WO2019170131A1 - 靶向cd73的抗体及抗体-药物偶联物、其制备方法和用途 - Google Patents

靶向cd73的抗体及抗体-药物偶联物、其制备方法和用途 Download PDF

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WO2019170131A1
WO2019170131A1 PCT/CN2019/077369 CN2019077369W WO2019170131A1 WO 2019170131 A1 WO2019170131 A1 WO 2019170131A1 CN 2019077369 W CN2019077369 W CN 2019077369W WO 2019170131 A1 WO2019170131 A1 WO 2019170131A1
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antibody
seq
variable region
chain variable
cells
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English (en)
French (fr)
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余科
金锐
刘亮
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Fudan University
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Fudan University
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Priority claimed from CN201810506111.8A external-priority patent/CN110240654A/zh
Application filed by Fudan University filed Critical Fudan University
Priority to CA3093327A priority Critical patent/CA3093327C/en
Priority to CN201980001728.7A priority patent/CN110869393B/zh
Priority to EP19763891.9A priority patent/EP3783025A4/en
Priority to JP2020546414A priority patent/JP7330996B2/ja
Priority to US16/978,995 priority patent/US12024565B2/en
Publication of WO2019170131A1 publication Critical patent/WO2019170131A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [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

Definitions

  • the present invention relates to the field of medicine, and in particular to an antibody and antibody-drug conjugate (ADC-targeting antibody and antibody-drug conjugate, preparation method and use thereof) that targets CD73.
  • ADC-targeting antibody and antibody-drug conjugate, preparation method and use thereof targets CD73.
  • CD73 is an extracellular 5-nuclease (NT5E) with a molecular weight of 70 kD anchored to the cell surface by glycosylphosphatidylinositol (GPI). Under physiological conditions, CD73 is mainly expressed in various tissues such as the large intestine, kidney, liver, lung, lymph nodes and other tissues.
  • N5E 5-nuclease
  • GPI glycosylphosphatidylinositol
  • Adenosine monophosphate AMP
  • NAD+ nicotinamide adenine dinucleotide
  • a large amount of adenosine is formed around the tissue, which participates in various physiological processes of the cell by binding to the corresponding adenosine receptors (A1AR, A2AR, A2BR, A3AR).
  • CD73 and adenosine pathway are closely related to the occurrence and development of tumors.
  • it can promote tumor immune escape.
  • Hypoxia activation of inflammatory factors (IFN- ⁇ , TNF- ⁇ , IL-1 ⁇ , TGF- ⁇ , etc.) and related signaling pathways (Wnt, cAMP) can induce abnormal expression of CD73 in tumor cells, which produces a large number of AMP ADO, surrounded by tumors, forms a "loop" type microenvironment that resists tumor immunosuppression and promotes tumor immune escape.
  • ADO acts on CD8+ T cell surface adenosine receptor (A2AR),
  • A2AR CD8+ T cell surface adenosine receptor
  • the cAMP signaling pathway inhibits its proliferation, amplification, and reduces the release of related pro-inflammatory cytokines IFN- ⁇ , TNF- ⁇ , etc., thereby reducing its cytotoxic effect.
  • ADO can interfere with the adhesion between NK cells and tumor cells, thereby reducing the ability of NK cells to exocytose cytotoxic particles, and the cytotoxicity is weakened.
  • ADO promotes its expansion and enhances its immunosuppressive and anti-inflammatory functions by binding to A2AR on the surface of regulatory T cells (Tregs).
  • CD73 catalyzed by CD73 on the surface of Tregs binds to CD8+ effector T cells A2AR inhibits NF-kB. Activation leads to a decrease in the secretion of pro-inflammatory cytokines and chemokines.
  • Preclinical studies have also shown that injection of CD4+CD25+Tregs from wild-type mice into Tregs-deficient mice promotes colon cancer development, whereas Tregs from CD73-deficient mice do not produce any effect, suggesting CD73 and Tregs play an important role in the immunosuppression of tumors.
  • ADO inhibits the differentiation of M1 macrophages and reduces the release of pro-inflammatory cytokines IL-12, TNF- ⁇ , iNOS, etc., which can activate M2 macrophages and produce a large number of anti-inflammatory cytokines (TGF- ⁇ ). , arginase1), thereby helping the tumor to produce immune escape.
  • CD73 can promote tumor growth and metastasis. Preclinical studies have shown that CD73 is abnormally expressed in a variety of tumor cells, such as breast cancer, bladder cancer, ovarian cancer, colon cancer, non-small cell lung cancer, etc., and clinical data show that high expression of CD73 is closely related to the poor prognosis of cancer patients [ Expert Rev Anticancer Ther.
  • CD73 can be used as a clinical treatment and prognostic target for a variety of tumors.
  • CD73 blockers can significantly inhibit the number of new blood vessels and the maturation of vascular beds.
  • ADO produced by CD73 can promote the proliferation of microvascular endothelial cells and the release of vascular endothelial growth factor (VEGF) by up-regulating cyclin D1 (Cyclin D1), promote tumor angiogenesis, and provide sufficient energy for tumor growth.
  • VEGF vascular endothelial growth factor
  • CD73 plays an important role in tumor metastasis.
  • CD73 Drug resistance is a major problem and challenge in cancer treatment.
  • pCR pathological complete response rate
  • Targeting CD73 monoclonal antibody can significantly enhance the anti-tumor immune response and anti-tumor activity of doxorubicin.
  • trastuzumab trastuzumab
  • high CD73 expression was significantly associated with poor prognosis; targeting CD73 monoclonal antibody in combination with Trastuzumab increased CD8+ T cells and reduced MDSC infiltration. Produces synergistic anti-tumor effects [Cancer Res. 2017; 77:5652].
  • Antibody-drug conjugate is a monoclonal antibody that specifically recognizes specific antigens on the surface of tumor cells, so as to accurately deliver anti-tumor drugs (such as small molecule chemotherapy drugs) to tumors. The target cells are released and achieve the purpose of accurately killing the tumor.
  • ADC is also considered to be the most potential anti-tumor drug because of its proper molecular weight, high stability, high targeting, and low toxicity.
  • the successful development of ADC also has many problems that must be considered and must be solved. For example, antibodies should specifically identify lesions, have low immunosensitivity, and can efficiently and rapidly undergo endocytosis; antibody-drug linkers are stable in blood. It is highly sexual and can be specifically activated in targeted cells and efficiently release small molecule drugs; the coupled small molecule drug cells have strong killing ability.
  • CD73 is abnormally expressed in a variety of tumor cells and is closely related to the poor prognosis of cancer patients.
  • CD73 mainly produces anti-tumor immunosuppressive effects through adenosine pathway, promoting tumor growth, metastasis and angiogenesis.
  • CD73 is also involved in the development of anti-cancer drug resistance, which poses great challenges for cancer treatment. Therefore, the development of a targeted CD73 monoclonal antibody provides a new approach for clinically alone or in combination with patients with abnormal expression of CD73 tumors.
  • there is currently a lack of highly specific antibody-drug conjugates against human CD73 there is currently a lack of highly specific antibody-drug conjugates against human CD73. Therefore, the development of antibody-drug conjugates targeting tumor CD73 and superior drug performance will exert its characteristics and Advantages provide new ideas and prospects for the treatment of cancers with abnormal expression of CD73.
  • the present invention provides an antibody targeting human CD73, which has the activity of blocking CD73 to catalyze the hydrolysis of adenosine monophosphate (AMP) to adenosine, inhibiting tumor growth and metastasis activity, and reducing anti-tumor resistance. The emergence of sex.
  • AMP adenosine monophosphate
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CD73 binding affinity.
  • the heavy chain variable region comprises the following complementarity determining regions:
  • SEQ ID NO.: 10 SEQ ID NO.: 11
  • SEQ ID NO.: 12 the heavy chain complementarity determining region HCDR1, HCDR2, HCDR3 of mAb002c; or
  • SEQ ID NO.: 1 SEQ ID NO.: 2, SEQ ID NO.: 3 heavy chain complementarity determining region HCDR1, HCDR2, HCDR3;
  • the heavy chain variable region further comprises a FR region of a human source or a FR region of a murine source.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 7.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.:27, SEQ ID NO.:28, SEQ ID NO.:29.
  • the heavy chain variable region has SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ ID NO.: 33, SEQ ID NO.: 34, SEQ ID NO.: 35, The amino acid sequence shown in SEQ ID NO.: 45, SEQ ID NO.: 46.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.:38, SEQ ID NO.:39, SEQ ID NO.:40, SEQ ID NO.:41.
  • a heavy chain of an antibody having the heavy chain variable region of the first aspect of the invention.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CD73 binding affinity.
  • the light chain variable region comprises the following complementarity determining region:
  • SEQ ID NO.: SEQ ID NO.: 14 shows the light chain complementarity determining region of mAb002c, LCDR1, LCDR2, LCDR3;
  • SEQ ID NO.: 4 SEQ ID NO.: 5
  • SEQ ID NO.: 6 light chain complementarity determining region LCDR1, LCDR2, LCDR3;
  • SEQ ID NO.: 24 SEQ ID NO.: 25, SEQ ID NO.: 26 light chain complementarity determining regions LCDR1, LCDR2, LCDR3 of mAb004c.
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 8, SEQ ID NO.: 9.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 19, SEQ ID NO.: 20.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.:30.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 36, SEQ ID NO.: 37, SEQ ID NO.: 47.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 42, SEQ ID NO.: 43, SEQ ID NO.: 44.
  • a light chain of an antibody having the light chain variable region of the third aspect of the invention.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • an antibody having:
  • the antibody has: the heavy chain of the second aspect of the invention; and/or the light chain of the fourth aspect of the invention.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the CDR regions of the humanized antibody comprise 1, 2, or 3 amino acid changes.
  • the animal is a non-human mammal, preferably a mouse, a sheep, or a rabbit.
  • 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 amino acids added, deleted, modified and/or substituted does not exceed 40%, preferably 20%, more preferably 10%, of the total amino acid number of the initial amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 7, preferably from 1 to 3, more preferably one.
  • the at least one amino acid sequence added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
  • the derivative sequence that adds, deletes, modifies, and/or substitutes at least one amino acid has a catalytic function of inhibiting cell surface CD73 or recombinant CD73 protease.
  • the antibody is in the form of a drug conjugate.
  • the affinity of EC-derived sequence of CD73 (e.g., human CD73 extracellular domain protein, CD73-ECD) 50 is 0.016 ⁇ 0.2nM, is preferably 0.016 ⁇ 0.03nM, more preferably 0.016 ⁇ 0.02 nM.
  • the antibody has one or more characteristics selected from the group consisting of:
  • CD73 that specifically binds to tumor cells, and/or immune/stromal cells in the tumor microenvironment
  • a recombinant protein having:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • a CAR construct wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to CD73, and the scFv has the first aspect of the invention The heavy chain variable region of one aspect and the light chain variable region of the third aspect of the invention.
  • a recombinant immune cell is provided, the immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.
  • the immune cells are selected from the group consisting of NK cells, T cells.
  • the immune cells are from a human or non-human mammal (e.g., a mouse).
  • an antibody drug conjugate comprising:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an anti-CD73 antibody
  • LU is a joint (also known as a linker);
  • D is a drug
  • subscript p is a value selected from 1-10, preferably 1-8.
  • LU is selected from the group consisting of 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), 6-Malay Amido hexanoyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MC-ala-phe-PAB), maleimidopropionyl-valine-citrulline-p-aminobenzyloxy Carbonyl (MP-val-cit-PAB), maleimidopropionyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MP-ala-phe-PAB), N-succinimidyl 4 -(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4- (2
  • LU is a disubstituted maleimide linker.
  • the structure of the antibody drug conjugate is as shown in Formula Ia, Ib:
  • Ar' is selected from the group consisting of substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted C6-C10 arylene, substituted or unsubstituted 5- 12-membered heteroarylene;
  • L 1 is -O(CH 2 CH 2 O) n - attached to the Ar' group, wherein n is selected from any of 1-20.
  • L 2 is a chemical bond, or an AA-PAB structure; wherein AA is a polypeptide fragment consisting of 2-4 amino acids, and PAB is p-aminobenzylcarbamoyl;
  • CTD is a cytotoxic small molecule drug that is bonded to L 2 via an amide bond.
  • m is from 1.0 to 5.0, preferably from 3.0 to 4.2; more preferably from 3.5 to 4.5; still more preferably from 3.8 to 4.2, still more preferably from 3.9 to 4.1, most preferably 4.0;
  • Ab is an antibody that targets CD73.
  • the formula Ib is a ring-opened product of N-phenylmaleimide of formula Ia.
  • the conjugate is covalently linked to one or more pharmaceutical components.
  • the antibody moiety is coupled to the coupled moiety by covalent means (e.g., by covalent attachment to a linker, respectively).
  • the ring-closed or ring-opened maleimide group is attached to the disulfide-reduced sulfhydryl group of the antibody hinge region.
  • the antibody-drug conjugate is formed by reduction of a disulfide chain of the hinge region of the antibody or antibody fragment to generate a pair of cysteine residues, and by the cysteine residue
  • the thiol group in the group is substituted with an aryl sulfide in the substituted maleimide linker-drug conjugate represented by the formula Ic, thereby obtaining the antibody-drug conjugates Ia and/or Ib.
  • the ring-closed or ring-opened maleimide group is attached to the fully reduced antibody, ie, the 2-pair disulfide chain of the hinge region is fully open, preferably m is 3.8-4.2, More preferably, it is 3.9-4.1, and most preferably 4.0.
  • the Ar' is selected from the group consisting of phenyl, halobenzene, C1-C4 alkylphenyl, C1-C4 alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl, Wherein W is an amine group R 1 attached to a carbonyl group, and R 1 is selected from -NH 2 , wherein: the C1-C4 alkylphenyl group is further preferably a 4-methylphenyl group; and the C1-C4 alkoxyphenyl group is further preferably a 4-methoxyphenyl group.
  • Ar' is selected from a substituted or unsubstituted phenylene group or a pyridyl group, and said substituent means that the hydrogen atom on the group is substituted with one or more substituents selected from the group consisting of halogen , C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile, amide.
  • the AA is selected from the group consisting of Val-Cit, Val-Ala, and Phe-Lys. -lysine), Ala-Ala-Asn (alanine-alanine-asparagine), D-Ala-Phe-Lys (D-alanine-phenylalanine-lysine), Gly -Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), or a combination thereof.
  • the drug D or CTD is selected from the group consisting of:
  • MMAE Monomethyl auristatin E
  • MMAF Monomethyl auristatin F
  • MMAD Monomethyl Dolastatin 10
  • DNA damage drug preferably, the DNA damage drug comprises docamycin, pyrrolo[2,1-c][1,4]benzodiazepine (PBD).
  • PBD pyrrolo[2,1-c][1,4]benzodiazepine
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the heavy chain variable region sequence of the antibody is selected from the group consisting of SEQ ID NO.: 7, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: And SEQ ID NO. ID NO.: 35, SEQ ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 38, SEQ ID NO.: 39, SEQ ID NO.: 40, SEQ ID NO.: 41; or
  • the light chain variable region sequence of the antibody is selected from the group consisting of SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 30 SEQ ID NO.: 36, SEQ ID NO.: 37, SEQ ID NO.: 47, SEQ ID NO.: 42, SEQ ID NO.: 43, SEQ ID NO.: 44.
  • the chimeric antibody is selected from the group consisting of mAb001c, mAb001c-VK-SGS, mAb002c, mAb002c-VH-QG, mAb002c-VH-NA, mAb002c-VK-SG, mAb002c-VH-QG /VK-SG, mAb004c, mAb004c-VH-QG, mAb004c-VH-NA (Table-1 of the specification);
  • the humanized antibody is selected from the group consisting of Hu001c-14, Hu001c-15, Hu001c-21, Hu001c-22, Hu001c-23, Hu001c-24, Hu001c-25, Hu001c-28, Hu001c-30, Hu001c-31, Hu001c-32, Hu002c-2, Hu002c-3, Hu002c-4, Hu002c-6, Hu002c- 7. Hu002c-8, Hu002c-10, Hu002c-11
  • an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, The light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the invention
  • an antibody drug conjugate for (i) preparing a diagnostic reagent; and/or (ii) preparing a prophylactic And/or drugs for treating diseases associated with CD73.
  • the detection reagent, test plate or kit is used to:
  • the detection reagent, test plate or kit is used to diagnose a CD73 related disease.
  • the medicament is for treating or preventing tumors, tumor migration, or tumor resistance that are highly expressed by CD73.
  • the tumor resistance includes: drug resistance of the tumor immunotherapy drug, drug resistance of the tumor targeted therapy drug, drug resistance of conventional tumor chemotherapy, and insensitivity to radiation therapy.
  • the medicament is for use in a group selected from the group consisting of:
  • CD73 that specifically binds to tumor cells, and/or immune/stromal cells in the tumor microenvironment
  • the CD73-associated disease is selected from the group consisting of cancer, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof.
  • the CD73-associated disease comprises: the occurrence, growth and/or metastasis of a tumor.
  • the cancer comprises a solid tumor, a blood cancer.
  • the cancer is a tumor with high expression of CD73.
  • the CD73-expressing tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma. Or a combination thereof.
  • the cancer is a drug resistant tumor.
  • the CD73-expressing tumor refers to the ratio of the level of CD73 transcript and/or protein in the tumor tissue to the level L0 of the transcript and/or protein in the normal tissue, L1/L0 ⁇ 2 Preferably ⁇ 3.
  • the metabolic related diseases include: diabetes, foodborne obesity, and fat inflammation.
  • the infectious disease comprises: bacterial and viral infections.
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, and the light chain of the third aspect of the invention
  • the variable region, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the immune cell of the eighth aspect of the invention, the invention The antibody drug conjugate of the ninth aspect, or a combination thereof;
  • a pharmaceutical composition comprising:
  • an active ingredient which is an antibody drug conjugate as described in the antibody drug conjugate of the ninth aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the fourth aspect of the invention a light chain, or an antibody of the fifth aspect of the invention;
  • the invention provides a vector comprising the polynucleotide of the twelfth aspect of the invention.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector or genome of the thirteenth aspect of the present invention, wherein the multinuclear according to the twelfth aspect of the present invention is integrated Glycosylate.
  • a method of detecting CD73 in a sample in vitro comprising the steps of:
  • test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody of the fifth aspect of the invention or the invention
  • the immunoconjugate of the ninth aspect comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody of the fifth aspect of the invention or the invention.
  • a kit comprising:
  • a first container comprising the antibody of the fifth aspect of the invention.
  • the kit contains the test plate of the sixteenth aspect of the invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a nineteenth aspect of the invention provides a method of treating a CD73-associated disease, the method comprising: administering to a subject in need thereof the antibody of the fifth aspect of the invention, the antibody-drug of the antibody of the ninth aspect A conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • the method further comprises administering to the subject in need of other drugs or treatments for combination therapy.
  • the other drugs or treatments include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiation therapy.
  • the anti-tumor immunotherapeutic agent comprises PD-1, PD-L1 mAb.
  • a method of preparing a chimeric antibody comprising the steps of:
  • a method of preparing a humanized antibody comprising the steps of:
  • the nucleotide sequence of the CDR region of the heavy chain variable region of the first aspect of the invention and/or the light chain variable region of the third aspect of the invention is implanted into a nucleoside comprising a FR region of a human antibody
  • the acid sequence template is then cloned into an expression vector containing the human antibody constant region, and the humanized antibody is expressed by transfecting the animal cells.
  • a method of inhibiting tumor cell growth and migration comprising the steps of: administering to a subject in need thereof the antibody of the fifth aspect of the invention, antibody-drug coupling of said antibody Or a CAR-T cell expressing the antibody, or a combination thereof.
  • a method of inhibiting growth of a tumor in a model animal comprising the steps of: administering to the subject in need thereof the antibody of the fifth aspect of the invention, the antibody-drug couple of the antibody A conjugate, or a CAR-T cell expressing the antibody.
  • the medicament may be administered alone or in combination, including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiation therapy.
  • a method for preparing an antibody-drug conjugate according to the ninth aspect of the present invention comprising the steps of:
  • crosslinking reaction of the preparation method is as follows:
  • the antibody in the step (1) is reduced by a reducing reagent such that the interchain disulfide bond of the antibody is reduced to produce a thiol group.
  • the reducing agent in the 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 Threitol
  • the buffer is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / sodium chloride (NaCl) / diethyltriamine pentaacetic acid (DTPA) Buffer, disodium hydrogen phosphate-citric acid/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), boric acid-borax/sodium chloride (NaCl)/diethyltriaminepentaacetic 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
  • NaCl sodium chloride
  • DTPA diethyltriamine penta
  • the volume of the organic solvent in the reaction liquid does not exceed 15%.
  • the organic solvent in the step (2) is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA), and dimethylene. Sulfone (DMSO).
  • the coupling reaction is carried out at 0 to 37 °C.
  • the step (1) is carried out using beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride or DTT, and steps are further included between the step (1) and the step (2).
  • the antibody-drug conjugate Ia is converted to the antibody-drug conjugate Ib in a pH 6-8 buffer.
  • Figure 1 shows the discovery of an anti-human CD73 antibody of the present invention.
  • Figure 1A is a flow cytometry fluorescence sorter (FACS) for the detection of a series of original anti-human CD73 monoclonal antibody (original hybridoma) culture supernatants for human CD73-high expression of MDA-MB-231 (CD73-P), CD73 - Binding activity of low expressed MDA-MB-453 (CD73-N) breast cancer cells.
  • the five antibodies shown are numbered mAb001, mAb002, mAb003, mAb004, mAb005.
  • 1B is identified five subtypes of purified antibodies, binding affinity for MDA-MB-231 cells, FACS were detected EC 50 1.24nM, 0.65nM, 10.7nM, 4.69nM, 26.07nM.
  • Figure 2 is a graph showing the results of agarose gel electrophoresis of PCR amplification of mAb001, mAb002, mAb004 heavy chain variable region (VH) and light chain variable region (VL) fragments.
  • VH heavy chain variable region
  • VL light chain variable region
  • FIG. 3 is a three individuals expressing HEK293T cells - mouse chimeric antibody (chimeric antibody) mAb001c, mAb002c, mAb004c, and then purified using MabSelect TM SuRe TM map column.
  • FIG 4 is an ELISA measuring human - mouse chimeric antibody mAb001c, mAb002c, mAb004c of CD73-ECD binding affinity (Binding affinity EC 50).
  • FIG 5 is a mAb001c, mAb002c, mAb004c inhibitory activity on recombinant human CD73 enzymatic function (IC 50).
  • Figure 6 shows flow cytometry (FACS) detection of mAb001c, mAb002c, mAb004c for breast cancer MDA-MB-453, MDA-MB-231, lung cancer NCI-H460, NCI-H1299, Calu-6, pancreatic cancer SW1990 , the binding rate (MFI) of CD73 receptor on the surface of glioma U87MG cells.
  • FACS flow cytometry
  • Figure 7 shows that the level of CD73 protein on the surface of tumor cells is closely related to its enzymatic activity.
  • CD73-high expression U87MG, Calu-1, NCI-H1299
  • CD73-low expression MDA-MB-453 cell lines were measured, and the number of cells was measured for 3 hours after incubation with adenosine monophosphate (AMP). .
  • AMP adenosine monophosphate
  • Figure 8 shows the results of binding affinity (Binding affinity EC 50 ) of mAb001c, mAb002c, mAb004c on the surface of MDA-MB-231 cells.
  • Binding affinity EC 50 Binding affinity EC 50
  • Figure 9 shows the results of binding affinity (Binding affinity EC 50 ) of mAb001c, mAb002c, mAb004c to NCI-H1299 cell surface CD73.
  • Binding affinity EC 50 Binding affinity EC 50
  • Figure 10 is a graph showing the inhibitory activity (IC 50 ) of mAb001c, mAb002c, and mAb004c on the catalytic function of CD73 enzyme on the surface of MDA-MB-231 cells.
  • Figure 11 is a graph showing the inhibitory activity (IC 50 ) of mAb001c, mAb002c, and mAb004c on the catalytic function of CD73 enzyme on the surface of NCI-H1299 cells.
  • Figure 12 is a graph showing the inhibitory activity (IC 50 ) of mAb001c, mAb002c, and mAb004c on the catalytic function of CD73 enzyme on the surface of Calu-1 cells.
  • FIG 16 is a detection of humanized antibody mAb001c series Hu001c-14 ⁇ 15, Hu001c- 21 ⁇ 28, inhibit the activity of the enzyme catalytic function Hu001c-30 ⁇ 32 recombinant human CD73 (IC 50).
  • FIG 17 is a detection of humanized antibody mAb002c series Hu002c-2 ⁇ 16 inhibitory activity on recombinant human enzymatic functions CD73 (IC 50).
  • FIG 18 is a series of FACS analysis mAb001c- mutants, mAb002c- mutant series of MDA-MB-231 cell surface CD73 binding affinity (Binding affinity EC 50). This test was performed by incubating 1 x 10 5 cells with the indicated concentration of antibody for 1 hour.
  • FIG 19 is a FACS analysis of humanized antibodies mAb001c series Hu001c-14, Hu001c-22 ⁇ 28, MDA-MB-231 cell surface Hu001c-30 ⁇ 32 binding affinity of CD73 (Binding affinity EC 50). This test was performed by incubating 1 x 10 5 cells with the indicated concentration of antibody for 1 hour.
  • FIG 20 is a FACS analysis of humanized antibodies mAb001c series Hu001c-14, Hu001c-22 ⁇ 28, the surface of CD73 NCI-H1299 cells Hu001c-30 ⁇ 32 binding affinity (Binding affinity EC 50). This test was performed by incubating 1 x 10 5 cells with the indicated concentration of antibody for 1 hour.
  • FIG 22 is a FACS analysis of humanized antibodies mAb002c series Hu002c-2 ⁇ 16 of the surface of NCI-H1299 cells CD73 binding affinity (Binding affinity EC 50). This test was performed by incubating 1 x 10 5 cells with the indicated concentration of antibody for 1 hour.
  • Figure 23 is a graph showing the inhibitory activity (IC 50 ) of the humanized antibody series Hu001c-14-15, Hu001c-21-28, and Hu001c-30-32 of mAb001c on the surface CD73 enzyme catalytic function of NCI-H1299 cells.
  • Figure 24 is a graph showing the inhibitory activity (IC 50 ) of the humanized antibody series Hu002c-2 to 16 of mAb002c on the surface CD73 enzyme catalytic function of NCI-H1299 cells.
  • Figure 25 shows that binding of mAb001c, mAb002c, mAb004c to MDA-MB-231 cells results in internalization to intracellular lysosomes.
  • the antibody (5 ⁇ g/mL) was incubated with the cells at 4 ° C for 1 hour, or at 37 ° C for 4 hours, and placed under a laser confocal microscope.
  • Figure 26 is a graph showing the anti-tumor activity of the CD73 humanized antibody in vivo.
  • the in vivo test used CD73-highly expressed U87MG glioma cells mixed with 50 ⁇ g of antibody and inoculated subcutaneously into the back of nude mice, observed 2 to 3 times a week, and measured tumor volume and mouse body weight.
  • Figure 27 is a graph showing the anti-tumor activity of the CD73 humanized antibody in vivo. In vivo experiments were performed by mixing U87MG glioma cells with 50 ⁇ g of antibody, inoculated into the back of nude mice, and observed 2 to 3 times a week to measure tumor volume and mouse body weight.
  • Figure 28 is a graph showing the anti-tumor activity of the CD73 humanized antibody in vivo.
  • the in vivo test used CD73-highly expressed NCI-H1299 non-small cell lung cancer cells mixed with 50 ⁇ g of antibody and inoculated subcutaneously into the back of nude mice, observed 2 to 3 times a week, and measured tumor volume and mouse body weight.
  • Figure 29 is a graph showing the anti-tumor activity of the CD73 humanized antibody in vivo. In vivo experiments were performed by mixing NCI-H1299 non-small cell lung cancer cells with 50 ⁇ g of antibody, inoculated into the back of nude mice, and observed 2 to 3 times a week, and the tumor volume and mouse body weight were measured.
  • Figure 30 shows the expression of CD73 protein in high-invasive, high-metastasis basal breast cancer and luminal breast cancer cell lines by Western blot.
  • Figure 31 is a graph showing the expression level of CD73 mRNA in the Cancer Cell Line Encyclopedia (CCLE) database in a highly invasive, high metastatic Basal-type versus Luminal-type breast cancer cell line.
  • CCLE Cancer Cell Line Encyclopedia
  • Figure 32 shows the expression of CD73 protein in different lung cancer cell lines by Western blot.
  • Figure 33 is a graph showing the expression levels of CD73 mRNA in the Cancer Cell Line Encyclopedia (CCLE) database in non-small cell lung cancer (NSCLC) versus small cell lung cancer (SCLC) cell lines.
  • CCLE Cancer Cell Line Encyclopedia
  • Figure 34 shows that the humanized antibody Hu001c-14 can effectively reverse the inhibitory effect of adenosine monophosphate (AMP) on proliferation of human T lymphocytes.
  • the test was performed by sorting to obtain CD3+ human T cells, and the cell proliferation rate was counted after 5 days of culture.
  • AMP adenosine monophosphate
  • Figure 35 shows that the humanized antibody Hu002c-3 can effectively reverse the inhibitory effect of AMP on proliferation of human T lymphocytes.
  • the test was performed by sorting to obtain CD3+ human T cells, and the cell proliferation rate was counted after 5 days of culture.
  • Figure 36 shows that the humanized antibody Hu001c-14 can effectively reverse the inhibitory effect of AMP on the expression of INF- ⁇ in human T lymphocytes.
  • CD3+ human T cells were obtained by sorting, and the T cell culture supernatant was detected after 5 days of culture.
  • Figure 37 shows that the humanized antibody Hu002c-3 can effectively reverse the inhibitory effect of AMP on the expression of INF- ⁇ in human T lymphocytes.
  • CD3+ human T cells were obtained by sorting, and the T cell culture supernatant was detected after 5 days of culture.
  • Figure 38 shows the detection of breast cancer MDA-MB-453, HCC1937, MDA-MB-231, lung cancer NCI-H460, NCI-H292, NCI-H441, Calu-6, NCI-H1299 by flow cytometry (FACS).
  • Calu-1 pancreatic cancer SW1990, expression level (MFI) of CD73 receptor on the surface of glioma U87MG cells.
  • MFI expression level
  • Figure 39 shows that binding of the humanized CD73 antibody Hu001c-14, Hu001c-15 to MDA-MB-231 cells results in internalization to intracellular lysosomes.
  • the antibody (5 ⁇ g/mL) was incubated with the cells at 4 ° C for 1 hour, or at 37 ° C for 4 hours, and placed under a laser confocal microscope.
  • Figure 40 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate Hu001c14-vcMMAE.
  • Figure 41 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate Hu001c14-BL20-MMAE.
  • Figure 42 is a mass spectrum of monoclonal antibody Hu001c-14.
  • Figure 43 is a mass spectrum of the antibody drug conjugate Hu001c14-vcMMAE.
  • Figure 44 is a mass spectrum of the antibody drug conjugate Hu001c14-BL20-MMAE.
  • Figure 45 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate Hu001c15-vcMMAE.
  • Figure 46 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate Hu001c15-BL20-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 47 is a mass spectrum of monoclonal antibody Hu001c-15.
  • Figure 48 is a mass spectrum of the antibody drug conjugate Hu001c15-vcMMAE.
  • Figure 49 is a mass spectrum of the antibody drug conjugate Hu001c15-BL20-MMAE.
  • Figure 50 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate on breast cancer cell MDA-MB-453.
  • Figure 51 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate on lung cancer cell Calu-1.
  • Figure 52 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate against glioma cell line U87MG.
  • Figure 53 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate on lung cancer cell Calu-6.
  • Figure 54 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate on lung cancer cell NCI-H441.
  • Fig. 55 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate on lung cancer cell NCI-H292.
  • Figure 56 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate against triple negative breast cancer cell MDA-MB-231.
  • Fig. 57 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate against lung cancer cell PC9.
  • Figure 58 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate against lung cancer cell line HCC827.
  • Fig. 59 shows the results of detection of proliferation inhibitory activity of CD73 antibody-drug conjugate against lung cancer cell NCI-H1975.
  • Figure 60 shows cytotoxic activity of CD73- drug conjugate (IC 50 value) is directly related to the level of CD73 expression in a test cell, the performance of target-specific cytotoxicity.
  • Figure 61 shows the growth of human T lymphocytes (obtained by CD3 positive sorting) after treatment with CD73 antibody, antibody-drug conjugate (both 10 nM), or AMP (0.3 mM). The number of viable cells was read by FACS at the selected time point, and a growth curve was drawn.
  • Figure 62 is a graph showing the inhibition of a fixed volume of human T lymphocytes after incubation with different concentrations of CD73 antibody-drug conjugate for 5 days, at a proliferation rate (relative to vehicle/buffer).
  • Figure 64 is an anti-tumor activity of a CD73 antibody-drug conjugate in vivo.
  • Figure 65 is an anti-tumor activity of a CD73 antibody-drug conjugate in vivo.
  • Figure 66 is an anti-tumor activity of a CD73 antibody-drug conjugate in vivo.
  • Figure 67 is an anti-tumor activity of a CD73 antibody-drug conjugate in vivo.
  • Non-small cell lung cancer NCI-H292 cells with high CD73 expression were inoculated subcutaneously in the back of nude mice.
  • FIG 68 is a series of detecting Hu001c inhibitory activity of the humanized antibody for cynomolgus monkey CD73 recombinant enzyme catalytic function (IC 50).
  • FIG 69 is a series of detecting Hu002c humanized antibody activity against recombinant cynomolgus monkey CD73 enzymatic function (IC 50).
  • Figure 70 is a test result of hematology index of Hu001c14-vcMMAE (subject No. FD114-ADC) in a cynomolgus monkey safety test.
  • Figure 71 is a test result of hemagglutination index of Hu001c14-vcMMAE (subject No. FD114-ADC) in a cynomolgus monkey safety test.
  • Figure 72 is a graph showing the results of plasma biochemical indicators of Hu001c14-vcMMAE (subject No. FD114-ADC) in a cynomolgus monkey safety test.
  • the inventors have unexpectedly obtained five anti-CD73 monoclonal antibodies, which are named mAb001 to mAb005, through extensive and intensive research.
  • human-mouse chimeric antibodies mAb001c, mAb002c, mAb004c were constructed by selecting mAb001 (IgG1- ⁇ ), mAb002 (IgG1- ⁇ ), mAb004 (IgG2b- ⁇ ).
  • the antibody was able to bind to the CD73 antigen with high specificity, and the EC 50 was determined by ELISA to be 0.024 nM, 0.016 nM, and 0.038 nM, respectively.
  • the antibody has significant antitumor activity without significant toxic side effects to the mammal itself.
  • humanized antibodies designed based on mAb001c, mAb002c, and corresponding antibody drug conjugates (ADCs) also have excellent properties.
  • the CD73 antibody drug conjugate product obtained by the novel linker of the present invention has the advantage of high homogeneity, further improving stability in vitro and in vivo. The present invention has been completed on this basis.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined 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, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a variable region having a monoclonal antibody from a murine, and a chimeric antibody from a constant region of a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent No. 4,816,397, incorporated herein by reference in its entirety herein.
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety.
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are at most 10, preferably at most 8, more preferably at most 5, most preferably at most as compared with the amino acid sequence of the antibody of the present invention.
  • the three amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention provides three broad classes of highly specific and high affinity antibodies targeting CD73 comprising heavy and light chains comprising a heavy chain variable region (VH) amino acid sequence comprising a light chain Variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the heavy chain variable region (VH) amino acid sequence, the light chain variable region (VL) amino acid sequence comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the following polypeptide sequences:
  • HCDR1 is SEQ ID NO.: 1: NYYIY, SEQ ID NO.: 10: SYWMH, or SEQ ID NO.: 21: DYNMD;
  • HCDR2 is SEQ ID NO.: 2: WIYPGNLNIKYNEKFKG, SEQ ID NO.: 11: EINPSNGRSNYNEKFKS, or SEQ ID NO.: 22: DINPNNGGSVYNQKFKG;
  • HCDR3 is SEQ ID NO.: 3: DDNYAWFAY, SEQ ID NO.: 12: RGVSGNYFDY, or SEQ ID NO.: 23: ITGTGYWSFDV;
  • LCDR1 is SEQ ID NO.: 4: KASQDVSTAVA, SEQ ID NO: 13: KASQDINTYLS, or SEQ ID NO.: 24: RASENIYSNLA;
  • LCDR2 is SEQ ID NO.: 5: WTNTRHT, SEQ ID NO.: 14: RSNILVD, or SEQ ID NO.: 25: GATNLAE;
  • LCDR3 is SEQ ID NO.: 6: QQHYSTPFT; SEQ ID NO.: 15: LQYDEFPYT, or SEQ ID NO.: 26: QHFWGIPWT;
  • a sequence having a CD73 binding affinity by adding, deleting, modifying and/or substituting at least one amino acid sequence of any one of the above amino acid sequences.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.
  • the antibody has an inhibitory function on the cell surface and a recombinant CD73 protease which is rapidly endocytosed into the lysosome by the cell.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, preferably a humanized antibody, and more preferably a fully humanized antibody.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • 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 a modified antibody that targets human CD73.
  • VH CDR1, CDR2, CDR3 are each independently selected from the group consisting of SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or selected from the group consisting of SEQ ID NO .:10, SEQ ID NO.: 11, SEQ ID NO.: 12, or a sequence selected from any one or more of SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, Or they are added, deleted, modified and/or substituted for at least one amino acid sequence having CD73 binding affinity;
  • VL CDR1, CDR2, CDR3 are each independently selected from SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, or selected from SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, or selected from SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO. Any one or more of the sequences of 26, or a sequence having a CD73 binding affinity for addition, deletion, modification and/or
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably It is 1-2.
  • the antibody is the original murine antibody mAb001, mAb002, mAb003, mAb004, mAb005.
  • the antibody is human-mouse chimeric antibody mAb001c, mAb001c-VK-SGS, mAb002c, mAb002c-VH-QG, mAb002c-VH-NA, mAb002c-VK-SG, mAb002c-VH-QG /VK-SG, mAb004c, mAb004c-VH-QG, mAb004c-VH-NA.
  • the antibodies are humanized antibodies Hu001c-14, Hu001c-15, Hu001c-21, Hu001c-22, Hu001c-23, Hu001c-24, Hu001c-25, Hu001c-28, Hu001c-30 , Hu001c-31, Hu001c-32.
  • the antibodies are humanized antibodies Hu002c-2, Hu002c-3, Hu002c-4, Hu002c-6, Hu002c-7, Hu002c-8, Hu002c-10, Hu002c-11, Hu002c-12 Hu002c-14, Hu002c-15, Hu002c-16.
  • amino acid sequence numbers of the heavy and light chain variable regions (VH/VL) of the chimeric antibody are listed in Table 1.
  • amino acid sequence numbers of the heavy and light chain variable regions (VH/VL) of the humanized antibody are set forth in Table 2.
  • the three broad classes of antibodies of the present invention can be used in combination for the construction of CAR constructs, recombinant immune cells comprising CAR constructs, antibody drug conjugates, and the like, and can also be used for (a) preparation of detection reagents, detection plates or a kit; and/or (b) a medicament for the prevention and/or treatment of a CD73-related disease.
  • Serial number Sequence name Serial number Sequence name SEQ ID NO.: 1 mAb001 HCDR1 SEQ ID NO.: 27 mAb004-VH SEQ ID NO.: 2 mAb001 HCDR2 SEQ ID NO.: 28 mAb004-VH-QG SEQ ID NO.: 3 mAb001 HCDR3 SEQ ID NO.: 29 mAb004-VH-NA SEQ ID NO.: 4 mAb001 LCDR1 SEQ ID NO.: 30 mAb004-VL SEQ ID NO.: 5 mAb001 LCDR2 SEQ ID NO.: 31 mAb001-VH_HuG.3 SEQ ID NO.: 6 mAb001 LCDR3 SEQ ID NO.: 32 mAb001-VH_HuG.5 SEQ ID NO.: 7 mAb001-VH SEQ ID NO.: 33 mAb001-VH_HuG.6 SEQ ID NO.: 8 mAb001-VL SEQ ID NO.: 34
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • 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 antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, 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.
  • ADC Antibody-drug conjugate
  • the invention also provides an antibody-drug conjugate (ADC) based on an antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-conjugated drug comprises the antibody, and an effector molecule, which is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a therapeutically active drug.
  • the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or a combination of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between an effector molecule and an antibody, such as glutaraldehyde or the like.
  • the coupling agent using a carboxyl group may be any one or more of an cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazine coupling agent (coupling site is an acylhydrazine).
  • Certain residues on the antibody are used to link to a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes). , stabilizers (such as ethylene glycol polymers) and therapeutic agents.
  • imaging agents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as ethylene glycol polymers
  • therapeutic agents such as ethylene glycol polymers
  • the antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly via a linker.
  • Typical coupling methods suitable for use in the present invention include both K-Lock and C-Lock coupling methods.
  • K lysine
  • C cysteine in the antibody sequence
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC comprises a linker between the drug and the antibody.
  • the linker can be a degradable or non-degradable linker.
  • Degradable linkers are typically susceptible to degradation under the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as barium splices) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
  • the linker Prior to attachment to an antibody, the linker has an reactive reactive group capable of reacting with certain amino acid residues, and attachment is achieved by reactive reactive groups.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (eg, iodine, bromine or chlorinated); haloesters (eg, iodine, bromine or chlorinated) Halogenated methyl ketone (eg iodine, bromine or chlorinated), benzyl halide (eg iodine, bromine or chlorinated); vinyl sulfone, pyridyl disulfide; mercury derivative such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker can include, for example, a maleimide attached to the antibody via
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker binds the antibody to the drug, and the drug has a functional group that can bond to the linker.
  • the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group which may be bonded to a linker.
  • the drug is directly attached to the linker, the drug has a reactive group that is reactive prior to attachment to the antibody.
  • Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , vinca alkaloids, etc.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( Taxanes), benzodiazepines or benzodiazepine containing drugs (eg pyrrolo[1,4]benzodiazepines (PBDs), porphyrin benzodiazepines Classes (indolinobenzodiazepines) and oxazolidinobenzodiazepines and vinca alkaloids.
  • typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), tax
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two or more step process. For example, a cysteine residue is reacted with a reactive moiety of the linker in a first step, and in a subsequent step, a functional group on the linker reacts with the drug to form an ADC.
  • a functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety.
  • a portion based on an azide compound can be used to specifically react with a reactive alkynyl group on a drug moiety.
  • the drug is covalently bound to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • 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 amines) And alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction 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 amines
  • activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols).
  • the invention also provides a method of making an ADC, which can further comprise: binding the antibody to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • subscript p is a value selected from 1-10, preferably from 1 to 8.
  • drug refers broadly to any compound having the desired biological activity and having reactive functional groups to prepare the conjugates of the invention. Desirable biological activities include, diagnosing, curing, ameliorating, treating, and preventing diseases in humans or other animals. Thus, as long as the necessary reactive functional groups are present, the term “drug” refers to compounds including the official National Pharmacopoeia, as well as, for example, the US Official Homeopathic Pharmacopoeia, the official National Formulary, or any of its supplements. Typical drugs are listed in the physician's desk medication reference (PDR) and the US Food and Drug Administration (FDA) Orange Book. It should be understood that as new drugs are continuously discovered and developed, these drugs should also be included in the "drugs" of the conjugated drugs of the present invention.
  • PDR physician's desk medication reference
  • FDA US Food and Drug Administration
  • Drugs that can be used to form the ADC of the invention include, but are not limited to, cytotoxic agents (e.g., cytotoxic small molecule drugs).
  • cytotoxic agent refers to a substance that inhibits or prevents the expression of cells, the function of cells, and/or the destruction of cells.
  • the term includes radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • cytotoxic agents include, but are not limited to, auristatins (eg, auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, etometanol, ricin, ricin A-chain, Butatin, doxymethine, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide , vincristine, vinblastine, colchicine, dihydroxy anthrax dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, abrin toxin A chain , lotus root toxin A chain, alpha-tripococcus, white toxin, mittollin, retstrictocin, phenolic acid, a
  • Preferred small molecule drugs are compounds having high cytotoxicity, preferably monomethyl auristatin, calicheamicin, maytansinoids, or combinations thereof; more preferably selected from: monomethyl ali Statin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.
  • MMAE monomethyl ali Statin-E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • the drug refers to: a cytotoxic drug for cancer treatment, or a protein or polypeptide having a desired biological activity, such as a toxin such as acacia toxin, ricin A, and pseudomonas Toxins, and diphtheria toxins; other suitable proteins include tumor necrosis factor, alpha-interferon, beta-interferon, neurogenic growth factor, platelet-derived growth factor, tissue-type fibrinolytic growth factor, and biological response modulation agents, For example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF) , granulocyte colony-stimulating factor, or other growth factors.
  • a cytotoxic drug for cancer treatment or a protein or polypeptide having a desired biological activity
  • a toxin such as acacia toxin, ricin A, and pseudomonas Toxins, and
  • a preferred agent of the invention is maytansine or maytansinoid.
  • Maytansin compounds inhibit cell proliferation by inhibiting microtubule formation by tubulin.
  • Maytansin is a derivative of maytansine. Both maytansine and maytansinoids are highly cytotoxic, but they have significant limitations in the clinical application of cancer therapy, mainly due to the low selectivity of such molecules for tumors. However, this high cytotoxicity has made them the drug of choice for antibody drug conjugates.
  • the structure of deacetylmaytansine is listed below.
  • the auristatin peptide drug is an analog of Dolastatin 10, which is a biologically active polypeptide isolated from marine mollusk sea rabbits. Dolon toxin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine).
  • the rabbit toxin 10, the auristatin peptide PE, and the auristatin peptide E are all linear polypeptides containing four amino acids (three of which are unique to the sea rabbit toxin compound) and a C-terminal amide group.
  • Two representative autin peptide compounds, monomethyl auratin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF) are the preferred drugs for antibody drug conjugates.
  • MMAE Monomethyl Auristatin E
  • MMAF Monomethyl Auristatin F
  • MMAD Monomethyl Dolastatin 10
  • PBD pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD dimers PBD dimers
  • PBD is a natural product produced by Streptomyces, and its unique feature is the ability to form non-twisted covalent additions in the DNA minor groove, specifically at the ⁇ -guanine- ⁇ sequence.
  • the use of PBD as a partial small molecule strategy to target locked DNA sequences and as a new type of anticancer and antibacterial drugs has attracted increasing interest.
  • a flexible carbon chain is used to link the C8/C8' hydroxyl groups of the two PBD units, and the resulting dimer has enhanced biological activity.
  • PBD dimers are thought to be DNA damage that can produce sequence selectivity, such as reversed 5'-Pu-GATC-Py-3' cross-linking, resulting in their biological activity. These compounds have proven to be highly potent cytotoxic drugs and can be used as an alternative to antibody drug conjugates.
  • Another preferred drug of the invention is a derivative of PNU-159682, which is the major active metabolite of Nemorubicin in human liver microsomes, with a 3000-fold increase in activity compared to MMDX and doxorubicin.
  • the drug is not limited to the above-mentioned categories, but also includes all drugs that can be used for antibody drug conjugates. And especially those which are capable of coordination by an amide bond to a linker, such as by a cytotoxin having a basic amine group (primary amine or secondary amine), such as the cytotoxin D1-D14 shown above. structure.
  • a linker such as by a cytotoxin having a basic amine group (primary amine or secondary amine), such as the cytotoxin D1-D14 shown above. structure.
  • the present invention relates to antibody-drug conjugates, and more particularly to CD73 antibody-drug conjugates having therapeutic applications.
  • the anti-CD73 antibody can be conjugated to a chemotherapeutic or small molecule toxin via a linker.
  • the invention also relates to methods of treating mammalian cells or related pathological conditions using an anti-CD73 antibody-drug conjugate.
  • the present invention employs a novel class of disubstituted maleimide linkers for targeting CD73 antibodies for coupling, which can be fully/partially cross-coupled to the light chain-heavy chain and heavy chain-heavy chain of the antibody.
  • Targeted CD73 antibody drug conjugates obtained by sulfur-reduced cysteine sulfhydryl groups and using such a coupling method have a narrower drug/antibody ratio (DAR) than conventional antibody drug conjugates distributed.
  • DAR drug/antibody ratio
  • Ar' is selected from the group consisting of substituted or unsubstituted C6-C10 arylene, substituted or unsubstituted 5-12 membered heteroarylene;
  • L 1 is -O(CH 2 CH 2 O) n - attached to the Ar' group, wherein n is selected from any of 1-20.
  • L 2 is a chemical bond, or an AA-PAB structure; wherein AA is a polypeptide fragment consisting of 2-4 amino acids, and PAB is p-aminobenzylcarbamoyl;
  • CTD is a cytotoxic small molecule drug that is bonded to L 2 via an amide bond.
  • Ab is an antibody that targets CD73.
  • the present invention provides a coupling method for coupling a small toxin molecule to a targeted CD73 antibody via a specific linker, which substantially increases the lethality of the antibody against tumor cells without altering the affinity of the antibody.
  • the invention provides a linker or coupling reagent comprising a diarylthiomaleimide unit and a coupling group.
  • the diarylthiomaleimide unit is used to crosslink the sulfhydryl group between the antibody chains (after reduction), while the coupling group is used to couple with the small molecule drug or drug-linker unit.
  • These ADCs are homogeneous due to the bidentate binding of the diarylthiomaleimide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody. It is more stable than an ADC with a single-toothed joint. Thus they will have a increased in vivo half-life, reduce the amount of systemically released cytotoxin, and be safer than the ADC with a single-toothed linker.
  • the produced drug-linker unit is coupled to the antibody via the linker to form a partial interchain cross-linking conjugate.
  • the antibody/antibody ratio (DAR) distribution of the antibody drug conjugate prepared by the method of the present invention is narrower than that of the conventional antibody drug conjugate, thereby greatly improving product uniformity and pharmacological property uniformity.
  • the antibody drug conjugate can be used to target delivery of a drug to a target cell population, such as a tumor cell.
  • the antibody drug conjugate can specifically bind to the cell surface protein, and the resulting conjugate is then endocytosed by the cell. Within the cell, the drug is released as an active drug to produce efficacy.
  • Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that bind to an antigen; or antibody Fc fusion proteins; or proteins.
  • a "drug” is a highly active drug (see definitions), and in some cases the drug may be polyethylene glycol.
  • the coupled product provided by the present invention although still a mixture, has a narrow DAR distribution range compared to the antibody drug conjugate obtained by conventional coupling.
  • the average DAR value is close to 4, which is close to the range of optimal DAR values (2-4) for optimal antibody drug conjugates.
  • the linker-drug conjugate comprises a substituted maleimide linker-drug conjugate of the formula Ic or a pharmaceutically acceptable salt or solvate thereof;
  • R is X or ArS-
  • X is selected from the group consisting of halogen, preferably bromine or iodine;
  • Ar' is selected from the group consisting of substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted C6-C10 arylene, substituted or unsubstituted 5- 12-membered heteroarylene;
  • L 1 is -O(CH 2 CH 2 O) n - attached to the Ar' group, wherein n is selected from any integer from 1 to 20, preferably any integer from 1 to 10;
  • L 2 is a chemical bond or an AA-PAB structure; wherein AA is a dipeptide or a tripeptide or a tetrapeptide fragment (ie, a fragment formed by ligating a 2-4 amino acid through a peptide bond), and PAB is a p-aminobenzylcarbamoyl group;
  • CTD is a cytotoxic small molecule drug that binds to L 2 via an amide bond and/or a drug that treats autoimmune diseases and anti-inflammatory.
  • the compound of formula Ic is selected from the group consisting of
  • Intermediate A is obtained by reaction of n-glycol with tert-butyl bromoacetate, followed by aromatic nucleophilic substitution with substituted nitrofluorobenzene to give intermediate B.
  • intermediate B can also be obtained by reacting p-toluenesulfonate protected intermediate F with a substituted nitrofluorophenol.
  • the nitro group in the intermediate B is reduced to an amino group to obtain an intermediate C, which is then cyclized with 2,3-dibromomaleic anhydride to obtain an intermediate D, which is then subjected to a substitution reaction with an arylthiophenol to obtain a linker fragment.
  • Molecular E A series of molecules F can be obtained by condensation with a dipeptide/tripeptide-PAB cytotoxic drug linker. The reaction route is as follows:
  • Triethylene glycol (92 g, 613 mmol) was dissolved in tBuOH (200 mL).
  • tBuOH a solution of t-butyl bromoacetate (39.8 g, 204 mmol.
  • TLC detected the end of the reaction.
  • dichloromethane 400 ml
  • the organic phase was washed with 400 ml of water, and the aqueous phase was extracted once with 300 ml of dichloromethane.
  • the organic phase was combined and washed once with saturated brine and dried over anhydrous sodium sulfate Steamed and dried.
  • the crude product was purified by EtOAc EtOAc EtOAc (EtOAc)
  • reaction system was extracted with 100 ml of dichloromethane, washed once with 200 ml of 1N diluted hydrochloric acid, twice with 200 ml of water, once with 200 ml of saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness.
  • the preparation route of the antibody drug conjugate is as follows.
  • the interchain disulfide bond of the antibody is reduced to yield 2n (e.g., 8) sulfhydryl groups.
  • the substituted maleimide linker-drug conjugate of the present invention (Formula Ic) is cross-linked with the reduced antibody thiol to form the corresponding antibody drug conjugate, wherein the antibody drug conjugate is present as follows One or two forms.
  • a typical preparation method comprises: diluting the antibody stock solution to 2-10 mg/mL with a reaction buffer, adding a 140-200 times excess molar ratio of dithiothreitol (DTT), or adding a 6.0-20 fold excess molar ratio.
  • DTT dithiothreitol
  • Tris(2-carboxyethyl)phosphine hydrochloride the reaction solution is stirred at 10-35 ° C for 2-48 hours;
  • the reaction buffer may be a buffer prepared in the following ratio: 50 mM phosphoric acid Potassium hydrogen-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate - citric acid / 150 mM Sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM boric acid - borax / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide/150 m
  • reaction solution is cooled to 0-10 ° C. If DTT reduction is used, excess DTT is removed by desalting column or ultrafiltration after completion of the reduction reaction, and then substituted maleimide compound (previously dissolved in 10 mg/ml) is added. Acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA), and ensure that the volume of organic solvent in the reaction solution does not exceed 15%, even The reaction was stirred at 0-37 ° C for 2-4 hours. If TCEP reduction is used, it is also possible to directly add a substituted maleimide compound for coupling without removing the remaining TCEP.
  • ACN acetonitrile
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • DMA diethyl acetamide
  • the coupling reaction mixture was purified by filtration using a sodium succinate/NaCl buffer or a histidine-acetic acid/sucrose gel using a desalting column, and a peak sample was collected based on the UV280 ultraviolet absorption value. Or ultrafiltration several times.
  • the bacteria were then sterilized by filtration and the resulting product was stored at a low temperature.
  • the temperature is from -100 to 60 ° C, and the pore size of the filtration device is preferably from 0.15 to 0.3 ⁇ m.
  • the drug antibody coupling ratio (DAR) of the obtained antibody drug conjugate was relatively uniform.
  • DAR drug antibody coupling ratio
  • the ADC product homogeneity is very high (typically DAR dominant products (such as DAR is about 4) account for at least 60%, at least 70%, of at least 70% of all ADCs. 80%, at least 90% or higher).
  • DAR hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • IEC ion exchange Chromatography
  • the antibodies of the invention or their ADCs can be used in detection applications, for example for detecting samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples, and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may include, for example, a resected sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
  • the antibody of the invention may be immobilized on a test plate.
  • the invention also provides the use of an antibody of the invention, for example for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a CD73-related disease.
  • the CD73-related diseases include tumorigenesis, growth and/or metastasis, tumor resistance-related diseases, inflammation, metabolism-related diseases, and the like.
  • the tumor includes, but is 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, and knot.
  • 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 esophageal cancer
  • kidney cancer esophageal cancer
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Lytle syndrome, psoriasis arthropathy, infectious arthritis, tuberculous arthritis, viral joints Inflammation, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolic related diseases include, but are not limited to, diabetes, foodborne obesity, and fat inflammation.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used for binding to a CD73 protein molecule, and thus can be used for the prevention and treatment of diseases such as tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also 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 typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibody-drug conjugate provided by the present invention can be targeted to a specific cell population, it binds to a cell surface specific protein (antigen), thereby releasing the drug in an active form by endocytosis or drug infiltration.
  • the antibody-drug conjugate of the present invention can be used for the treatment of a target disease, and the antibody-drug conjugate mentioned above can be administered to a subject (for example, a human) by a suitable route in a therapeutically effective amount.
  • a subject in need of treatment can be a patient at risk or suspected of having a condition associated with the activity or amount of expression of a particular antigen. Such patients can be identified by routine physical examination.
  • delivery can be by conventional methods in the art.
  • it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.
  • the antibody of the present invention has excellent biological activity and specificity, and has high affinity (EC 50 of 0.016 to 0.038 nM by ELISA), and has an inhibitory activity against CD73 enzyme function (the enzyme activity is determined to have an IC 50 of 0.025). ⁇ 0.039nM).
  • CD73 has good binding affinity to tumor cells (FACS has an EC 50 of 0.35-2.5 nM) and inhibits the function of tumor CD73 enzyme (IC 50 value is 0.2 nM-0.6 nM), which can be used as a target CD73. Therapeutic antibodies.
  • the humanized antibody of the present invention has not only activity comparable to that of a murine antibody but also lower immunogenicity.
  • Both the antibody and the ADC of the present invention have significant in vivo antitumor activity, while there are no visible side effects to mammals such as model mice themselves.
  • the antibody of the present invention has a significant proliferation protective effect on human lymphocytes, and can effectively reverse the inhibition of proliferation of T lymphocytes by adenosine monophosphate (AMP) and promote the expression and secretion of INF- ⁇ , and the EC 50 is 0.01. ⁇ 0.08nM.
  • AMP adenosine monophosphate
  • the antibody-drug conjugate (ADC) of the present invention has excellent CD73-dependent antitumor activity, that is, no significant toxic side effects on CD73-normal or low-expressing cells, and high expression of CD73- Tumor cells have extremely high killing activity, and the cell proliferation inhibition assay has an IC 50 of 0.02 nM to 0.05 nM.
  • the antibody-drug conjugate (ADC) of the present invention has no significant toxic side effects on the proliferation of normal human T lymphocytes, and the cell proliferation inhibition assay measures an IC 50 of >100 nM.
  • the antibody-drug conjugate (ADC) of the present invention does not exhibit high or unexpected toxic side effects on mammals such as cynomolgus monkeys, and has potential prospects for clinical drug applications.
  • the novel linker provided by the present invention can be coupled to a targeted CD73 antibody by a simple chemical method, and the DAR value distribution of the CD73 antibody drug conjugate obtained by using the linker is compared with the conventional coupling method. Very narrow, so the product produced is highly uniform, and the obtained single component of the cross-linker (DAR is 4) accounts for more than 80%.
  • the in vitro tumor cell proliferation inhibitory activity of the cross-linker is higher than that of the traditional mcVC-PAB cross-linking organism. The activity and safety of the drug are improved or maintained.
  • the maleimide-based disulfide bridge of the present invention has better stability, and the introduction of a substituent at the Ar' site can mediate the reaction rate of maleimide ring-opening hydrolysis and slow down the maleimide opening.
  • the cyclization secondary hydrolysis reaction after the ring is less prone to thiol ether exchange and ring-opening secondary hydrolysis reaction after ring opening, further enhancing the stability of the CD73 antibody-drug conjugate in vitro and in vivo.
  • Step 1 preparation of hybridoma cells:
  • CD73-ECD The extracellular region of human CD73 protein (CD73-ECD) was first prepared as an antigen.
  • NCBI NCBI: NP_002517.1 amino acid positions 27 to 547, gene-cloning technology and mammalian vector expression system were used to obtain C-terminus polyhistidine-tagged antigens.
  • the specific amino acid sequence is as follows ( SEQ ID NO.: 48):
  • the Balb/c mouse was immunized with the CD73 extracellular domain protein prepared above, and the amount of CD73 extracellular domain protein was 50 ⁇ g/head to prepare immune spleen cells; the mouse myeloma cells (SP2/0) and feeder cells were prepared at appropriate time. Need for integration.
  • the spleen cells and SP2/0 cells are fused by PEG-mediated fusion, PEG is removed, resuspended in HAT complete medium containing feeder cells, inoculated into 96-well plates, and cultured by ELISA/FACS method. Positive well screening was performed. Finally, the cells of the positive wells were cloned and cultured by limiting dilution method, and the cells with high titer, good morphology and monoclonal growth were screened by ELSIA or FASCS, and the subcloning screening was continued until the positive cloning rate was 100 for three consecutive screenings. %, the cell strain can be expanded and constructed.
  • Step 2 Purification of human CD73 murine monoclonal antibody:
  • the cell culture supernatant was collected, filtered through a 0.22 ⁇ m filter, and the obtained culture supernatant was added at a constant rate to a previously equilibrated Protein A resin column.
  • the equilibration column was then eluted with 0.1 M sodium citrate buffer, and the eluate was collected and quantified for SDS-PAGE electrophoresis, SEC-HPLC, and endotoxin detection.
  • the obtained purified antibody was dispensed and stored at -80 ° C until use.
  • Step 3 Determination of the biological activity and specificity of a monoclonal antibody targeting human CD73 murine:
  • the selected five hybridoma monoclonal antibodies were assayed for biological activity and specificity.
  • the supernatant of the monoclonal cell culture solution was detected by flow cytometry (FACS), and all of the five antibodies specifically bind to human CD73-highly expressed MDA-MB-231 cells ( CD73-P), but no significant binding activity to CD73-low expressed MDA-MB-453 cells (CD73-N).
  • FACS flow cytometry
  • CD73-P human CD73-highly expressed MDA-MB-231 cells
  • CD73-N CD73-low expressed MDA-MB-453 cells
  • mAb001, mAb002, mAb003, mAb004, mAb005 had excellent binding affinity to MDA-MB-231 cells, and the EC 50 of FACS was 1.24 nM, 0.65 nM, 10.7 nM, 4.69 nM, 26.07 nM.
  • mAb001, mAb002 and mAb004 were preferentially selected for antibody sequencing.
  • Primers were designed to amplify heavy chain (VH), light chain (VL) variable region fragments (see Figure 2) by conventional PCR techniques, cloned into vectors, and sequenced.
  • the following heavy chain variable region (VH), light chain variable region (VL) amino acid sequence, and complementarity determining region (CDR) information were obtained by routine sequencing and analysis by Kabat database (underlined "_" shows CDR-1/ 2/3 amino acid sequence).
  • variable region sequences Three sets of variable region sequences are identified by genetic recombination techniques (see SEQ ID NO.: 7, SEQ ID NO.: 16, SEQ ID NO.: 27, SEQ ID NO.: 8, SEQ ID NO.: 19, SEQ ID NO.:30) was cloned into a vector containing the human IgG1 heavy chain constant region and Kappa chain constant region, was sequenced correctly, chimeric antibody expression using the transfection and mammalian expression systems (FreeStyle TM 293T cells) will be constructed And purified (see Figure 3), the obtained human-mouse chimeric antibodies were numbered mAb001c, mAb002c, mAb004c, respectively.
  • variable region sequence of the antibody contains several unfavorable amino acids which have been subjected to point mutation modification.
  • the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) after point mutation are listed below ("_" shows the CDR amino acid sequence).
  • the point mutation (PTM) was cloned by the above point mutation template to obtain a corresponding chimeric antibody mutant obtained by point mutation to the hIgG1 vector.
  • the human-mouse chimeric antibodies and the numbering of the antibody mutants, the heavy and light chain numbers of the antibodies are summarized in Table-1.
  • CD73 protein extracellular domain (CD73-ECD) was diluted to 1 ⁇ g/mL with a coating solution, coated with an ELISA plate, 100 ⁇ L/well, 4 ° C, overnight. Wash off the excess antigen, block with 1% BSA for 2h at room temperature, then add 3 times serial dilution of each monoclonal antibody, 100 ⁇ L/well, incubate for 1h at room temperature; wash away unbound antibody, add appropriate concentration of horseradish peroxidase Labeled anti-mouse secondary antibody, 100 ⁇ L/well, incubated for 0.5 h at room temperature.
  • the unbound secondary antibody was washed away, reacted with TMB coloring solution for about 15 min, 1N HCL was added, 50 ⁇ L/well, the color reaction was terminated, and the absorbance was measured at 450 nm, and the data was analyzed.
  • mAb001c, mAb002c, and mAb004c have strong affinity for CD73-ECD with EC 50 of 0.024 nM, 0.016 nM, and 0.038 nM, respectively.
  • the human recombinant CD73 enzyme (CD73 extracellular region) was diluted to 0.1 ⁇ g/mL with an antigen dilution and evenly spread into a 96-well low-adsorption culture plate at 25 ⁇ L/well.
  • 50 ⁇ L of CD73 antibody diluted from 2 nM to 0.0009 nM in a 3-fold gradient was added to the culture plate, mixed (final concentration 1 nM to 0.00045 nM), and after incubation at 37 ° C for 1 h, 25 ⁇ L of a mixture containing 1.2 mM AMP and 0.4 mM ATP was added. The solution was incubated at 37 ° C for 1 h.
  • mAb001c, mAb002c, and mAb004c all significantly inhibited the activity of recombinant CD73 protease to hydrolyze AMP, and the IC 50 thereof were 0.025 nM, 0.031 nM, and 0.039 nM, respectively.
  • the IC 50 of mAb002c-VH-QG/VK-SG was 0.038 nM and 0.06 nM.
  • the cancer cells MDA-MB-453 and non-small cell lung cancer cells NCI-H460 were assayed for binding of chimeric antibodies to cell surface CD73.
  • 3x10 5 tumor cells were mixed with the antibody (final concentration 5 ⁇ g/mL), then incubated at 4 ° C for 1 h, the cells were washed twice with PBS to remove unbound primary antibody, and the target cells were labeled with PE-labeled secondary antibody at 4 ° C. After incubation for 30 min, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200 ⁇ L of PBS, and the binding rate was measured by flow cytometry (FACS).
  • FACS flow cytometry
  • the chimeric antibodies can specifically recognize and bind to CD73-highly expressed tumor cells, and the binding rate fluorescence intensity order is Calu-1, NCI-H1299, U87MG, SW1990, MDA-MB. -231, while the CD73-low expressed tumor cells MDA-MB-453, NCI-H460 showed very weak binding fluorescence intensity. Comparing Calu-1, MDA-MB-453 with antibody binding rate (MFI), the difference in mAb001c binding rate was 250 times, mAb002c was 978 times, and mAb004c was 856 times.
  • MFI antibody binding rate
  • Example 7 The level of CD73 protein on the surface of tumor cells is closely related to its enzyme activity.
  • CD73-high expression (U87MG, Calu-1, NCI-H1299) and CD73-low expression (MDA-MB-453) cell lines were used to study the correlation between cell surface CD73 protein content and enzyme activity.
  • 100 ⁇ L of each cell strain containing the above-mentioned cell lines was released from 20000 to 625 according to a double gradient, uniformly spread into a 96-well cell culture plate, cultured at 37 ° C for 16 hours, and then washed three times with serum-free medium to remove residual serum, slowly. 50 ⁇ L of 300 ⁇ M AMP was added, mixed, and cultured at 37 ° C for 3 h.
  • CD73-lowly expressed MDA-MB-453 cells produced only a very low amount (background level) of enzyme activity, while CD73-highly expressed three cells. Both showed high enzymatic activity, confirming that the level of CD73 protein on the surface of tumor cells is closely related to its enzyme activity.
  • the secondary antibody was incubated at 4 ° C for 30 min, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200 ⁇ L of PBS, and the binding affinity of the test antibody to the corresponding cell surface CD73 was determined by flow cytometry (Binding affinity). ).
  • mAb001c, mAb002c, and mAb004c have excellent binding affinity to MDA-MB-231 with EC 50 of 0.7 nM, 0.36 nM, and 2.5 nM, respectively.
  • mAb001c, mAb002c, and mAb004c have the same excellent binding affinity to NCI-H1299, and the EC 50 is 1.0 nM, 0.39 nM, and 2.2 nM, respectively;
  • the derived mutant of mAb001c/mAb002c also has excellent binding affinity to MDA-MB-231, and the EC 50 value of mAb001c-VK-SGS is 1.39 nM; mAb002c-VH-QG/NA the EC 50 value of 0.43nM; mAb002c-VK-SG EC 50 value of 0.46nM.
  • the monoclonal antibody of the present example is capable of targeting CD73 of human tumor cells.
  • CD73-highly expressed triple negative breast cancer cells MDA-MB-231, non-small cell lung cancer cells NCI-H1299 and Calu-1 were used as target cells. Place the appropriate number of tumor cells (pre-experimentally confirmed) in 96-well plates, and after 16 hours of incubation at 37 ° C, wash the cells 3 times with serum-free RPMI-1640 medium, and dilute 50 ⁇ L from 200 nM to 0.091 nM in a 3-fold gradient.
  • test antibody was added to a 96-well plate, and after incubation at 37 ° C for 30 min, 25 ⁇ L of 0.9 mM AMP was added, and cultured at 37 ° C, 5% CO 2 for 3 h (final antibody concentration of 133.3 nM to 0.06 nM). 25 ⁇ L of the above culture supernatant was taken out and added to another 96-well white plate, and 25 ⁇ L of 0.1 mM ATP was added thereto, followed by mixing. Add 50 ⁇ L of CellTiter-Glo reagent to each well, mix and protect from light for 3 to 5 minutes, and measure the fluorescence signal intensity with a microplate reader.
  • mAb001c, mAb002c and mAb004c significantly inhibited the function of CD73-catalyzed hydrolysis of AMP on the surface of MDA-MB-231 cells with IC 50 of 1.858 nM, 0.791 nM and 4.164 nM, respectively.
  • mAb001c, mAb002c, and mAb004c inhibited the function of CD73 catalyzed hydrolysis of AMP on the surface of NCI-H1299 cells with IC 50 of 0.236 nM, 0.191 nM, and 0.385 nM, respectively.
  • mAb001c, mAb002c, and mAb004c inhibited the function of CD73 catalyzed hydrolysis of AMP on the surface of Calu-1 cells, and the IC 50 was 0.506 nM, 0.281 nM, and 0.630 nM, respectively.
  • the humanized template matching the non-CDR region of mAb001c and mAb002c was selected in the Germline database, and then the CDR region of the antibody was transplanted onto the selected humanized template to 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 residue having an important interaction with the CDR and the CDR region and the residue having a significant influence on the conformation of the VL and VH are subjected to back mutation.
  • variable regions of 7 humanized heavy chains SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ ID NO.: 33, SEQ ID NO.: 34, SEQ) ID NO.: 35, SEQ ID NO.: 45, SEQ ID NO.: 46
  • variable region of the 3 humanized light chain SEQ ID NO.: 36, SEQ ID NO.: 37, SEQ ID NO) .:47.
  • variable regions of four humanized heavy chains SEQ ID NO.: 38, SEQ ID NO.: 39, SEQ ID NO.: 40, SEQ ID NO.: 41
  • the variable region of the 3 humanized light chain SEQ ID NO.: 42, SEQ ID NO.: 43, SEQ ID NO.: 44.
  • the designed humanized variable region sequence was cloned into a vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region by genetic recombination technology, and after sequencing, the transfection technique and the mammalian expression system (FreeStyleTM 293 ) were utilized.
  • the humanized antibody expression vector to be constructed.
  • the humanized heavy and light chains were separately expressed, and finally 11 humanized antibodies were obtained in the mAb001c-series, 12 humanized antibodies were obtained in the mAb002-series, and the corresponding heavy and light chain combinations of each antibody were as follows. -2 is shown.
  • the humanized antibody in Table 2 was serially diluted, and its affinity for CD73 protein was determined by ELISA.
  • the humanized antibody of Table 2 was subjected to gradient dilution, and the effect of the antibody on the activity of the recombinant CD73 enzyme was measured by the method of Example 5.
  • both of the humanized antibodies have a strong inhibitory effect on the CD73 enzyme, and the IC 50 value thereof was 0.02 nM to 0.3 nM.
  • the affinity of the humanized antibody in Table 2 to the surface CD73 of MDA-MB-231 and NCI-H1299 lung cancer cells was determined by flow cytometry.
  • the experimental method is as described in Example 6.
  • the two groups of humanized antibodies have high affinity for CD73 on the surface of MDA-MB-231 cells, and the EC 50 value is 0.2 nM to 0.8 nM.
  • the two groups of humanized antibodies have high affinity for CD73 on the surface of NCI-H1299 cells, and the EC 50 value is from 0.3 nM to 1.4 nM.
  • the two groups of humanized antibodies have high inhibitory activity against the CD73 enzyme on the surface of NCI-H1299 cells, and the IC 50 value is 0.2 nM to 0.6 nM.
  • the 50% density MDA-MB-231 cells were plated in a laser confocal culture dish, and cultured at 37 ° C for 16 h, then 5 ⁇ g / mL CD73 antibody was added, and the cells were incubated at 37 ° C for 4 h or 4 ° C for 1 h, and washed with PBS three times to remove.
  • the antibody that did not bind to the cells was fixed with 4% paraformaldehyde for 30 min at room temperature.
  • the cells were washed three times with PBS and permeabilized with 0.4% Triton X-100 for 10 min.
  • Lamp-2 (rabbit anti-human) antibody was incubated for 1 h at 37 ° C to label the position of the cell lysosome. Unbound antibody was washed away with PBS, and R-PE-labeled goat anti-human and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody were incubated for 30 min at 37 °C. The unbound secondary antibody was washed away, stained with DAPI for 10 min to label the nuclear position, and then the antibody endocytosis of the antibody was observed by laser confocal microscopy (20 ⁇ ).
  • mAb001c, mAb002c, and mAb004c were rapidly and largely engulfed into lysosomes by MDA-MB-231 cells.
  • ADC antibody-drug conjugate
  • Immunodeficient nude mice (Balb/c, nude) were randomly divided into several groups, and 100 ⁇ L of cell suspension containing 5 ⁇ 10 6 U87MG, or 9 ⁇ 10 6 NCI-H1299 was mixed with 100 ⁇ L of the indicated humanized antibody (final concentration)
  • 100 ⁇ L of the indicated humanized antibody (final concentration)
  • a negative control with hIgG1 as a subtype match was used.
  • the inhibitory effect of the antibody on the growth of subcutaneous tumors was observed.
  • the body weight and tumor size of nude mice were measured 2-3 times a week, and the tumor growth curve was drawn to evaluate the activity.
  • both the humanized antibody Hu001c-14 and the humanized antibody Hu002c-3 to 8 significantly inhibited the growth of U87MG tumor in nude mice.
  • both the humanized antibody Hu001c-14 and the humanized antibody Hu001c-24 to 32 significantly inhibited the growth of U87MG tumor in nude mice.
  • both the humanized antibodies Hu001c-14 to 15 and the humanized antibodies Hu001c-23 to 32 significantly inhibited the growth of NCI-H1299 tumors in nude mice.
  • Example 17 CD73 is highly abnormally activated in triple negative mammary glands
  • the total cell protein was prepared for a variety of different molecular typing breast cell lines. After accurate quantification, the expression level of CD73 protein was detected by Western blot.
  • CD73 protein is highly aberrantly activated in some high-invasive, high-metastasis basal breast cancer (Basal-type, clinically mostly triple-negative breast cancer) cell lines, but in relative malignancy Low or weak expression of Luminal-type (mostly clinically expressed as hormone receptor-positive breast cancer) cell lines.
  • CD73 mRNA expression level of the breast cancer cell line in the Cancer Cell Line Encyclopedia (CCLE) database was analyzed. The results are shown in Figure 31.
  • the expression level of CD73 mRNA in the highly invasive and highly metastatic Basal-type breast cancer cell lines was generally higher than that in the Luminal-type breast cancer cell line and was statistically significant. Therefore, the antibody targeting CD73 of the present invention will have a more remarkable effect in the diagnosis, prevention and treatment of triple negative breast cancer.
  • Example 18 CD73 is highly aberrantly activated in lung cancer
  • the total protein was prepared from a variety of lung cancer cell lines with different tissue sources and different molecular types. After accurate quantification, the expression level of CD73 protein was detected by Western blot.
  • CD73 protein was abnormally activated and expressed in a plurality of non-small cell lung cancer (NSCLC) cell lines.
  • NSCLC non-small cell lung cancer
  • the CD73 mRNA level of the lung cancer cell line in the CCLE database was analyzed.
  • the expression level of CD73 mRNA in the non-small cell lung cancer (NSCLC) cell line was significantly higher than that of small cell lung cancer (SCLC), suggesting that the antibody targeting CD73 of the present invention is useful for diagnosing, preventing and treating non-small cells.
  • the application of lung cancer (NSCLC) has a more significant effect.
  • PBMCs were first cultured for 3-4 days in medium containing 500 ng/m LCD3/CD28 antibody and 100 IU/mL IL-2, followed by sorting kit (Stemcell, Cat#1795) CD3 positive T lymphocytes were obtained after sorting PBMC.
  • CFSE carboxyfluorescein succinimidyl ester
  • adenosine monophosphate (AMP, final concentration of 0.2 mM)
  • mix 50 ⁇ L of adenosine monophosphate (AMP, final concentration of 0.2 mM)
  • AMP adenosine monophosphate
  • FACS flow cytometry
  • Detection of T cell IFN- ⁇ Uptake 50 ⁇ L/well of T cell culture supernatant for detection of IFN- ⁇ protein concentration, using ELISA kit (Lianke Biotechnology Co., Ltd., Cat# EK180HS-48), and refer to the kit The technical steps provided.
  • the results are shown in Fig. 34 and Fig. 35.
  • the humanized CD73 antibodies Hu001c-14 and Hu002c-3 have significant proliferative protective effects on human T lymphocytes, which can effectively reverse the proliferation inhibition of AMP on T cells, and the EC 50 is 0.08. ⁇ 0.01 nM and 0.01 ⁇ 0.001 nM.
  • the results are shown in Fig. 36 and Fig. 37.
  • the humanized CD73 antibodies Hu001c-14 and Hu002c-3 can effectively reverse the inhibitory effect of AMP on T cell expression/secretion INF- ⁇ .
  • the heavy and light chain variable region sequences (VH/VL) of the MEDI9447 antibody disclosed in US20160194407 were artificially synthesized into their heavy and light chain variable regions, and cloned into a vector containing the human IgG1 heavy chain constant region, respectively, containing Kappa.
  • experimental chain constant region or vector containing Lambda chain constant region was sequenced correctly obtained MEDI9447- ⁇ (consistent with the present invention, the CD73 antibody) or MEDI9447- ⁇ after 293T cells system expression and purification of FreeStyle TM, respectively, the preparation of antibodies
  • the conditions are the same as those of the third embodiment and the tenth embodiment.
  • VH Heavy chain variable region
  • VL Light chain variable region
  • the prepared MEDI9447- ⁇ antibody was used for in vivo anti-tumor activity test.
  • CD73-highly expressed U87MG glioma was selected as an in vivo tumor model.
  • the antibody was mixed with 5 ⁇ 10 6 cells and inoculated into the back of nude mice (50 ⁇ g).
  • Antibody/tumor), tumor growth and body weight changes were observed for 31 days.
  • Figure 26 shows the tumor growth curves of each group in vivo efficacy experiments, and the antitumor activities on day 31 are summarized in Table-4.
  • Figure 27 shows the tumor growth curves of another group of humanized antibodies in U87MG glioma in vivo experiments, and the antitumor activities on day 33 are summarized in Table-5.
  • the CD73 antibody of the present invention has a high affinity, and the Hu002c-series and Hu001c-series humanized antibodies of the present invention have good or better anti-tumor activity in vitro and/or in vivo as compared with the prior art. .
  • 1x10 5 tumor cells were mixed with antibody mAb001c (final concentration 10 ⁇ g/mL), then incubated at 4 ° C for 1 h, the cells were washed twice with PBS to remove unbound primary antibody, and the target cells were labeled with PE-labeled secondary antibody 4 Incubate for 30 min at °C, wash the cells twice with PBS to remove unbound secondary antibody, and finally resuspend the cells in 200 ⁇ L of PBS and measure the binding rate by flow cytometry (FACS).
  • FACS flow cytometry
  • mAb001c specifically recognizes and binds to CD73-highly expressed tumor cells, and the binding rate of fluorescence intensity is Calu-1, NCI-H1299, U87MG, Calu-6, NCI-H441, NCI-. H292, SW1990, MDA-MB-231 showed very weak binding fluorescence intensity to CD73-low expressed tumor cells MDA-MB-453 and NCI-H460.
  • the 50% density MDA-MB-231 cells were plated in a laser confocal culture dish, and cultured at 37 ° C for 16 h, then 5 ⁇ g / mL CD73 antibody was added, and the cells were incubated at 37 ° C for 4 h or 4 ° C for 1 h, and washed with PBS three times to remove.
  • the antibody that did not bind to the cells was fixed with 4% paraformaldehyde for 30 min at room temperature.
  • the cells were washed three times with PBS and permeabilized with 0.4% Triton X-100 for 10 min.
  • Lamp-2 (rabbit anti-human) antibody was incubated for 1 h at 37 ° C to label the position of the cell lysosome. Unbound antibody was washed away with PBS, and R-PE-labeled goat anti-human and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody were incubated for 30 min at 37 °C. The unbound secondary antibody was washed away, stained with DAPI for 10 min to label the nuclear position, and then the antibody endocytosis of the antibody was observed by laser confocal microscopy (20 ⁇ ).
  • the antibody conjugate was named Hu001c14-vcMMAE.
  • Hu001c14-vcMMAE the molecular weight of the conjugate is consistent with the expected value and the average DAR value is about 4.0.
  • the above reaction solution was cooled to 20 ° C, an appropriate amount of diethyl acetamide (DMA) was added, and a compound 6c-4 (10 mg/ml pre-dissolved in DMA) was added in a 6-fold excess molar ratio to ensure DMA in the reaction system.
  • the volume ratio was not more than 10%, and the coupling was carried out by stirring at 20 ° C for 2.0 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a Tris-hydrochloric acid/sucrose gel of pH 7.5, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized via a 0.22 micron pore size filter device and stored at -80 ° C.
  • the resulting antibody conjugate was designated Hu001c14-BL20-MMAE.
  • the mass spectrum of the humanized antibody Hu001c-14 (Fig. 42) and the HIC and mass spectrum of its antibody conjugate Hu001c14-BL20-MMAE (Fig. 41, Fig. 44) indicate that the antibody Hu001c-14 is formed by coupling reaction.
  • the antibody conjugate Hu001c14-BL20-MMAE, the molecular weight of the conjugate was in agreement with the expected value, and the DAR was about 4.0.
  • the antibody conjugate was named Hu001c15-vcMMAE.
  • the mass spectrum of the antibody Hu001c-15 (Fig. 47) and the HIC and mass spectrum of its antibody conjugate Hu001c15-vcMMAE (Fig. 45, Fig. 48) indicate that the antibody Hu001c-15 forms an antibody conjugate after coupling reaction.
  • Hu001c15-vcMMAE the molecular weight of the conjugate was consistent with the expected value and the average DAR value was approximately 4.0.
  • Hu001c-15 stock solution with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH 2 PO 4 -Na 2 HPO 4 ) / 150 mM sodium chloride (NaCl) / 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0
  • the reaction buffer was adjusted to a concentration of 10 mg/mL, and a 10-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 25 ° C for 4 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the above reaction solution was cooled to 20 ° C, an appropriate amount of diethyl acetamide (DMA) was added, and a compound 6c-4 (10 mg/ml pre-dissolved in DMA) was added in a 6-fold excess molar ratio to ensure DMA in the reaction system.
  • the volume ratio was not more than 10%, and the coupling was carried out by stirring at 20 ° C for 2.0 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a Tris-hydrochloric acid/sucrose gel of pH 7.5, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized via a 0.22 micron pore size filter device and stored at -80 ° C.
  • the resulting antibody conjugate was designated Hu001c15-BL20-MMAE.
  • the mass spectrum of the antibody Hu001c-15 (Fig. 47) and the HIC and mass spectrum of its antibody conjugate Hu001C15-BL20-MMAE (Fig. 46, Fig. 49) indicate that the antibody Hu001c-15 forms an antibody couple after coupling reaction.
  • the conjugate conjugated Hu001c15-BL20-MMAE, the molecular weight of the conjugate was consistent with the expected value, and the DAR was about 4.0.
  • the cell lines used in this example were purchased from the American Type Culture Collection (ATCC) or the Chinese Academy of Sciences Cell Bank and cultured according to the corresponding instructions, including: MDA-MB-453, Calu-1, U87MG, Calu-6, NCI-H441, NCI-H292, MDA-MB-231, PC9, HCC827, NCI-H1975.
  • the cells in the logarithmic growth phase were inoculated into 96-well cell culture plates at a density of 800-2500 cells per well (depending on the growth rate of different cells), 150 ⁇ L/well, 37 ° C, 5% CO 2 .
  • CD73-ADCs had no significant inhibitory effect on the proliferation of CD73-expressing cells MDA-MB-453, while Calu-1 (Fig. 51), U87MG (Fig. 52), and Calu-6, which are highly expressed on CD73.
  • Fig. 53 NCI-H441 (Fig. 54), NCI-H292 (Fig. 55), MDA-MB-231 (Fig. 56), PC9 (Fig. 57), HCC827 (Fig. 58), NCI-H1975 (Fig. 59) Both showed strong inhibition of cell proliferation.
  • CD73-ADC cytotoxicity indicates that CD73 expression level is directly related to the cytotoxic activity of CD73- cells with a test drug conjugates, it is determined as a target-specific cytotoxicity of CD73 (FIG. 60 ).
  • Table-6 summarizes the partial inhibition of cell proliferation IC 50 values tested.
  • PBMC Peripheral blood mononuclear cells
  • CFSE-labeled T cells were plated into 96-well plates (5000 cells/well), and vehicle (buffer), CD73 antibody, CD73-ADC, control hIgG1-ADC were added. (all 10nM), or add 0.3mM adenosine monophosphate (AMP), use the flow cytometry (FACS) to read and count the number of viable cells on the 3rd, 6th, 9th and 12th day after culture, and draw the growth curve. . As shown in Figure 61, 10 nM of Hu001c14-BL20-MMAE did not significantly alter the proliferation curve of T cells compared to vehicle and hIgG1-BL20-MMAE. However, consistent with expectations, AMP significantly reduced the proliferation rate of T cells.
  • Hu001c14-BL20-MMAE and Hu001c15-BL20-MMAE showed no significant toxic side effects (IC 50 >100 nM) in the test concentration range.
  • a cell suspension containing 5 ⁇ 10 6 U87MG, NCI-H441, and NCI-H292 was inoculated subcutaneously into the back of immunodeficient mice (Balb/c, nude).
  • hIgG hIgG1-MMAE
  • Docetaxel docetaxel
  • Tumor volume and nude mouse body weight were measured 2-3 times per week and recorded to plot tumor growth curves.
  • Hu001c14-BL20-MMAE showed a dose-related therapeutic effect at administration of 3 mg/kg and 1 mg/kg. And at the same dose of 3mg/kg, Hu001c14-BL20-MMAE showed stronger antitumor activity than Hu001c14-vcMMAE, indicating that the BL20-MMAE linker is superior.
  • Hu001c14-BL20-MMAE and Hu001c15-BL20-MMAE can cause significant tumor regression after administration of 1 mg/kg, and it is still obvious after administration of 0.3 mg/kg.
  • the anti-tumor activity further clarifies the high sensitivity of NCI-H441 tumors to CD73-ADC.
  • a carbon-terminal polyhistidine-tagged recombinant cynomolgus CD73 enzyme was prepared using the cynomolgus cynomolgus CD73/NT5E extracellular domain sequence (EHH53214.1; Met1-Lys547), and the specific amino acid sequence is shown in SEQ ID No.: 48. .
  • the humanized antibody of Table 2 was subjected to gradient dilution, and the effect of the antibody on the activity of the recombinant cynomolgus CD73 enzyme was determined by the method of Example 5.
  • the two groups of humanized antibodies all had the desired inhibitory effects on the cynomolgus CD73 enzyme, and the range of the IC 50 value was roughly equivalent to the inhibitory activity against the human CD73 enzyme.
  • cynomolgus monkeys were given a single dose escalation method with 3 and 6 mg/kg of Hu001c14-vcMMAE, and the animals were well tolerated.
  • the number of erythroid/granulocyte cells was mainly observed after administration.
  • the reduction and increase in fibrinogen can be restored after stopping the drug.
  • the maximum tolerated dose (MTD) is greater than 6 mg/kg.
  • Hu001c14-vcMMAE and Hu001c15-vcMMAE of traditional mcVC-PAB cross-linking technology Hu001c14-BL20-MMAE and Hu001c15-BL20-MMAE prepared by the novel linker of the invention have comparable or higher antitumor activity. (Table-6).
  • Hu001c14-BL20-MMAE and Hu001c15-BL20-MMAE all showed lower non-specific (caused by target shedding) side effects, for example, on CD73 - MDA-MB-453 low expression of the IC 50 value cytotoxic further increased (Fig. 50, table-6).
  • CD73-ADC Based on Hu001c14-vcMMAE intravenous administration of cynomolgus monkeys (3mg/kg, 6mg/kg) preliminary toxicology test, CD73-ADC showed satisfactory and controllable safety, so it has potential prospects for clinical application.

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Abstract

本发明公开了靶向CD73的抗体及抗体-药物偶联物(ADC)、其制备方法和用途。还公开了制备所述的单克隆抗体和ADC的方法。本发明的单克隆抗体以及相应的ADC能够高效、高特异性地结合纯化的CD73蛋白和多种肿瘤细胞表面的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的抗体及抗体-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的抗体及抗体-药物偶联物(ADC)、其制备方法和用途(CD73-targeting antibody and antibody-drug conjugate,preparation method and use thereof)。
背景技术
CD73是一种分子量为70KD的胞外5-核酸酶(NT5E)，被糖磷脂酰肌醇(GPI)锚定于细胞表面。生理状态下，CD73主要表达于多种组织，如大肠，肾脏，肝脏，肺，淋巴结等组织。细胞外释放的ATP/ADP经CD39水解产生的腺苷单磷酸(AMP)或烟酰胺腺嘌呤二核苷酸(NAD+)经一系列代谢产生的AMP，在CD73的催化作用下脱去磷酸基团生成大量的腺苷(Adenosine,ADO)暴露在组织周围，后者通过结合相应的腺苷受体(A1AR、A2AR、A2BR、A3AR)参与细胞多种生理进程。
研究表明，CD73及腺苷通路与肿瘤的发生发展密切相关。首先，其能够促进肿瘤发生免疫逃逸。低氧，炎症因子(IFN-γ、TNF-α、IL-1β、TGF-β等)及相关信号通路(Wnt,cAMP)的激活能够诱导肿瘤细胞异常表达CD73，后者通过催化AMP产生大量的ADO，包围在肿瘤周围，形成抗肿瘤免疫抑制的“环”型微环境，促进肿瘤发生免疫逃逸，其主要机制为：1)ADO作用于CD8+T细胞表面腺苷受体(A2AR)，通过cAMP信号通路抑制其增殖，扩增，并且减少相关促炎细胞因子IFN-γ、TNF-α等的释放，从而降低其细胞毒性作用。2)ADO能够干扰NK细胞与肿瘤细胞之间的粘附，从而降低NK细胞胞吐细胞毒性颗粒的能力，细胞毒性作用减弱。3)ADO通过结合调节性T细胞(Tregs)表面的A2AR促进其扩增，增强其免疫抑制及抗炎功能；同时，Tregs表面的CD73催化产生的ADO结合CD8+效应T细胞A2AR抑制NF-kB的激活，导致促炎细胞因子及趋化因子的分泌减少。临床前研究也表明，将来源于野生型小鼠的CD4+CD25+Tregs注入Tregs缺失的小鼠体内将促进结肠癌的发展，而来源于CD73缺陷小鼠的Tregs不产生任何作用，提示CD73及Tregs对肿瘤产生免疫抑制具有重要作用。4)ADO抑制M1型巨噬细胞的分化，减少促炎细胞因子IL-12、TNF-α、iNOS等的释放，其能够激活M2型巨噬细胞，产生大量的抗炎细胞因子(TGF-β,arginase1)，从而帮助肿瘤产生免疫逃逸。其次，CD73能够促进肿瘤的生长和转移进程。临床前研究结果显示，CD73异常表达于多种肿瘤细胞，如乳腺癌，膀胱癌，卵巢癌，结肠癌，非小细胞肺癌等，且临床数据显示高表达CD73与肿瘤病人的不良预后密切相关[Expert Rev Anticancer Ther.2017；17:527]，提示CD73可作为多种肿瘤临床治疗及预后靶标。体外研究指出CD73能够促进荷有B16F10黑色素瘤细胞的C57BL/6小鼠新生血管的形成，采用CD73阻滞药物能够显著抑制新生血管的数量及血管床的成熟。另外，CD73产生的ADO能够通过上调细胞周期蛋白D1(Cyclin D1)促进微血管内皮细胞的增殖，以及血管内皮生长因子(VEGF)的释放，促进肿瘤血管形成，为肿瘤的生长提供了充足的能量。体内研究表明CD73缺陷的小鼠表现为肿瘤血管的减少，提示靶向CD73有望联合抗血管新生类药物(Bevacizumab)，为临床治疗肿瘤提供新的方案。大量研究表明ADO与肿瘤细胞的转移密切相关。通过分析膀胱癌肿瘤样本发现，与非淋巴结转移的肿瘤样本相比，淋巴结转移样本中CD73表达显著升高。Stagg 等采用CD73单抗[TY/23]处理小鼠能够有效的抑制4T1.2自发性肺转移，另一项结果也证实了CD73单抗AD2通过促进CD73的聚集和内吞，从而阻止了循环肿瘤细胞建立继发性肿瘤灶，极大程度的限制了肿瘤细胞的渗出和定殖于其他组织的能力。同样，临床数据分析表明高表达的CD73与胃癌，膀胱癌，恶性黑色素瘤等转移密切相关，进一步提示CD73对肿瘤转移具有重要作用。
耐药是肿瘤治疗中的一大难题和挑战。研究表明，CD73参与肿瘤化疗，超声治疗，靶向治疗及免疫治疗等的耐药性产生，极大的阻碍了肿瘤治疗的有效性[Discovery Today 2017；22:1686]。研究表明，采用阿霉素(Anthracycline)治疗三阴性乳腺癌(TNBC)病人时，高表达的CD73与病理完全应答率(pCR)密切相关，即低表达CD73的病人呈现出对阿霉素治疗表现出更好的响应，机制研究显示，阿霉素通过上调CD73/CD39表达，抑制CD8+T细胞IFN-γ等分泌，抑制其抗肿瘤免疫作用。采用靶向CD73单抗能够显著增强阿霉素抗肿瘤免疫应答及抗肿瘤活性。此外，在一项曲妥珠单抗(Trastuzumab)治疗Her2+乳腺癌的临床试验中，CD73高表达与不良预后显著相关；靶向CD73单抗联合Trastuzumab可通过增加CD8+T细胞、减少MDSC浸润从而产生协同抗肿瘤作用[Cancer Res.2017；77:5652]。
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也因为其分子量大小合适，稳定性高，靶向性强，毒副作用小被认为是最具潜力的抗肿瘤药物。但成功开发ADC也存在诸多必须考虑且必须解决的问题，如抗体要特异性的识别病变部位，免疫致敏性低，能够高效迅速的发生细胞内吞作用；抗体-药物接头，在血液中稳定性要高并能在靶向细胞中特异的被激活并高效释放小分子药物；所偶联的小分子药物细胞杀伤能力要强等。
综上，CD73异常表达于多种肿瘤细胞，与肿瘤病人的不良预后密切相关。CD73主要通过腺苷通路产生抗肿瘤免疫抑制作用，促进肿瘤的生长，转移及血管新生。此外，CD73还参与抗肿瘤药物耐药性产生，为肿瘤治疗带来极大挑战。因此，开发靶向CD73单抗药物为临床上单独或联合治疗CD73异常表达肿瘤患者提供新的方案。同时，目前临床上尚缺乏高特异性的针对人CD73的抗体-药物偶联物,因此，开发靶向肿瘤CD73的并且药物性能更优异的抗体-药物偶联物，将发挥其具有的特点和优势，为治疗CD73异常表达的癌症提供新的思路和前景。
发明内容
本发明提供了一种靶向人源CD73的抗体，其具有阻断CD73催化腺苷一磷酸(AMP)水解生成腺苷的活性，具有抑制肿瘤生长和转移活性，并可以减少抗肿瘤治疗耐药性的出现。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:10所示的CDR1，
SEQ ID NO.:11所示的CDR2，和
SEQ ID NO.:12所示的CDR3；
或者，
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，
SEQ ID NO.:21的CDR1，
SEQ ID NO.:22所示的CDR2，和
SEQ ID NO.:23所示的CDR3，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述重链可变区包括以下互补决定区：
SEQ ID NO.:10、SEQ ID NO.:11、SEQ ID NO.:12所示mAb002c的重链互补决定区HCDR1、HCDR2、HCDR3；或
SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:3所示mAb001c的重链互补决定区HCDR1、HCDR2、HCDR3；或
SEQ ID NO.:21、SEQ ID NO.:22、SEQ ID NO.:23所示mAb004c的重链互补决定区HCDR1、HCDR2、HCDR3。
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.:7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.:16、SEQ ID NO.:17、SEQ ID NO.:18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.:27、SEQ ID NO.:28、SEQ ID NO.:29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.:31、SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35、SEQ ID NO.:45、SEQ ID NO.:46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.:38、SEQ ID NO.:39、SEQ ID NO.:40、SEQ ID NO.:41所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区包括以下三个互补决定区CDR：
或者，
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，
SEQ ID NO.:4所示的CDR1'，
SEQ ID NO.:5所示的CDR2'，和
SEQ ID NO.:6所示的CDR3'；
或者，
SEQ ID NO.:24所示的CDR1'，
SEQ ID NO.:25所示的CDR2'，和
SEQ ID NO.:26所示的CDR3'；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区包括以下互补决定区：
SEQ ID NO.:13、SEQ ID NO.:14、SEQ ID NO.:15所示mAb002c的轻链互补决定区LCDR1、LCDR2、LCDR3；或
SEQ ID NO.:4、SEQ ID NO.:5、SEQ ID NO.:6所示mAb001c的轻链互补决定区LCDR1、LCDR2、LCDR3；或
SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26所示mAb004c的轻链互补决定区LCDR1、LCDR2、LCDR3。
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.:8、SEQ ID NO.:9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.:19、SEQ ID NO.:20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.:30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.:36、SEQ ID NO.:37、SEQ ID NO.:47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.:42、SEQ ID NO.:43、SEQ ID NO.:44所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有本发明第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)本发明第一方面所述的重链可变区；和/或
(2)本发明第三方面所述的轻链可变区；
或者，所述抗体具有：本发明第二方面所述的重链；和/或本发明第四方面所述的轻链。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
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。
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，所述衍生序列对CD73(如人CD73蛋白胞外区,CD73-ECD)的亲和力EC 50为0.016～0.2nM，较佳地为0.016～0.03nM，更佳地为0.016～0.02nM。
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。
本发明的第六方面，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重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
本发明的第七方面，提供了一种CAR构建物，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于CD73的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
本发明的第八方面，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 明第七方面所述的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)。
本发明的第九方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在另一优选例中，所述抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019077369-appb-000001
其中：
Ab是抗CD73的抗体，
LU是接头(又称连接子)；
D是药物；
而且下标p是选自1-10，较佳地1-8的值。
在另一优选例中，LU选自下组：6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)、6-马来酰亚氨基己酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MC-ala-phe-PAB)、马来酰亚氨基丙酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MP-val-cit-PAB)、马来酰亚氨基丙酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MP-ala-phe-PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫基)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、4-(2-吡啶基二硫代)丁酸-N-羟基琥珀酰亚胺酯(SPDB)或N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)和双取代马来酰亚胺类连接子。
在另一优选例中，LU为双取代马来酰亚胺类连接子。
在另一优选例中，所述的抗体药物偶联物的结构如式Ia、Ib所示：
Figure PCTCN2019077369-appb-000002
Figure PCTCN2019077369-appb-000003
其中，
Ar'选自下组：取代或未取代的C6-C10芳基，取代或未取代的5-12元杂芳基，取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；
L 1为连接于Ar'基团上的-O(CH 2CH 2O) n-，其中n选自1-20中任一整数。
L 2为化学键，或AA-PAB结构；其中，AA为2-4个氨基酸组成的多肽片断，PAB为对-氨基苄基氨甲酰基；
CTD为通过酰胺键键合于L 2的细胞毒类小分子药物。
m为1.0～5.0，优选为3.0-4.2；更优选为3.5-4.5；又更优选为3.8-4.2，又更优选为3.9-4.1，最优选为4.0；
Ab为靶向CD73的抗体。
在另一优选例中，所述的式Ib为式Ia中N-苯基马来酰亚胺开环后产物。
在另一优选例中，所述的偶联物共价连接有一个或多个药物组分。
在另一优选例中，所述的抗体部分与所述的偶联部分通过共价方式(如通过分别共价连接于连接子上)进行偶联。
在另一优选例中，所述的闭环或开环的马来酰亚胺基团连接于抗体铰链区的二硫链还原后的巯基上。
在另一优选例中，所述的抗体-药物偶联物是通过所述抗体或抗体片断铰链区的二硫链还原生成一对半胱氨酸残基，并通过所述半胱氨酸残基中巯基与式Ic表示的取代马来酰亚胺类连接子-药物缀合物中的芳基硫醚发生取代反应，从而获得抗体-药物偶联物Ia和/或Ib。
在另一优选例中，所述的闭环或开环的马来酰亚胺基团连接于完全还原后的抗体上，即铰链区的2对二硫链完全打开，优选m为3.8-4.2，更优选3.9-4.1，最优选4.0。
在另一优选例中，所述Ar'选自下组：苯基、卤代苯、C1-C4烷基苯基、C1-C4烷氧基苯基、2-吡啶基、2-嘧啶基、1-甲基咪唑-2-基、
Figure PCTCN2019077369-appb-000004
其中W为与羰基连接的胺基R 1，R 1选自-NH 2、
Figure PCTCN2019077369-appb-000005
Figure PCTCN2019077369-appb-000006
其中：C1-C4烷基苯基进一步优选为4-甲基苯基；C1-C4烷氧基苯基进一步优选为4-甲氧基苯基。
在另一优选例中，Ar'选自取代或未取代的亚苯基或吡啶基，所述的取代指基团上的氢原子被选自下组的一个或多个取代基所取代：卤素、C1-C4烷基、C1-C4烷氧基、三氟甲基、腈基、酰胺基。
在另一优选例中，所述的AA选自下组：Val-Cit(缬氨酸-瓜氨酸)、Val-Ala(缬氨酸-丙氨酸)、Phe-Lys(苯丙氨酸-赖氨酸)、Ala-Ala-Asn(丙氨酸-丙氨酸-天冬酰胺)、D-Ala-Phe-Lys(D型丙氨酸-苯丙氨酸-赖氨酸)、Gly-Gly-Phe-Gly(甘氨酸-甘氨酸-苯丙氨酸-甘氨酸)、或其组合。
在另一优选例中，其中药物D或CTD选自下组：
(i)美登素衍生物(DM1、DM4)，auristatin和多拉司他汀；
(ii)Monomethyl auristatin E(MMAE)，Monomethyl auristatin F(MMAF)，Monomethyl Dolastatin 10(MMAD)类衍生物或其组合；和
(iii)DNA损伤药物，较佳地，所述的DNA损伤药物包括多卡霉素、吡咯并[2,1-c][1,4]苯二氮卓(PBD)。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述抗体的重链可变区序列选自下组：SEQ ID NO.:7、SEQ ID NO.:16、SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:27、SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:31、SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35、SEQ ID NO.:45、SEQ ID NO.:46、SEQ ID NO.:38、SEQ ID NO.:39、SEQ ID NO.:40、SEQ ID NO.:41；和/或
所述的抗体的轻链可变区序列选自下组：SEQ ID NO.:8、SEQ ID NO.:9、SEQ ID NO.:19、SEQ ID NO.:20、SEQ ID NO.:30、SEQ ID NO.:36、SEQ ID NO.:37、SEQ ID NO.:47、SEQ ID NO.:42、SEQ ID NO.:43、SEQ ID NO.:44。
在另一优选例中，所述嵌合抗体选自下组：mAb001c、mAb001c-VK-SGS、mAb002c、mAb002c-VH-QG、mAb002c-VH-NA、mAb002c-VK-SG、mAb002c-VH-QG/VK-SG、mAb004c、mAb004c-VH-QG、mAb004c-VH-NA(说明书的表-1)；所述的人源化抗体选自下组：Hu001c-14、Hu001c-15、Hu001c-21、Hu001c-22、Hu001c-23、Hu001c-24、Hu001c-25、Hu001c-28、Hu001c-30、Hu001c-31、Hu001c-32、Hu002c-2、Hu002c-3、Hu002c-4、Hu002c-6、Hu002c-7、Hu002c-8、Hu002c-10、Hu002c-11、Hu002c-12、Hu002c-14、Hu002c-15、Hu002c-16(说明书的表-2)。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗体药物偶联物、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。
本发明的另一方面，提供了一种本发明第九所述的抗体药物偶联物的应用，所述抗体药物偶联物用于(i)制备诊断试剂；和/或(ii)制备预防和/或治疗CD73相关的疾病的药物。
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。
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，所述感染疾病包括：细菌和病毒感染。
本发明的第十一方面，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，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
本发明另一方面，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，所述活性成分为如本发明第九方面所述的抗体药物偶联物所述的抗体药物偶联物或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)本发明第六方面所述的重组蛋白；
(3)本发明第七方面所述的CAR构建物。
本发明的第十三方面，提供了一种载体，所述的载体含有本发明第十二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。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有本发明第十三方面所述的载体或基因组中整合有本发明第十二方面所述的多核苷酸。
本发明的第十五方面，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，所述方法包括步骤：
(1)在体外，将所述样品与本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD73。
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有本发明第五方面所述的抗体或本发明第九方面所述的免疫偶联物。
本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有本发明第十六方面所述的检测板。
本发明的第十八方面，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，培养本发明第十四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五方面所述的抗体或本发明第六方面所述的重组蛋白。
本发明的第十九方面，提供了一种治疗CD73相关疾病的方法，所述方法包括：给需要的对象施用本发明第五方面所述的抗体、第九方面所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、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。
在本发明的第二十方面，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重链可变区和/或本发明第三方面所述的轻链可变区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-鼠嵌合抗体。
在本发明的第二十一方面，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重链可变区和/或本发明第三方面所述的轻链可变区中的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。
在本发明的第二十二方面，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第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在本发明的第二十三方面，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第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的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。
在本发明的第二十四方面，提供了一种本发明第九方面所述的抗体-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Ic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)，得到抗体-药物偶联物1a和/或1b。
在另一优选例中，所述制备方法的交联反应如下式所示：
Figure PCTCN2019077369-appb-000007
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(KH 2PO 4-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)。
在另一优选例中，所述的步骤(2)中，所述的偶联反应在0-37℃下进行。
在另一优选例中，所述的步骤(1)采用beta-巯基乙醇、beta-巯基乙胺盐酸盐或DTT还原，在所述的步骤(1)和步骤(2)之间还包括步骤(1a)：在还原反应完成后，对产物进行过脱盐柱或超滤以去除还原剂。
在另一优选例中，在pH 6-8缓冲液中，抗体-药物偶联物Ia内转化为抗体-药物偶联物Ib。
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为本发明的抗人CD73抗体的发现。图1A为流式细胞荧光分选仪(FACS)检测一系列原始抗人CD73单克隆抗体(original hybridoma)培养上清对人源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。所示5个抗体被编号为mAb001、mAb002、mAb003、mAb004、mAb005。图1B为纯化后5个抗体的亚型鉴定，针对MDA-MB-231细胞的结合亲和力，FACS检测其EC 50分别为1.24nM、0.65nM、10.7nM、4.69nM、26.07nM。
图2为PCR扩增mAb001、mAb002、mAb004重链可变区(VH)、轻链可变区(VL)片段琼脂糖凝胶电泳结果图。VH/VL片段测序鉴定后用于克隆和组装人-鼠嵌合抗体表达载体。
图3为HEK293T细胞表达的3个人-鼠嵌合抗体(chimeric antibody)mAb001c、mAb002c、mAb004c，然后采用MabSelect TM SuRe TM柱子的纯化图谱。
图4为ELISA测定人-鼠嵌合抗体mAb001c、mAb002c、mAb004c对CD73-ECD的结合亲和力(Binding affinity EC 50)。
图5为mAb001c、mAb002c、mAb004c对重组人CD73酶催化功能的抑制活性(IC 50)。
图6为流式细胞荧光分选仪(FACS)检测mAb001c、mAb002c、mAb004c对乳腺癌MDA-MB-453、MDA-MB-231，肺癌NCI-H460、NCI-H1299、Calu-6，胰腺癌SW1990，脑胶质瘤U87MG细胞表面CD73受体的结合率(MFI)。本试验采用3x10 5个细胞与5μg/mL抗体混合， 孵育1小时后检测。
图7为肿瘤细胞表面的CD73蛋白水平与其酶活性紧密相关。采用CD73-高表达(U87MG、Calu-1、NCI-H1299)以及CD73-低表达(MDA-MB-453)细胞株，所示细胞数与腺苷单磷酸(AMP)孵育3小时后测定酶活性。
图8为mAb001c、mAb002c、mAb004c对MDA-MB-231细胞表面CD73的结合亲和力(Binding affinity EC 50)检测结果。本试验采用1x10 5个细胞与所示浓度梯度的抗体混合，孵育1小时后检测。
图9为mAb001c、mAb002c、mAb004c对NCI-H1299细胞表面CD73的结合亲和力(Binding affinity EC 50)检测结果。本试验采用1x10 5个细胞与所示浓度梯度的抗体混合，孵育1小时后检测。
图10为检测mAb001c、mAb002c、mAb004c对MDA-MB-231细胞表面CD73酶催化功能的抑制活性(IC 50)。
图11为检测mAb001c、mAb002c、mAb004c对NCI-H1299细胞表面CD73酶催化功能的抑制活性(IC 50)。
图12为检测mAb001c、mAb002c、mAb004c对Calu-1细胞表面CD73酶催化功能的抑制活性(IC 50)。
图13为ELISA检测一系列mAb001c-点突变体、mAb002c-点突变体、mAb004c-点突变体对CD73-ECD的结合亲和力(Binding affinity EC 50)。
图14为ELISA检测mAb001c的人源化抗体系列Hu001c-14～15、Hu001c-21～28、Hu001c-30～32对CD73-ECD的结合亲和力(Binding affinity EC 50)。
图15为ELISA检测mAb002c的人源化抗体系列Hu002c-2～16对CD73-ECD的结合亲和力(Binding affinity EC 50)。
图16为检测mAb001c的人源化抗体系列Hu001c-14～15、Hu001c-21～28、Hu001c-30～32对重组人CD73酶催化功能的抑制活性(IC 50)。
图17为检测mAb002c的人源化抗体系列Hu002c-2～16对重组人CD73酶催化功能的抑制活性(IC 50)。
图18为FACS检测mAb001c-突变体系列、mAb002c-突变体系列对MDA-MB-231细胞表面CD73的结合亲和力(Binding affinity EC 50)。本试验采用1x10 5个细胞与所示浓度梯度的抗体混合孵育1小时后检测。
图19为FACS检测mAb001c的人源化抗体系列Hu001c-14、Hu001c-22～28、Hu001c-30～32对MDA-MB-231细胞表面CD73的结合亲和力(Binding affinity EC 50)。本试验采用1x10 5个细胞与所示浓度梯度的抗体混合孵育1小时后检测。
图20为FACS检测mAb001c的人源化抗体系列Hu001c-14、Hu001c-22～28、Hu001c-30～32对NCI-H1299细胞表面CD73的结合亲和力(Binding affinity EC 50)。本试验采用1x10 5个细胞与所示浓度梯度的抗体混合孵育1小时后检测。
图21为FACS检测mAb002c的人源化抗体系列Hu002c-2～16对MDA-MB-231细胞表面CD73的结合亲和力(Binding affinity EC 50)。本试验采用1x10 5细细胞与所示浓度梯度的抗体混合孵育1小时后检测。
图22为FACS检测mAb002c的人源化抗体系列Hu002c-2～16对NCI-H1299细胞表面CD73 的结合亲和力(Binding affinity EC 50)。本试验采用1x10 5细胞与所示浓度梯度的抗体混合孵育1小时后检测。
图23为检测mAb001c的人源化抗体系列Hu001c-14～15、Hu001c-21～28、Hu001c-30～32对NCI-H1299细胞表面CD73酶催化功能的抑制活性(IC 50)。
图24为检测mAb002c的人源化抗体系列Hu002c-2～16对NCI-H1299细胞表面CD73酶催化功能的抑制活性(IC 50)。
图25为mAb001c、mAb002c、mAb004c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)与细胞孵育4℃1小时，或37℃4小时后置于激光共聚焦显微镜观察结果。
图26为所述CD73人源化抗体在体内抗肿瘤活性测试。体内试验采用CD73-高表达的U87MG脑胶质瘤细胞与50μg抗体混匀后接种至裸鼠背部皮下，每周观察2～3次，测量肿瘤体积和小鼠体重。
图27为所述CD73人源化抗体在体内抗肿瘤活性测试。体内试验采用U87MG脑胶质瘤细胞与50μg抗体混匀后接种至裸鼠背部皮下，每周观察2～3次，测量肿瘤体积和小鼠体重。
图28为所述CD73人源化抗体在体内抗肿瘤活性测试。体内试验采用CD73-高表达的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，测量肿瘤体积和小鼠体重。
图29为所述CD73人源化抗体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，测量肿瘤体积和小鼠体重。
图30为免疫印迹试验(Western blot)检测CD73蛋白在高侵袭、高转移的基底型乳腺癌和管腔型乳腺癌细胞株中的表达情况。
图31为分析Cancer Cell Line Encyclopedia(CCLE)数据库CD73mRNA在高侵袭、高转移的基底型(Basal-type)对比管腔型(Luminal-type)乳腺癌细胞株群中的表达水平。
图32为免疫印迹试验(Western blot)检测CD73蛋白在不同肺癌细胞株中的表达情况。
图33为分析Cancer Cell Line Encyclopedia(CCLE)数据库CD73mRNA在非小细胞肺癌(NSCLC)对比小细胞肺癌(SCLC)细胞株中的表达水平。
图34为人源化抗体Hu001c-14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。
图35为人源化抗体Hu002c-3能有效逆转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36为人源化抗体Hu001c-14能有效逆转AMP对人T淋巴细胞INF-γ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37为人源化抗体Hu002c-3能有效逆转AMP对人T淋巴细胞INF-γ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38为流式细胞荧光分选仪(FACS)检测乳腺癌MDA-MB-453、HCC1937、MDA-MB-231，肺癌NCI-H460、NCI-H292、NCI-H441、Calu-6、NCI-H1299、Calu-1，胰腺癌SW1990，脑胶质瘤U87MG细胞表面CD73受体的表达水平(MFI)。本实验采用1x10 5细胞与10μg/mL的mAb001c混合孵育1h后检测。
图39为人源化CD73抗体Hu001c-14、Hu001c-15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)与细胞孵育4℃1小时，或37℃4小时后置于激光共聚焦显微镜观察结果。
图40为抗体药物偶联物Hu001c14-vcMMAE的疏水作用层析(HIC)图谱。
图41为抗体药物偶联物Hu001c14-BL20-MMAE的疏水作用层析(HIC)图谱。
图42为单克隆抗体Hu001c-14的质谱图谱。
图43为抗体药物偶联物Hu001c14-vcMMAE的质谱图谱。
图44为抗体药物偶联物Hu001c14-BL20-MMAE的质谱图谱。
图45为抗体药物偶联物Hu001c15-vcMMAE的疏水作用层析(HIC)图谱。
图46为抗体药物偶联物Hu001c15-BL20-MMAE的疏水作用层析(HIC)图谱。
图47为单克隆抗体Hu001c-15的质谱图谱。
图48为抗体药物偶联物Hu001c15-vcMMAE的质谱图谱。
图49为抗体药物偶联物Hu001c15-BL20-MMAE的质谱图谱。
图50为CD73抗体-药物偶联物对乳腺癌细胞MDA-MB-453的增殖抑制活性的检测结果。
图51为CD73抗体-药物偶联物对肺癌细胞Calu-1的增殖抑制活性的检测结果。
图52为CD73抗体-药物偶联物对胶质瘤细胞U87MG的增殖抑制活性的检测结果。
图53为CD73抗体-药物偶联物对肺癌细胞Calu-6的增殖抑制活性的检测结果。
图54为CD73抗体-药物偶联物对肺癌细胞NCI-H441的增殖抑制活性的检测结果。
图55为CD73抗体-药物偶联物对肺癌细胞NCI-H292的增殖抑制活性的检测结果。
图56为CD73抗体-药物偶联物对三阴性乳腺癌细胞MDA-MB-231的增殖抑制活性的检测结果。
图57为CD73抗体-药物偶联物对肺癌细胞PC9的增殖抑制活性的检测结果。
图58为CD73抗体-药物偶联物对肺癌细胞HCC827的增殖抑制活性的检测结果。
图59为CD73抗体-药物偶联物对肺癌细胞NCI-H1975的增殖抑制活性的检测结果。
图60显示CD73-药物偶联物的细胞毒活性(IC 50值)与受试细胞的CD73表达水平直接相关，表现为靶标特异性细胞毒性。
图61为人T淋巴细胞(CD3阳性分选获得)经CD73抗体、抗体-药物偶联物(均为10nM)、或AMP(0.3mM)处理后的生长情况。在选取的时间点用FACS读取活细胞数，绘制生长曲线。
图62为人T淋巴细胞与不同浓度的CD73抗体-药物偶联物孵育5天后读取固定体积的的活细胞数，以增殖率(相对于溶媒/缓冲液)绘制抑制曲线图。
图63为CD73抗体-药物偶联物在体内的抗肿瘤活性。采用CD73高表达的U87MG脑胶质瘤细胞接种裸鼠背部皮下第10天时测量肿瘤体积并随机分组(n＝8)，静脉给药共计2次(第10、17天)，剂量均为5mg/kg。
图64为CD73抗体-药物偶联物在体内的抗肿瘤活性。U87MG脑胶质瘤细胞接种后第22天时随机分组(n＝8)，静脉给药共计2次(第22、29天)，Hu001c14-BL20-MMAE(3mg/kg、1mg/kg)；Hu001c14-vc-MMAE(3mg/kg)。
图65为CD73抗体-药物偶联物在体内的抗肿瘤活性。采用CD73高表达的非小细胞肺癌NCI-H441细胞接种裸鼠背部皮下后第19天时随机分组(n＝8)，静脉给药共计2次(第19、26 天)，剂量为3mg/kg、1mg/kg。
图66为CD73抗体-药物偶联物在体内的抗肿瘤活性。NCI-H441细胞接种后第12天时随机分组(n＝8)，静脉给药共计2次(第12、19天)，剂量分别1mg/kg、0.3mg/kg。
图67为CD73抗体-药物偶联物在体内的抗肿瘤活性。采用CD73高表达的非小细胞肺癌NCI-H292细胞接种裸鼠背部皮下。图67A为观察肿瘤的生长，接种第11天时分组(n＝8)和给药(第11、18天)，两组抗体-偶联物剂量均为3mg/kg、1mg/kg。图67B为观察大体积肿瘤的消退，接种第23天分组(n＝8)和给药(第23、30天)，抗体-偶联物剂量为5mg/kg，多烯紫杉醇(docetaxel)为15mg/kg。
图68为检测Hu001c系列的人源化抗体对重组食蟹猴CD73酶催化功能的抑制活性(IC 50)。
图69为检测Hu002c系列的人源化抗体对重组食蟹猴CD73酶催化功能的抑制活性(IC 50)。
图70为Hu001c14-vcMMAE(受试物编号FD114-ADC)在食蟹猴安全性试验的血液学指标测试结果。
图71为Hu001c14-vcMMAE(受试物编号FD114-ADC)在食蟹猴安全性试验的血凝指标测试结果。
图72为Hu001c14-vcMMAE(受试物编号FD114-ADC)在食蟹猴安全性试验的血浆生化指标测试结果。
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5个抗CD73单克隆抗体，分别命名为mAb001至mAb005。依据活性测试结果，选择mAb001(IgG1-κ)、mAb002(IgG1-κ)、mAb004(IgG2b-κ)构建人-鼠嵌合抗体mAb001c、mAb002c、mAb004c。所述的抗体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。并且，所述的抗体具有显著的抗肿瘤活性，而对于哺乳动物本身没有明显的毒副作用。此外，基于mAb001c、mAb002c设计的人源化抗体以及相应的抗体药物偶联物(ADC)也具有优异的特性。此外，通过本发明的新型连接子得到的CD73抗体药物偶联物产品具有均一性高，进一步提高体外和体内的稳定性的优势。在此基础上完成了本发明。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体 可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗CD73的抗体
本发明提供3大类靶向CD73的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
1.优选地，重链可变区(VH)氨基酸序列、轻链可变区(VL)氨基酸序列包含具有以下多肽序列的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
a1)HCDR1为SEQ ID NO.:1：NYYIY，SEQ ID NO.:10:SYWMH，或SEQ ID NO.:21：DYNMD；
a2)HCDR2为SEQ ID NO.:2：WIYPGNLNIKYNEKFKG，SEQ ID NO.:11:EINPSNGRSNYNEKFKS，或SEQ ID NO.:22：DINPNNGGSVYNQKFKG；
a3)HCDR3为SEQ ID NO.:3：DDNYAWFAY，SEQ ID NO.:12:RGVSGNYFDY，或SEQ ID NO.:23：ITGTGYWSFDV；
a4)LCDR1为SEQ ID NO.:4：KASQDVSTAVA，SEQ ID NO:13:KASQDINTYLS，或SEQ ID NO.:24：RASENIYSNLA；
a5)LCDR2为SEQ ID NO.:5：WTNTRHT，SEQ ID NO.:14：RSNILVD，或SEQ ID NO.:25：GATNLAE；或者
a6)LCDR3为SEQ ID NO.:6：QQHYSTPFT；SEQ ID NO.:15：LQYDEFPYT，或SEQ ID NO.:26：QHFWGIPWT；
a7)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.:10～12、SEQ ID NO.:21～23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，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID NO.:4～6、SEQ ID NO.:13～15、SEQ ID NO.:24～26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，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:3，或选自SEQ ID NO.:10、SEQ ID NO.:11、SEQ ID NO.:12，或选自SEQ ID NO.:21、SEQ ID NO.:22、SEQ ID NO.:23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；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:4、SEQ ID NO.:5、SEQ ID NO.:6，或选自SEQ ID NO.:13、SEQ ID NO.:14、SEQ ID NO.:15，或选自SEQ ID NO.:24、SEQ ID NO.:25、SEQ ID NO.:26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。
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，所述的抗体为原始的鼠源抗体mAb001、mAb002、mAb003、mAb004、mAb005。
在另一优选例中，所述的抗体为人-鼠嵌合抗体mAb001c、mAb001c-VK-SGS、mAb002c、mAb002c-VH-QG、mAb002c-VH-NA、mAb002c-VK-SG、mAb002c-VH-QG/VK-SG、mAb004c、mAb004c-VH-QG、mAb004c-VH-NA。
在另一优选例中，所述的抗体为人源化抗体Hu001c-14、Hu001c-15、Hu001c-21、Hu001c-22、Hu001c-23、Hu001c-24、Hu001c-25、Hu001c-28、Hu001c-30、Hu001c-31、Hu001c-32。
在另一优选例中，所述的抗体为人源化抗体Hu002c-2、Hu002c-3、Hu002c-4、Hu002c-6、Hu002c-7、Hu002c-8、Hu002c-10、Hu002c-11、Hu002c-12、Hu002c-14、Hu002c-15、Hu002c-16。
在另一优选例中，所述嵌合抗体的重链和轻链可变区(VH/VL)的氨基酸序列编号由表1列出。
在另一优选例中，所述人源化抗体的重链和轻链可变区(VH/VL)的氨基酸序列编号由表2列出。
本发明的3大类抗体可以联合应用，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。
本发明序列表中涉及的各序列的代表含义如下：
序列编号 序列名称 序列编号 序列名称
SEQ ID NO.:1 mAb001 HCDR1 SEQ ID NO.:27 mAb004-VH
SEQ ID NO.:2 mAb001 HCDR2 SEQ ID NO.:28 mAb004-VH-QG
SEQ ID NO.:3 mAb001 HCDR3 SEQ ID NO.:29 mAb004-VH-NA
SEQ ID NO.:4 mAb001 LCDR1 SEQ ID NO.:30 mAb004-VL
SEQ ID NO.:5 mAb001 LCDR2 SEQ ID NO.:31 mAb001-VH_HuG.3
SEQ ID NO.:6 mAb001 LCDR3 SEQ ID NO.:32 mAb001-VH_HuG.5
SEQ ID NO.:7 mAb001-VH SEQ ID NO.:33 mAb001-VH_HuG.6
SEQ ID NO.:8 mAb001-VL SEQ ID NO.:34 mAb001-VH_HuG.7
SEQ ID NO.:9 mAb001-VL-SGS SEQ ID NO.:35 mAb001-VH_HuG.8
SEQ ID NO.:10 mAb002 HCDR1 SEQ ID NO.:36 mAb001-VK_HuG.1
SEQ ID NO.:11 mAb002 HCDR2 SEQ ID NO.:37 mAb001-VK_HuG.2
SEQ ID NO.:12 mAb002 HCDR3 SEQ ID NO.:38 mAb002-VH_HuG0
SEQ ID NO.:13 mAb002 LCDR1 SEQ ID NO.:39 mAb002-VH_HuG1
SEQ ID NO.:14 mAb002 LCDR2 SEQ ID NO.:40 mAb002-VH_HuG2
SEQ ID NO.:15 mAb002 LCDR3 SEQ ID NO.:41 mAb002-VH_HuG3
SEQ ID NO.:16 mAb002-VH SEQ ID NO.:42 mAb002-VK_HuG1
SEQ ID NO.:17 mAb002-VH-QG SEQ ID NO.:43 mAb002-VK_HuG2
SEQ ID NO.:18 mAb002-VH-NA SEQ ID NO.:44 mAb002-VK_HuG3
SEQ ID NO.:19 mAb002-VL SEQ ID NO.:45 mAb001-VH_HuG.9
SEQ ID NO.:20 mAb002-VL-SG SEQ ID NO.:46 mAb001-VH_HuG.10
SEQ ID NO.:21 mAb004 HCDR1 SEQ ID NO.:47 mAb001-VK_HuG.0
SEQ ID NO.:22 mAb004 HCDR2 SEQ ID NO.:48 人CD73蛋白的胞外区
SEQ ID NO.:23 mAb004 HCDR3 SEQ ID NO.:49 MEDI9447VH
SEQ ID NO.:24 mAb004 LCDR1 SEQ ID NO.:50 MEDI9447VL
SEQ ID NO.:25 mAb004 LCDR2    
SEQ ID NO.:26 mAb004 LCDR3    
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
典型的适用于本发明的偶联方式，包括K-Lock和C-Lock两种偶联方式。在K-Lock偶联方式中，药物分子偶联于抗体序列中赖氨酸(K)残基，在C-Lock偶联方式中，药物分子偶联于抗体序列中的半胱氨酸(C)残基。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合 适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能 基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019077369-appb-000008
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1-10，较佳地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ADC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。
优选的小分子药物为具有高细胞毒性的化合物，优选单甲基澳瑞他汀(monomethyl auristatin)、加利车霉素、美登素类、或其组合；更佳地选自：单甲基阿里他汀-E(MMAE)、单甲基阿里他汀-D(MMAD)、单甲基阿里他汀-F(MMAF)、或其组合。
较佳地，所述的药物是指：用于癌症治疗的细胞毒性药物，或具有期望生物活性的蛋白或多肽，例如一种毒素，如相思子毒素，蓖麻毒素A，假单胞菌外毒素，和白喉毒素；其他合适的蛋白包括肿瘤坏死因子，α-干扰素，β-干扰素，神经原生长因子，血小板衍生生长因子，组织型纤酶溶原生长因子，以及生物反应调节制剂，例如淋巴因子，白细胞介素-1(IL-1),白细胞介素-2(IL-2),白细胞介素-6(IL-6),粒细胞巨噬细胞集落刺激因子(GM-CSF),粒细胞集落刺激因子，或其它生长因子。
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。
Figure PCTCN2019077369-appb-000009
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。
Figure PCTCN2019077369-appb-000010
Monomethyl Auristatin E(MMAE)
Figure PCTCN2019077369-appb-000011
Monomethyl Auristatin F(MMAF)
Figure PCTCN2019077369-appb-000012
Monomethyl Dolastatin 10(MMAD)
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。
Figure PCTCN2019077369-appb-000013
PBD二聚体
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。
Figure PCTCN2019077369-appb-000014
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-D14的结构。
CD73抗体-药物偶联物
本发明涉及抗体-药物偶联物，更具体地说，本发明涉及具有治疗应用的CD73抗体-药物偶联物。可以通过连接子将抗CD73抗体与化疗药或者小分子毒素偶联。本发明还涉及及使用抗CD73抗体-药物偶联物治疗哺乳动物细胞或相关病理性情况的方法。
本发明采用一类新型双取代马来酰亚胺类连接子应用于靶向CD73抗体进行偶联，该连接子可以全部/部分交叉偶联抗体的轻链-重链及重链-重链二硫键还原的半胱氨酸巯基上，且应用此种偶联方法得到的靶向CD73抗体药物偶联物，与传统抗体药物偶联物相比，具有更窄的药物/抗体比值(DAR)分布。
Figure PCTCN2019077369-appb-000015
其中，
Ar'选自下组：取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；
L 1为连接于Ar'基团上的-O(CH 2CH 2O) n-，其中n选自1-20中任一整数。
L 2为化学键，或AA-PAB结构；其中，AA为2-4个氨基酸组成的多肽片断，PAB为对-氨基苄基氨甲酰基；
CTD为通过酰胺键键合于L 2的细胞毒类小分子药物。
m为3.8-4.2；
Ab为靶向CD73的抗体。
本发明提供了一种偶联方法，将毒素小分子通过特定连接物偶联到靶向CD73抗体上，在不改变抗体亲和性的基础上大幅提高抗体对肿瘤细胞的杀伤力。
本发明提供了连接子或偶联试剂，包含二芳硫基马来酰亚胺单元和一个偶联基团。二芳硫基马来酰亚胺单元用于交联抗体链间的巯基基团(还原后)，而偶联基团用于与小分子药物或药物-连接子单元偶联。由于该二芳硫基马来酰亚胺单元与抗体中的开放半胱氨酸-半胱氨酸二硫键的两个硫原子的二齿结合(bidentate binding)，因此这些ADC是均质的并比含有单齿接头的ADC具有更强的稳定性。因此它们将具有增长的体内半衰期，减少全身性释放的细胞毒素的量，并且比具有单齿接头的ADC更加安全的药物性质。
在另一个方面，所产生的药物-连接子单元通过该连接子与抗体偶联，生成部分链间交联的偶联物。与传统的抗体药物偶联物相比，应用本发明方法制备的抗体药物偶联物的药物/抗体比值(DAR)分布更窄，从而大幅提升了产品均一性及药理学特性均一性。该抗体药物偶联物可用于靶向输送药物到达目标细胞群体，例如肿瘤细胞。抗体药物偶联物可以特异性的与细胞表面蛋白结合，所产生的结合物随即被细胞内吞。在细胞内，药物以活性药物的方式释放出来产生功效。抗体包括嵌合抗体，人源化抗体，人抗体；可与抗原结合的抗体片段；或者抗体Fc融合蛋白；或者蛋白。“药物”是高活性药物(见定义部分)，在某种情况下，药物可以是聚乙二醇。
本发明提供的偶联产品，尽管仍然是混合物，但与传统方式偶联得到的抗体药物偶联物相比，其DAR分布范围很窄。其平均DAR值接近4，接近最佳抗体药物偶联物平均DAR值(2-4)范围。此外，偶联产品极少不含有裸抗(DAR＝0)，这一组分对细胞毒杀不起 作用。同时，偶联产品也不含有重度偶联产品(DAR＝8),这一组分在体内的清除速度很快，相对于低DAR的组分而言。因此，本发明提供的抗体药物偶联物产品非均一性得到很大的改善。
连接子-药物缀合物
在本发明中，连接子-药物缀合物包括式Ic所示的取代马来酰亚胺类连接子-药物缀合物或其药学上可接受的盐或溶剂化合物；
Figure PCTCN2019077369-appb-000016
其中，
R为X或ArS-，
X选自下组：卤素，优选为溴或碘；
Ar'选自下组：取代或未取代的C6-C10芳基，取代或未取代的5-12元杂芳基，取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；
L 1为连接于Ar'基团上的-O(CH 2CH 2O) n-，其中n选自1-20中任一整数，优选为1-10中任一整数；
L 2为化学键或AA-PAB结构；其中，AA为二肽或三肽或四肽片断(即2-4个氨基酸通过肽键连接形成的片段)，PAB为对-氨基苄基氨甲酰基；
CTD为通过酰胺键键合于L 2的细胞毒类小分子药物和/或治疗自身免疫疾病和抗炎症的药物。
所述的式Ic化合物选自下组：
Figure PCTCN2019077369-appb-000017
Figure PCTCN2019077369-appb-000018
Figure PCTCN2019077369-appb-000019
等。
式Ic所示的化合物的合成与制备
式Ic所示的化合物的通用型制备流程如下：
通过n甘醇与溴乙酸叔丁酯反应获得中间体A，后与取代的硝基氟苯进行芳香亲核取代得到中间体B。此外，中间体B也可通过对甲苯磺酸酯保护的中间体F与取代的硝基氟酚反应获得。中间体B中硝基基团还原为氨基得到中间体C，后与2,3-二溴马来酸酐环合反应得中间体D，后再与芳基硫酚进行取代反应后获得连接子片断分子E。通过与带有二肽/三肽-PAB细胞毒药物连接子进行缩合，可获得系列分子F。反应路线如下：
Figure PCTCN2019077369-appb-000020
以Ic-4为例，具体说明其制备流程：
Figure PCTCN2019077369-appb-000021
1.1.1中间体A-1(步骤a)
Figure PCTCN2019077369-appb-000022
将三甘醇(92g,613mmol)溶于tBuOH(200ml)中。冰浴下加入KOtBu(22.91g,204mmol)搅拌半小时，氩气保护下，滴加溴乙酸叔丁酯(39.8g,204mmol)在tBuOH(40ml)的溶液，室温搅拌过夜。第二天，TLC检测反应结束。旋蒸除去叔丁醇后，剩余物加入400ml二氯甲烷，有机相用400ml水洗，该水相用300ml二氯甲烷萃取一次，合并有机相后用饱和食盐水洗一次，无水硫酸钠干燥，旋蒸蒸干。粗产物经石油醚：乙酸乙酯＝3:1-->1:1柱层析，得中间体A-1(24g,44.5％yield)，为黄色油状物。
1.1.2中间体B-1(步骤b)
Figure PCTCN2019077369-appb-000023
在250ml圆底瓶中将中间体A-1(4g,15.13mmol),三乙胺(2.53ml,18.16mmol)和二甲胺基吡啶(0.370g,3.03mmol)溶于100毫升分子筛干燥二氯甲烷中搅拌，冰浴下分批加入对甲基苯磺酰氯(3.17g,16.65mmol)，移至室温氩气保护搅拌过夜。
将反应体系加入100ml二氯甲烷萃取，用200ml 1N稀盐酸洗一次，200ml水洗两次，200ml饱和盐水洗一次，无水硫酸钠干燥，旋蒸蒸干有机相。用200-300目硅胶装柱， PE:EA＝5:1-2:1洗脱进行柱层析分离。旋蒸蒸干得中间体B-1(2.8g,收率44.2％)。
1.1.3中间体C-1(步骤c)
Figure PCTCN2019077369-appb-000024
将中间体B-1(3g,7.19mmol)，2,6-二氟-4-硝基苯酚(1g,7.19mmol)，溶于20ml DMF中，加入K 2CO 3(1.9g,14.4mmol)，加热至100度搅拌5小时。旋蒸蒸干溶剂，加入200ml二氯甲烷溶解，萃取，分别用200ml 1N稀盐酸，200ml水和200ml饱和盐水洗各洗一次，无水硫酸钠干燥，旋蒸蒸干，用200-300目硅胶装柱，PE：EA＝5:1-3:1洗脱柱层析纯化，旋蒸蒸干得中间体C-1(2g,收率72％)
1.1.4中间体D-1(步骤d)
Figure PCTCN2019077369-appb-000025
将中间体B-1(6g,15.57mmol)溶于100毫升无水乙醇中并且将溶液，加入装有10％Pd-C 1.2g的反应瓶中。加氢反应6小时(1atm,38℃)，TLC检测反应完全。硅藻土过滤反应液，滤饼用乙醇淋洗，滤液旋蒸蒸干，得中间体D-1(4.8g,87％yield)为黄色油状物。
1.1.5化合物E-1(步骤e)
Figure PCTCN2019077369-appb-000026
称取中间体D-1(1.0g,2.81mmol)于平行反应管中，氮气保护下加入AcOH(3ml)，搅拌溶解。后慢慢加入3,4-二溴马来酸酐(0.72g,2.81mmol)。氮气保护下加热到110℃搅拌过夜。TLC检测反应。将反应液冷却到室温后，旋蒸蒸干溶剂，并加入甲苯旋蒸蒸干两次，得棕色油状化合物E-1。无需纯化直接用于下步反应。
1.1.6化合物F-1的合成(步骤f)
Figure PCTCN2019077369-appb-000027
称取化合物E-1(2.0g,3.72mmol)于100毫升圆底瓶中,氮气保护下加入30ml无水二氯甲烷搅拌溶解。称取4-(N-吗啉甲酰胺)苯硫酚(1.66g,7.45mmol)氮气保护下加入反应液中,溶解后在冰浴下慢慢滴加DIPEA(1.3mL ml,7.45mmol)，完毕后搅拌5分钟，撤去冰浴。在氮气保护下室温搅拌2小时，TLC检测反应结束。
减压蒸干溶剂后，柱层析(200目～300目硅胶)分离纯化，二氯甲烷装柱和淋洗，然后慢慢加大极性从2％至10％甲醇淋洗，收集蒸干溶剂得橘黄色油状产物F-1(2.2g,72％yield)。LC-MS(M +)理论值:821.2，实测值:821.3(ESI,M+H +)。
1.1.7化合物G-1(Ic-1)的合成(步骤g)
Figure PCTCN2019077369-appb-000028
向100毫升圆底瓶中称入化合物E-9(300mg,0.365mmol)，氮气保护下加入无水DMF(20mL)使其完全溶解后,依次称取HATU(166mg,0.438mmol)和DIEA(0.127ml,0.730mmol)加入瓶中。室温搅拌15分钟后加入化合物VC-PAB-MMAE(416mg,0.365mmol),氮气保护下室温搅拌过夜。TLC与HPLC跟踪反应过夜，原料F-1消失。减压蒸干溶剂，做定量分析，后经反相HPLC纯化，得产物为得到产物为黄色无定形粉末。LC-MS(M +)理论值:1961.9，实测值:1962.7(ESI,M+H +)。
CD73抗体-药物偶联物的制备
抗体药物偶联物制备路线如下所示。抗体链间二硫键被还原，产生2n个(如8个)巯基基团。本发明的取代马来酰亚胺类连接子-药物缀合物(式Ic)与还原后的抗体巯基交联，生成相应的抗体药物偶联物，其中该抗体药物偶联物存在如下所示中的一种或二种形式。
Figure PCTCN2019077369-appb-000029
一种典型的制备方法包括：将抗体原液用反应缓冲液稀释至2-10mg/mL，加入140-200倍过量摩尔比的二硫苏糖醇(DTT)，或加入6.0-20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于10-35℃搅动2-48小时；在此所述反应缓冲液可以是按以下比例制备的缓冲液：50mM磷酸二氢钾-氢氧化钠(KH 2PO 4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9和PBS//1mM二乙基三胺五乙酸(DTPA)，pH 6-9。
将上述反应液冷至0-10℃，若采用DTT还原，需在还原反应完成后过脱盐柱或超滤除去过量的DTT，再加入取代马来酰亚胺类化合物(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。优选温度为-100-60℃，过滤装置的孔径优选0.15-0.3微米。
所得抗体药物偶联物的药物抗体偶联比(DAR)较为均一。采用本发明不同取代马来酰 亚胺连接头(连接子片断)时，ADC产物均一性非常高(通常DAR优势产物(如DAR约为4)占所有ADC的至少60％，至少70％，至少80％，至少90％或更高)。对于DAR有一定差别的ADC，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)。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
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、ADC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ADC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ADC而言，由于本发明提供的抗体-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EC 50为0.016～0.038nM)，具有CD73酶功能的抑制活性(酶活性测定其IC 50为0.025～0.039nM)。此外，对肿瘤细胞的CD73具有良好的结合亲合力(FACS测定其EC 50为0.35～2.5nM)，并抑制肿瘤CD73酶的功能(IC 50值为0.2nM～0.6nM),可用做靶向CD73的治疗抗体。
(b)本发明的人源化抗体不仅具有与鼠源抗体相当的活性，而且具有更低的免疫原性。
(c)本发明的抗体和ADC均具有显著的体内抗肿瘤活性，而对于哺乳动物如模式小鼠本身没有可见的毒副作用。
(d)本发明的抗体对人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INF-γ的表达和分泌，其EC 50为0.01～0.08nM。
(e)本发明所述的抗体-药物偶联物(ADC)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-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IC 50为0.02nM～0.05nM。
(f)本发明所述的抗体-药物偶联物(ADC)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IC 50为>100nM。
(g)本发明所述的抗体-药物偶联物(ADC)对哺乳动物如食蟹猴未呈现高度或意外的毒副作用，具有临床药物应用的潜在前景。
(h)本发明提供的新型连接子，可通过简单的化学方法与靶向CD73抗体偶联，与传统的偶联方式相比，应用这种连接子得到的CD73抗体药物偶联物DAR值分布非常窄，因此生成的产品均一性高，获得的交联物单一分布的组份(DAR为4)占比80％以上，交联物的体外肿瘤细胞增殖抑制活性较传统mcVC-PAB交联生物学活性、安全性等成药性质方面有所提高或保持。
(i)本发明基于马来酰亚胺的二硫链桥接具有更好的稳定性，Ar'部位引入取代基可以调解马来酰亚胺开环水解的反应速度与减缓马来酰亚胺开环后的环合二次水解反应，在体内不易发生巯醚交换和开环后的环合二次水解反应，进一步加强了CD73抗体-药物偶联物在体外和体内的稳定性。
下面结合具体实施例，进一步阐述本发明。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具体条件的实验方法，通常按照常规条件，例如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，否则百分比和份数是重量百分比和重量份数。细胞株为常规的市售产品或购自ATCC，质粒均为市售产品。
实施列1 靶向人CD73单克隆抗体的发现和制备
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.:48)：
Figure PCTCN2019077369-appb-000030
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，去除PEG，用含有饲养细胞的HAT完全培养基重悬，接种到96孔板中培养，通过ELISA/FACS法进行阳性孔筛选。最后再对阳性孔的细胞通过有限稀释法进行克隆化培养，通过ELSIA或FASCS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％，即可对该细胞株进行扩大培养和建库。
步骤②，靶向人CD73鼠源单克隆抗体的纯化：
将步骤①中筛选出来的杂交瘤细胞在滚瓶中扩大培养14d后，收集细胞培养上清，经0.22μm滤膜过滤后，将所得培养上清恒速加入事先平衡好的Protein A树脂柱中，并用0.1M Tris-HCl(PH＝8.0,含有1.5M NaCl)平衡柱子。然后采用0.1M柠檬酸钠缓冲液洗脱平衡柱，收集洗脱液并定量并进行SDS-PAGE电泳、SEC-HPLC及内毒素检测。所得纯化抗体分装、-80℃冻存备用。
步骤③，靶向人CD73鼠源单克隆抗体的生物活性和特异性的确定：
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测定定。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。
实施例2 抗体测序、互补决定区(CDR)的鉴定
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(见图2)，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.:7mAb001重链可变区(VH)氨基酸序列
Figure PCTCN2019077369-appb-000031
SEQ ID NO.:16mAb002重链可变区(VH)氨基酸序列
Figure PCTCN2019077369-appb-000032
SEQ ID NO.:27mAb004重链可变区(VH)氨基酸序列
Figure PCTCN2019077369-appb-000033
SEQ ID NO.:8mAb001轻链可变区(VL)氨基酸序列
Figure PCTCN2019077369-appb-000034
SEQ ID NO.:19mAb002轻链可变区(VL)氨基酸序列
Figure PCTCN2019077369-appb-000035
SEQ ID NO.:30mAb004轻链可变区(VL)氨基酸序列
Figure PCTCN2019077369-appb-000036
实施例3 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.:7、SEQ ID NO.:16、SEQ ID NO.:27、SEQ ID NO.:8、SEQ ID NO.:19、SEQ ID NO.:30)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系统(FreeStyle TM293T细胞)将构建的嵌合型抗体表达和纯化(见图3)，所获得的人-鼠嵌合型抗体，分别编号为mAb001c、mAb002c、mAb004c。
所述抗体的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.:9mAb001-VL-SGS
Figure PCTCN2019077369-appb-000037
SEQ ID NO.:17mAb002-VH-QG
Figure PCTCN2019077369-appb-000038
SEQ ID NO.:18mAb002-VH-NA
Figure PCTCN2019077369-appb-000039
SEQ ID NO.:20mAb002-VL-SG
Figure PCTCN2019077369-appb-000040
SEQ ID NO.:28mAb004-VH-QG
Figure PCTCN2019077369-appb-000041
SEQ ID NO.:29mAb004-VH-NA
Figure PCTCN2019077369-appb-000042
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 PCTCN2019077369-appb-000043
Figure PCTCN2019077369-appb-000044
实施例4 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4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；
检测结果如图13所示，所述mAb001c/mAb002c/mAb004c突变体的ELISA EC 50值为0.05nM～0.093nM。
实施例5 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50μLCellTiter-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5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；
检测结果如图16所示，mAb001c-Vk-SGS的IC 50为0.02nM；
检测结果如图17所示，mAb002c-VH-QG/VK-SG的IC 50为0.038nM、0.06nM。
实施例6 嵌合抗体对肿瘤细胞表面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、Calu-1、脑胶质瘤细胞U87MG、胰腺癌细胞SW1990，同时采用CD73-低表达的乳腺癌细胞MDA-MB-453、非小细胞肺癌细胞NCI-H460，测定嵌合抗体对细胞表面CD73的结合情况。采用3x10 5个肿瘤细胞与抗体混匀(终浓度5μg/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200μLPBS中，通过流式细胞荧光分选仪(FACS)检测结合率。
检测结果如图6所示，所述嵌合抗体均能特异性识别和结合CD73-高表达的肿瘤细胞，结合率荧光强度顺序依次为Calu-1、NCI-H1299、U87MG、SW1990、MDA-MB-231，而对CD73-低表达的肿瘤细胞MDA-MB-453、NCI-H460显示极微弱的结合荧光强度。对比Calu-1、MDA-MB-453与抗体的结合率(MFI)，mAb001c结合率的差异为250倍，mAb002c为978倍，mAb004c为856倍。
实施例7 肿瘤细胞表面的CD73蛋白水平与其酶活性紧密相关
采用CD73-高表达(U87MG、Calu-1、NCI-H1299)、CD73-低表达(MDA-MB-453)细胞株来研究细胞表面CD73蛋白量与酶活性的相关性。首先将100μL含有上述各细胞株数目按照两倍梯度从20000释到625，均匀铺到96孔细胞培养板中，37℃培养16h后，用无血清的培养基洗3次以去除残留血清，缓慢加入50μL 300μM AMP，混匀，37℃培养3h。小心取出25μL培液上清，置于另一96孔白板中，加入25μL 100μM 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。
结果如图7所示，在检测试验的线性范围内，CD73-低表达的MDA-MB-453细胞仅产出极低量(本底水平)的酶活性，而CD73-高表达的三株细胞均显示了很高的酶活性，从而确认了肿瘤细胞表面的CD73蛋白水平与其酶活性紧密相关。
实施例8 嵌合抗体对肿瘤细胞表面CD73的结合亲和力的测定
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100μLRPMI-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200μLPBS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8所示，mAb001c、mAb002c、mAb004c对MDA-MB-231具有优异的结合亲和力，EC 50分别为0.7nM、0.36nM、2.5nM；
测结果如图9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；
测结果如图18所示，所述mAb001c/mAb002c的衍生突变体对MDA-MB-231同样具有优异的结合亲和力，mAb001c-VK-SGS的EC 50值为1.39nM；mAb002c-VH-QG/NA的EC 50值为0.43nM；mAb002c-VK-SG的EC 50值为0.46nM。
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。
实施例9 嵌合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、非小细胞肺癌细胞NCI-H1299和Calu-1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25μL0.9mM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10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1.858nM、0.791nM和4.164nM。
检测结果如图11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0.236nM、0.191nM、0.385nM。
检测结果如图12所示，mAb001c、mAb002c、mAb004c抑制Calu-1细胞表面CD73催化水解AMP的功能，IC 50分别为0.506nM、0.281nM、0.630nM。
实施例10 人源化抗体的制备
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.:31、SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35、SEQ ID NO.:45、SEQ ID NO.:46)，以及3个人源化轻链的可变区(SEQ ID NO.:36、SEQ ID NO.:37、SEQ ID NO.:47)。
SEQ ID NO.:31mAb001-VH_HuG.3
Figure PCTCN2019077369-appb-000045
SEQ ID NO.:32mAb001-VH_HuG.5
Figure PCTCN2019077369-appb-000046
SEQ ID NO.:33mAb001-VH_HuG.6
Figure PCTCN2019077369-appb-000047
SEQ ID NO.:34mAb001-VH_HuG.7
Figure PCTCN2019077369-appb-000048
SEQ ID NO.:35mAb001-VH_HuG.8
Figure PCTCN2019077369-appb-000049
SEQ ID NO.:45mAb001-VH_HuG.9
Figure PCTCN2019077369-appb-000050
SEQ ID NO.:46mAb001-VH_HuG.10
Figure PCTCN2019077369-appb-000051
Figure PCTCN2019077369-appb-000052
SEQ ID NO.:36mAb001-VK_HuG.1
Figure PCTCN2019077369-appb-000053
SEQ ID NO.:37mAb001-VK_HuG.2
Figure PCTCN2019077369-appb-000054
SEQ ID NO.:47mAb001-VK_HuG.0
Figure PCTCN2019077369-appb-000055
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.:38、SEQ ID NO.:39、SEQ ID NO.:40、SEQ ID NO.:41)以及3个人源化轻链的可变区(SEQ ID NO.:42、SEQ ID NO.:43、SEQ ID NO.:44)。
SEQ ID NO.:38mAb002-VH_HuG0
Figure PCTCN2019077369-appb-000056
SEQ ID NO.:39mAb002-VH_HuG1
Figure PCTCN2019077369-appb-000057
SEQ ID NO.:40mAb002-VH_HuG2
Figure PCTCN2019077369-appb-000058
SEQ ID NO.:41mAb002-VH_HuG3
Figure PCTCN2019077369-appb-000059
SEQ ID NO.:42mAb002-VK_HuG1
Figure PCTCN2019077369-appb-000060
SEQ ID NO.:43mAb002-VK_HuG2
Figure PCTCN2019077369-appb-000061
SEQ ID NO.:44mAb002-VK_HuG3
Figure PCTCN2019077369-appb-000062
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系统(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，mAb002-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 PCTCN2019077369-appb-000063
实施例11 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4。
实验结果如图14、图15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。
实施例12 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，按照实施例5的方法测定抗体对重组CD73酶活性的影响。
实验结果如图16、图17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。
实施例13 人源化抗体对肿瘤细胞CD73的结合
通过流式细胞仪测定将表2中的人源化抗体对MDA-MB-231、NCI-H1299肺癌细胞表面CD73的亲和力，实验方法参照实施例6。
试验结果如图19、图21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0.2nM～0.8nM。
试验结果如图20、图22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0.3nM～1.4nM。
实施例14 人源化抗体对肿瘤细胞CD73酶功能的抑制活性
测定了表2中的人源化抗体对NCI-H1299细胞表面CD73酶功能的影响，实验方法参照
实施例8。
实验结果如图23、图24所示，所述2组人源化抗体对NCI-H1299细胞表面的CD73酶具有很高的抑制活性，IC 50值为0.2nM～0.6nM。
实施例15 CD73嵌合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细胞铺于激光共聚焦培养皿中，于37℃培养16h后加入5μg/mL的CD73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 Fluor 488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，之后用激光共聚焦显微镜(20×)观察抗体的抗体内吞情况。
结果如图25所示，mAb001c、mAb002c、mAb004c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抗体-药物偶联物(ADC)，提示CD73-ADC将具有良好的ADC药物特性，可用于广谱和高特异性靶向CD73-阳性肿瘤治疗药物的前景。
实施例16 人源化CD73抗体在裸鼠移植瘤模型中的抗肿瘤活性
随机将免疫缺陷型裸小鼠(Balb/c,nude)分为若干组，将100μL含有5x10 6U87MG，或9x10 6NCI-H1299的细胞悬液与100μL所示人源化抗体混匀(终浓度为50μg/瘤)，然后将200μL细胞-抗体混合液接种入裸鼠背部皮下(n＝4)。采用hIgG1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，评定活性。
结果如图26所示,人源化抗体Hu001c-14、人源化抗体Hu002c-3～8均能显著抑制U87MG肿瘤在裸鼠体内的生长。
结果如图27所示,人源化抗体Hu001c-14、人源化抗体Hu001c-24～32均能显著抑制U87MG肿瘤在裸鼠体内的生长。
结果如图28所示,人源化抗体Hu001c-14～15、人源化抗体Hu002c-3～8均能显著抑制NCI-H1299肿瘤在裸鼠体内的生长。
结果如图29所示,人源化抗体Hu001c-14～15、人源化抗体Hu001c-23～32均能显著抑制NCI-H1299肿瘤在裸鼠体内的生长。
实施例17 CD73在三阴性乳腺中高度异常激活
首先针对多种不同分子分型的乳腺细胞株，制备细胞总蛋白，精确定量后，通过免疫印迹试验(Western blot)检测CD73蛋白的表达水平。
结果如图30显示，CD73蛋白在一些高侵袭、高转移的基底型乳腺癌(Basal-type，临床上大多表现为三阴性乳腺癌)细胞株群呈高度异常激活表达，而在相对恶性程度较低的官腔型乳腺癌(Luminal-type，临床上大多表现为激素受体阳性乳腺癌)细胞株中呈阴性或微弱表达。
然后，对Cancer Cell Line Encyclopedia(CCLE)数据库中乳腺癌细胞株的CD73mRNA表达水平进行分析。结果如图31显示，高侵袭、高转移的Basal-type乳腺癌细胞株群中CD73mRNA的表达水平普遍高于Luminal-type乳腺癌细胞株群且具有统计学意义。因此，本发明 以CD73为靶点的抗体，在诊断、预防和治疗三阴性乳腺癌的应用中将具有更为显著的效果。
实施例18 CD73在肺癌中高度异常激活表达
首先针对多种不同组织来源、不同分子分型的肺癌细胞株，制备细胞总蛋白，精确定量后，通过免疫印迹试验(Western blot)检测CD73蛋白的表达水平。
结果如图32显示，CD73蛋白在众多非小细胞肺癌(NSCLC)细胞株中异常激活表达。
然后，对CCLE数据库中肺癌细胞株的CD73mRNA水平进行分析。结果如图33表明，在非小细胞肺癌(NSCLC)细胞株中CD73mRNA表达水平显著高于小细胞肺癌(SCLC)，提示本发明以CD73为靶点的抗体，在诊断、预防和治疗非小细胞肺癌(NSCLC)的应用中具有更为显著的效果。
实施例19 人源化CD73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/mLCD3/CD28抗体和100IU/mLIL-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(终浓度10nM～0.0001nM，n＝4)，并加入50μL腺苷单磷酸(AMP，终浓度为0.2mM)，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EC 50值。
T细胞IFN-γ的检测：摄取50μL/每孔T细胞培养上清用于检测IFN-γ蛋白浓度，采用ELISA试剂盒(联科生物技术有限公司，Cat#EK180HS-48)，并参照试剂盒所提供的技术操作步骤。
结果如图34、图35显示，人源化CD73抗体Hu001c-14、Hu002c-3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，其EC 50分别为0.08±0.01nM和0.01±0.001nM。
结果如图36、图37显示，人源化CD73抗体Hu001c-14、Hu002c-3能有效逆转AMP对T细胞表达/分泌INF-γ的抑制作用。
实施例20 所述CD73抗体与现有技术的比较
以US20160194407中公开的MEDI9447抗体的重链、轻链可变区序列(VH/VL)，人工合成其重链、轻链可变区，分别克隆入含有人IgG1重链恒定区的载体，含有Kappa链恒定区或含有Lambda链恒定区的载体，经测序无误后在FreeStyle TM293T细胞体系表达和纯化后分别获得MEDI9447-κ(与本发明的CD73抗体一致)或者MEDI9447-λ，将抗体制备的实验条件与实施例3、实施例10保持一致。
重链可变区(VH)SEQ ID NO.:49
Figure PCTCN2019077369-appb-000064
轻链可变区(VL)SEQ ID NO.:50
Figure PCTCN2019077369-appb-000065
参照实施例4-8中的方法，检测上述制备的MEDI9447-κ以及mAb001c和mAb002c抗体的CD73-ECD蛋白的ELISA亲和力、重组人CD73酶的抑制活性、肿瘤细胞的结合亲和力、肿瘤细胞CD73酶的抑制活性，研究结果汇总于表-3。
表-3：抗体的测试活性
Figure PCTCN2019077369-appb-000066
同时，将制备的MEDI9447-κ抗体用于体内抗肿瘤活性试验，选取CD73-高表达的U87MG脑胶质瘤为体内肿瘤模型，将抗体与5x10 6个细胞混合后接种入裸鼠背部皮下(50μg抗体/瘤)，观察肿瘤生长和体重变化共31天。图26显示体内药效实验各组的肿瘤生长曲线，第31天的抗肿瘤活性汇总于表-4。
表-4：人源化抗体在裸鼠体内的抗肿瘤活性
Figure PCTCN2019077369-appb-000067
类似地，图27显示另一组人源化抗体在U87MG胶质瘤体内实验中的肿瘤生长曲线，第33天的抗肿瘤活性汇总于表-5。
表-5：人源化抗体在裸鼠体内的抗肿瘤活性
Figure PCTCN2019077369-appb-000068
Figure PCTCN2019077369-appb-000069
综上，本发明的CD73抗体具有很高的亲和力，与现有技术对比，本发明的Hu002c-系列、Hu001c-系列的人源化抗体均具有良好或更好的体外和/或体内抗肿瘤活性。
实施例21 筛选细胞表面高表达CD73的肿瘤细胞株用于ADC药效测定
采用1x10 5个肿瘤细胞与抗体mAb001c混匀(终浓度10μg/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200μLPBS中，通过流式细胞荧光分选仪(FACS)检测结合率。
检测结果如图38所示，mAb001c能特异性识别和结合CD73-高表达的肿瘤细胞，结合率荧光强度顺序依次为Calu-1、NCI-H1299、U87MG、Calu-6、NCI-H441、NCI-H292、SW1990、MDA-MB-231，而对CD73-低表达的肿瘤细胞MDA-MB-453、NCI-H460显示极微弱的结合荧光强度。
实施例22 CD73人源化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细胞铺于激光共聚焦培养皿中，于37℃培养16h后加入5μg/mL的CD73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 Fluor 488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，之后用激光共聚焦显微镜(20×)观察抗体的抗体内吞情况。
结果如图39所示，Hu001c-14、Hu001c-15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抗体-药物偶联物(ADC)，提示CD73-ADC将具有良好的ADC药物特性，可用于广谱和高特异性靶向CD73-阳性肿瘤治疗药物的前景。
实施例23 Hu001c14-vcMMAE、Hu001c14-BL20-MMAE的制备
在靶向CD73的人源化抗体Hu001c-14原液中加入PBS/EDTA(pH＝7.4)缓冲液使其浓度在20mg/ml，然后用2.6eq的TCEP于25℃还原2小时，取出后置于冰上冷却，未纯化直接加入6.0eq的mc-VC-PAB-MMAE(购自上海皓元化学，预先溶在DMA中)，0℃反应1小时，加半胱氨酸终止反应。采用G25脱盐柱除去过量的小分子，并置换至20mM枸橼酸-枸橼酸钠/6％蔗糖，pH 6.6的缓冲液中，经0.22微米孔径的过滤装置除菌，-80℃保存，所得抗 体偶联物命名为Hu001c14-vcMMAE。人源化抗体Hu001c-14的质谱图谱(图42)与其抗体偶联物Hu001c14-vcMMAE的HIC和质谱图谱(图40、图43)均表明，抗体经偶联反应后，形成了抗体偶联物Hu001c14-vcMMAE，偶联物的分子量与预期值相符，平均DAR值约为4.0。
将人源化抗体Hu001c-14原液置换至50mM磷酸二氢钠-磷酸氢二钠(NaH 2PO 4-Na 2HPO 4)/150mM氯化钠(NaCl)/2mM乙二胺四乙酸(EDTA)，pH 7.0的反应缓冲液中，使其浓度为10mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动4小时。将上述反应液冷却至20℃，加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物Ic-4(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20℃搅动2.0小时进行偶联。采用脱盐柱将偶联反应混合物用pH 7.5的Tris-盐酸/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0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为Hu001c14-BL20-MMAE。人源化抗体Hu001c-14的质谱图谱(图42)与其抗体偶联物Hu001c14-BL20-MMAE的HIC和质谱图谱(图41、图44)均表明，抗体Hu001c-14经偶联反应后，形成了抗体偶联物Hu001c14-BL20-MMAE，偶联物的分子量与预期值相符，DAR约为4.0。
实施例24 Hu001c15-vcMMAE、Hu001c15-BL20-MMAE的制备
在靶向CD73的人源化抗体Hu001c-15原液中加入PBS/EDTA(pH＝7.4)缓冲液使其浓度在20mg/ml，然后用2.6eq的TCEP于25℃还原2小时，取出后置于冰上冷却，未纯化直接加入6.0eq的mc-VC-PAB-MMAE(购自上海皓元化学，预先溶在DMA中)，0℃反应1小时，加半胱氨酸终止反应。采用G25脱盐柱除去过量的小分子，并置换至20mM枸橼酸-枸橼酸钠/6％蔗糖，pH 6.6的缓冲液中，经0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为Hu001c15-vcMMAE。抗体Hu001c-15的质谱图谱(图47)与其抗体偶联物Hu001c15-vcMMAE的HIC和质谱图谱(图45、图48)均表明，抗体Hu001c-15经偶联反应后，形成了抗体偶联物Hu001c15-vcMMAE，偶联物的分子量与预期值相符，平均DAR值约为4.0。
将Hu001c-15原液置换至50mM磷酸二氢钠-磷酸氢二钠(NaH 2PO 4-Na 2HPO 4)/150mM氯化钠(NaCl)/2mM乙二胺四乙酸(EDTA)，pH 7.0的反应缓冲液中，使其浓度为10mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动4小时。将上述反应液冷却至20℃,加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物Ic-4(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20℃搅动2.0小时进行偶联。采用脱盐柱将偶联反应混合物用pH 7.5的Tris-盐酸/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0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为Hu001c15-BL20-MMAE。抗体Hu001c-15的质谱图谱(图47)与其抗体偶联物Hu001C15-BL20-MMAE的HIC和质谱图谱(图46、图49)均表明，抗体Hu001c-15经偶联反应后，形成了抗体偶联物Hu001c15-BL20-MMAE，偶联物的分子量与预期值相符，DAR约为4.0。
实施例25 CD73抗体-药物偶联物(CD73-ADC)针对CD73高表达的肿瘤细胞的体外抗肿瘤活性
本实例所使用细胞系购自于美国典型培养物保藏中心(ATCC)或中国科学院细胞库,并按照相应的说明进行培养，包括：MDA-MB-453、Calu-1、U87MG、Calu-6、NCI-H441、NCI-H292、MDA-MB-231、PC9、HCC827、NCI-H1975。将上述处于对数生长期的细胞，分别以每孔800-2500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5-12h后，分别加入不同浓度的CD73-ADCs，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5-6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 5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IC 50值。
四种优选的人源化CD73-ADC：Hu001c14-BL20-MMAE、Hu001c14-vcMMAE、Hu001c15-BL20-MMAE、Hu001c15-vcMMAE的体外抗肿瘤活性结果分别如图50至图59所示。
如图50所示，CD73-ADCs对CD73低表达的细胞MDA-MB-453的增殖抑制作用不明显，而对CD73高表达的Calu-1(图51)、U87MG(图52)、Calu-6(图53)、NCI-H441(图54)、NCI-H292(图55)、MDA-MB-231(图56)、PC9(图57)、HCC827(图58)、NCI-H1975(图59)均显示很强的细胞增殖抑制作用。
总体而言，CD73-ADC的细胞毒性(IC 50值)表明CD73-药物偶联物的细胞毒活性与受试细胞的CD73表达水平直接相关，因此判断为为CD73靶标特异性细胞毒性(图60)。表-6汇总了部分细胞增殖抑制测试的IC 50值。
表-6：CD73人源化抗体-药物偶联物的体外抗肿瘤活性
Figure PCTCN2019077369-appb-000070
实施例26 CD73-ADC对人T淋巴细胞增殖的影响
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/mLCD3/CD28抗体和100IU/mLIL-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。
先是观查对T细胞增殖曲线的影响，将CFSE-标记的T细胞铺至96孔板中(5000个细胞/孔)，加入溶媒(缓冲液)、CD73抗体、CD73-ADC、对照hIgG1-ADC(均为10nM)，或加入0.3mM腺苷单磷酸(AMP)，培养后第3、6、9、12天采用流式细胞分选仪(FACS)读取并统计活细胞数，绘制生长曲线。如图61所示，与溶媒和hIgG1-BL20-MMAE相比，10nM的Hu001c14-BL20-MMAE并未明显改变T细胞的增殖曲线。但是，与预期的一致，AMP显著降低了T细胞的增殖率。
然后在另一组试验中，将CFSE-标记的T细胞铺至96孔板中(2x10 4个细胞/孔),每孔加入按照梯度稀释的CD73-ADC(终浓度100nM～0.00128nM，n＝3)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IC 50值。如图62所示，Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE在受试浓度区间未显示明显的毒副作用(IC 50>100nM)。
实施例27 CD73-ADC的体内抗肿瘤活性
分别将200μL含有5×10 6U87MG、NCI-H441、NCI-H292的细胞悬液接种到免疫缺陷小鼠(Balb/c,nude)背部皮下。待肿瘤体积长至100～300mm 3,根据肿瘤体积大小及裸鼠体重随机分组(n＝8),分别采用5mg/kg、3mg/kg、1mg/kg、0.3mg/kg剂量，每周尾静脉给药一次，共计给药2周；同时设置hIgG(hIgG1-MMAE)作为阴性对照，15mg/kg多烯紫杉醇(Docetaxel)作为阳性对照。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。肿瘤体积(V)计算公式为：V＝1/2×a×b 2，其中a、b分别表示肿瘤的长、宽。
三种优选的人源化CD73-ADC：Hu001c14-BL20-MMAE、Hu001c14-vcMMAE、Hu001c15-BL20-MMAE的体内抗肿瘤活性结果分别如图63至图67所示。
如图63所示，5mg/kg的Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE能完全抑制CD73高表达的U87MG肿瘤的生长，肿瘤消退，停药>30天未见肿瘤回复生长。
如图64所示，在U87MG肿瘤模型中，Hu001c14-BL20-MMAE在给药3mg/kg、1mg/kg时呈剂量相关的治疗效果。并且在同等3mg/kg剂量下，Hu001c14-BL20-MMAE相比Hu001c14-vcMMAE显示出更强的抗肿瘤活性，说明BL20-MMAE连接子更具优越性。
如图65所示，在NCI-H441肿瘤模型中，Hu001c14-BL20-MMAE在给药3mg/kg、1mg/kg后均能导致明显的肿瘤消退，说明NCI-H441肿瘤对CD73-ADC的高度敏感性。
如图66所示，在NCI-H441肿瘤模型中，Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE在给药1mg/kg后均可导致明显的肿瘤消退，给药0.3mg/kg后依然具有明显的抗肿瘤活性，更明确了NCI-H441肿瘤对CD73-ADC的高度敏感性。
如图67所示，在NCI-H292肿瘤模型中，Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE在给药3mg/kg、1mg/kg后均能完全抑制肿瘤的生长(图67A)。在另一组独立的肿瘤消退试验中，待肿瘤体积达到～700mm 3时给药，5mg/kg Hu001c14-BL20-MMAE的治疗效果明显优于15mg/kg Docetaxel(图67B)。
实施例28 CD73人源化抗体对食蟹猴CD73酶活性的抑制作用
采用食蟹猴cynomolgus CD73/NT5E胞外区序列(EHH53214.1；Met1-Lys547)制备获得碳末端多组氨酸标记的重组食蟹猴CD73酶，具体氨基酸序列如SEQ ID No.:48所示。
将表2中的人源化抗体进行梯度稀释，按照实施例5的方法测定抗体对重组食蟹猴CD73酶活性的影响。
试验结果如图68、图69所示，所述2组人源化抗体均对食蟹猴CD73酶具有预期的的抑制效果，IC 50值的范围与其针对人CD73酶的抑制活性大致相当。
实施例29 CD73-ADC针对食蟹猴的安全性试验
采用雌性食蟹猴2只，单次静脉输注给予3mg/kg的Hu001c14-vcMMAE(受试物编号为FD114-ADC)后连续观察21天，第22天再次静脉输注给予6mg/kg的Hu001c14-vcMMAE(受试物编号为FD114-ADC)，再次连续观察21天后(总第42天后)安乐死并解剖。试验期间对如下指标进行了评价：笼旁观察、体重、耗食量、血液学、血凝、血浆生化、免疫表型(仅6mg/kg给药后第21天检测)、肉眼形态学观察以及生物分析。
结果显示在各给药剂量下，动物在临床症状、体重、耗食量和肉眼形态学方面均未见明显药物相关性改变。血液学指标：3和6mg/kg给药后第7天均可见RBC、HGB和HCT轻微减少，RET、WBC及其分类(主要为NEUT、MONO和EOS)明显减少；3mg/kg给药后第14天和第21天仍可见RBC、HGB和HCT轻微减少，6mg/kg给药后第14天和/或第21天上述改变完全恢复或有恢复趋势(图70)；血凝指标：3mg/kg给药后第14天可见2/2动物FIB一过性轻微增加；6mg/kg给药后1/2动物可见FIB轻微增加，至给药后第21天可见恢复趋势(图71)；血浆生化指标：3和6mg/kg给药后均未见明显药物相关性血浆生化改变(图72)；免疫表型：6mg/kg给药后第21天1/2动物可见CD3+CD4+轻微增加，CD3-CD20+轻微减少。总体而言，在本试验条件下，食蟹猴单剂量递增方式静脉输注3和6mg/kg的Hu001c14-vcMMAE，动物均能较好耐受，给药后主要可见红系/粒系细胞数量的减少以及纤维蛋白原的增加，停药后可恢复。最大耐受剂量(MTD)大于6mg/kg。
综上，CD73-ADC相关实施例的研究明确表明：
1、CD73人源化抗体-药物偶联物Hu001c14-vcMMAE、Hu001c15-vcMMAE、Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE均具有良好的CD73特异的肿瘤细胞杀伤活性，即针对CD73-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人源化抗体-药物偶联物Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE针对正常人的T淋巴细胞的细胞增殖无明显毒性；
3、与传统mcVC-PAB交联技术的Hu001c14-vcMMAE、Hu001c15-vcMMAE相比，采用本发明新型连接子所制备的Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE具有相当或更高的抗肿瘤活性(表-6)。
4、基于新型连接子更优异的均一性和稳定性，Hu001c14-BL20-MMAE、Hu001c15-BL20-MMAE均显示出更低的非特异性(靶头脱落所致)的毒副作用，例如，其对CD73-低表达的MDA-MB-453的细胞毒IC 50值进一步升高(图50、表-6)。
5、基于Hu001c14-vcMMAE静脉注射给予食蟹猴(3mg/kg、6mg/kg)的初步毒理试验，CD73-ADC显示了符合预期和可控的安全性，因此具有临床应用的潜在前景。
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。
Figure PCTCN2019077369-appb-000071
Figure PCTCN2019077369-appb-000072
Figure PCTCN2019077369-appb-000073
Figure PCTCN2019077369-appb-000074

Claims (18)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.:10所示的CDR1，
    SEQ ID NO.:11所示的CDR2，和
    SEQ ID NO.:12所示的CDR3；
    或者，
    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，
    SEQ ID NO.:21的CDR1，
    SEQ ID NO.:22所示的CDR2，和
    SEQ ID NO.:23所示的CDR3，
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    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，
    SEQ ID NO.:4所示的CDR1'，
    SEQ ID NO.:5所示的CDR2'，和
    SEQ ID NO.:6所示的CDR3'；
    或者，
    SEQ ID NO.:24所示的CDR1'，
    SEQ ID NO.:25所示的CDR2'，和
    SEQ ID NO.:26所示的CDR3'；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CD73结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 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所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  7. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于CD73的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
  8. 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7所述的CAR构建物。
  9. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  10. 如权利要求9所述的抗体-药物偶联物，其特征在于，所述抗体药物偶联物ADC如下分子式所示：
    Figure PCTCN2019077369-appb-100001
    其中：
    Ab是抗CD73的抗体，
    LU是接头(又称连接子)；
    D是药物；
    而且下标p是选自1-10，较佳地1-8的值。
  11. 如权利要求10所述的抗体-药物偶联物，其特征在于，LU选自下组：6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)、6-马来酰亚氨基己酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MC-ala-phe-PAB)、马来酰亚氨基丙酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MP-val-cit-PAB)、马来酰亚氨基丙酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MP-ala-phe-PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫基)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、4-(2-吡啶基二硫代)丁酸-N-羟基琥珀酰亚胺酯(SPDB)或N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)和双取代马来酰亚胺类连接子。
  12. 如权利要求10所述的抗体-药物偶联物，其特征在于，其中D选自下组：
    (i)美登素衍生物(DM1,DM4)，auristatin和多拉司他汀；
    (ii)Monomethyl auristatin E(MMAE)，Monomethylauristatin F(MMAF)，Monomethyl  Dolastatin 10(MMAD)类衍生物或其组合；和
    (iii)DNA损伤药物，较佳地，所述的DNA损伤药物包括多卡霉素、吡咯并[2,1-c][1,4]苯二氮卓(PBD)。
  13. 如权利要求9所述的抗体-药物偶联物，其特征在于，所述抗体的重链可变区序列选自下组：SEQ ID NO.:7、SEQ ID NO.:16、SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:27、SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:31、SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35、SEQ ID NO.:45、SEQ ID NO.:46、SEQ ID NO.:38、SEQ ID NO.:39、SEQ ID NO.:40、SEQ ID NO.:41；和/或
    所述的抗体的轻链可变区序列选自下组：SEQ ID NO.:8、SEQ ID NO.:9、SEQ ID NO.:19、SEQ ID NO.:20、SEQ ID NO.:30、SEQ ID NO.:36、SEQ ID NO.:47、SEQ ID NO.:37、SEQ ID NO.:42、SEQ ID NO.:43、SEQ ID NO.:44。
  14. 如权利要求9所述的抗体-药物偶联物，其特征在于，所述嵌合抗体选自下组：mAb001c、mAb001c-VK-SGS、mAb002c、mAb002c-VH-QG、mAb002c-VH-NA、mAb002c-VK-SG、mAb002c-VH-QG/VK-SG、mAb004c、mAb004c-VH-QG、mAb004c-VH-NA(说明书的表-1)；所述的人源化抗体选自下组：Hu001c-14、Hu001c-15、Hu001c-21、Hu001c-22、Hu001c-23、Hu001c-24、Hu001c-25、Hu001c-28、Hu001c-30、Hu001c-31、Hu001c-32、Hu002c-2、Hu002c-3、Hu002c-4、Hu002c-6、Hu002c-7、Hu002c-8、Hu002c-10、Hu002c-11、Hu002c-12、Hu002c-14、Hu002c-15、Hu002c-16(说明书的表-2)。
  15. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求6所述的重组蛋白、如权利要求8所述的免疫细胞、如权利要求9所述的抗体药物偶联物、或其组合，其特征在于，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。
  16. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于CD73的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
  17. 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7所述的CAR构建物。
  18. 一种药物组合物，其特征在于，它含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求6所述的重组蛋白、如权利要求8所述的免疫细胞、如权利要求9所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021138498A1 (en) 2020-01-03 2021-07-08 Incyte Corporation Cd73 inhibitor and a2a/a2b adenosine receptor inhibitor combination therapy
CN114380915A (zh) * 2020-10-19 2022-04-22 中山康方生物医药有限公司 抗cd73的抗体及其用途
WO2022147092A1 (en) 2020-12-29 2022-07-07 Incyte Corporation Combination therapy comprising a2a/a2b inhibitors, pd-1/pd-l1 inhibitors, and anti-cd73 antibodies
US11673894B2 (en) 2018-02-27 2023-06-13 Incyte Corporation Imidazopyrimidines and triazolopyrimidines as A2A / A2B inhibitors
WO2023174213A1 (zh) * 2022-03-14 2023-09-21 上海华奥泰生物药业股份有限公司 抗体药物偶联物及其应用
WO2023201267A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating trop-2 expressing cancers
CN114456267B (zh) * 2020-11-03 2023-12-01 浙江大学医学院附属第二医院 一种抗cd73人源化单克隆抗体及其应用
US20240002527A1 (en) * 2020-11-20 2024-01-04 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Anti-cd73 antibody, antibody-drug conjugate, and use thereof
US11873304B2 (en) 2018-05-18 2024-01-16 Incyte Corporation Fused pyrimidine derivatives as A2A/A2B inhibitors
US11884665B2 (en) 2019-01-29 2024-01-30 Incyte Corporation Pyrazolopyridines and triazolopyridines as A2A / A2B inhibitors
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
JP2024516581A (ja) * 2021-02-24 2024-04-16 蘇州近岸蛋白質科技股▲ふん▼有限公司 抗ヒトｃｄ７３抗体およびその適用
US11999740B2 (en) 2018-07-05 2024-06-04 Incyte Corporation Fused pyrazine derivatives as A2A / A2B inhibitors
US12018089B2 (en) 2020-01-03 2024-06-25 Incyte Corporation Anti-CD73 antibodies and uses thereof
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
WO2024251149A1 (zh) * 2023-06-05 2024-12-12 江苏迈威康新药研发有限公司 抗b7-h3的抗体或其片段以及靶向b7-h3的抗体药物偶联物
WO2025076127A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025137640A1 (en) 2023-12-22 2025-06-26 Gilead Sciences, Inc. Azaspiro wrn inhibitors
WO2025179294A2 (en) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US20160194407A1 (en) 2014-11-10 2016-07-07 Medimmune Limited Binding molecules specific for cd73 and uses thereof
CN107001474A (zh) * 2014-11-21 2017-08-01 百时美施贵宝公司 抗cd73抗体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US20160194407A1 (en) 2014-11-10 2016-07-07 Medimmune Limited Binding molecules specific for cd73 and uses thereof
CN107001474A (zh) * 2014-11-21 2017-08-01 百时美施贵宝公司 抗cd73抗体及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANCER RES., vol. 77, 2017, pages 5652
EXPERT REV ANTICANCER THER., vol. 17, 2017, pages 527
MUNSON ET AL., ANAL. BIOCHEM., vol. 107, 1980, pages 220
SAMBROOK ET AL.: "Molecule Clone: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS

Cited By (28)

* Cited by examiner, † Cited by third party
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US11673894B2 (en) 2018-02-27 2023-06-13 Incyte Corporation Imidazopyrimidines and triazolopyrimidines as A2A / A2B inhibitors
US11873304B2 (en) 2018-05-18 2024-01-16 Incyte Corporation Fused pyrimidine derivatives as A2A/A2B inhibitors
US11999740B2 (en) 2018-07-05 2024-06-04 Incyte Corporation Fused pyrazine derivatives as A2A / A2B inhibitors
US11884665B2 (en) 2019-01-29 2024-01-30 Incyte Corporation Pyrazolopyridines and triazolopyridines as A2A / A2B inhibitors
US12018089B2 (en) 2020-01-03 2024-06-25 Incyte Corporation Anti-CD73 antibodies and uses thereof
US12060433B2 (en) 2020-01-03 2024-08-13 Incyte Corporation CD73 inhibitor and A2A/A2B adenosine receptor inhibitor combination therapy
WO2021138498A1 (en) 2020-01-03 2021-07-08 Incyte Corporation Cd73 inhibitor and a2a/a2b adenosine receptor inhibitor combination therapy
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WO2022083049A1 (zh) * 2020-10-19 2022-04-28 中山康方生物医药有限公司 抗cd73的抗体及其用途
CN114380915A (zh) * 2020-10-19 2022-04-22 中山康方生物医药有限公司 抗cd73的抗体及其用途
CN114456267B (zh) * 2020-11-03 2023-12-01 浙江大学医学院附属第二医院 一种抗cd73人源化单克隆抗体及其应用
US20240002527A1 (en) * 2020-11-20 2024-01-04 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Anti-cd73 antibody, antibody-drug conjugate, and use thereof
WO2022147092A1 (en) 2020-12-29 2022-07-07 Incyte Corporation Combination therapy comprising a2a/a2b inhibitors, pd-1/pd-l1 inhibitors, and anti-cd73 antibodies
JP2024516581A (ja) * 2021-02-24 2024-04-16 蘇州近岸蛋白質科技股▲ふん▼有限公司 抗ヒトｃｄ７３抗体およびその適用
EP4299589A4 (en) * 2021-02-24 2025-06-11 Novoprotein Scientififc Inc. HUMAN ANTI-CD73 ANTIBODY AND ITS USE
JP7691720B2 (ja) 2021-02-24 2025-06-12 蘇州近岸蛋白質科技股▲ふん▼有限公司 抗ヒトｃｄ７３抗体およびその適用
WO2023174213A1 (zh) * 2022-03-14 2023-09-21 上海华奥泰生物药业股份有限公司 抗体药物偶联物及其应用
WO2023201267A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating trop-2 expressing cancers
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
WO2024251149A1 (zh) * 2023-06-05 2024-12-12 江苏迈威康新药研发有限公司 抗b7-h3的抗体或其片段以及靶向b7-h3的抗体药物偶联物
WO2025076127A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025137640A1 (en) 2023-12-22 2025-06-26 Gilead Sciences, Inc. Azaspiro wrn inhibitors
WO2025179294A2 (en) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles

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