WO2019170131A1 - Anticorps cd73 ciblé et conjugué anticorps-médicament, procédé de préparation associé et utilisations correspondantes - Google Patents

Anticorps cd73 ciblé et conjugué anticorps-médicament, procédé de préparation associé et utilisations correspondantes 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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Prior art keywords
antibody
seq
variable region
chain variable
cells
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PCT/CN2019/077369
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English (en)
Chinese (zh)
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余科
金锐
刘亮
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复旦大学
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Priority claimed from CN201810506111.8A external-priority patent/CN110240654A/zh
Application filed by 复旦大学 filed Critical 复旦大学
Priority to US16/978,995 priority Critical patent/US20210024646A1/en
Priority to CN201980001728.7A priority patent/CN110869393B/zh
Priority to EP19763891.9A priority patent/EP3783025A4/fr
Priority to JP2020546414A priority patent/JP7330996B2/ja
Priority to CA3093327A priority patent/CA3093327C/fr
Publication of WO2019170131A1 publication Critical patent/WO2019170131A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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

La présente invention concerne un anticorps CD73 ciblé et un conjugué anticorps-médicament (ADC), un procédé de préparation associé et des utilisations correspondantes. L'invention concerne en outre un procédé de préparation de l'anticorps monoclonal et de l'ADC. L'anticorps monoclonal et l'ADC correspondant selon la présente invention peuvent être combinés de manière efficace et hautement spécifique avec la protéine CD73 purifiée et CD73 sur les surfaces de multiples cellules tumorales pour bloquer l'activité catalytique de l'enzyme CD73, et ont une affinité élevée, une faible immunogénicité et un effet anti-tumoral significatif.
PCT/CN2019/077369 2018-03-07 2019-03-07 Anticorps cd73 ciblé et conjugué anticorps-médicament, procédé de préparation associé et utilisations correspondantes WO2019170131A1 (fr)

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EP19763891.9A EP3783025A4 (fr) 2018-03-07 2019-03-07 Anticorps cd73 ciblé et conjugué anticorps-médicament, procédé de préparation associé et utilisations correspondantes
JP2020546414A JP7330996B2 (ja) 2018-03-07 2019-03-07 Ｃｄ７３を標的とする抗体および抗体－薬物複合体、その製造方法と使用
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WO2021138498A1 (fr) 2020-01-03 2021-07-08 Incyte Corporation Polythérapie à base d'un inhibiteur de cd73 et d'inhibiteurs du récepteur de l'adénosine a2a/a2b
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 (fr) 2020-12-29 2022-07-07 Incyte Corporation Polythérapie comprenant des inhibiteurs a2a/a2b, des inhibiteurs pd-1/pd-l1 et des anticorps anti-cd73
US11673894B2 (en) 2018-02-27 2023-06-13 Incyte Corporation Imidazopyrimidines and triazolopyrimidines as A2A / A2B inhibitors
WO2023174213A1 (fr) * 2022-03-14 2023-09-21 上海华奥泰生物药业股份有限公司 Conjugué anticorps-médicament et son utilisation
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2
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
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
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
US11999740B2 (en) 2021-10-13 2024-06-04 Incyte Corporation Fused pyrazine derivatives as A2A / A2B inhibitors

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Cited By (14)

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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
US11884665B2 (en) 2019-01-29 2024-01-30 Incyte Corporation Pyrazolopyridines and triazolopyridines as A2A / A2B inhibitors
WO2021138498A1 (fr) 2020-01-03 2021-07-08 Incyte Corporation Polythérapie à base d'un inhibiteur de cd73 et d'inhibiteurs du récepteur de l'adénosine a2a/a2b
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
WO2022083049A1 (fr) * 2020-10-19 2022-04-28 中山康方生物医药有限公司 Anticorps anti-cd73 et son utilisation
CN114380915B (zh) * 2020-10-19 2024-03-22 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
WO2022147092A1 (fr) 2020-12-29 2022-07-07 Incyte Corporation Polythérapie comprenant des inhibiteurs a2a/a2b, des inhibiteurs pd-1/pd-l1 et des anticorps anti-cd73
US11999740B2 (en) 2021-10-13 2024-06-04 Incyte Corporation Fused pyrazine derivatives as A2A / A2B inhibitors
WO2023174213A1 (fr) * 2022-03-14 2023-09-21 上海华奥泰生物药业股份有限公司 Conjugué anticorps-médicament et son utilisation
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo

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