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WO2025040000A1 - Conjugué anticorps anti-5t4-médicament et son utilisation - Google Patents

Conjugué anticorps anti-5t4-médicament et son utilisation Download PDF

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Publication number
WO2025040000A1
WO2025040000A1 PCT/CN2024/112592 CN2024112592W WO2025040000A1 WO 2025040000 A1 WO2025040000 A1 WO 2025040000A1 CN 2024112592 W CN2024112592 W CN 2024112592W WO 2025040000 A1 WO2025040000 A1 WO 2025040000A1
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Prior art keywords
antibody
cancer
solvate
variable region
drug conjugate
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PCT/CN2024/112592
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English (en)
Chinese (zh)
Inventor
王延春
李鸿峰
刘文超
刘芳
巩文词
蔡知见
杨柳
高珊
蒋雯卿
方磊
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Shanghai Miracogen Inc
Concept to Medicine Biotech Co Ltd
Lepu Biopharma Co Ltd
Original Assignee
Shanghai Miracogen Inc
Concept to Medicine Biotech Co Ltd
Lepu Biopharma Co Ltd
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Application filed by Shanghai Miracogen Inc, Concept to Medicine Biotech Co Ltd, Lepu Biopharma Co Ltd filed Critical Shanghai Miracogen Inc
Priority to CN202480003886.7A priority Critical patent/CN119816522B/zh
Priority to CN202510733151.6A priority patent/CN120361243A/zh
Publication of WO2025040000A1 publication Critical patent/WO2025040000A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a 5T4 antibody-drug conjugate and applications thereof.
  • Oncofetal protein 5T4 also known as Trophoblast glycoprotein (TPBG) and Wnt activation inhibitory factor 1 (WAIF1), is a highly glycosylated transmembrane glycoprotein encoded by the TPBG gene.
  • the molecular weight of TPBG protein is about 72kDa, containing 420 amino acids, composed of an extracellular domain of 310 amino acids, a transmembrane region of 20 amino acids and a cytoplasmic domain of 44 amino acids. It can affect the reconstruction of the cytoskeleton and the integrity of cells by binding to proteins containing PDZ domains in the cytoplasm.
  • 5T4 is closely related to different physiological and pathological processes, such as cell-to-cell connections, cell morphology and movement, cell adhesion ability, and cell membrane integrity. 5T4 is relatively widely expressed during embryonic development, while normal tissues only exist in certain special epithelia, such as basal stratified squamous epithelium, glandular and ductal epithelium, as well as retinal secondary neurons and olfactory bulbs.
  • 5T4 is highly expressed in many malignant solid tumors, including pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, non-small cell lung cancer, breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer and colorectal cancer.
  • pancreatic cancer prostate cancer, ovarian cancer, mesothelioma
  • non-small cell lung cancer breast cancer, head and neck cancer
  • cervical cancer cervical cancer
  • kidney cancer kidney cancer
  • gastric cancer and colorectal cancer in colon cancer, gastric cancer or ovarian cancer
  • the expression of 5T4 is as high as more than 95%.
  • 5T4 may exert its effects through the following three mechanisms: 1) epithelial-mesenchymal transition (EMT); 2) regulation of the CXCL12/CXCR4 biological axis; and 3) inhibition of Wnt signal transduction.
  • EMT epithelial-mesenchymal transition
  • CXCL12/CXCR4 biological axis regulation of the CXCL12/CXCR4 biological axis
  • Wnt signal transduction inhibition of Wnt signal transduction.
  • 5T4 is highly expressed in a variety of solid tumor tissues, but rarely exists in normal mature tissues, and its correlation with tumor metastasis and poor prognosis makes it one of the ideal targets for tumor drugs.
  • the clinical drugs under development involve a variety of immunotherapies including monoclonal antibodies, bispecific antibodies, trispecific antibodies, ADCs, tumor vaccines, CAR-raNK, etc.
  • the ADC drugs entering clinical research include PF-06263507 (A1-mcMMAF) developed by Pfizer, ASN-004 developed by Asana BioSciences, and SYD-1875 developed by Byondis BV.
  • PF-06263507 is composed of 5T4-specific humanized antibody PF-06281192, non-cleavable maleimidohexyl (mc) linker and microtubule inhibitor monomethyl auristatin F (MMAF).
  • ASN-004 is composed of a 5T4-specific single-chain scFv-Fc antibody, a Dolaflexin drug linker (Mersana Therapeutics), and a tubulin inhibitor auristatin derivative (AF-HPA).
  • SYD-1875 is composed of a 5T4-specific engineered cysteine residue antibody HCP41C and a linker payload vc-seco-DUBA.
  • the inventors of the present application prepared a new anti-5T4 antibody-drug conjugate (ADC) through a large number of experiments and creative work, and confirmed that it has good biological activity, thereby completing the present invention.
  • ADC anti-5T4 antibody-drug conjugate
  • the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody drug conjugate has a structure as shown in Formula I,
  • Ab is an anti-5T4 antibody or an antigen-binding fragment thereof, wherein the anti-5T4 antibody comprises a heavy chain variable region and a light chain variable region,
  • the heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 3,
  • the heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 4,
  • the heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 5,
  • the light chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 6,
  • the light chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 7;
  • the light chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 8;
  • D is cytotoxin
  • p is any value between 1 and 10 (e.g., 1, 1.5, 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.
  • LD indicates that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule
  • Ab-(LD) p indicates that p LD molecules are coupled to Ab by a covalent bond.
  • the drug-antibody ratio refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I).
  • the number of drug molecules contained in the antibody-drug conjugate described herein may be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody.
  • "p is any value between 1-10” which means that p can be any integer selected from 1-10 (including endpoints 1 and 10), or any decimal selected from 1-10.
  • the DAR values of antibody-drug conjugates prepared in different batches may not be exactly the same, for example, they may fluctuate within a range of no more than 0.5.
  • the drug-antibody ratio can be determined by conventional means, such as mass spectrometry, ELISA assays, HIC, and HPLC.
  • the quantitative distribution of ADC in terms of p can also be determined.
  • separation, purification, and verification of homogenous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as HIC, reverse phase HPLC, or electrophoresis.
  • the linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).
  • the linker is selected from the group consisting of: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).
  • the cytotoxin is selected from: topoisomerase inhibitors, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, gemcitabine, maytansine alkaloids (such as Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (such as duocarmycin), doxorubicin, ricin, diphtheria toxin, Duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.
  • MMAE Monomethyl auristatin E
  • MMAF Monomethyl auristatin F
  • SN-38 gemcitabine
  • maytansine alkaloids such as Maytansine DM1, Maytansine DM4
  • calicheamicin such as duocarmycin
  • MGBA such as duocarmycin
  • doxorubicin doxorubicin
  • ricin diphtheria toxin
  • the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.
  • exemplary topoisomerase I inhibitors include camptothecin and its derivatives.
  • Exemplary camptothecin derivatives include Exatecan, Belotecan, SN-38, Topotecan, Irinotecan (cpt-11), Deruxtecan, etc.
  • the cytotoxin is selected from: Exatecan (abbreviated as Exa), Monomethyl auristatin E (MMAE), and Monomethyl auristatin F (MMAF).
  • Exa Exatecan
  • MMAE Monomethyl auristatin E
  • MMAF Monomethyl auristatin F
  • L-D is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF.
  • the structural formula of LD is as follows:
  • the succinimide at the end of the LD is coupled to the thiol group in the antibody.
  • the structural formula of the formed ADC is as follows:
  • the antibody Ab is connected to the carbon atom of the succinimide at the end of L-D through -S-, and the -S- is not a thiol group introduced into Ab separately, but a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.
  • the sequence of the heavy chain variable region CDR1 of the anti-5T4 antibody is as shown in SEQ ID NO: 3
  • the sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO: 4
  • the sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO: 5
  • the sequence of the light chain variable region CDR1 is as shown in SEQ ID NO: 6
  • the sequence of the light chain variable region CDR2 is as shown in SEQ ID NO: 7
  • the sequence of the light chain variable region CDR3 is as shown in SEQ ID NO: 8.
  • sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 1
  • sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 2.
  • sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 9
  • sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 10.
  • sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 11
  • sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 10.
  • the light chain constant region of the anti-5T4 antibody is selected from a human lambda constant region, kappa constant region or a mutant of the above constant region.
  • p is any number between 3-8 (eg, 3.8, 4.1, 4.2, 7.9, or 8.0).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
  • the chemotherapy drug is, for example, doxorubicin (Adriamycin), cyclophosphamide, taxanes [such as paclitaxel (Taxol), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Furlong, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.
  • doxorubicin Adriamycin
  • cyclophosphamide taxanes [such as paclitaxel (Taxol),
  • the immunotherapy drug is, for example, PD-1 monoclonal antibody (e.g., pembrolizumab, nivolumab), PD-L1 monoclonal antibody (e.g., atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclonal antibody (e.g., ramucirumab, apatinib), HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), etc., or a combination thereof.
  • PD-1 monoclonal antibody e.g., pembrolizumab, nivolumab
  • PD-L1 monoclonal antibody e.g., atezolizumab
  • TIGIT monoclonal antibody e.g., 4-1BB monoclonal antibody
  • VEGFR2 monoclonal antibody e.g.,
  • the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide.
  • glucocorticoids such as cortisone and prednisone
  • microbial metabolites such as cyclosporine and tacrolimus
  • antimetabolites such as azathioprine and 6-mercaptopurine
  • polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3
  • alkylating agents such as cyclophosphamide.
  • the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prograf, zenapax, sulelac, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • the present invention provides the use of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and/or treating a disease associated with 5T4.
  • the present invention provides the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition for use in preventing and/or treating diseases associated with 5T4.
  • the present invention provides a method for treating and/or preventing a disease associated with 5T4, comprising: administering to a subject in need thereof an effective amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt, or the aforementioned pharmaceutical composition.
  • the disease associated with 5T4 is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.
  • the disease associated with 5T4 is breast cancer, non-small cell lung cancer, or colorectal cancer.
  • the present invention provides an in vitro non-therapeutic method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition, wherein the tumor is a tumor expressing 5T4.
  • the 5T4-expressing tumor is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.
  • the 5T4-expressing tumor is selected from breast cancer, non-small cell lung cancer, and colorectal cancer.
  • the newly developed 5T4-ADC of the present invention exhibits a strong cell-killing effect in various tumor cells (such as breast cancer and non-small cell lung cancer).
  • the newly developed 5T4-ADC of the present invention showed significant pharmacological effects in inhibiting tumor cell growth in various tumor models (such as the human colorectal cancer nude mouse CRC#047PDX model).
  • FIG1-1 shows the binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein;
  • Figure 1-2 shows the binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells
  • Figures 1-3 show the activity of humanized antibodies 14G12z28 and 14G12z43 in binding to human 5T4-His protein and CHOK1-hu5T4 cells, as well as the activity of humanized antibody 14G12z28 in binding to MCF-7 cells;
  • FIG2 shows a hydrophobic interaction chromatogram of 14G12-vcMMAE
  • FIG3 shows a hydrophobic interaction chromatogram of 14G12-MC-MMAF
  • FIG4 shows a hydrophobic interaction chromatogram of 14G12z28-MC-MMAF
  • FIG5 shows a hydrophobic interaction chromatogram of 14G12z43-MC-MMAF
  • FIG6 shows a hydrophobic interaction chromatogram of 14G12z28-MC-AAN-Exa
  • FIG7 shows a hydrophobic interaction chromatogram of 14G12z28-MCC-AAQ-Exa
  • FIG8 shows the binding affinity of each test substance to tumor cells NCI-H1975
  • FIG9 shows the binding affinity of each test substance to tumor cells HCT116
  • FIG10 shows the internalization results of each test substance in HCT116 cells
  • FIG11 is a representative graph showing the killing of tumor cells MCF7 by different 5T4-ADCs
  • FIG12 is a representative graph showing the killing effect of different 5T4-ADCs on tumor cells NCI-H1975;
  • FIG13 is a representative graph showing the killing of tumor cells MDA-MB-468 by different 5T4-ADCs
  • FIG14 shows the in vivo anti-tumor activity of different 5T4-ADCs in the CRC#047PDX mouse model
  • FIG. 15 shows the effects of different 5T4-ADCs on body weight in CRC#047PDX mouse model.
  • any numerical range should be understood to include any value or any sub-range within the range.
  • the term "antibody” refers to an immunoglobulin molecule generally composed of two pairs of identical polypeptide chains (each pair having a "light” (L) chain and a "heavy” (H) chain).
  • the light chain of an antibody can be divided into two types: ⁇ and ⁇ .
  • the heavy chain can be divided into five types: ⁇ , ⁇ , ⁇ , ⁇ or ⁇ .
  • antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE.
  • the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of three domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain , CL .
  • the constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system.
  • the VH and VL regions can be further subdivided into regions of high variability called complementarity determining regions (CDRs).
  • variable regions of each heavy chain/light chain pair form the antibody binding site.
  • the assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md . (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.
  • humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins.
  • humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibodies are replaced by CDR residues from non-human species (donor antibodies) such as mice, rats, or rabbits having the desired specificity, affinity, and capacity.
  • telomeres in the CDR1, CDR2 and/or CDR3 regions of VH and/or VL can be mutated to improve one or more binding properties (e.g., affinity) of the antibody.
  • PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • the mutations in the CDRs are usually no more than one or two.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region
  • the term “Fab'fragment” means a fragment obtained after reducing the disulfide bonds connecting two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable region (e.g., a Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • the term "scFv” refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 - VL-linker-VH-COOH or NH2 - VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv may form a di-scFv, which refers to two or more single scFvs in series to form an antibody.
  • scFv may form a (scFv) 2 , which refers to two or more single scFvs in parallel to form an antibody.
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning generally understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody.
  • Single-domain antibodies are also called nanobodies.
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antibody antigen-binding fragments can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992); Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (see Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)).
  • Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the culture medium of recombinant host cells.
  • Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.
  • BLAST and BLAST 2.0 algorithms used to determine the percentage of sequence homology and sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively.
  • BLAST and BLAST 2.0 can be used to determine the percentage of amino acid sequence homology of the present invention, using, for example, the parameters described in the literature or the default parameters.
  • Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).
  • the mutant of the amino acid sequence refers to a sequence having a homology with the amino acid sequence of greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, such as a sequence having 3, 2 or 1 substituted, deleted or added amino acids.
  • the substituted, added or deleted amino acids do not exceed 3 amino acids. More preferably, the substituted, added or deleted amino acids do not exceed 2 amino acids. Most preferably, the substituted, added or deleted amino acids do not exceed 1 amino acid.
  • substitution variant is a variant in which at least one amino acid residue in the native sequence is removed and inserted into the same position by a different amino acid.
  • the substitution may be single, in which only one amino acid is substituted in the molecule, or multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions may be located at consecutive sites.
  • an amino acid may be substituted by multiple residues, in which such variants include both substitutions and insertions.
  • An “insertion” (or “addition”) variant is a variant in which one or more amino acids are inserted into an amino acid at a specific position adjacent to a native sequence. Adjacent amino acids means connected to the ⁇ -carboxyl or ⁇ -amino functional group of the amino acid.
  • a “deletion” variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of the molecule.
  • antibodies do not contain many free and reactive cysteine thiol groups to which drug moieties can be attached; in fact, most cysteine thiol groups in antibodies exist as disulfide bridges.
  • antibodies can be reduced under partial or full reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • the term "pharmaceutically acceptable salt” refers to (i) a salt formed by an acidic functional group present in the conjugate provided by the present invention and a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, trieth
  • Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic substance with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic substance with a suitable base to provide a pharmaceutically acceptable cation.
  • solvate refers to these forms of the antibody drug conjugate of the present invention: the antibody drug conjugate forms a complex in a solid or liquid form by coordination with solvent molecules.
  • Hydrate is a specific form of solvate, which has coordinated water molecules.
  • hydrate is a preferred solvate.
  • the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations used.
  • the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to active ingredients. In addition to excipients, carriers, and stability improvement, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained and controlled release. They are important components that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products.
  • solvents propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.; according to their route of administration, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc.
  • the same pharmaceutical excipients can be used in drug preparations with different routes of administration and
  • the pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc.
  • the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.
  • the term "treat” generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and/or in terms of partially or completely stabilizing or curing a disease and/or As a side effect of a disease, it can be therapeutic.
  • treatment encompasses any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of the disease, i.e., preventing its development; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.
  • a vertebrate refers to a mammal.
  • Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats.
  • a mammal refers to a human.
  • an amount refers to an amount that is effective in achieving the desired therapeutic or preventive effect at the necessary dose and time.
  • the "therapeutically effective amount” of the substance/molecule of the present invention may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance/molecule to induce the desired response in the individual.
  • the therapeutically effective amount also encompasses the amount in which the therapeutic beneficial effects of the substance/molecule outweigh any toxic or harmful consequences.
  • Preventively effective amount refers to an amount that is effective in achieving the desired preventive effect at the necessary dose and time.
  • human 5T4-His protein was first used for primary immunization in SJL mice, Balb/c mice, and C57BL/6 mice, and then human 5T4-His protein or CHO-K1 cells overexpressing human 5T4 (CHOK1-hu5T4) were used for enhanced immunization in immunized mice.
  • the antibody titer against human 5T4-his protein in the sera of immunized mice was detected by ELISA and FACS experiments. Hybridoma cells were prepared from immunized mice with high serum antibody titers.
  • Table 1-1 14G12 antibody variable region sequence
  • the chimeric antibody 14G12 was constructed by linking the variable region sequences of the 14G12 antibody to a human IgG1/C ⁇ backbone.
  • the affinity characteristics of chimeric antibody 14G12 binding to human 5T4-His protein were detected by SPR method.
  • the chimeric antibody 14G12 was fixed to the Protein A chip, and the affinity of chimeric antibody 14G12 binding to human 5T4-His protein was detected by fitting the binding curve under different antigen concentration conditions, as shown in Table 1-4.
  • the binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein was detected by ELISA experiment. After the 96-well plate was coated with human 5T4-His protein or cynomolgus monkey 5T4-His protein, the chimeric antibody 14G12 with gradient dilution was added and incubated at room temperature for 60 minutes. After washing with PBST, mouse-anti-human IgG Fc antibody conjugated with HRP was added and incubated at room temperature for 30 minutes. After washing with PBST, TMB substrate was added and the OD 450nm absorbance was analyzed.
  • the binding affinity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein is shown in Table 1-2 and Figure 1-1.
  • the chimeric antibody 14G12 had comparable human 5T4-His binding activity ( FIG. 1-1A ) and significantly superior cynomolgus monkey 5T4-His binding activity ( FIG. 1-1B ).
  • the binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells or MCF-7 cells was detected by FACS experiment.
  • CHOK1-hu5T4 cells or MCF-7 cells growing in logarithmic phase were collected. After centrifugation, the cells were resuspended in FACS buffer and divided into 96-well U-bottom cell culture plates.
  • Antibodies diluted in FACS buffer were added and incubated at 4°C for 30 minutes. After washing the cells in the wells with FACS buffer, PE Goat anti-Human IgG Fc Secondary Antibody (eBioscience TM , Invitrogen) diluted in FACS buffer was added and incubated at 4°C for 30 minutes in the dark.
  • the chimeric antibody 14G12 has good binding affinity to CHOK1-hu5T4 cells.
  • variable region framework sequence of 14G12 antibody By comparing the variable region framework sequence of 14G12 antibody with the existing human IgG variable region framework sequence, a human sequence with a high degree of match was screened.
  • the 14G12 antibody CDR sequence was transplanted into the selected human IgG variable region framework sequence, and after back mutation, a series of 14G12 humanized antibodies were obtained.
  • the humanized antibodies 14G12z28 and 14G12z43 have comparable binding activity to the chimeric antibody 14G12 in terms of human 5T4-His protein ( Figure 1-3A, B), and slightly better binding activity to CHOK1-hu5T4 cells than the chimeric antibody 14G12 ( Figure 1-3C, D).
  • the humanized antibody 14G12z28 has good binding affinity to MCF-7 cells ( Figure 1-3E).
  • Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated by HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain intermediate 1; the specific steps are: Fmoc-Ala-OH (3 g, 1.0 eq) and HOSu (1.45 g, 1.3 eq) were added to the reaction bottle, 21 mL of THF was added, the temperature was controlled at room temperature, DCC (2.59 g, 1.3 eq) was slowly added under stirring, and then the reaction was carried out at room temperature, monitored by HPLC, and after the reaction was completed, the reaction solution was filtered and the filter cake was rinsed with THF (6 mL).
  • the intermediate 1 (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) is activated by HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain the intermediate 2; the specific steps are: adding the intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) into the reaction flask, controlling the temperature at room temperature, slowly adding DCC (0.61 g, 1.3 eq) under stirring conditions, reacting at room temperature, and monitoring by HPLC.
  • reaction solution is filtered, and the filter cake is rinsed with THF (2 mL).
  • Purified water (10 mL) was added to the filtrate, and L-Asn (0.34 g, 1.1 eq) and solid sodium bicarbonate (0.19 g, 1.0 eq) were added.
  • the reaction was stirred at room temperature and monitored by HPLC.
  • citric acid monohydrate (0.48 g, 1.0 eq) was added and stirred.
  • the reaction solution was concentrated to remove most of the solvent, and a purified residue was prepared. The prepared solution was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The organic phase was concentrated to dryness to obtain Intermediate 2.
  • the reaction formula is as follows:
  • the intermediate 3 is obtained; the specific steps are: add the intermediate 2 (100 mg) and DMF (1.5 mL) into the reaction bottle, control the temperature at room temperature, add DEA (300 ⁇ L) dropwise, react at room temperature, monitor by HPLC, until there is no residue of the intermediate 2, concentrate to remove DMF, add DCM (4 mL), purified water (4 mL), stir and separate, concentrate the aqueous phase to dryness to obtain the intermediate 3, the reaction formula is as follows:
  • Intermediate three reacts with 6-(maleimido)hexanoic acid succinimidyl ester (CAS No.: 55750-63-5) to obtain intermediate four; the specific steps are: adding intermediate three (92 mg, 1.0 eq), 6-(maleimido)hexanoic acid succinimidyl ester (135 mg, 1.3 eq), DMF (1.5 mL), DIPEA (0.059 mL, 1.0 eq) into a reaction flask, HPLC monitoring, after the reaction is completed, preparing and purifying, concentrating the preparation liquid, and obtaining intermediate four, the reaction formula is as follows:
  • the intermediate 4 and exotecan mesylate (CAS No.: 169869-90-3) are subjected to condensation reaction to obtain the product; the specific steps are: under room temperature, the intermediate 4 (26 mg, 1.0 eq) is added to the reaction bottle, DMF (1.5 mL) is added, and exotecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg,
  • reaction formula is as follows:
  • 5T4 antibody such as 14G12, 14G12z28 or 14G12z43
  • Adjust the pH of the antibody to about 7.5 with Tris-EDTA solution
  • use Nanodrop to detect protein concentration
  • weigh the net weight of the antibody solution and calculate the total amount of protein.
  • Add TCEP solution to the antibody place on a 3D shaker, react for more than 120 minutes at room temperature, and continuously mix to partially reduce the disulfide bonds between the antibody chains.
  • the protein concentration of the antibody drug conjugate was detected by UV/BCA method, and the DAR was detected by HIC (as shown in Figures 2 to 5).
  • the DARs were 3.8, 3.8, 4.2 and 4.1, respectively.
  • the purity was detected by SEC.
  • the protein concentration of the antibody drug conjugate was detected by UV/BCA method, and the DAR was detected by HIC (as shown in Figures 6 and 7).
  • the DARs were 8.0 and 8.0, respectively.
  • the purity was detected by SEC.
  • the chimeric antibody 14G12 and the humanized antibody 14G12z28 did not show significant changes in their binding affinity to NCI-H1975 or HCT116 cells expressing 5T4 before and after coupling with cytotoxins, indicating that the antibody and toxin coupling basically did not affect their cell binding activity.
  • the binding affinity of each test substance to tumor cells is shown in Table 2 and Figures 8 and 9 .
  • HCT116 colorectal cells growing in the logarithmic phase After centrifugation, wash the cells once, resuspend the cells in pre-cooled cell culture medium containing 3% FBS, adjust the cell density, and divide the cells into 96-well U-bottom cell culture plates so that each well contains 500,000 to 800,000 cells. Add antibodies or ADCs diluted in DMEM medium containing 3% FBS and mix well to make the final concentration of the sample 10 ⁇ g/mL. Incubate the 96-well plate on ice for 60min. Wash the cells in the wells thoroughly with pre-cooled FACS buffer (PBS+3% FBS) to remove unbound antibodies or ADCs.
  • pre-cooled FACS buffer PBS+3% FBS
  • the fluorescence signal of the cell samples was analyzed using CytoFLEX flow cytometer (Beckman Coulter), and MFI (GeoMean fluorescence intensity) was used to represent the fluorescence signal. Except for incubation, the whole experiment was kept on ice or at 4°C.
  • % reduction of cell surface molecules (MFI on ice - MFI at 37°C ) / MFI on ice * 100%.
  • MMAE or MMAF ADC Cultured in a cell culture incubator for 4 days (MMAE or MMAF ADC) or 6 days (Exatecan ADC), then added 1/10 of the well volume of PrestoBlue reagent (Invitrogen) and incubated in a cell culture incubator for 1h.
  • PrestoBlue reagent Invitrogen
  • the fluorescence signal was read using a SpectraMax M5 plate reader, and the excitation and emission wavelengths of the instrument were set to 560nm and 590nm, respectively.
  • the obtained fluorescence signal data was analyzed using SoftMax Pro 6.5 software.
  • Table 6 EC50 values of cell killing activity of different 5T4-ADCs in tumor cells
  • the establishment process of the human colorectal cancer nude mouse CRC#047PDX model was as follows: tumor tissue with a volume of about 30 mm 3 was transplanted subcutaneously on the right side of the back of BALB/c nude mice. When the tumor volume reached 200-300 mm 3 , the mice were grouped by random block method, and the day of grouping was Day 0. There were 6 mice in each group to ensure that the tumor volume between groups was uniform and the body weight was taken into account.
  • Non-binding-MC-AAN-Exa 10 mg/kg control administration group
  • 14G12z28-MC-AAN-Exa 10 and 3 mg/kg administration group.
  • the tail vein was administered once.
  • Non-binding means that the antibody is a non-binding human IgG1 isotype control, which has no binding to the target on the surface of the tumor cells used in the experiment and is used as a negative control antibody.
  • T/C (%) (RTV of drug administration group/RTV of vehicle group) ⁇ 100%.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
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  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente demande concerne un conjugué anticorps anti-5T4-médicament et son utilisation, et concerne spécifiquement un conjugué anticorps-médicament, ou un sel ou solvate pharmaceutiquement acceptable de celui-ci ou un solvate du sel. Le conjugué anticorps-médicament a une structure telle que représentée dans la formule I, Ab étant un anticorps anti-5T4. Le conjugué anticorps-médicament a une bonne activité d'inhibition de la croissance des cellules tumorales à la fois in vivo et in vitro, et a une bonne perspective d'application. Ab-(L-D)p Formule I
PCT/CN2024/112592 2023-08-18 2024-08-16 Conjugué anticorps anti-5t4-médicament et son utilisation Pending WO2025040000A1 (fr)

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CN202480003886.7A CN119816522B (zh) 2023-08-18 2024-08-16 5t4抗体药物偶联物及其应用
CN202510733151.6A CN120361243A (zh) 2023-08-18 2024-08-16 5t4抗体药物偶联物及其应用

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102282168A (zh) * 2008-11-18 2011-12-14 梅里麦克制药股份有限公司 人血清白蛋白接头以及其结合物
WO2017180813A1 (fr) * 2016-04-15 2017-10-19 Macrogenics, Inc. Nouvelles molécules de liaison à b7-h3, leurs conjugués anticorps-médicaments et leurs procédés d'utilisation
CN108187065A (zh) * 2017-12-29 2018-06-22 广东众生药业股份有限公司 抗5t4抗体与美登素衍生物dm4偶联复合物及制备方法和用途
CN108285487A (zh) * 2017-01-08 2018-07-17 浙江昭华生物医药有限公司 抗5t4抗体-药物偶联物及其应用
US20200369765A1 (en) * 2017-07-10 2020-11-26 Innate Pharma Siglec-9-neutralizing antibodies
CN115337406A (zh) * 2021-05-13 2022-11-15 清华大学 一种抗体药物偶联物及其制备方法和应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2596194T3 (es) * 2011-04-01 2017-01-05 Wyeth Llc Conjugados de anticuerpo-fármaco
EP4148070A4 (fr) * 2020-05-03 2024-04-24 Shanghai Miracogen Inc. Conjugué anticorps-médicament et sa préparation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102282168A (zh) * 2008-11-18 2011-12-14 梅里麦克制药股份有限公司 人血清白蛋白接头以及其结合物
WO2017180813A1 (fr) * 2016-04-15 2017-10-19 Macrogenics, Inc. Nouvelles molécules de liaison à b7-h3, leurs conjugués anticorps-médicaments et leurs procédés d'utilisation
CN108285487A (zh) * 2017-01-08 2018-07-17 浙江昭华生物医药有限公司 抗5t4抗体-药物偶联物及其应用
US20200369765A1 (en) * 2017-07-10 2020-11-26 Innate Pharma Siglec-9-neutralizing antibodies
CN108187065A (zh) * 2017-12-29 2018-06-22 广东众生药业股份有限公司 抗5t4抗体与美登素衍生物dm4偶联复合物及制备方法和用途
CN115337406A (zh) * 2021-05-13 2022-11-15 清华大学 一种抗体药物偶联物及其制备方法和应用

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