβR融合蛋白对重组人CD73酶催化功能的抑制作用。
检测结果如图14所示，Hu001-14-TGFβR、Hu001-32-TGFβR、Hu002-3-TGFβR均具 有显著抑制重组CD73蛋白酶水解AMP的活性，其IC 50分别为0.052nM、0.083nM、0.032nM；
步骤④ELISA检测融合蛋白对TGFβ的结合亲和力
将人TGFβ1(Sino biological,Cat#10804-HNAC)作为抗原包被在酶标板上，TGFβ1的浓度为0.5μg/mL。实验方法参考步骤②，其中一抗终浓度为27μg/mL，3倍梯度稀释。
检测结果如图15所示，Hu001-14-TGFβR、Hu001-32-TGFβR、Hu002-3-TGFβR对TGFβ有很强的亲和性，EC 50分别为0.4nM、0.26nM、0.55nM。
步骤⑤融合蛋白对肿瘤细胞表面CD73的特异性结合
采用CD73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，测定融合蛋白对细胞表面CD73的结合情况。采用10 5个肿瘤细胞与融合蛋白(一抗)混匀(终浓度12.5μg/mL,3倍梯度稀释)，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PE标记的二抗在4℃孵育30min，PBS洗涤细胞三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(FACS)检测结合率。
检测结果如图16和图17所示，Hu001-14-TGFβR、Hu001-32-TGFβR、Hu002-3-TGFβR对肿瘤细胞表面的CD73有很强的亲和性，对H1299的EC 50分别为1.6nM、0.9nM和1.0nM；对MDA-MB-231的EC 50分别为1.5nM、1.4nM；。
实施例6抗CD73-TGFβR融合蛋白对T淋巴细胞增殖的保护作用
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细胞增殖试验：将上述分选得到的T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(1～2x10 4个细胞/孔)，每孔加入50μL按照梯度稀释的抗体和融合蛋白(终浓度10nM～0.0001nM，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，采用流式细胞仪(FACS)读取并统计固定体积的细胞数。
结果如图18显示，融合蛋白Hu001-14-TGFβR、Hu002-3-TGFβR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细胞的增殖抑制，并且效果相对于Hu001-14抗体有显著优势。
实施例7抗CD73-TGFβR融合蛋白的体内抗NCI-H1299肿瘤活性
步骤①抗CD73-TGFβR在裸鼠移植NCI-H299肿瘤模型中的抗肿瘤活性
随机将免疫缺陷型裸鼠(Balb/c,nude)分为若干组，将50μL含有9x10 6NCI-H1299的细胞悬液与50μL Hu001-14-TGFβR混匀(62.5μg/瘤，相当于抗体浓度50μg/瘤)后再与100μL基质胶(BD/Corning,Cat#354248)混匀后接种入裸鼠背部皮下(n＝6～8)。采用hIgG1(终浓度为50μg/瘤)为亚型匹配的阴性对照。观察抗体对皮下瘤生长的抑制作用，每周2～3次测量裸鼠体重及肿瘤大小，绘制肿瘤生长曲线，并最终称取肿瘤的重量，评定活性。
结果如图19所示，Hu001-14-TGFβR能显著抑制NCI-H1299肿瘤在裸鼠体内的生长。
步骤②抗CD73-TGFβR对肿瘤组织中TGFβ1生成的抑制活性
将步骤①获取的肿瘤样本进行石蜡包埋并切片，将hIgG1组肿瘤和Hu001-14-TGFβR给药组肿瘤放在同一张石蜡切片上，对获取的切片进行免疫组化(IHC)染色。首先进行脱蜡和水化，然后利用柠檬酸进行抗原修复，再进行封闭，TGFβ1一抗(Proteintech，Cat#21898-1-AP)孵育过夜，一抗使用浓度为1：50，二抗(Jackson Immuno,Cat#111-035-003)使用浓度为1：200稀释，最后加入DAB显色液显色之后，再利用苏木素复染，脱水封片。利用显微镜进行成像记录。将获得的IHC成像图片，利用ImageJ软件进行分析，对大约10个代表性的视野进行统计灰度值，最终统计平均值±S.E。
如图20所示，TGFβ1的表达堆积水平在hIgG1组肿瘤中显著高于Hu001-14-TGFβR给药组。
步骤③抗CD73-TGFβR对肿瘤微环境中MII型巨噬细胞的抑制
对本实施例步骤②使用的相同的石蜡切片进行IHC染色，实验方法参照步骤②，其中CD206(MII-型巨噬细胞标志物)的一抗使用浓度为1：200稀释，F4/80(巨噬细胞共标志物)的一抗使用浓度为1：200稀释，二抗(Jackson Immuno,Cat#111-035-003)使用浓度为1:200稀释。
如图21所示，F480/CD206比值在hIgG1组肿瘤中要显著低于Hu001-14-TGFβR给药组，说明Hu001-14-TGFβR能够降低肿瘤微环境中的MII-型巨噬细胞水平。
实施例8静脉注射给药抗CD73-TGFβR能显著抑制NCI-H441肿瘤生长并改善肿瘤免疫微环境
步骤①静脉注射抗CD73-TGFβR对肿瘤生长的抑制活性
将200μL含有5×10 6NCI-H441的细胞悬液接种到免疫缺陷小鼠(Balb/c,nude)背部皮下。接种当天根据裸鼠体重随机分组(n＝6)，Hu001-14采用10mg/kg剂量，Hu001-14-TGFβR采用12.5mg/kg(相当于抗体10mg/kg剂量)，每周尾静脉给药两次，共计给药4周；同时设置hIgG1作为阴性对照。每周测量2～3次肿瘤体积及裸鼠体重并记录以绘制肿瘤生长曲线。
结果如图22所示，相比Hu001-14抗体组，Hu001-14-TGFβR组能更显著的抑制NCI-H441肿瘤在裸鼠体内的生长。
步骤②静脉注射抗CD73-TGFβR提高了肿瘤微环境中MI型巨噬细胞的浸润水平
将步骤①获取的肿瘤样本进行石蜡包埋并切片，将hIgG1给药组肿瘤、Hu001-14给药组和Hu001-14-TGFβR给药组肿瘤放在同一张石蜡切片上，然后对石蜡切片进行免疫荧光(IF)染色。首先进行脱蜡和水化，然后利用柠檬酸进行抗原修复，再进行封闭，anti-CD86的PE标记的一抗(Biolegend)和anti-F4/80的488标记的一抗(Biolegend)孵育过夜，一抗使用浓度皆为1：100稀释，利用DAPI染细胞核之后利用抗淬灭剂封片。利用荧光显微镜进行成像记录。将获得的IF成像图片，利用ImageJ软件进行分析，对代表性的视野进行统计，最终统计平均值±S.E。
结果如图23所示，在IgG1组肿瘤中的MI-型(CD86 +F4/80 +双阳性)巨噬细胞的总体数量很低，CD73抗体Hu001-14治疗组有一定的提高。相比Hu001-14组，Hu001-14-TGFβR治疗组能更显著且大幅度提高MI-型巨噬细胞在肿瘤微环境中的浸润/扩增数量，从而实现抗肿瘤的作用。
步骤③静脉注射抗CD73-TGFβR降低了肿瘤微环境中MII型巨噬细胞的浸润水平
对本实施例步骤②使用的相同的石蜡切片分别进行IHC和IF染色，方法分别参照实施例7步骤②和本实施例步骤②。其中anti-CD206的PE标记的一抗(Biolegend)和anti-F4/80的488标记的一抗(Biolegend)浓度为1：100稀释。
结果如图24所示，免疫组化(IHC)检测表明Hu001-14组和Hu001-14-TGFβR组均降低了瘤内CD206的表达。但与Hu001-14不同，Hu001-14-TGFβR还显著提高了瘤内总体F4/80阳性细胞的数量，这一结果结合图23说明了抗CD73-TGFβR融合蛋白具有降低MII-型巨噬细胞并且扩增MI-型巨噬细胞的双重药理作用。
结果如图25所示，为进一步验证上述结果，对肿瘤进行CD206和F4/80免疫双荧光共染检测。结果证明，Hu001-14和Hu001-14-TGFβR均能够降低瘤内MII-型巨噬细胞(CD206 +F4/80 +双阳性细胞)，其中Hu001-14-TGFβR对MII-型巨噬细胞的抑制作用更显著。
步骤④静脉注射抗CD73-TGFβR提高了肿瘤微环境中成熟树突状细胞的水平
对本实施例步骤②使用的相同的石蜡切片进行免疫荧光(IF)染色，方法参照步骤②，其中anti-CD86的PE标记的一抗(Biolegend)和anti-CD11c的FITC标记的一抗(Biolegend)浓度为1：100稀释。
结果如图26所示，Hu001-14给药组的肿瘤中，树突状细胞的水平相对于hIgG1组未见明显提高，而Hu001-14-TGFβR给药能够显著提高肿瘤中的成熟树突状细胞的水平，从而提高肿瘤微环境中抗原呈递能力和抗肿瘤的能力。
实施例9抗CD73-TGFβR能促进免疫重建NSG鼠移植瘤中人CD45 +免疫细胞和CD8 +T淋巴细胞的浸润
步骤①NSG鼠人源化免疫重建和抗CD73-TGFβR对NCI-H1299肿瘤的抑制作用
为研究抗CD73-TGFβR对肿瘤中人CD45 +免疫细胞和CD8 +T细胞浸润的作用，建立了NSG鼠(南方模式动物)的人免疫重建鼠NCI-H1299肺癌细胞移植瘤模型。
在试验的第0天，将冻存的人PBMC复苏，离心去除上清，将细胞重悬在含100IU/mL IL-2的PBMC培养基中，放入细胞培养箱中使其恢复6小时。将细胞离心，弃培养液，再用PBS清洗并重悬至细胞浓度2.5x10 7/mL。每只NSG鼠尾静脉接种5x10 6PBMC，同时设置不接种PBMC的组。
在试验的第1天，将NCI-H1299细胞消化之后，离心去上清，用PBS重悬细胞至所需细胞密度。将50μL细胞悬液(9×10 6)与50μL预先配置的hIgG1(0.5mg/mL，25μg/瘤)，Hu001-14(0.5mg/mL，25μg/瘤)和Hu001-14-TGFβR(0.625mg/mL，31.25μg/瘤)混合，置4℃孵育30min后与100μL基质胶混匀，接种到NSG鼠背部皮下。
在试验的第2天，按照day1的分组(n＝6)，分别尾静脉注射对应的hIgG1(10mg/kg)，Hu001-14(10mg/kg)和Hu001-14-TGFβR(12.5mg/kg)。
在试验的第28天，经小鼠眼眶取血，收取血细胞经裂解红细胞之后分别使用人CD45和CD3抗体染色，使用流式细胞仪(FACS)读取活细胞中人CD45 +及CD3 +细胞所占的比例。
结果如图27(A)所示，与未接种PBMC的组相比，在28天时，接种人PBMC的小鼠血液中人CD45 +细胞比例为61.1％，人CD3 +细胞比例为51.0％，说明人源化NSG鼠免 疫重建成功。结果如图27(B)所示，与hIgG1组相比，Hu001-14和Hu001-14-TGFβR治疗组的肿瘤重量较低，其中Hu001-14-TGFβR组最低，显示更强的体内抑瘤效果。
步骤②抗CD73-TGFβR对NCI-H1299肿瘤中的CD45 +细胞和CD8 +T细胞的浸润的作用
在第28天试验终止时收取NCI-H1299肿瘤，采用4％中性多聚甲醛固定、脱水、石蜡包埋、切片及免疫组织化学染色，观察hIgG1，Hu001-14和Hu001-14-TGFβR对肿瘤中人CD45 +免疫细胞和CD8 +T细胞浸润的影响。
结果如图28所示，与hIgG1组相比，Hu001-14和Hu001-14-TGFβR均能显著促进肿瘤组织中人CD45 +免疫细胞的浸润。与Hu001-14相比，Hu001-14-TGFβR组肿瘤中的CD45 +免疫细胞的浸润程度更高。结果同时显示，与hIgG1组相比，Hu001-14和Hu001-14-TGFβR均能显著促进肿瘤组织中人CD8 +T细胞的浸润。与Hu001-14相比，Hu001-14-TGFβR肿瘤中人CD8 +T细胞的浸润程度更高。
综上，本实施例结果说明，Hu001-14和Hu001-14-TGFβR在人源化NSG免疫重建鼠NCI-H1299移植瘤模型中均具有明确的肿瘤免疫治疗效果；相比Hu001-14，Hu001-14-TGFβR更进一步提高了免疫效应细胞对肿瘤组织的浸润程度，改善肿瘤免疫微环境，以呈现更显著的抗肿瘤治疗效果。
实施例10抗TF-TGFβR融合蛋白的制备和检测
步骤①抗TF-TGFβR融合蛋白的制备
参照实施例4，抗TF-TGFβR融合蛋白HuSC1-39-TGFβR由重链和轻链组成。重链部分由抗TF的人源化抗体HuSC1-39与TGFβRII胞外区通过Linker组成，HuSC1-39的重链可变区为SEQ ID NO.:37。HuSC1-39-TGFβR的轻链可变区与HuSC1-39的轻链可变区(SEQ ID NO.:38)相同。HuSC1-39的重链可变区和轻链可变区引自CN201610705557.4。
如表-8所示，构建出融合蛋白HuSC1-39-TGFβR。
表-8抗TF-TGFβR融合蛋白
Figure PCTCN2020113357-appb-000051
SEQ ID NO.:37抗TF抗体HuSC1-39的重链可变区氨基酸序列
Figure PCTCN2020113357-appb-000052
SEQ ID NO.:38抗TF抗体HuSC1-39的轻链可变区氨基酸序列
Figure PCTCN2020113357-appb-000053
SEQ ID NO.:39 HuSC1-39-TGFβR融合蛋白重链的氨基酸序列
Figure PCTCN2020113357-appb-000054
Figure PCTCN2020113357-appb-000055
融合蛋白重链载体构建结果如图29A所示，通过对获取的载体分别进行BspQI、BamHI单酶切以及BspQI/BamHI双酶切，确认酶切后片段与预期的结果一致。
融合重链和轻链的表达载体质粒通过脂质体转染到293T细胞中进行蛋白表达。在培养皿中培养5d之后收集表达上清，然后利用蛋白A进行纯化，纯化的蛋白通过SDS-PAGE进行分析。
蛋白纯化的结果如图29B所示，对还原和非还原状态的蛋白进行SDS-PAGE分析之后发现其电泳呈现的分子量与理论分子量基本一致，说明纯化出的蛋白是目的蛋白；HuSC1-39-TGFβR的蛋白检测纯度为91.7％。
步骤②HuSC1-39-TGFβR对人TF抗原亲和力的ELISA测定
用包被液将TF蛋白胞外区(TF-ECD)稀释成2μg/mL，包被ELISA板，ELISA测定的具体方法参照实施例5。
检测结果如图30所示，HuSC1-39-TGFβR对TF-ECD有很强的亲和性，EC 50为0.095nM。
步骤③HuSC1-39-TGFβR对TGFβ1亲和力的ELISA测定
将TGFβ1作为抗原包被在酶标板上，TGFβ1的浓度为0.5μg/mL。实验方法参考实施例5，其中一抗终浓度为27μg/mL，3倍梯度稀释。
检测结果如图31所示，HuSC1-39-TGFβR对TGFβ1有很强的亲和性，EC 50为0.61nM。
步骤④融合蛋白对肿瘤细胞表面TF的特异性结合
参照实施例5，采用TF高表达的三阴性乳腺癌细胞MDA-MB-231、胰腺癌细胞BxPC3，测定融合蛋白对细胞表面TF的结合亲和力。
检测结果如图32、图33所示，HuSC1-39-TGFβR对肿瘤细胞表面的TF有很强的特异结合，对BxPC3细胞结合的EC 50为5.6nM；对MDA-MB-231细胞结合的EC 50为4.7nM。
实施例11抗TF-TGFβR能更有效抑制HCC1806肿瘤生长并改善肿瘤免疫微环境
步骤①抗TF-TGFβR在NSG鼠移植瘤模型中的抗肿瘤活性
随机将免疫缺陷型NSG鼠(南方模式动物)分为3组，将100μL含有2.5x10 6NCI-H1806的细胞悬液与100μL hIgG1(20μg/瘤)，HuSC1-39(20μg/瘤)，HuSC1-39-TGFβR (25μg/瘤)混匀，孵育30分钟后接种入NSG鼠乳垫处(n＝4)，观察对肿瘤生长的抑制作用。每周2-3次测量动物体重及肿瘤大小，试验终止时(第16天)称取肿瘤的重量，评定药效活性。
结果如图34所示，HuSC1-39-TGFβR能显著抑制NCI-H1806肿瘤在NSG鼠体内的生长，其效果优于TF抗体HuSC1-39。
步骤②抗TF-TGFβR提高了肿瘤微环境中MI型巨噬细胞的水平
将步骤①获取的肿瘤样本进行石蜡包埋并切片，将hIgG1组、HuSC1-39组和HuSC1-39-TGFβR组肿瘤石蜡切片制作在同一张载片上，进行免疫荧光(IF)染色。首先进行脱蜡和水化，然后利用柠檬酸进行抗原修复，再进行封闭。CD86和F4/80双荧光染色采用anti-CD86的PE标记的一抗(Biolegend)和anti-F4/80的488标记的一抗(Biolegend)，浓度为1：100稀释，孵育过夜，DAPI染细胞核之后利用抗淬灭剂封片。利用荧光显微镜进行成像记录。将获得的IF成像图片，利用ImageJ软件进行分析，对代表性的视野进行统计，最终统计平均值±S.E。
结果如图35所示，hIgG1组中CD86 +F4/80 +表达水平较低，说明hIgG1组的MI-型巨噬细胞较少，TF抗体HuSC1-39给药组明显提高了MI-型巨噬细胞的水平，并且HuSC1-39-TGFβR组中MI-巨噬细胞在肿瘤微环境中的水平最高，说明HuSC1-39-TGFβR可能是通过降低TGFβ对MI-型巨噬细胞的抑制，进而进一步提高了肿瘤中MI-型巨噬细胞浸润程度，达到肿瘤杀伤作用。
步骤③抗TF-TGFβR降低了肿瘤微环境中MII型巨噬细胞的水平
对本实施例步骤②使用的相同的石蜡切片进行IHC染色和免疫荧光(IF)，方法分别参照实施例7的步骤②和本实施例的步骤②，其中anti-CD206的PE标记的一抗(Biolegend)和anti-F4/80的488标记的一抗(Biolegend)浓度为1：100稀释。
结果如图36所示，免疫组化(IHC)检测表明，HuSC1-39和HuSC1-39-TGFβR均降低了CD206的表达，并且提高了F4/80的水平。相比HuSC1-39，HuSC1-39-TGFβR治疗组的整体CD206/F4/80比值更低，说明HuSC1-39-TGFβR对肿瘤中MII-型巨噬细胞浸润的抑制活性更显著。
如图37所示，为进一步验证上述结果，对肿瘤进行CD206和F4/80免疫双荧光共染检测，结果证明HuSC1-39和HuSC1-39-TGFβR都能够降低瘤内MII-型巨噬细胞(CD206 +F4/80 +双阳性细胞)，其中HuSC1-39-TGFβR的抑制活性最为显著。
综上，本实施例结果说明，HuSC1-39和HuSC1-39-TGFβR在NSG鼠HCC1806乳腺癌移植瘤模型中均具有明确的肿瘤抑制作用和改善肿瘤微环境的效果；相比HuSC1-39，HuSC1-39-TGFβR更进一步提高了抗肿瘤免疫效应细胞对肿瘤组织的浸润程度，下调免疫抑制细胞的水平，从而改善肿瘤免疫微环境。
Figure PCTCN2020113357-appb-000056
Figure PCTCN2020113357-appb-000057
Figure PCTCN2020113357-appb-000058
Figure PCTCN2020113357-appb-000059

Claims (24)

  1. 一种重组双功能融合蛋白，其特征在于，所述重组双功能融合蛋白包含：
    第一结合结构域(D1)；和
    第二结合结构域(D2)；
    其中，所述第一结合结构域特异性结合靶分子CD73或TF蛋白；
    所述第二结合结构域特异性结合靶分子TGFβ蛋白。
  2. 如权利要求1所述的重组双功能融合蛋白，其特征在于，所述D1为特异性结合CD73或TF蛋白的抗体或抗体片段，所述的抗体片段包含重链可变区和轻链可变区，和/或，
    所述D2为特异性结合TGFβ蛋白的多肽片段，并且所述多肽片段衍生自TGFβ受体。
  3. 如权利要求2所述的重组双功能融合蛋白，其特征在于，所述D1为抗CD73单克隆抗体或抗TF单克隆抗体，并且D2通过连接肽连接在D1的选自下组的区域：重链可变区、重链恒定区、轻链可变区、或其组合。
  4. 如权利要求1所述的重组双功能融合蛋白，其特征在于，所述双功能融合蛋白(单体)从N端到C端具有式I所示的结构：
    Figure PCTCN2020113357-appb-100001
    其中，
    T1、T2、T3各自独立地为无或TGFβ受体II的胞外区，且至少一个不为无；
    L1、L2、L3各自独立地为键或接头元件；
    VL代表抗CD73或TF抗体的轻链可变区；
    CL代表抗CD73或TF抗体的轻链恒定区；
    VH代表抗CD73或TF抗体的重链可变区；
    CH代表抗CD73或TF抗体的重链恒定区；
    “～”代表二硫键或共价键；
    “-”代表肽键；
    其中，所述双功能融合蛋白(单体)具有同时结合CD73或TF以及结合TGFβ的活性。
  5. 如权利要求4所述的重组双功能融合蛋白，其特征在于，所述的重链可变区具有SEQ ID NO.:2、3、4、9、10、12、13、14、15、16、17、18、19、20、24、25、26或27所示的氨基酸序列。
  6. 如权利要求4所述的重组双功能融合蛋白，其特征在于，所述的轻链可变区具 有SEQ ID NO.:5、6、7、8、11、21、22、23、28、29或30所示的氨基酸序列。
  7. 如权利要求4所述的重组双功能融合蛋白，其特征在于，所述的TGFβ受体II的胞外区具有SEQ ID NO.:33所示的氨基酸序列。
  8. 一种抗CD73的抗体，其特征在于，所述的抗体含有：
    (e)抗体重链可变区；和
    (f)抗体轻链可变区；
    所述的抗体重链可变区具有SEQ ID NO.:2、3、4、9、10、12、13、14、15、16、17、18、19、20、24、25、26或27所示的氨基酸序列；
    所述的抗体轻链可变区具有SEQ ID NO.:5、6、7、8、11、21、22、23、28、29或30所示的氨基酸序列。
  9. 根据权利要求8所述一种抗CD73的抗体，其特征在于，所述的抗体还含有重链恒定区，所述的重链恒定区为人源、鼠源或兔源的。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素或酶。
  11. 一种药物组合物，其特征在于，它含有：
    (i)如权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体；以及
    (ii)药学上可接受的载体。
  12. 如权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体的用途，其特征在于，用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗CD73或TF、和/或TGFβ相关疾病的药物。
  13. 如权利要求12所述的用途，其特征在于，
    所述检测试剂或试剂盒用于：
    (1)检测样品中的CD73蛋白和/或TGFβ蛋白；和/或
    (2)检测肿瘤细胞中内源性的CD73蛋白和/或肿瘤细胞分泌的TGFβ蛋白；和/或
    (3)检测表达CD73蛋白和/或分泌TGFβ蛋白的肿瘤细胞；和/或，
    所述的药物用于治疗或预防CD73和/或TGFβ高表达的肿瘤、肿瘤迁移、或肿瘤耐药；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。
  14. 如权利要求12所述的用途，其特征在于，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)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
    (e)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；
    (f)抑制TGFβ诱导的能够促进肿瘤的免疫细胞的功能；
    (g)抑制TGFβ诱导产生的肿瘤微环境的免疫逃逸作用和纤维化等；
    (h)抑制肿瘤的耐药；
    (i)抑制肿瘤细胞迁移或转移。
  15. 一种治疗与CD73和/或TGFβ相关疾病的方法，其特征在于，所述方法给需要的对象施用权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体、或如权利要求11所述的药物组合物。
  16. 一种制备权利要求1～7中任一项所述的重组双功能融合蛋白的方法，其特征在于，包括步骤：
    (a)将如权利要求8或9所述抗CD73的抗体或TF抗体的重链的表达载体进行双酶切，获取线性载体，再将带有相同酶切位点的Linker和TGFβRII胞外区的DNA片段***到线性载体中，获得融合蛋白重链的表达载体；
    (b)将融合蛋白重链的表达载体和如权利要求8或9所述抗CD73的抗体或TF抗体的轻链的表达载体一同转染动物细胞表达融合蛋白。
  17. 一种多核苷酸，其特征在于，它编码权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体。
  18. [根据细则26改正19.10.2020]　
    一种载体，其特征在于，它含有如权利要求17所述的多核苷酸。
  19. [根据细则26改正19.10.2020]　
    一种遗传工程化的宿主细胞，其特征在于，它含有如权利要求18所述的载体或基因组中整合有如权利要求17所述的多核苷酸。
  20. 一种重组蛋白，其特征在于，该重组蛋白含有：
    (a)如权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体；和
    (b)任选的协助表达和/或纯化的标签序列。
  21. 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，培养如权利要求19所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体。
  22. 一种抑制肿瘤细胞生长和迁移的方法，其特征在于，包括步骤：给需要的对象施用权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体、或如权利要求11所述的药物组合物。
  23. 一种对T淋巴细胞增殖保护作用的方法，其特征在于，包括步骤：给需要的对象施用权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体、或如权利要求11所述的药物组合物。
  24. 一种抑制肿瘤在模型动物体内生长的方法，其特征在于，包括步骤：给需要的对象施用权利要求1～7中任一项所述的重组双功能融合蛋白、或如权利要求8或9所述抗CD73的抗体、或如权利要求11所述的药物组合物。
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