WO2022194078A1 - 用于靶分子修饰的缀合物及其制备方法 - Google Patents

用于靶分子修饰的缀合物及其制备方法 Download PDF

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WO2022194078A1
WO2022194078A1 PCT/CN2022/080594 CN2022080594W WO2022194078A1 WO 2022194078 A1 WO2022194078 A1 WO 2022194078A1 CN 2022080594 W CN2022080594 W CN 2022080594W WO 2022194078 A1 WO2022194078 A1 WO 2022194078A1
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target molecule
targeting moiety
amino acid
conjugate
targeting
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PCT/CN2022/080594
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English (en)
French (fr)
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陈鹏
张衡
韩雨
林锋
林坚
杨远帆
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北京大学
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A61K47/62—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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/62—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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present application relates to the field of biomedicine, and in particular to a conjugate for target molecule modification and a preparation method thereof.
  • Proteolysis Targeting Chimera can simultaneously target intracellular tumor-associated proteins and E3 ligase to degrade intracellular proteins after entering cells.
  • PROTAC Proteolysis Targeting Chimera
  • the lysosome targeting chimera (LYTAC) technology couples the oligosaccharide targeting the cell surface receptor CI-M6PR to the antibody of the target protein through chemical reaction.
  • LYTAC lysosome targeting chimera
  • LYTAC The binding ability and degradation efficiency of LYTAC compounds to target molecules also need to be improved.
  • AbTAC has developed bispecific antibodies, in which one domain targets the endocytic receptor RNF43 and the other domain targets the target protein.
  • the preparation of uniform bispecific antibodies is challenging and the technology is limited. Expression of the endocytic receptor RNF43 in target cells. Therefore, it is urgent to develop a specific receptor-independent and efficient membrane protein degradation technology.
  • the present application provides a conjugate for target molecule modification and a preparation method thereof.
  • the conjugate used for the modification of the target molecule of the present application may have the characteristics of simple synthesis, single product, high binding ability to the target molecule and/or high degradation efficiency of the target molecule, and can be used for in vivo and/or in vitro modification of the target molecule. Specific degradation can be used for disease prevention and treatment.
  • the present application provides a conjugate comprising: a target molecule targeting moiety and a protein degrader targeting moiety; wherein the target molecule targeting moiety can specifically recognize the target molecule and be covalently with the target molecule
  • the protein degrader targeting moiety is directly or indirectly linked to the target molecule targeting moiety.
  • the targeting moiety of the target molecule comprises a first functional group
  • the target molecule comprises a second functional group
  • the first functional group can Reacts with the second functional group to form a covalent bond
  • the first functional group when the distance between the first functional group and the second functional group is less than about 5E-10 meters, the first functional group is capable of reacting with the second functional group to form a covalent bond.
  • the second functional group comprises an electrophilic group.
  • the second functional group comprises a side chain group of a natural amino acid.
  • the second functional group is selected from the group consisting of amino, imidazolyl, sulfhydryl, hydroxyl, carboxyl, methylthio, and derivatives of the foregoing.
  • the target molecule comprises a cell surface molecule, an intracellular molecule and/or an extracellular molecule.
  • the target molecule is selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the target molecule is selected from the group consisting of immune checkpoint molecules, tumor-associated molecules, and signaling pathway proteins.
  • the target molecule is selected from the group consisting of PD-L1 (apoptotic-ligand 1), EGFR (epidermal growth factor receptor), HER-2 (human epidermal growth factor receptor-2) ), Ras protein, CD38 (lymphocyte differentiation antigen CD38), and the aforementioned functional fragments.
  • the first functional group is selected from the group consisting of:
  • R 1 is O, N or absent
  • R 2 is halogen, alkenyl or alkynyl
  • R 3 is halogen, alkenyl or alkynyl
  • R 4 is halogen
  • n1 and n2 are each independently ground is at least 0.
  • the first functional group is selected from the group consisting of:
  • the targeting moiety of the target molecule comprises a residue of an unnatural amino acid comprising the first functional group selected from the group consisting of:
  • the targeting moiety of the target molecule comprises a residue of an unnatural amino acid comprising the first functional group, the unnatural amino acid being selected from the group consisting of fluorosulfate-L - Tyrosine, 6-bromo-hexanoyllysine, and derivatives of the foregoing.
  • the targeting moiety of the target molecule is selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the targeting portion of the target molecule comprises an antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of VHH, Fab, Fab', Fv fragment, (Fab') 2 , (Fab) 2 , scFv, di-scFv, affibody and dAbs.
  • the targeting portion of the target molecule comprises a single domain antibody or antigen-binding fragment thereof.
  • the targeting portion of the target molecule comprises an amino acid sequence that is at the 108th amino acid, the 110th amino acid and/or the 113th amino acid compared with the corresponding amino acid sequence shown in SEQ ID NO: 13
  • the first functional group is included.
  • the targeting moiety of the target molecule is capable of specifically recognizing the PD-L1.
  • the amino acid sequence contained in the targeting part of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the 108th amino acid and the 110th amino acid of the target molecule targeting part and/or amino acid at position 113 is replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid of the targeting portion of the target molecule is replaced by the 6- bromo-hexanoyl lysine replacement, amino acid 110 replaced by said 6-bromo-hexanoyl lysine, amino acid 113 replaced by said fluorosulfate-L-tyrosine and/or 108 The amino acid at position 1 was replaced by the fluorosulfate-L-tyrosine.
  • the targeting portion of the target molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
  • the targeting moiety of the target molecule comprises an amino acid sequence comprising the first functional group at the 116th amino acid compared to the corresponding amino acid sequence shown in SEQ ID NO: 18.
  • the targeting moiety of the target molecule is capable of specifically recognizing the EGFR.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule is replaced by the non-natural amino acid. Amino acid substitution.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule is fluorinated Sulfate-L-tyrosine substitution.
  • the targeting portion of the target molecule comprises the amino acid sequence shown in SEQ ID NO:12.
  • the protein degrader comprises lysosomes and/or proteasomes.
  • the protein degrader targeting moiety is selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the protein degrader targeting moiety comprises a molecule or a functionally active fragment thereof capable of targeting the lysosome or proteasome.
  • the protein degrader targeting moiety is selected from the group consisting of: lysosomal targeting peptide (LTP), oligosaccharide peptide, mannose 6-phosphate (M6P), N-acetylgalactose Amine (GalNAc), galactose, and any combination of the foregoing.
  • LTP lysosomal targeting peptide
  • M6P mannose 6-phosphate
  • GalNAc N-acetylgalactose Amine
  • galactose galactose
  • the protein degrader targeting moiety comprises the lysosomal targeting peptide, the amino acid sequence of the lysosomal targeting peptide is set forth in any one of SEQ ID NOs: 63-77 .
  • the conjugate further comprises a transmembrane moiety capable of facilitating the entry of the target molecule into cells, wherein the transmembrane moiety is directly or directly associated with the targeting moiety of the target molecule. Indirectly linked, and/or the transmembrane moiety is linked directly or indirectly to the protein degrader targeting moiety.
  • the transmembrane moiety is selected from the group consisting of penetrating peptide (CPP), oligosaccharide peptide, mannose 6-phosphate (M6P), N-acetylgalactosamine (GalNAc), galactosamine Lactose, and any combination of the foregoing.
  • CPP penetrating peptide
  • M6P mannose 6-phosphate
  • GalNAc N-acetylgalactosamine
  • galactosamine Lactose galactosamine Lactose
  • the transmembrane moiety comprises polyarginine, and the number of repeating units of the polyarginine is at least 2.
  • the polymeric monomers of polyarginine comprise D-configuration arginine and/or L-configuration arginine.
  • the conjugate comprises the penetrating peptide and the lysosome-targeting peptide, the penetrating peptide and the lysosome-targeting peptide being linked directly or indirectly.
  • the protein degrader targeting moiety comprises a molecule or a functionally active fragment thereof capable of targeting the lysosome or proteasome and/or capable of binding to a cell surface receptor that promotes endocytosis.
  • the protein degrader targeting moiety comprises a molecule capable of binding the mannose-6-phosphate receptor (M6PR).
  • M6PR mannose-6-phosphate receptor
  • the protein degrader targeting moiety comprises one or more mannose 6-phosphate (M6P) or functionally active fragments thereof.
  • M6P mannose 6-phosphate
  • the protein degrader targeting moiety comprises from about 1 to about 500 of the mannose 6-phosphate or functionally active fragment thereof.
  • the protein degrader targeting moiety comprises a scaffold molecule directly or indirectly linked to the one or more mannose 6-phosphate or functionally active fragments thereof.
  • the scaffold molecule comprises a glycan structure.
  • the scaffold molecule comprises a glycoprotein comprising one or more amino acids modified with the one or more mannose 6-phosphate or functionally active fragments thereof.
  • the glycoprotein comprises a group of N-carboxy anhydride (NCA) or a derivative thereof.
  • NCA N-carboxy anhydride
  • the protein degrader targeting moiety comprises a molecule capable of binding the asialoglycoprotein receptor (ASGPR) or a functionally active fragment thereof.
  • ASGPR asialoglycoprotein receptor
  • the protein degrader targeting moiety comprises one or more ligands of the asialoglycoprotein receptor.
  • the protein degrader targeting moiety comprises one or more N-acetylgalactosamine (GalNAc).
  • the protein degrader targeting moiety comprises one or more galactoses.
  • the protein degrader targeting moiety comprises one or more glucoses.
  • the protein degrader targeting moiety comprises from about 1 to about 500 ligands of the asialoglycoprotein receptor.
  • the protein degrader targeting moiety comprises a scaffold molecule directly or indirectly linked to the one or more ligands of the asialoglycoprotein receptor.
  • the protein degrader targeting moiety comprises the one or more N-acetylgalactosamine-modified one or more alanines.
  • the scaffold molecule comprises one, two or three of the N-acetylgalactosamine.
  • the scaffold molecule comprises one, two or three of the galactose.
  • the conjugate comprises a fusion protein.
  • the conjugate comprises the target molecule targeting moiety, the protein degrader targeting moiety and the transmembrane moiety, the target molecule targeting moiety, the protein degrader target
  • the facing portion and the transmembrane portion are directly or indirectly connected independently of each other.
  • the indirect linking comprises linking through a linker.
  • the linker comprises a peptide linker.
  • the linker comprises a cleavable or non-cleavable linker.
  • the cleavable linker comprises a linker cleavable by a protease.
  • the conjugate comprises the targeting moiety of the target molecule, the penetrating peptide, and the lysosomal targeting peptide.
  • the present application provides a polypeptide comprising a targeting moiety of a target molecule capable of specifically recognizing the target molecule and covalently binding to the target molecule.
  • the targeting moiety of the target molecule comprises a first functional group
  • the target molecule comprises a second functional group
  • the first functional group can Reacts with the second functional group to form a covalent bond
  • the first functional group when the distance between the first functional group and the second functional group is less than about 5E-10 meters, the first functional group is capable of reacting with the second functional group to form a covalent bond.
  • the second functional group comprises an electrophilic group.
  • the second functional group comprises a side chain group of a natural amino acid.
  • the second functional group is selected from the group consisting of amino, imidazolyl, sulfhydryl, hydroxyl, carboxyl, methylthio, and derivatives of the foregoing.
  • the target molecule comprises a cell surface molecule, an intracellular molecule and/or an extracellular molecule.
  • the target molecule is selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the target molecule is selected from the group consisting of immune checkpoint molecules, tumor-associated molecules, and signaling pathway proteins.
  • the target molecule is selected from the group consisting of PD-L1 (apoptotic-ligand 1), EGFR (epidermal growth factor receptor), HER-2 (human epidermal growth factor receptor-2) ), Ras protein, CD38 (lymphocyte differentiation antigen CD38), and the aforementioned functional fragments.
  • the first functional group is selected from the group consisting of:
  • R 1 is O, N or absent
  • R 2 is halogen, alkenyl or alkynyl
  • R 3 is halogen, alkenyl or alkynyl
  • R 4 is halogen
  • n1 and n2 are each independently ground is at least 0.
  • the first functional group is selected from the group consisting of:
  • the targeting moiety of the target molecule comprises a residue of an unnatural amino acid comprising the first functional group selected from the group consisting of:
  • the targeting moiety of the target molecule comprises a residue of an unnatural amino acid comprising the first functional group, the unnatural amino acid being selected from the group consisting of fluorosulfate-L - Tyrosine, 6-bromo-hexanoyllysine, and derivatives of the foregoing.
  • the targeting moiety of the target molecule is selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the targeting portion of the target molecule comprises an antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of VHH, Fab, Fab', Fv fragment, (Fab') 2 , (Fab) 2 , scFv, di-scFv, affibody and dAbs.
  • the targeting portion of the target molecule comprises a single domain antibody or antigen-binding fragment thereof.
  • the targeting portion of the target molecule comprises an amino acid sequence that is at the 108th amino acid, the 110th amino acid and/or the 113th amino acid compared with the corresponding amino acid sequence shown in SEQ ID NO: 13
  • the first functional group is included.
  • the targeting moiety of the target molecule is capable of specifically recognizing the PD-L1.
  • the amino acid sequence contained in the targeting part of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the 108th amino acid and the 110th amino acid of the target molecule targeting part and/or amino acid at position 113 is replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid of the targeting portion of the target molecule is replaced by the 6- bromo-hexanoyl lysine replacement, amino acid 110 replaced by said 6-bromo-hexanoyl lysine, amino acid 113 replaced by said fluorosulfate-L-tyrosine and/or 108 The amino acid at position 1 was replaced by the fluorosulfate-L-tyrosine.
  • the targeting portion of the target molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
  • the targeting moiety of the target molecule comprises an amino acid sequence comprising the first functional group at the 116th amino acid compared to the corresponding amino acid sequence shown in SEQ ID NO: 18.
  • the targeting moiety of the target molecule is capable of specifically recognizing the EGFR.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule is replaced by the non-natural amino acid. Amino acid substitution.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule is fluorinated Sulfate-L-tyrosine substitution.
  • the targeting portion of the target molecule comprises the amino acid sequence shown in SEQ ID NO:12.
  • the present application provides a nucleic acid molecule encoding a conjugate of the present application and/or a polypeptide of the present application.
  • the present application provides a vector comprising the nucleic acid molecule of the present application.
  • the present application provides an immunoconjugate comprising the conjugate of the present application and/or the polypeptide of the present application.
  • the application provides a cell comprising the conjugate of the application, the nucleic acid molecule of the application, the vector of the application, and/or the immunoconjugate of the application.
  • the application provides a composition comprising the conjugate of the application, the polypeptide of the application, the nucleic acid molecule of the application, the carrier of the application, the immunoconjugate of the application and/or the application cells, and optionally a pharmaceutically acceptable carrier.
  • the application provides a kit comprising the conjugate of the application, the polypeptide of the application, the nucleic acid molecule of the application, the carrier of the application, the immunoconjugate of the application, the cell of the application , and/or the composition of the present application.
  • the application provides conjugates of the application, polypeptides of the application, nucleic acid molecules of the application, vectors of the application, immunoconjugates of the application, cells of the application, compositions of the application, And/or the use of the kit of the present application in the preparation of medicaments for preventing and/or treating diseases and/or disorders.
  • the disease and/or disorder comprises a tumor.
  • the disease and/or disorder is selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, CD38 positive tumors and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder comprises a solid tumor.
  • the disease and/or disorder comprises breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the application provides conjugates of the application, polypeptides of the application, nucleic acid molecules of the application, vectors of the application, immunoconjugates of the application, cells of the application, compositions of the application, and/or kits of the present application for the prevention and/or treatment of diseases and/or disorders.
  • the disease and/or disorder comprises a tumor.
  • the disease and/or disorder is selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, CD38 positive tumors and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder comprises a solid tumor.
  • the disease and/or disorder comprises breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the present application provides a method of preventing and/or treating a disease and/or disorder, comprising administering to a subject in need thereof a conjugate of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, the present The carrier of the application, the immunoconjugate of the application, the cell of the application, the composition of the application, and/or the kit of the application.
  • the disease and/or disorder comprises a tumor.
  • the disease and/or disorder is selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, CD38 positive tumors and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder comprises a solid tumor and/or a hematological tumor.
  • the disease and/or disorder comprises breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the present application provides a method of affecting the content of a target molecule by administering a conjugate of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a carrier of the present application, an immunoconjugate of the present application, the present The cells of the application, the compositions of the application and/or the kits of the application.
  • the method comprises an in vitro method, an ex vivo method and/or an in vivo method.
  • the target molecule is a cell surface molecule and/or an intracellular molecule.
  • the target molecule is a molecule on the surface of skin cells, bone marrow cells, cervical cells, lung cells and/or breast cells.
  • the target molecule is a molecule on the surface of skin cancer cells, myeloma cells, cervical cancer cells, lung cancer cells and/or breast cancer cells.
  • the cells are selected from the group consisting of PD-L1 positive cells, EGFR positive cells, HER-2 positive cells, CD38 positive cells and cells with abnormal Ras function and/or activity.
  • the present application provides a method for preparing a conjugate of the present application and/or a polypeptide of the present application, comprising culturing a cell of the present application under conditions such that the conjugate and/or the polypeptide of the present application are expressed.
  • the targeting moiety of the target molecule and the protein degrader targeting moiety are linked by transpeptidase A (Srt A).
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93 and/or the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, wherein X is any amino acid.
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92 and/or the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93 and/or the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence , where X is any amino acid.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPETG as set forth in SEQ ID NO: 92 and/or the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence .
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPXTG as shown in SEQ ID NO: 93
  • the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) connects the targeting part of the target molecule and the targeting part of the protein degrader, wherein X is any amino acid.
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92
  • the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) links the targeting moiety of the target molecule with the targeting moiety of the protein degrader.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93
  • the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) connects the targeting part of the target molecule and the targeting part of the protein degrader, wherein X is any amino acid.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92
  • the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) links the targeting moiety of the target molecule with the targeting moiety of the protein degrader.
  • the present application provides a method for preparing a conjugate of the present application, comprising directly or indirectly linking the target molecule targeting moiety of the conjugate to the protein degrader targeting moiety.
  • the targeting moiety of the target molecule and the protein degrader targeting moiety are linked by transpeptidase A (Srt A).
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93 and/or the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, wherein X is any amino acid.
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92 and/or the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93 and/or the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence , where X is any amino acid.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPETG as set forth in SEQ ID NO: 92 and/or the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence .
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPXTG as shown in SEQ ID NO: 93
  • the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) connects the targeting part of the target molecule and the targeting part of the protein degrader, wherein X is any amino acid.
  • the C-terminus of the targeting moiety of the target molecule comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92
  • the N-terminus of the protein degrader targeting moiety comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) links the targeting moiety of the target molecule with the targeting moiety of the protein degrader.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPXTG as set forth in SEQ ID NO: 93
  • the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) connects the targeting part of the target molecule and the targeting part of the protein degrader, wherein X is any amino acid.
  • the C-terminus of the protein degrader targeting moiety comprises the amino acid sequence LPETG as shown in SEQ ID NO: 92
  • the N-terminus of the targeting moiety of the target molecule comprises an oligoglycine sequence, through which The transpeptidase A (Srt A) links the targeting moiety of the target molecule with the targeting moiety of the protein degrader.
  • Figure 1 shows a schematic diagram of a degradation pathway that can be achieved by the conjugates of the present application to degrade target molecules.
  • Figures 2A and 2B show primary mass spectrograms of wild-type single domain antibodies that can be used as target molecules to target partial precursors.
  • Figure 3A and Figure 3B show the mass spectra of the resulting compound library after different sites of PD-L1 single domain antibody were replaced by BrC6K or FSY.
  • Figure 4A and Figure 4B show the cross-linking results of the obtained compound library with the target molecule after different sites of PD-L1 single-domain antibody were replaced by BrC6K or FSY by SDS-PAGE analysis.
  • Figure 5A, Figure 5B and Figure 5C show the tandem mass spectrometry of the cross-linked peptide segment after the targeting moiety of the target molecule with high adjacent cross-linking reactivity is cross-linked with the target molecule.
  • Figure 6A, Figure 6B, Figure 6C and Figure 6D show the mass spectrograms of the targeting moieties of the resulting target molecules after different sites in PD-L1 single domain antibody or EGFR single domain antibody are replaced by BrC6K or FSY.
  • Figure 7A, Figure 7B, and Figure 7C show the results of in vitro crosslinking detection of the obtained target molecule targeting moiety and the target molecule after different sites in PD-L1 single domain antibody or EGFR single domain antibody are replaced by BrC6K or FSY.
  • Figure 8A, Figure 8B, Figure 8C and Figure 8D show the detection results of the binding of the targeting moiety of the target molecule to the cell surface target molecule.
  • Figure 9A and Figure 9B are schematic diagrams showing the preparation of the conjugate of the present application by linking the targeting moiety of the transpeptidase-mediated protein degrader and the targeting moiety of the target molecule.
  • Figure 10 shows the primary mass spectrometry of the conjugate GlueTAC FSY /PD-L1 obtained by the targeting moiety of PD-L1 target molecule ⁇ PD-L1 L108FSY and the targeting moiety of PD-L1 target molecule linked to the targeting moiety of the protein degrader picture.
  • Figure 11 shows the conjugate NbTAC-EGFR obtained by linking the protein degrader targeting moiety of a wild-type EGFR single domain antibody, and the conjugate GlueTAC-EGFR obtained by linking the EGFR target molecule targeting moiety to the protein degrader targeting moiety of the first-order mass spectrum.
  • Figure 12 shows the live cell confocal fluorescence imaging of the control group and the GlueTAC test group.
  • Figure 13A, Figure 13B, Figure 13C and Figure 13D show the results of immunoblotting assays to characterize the conjugates of the present application to promote the degradation of target molecules.
  • Figure 14 shows the results of immunoblotting assays to characterize the degradation of target molecules by the conjugates of the present application.
  • Figure 15 shows the results of application of conjugates to promote degradation of target molecules in the presence of ammonium chloride.
  • Figure 16 shows the result that the conjugate of the present application promotes the degradation of target molecules in tumor tissue in vivo.
  • the term "pharmaceutically acceptable carrier” generally refers to a pharmaceutically acceptable carrier, excipient or stabilizer which, at the doses and concentrations employed, is sufficient for the cells or mammals to which it is exposed. is non-toxic. Physiologically acceptable carriers may include suitable substances. A pharmaceutically acceptable carrier is usually not the same substance as a vector used to insert nucleic acids in genetic engineering.
  • tumor generally refers to any new pathological tissue proliferation. Tumor cells can spread to other parts of the body locally or through the bloodstream and lymphatic system.
  • the tumors may include benign tumors and malignant tumors.
  • the tumor may include a solid tumor.
  • the tumor may include cancer.
  • examples of such tumors include, but are not limited to, breast cancer.
  • solid tumor generally refers to a tangible tumor that can be detected by means of clinical examination (eg, X-ray irradiation, CT scan, B-ultrasound or palpation, etc.).
  • the tumor may comprise a neoplasm or solid lesion formed by abnormal cell growth.
  • conjugate generally refers to any substance formed by the joining together of two or more separate moieties.
  • a conjugate may comprise a substance formed by linking one segment of polypeptide to another segment or segments of polypeptide.
  • conjugates can be linked by covalent bonds.
  • the conjugate can be a substance formed by linking a single domain antibody to a penetrating peptide and a lysosomal targeting peptide by transpeptidase.
  • targeting moiety of a target molecule generally refers to a moiety capable of specifically recognizing and/or specifically binding a target molecule.
  • the targeting moiety of the target molecule can be an antigen-binding protein, an antibody, or an antigen-binding fragment of an antibody.
  • the targeting moiety of the target molecule may also have the ability to form a covalent bond with the target molecule.
  • the term "specific recognition” generally refers to the recognition of a specific target molecule, but a substance or moiety that does not substantially recognize or bind to other molecules in a sample.
  • antibodies that specifically bind to an antigen may also bind to different allelic forms of the target molecule.
  • protein degrader generally refers to a specific structure within a cell that is involved in protein degradation.
  • a protein degrader can comprise a specific morphological and functional micro-organ within a cell that has the ability to degrade proteins.
  • a protein degrader can contain intracellular lysosomes.
  • protein degrader targeting moiety generally refers to a moiety capable of targeting a protein degrader.
  • a substance with a protein degrader targeting moiety can enter the protein degrader at a higher quantitative rate than that substance without a protein degrader targeting moiety.
  • the rate of that substance entering the protein degrader can be increased.
  • the protein degrader targeting moiety can be a lysosomal targeting peptide or can be a molecule capable of binding the mannose-6-phosphate receptor (M6PR).
  • penetrating peptide generally refers to a class of short peptides capable of passing through cell membranes or tissue barriers.
  • penetrating peptides can transport biological macromolecules such as proteins, RNAs, and DNAs into cells to exert their effector functions through mechanisms such as endocytosis and direct penetration.
  • lysosome targeting peptide generally refers to a class of peptides capable of targeting lysosomes.
  • lysosomal targeting peptides can carry protein, RNA, DNA and other target molecules into lysosomes.
  • the term "functional group” generally refers to a part of a substance that performs some function or activity and reacts with other molecules.
  • one functional group can chemically react with another functional group to form a covalent linkage.
  • electrophilic group generally refers to an electron deficient or electron deficient molecule or portion thereof.
  • the electrophilic group may be selected from the group consisting of amino, imidazolyl, sulfhydryl, hydroxyl, carboxyl, methylthio, and derivatives of the foregoing.
  • amino generally refers to -NH2 .
  • the amino group can be optionally substituted.
  • imidazolyl generally refers to 1,3 diazolyl (Imidazolyl).
  • imidazolyl can be optionally substituted.
  • thiol generally refers to -SH.
  • the thiol group can be optionally substituted.
  • hydroxyl generally refers to -OH.
  • hydroxyl groups can be optionally substituted.
  • carboxy generally refers to -COOH.
  • carboxyl group may be optionally substituted.
  • methylthio generally refers to -SCH3 .
  • the methylthio group can be optionally substituted.
  • halogen generally refers to fluorine, chlorine, bromine and/or iodine.
  • alkyl alkenyl
  • cycloalkyl may generally be preceded by a designation to indicate the number of atoms present in the group in a particular case, eg, C1 - C4alkane group, C 3 -C 7 cycloalkoxy, C 1 -C 4 alkylcarbonylamino, etc.
  • the subscript numbers following "C” indicate the number of carbon atoms present in the group.
  • a C3 alkyl group refers to an alkyl group having three carbon atoms (eg, n-propyl, isopropyl); members of a group in C1-4 can have any number falling within the range 1-4 carbon atom.
  • the terms “alkyl”, “alkenyl”, “cycloalkyl” can generally be optionally substituted.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the term "PD-L1" generally refers to one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2).
  • PD-L1 can downregulate T cell activation and cytokine secretion upon binding to PD-1.
  • the term “PD-L1” can include human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, as well as substances comprising functionally active fragments of PD-L1.
  • the complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.
  • EGFR generally refers to human epidermal growth factor receptor 1 (also known as HER-1).
  • GenBank accession number for EGFR may be AF125253.
  • EGFR can include human EGFR, variants, isotypes and species homologs of EGFR, as well as substances comprising functionally active fragments of EGFR.
  • HER-2 generally refers to human epidermal growth factor receptor 2.
  • GenBank accession number for HER-2 may be P04626.
  • HER-2 can include human HER-2, variants, isoforms and species homologs of HER-2, as well as substances comprising functionally active fragments of HER-2.
  • Ras generally refers to a GTP binding protein.
  • GenBank accession number for Ras can be P01112.
  • Ras can include human Ras, variants, isoforms and species homologs of Ras, Ras that has lost the ability to hydrolyze GTP, and substances comprising functionally active fragments of Ras.
  • CD38 generally refers to the lymphocyte surface antigen CD38.
  • GenBank accession number for CD38 may be P28907.
  • CD38 can include human CD38, variants, isoforms and species homologs of CD38, as well as substances comprising functionally active fragments of CD38.
  • immune checkpoint molecule generally refers to a molecule that modulates the immune system.
  • immune checkpoint molecules prevent the immune system from attacking cells.
  • the immune checkpoint molecule can be PD-L1.
  • tumor-associated molecule generally refers to a molecule associated with the occurrence and/or progression of a tumor.
  • a tumor-associated molecule can be a molecule associated with the growth and/or metastasis of a tumor.
  • a tumor-associated molecule can be a molecule that is highly expressed by tumor cells.
  • signaling pathway protein generally refers to proteins associated with cellular signaling pathways.
  • signaling pathway proteins can activate proteins of the MAPK signaling pathway.
  • CDR also referred to as "complementarity determining regions” generally refers to regions in the variable domains of antibodies, the sequences of which are highly variable and/or form structurally defined loops.
  • an antibody typically includes six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • VH HCDR1, HCDR2, HCDR3
  • LCDR1, LCDR2, LCDR3 three in the VL
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art.
  • the amino acid sequence numbering of the targeting portion of the target molecule can be according to the IMGT numbering scheme.
  • the CDRs of the targeting moiety of the target molecule can be determined according to the Kabat numbering system.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, and can encompass any polypeptide that includes an antigen binding site, whether produced in vitro or in vivo.
  • the term may include, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutant and transplanted antibodies.
  • the term “antibody” may also include antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd , affinity antibodies, dAbs, and/or other antibody fragments that retain antigen-binding function. Typically, such fragments may include an antigen binding domain.
  • the basic 4-chain antibody unit may be a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies can be composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contain 10 antigen-binding sites, while IgA antibodies can include 2-5 that can be combined with the J chain to form a polymerization Basic 4-chain unit of multivalent combination.
  • the 4-chain unit may typically be about 150,000 Daltons.
  • Each L chain can be linked to the H chain by one covalent disulfide bond, while the two H chains can be connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain may also have regularly spaced intrachain disulfide bridges.
  • Each H chain may have a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, and four CH domains for the mu and epsilon isoforms each .
  • Each L chain may have a variable domain (VL) at the N-terminus and a constant domain at the other end. VL corresponds to VH, and CL may correspond to the first constant domain (CH1) of the heavy chain. Particular amino acid residues can be considered to form the interface between the light and heavy chain variable domains.
  • the VH and VL pairings can together form a single antigen binding site. For the structure and properties of different classes of antibodies.
  • immunoglobulins from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Based on the amino acid sequence of the heavy chain (CH) constant domains, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which can have heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • polypeptide generally refers to a polymer of amino acids.
  • a polypeptide may not refer to a product of a particular length; thus, peptides, oligopeptides, and proteins may be included in the definition of polypeptide.
  • This term may also not refer to or exclude post-expression modifications of the polypeptide, eg, glycosylation, acetylation, phosphorylation, and the like.
  • a polypeptide may comprise analogs of one or more amino acids (including, for example, unnatural amino acids, etc.), polypeptides with substituted bonds, and modifications known in the art, both naturally occurring and non-natural Naturally occurring modifications.
  • the term "antigen-binding fragment” generally refers to one or more polypeptide fragments that have the ability to specifically bind an antigen.
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, affibody and/or dAb.
  • variable region generally refers to a region where certain segments of the variable domain may differ significantly in sequence between antibodies.
  • a “variable region” in a light chain may comprise a light chain variable region VL; a “variable region” in a heavy chain may comprise the heavy chain variable region VH.
  • Variable domains can mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability may not be evenly distributed across the variable domain. It can generally be concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of variable domains may be referred to as framework regions (FRs).
  • CDRs or HVRs hypervariable regions
  • variable domains of native heavy and light chains can each comprise four FR regions, most in a ⁇ -sheet configuration, connected by three CDRs that form circular connections and, in some cases, can form a ⁇ -sheet structure a part of.
  • the CDRs in each chain can be held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antigen-binding site of the antibody.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) of the H chain (VH).
  • Fab' or "Fab' fragment” generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment can include all of the light chain, all of the variable regions of the heavy chain, and all or part of the first and second constant regions of the heavy chain.
  • Fab' fragments may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • (Fab')2 generally refers to antibody fragments produced by pepsin digestion of whole antibodies.
  • F(ab')2 fragments may contain two Fab fragments and part of the hinge region held together by a disulfide bond.
  • F(ab')2 fragments can have bivalent antigen-binding activity and are capable of cross-linking antigens.
  • Fv or “Fv fragment” generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which may include all or part of the heavy chain variable region and light chain variable region, and may lack heavy chain constant and light chain constant regions.
  • the heavy and light chain variable regions can include, for example, CDRs.
  • Fv fragments can include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains can be
  • the variable regions can be contiguous (eg, via synthetic linkers such as short flexible polypeptide linkers) and can be expressed as a single-chain polypeptide, and wherein the scFv can retain the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N- and C-termini of the polypeptide), and the scFv may include a VL-linker-VH or VH-linker-VL can be included.
  • di-scFv generally refers to a substance in which two scFvs can be linked in any order (eg, relative to the N-terminal and C-terminal ends of the polypeptide).
  • affinity antibody generally refers to an affinity protein.
  • an affinity antibody (Affibody) molecule can be an affinity protein based on a 58 amino acid residue protein domain, which can be derived from the triple helix bundle IgG binding domain of Staphylococcus protein A.
  • Affinity antibody variants targeting the desired molecule can be selected from the library, eg, using phage display technology.
  • Affinity antibody molecules can be characterized by a simple and robust structure and low molecular weight (6 kDa) for a variety of applications, eg, as detection reagents and inhibitors of receptor interactions.
  • 6 kDa low molecular weight
  • dAb generally refers to antigen-binding fragments with VH or VL domains, reference may be made to, eg, WO2006030220A1, WO2006003388A2 or other published patent applications.
  • VHH generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody. VHHs may also be referred to as Nanobodies (Nb) and/or single domain antibodies.
  • the term "monoclonal antibody” generally refers to a preparation of antibody molecules of single molecular composition.
  • Monoclonal antibodies can often be highly specific for a single antigenic site.
  • each monoclonal antibody can be directed against a single determinant on an antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” may denote a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions may be derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is less robust in human subjects than the parental (eg, mouse-derived) antibody. The possibility of triggering an adverse immune response can be reduced.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” may retain antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody comprising only human immunoglobulin protein sequences.
  • a fully human antibody may contain murine sugar chains if it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • mouse antibody or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Fully human antibodies can be generated in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary and other techniques that can be used to make antibodies, such as the use of libraries, are known in the art.
  • 6-bromo-hexanoyllysine or generally refers to a lysine containing a BrC6K group, which can be, for example,
  • 6-bromo-hexanoyllysine can include derivatives thereof.
  • fluorosulfate-L-tyrosine generally refers to a tyrosine containing an FSY group, which can be, for example,
  • fluorosulfate-L-tyrosine can include derivatives thereof.
  • the application provides a conjugate.
  • Figure 1 shows a schematic diagram of one of the degradation pathways that can be achieved by the conjugate of the present application to degrade the target molecule.
  • the conjugate of the present application can comprise: a target molecule targeting moiety and a protein degrader targeting moiety, the target molecule targeting moiety can specifically recognize the target molecule and can covalently bind to the target molecule; wherein , the protein degrader targeting moiety can be directly or indirectly linked to the target molecule targeting moiety.
  • the targeting moiety of the target molecule may contain a first functional group
  • the target molecule may contain a second functional group
  • the first functional group may interact with the target molecule when the targeting moiety of the target molecule is in proximity to the target molecule.
  • the second functional group reacts to form a covalent bond.
  • the second functional group may comprise an electrophilic group
  • the first functional group may undergo a cross-linking reaction with the second functional group, such as a substitution reaction and/or an addition reaction.
  • the targeting moiety of the target molecule and the target molecule can be linked by a substitution reaction and/or an addition reaction to form a covalent bond.
  • the first functional group may react with the second functional group to form a covalent bond when the targeting moiety of the target molecule and the target molecule are approached by non-covalent antibody-antigen interaction.
  • the distance between the backbone of the targeting moiety of the target molecule and the backbone of the target molecule may be approximately equal to the length of the side chain with the first functional group plus the length of the side chain with the first functional group The sum of the side chain lengths of the second functional group.
  • the orientation of the first functional group and the second functional group can be head-to-head, or the side chain with the first functional group and the side chain with the second functional group can be rotated to achieve a head-to-head orientation.
  • the first functional group can react with the second functional group to form a covalent bond.
  • the distance between the first functional group and the second functional group can be achieved by adjusting the length of the side chain of the targeting moiety of the target molecule with the first functional group.
  • the cross-linking efficiency or the proximity reactivity can be judged by detecting the time at which more than 50% of the first functional group and the second functional group are cross-linked.
  • more than 50% of the crosslinking between the first functional group and the second functional group occurs in less than about 72 hours, less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 8 hours, or less than about 4 hours , it can be considered that the first functional group has the activity of adjacent reaction with the second functional group.
  • the targeting moiety of the target molecule is brought into proximity with the target molecule by non-covalent interaction, eg, antibody-antigen interaction
  • the first functional group can be imparted with conditions and/or times that allow proximity reaction with the second functional group.
  • the second functional group comprises a side chain group of a natural amino acid.
  • natural amino acids may include Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan Tryptophan, Tyrosine, Aspartate, Histidine, Asparagine, Glutamate, Lysine, Glutamine Glutamine, Methionine, Arginine, Serine, Threonine, Cysteine, and/or Proline.
  • the second functional group may also comprise side chain groups of unnatural amino acids, eg some target molecules may comprise selenocysteine, pyrrolysine and/or unnatural amino acids.
  • the second functional group can also be any side chain group, for example, the target molecule can have any structure.
  • the second functional group can be an electrophilic group.
  • it may be a group containing a highly electronegative atom such as a halogen atom, an oxygen atom, a nitrogen atom, and a sulfur atom.
  • the second functional group may comprise an amino group, an imidazolyl group, a thiol group, a hydroxyl group, a carboxyl group and/or a methylthio group.
  • the second functional group may contain and / or
  • the side chain comprising the second functional group may be selected from the group: -CH2SH , -CH2CH2COOH , -CH2CH2CONH2 , -CH2CH2SCH3 , - ( CH2 ) 3NH -C (NH) NH2 , -( CH2 ) 4NH2 , -CH2 - C6H4OH , -CH2OH , -CH(OH) CH3 , -CH2COOH , -CH2CONH2 , and -CH 2 -C 3 H 3 N 2 .
  • the amino acid comprising the second functional group may be selected from the group consisting of cysteine, glutamic acid, glutamine, methionine, arginine, lysine, tyrosine, serine, threonine, Aspartic acid, asparagine and histidine.
  • the amino acid comprising the second functional group may be selected from the group consisting of histidine, lysine, tyrosine, cysteine, serine, threonine, glutamic acid and methionine.
  • a target molecule may comprise a cell surface molecule, an intracellular molecule, and/or an extracellular molecule.
  • the target molecule can be a receptor molecule on the cell surface.
  • the target molecule can be selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the target molecule can comprise a polypeptide.
  • the target molecule can be selected from the group consisting of immune checkpoint molecules, tumor-associated molecules, and signaling pathway proteins.
  • the target molecule may comprise a member of the epidermal growth factor receptor (HER) family.
  • HER epidermal growth factor receptor
  • the target molecule may comprise a member of the mitogen-activated protein kinase (MAPK) signaling pathway.
  • the target molecule may be selected from the group consisting of PD-L1, EGFR, HER-2, Ras, CD38, and functional fragments of the foregoing.
  • the target molecule can comprise the amino acid sequence set forth in SEQ ID NO:98.
  • PD-L1 may contain the second functional group, eg, histidine, at amino acid position 69.
  • the first functional group may be selected from the group consisting of sulfuryl fluoride, halogenated hydrocarbons, carbonyl halogenated hydrocarbons, alpha carbonyl unsaturated hydrocarbons, and derivatives of the foregoing groups.
  • the first functional group can be selected from the following group:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0.
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • the first functional group can be
  • R 3 can be halogen, alkenyl or alkynyl
  • n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6.
  • the first functional group can be
  • R 4 can be halogen, alkenyl or alkynyl
  • n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6.
  • the first functional group may be selected from the following group:
  • a derivative may refer to the functional group or compound which may be substituted or unsubstituted by one or more additional groups, which may be individually and independently selected from alkyl, alkenyl, alkynyl , cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercapto, cyano, halogen, carbonyl, thiocarbonyl, isocyanato, thiocyanate, isothiocyanate alkyl, nitro, perhaloalkyl, perfluoroalkyl and amino groups including mono- and di-substituted amino groups, and protected derivatives thereof.
  • the targeting moiety of the target molecule may comprise a residue of an unnatural amino acid, which may comprise the first functional group, and the unnatural amino acid may be selected from the group consisting of:
  • the targeting moiety of the target molecule may comprise a residue of an unnatural amino acid, which may comprise the first functional group, and the unnatural amino acid may be selected from the group consisting of: fluorosulfate-L-phenol amino acid, 6-bromo-hexanoyllysine, and derivatives of the foregoing.
  • the targeting moiety of the target molecule can be selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the targeting moiety of the target molecule may comprise a polypeptide.
  • the targeting moiety of the target molecule can comprise an antibody or antigen-binding fragment thereof.
  • the antibody may be selected from the group consisting of murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment can be selected from the group consisting of VHH, Fab, Fab', Fv fragments, (Fab') 2 , (Fab) 2 , scFv, di-scFv, affibodies and dAbs.
  • the targeting moiety of the target molecule may comprise a single domain antibody or antigen-binding fragment thereof.
  • the CDRs of antibodies are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia in combination, and the like. These coding systems are known in the art. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions.
  • the CDRs encompass CDR sequences that can be divided according to any CDR division; variants thereof are also encompassed, and the variants include substitutions, deletions and/or additions of one or more of the amino acid sequences of the CDRs amino acid.
  • the variants include substitutions, deletions and/or additions of one or more of the amino acid sequences of the CDRs amino acid.
  • homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the CDRs can be identified by the Kabat numbering scheme.
  • the targeting moiety of the target molecule described in this application may comprise heavy chain and/or light chain sequences modified by one or more conservative sequences.
  • conservative sequence modifications is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody.
  • Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the targeting moieties of the target molecules described herein by standard techniques known in the art, such as point mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions may be substitution of amino acid residues with amino acid residues having similar side chains. Sets of amino acid residues with similar side chains are known in the art.
  • These groups of amino acid residues may include those with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine amino acid, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, gly
  • one or more amino acid residues in the CDR region of the targeting portion of the target molecule of the present application may be replaced with other amino acid residues of the same side chain group.
  • the targeting moiety of the target molecule can be an antibody.
  • the targeting moiety of the target molecule may comprise the first functional group in the variable region of the antibody.
  • the targeting moiety of the target molecule of the present application may comprise a first functional group in the amino acid of the variable region of the antibody compared to the targeting moiety of the wild-type target molecule.
  • the targeting moiety of the target molecule may comprise the first functional group in the CDR region.
  • the targeting moiety of the target molecule of the present application may comprise a first functional group in the amino acid of the CDR region.
  • the targeting moiety of the target molecule may comprise the first functional group in any amino acid in the first 5 amino acids of the N-terminal and/or any amino acid in the last 5 amino acids in the C-terminal of the CDR region.
  • the targeting moiety of the target molecule may comprise the first functional group at any amino acid in the first 5 amino acids of the N-terminal and/or any amino acid in the last 5 amino acids in the C-terminal of the CDR3 region.
  • the CDR regions can be divided according to any CDR region division method, such as the Kabat division method.
  • CDR1, CDR2 and CDR3 of the targeting moiety of the target molecule of the present application can be shown as SEQ ID NOs: 1, 2 and 3, respectively.
  • the targeting moiety of the target molecule of the present application can specifically bind to PD-L1.
  • CDR1, CDR2 and CDR3 of the targeting moiety of the target molecule of the present application may be shown in SEQ ID NOs: 4, 5 and 6, respectively.
  • the targeting moiety of the target molecule of the present application can specifically bind to EGFR.
  • the targeting moiety of the target molecule can be an antibody.
  • the targeting moiety of the target molecule may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine in the variable region of the antibody.
  • the amino acid of the targeting moiety of the present application in the variable region of the antibody can be replaced by the fluorosulfate-L-tyrosine and/or by the 6 -Bromo-hexanoyllysine substitution.
  • the targeting moiety of the target molecule may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine in the CDR regions.
  • the amino acid in the CDR region of the targeting moiety of the target molecule of the present application can be replaced by the fluorosulfate-L-tyrosine and/or by the 6-bromo - Hexoyl lysine substitution.
  • any amino acid in the first 5 amino acids of the N-terminal of the CDR region and/or any amino acid in the last 5 amino acids of the C-terminal of the targeting moiety of the target molecule can be replaced by the fluorosulfate-L-tyrosine and /or replaced by said 6-bromo-hexanoyllysine.
  • any amino acid in the first 5 amino acids of the N-terminal of the CDR3 region and/or any amino acid in the last 5 amino acids of the C-terminal of the targeting moiety of the target molecule can be replaced by the fluorosulfate-L-tyrosine and /or replaced by said 6-bromo-hexanoyllysine.
  • the amino acid sequence contained in the targeting portion of the target molecule may comprise the amino acid at the 108th amino acid, the 110th amino acid and/or the 113th amino acid. a functional group.
  • the amino acid sequence included in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid, the 110th amino acid and/or the 113th amino acid can be included in the present application.
  • the side chain of the targeting moiety of the target molecule at amino acid 108, amino acid 110 and/or amino acid 113 may comprise the first functional group of the present application.
  • the first functional group contained in the targeting moiety of the target molecule at amino acid 108, amino acid 110 and/or amino acid 113 can react adjacently with the second functional group on the target molecule to form a covalent bond.
  • the targeting moiety of the target molecule can specifically recognize the PD-L1.
  • the amino acid sequence contained in the targeting part of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the 108th amino acid, the 110th amino acid and/or the 113th amino acid of the target molecule targeting part Can be replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the targeting portion of the target molecule has the 108th amino acid, the 110th amino acid and/or the 113th amino acid.
  • the amino acids at position 1 can each independently be replaced by the fluorosulfate-L-tyrosine and/or by the 6-bromo-hexanoyllysine.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine replacement.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the targeting portion of the target molecule has the 108th amino acid, the 110th amino acid and/or the 113th amino acid. position can be replaced by the 6-bromo-hexanoyllysine.
  • the targeting moiety of the target molecule may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
  • the amino acid sequence contained in the targeting portion of the target molecule may contain the first functional group at the 116th amino acid compared with the corresponding amino acid sequence shown in SEQ ID NO: 18.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid can be a substance containing the first functional group of the present application, such as the first functional group of the present application. of unnatural amino acids.
  • the side chain of the targeting moiety of the target molecule at the 116th amino acid may comprise the first functional group of the present application.
  • the first functional group contained in the targeting moiety of the target molecule at the 116th amino acid can react adjacently with the second functional group on the target molecule to form a covalent bond.
  • the targeting moiety of the target molecule can specifically recognize the EGFR.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine and/or 6-bromo-hexanoyllysine substitution.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine replacement.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the 6-bromo-hexanoyl Lysine replacement.
  • the targeting moiety of the target molecule may comprise the amino acid sequence shown in SEQ ID NO:12.
  • protein degraders can contain lysosomes.
  • the protein degrader targeting moiety can be selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • a protein degrader targeting moiety may comprise a molecule capable of targeting a lysosome or a functionally active fragment thereof.
  • the protein degrader targeting moiety may further comprise a transmembrane moiety capable of facilitating the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the target molecule targeting moiety, And/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • a protein degrader targeting moiety may comprise the following group: lysosomal targeting peptide (LTP), oligosaccharide peptide, mannose 6-phosphate (M6P), N-acetylgalactosamine (GalNAc), galactose , and any combination of the foregoing.
  • the conjugate may further comprise a transmembrane moiety that can facilitate entry of the target molecule into a cell, wherein the transmembrane moiety may be directly or indirectly linked to the targeting moiety of the target molecule, and/or Or the transmembrane moiety may be linked directly or indirectly to the protein degrader targeting moiety.
  • the transmembrane moiety can be selected from the group consisting of penetrating peptide (CPP), oligopeptide, mannose 6-phosphate (M6P), N-acetylgalactosamine (GalNAc), galactose, and any of the foregoing combination.
  • CPP penetrating peptide
  • M6P mannose 6-phosphate
  • GalNAc N-acetylgalactosamine
  • galactose galactose
  • the penetrating peptide may comprise polyarginine, and the number of repeating units of the polyarginine may be at least 2.
  • the protein degrader targeting moiety can comprise preprotein convertase subtilisin 9 (PCSK9) or a functionally active fragment thereof, which can bind to receptors on the cell surface and can promote PCSK9 and degradation of PCSK9-containing complexes.
  • PCSK9 preprotein convertase subtilisin 9
  • the protein degrader targeting moiety can comprise a tyrosine containing cytoplasmic motif or a functionally active fragment thereof, such as can be NPX 1 Y (as shown in SEQ ID NO: 63) or FX 2 NPX 3 Y (shown in SEQ ID NO: 65 ) , wherein X1, X2 or X3 can be any amino acid , eg, X1 , X2 or X3 can each independently be Glycine G and/or Alanine A.
  • the protein degrader targeting moiety may comprise polylactic-co-glycolic acid and/or polylactic acid, the polymerized monomers of the poly-lactic-co-glycolic acid may comprise lactic acid and glycolic acid, and the ratio of lactic acid and glycolic acid may be any of.
  • the protein degrader targeting moiety may comprise epidermal growth factor EGF or a functionally active fragment thereof, the sequence of which may be set forth in SEQ ID NO:67.
  • the protein degrader targeting moiety may comprise an RGD sequence, which may be a tripeptide sequence consisting of L-arginine, glycine, and L-aspartic acid, and the sequence may be as indicated by RGD.
  • a protein degrader targeting moiety may comprise the following group: Lysosomotropic octadecyl-rhodamine-B, dipyridyl-thio-semi-carbazones, Iron chelator Dp44mT (di-2-pyridylketone 4,4,-di-methyl-3-thiosemicarbazone), iron chelator Bp4eT (2-benzoylpyridine-4-ethyl-3thiosemicarbazone), iron chelator PIH (Pyridoxal isonicotinoyl hydrazone), and their derivatives.
  • the protein degrader targeting moiety can comprise polyarginine, such as poly-L-arginine, poly-D-arginine, or poly-L-arginine-D-arginine copolymer
  • the number of repeating units of a compound, such as polymeric arginine, may be at least 2.
  • the protein degrader targeting moiety can be selected from the group consisting of: octamer L-arginine, octamer D-arginine, nonamer L-arginine, nonamer D-arginine, dodecamer L-arginine -Arginine, and dodecameric D-arginine.
  • the protein degrader targeting moiety may be selected from the group consisting of Substance P (Substance P, SP, the sequence may be as shown in SEQ ID NO: 68), Neuropeptide Y (Neuropeptide Y, the sequence may be as shown in SEQ ID NO: 69) shown), TP penetrating peptide (TP transportan, the sequence can be shown as SEQ ID NO: 70), penetrating peptide TAT (the sequence can be shown as SEQ ID NO: 71), penetrating peptide (pAntp, Penetratin, the sequence can be shown as SEQ ID NO: 71) Can be as shown in SEQ ID NO: 72), low pH insertion peptide (pH low-insertion peptide, pHLIP, sequence can be as shown in SEQ ID NO: 73), transmembrane sequence ATRAM (acidity-triggered rational membrane, sequence can be as shown in SEQ ID NO: 74), activatable cell penetrating peptide (ACPP, the sequence may be
  • a protein degrader targeting moiety can target a substance selected from the group consisting of ion-independent mannose 6-phosphate receptor (CI-M6PR, also known as M6PR, the uniprot accession number can be P20645), caveolin- 1 (Caveolin-1, uniprot accession number can be Q03135), clathrin (Clathrin, uniprot accession number can be Q00610), prolactin receptor (Prolactin receptor, PRLR, uniprot accession number can be P16471), amyloid precursor Like protein 2 (Amyloid Precursor Like Protein 2, APLP2, uniprot accession number can be Q06481), lysosome-associated membrane glycoprotein 3 (Lysosome-associated membrane glycoprotein 3, LAMP3, CD63, uniprot accession number can be Q9UQV4), Galan Peptide receptor-1 (GALR-1receptor, uniprot accession number can be P47211), clathrin
  • a protein degrader targeting moiety may comprise a molecule or a functionally active fragment thereof capable of targeting the lysosome or proteasome and/or capable of binding to a cell surface receptor that promotes endocytosis.
  • the protein degrader targeting moiety comprises a molecule capable of binding the mannose-6-phosphate receptor (M6PR).
  • M6PR mannose-6-phosphate receptor
  • the protein degrader targeting moiety may comprise one or more mannose 6-phosphate (M6P) or functionally active fragments thereof.
  • the protein degrader targeting moiety may comprise from about 1 to about 500 of the mannose 6-phosphate or functionally active fragment thereof.
  • the protein degrader targeting moiety may comprise a scaffold molecule directly or indirectly linked to the one or more mannose 6-phosphate or functionally active fragments thereof.
  • the scaffold molecule may comprise a glycan structure.
  • the scaffold molecule can comprise a glycoprotein, which can comprise one or more amino acids modified with the one or more mannose 6-phosphate or functionally active fragments thereof.
  • the glycoprotein may comprise a group of N-carboxy anhydride (NCA) or a derivative thereof.
  • the protein degrader targeting moiety may comprise a molecule capable of binding to the asialoglycoprotein receptor (ASGPR) or a functionally active fragment thereof.
  • ASGPR asialoglycoprotein receptor
  • the protein degrader targeting moiety may comprise one or more ligands for the asialoglycoprotein receptor.
  • the protein degrader targeting moiety may comprise one or more N-acetylgalactosamine (GalNAc).
  • the protein degrader targeting moiety may comprise one or more galactoses.
  • the protein degrader targeting moiety may comprise one or more glucoses.
  • the protein degrader targeting moiety may comprise from about 1 to about 500 ligands of the asialoglycoprotein receptor.
  • the protein degrader targeting moiety can comprise a scaffold molecule that can directly or indirectly link the one or more ligands of the asialoglycoprotein receptor.
  • the protein degrader targeting moiety may comprise the one or more N-acetylgalactosamine-modified one or more alanines.
  • the scaffold molecule may comprise one, two or three of the N-acetylgalactosamine.
  • the scaffold molecule may comprise one, two or three of the galactoses.
  • the conjugate can comprise a fusion protein.
  • the conjugate may comprise the target molecule targeting moiety, the protein degrader targeting moiety and the transmembrane moiety, the target molecule targeting moiety, the protein degrader targeting moiety and the The transmembrane parts can be directly or indirectly connected to each other independently of each other.
  • the indirect linkage may include linkage through a linker.
  • the linker may comprise a peptide linker.
  • the linker may comprise a cleavable or non-cleavable linker.
  • the cleavable linker may comprise a linker cleavable by a protease.
  • the conjugate may comprise the target molecule targeting moiety, the transmembrane moiety, and the lysosomal targeting peptide.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosome targeting peptide, wherein the target molecule targeting moiety may comprise a first functional group, the target molecule may Contains a second functional group, when the targeting moiety of the target molecule is close to the target molecule, the first functional group can react with the second functional group to form a covalent bond, and the transmembrane moiety can promote the target The molecule enters the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosome targeting peptide, wherein the target molecule targeting moiety may comprise a first functional group, and the first functional group may be selected from the following groups:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0;
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl;
  • the first functional group can be wherein, R 3 can be halogen, alkenyl or alkynyl, and n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6;
  • the first functional group can be wherein, R 4 can be halogen, alkenyl or alkynyl, and n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6;
  • the transmembrane moiety can facilitate the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly connected to the targeting moiety of the target molecule, and/or the transmembrane moiety is The membrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosome targeting peptide, wherein the target molecule targeting moiety may comprise a first functional group, the first functional group Can be selected from the following group:
  • the transmembrane moiety can promote the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is linked to the target molecule.
  • the protein degrader targeting moiety is attached directly or indirectly.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, and the target molecule targeting moiety may comprise a residue of an unnatural amino acid, the unnatural amino acid Can be selected from the following group:
  • the transmembrane moiety can promote the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is linked to the target molecule.
  • the protein degrader targeting moiety is attached directly or indirectly.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, the target molecule targeting moiety may be an antibody, wherein the target molecule targeting moiety may be in Among the amino acids of the CDR region, the first 5 amino acids of the N-terminus of the CDR region and/or the last 5 amino acids of the C-terminus of the CDR region contain a first functional group, and the first functional group can be selected from the following group:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0;
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl;
  • the first functional group can be wherein, R 3 can be halogen, alkenyl or alkynyl, and n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6;
  • the first functional group can be wherein, R 4 can be halogen, alkenyl or alkynyl, and n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6;
  • the transmembrane moiety can facilitate the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly connected to the targeting moiety of the target molecule, and/or the transmembrane moiety is The membrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, the target molecule targeting moiety may be an antibody, wherein the target molecule targeting moiety may be in Among the amino acids of the CDR region, the first 5 amino acids of the N-terminus of the CDR region and/or the last 5 amino acids of the C-terminus of the CDR region contain a first functional group, and the first functional group can be selected from the following group:
  • the transmembrane moiety can promote the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is linked to the target molecule.
  • the protein degrader targeting moiety is attached directly or indirectly.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, the target molecule targeting moiety may be an antibody, wherein the target molecule targeting moiety may be in The CDR region, the first 5 amino acids of the N-terminus of the CDR region and/or the last 5 amino acids of the C-terminus of the CDR region comprise residues of unnatural amino acids, and the unnatural amino acids can be selected from the following group:
  • the transmembrane moiety can promote the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is linked to the target molecule.
  • the protein degrader targeting moiety is attached directly or indirectly.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide
  • the target molecule targeting moiety may be an antibody
  • the target molecule targeting moiety may be in The CDR region
  • the first 5 amino acids of the N-terminus of the CDR region and/or the last 5 amino acids of the C-terminus of the CDR region comprise residues of unnatural amino acids, which may comprise fluorosulfate-L-tyrosine and /or 6-bromo-hexanoyllysine
  • the transmembrane moiety can facilitate the entry of the target molecule into cells, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or The transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide
  • the target molecule targeting moiety may be an antibody
  • the target molecule targeting moiety may be in Among the amino acids of the CDR region, the first 5 amino acids of the N-terminus of the CDR3 region and/or the last 5 amino acids of the C-terminus of the CDR3 region contain unnatural amino acid residues
  • the unnatural amino acid may comprise fluorosulfate-L- Tyrosine and/or 6-bromo-hexanoyllysine
  • the transmembrane moiety can facilitate the entry of the target molecule into cells, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule , and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide
  • the target molecule targeting moiety may be an antibody
  • the target molecule targeting moiety may be in
  • the CDR regions comprise residues of unnatural amino acids, which may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine
  • the transmembrane moieties may facilitate the target
  • the molecule enters the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosome targeting peptide
  • the target molecule targeting moiety may be a PD-L1 single domain antibody, wherein the target molecule Compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the targeting moiety comprises the amino acid sequence, the amino acid at position 108, amino acid at position 110 and/or amino acid at position 113 may be replaced by fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine replacement
  • the transmembrane moiety can facilitate the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and /or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, and the target molecule targeting moiety may be a PD-L1 single domain antibody, wherein the target molecule Compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the amino acid sequence contained in the targeting moiety, the 108th amino acid can be replaced by fluorosulfate-L-tyrosine, and the transmembrane moiety can promote the target The molecule enters the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, and the target molecule targeting moiety may be an EGFR single domain antibody, wherein the target molecule targets Part of the included amino acid sequence is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid can be replaced by fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyl lysine,
  • the transmembrane moiety can facilitate the entry of the target molecule into the cell, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is linked to the protein degrader target Connect directly or indirectly to the part.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, and the target molecule targeting moiety may be an EGFR single domain antibody, wherein the target molecule targets Compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, the amino acid sequence contained in the part can be replaced by fluorosulfate-L-tyrosine, and the transmembrane part can promote the entry of the target molecule Intracellular, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • the conjugate of the present application may comprise a target molecule targeting moiety, the transmembrane moiety and the lysosomal targeting peptide, and the amino acid sequence of the protein degrader targeting moiety may be as in SEQ ID NOs: 63-91
  • the amino acid sequence of the transmembrane part can be as shown in any one of SEQ ID NOs: 78-79
  • the amino acid sequence of the lysosomal targeting peptide can be as shown in SEQ ID NOs: 63-77 shown, wherein the transmembrane moiety is directly or indirectly linked to the targeting moiety of the target molecule, and/or the transmembrane moiety is directly or indirectly linked to the protein degrader targeting moiety.
  • a polypeptide in another aspect, can comprise a targeting moiety of a target molecule that can specifically recognize and covalently bind to the target molecule.
  • the targeting moiety of the target molecule may contain a first functional group
  • the target molecule may contain a second functional group
  • the first functional group may interact with the target molecule when the targeting moiety of the target molecule is in proximity to the target molecule.
  • the second functional group reacts to form a covalent bond.
  • the second functional group may comprise an electrophilic group
  • the first functional group may undergo a cross-linking reaction with the second functional group, such as a substitution reaction and/or an addition reaction.
  • the targeting moiety of the target molecule and the target molecule can be linked by a substitution reaction and/or an addition reaction to form a covalent bond.
  • the first functional group may react with the second functional group to form a covalent bond when the targeting moiety of the target molecule and the target molecule are approached by non-covalent antibody-antigen interaction.
  • the distance between the backbone of the targeting moiety of the target molecule and the backbone of the target molecule may be approximately equal to the length of the side chain with the first functional group plus the length of the side chain with the first functional group The sum of the side chain lengths of the second functional group.
  • the orientation of the first functional group and the second functional group can be head-to-head, or the side chain with the first functional group and the side chain with the second functional group can be rotated to achieve a head-to-head orientation.
  • the first functional group can react with the second functional group to form a covalent bond.
  • the distance between the first functional group and the second functional group can be achieved by adjusting the length of the side chain of the targeting moiety of the target molecule with the first functional group.
  • the cross-linking efficiency or the proximity reactivity can be judged by detecting the time at which more than 50% of the first functional group and the second functional group are cross-linked.
  • more than 50% of the crosslinking between the first functional group and the second functional group occurs in less than about 72 hours, less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 8 hours, or less than about 4 hours , it can be considered that the first functional group has the activity of adjacent reaction with the second functional group.
  • the targeting moiety of the target molecule is brought into proximity with the target molecule by non-covalent interaction, eg, antibody-antigen interaction
  • the first functional group can be imparted with conditions and/or times that allow proximity reaction with the second functional group.
  • the second functional group comprises a side chain group of a natural amino acid.
  • natural amino acids may include Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan Tryptophan, Tyrosine, Aspartate, Histidine, Asparagine, Glutamate, Lysine, Glutamine Glutamine, Methionine, Arginine, Serine, Threonine, Cysteine, and/or Proline.
  • the second functional group may also comprise side chain groups of unnatural amino acids, eg some target molecules may comprise selenocysteine, pyrrolysine and/or unnatural amino acids.
  • the second functional group can also be any side chain group, for example, the target molecule can have any structure.
  • the second functional group can be an electrophilic group.
  • it may be a group containing a highly electronegative atom such as a halogen atom, an oxygen atom, a nitrogen atom, and a sulfur atom.
  • the second functional group may comprise an amino group, an imidazolyl group, a thiol group, a hydroxyl group, a carboxyl group and/or a methylthio group.
  • the second functional group may contain and / or
  • the side chain comprising the second functional group may be selected from the group: -CH2SH , -CH2CH2COOH , -CH2CH2CONH2 , -CH2CH2SCH3 , - ( CH2 ) 3NH -C (NH) NH2 , -( CH2 ) 4NH2 , -CH2 - C6H4OH , -CH2OH , -CH(OH) CH3 , -CH2COOH , -CH2CONH2 , and -CH 2 -C 3 H 3 N 2 .
  • the amino acid comprising the second functional group may be selected from the group consisting of cysteine, glutamic acid, glutamine, methionine, arginine, lysine, tyrosine, serine, threonine, Aspartic acid, asparagine and histidine.
  • the amino acid comprising the second functional group may be selected from the group consisting of histidine, lysine, tyrosine, cysteine, serine, threonine, glutamic acid and methionine.
  • a target molecule may comprise a cell surface molecule, an intracellular molecule, and/or an extracellular molecule.
  • the target molecule can be a receptor molecule on the cell surface.
  • the target molecule can be selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the target molecule can comprise a polypeptide.
  • the target molecule can be selected from the group consisting of immune checkpoint molecules, tumor-associated molecules, and signaling pathway proteins.
  • the target molecule may comprise a member of the epidermal growth factor receptor (HER) family.
  • HER epidermal growth factor receptor
  • the target molecule may comprise a member of the mitogen-activated protein kinase (MAPK) signaling pathway.
  • the target molecule may be selected from the group consisting of PD-L1, EGFR, HER-2, Ras, CD38, and functional fragments of the foregoing.
  • the target molecule can comprise the amino acid sequence set forth in SEQ ID NO:98.
  • PD-L1 may contain the second functional group, eg, histidine, at amino acid position 69.
  • the first functional group may be selected from the group consisting of sulfuryl fluoride, halogenated hydrocarbons, carbonyl halogenated hydrocarbons, alpha carbonyl unsaturated hydrocarbons, and derivatives of the foregoing groups.
  • the first functional group can be selected from the following group:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0.
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • the first functional group can be
  • R 3 can be halogen, alkenyl or alkynyl
  • n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6.
  • the first functional group can be
  • R 4 can be halogen, alkenyl or alkynyl
  • n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6.
  • the first functional group may be selected from the following group:
  • a derivative may refer to the functional group or compound which may be substituted or unsubstituted by one or more additional groups, which may be individually and independently selected from alkyl, alkenyl, alkynyl , cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercapto, cyano, halogen, carbonyl, thiocarbonyl, isocyanato, thiocyanate, isothiocyanate alkyl, nitro, perhaloalkyl, perfluoroalkyl and amino groups including mono- and di-substituted amino groups, and protected derivatives thereof.
  • the targeting moiety of the target molecule may comprise a residue of an unnatural amino acid, which may comprise the first functional group, and the unnatural amino acid may be selected from the group consisting of:
  • the targeting moiety of the target molecule may comprise a residue of an unnatural amino acid, which may comprise the first functional group, and the unnatural amino acid may be selected from the group consisting of: fluorosulfate-L-phenol amino acid, 6-bromo-hexanoyllysine, and derivatives of the foregoing.
  • the targeting moiety of the target molecule can be selected from the group consisting of polypeptides, nucleic acids, small molecules, polysaccharides, lipids, nanoparticles, and any combination of the foregoing.
  • the targeting moiety of the target molecule may comprise a polypeptide.
  • the targeting moiety of the target molecule can comprise an antibody or antigen-binding fragment thereof.
  • the antibody may be selected from the group consisting of murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment can be selected from the group consisting of VHH, Fab, Fab', Fv fragments, (Fab') 2 , (Fab) 2 , scFv, di-scFv, affibodies and dAbs.
  • the targeting moiety of the target molecule may comprise a single domain antibody or antigen-binding fragment thereof.
  • the CDRs of antibodies are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions.
  • the CDRs cover CDR sequences that can be divided according to any CDR division; also cover variants thereof, which include substitutions, deletions and/or additions of one or more amino acid sequences of the CDRs amino acid.
  • substitutions, deletions and/or additions of one or more amino acid sequences of the CDRs amino acid For example 1-30, 1-20 or 1-10, also for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or Or insertions; homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the CDRs can be identified by the Kabat numbering scheme.
  • the targeting moiety of the target molecule described in this application may comprise heavy chain and/or light chain sequences modified by one or more conservative sequences.
  • conservative sequence modifications are meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody.
  • Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the targeting moieties of the target molecules described herein by standard techniques known in the art, such as point mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions may be substitution of amino acid residues with amino acid residues having similar side chains. Sets of amino acid residues with similar side chains are known in the art.
  • These groups of amino acid residues may include those with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine amino acid, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, gly
  • one or more amino acid residues in the CDR region of the targeting portion of the target molecule of the present application may be replaced with other amino acid residues of the same side chain group.
  • the targeting moiety of the target molecule can be an antibody.
  • the targeting moiety of the target molecule may comprise the first functional group in the variable region of the antibody.
  • the targeting moiety of the target molecule of the present application may comprise a first functional group in the amino acid of the variable region of the antibody compared to the targeting moiety of the wild-type target molecule.
  • the targeting moiety of the target molecule may comprise the first functional group in the CDR region.
  • the targeting moiety of the target molecule of the present application may comprise a first functional group in the amino acid of the CDR region.
  • the targeting moiety of the target molecule may comprise the first functional group in any amino acid in the first 5 amino acids of the N-terminal and/or any amino acid in the last 5 amino acids in the C-terminal of the CDR region.
  • the targeting moiety of the target molecule may comprise the first functional group at any amino acid in the first 5 amino acids of the N-terminal and/or any amino acid in the last 5 amino acids in the C-terminal of the CDR3 region.
  • the CDR region may be divided according to any CDR region division method, such as the Kabat division method.
  • CDR1, CDR2 and CDR3 of the targeting moiety of the target molecule of the present application can be shown as SEQ ID NOs: 1, 2 and 3, respectively.
  • the targeting moiety of the target molecule of the present application can specifically bind to PD-L1.
  • CDR1, CDR2 and CDR3 of the targeting moiety of the target molecule of the present application may be shown in SEQ ID NOs: 4, 5 and 6, respectively.
  • the targeting moiety of the target molecule of the present application can specifically bind to EGFR.
  • the targeting moiety of the target molecule can be an antibody.
  • the targeting moiety of the target molecule may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine in the variable region of the antibody.
  • the amino acid of the targeting moiety of the present application in the variable region of the antibody can be replaced by the fluorosulfate-L-tyrosine and/or by the 6 -Bromo-hexanoyllysine substitution.
  • the targeting moiety of the target molecule may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine in the CDR regions.
  • the amino acid in the CDR region of the targeting moiety of the target molecule of the present application can be replaced by the fluorosulfate-L-tyrosine and/or by the 6-bromo - Hexoyl lysine substitution.
  • any amino acid in the first 5 amino acids of the N-terminal of the CDR region and/or any amino acid in the last 5 amino acids of the C-terminal of the targeting moiety of the target molecule can be replaced by the fluorosulfate-L-tyrosine and /or replaced by said 6-bromo-hexanoyllysine.
  • any amino acid in the first 5 amino acids of the N-terminal of the CDR3 region and/or any amino acid in the last 5 amino acids of the C-terminal of the targeting moiety of the target molecule can be replaced by the fluorosulfate-L-tyrosine and /or replaced by said 6-bromo-hexanoyllysine.
  • the amino acid sequence contained in the targeting portion of the target molecule may comprise the amino acid at the 108th amino acid, the 110th amino acid and/or the 113th amino acid. a functional group.
  • the amino acid sequence included in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid, the 110th amino acid and/or the 113th amino acid can be included in the present application.
  • the side chain of the targeting moiety of the target molecule at amino acid 108, amino acid 110 and/or amino acid 113 may comprise the first functional group of the present application.
  • the first functional group contained in the targeting moiety of the target molecule at amino acid 108, amino acid 110 and/or amino acid 113 can react adjacently with the second functional group on the target molecule to form a covalent bond.
  • the targeting moiety of the target molecule can specifically recognize the PD-L1.
  • the amino acid sequence contained in the targeting part of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, the 108th amino acid, the 110th amino acid and/or the 113th amino acid of the target molecule targeting part Can be replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the targeting portion of the target molecule has the 108th amino acid, the 110th amino acid and/or the 113th amino acid.
  • the amino acids at position 1 can each independently be replaced by the fluorosulfate-L-tyrosine and/or by the 6-bromo-hexanoyllysine.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the 108th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine replacement.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 13, and the targeting portion of the target molecule has the 108th amino acid, the 110th amino acid and/or the 113th amino acid. position can be replaced by the 6-bromo-hexanoyllysine.
  • the targeting moiety of the target molecule may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
  • the amino acid sequence contained in the targeting portion of the target molecule may contain the first functional group at the 116th amino acid compared with the corresponding amino acid sequence shown in SEQ ID NO: 18.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid can be a substance containing the first functional group of the present application, such as the first functional group of the present application. of unnatural amino acids.
  • the side chain of the targeting moiety of the target molecule at the 116th amino acid may comprise the first functional group of the present application.
  • the first functional group contained in the targeting moiety of the target molecule at the 116th amino acid can react adjacently with the second functional group on the target molecule to form a covalent bond.
  • the targeting moiety of the target molecule can specifically recognize the EGFR.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the unnatural amino acid.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine and/or 6-bromo-hexanoyllysine substitution.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the fluorosulfate-L -Tyrosine replacement.
  • the amino acid sequence contained in the targeting portion of the target molecule is compared with the corresponding amino acid sequence shown in SEQ ID NO: 18, and the 116th amino acid of the targeting portion of the target molecule can be replaced by the 6-bromo-hexanoyl Lysine replacement.
  • the targeting moiety of the target molecule may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, wherein the targeting moiety of the target molecule may comprise a first functional group, and the target molecule may comprise a second functional group, when the targeting moiety of the target molecule and the target When the molecules are in proximity, the first functional group can react with the second functional group to form a covalent bond.
  • the polypeptide of the present application may comprise a target molecule targeting moiety, wherein the target molecule targeting moiety may comprise a first functional group, and the first functional group may be selected from the group consisting of:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0;
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl;
  • the first functional group can be wherein, R 3 can be halogen, alkenyl or alkynyl, and n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6;
  • the first functional group can be
  • R 4 can be halogen, alkenyl or alkynyl
  • n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6.
  • the polypeptides of the present application may comprise a target molecule targeting moiety, wherein the target molecule targeting moiety may comprise a first functional group, and the first functional group may be selected from the group consisting of:
  • polypeptides of the present application may comprise a targeting moiety of a target molecule, which may comprise residues of unnatural amino acids, which may be selected from the group consisting of:
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be an antibody, wherein the targeting moiety of the target molecule may be in the amino acids of the CDR region, the first 5 amino acids of the N-terminal of the CDR region and/or Or the C-terminal rear 5 amino acids of the CDR region comprise a first functional group, and the first functional group can be selected from the following group:
  • R 1 can be O, N or absent
  • R 2 can be halogen, alkenyl or alkynyl
  • R 3 can be halogen, alkenyl or alkynyl
  • R 4 can be halogen
  • n1 and n2 can be each independently at least 0;
  • the first functional group can be Wherein, R 1 can be O, N or absent, and R 2 can be halogen, alkenyl or alkynyl.
  • R 2 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl;
  • the first functional group can be wherein, R 3 can be halogen, alkenyl or alkynyl, and n1 can be at least 0.
  • R 3 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n1 can be 0 to 6;
  • the first functional group can be
  • R 4 can be halogen, alkenyl or alkynyl
  • n2 can be at least 0.
  • R 4 can be F, Cl, Br or I, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • n2 can be 0 to 6.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be an antibody, wherein the targeting moiety of the target molecule may be in the amino acids of the CDR region, the first 5 amino acids of the N-terminal of the CDR region and/or Or the C-terminal rear 5 amino acids of the CDR region comprise a first functional group, and the first functional group can be selected from the following group:
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be an antibody, wherein the targeting moiety of the target molecule may be in the CDR region, the first 5 amino acids of the N-terminal of the CDR region and/or the CDR region
  • the rear 5 amino acids of the C-terminus comprise residues of unnatural amino acids, and the unnatural amino acids can be selected from the following group:
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be an antibody, wherein the targeting moiety of the target molecule may be in the CDR region, the first 5 amino acids of the N-terminal of the CDR region and/or the CDR region
  • the last 5 amino acids at the C-terminus of C-terminal comprise residues of unnatural amino acids, which may comprise fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be an antibody, wherein the targeting moiety of the target molecule may be in the amino acids of the CDR region, the first 5 amino acids of the N-terminus of the CDR3 region and/or Or residues of unnatural amino acids are included in the last 5 amino acids of the C-terminus of the CDR3 region, and the unnatural amino acids may include fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, which may be an antibody, wherein the targeting moiety of the target molecule may comprise residues of a non-natural amino acid in the CDR region, and the non-natural amino acid may comprise Fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be a PD-L1 single domain antibody, wherein the targeting moiety of the target molecule comprises an amino acid sequence corresponding to that shown in SEQ ID NO: 13 Compared with the amino acid sequence, amino acid 108, amino acid 110 and/or amino acid 113 may be replaced by fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine.
  • the polypeptide of the present application may comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule may be a PD-L1 single domain antibody, wherein the targeting moiety of the target molecule comprises an amino acid sequence corresponding to that shown in SEQ ID NO: 13 Compared with the amino acid sequence, the 108th amino acid can be replaced by fluorosulfate-L-tyrosine.
  • the polypeptide of the present application can comprise a targeting moiety of a target molecule
  • the targeting moiety of the target molecule can be an EGFR single-domain antibody, wherein the targeting moiety of the target molecule comprises an amino acid sequence with the corresponding amino acid sequence shown in SEQ ID NO: 18
  • amino acid 116 can be replaced by fluorosulfate-L-tyrosine and/or 6-bromo-hexanoyllysine.
  • the polypeptide of the present application can comprise a targeting moiety of a target molecule, and the targeting moiety of the target molecule can be an EGFR single-domain antibody, wherein the targeting moiety of the target molecule comprises an amino acid sequence with the corresponding amino acid sequence shown in SEQ ID NO: 18
  • amino acid 116 can be replaced by fluorosulfate-L-tyrosine.
  • polypeptide of the present application may comprise a targeting moiety of a target molecule, and the amino acid sequence of the targeting moiety of the target molecule may be as shown in any one of SEQ ID NOs: 9-12.
  • the polypeptide may also comprise a small molecule species, which may be linked directly or indirectly to the targeting moiety of the target molecule.
  • the small molecule substance can be a fluorescent molecule, such as a fluorescent probe TAMRA (5-Carboxytetramethylrhodamine).
  • the application provides isolated nucleic acid molecules that can encode the conjugates and/or polypeptides described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, eg, by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified (iv) synthetic, such as by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector can be an expression vector.
  • the carrier may also include components to facilitate its entry into the cell, such as viral particles, liposomes or protein coats.
  • the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the cells can be bacterial cells (eg, E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2/0 cells, NSO cells or myeloma cells.
  • the vectors described in this application can be introduced into the host cells by methods known in the art, such as thermal transformation, electroporation, virus infection, Ca ion transformation, lipofectine transfection, lipofectamin transfection or other transfection reagents, etc. .
  • the present application also provides immunoconjugates, which may comprise the conjugates and/or polypeptides described herein.
  • the conjugates and/or polypeptides described herein, or fragments thereof can be combined with another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, etc. connect.
  • the attachment can be through one or more covalent bonds, or non-covalent interactions, and can include chelation.
  • linkers which may be known in the art, can be used to form immunoconjugates.
  • immunoconjugates can be provided in the form of fusion proteins, which can be expressed from polynucleotides encoding the immunoconjugates.
  • the immunoconjugate may also comprise, for example, an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody and the therapeutic agent can be cross-linked by a linker, which can be a cleavable linker, eg, a linker cleavable in vivo and/or in vitro, eg, a peptidic linker, a disulfide linker, or a hydrazone class connector.
  • a linker which can be a cleavable linker, eg, a linker cleavable in vivo and/or in vitro, eg, a peptidic linker, a disulfide linker, or a hydrazone class connector.
  • the present application also provides pharmaceutical compositions, which may comprise the conjugates and/or polypeptides described in the present application, the polypeptide molecules described in the present application, the immunoconjugates described in the present application, the The nucleic acid molecule described herein, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • Acceptable ingredients of the compositions may be nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical compositions of the present invention may include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, typically those active compounds with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as on the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with pharmaceutical administration, and is generally safe and nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. The uninterrupted (or continuous) administration can be accomplished by a small pump system worn by the patient to measure the flow of the therapeutic agent into the patient.
  • the present application also provides a kit, which can include the conjugate of the present application, the polypeptide of the present application, the nucleic acid molecule of the present application, the carrier of the present application, the immunoconjugate of the present application, the cells, and/or compositions of the present application.
  • the kits of the present application can be used to prevent and/or treat diseases and/or disorders, affect target molecule content, and/or prepare conjugates of the present application and/or polypeptides of the present application.
  • the application also provides the conjugate of the application, the polypeptide of the application, the nucleic acid molecule of the application, the carrier of the application, the immunoconjugate of the application, the cell of the application, the composition of the application , and/or the use of the kit of the present application in the preparation of medicaments that can be used to prevent and/or treat diseases and/or disorders.
  • the disease and/or disorder may comprise a tumor.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, CD38 positive tumors, and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 overexpressing tumors, EGFR overexpressing tumors, HER-2 overexpressing tumors, and Ras overexpressing tumors.
  • the disease and/or disorder may comprise a solid tumor.
  • the disease and/or disorder may comprise breast cancer, lung cancer, skin tumor and/or cervical cancer.
  • the disease and/or disorder may comprise breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the application also provides the conjugate of the application, the polypeptide of the application, the nucleic acid molecule of the application, the carrier of the application, the immunoconjugate of the application, the cell of the application, the composition of the application , and/or the kits of the present application, which can be used to prevent and/or treat diseases and/or disorders.
  • the disease and/or disorder may comprise a tumor.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 high expressing tumors, EGFR high expressing tumors, HER-2 high expressing tumors, CD38 high expressing tumors and Ras high expressing tumors.
  • the disease and/or disorder may comprise a solid tumor.
  • the disease and/or disorder may comprise breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the present application also provides a method of preventing and/or treating a disease and/or disorder, which may comprise administering the conjugate of the present application, the polypeptide of the present application, the nucleic acid molecule of the present application to a subject in need thereof , the carrier of the present application, the immunoconjugate of the present application, the cell of the present application, the composition of the present application, and/or the kit of the present application.
  • the disease and/or disorder may comprise a tumor.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 positive tumors, EGFR positive tumors, HER-2 positive tumors, and tumors with abnormal Ras function and/or activity.
  • the disease and/or disorder may be selected from the group consisting of PD-L1 high expressing tumors, EGFR high expressing tumors, HER-2 high expressing tumors, CD38 high expressing tumors and Ras high expressing tumors.
  • the disease and/or disorder may comprise a solid tumor.
  • the disease and/or disorder may comprise breast cancer, lung cancer, skin cancer, myeloma and/or cervical cancer.
  • the present application also provides a method for affecting the content of a target molecule, which may comprise administering the conjugate of the present application, the polypeptide of the present application, the nucleic acid molecule of the present application, the carrier of the present application, and the immunoconjugation of the present application. substances, cells of the present application, compositions of the present application and/or kits of the present application.
  • the method may comprise reducing the content of the target molecule.
  • the methods may comprise in vitro methods, ex vivo methods and/or in vivo methods.
  • the target molecule can be a cell surface molecule and/or an intracellular molecule.
  • the target molecule can be a molecule on the surface of skin cells, bone marrow cells, cervical cells, lung cells and/or breast cells.
  • the target molecule can be a molecule on the surface of skin cancer cells, myeloma cells, cervical cancer cells, lung cancer cells and/or breast cancer cells.
  • the cells may be selected from the group consisting of PD-L1 positive cells, EGFR positive cells, HER-2 positive cells, CD38 positive cells, and tumors with abnormal Ras function and/or activity.
  • the cells may be selected from the group consisting of PD-L1 high expressing cells, EGFR high expressing cells, HER-2 high expressing cells, CD38 high expressing cells and Ras high expressing cells.
  • the present application also provides a method for preparing the conjugate of the present application and/or the polypeptide of the present application, which may comprise culturing the cell.
  • target molecule targeting moiety and the protein degrader targeting moiety can be linked by transpeptidase A (Srt A).
  • the C-terminus of the targeting moiety of the target molecule may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and/or the N of the targeting moiety of the target molecule
  • the termini may contain oligoglycine sequences.
  • the C-terminus of the protein degrader targeting moiety may comprise the amino acid sequence LPETG or LPXTG as set forth in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and/or the protein degrader targeting moiety
  • the N-terminus of can contain oligoglycine sequences.
  • the C-terminus of the targeting moiety of the target molecule may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and the N-terminus of the protein degrader targeting moiety may Containing an oligoglycine sequence, the targeting moiety of the target molecule and the protein degrader targeting moiety can be linked by the transpeptidase A (Srt A).
  • Srt A transpeptidase A
  • the C-terminus of the protein degrader targeting moiety may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and the N-terminus of the targeting moiety of the target molecule may Containing an oligoglycine sequence, the targeting moiety of the target molecule and the protein degrader targeting moiety can be linked by the transpeptidase A (Srt A).
  • the present application also provides a method for screening targeting moieties of target molecules with high proximity reactivity.
  • covalent antibodies can be prepared by introducing unnatural amino acids with adjacent reactivity into single-domain antibodies.
  • the site for introducing the non-natural amino acid with adjacent reactivity can be the CDR region of a single domain antibody, the first 5 amino acid positions of the N-terminal of the CDR region and/or the first 5 amino acid positions of the C-terminal of the CDR region.
  • the following screening method can be used: the CDR region of IgG, scFv, Fab or single domain antibody, the first 5 amino acid sites of the N-terminal of the CDR region and / or insert an unnatural amino acid with adjacent reactivity into the first 5 amino acid sites of the C-terminal of the CDR region, or according to the crystal structure of the antibody and antigen complex, search for the 5 angstroms of the interface amino acid of the antigen, which can be used on the antibody.
  • a candidate site for inserting an unnatural amino acid with adjacent reactivity for example, the interface amino acid of the antigen can be the closest amino acid to the antibody on the antigen, for example, it can be the interface amino acid with a nucleophilic group, and the candidate site of the antibody can be the distance All sites within 5 angstroms of the interface amino acid; after mutating the candidate site into an unnatural amino acid, a covalent antibody library with proximity reactivity can be obtained for screening the targeting moiety of the target molecule with higher proximity reactivity.
  • an amino acid at a candidate site can be replaced by a non-natural amino acid with adjacent reactivity by genetic code expansion techniques.
  • the cross-linked band between the targeting moiety of the target molecule and the antigen with higher adjacent reactivity can be separated by denaturing gel electrophoresis.
  • tandem mass spectrometry analysis, and the insertion position of adjacent reactive unnatural amino acids and nucleophilic residues reacting on the antigen in covalent antibodies with higher adjacent reactivity can be obtained by the analysis of cross-linked peptide fragments.
  • the method of verifying whether the targeting moiety of the target molecule of the present application can be covalently bound to the target molecule may be, incubating the targeting moiety of the target molecule and the target molecule together, washing with a denaturant, and detecting the binding of the targeting moiety of the target molecule to the target molecule. Whether the molecule can still bind.
  • a fluorescently labeled ⁇ PD-L1 covalent antibody can be incubated with another fluorescently labeled target molecule, and confocal fluorescence imaging can be used to characterize whether the targeting moiety of the target molecule can still bind to the target molecule.
  • protein cross-linking can be tested by western blot to produce higher molecular weight cross-linked bands.
  • a conjugate can be obtained by attaching a functional polypeptide to the C-terminus or N-terminus of a covalent antibody by transpeptidase.
  • the degradation process of degrons can be characterized by co-localized imaging of fluorescently labeled conjugates, target molecule white tandem green fluorescent protein, and lysosomal tracers.
  • conjugates of different structural forms can be incubated with tumor cells, and optionally lysosomal inhibitors can be added to a portion of the culture medium, after which the cells are harvested, proteins extracted, lysed at high temperature and denatured, and western blotted. The degree of degradation of the target molecule by the conjugate is quantitatively examined.
  • the targeting moiety of the target molecule described in this application can be linked by covalent bonds, which can reduce the dissociation value of the antibody to the antigen indefinitely, and can solve the shortcomings of short half-life and easy off-target of the fragment antibody.
  • Conjugates prepared by coupling functional molecules with transpeptidase can be rapidly endocytosed after binding to target molecules and finally enter lysosomes for degradation. Using the characteristics of irreversible binding between the targeting part of the target molecule and the target molecule, the conjugate-target molecule complex can not be dissociated during the transport process, avoiding the dissociation of the target molecule before reaching the lysosome, which cannot be degraded .
  • the present application can achieve efficient degradation of membrane proteins.
  • LYTAC and AbTAC the present application can achieve the same degradation efficiency for membrane protein degradation without relying on specific tumor surface receptors.
  • the conjugates can have broader application prospects in the fields of basic scientific research and anti-tumor therapy.
  • the present application also provides a method for preparing the conjugate of the present application, which may comprise directly or indirectly linking the target molecule targeting moiety of the conjugate to the protein degrader targeting moiety.
  • target molecule targeting moiety and the protein degrader targeting moiety can be linked by transpeptidase A (Srt A).
  • the C-terminus of the targeting moiety of the target molecule may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and/or the N of the targeting moiety of the target molecule
  • the termini may contain oligoglycine sequences.
  • the C-terminus of the protein degrader targeting moiety may comprise the amino acid sequence LPETG or LPXTG as set forth in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and/or the protein degrader targeting moiety
  • the N-terminus of can contain oligoglycine sequences.
  • the C-terminus of the targeting moiety of the target molecule may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and the N-terminus of the protein degrader targeting moiety may Containing an oligoglycine sequence, the targeting moiety of the target molecule and the protein degrader targeting moiety can be linked by the transpeptidase A (Srt A).
  • Srt A transpeptidase A
  • the C-terminus of the protein degrader targeting moiety may comprise the amino acid sequence LPETG or LPXTG as shown in SEQ ID NO: 92 or 93, wherein X may be any amino acid, and the N-terminus of the targeting moiety of the target molecule may Containing an oligoglycine sequence, the targeting moiety of the target molecule and the protein degrader targeting moiety can be linked by the transpeptidase A (Srt A).
  • a target molecule targeting moiety precursor capable of binding the target molecule can be prepared first.
  • wild-type single domain antibodies can be used as target molecules to target partial precursors.
  • the expression vector pET 20b(+) (Novagen, Cat. No. 69739-3) was transformed by homologous recombination using a recombinase (Novagen, Cat. No. C112-01), and an enzyme cleavage site Not was introduced into it. 1, obtain the expression vector Nb vector specially used for wild-type single domain antibody, wherein the primer pair used for linearization pET 20b (+) is as follows:
  • Upstream primer GAAGCTGAGTGCGGCCGCAGGAAGCTGAGTTGGCTGC (as shown in SEQ ID NO: 7);
  • Downstream primer CTGCGGCCGCACTCAGCTTCCTTTCGGGCTTTGTTAGC (as shown in SEQ ID NO: 8);
  • a double-stranded nucleic acid comprising a PD-L1 single-domain antibody expression sequence (the PD-L1 single-domain antibody amino acid sequence can be shown in SEQ ID NO: 14 or 16, and the nucleic acid sequence can be shown in SEQ ID NO: 15 or 17), and a double-stranded nucleic acid comprising an EGFR single-domain antibody expression sequence (the EGFR single-domain antibody amino acid sequence can be shown in SEQ ID NO: 19, and the nucleic acid sequence can be shown in SEQ ID NO: 20).
  • a transpeptidase recognition sequence such as the amino acid sequence LPETG shown in SEQ ID NO: 92, can be added to the C-terminus of the single-domain antibody, followed by restriction The endonucleases Nco I and Not I subcloned the coding sequence of the single domain antibody into the Nb vector.
  • the Nb vector plasmid containing the single domain antibody was transformed into the expression host strain BL 21 (DE3) (TIANGEN, Cat. No. CB105-02) by heat shock method, which was coated with ampicillin (100 ⁇ g/mL) Plates of solid medium at 37 °C overnight.
  • a single colony was selected for inoculation and cultured overnight, and the overnight strain was transferred to a fresh auto-induction medium at a ratio of 1:100 the next day (each liter contained 6g Na 2 HPO 4 , 3g KH 2 PO 4 , 20g peptone ( typtone), 5g yeast extract (yeast extract), 5g NaCl, 200mg MgCl 2 , 200mg CaCl 2 , 0.5g glucose, 2.5g lactose, 6mL glycerol), shaken at 37°C until the OD value reaches 0.6-0.8, transfer Incubate at 27°C on a shaker for 12-16 hours. Centrifuge at 4000 rpm, discard the supernatant medium, and collect the bacterial cells.
  • the purified protein was concentrated and stored in a In the PBS (pH 7.4) buffer solution of glycerol, the purification effect was characterized by UPLC/MS. The results showed that the molecular weight of the single domain antibody was correct and the purity was above 95%.
  • Figures 2A and 2B show primary mass spectrograms of wild-type single domain antibodies that can be used as target molecules to target partial precursors.
  • Figure 2A is the mass spectrum of the wild-type single-domain antibody ⁇ PD-L1, the theoretical value is 16016Da, and the measured value is 16015Da
  • Figure 2B is the mass spectrum of the wild-type single-domain antibody ⁇ EGFR, the theoretical value is 15603Da, and the measured value is 15601Da.
  • a first functional group that can form a covalent bond to the target molecule can be introduced into the amino acid site of the precursor of the target molecule targeting moiety.
  • the interfacial amino acids of single domain antibodies can be replaced with unnatural amino acids.
  • the method for determining the interface amino acid site may be to first divide the single-domain antibody into the CDR region according to the CDR division method commonly used in the art.
  • the interface amino acid can be any amino acid in the CDR region, and the interface amino acid can also be the first 5 N-terminus of the CDR.
  • any amino acid among these amino acids, and the interface amino acid can also be any amino acid among the five amino acids after the C-terminus of the CDR.
  • the amino acid site of the precursor of the targeting moiety of the target molecule can be replaced by a non-natural amino acid that can form a covalent link with the target molecule by means of amber codon TAG mutation.
  • Upstream primer AGTTGGCTGCTGCCACCAGAATTTGCCTGGCGGCA (as shown in SEQ ID NO:21);
  • Downstream primer GCAGGTATTTCATATGGTTAATTCCTCCTGTTAGCCC (as shown in SEQ ID NO:22);
  • Amber codon TAG mutations were introduced into the expressed sequence of single domain antibodies by overlay linear amplification PCR using PCR polymerase (Novizan, Cat. No. P505-d1) for insertion of unnatural amino acids.
  • Table 1 shows the primers used for the overlaid linear amplification PCR that introduced TAG mutations at different sites.
  • Table 1 The primers used in the coverage linear amplification PCR for introducing TAG mutations at different sites
  • the single-domain antibody expression vector plasmid with amber codon TAG mutation was co-transformed into the host strain DH10B (Zhuangmeng Biotechnology, Cat. No. ZC112-2) by heat shock method with tRNA Synthetase/tRNA vector plasmid respectively. ), spread on solid medium plates containing ampicillin (100 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL) overnight at 37°C. A single colony was picked from each plate and inoculated into LB medium containing antibiotics, and cultured at 37°C and 220rpm for 12 hours.
  • the purified protein was stored in a glycerol-containing In PBS (pH 7.4) buffer solution, the purification effect was characterized by UPLC/MS.
  • the synthesis of unnatural amino acids can be known in the art, for example, the synthase of 6-bromo-hexanoyllysine (BrC6K) can be described in Angew.Chem.Int.Ed.Engl.56, 14521-14525 (2017).
  • the synthase of fluorosulfate-L-tyrosine (FSY) can be described in J. Am. Chem. Soc. 140, 4995-4999 (2016).
  • two adjacent cross-linking reactive unnatural amino acids can be introduced into single-domain antibodies, respectively.
  • the results show that the molecular weights of the unnatural amino acids introduced at different sites are reflected in the mass spectrometry results.
  • This method can obtain the target molecule targeting partial compound library of the unnatural amino acids inserted at different sites.
  • Table 2 shows the molecular weight of PD-L1 single-domain antibody after different sites were replaced by BrC6K or FSY.
  • Figure 3A and Figure 3B show the mass spectra of the resulting compound library after different sites of PD-L1 single domain antibody were replaced by BrC6K or FSY.
  • Figure 3A shows the mass spectrum of the obtained compound library after different sites of PD-L1 single domain antibody were replaced by BrC6K
  • Figure 3B shows the obtained compound library after different sites of PD-L1 single domain antibody were replaced by FSY mass spectrum.
  • the compound library obtained after being replaced by BrC6K or FSY (the compound library contains the complexes of multiple single-domain antibodies replaced by BrC6K or FSY at different sites) and 70 ⁇ L PBS buffer (pH 8.0), 37 °C after a certain time reaction , lyophilized to remove solvent.
  • Add 40 ⁇ L of 1x loading buffer Karl Wei Century, Cat. No.
  • Figure 4A and Figure 4B show the cross-linking results of the obtained compound library with the target molecule after different sites of PD-L1 single-domain antibody were replaced by BrC6K or FSY by SDS-PAGE analysis.
  • Figure 4A shows the cross-linking results of the obtained compound library after the different sites of PD-L1 single domain antibody were replaced by BrC6K and the target molecule incubated for 0 hours and after 72 hours of incubation;
  • Figure 4B shows the results of PD-L1 single domain Cross-linking results after incubation of the resulting compound library with the target molecule after the different sites of the domain antibody were replaced by FSY.
  • the targeting part of the target molecule can form a new covalent interaction with the antigen protein, and this covalent binding is not affected by the denaturing agent SDS.
  • the target molecule targeting moieties in the obtained compound library all have adjacent cross-linking reactivity.
  • the PD-L1 single-domain antibody band and the PD-L1 band were separated from the gel.
  • the cut peptides were identified by multi-stage mass spectrometry.
  • the p-Find software searched for the peptides, and the obtained results were used as Control analysis.
  • the target molecules with high adjacent cross-linking reactivity can be determined. Unnatural amino acid insertion sites, and where cross-linking on the target molecule can be determined.
  • Figure 5A, Figure 5B and Figure 5C show the tandem mass spectrometry of the cross-linked peptide segment after the targeting moiety of the target molecule with high adjacent cross-linking reactivity is cross-linked with the target molecule.
  • Figure 5A shows that when the PD-L1 single domain antibody is replaced by BrC6K at position 110, it can have higher adjacent cross-linking reactivity with the 69th histidine of the target molecule PD-L1;
  • Figure 5B shows that, When position 113 of PD-L1 single-domain antibody is replaced by BrC6K, it can have higher adjacent cross-linking reactivity with histidine 69 of the target molecule PD-L1;
  • Figure 5C shows that PD-L1 single-domain antibody When the 108 position in the middle is replaced by FSY, it can have a higher adjacent cross-linking reactivity with the 69th histidine of the target molecule PD-L1.
  • the target molecule is expressed and purified: PD-L1 single domain antibody ( ⁇ PD-L1 T110BrC6K) after the 110th position is replaced by BrC6K, PD-L1 single domain antibody ( ⁇ PD-L1 G113BrC6K) after the 113th position is replaced by BrC6K ), a PD-L1 single-domain antibody ( ⁇ PD-L1 L108FSY) with position 108 replaced by FSY, and an EGFR single-domain antibody ( ⁇ EGFR Q116FSY) with position 116 replaced by FSY.
  • Figure 6A, Figure 6B, Figure 6C and Figure 6D show the mass spectrograms of the targeting moieties of the resulting target molecules after different sites in PD-L1 single domain antibody or EGFR single domain antibody are replaced by BrC6K or FSY.
  • Figure 6A shows the PD-L1 single domain antibody ( ⁇ PD-L1 T110BrC6K) after the 110th position was replaced by BrC6K
  • Figure 6B shows the PD-L1 single domain antibody ( ⁇ PD-L1 single domain antibody) after the 113th position was replaced by BrC6K L1 G113BrC6K
  • Figure 6C shows the PD-L1 single-domain antibody ( ⁇ PD-L1 L108FSY) after the 108th position was replaced by FSY
  • Figure 6D shows the EGFR single-domain antibody after the 116th position was replaced by FSY ( ⁇ EGFR Q116FSY).
  • Figure 7A, Figure 7B, and Figure 7C show the results of in vitro crosslinking detection of the obtained target molecule targeting moiety and the target molecule after different sites in PD-L1 single domain antibody or EGFR single domain antibody are replaced by BrC6K or FSY.
  • Figure 7A shows the SDS-PAGE analysis of wild-type PD-L1 single domain antibody, the target molecule targeting part ⁇ PD-L1 T110BrC6K, and the target molecule targeting part ⁇ PD-L1 G113BrC6K were incubated with PD-L1 protein for 24 hours.
  • the targeting part of the target molecule of the present application can form a cross-linked band with the target molecule;
  • Figure 7B shows that SDS-PAGE analysis of the mixture of ⁇ PD-L1 L108FSY and PD-L1 protein co-incubated for 24 hours.
  • the targeting part of the target molecule can form a cross-linked band with the target molecule;
  • Figure 7C shows that the wild-type EGFR single-domain antibody was analyzed by SDS-PAGE, and ⁇ EGFR Q116FSY was incubated with the target molecule EGFR protein for 12 hours.
  • the picture shows the results of fluorescent scanning gel showing that the targeting moieties of the target molecules of the present application can form cross-linked bands with the target molecules.
  • the targeting moieties of the target molecules obtained in the present application can all have the activity of cross-linking adjacent to the target molecules, and the targeting moieties ⁇ PD-L1 L108FSY obtained in the present application can have higher cross-linking reactions adjacent to PD-L1.
  • ⁇ EGFR Q116FSY was able to have higher cross-linking reactivity adjacent to EGFR.
  • the C-terminal of the targeting moiety of the target molecule is labeled with a fluorescent dye, for example, a carboxyrhodamine fluorescent substrate, using the transpeptidase Sortase A.
  • the targeting part of the target molecule with the LPETG transpeptidase linking sequence was replaced by ultrafiltration into the reaction solution (25mM Tris, 150mM NaCl, pH 7.4), the antibody concentration was adjusted to 100 ⁇ M, and 10 times the mass equivalent of GGG- TAMRA substrate and 0.1 times mass equivalent of transpeptidase Sortase A were incubated at 37°C for 1 hour to connect the fluorescent substrate to the targeting moiety of the target molecule, and MTSET reagent (Biorigin, Cat#BN15003) at a final concentration of 5mM was added, Biorigin -rad desalting column centrifuged to remove the substrate while replacing it with PBS (pH 7.4) solution, adding 10% glycerol at -80 °C for storage or use.
  • the method for constructing the cells expressing PD-L1 or expressing PD-L1-GFP can be a construction method commonly used in the art.
  • 3 plasmids pLV-PD-L1-C-GFP Spark (Yiqiao Shenzhou, Cat. No. HG10084-ACGLN), psPAX2 (Miao Ling Bio, Cat. No.
  • pMD2.G (Miao Ling Bio) can be combined Bio, Cat.No.P0262) was co-transfected into HEK 293T cells with a growth density of 80%, using DMEM medium containing 10% fetal bovine serum, and placed at a constant temperature of 37 °C and containing 5% CO 2 culture The fresh DMEM medium was replaced after 24 hours of culture in the incubator, the first virus was collected and replaced with fresh medium 48 hours after transfection, and the second and third virus particles were collected after 72 hours and 96 hours.
  • MDA-MB-231 Luc breast cancer cells Novo Bio, MDA-MB-231+Luciferase were grown in a 24-well plate.
  • the transfection of PD-L1 can be characterized by photographing fluorescent antibody-labeled cells by confocal fluorescence microscopy.
  • the human breast cancer MDA cells (PD-L1/MDA) expressing PD-L1 (PD-L1/MDA) in the culture dishes were digested and blown off, plated into 8-well plates at a ratio of 1:5, and cultured at 37°C for 12 hours. The final concentration of 200nM fluorescently labeled target molecule targeting moiety or wild-type single domain antibody was added, and the incubation was continued for 5 hours.
  • the normal washing group was washed three times with PBS solution (pH 7.4) for 15 min each time; the strong denaturing washing group was washed with strong denaturing solution (Stringent solution, 500mM NaCl, 3% Tween 20, 100mM glycine, pH 3.0), washed three times, each time For 15 min, the solution is under intense denaturing conditions, so that substances bound to the cell surface by non-covalent interactions (eg, wild-type single domain antibodies) will dissociate from the cells. Fluorescence imaging of the TAMRA channel using a Zeiss confocal fluorescence microscope.
  • Figure 8A, Figure 8B, Figure 8C and Figure 8D show the detection results of the binding of the targeting moiety of the target molecule to the cell surface target molecule.
  • Figure 8A shows that the target molecule targeting part ⁇ PD-L1 BrC6K after the PD-L1 single-domain antibody is replaced by BrC6K still binds to the target molecule after washing with strong denaturing solution
  • Figure 8B shows that the immunoblotting test characterizes ⁇ PD-L1 BrC6K and the target Molecular PD-L1 undergoes a cross-linking reaction
  • Figure 8C shows that the target molecule targeting part ⁇ EGFR FSY after the EGFR single domain antibody is replaced by FSY still binds to the target molecule after washing with strong denaturing solution
  • Figure 8D shows that the immunoblotting test characterizes ⁇ EGFR FSY cross-links with the target molecule PD-L1.
  • the targeting moiety of the target molecule ( ⁇ PD-L1 BrC6K or ⁇ EGFR FSY ) could still be detected on the cell surface after treatment with strong denaturing solution, while the wild-type single-domain antibody ( ⁇ PD-L1 or ⁇ EGFR) basically disappeared.
  • the targeting moiety of the target molecule binds to the target molecule on the surface of the cell membrane, under the promotion of the proximity effect, the unnatural amino acid with proximity cross-linking activity on the targeting moiety of the target molecule will interact with the nucleophilic residue on the target molecule. reaction, the binding mode of the two is changed from non-covalent binding to covalent cross-linking.
  • the present application provides a method for preparing a conjugate with degradation ability by linking a protein degrader targeting moiety to a target molecule targeting moiety.
  • a cell penetrating peptide and a lysosomal targeting peptide can be attached to the N- or C-terminus of the targeting moiety of the target molecule through an enzymatic coupling reaction, while maintaining the target molecule targeting Part of the ability of targeted binding and proximity cross-linking reactions to target molecules.
  • the penetrating peptide mentioned in this application refers to a polypeptide that can help extracellular substances to pass through the cell membrane.
  • a polypeptide obtained by connecting multiple positively charged arginines in series has a good ability to penetrate the membrane.
  • the penetrating peptide can be multiple Polyarginine; another example, NPXY (as shown in SEQ ID NO:64, wherein X can be glycine G, or alanine A), ALAPYIP (as shown in SEQ ID NO:77) can be well targeted lysosome.
  • transpeptidation reaction substrate sequences for the coupling reaction composed of the two include but are not limited to the protein degraders shown in Table 3. targeting part.
  • polypeptide substrates are handed over to a polypeptide synthesis company (Beijing Zhongke Yaguang Biotechnology Co., Ltd.), and can be synthesized by a standard solid-phase peptide synthesis method. After purification by HPLC, the purity is >95%.
  • G or G', L or L' represent the sequence recognized and connected by the transpeptidase Sortase A
  • CPP can represent polyarginine, for example, a polypeptide formed by connecting 9 D-configuration arginines
  • LTP can represent Represents NPXY targeting lysosome sequence (as shown in SEQ ID NO:64, wherein X can be glycine G, or alanine A)
  • LTS can represent ALAPYIP targeting lysosome sequence (as shown in SEQ ID NO:77 shown)
  • MMP-B or B-MMP can represent a negatively charged sequence that shields polyarginine, wherein the MMP sequence can be cleaved by proteases on the cell surface, so that the negatively charged sequence of the B sequence is cleaved, making the polyarginine cleaved Amino acid exerts the ability to penetrate the membrane.
  • Figure 9A and Figure 9B are schematic diagrams showing the preparation of the conjugate of the present application by linking the targeting moiety of the transpeptidase-mediated protein degrader and the targeting moiety of the target molecule.
  • Figure 9A shows that the C-terminal connexin degrader targeting moiety of the target molecule prepares the conjugate of the present application;
  • Figure 9B shows that the N-terminal connexin degrader targeting moiety of the target molecule prepares the present application conjugate.
  • the targeting moiety of the target molecule with the LPETG transpeptidase linking sequence was replaced with the reaction solution (25mM Tris, 150mM NaCl, 5mM CaCl 2 , pH 7.4) by ultrafiltration or desalting, and the final concentration of the antibody was adjusted to 100 ⁇ M, and the final concentration was added.
  • Protein degrader targeting moiety e.g. Gn- CPP+LTS, where n can be 1 to 10
  • transpeptidase Sortase A at a final concentration of 10 ⁇ M, incubate at 30°C for 1 hour, and monitor the reaction by LC-MS process.
  • MTSET reagent with a final concentration of 5 mM was added, mixed and incubated at room temperature for 10 min.
  • the substrate was removed using a desalting column (Bio-rad, Cat. No. 7326221) or PD-10 (GE, Cat. No. 52-1308-00) while changing into a PBS (pH 7.4) solution.
  • His-tag can be composed of 6-10 consecutive histidine residues, and the His-tag can be adsorbed by the nickel column; because the His-tag at the C-terminus of the targeting part of the target molecule involved in the ligation reaction is excised during the reaction , and the C-terminus of the transpeptidase has a His tag, so nickel magnetic beads or resin can be used to remove the remaining unreacted target molecule targeting moiety and transpeptidase in the system. Because it is a product obtained after site-specific linkage and purification, one molecule of protein degrader targeting moiety can be attached to each targeting moiety of the target molecule. The resulting conjugate was characterized by LC-MS mass spectrometry (LC-MS).
  • Figure 10 shows the primary mass spectrometry of the conjugate GlueTAC FSY /PD-L1 obtained by the targeting moiety of PD-L1 target molecule ⁇ PD-L1 L108FSY and the targeting moiety of PD-L1 target molecule linked to the targeting moiety of the protein degrader picture.
  • Figure 11 shows the conjugate NbTAC-EGFR obtained by linking the protein degrader targeting moiety of a wild-type EGFR single domain antibody, and the conjugate GlueTAC-EGFR obtained by linking the EGFR target molecule targeting moiety to the protein degrader targeting moiety of the first-order mass spectrum.
  • N-terminal linker targeting moiety of target molecule targeting moiety N-terminal linker targeting moiety of target molecule targeting moiety
  • the present application adds an oligoglycine sequence (eg using triglycine) to the N-terminal of the targeting moiety of the target molecule.
  • an oligoglycine sequence eg using triglycine
  • Upstream primer GATCCGACCTGTACCTAGGTTACCAGCAGCGGTG (as shown in SEQ ID NO:94);
  • Downstream primer ACCGCTGCTGGTAACCTAGGTACAGGTCGGATCTTC (as shown in SEQ ID NO:95).
  • Upstream primer CCTGGTTACCAGCAGCTAGGCATTTCAGTATTGGG (as shown in SEQ ID NO:96);
  • Downstream primer CCAATACTGAAATGCCTAGCTGCTGGTAACCAGGG (as shown in SEQ ID NO:97).
  • the wild-type single-domain antibody NG 3 - ⁇ PD-L1 with triglycine at the N-terminus was purified by the expression and purification methods of the present application, and the target molecule targeting part NG 3 - ⁇ PD-L1 L108FSY and target molecule targeting moiety NG3 - ⁇ PD-L1 G113BrC6K.
  • MTSET reagent with a final concentration of 5 mM was added, mixed and incubated at room temperature for 10 min.
  • a cation exchange resin to purify the target product from the reaction system, and use a desalting column (Bio-rad, Cat.No.7326221) or PD-10 (GE, Cat.No.52-1308-00) to remove the substrate after concentration.
  • PBS pH 7.4
  • the conjugate of the present application undergoes an adjacent cross-linking reaction after targeting the target molecule, resulting in a covalently linked complex, which enters the cell and is degraded together in the protein degrader.
  • the conjugate and the lysosomal localization probe With the fusion of green fluorescent protein, by monitoring the fluorescent signals of the target molecule, the conjugate and the lysosomal localization probe, the process of the conjugate promoting the degradation of the target molecule can be observed.
  • Human breast cancer MDA cells (PD-L1-GFP/MDA, constructed by the method of the present application) expressing PD-L1 and GFP fusion protein in a petri dish were digested and blown, and plated into 8-well plates at a ratio of 1:5 , 37 °C for 12 hours.
  • the final concentration of 200nM TAMRA fluorescently labeled wild-type PD-L1 single-domain antibody (control group) and the final concentration of 200nM TAMRA fluorescently labeled PD-L1 target molecule targeting part were added to the protein degrader targeting part.
  • GlueTAC test group Conjugate GlueTAC-PD-L1 (GlueTAC test group), add lysosomal fluorescent probe (Lysotracker Deepred, Thermo, Cat. No. L12492), and continue to culture for 5 hours. After three washes with PBS buffer (pH 7.4), the three channels of GFP, TAMRA and Deepred were subjected to fluorescence imaging using a Zeiss confocal fluorescence microscope.
  • Figure 12 shows the live cell confocal fluorescence imaging of the control group and the GlueTAC test group.
  • the results showed that there was no obvious consistency in the spatial distribution of the fluorescently labeled single domain antibody and the target molecule in the control group; the fluorescently labeled conjugate and the target molecule had a certain degree of consistency in the spatial distribution, and their fluorescence showed aggregation.
  • the conjugate and the target molecule exist in the form of a combined complex.
  • the fluorescence merge map shows that the spatial distribution of the red fluorescent lysosomal localization probe and the conjugate-target molecule complex are also consistent to a certain extent. It can be considered that the target molecule is mediated by the conjugate of the present application. sent to the lysosome for degradation.
  • human breast cancer MDA-MB-231 cells (PD-L1-GFP/MDA) expressing PD-L1 and GFP fusion proteins were digested and blown off in petri dishes. , constructed by the method of the present application) or human non-small cell lung cancer HCC827 cells (Procell, Cat. No. CL0094) were plated into 12-well plates at a ratio of 1:3 and cultured at 37°C for 12 hours.
  • Blank control group add buffer, such as Hank's Balanced Salt Solution (HBSS); test group A: add wild-type PD-L1 single domain antibody ( ⁇ PD-L1) at a final concentration of 200nM; test group B: The conjugate obtained by adding the N-terminal junction protein degrader targeting part of the wild-type PD-L1 single-domain antibody at a final concentration of 200nM (NbTAC-N/PD-L1); test group C: adding the wild-type PD-L1 single-domain antibody at a final concentration of 200nM The conjugate obtained from the targeting moiety of the C-terminal linker protein degrader of the type PD-L1 single-domain antibody (NbTAC-C/PD-L1); test group D: PD-L1 single-domain antibody No.
  • HBSS Hank's Balanced Salt Solution
  • test group E the final concentration of 200nM PD-L1 target molecule targeting moiety ⁇ PD-L1 G113BrC6K ligation protein degrader targeting moiety obtained Compound GlueTAC BrC6K /PD-L1;
  • Test Group F The conjugate GlueTAC FSY /PD-L1 obtained by adding the PD-L1 target molecule targeting moiety ⁇ PD-L1 L108FSY at a final concentration of 200nM.
  • the primary antibodies were mouse anti-human PD-L1 (CST) and mouse anti-human Tubulin (Cell Signaling Technologies, Cat#). 2125S) and mouse anti-His (Cell Signaling Technologies, Cat#12698S), the secondary antibody is rabbit anti-mouse IgG (Abcam, Cat.No.ab6728), and the image obtained by the final development uses Image Lab software to calculate the grayscale of each band value to quantitatively calculate the relative content of PD-L1.
  • Figure 13A, Figure 13B, Figure 13C and Figure 13D show the results of immunoblotting assays to characterize the conjugates of the present application to promote the degradation of target molecules.
  • Figure 13A, Figure 13B and Figure 13D show the results of promoting the degradation of the target molecule PD-L1 after the conjugate of the present application is incubated with human non-small cell lung cancer HCC827 cells for 24 hours
  • Figure 13C shows the conjugate of the present application with Human breast cancer MDA-MB-231 cells were incubated for 24 hours to promote the degradation of the target molecule PD-L1.
  • the conjugate obtained from the N-terminal or C-terminal linker targeting moiety of wild-type PD-L1 single-domain antibody could promote the degradation of target molecules.
  • the conjugate of the present application obtained by connecting the targeting moiety of the target molecule with the adjacent cross-linking reaction activity to the targeting moiety of the protein degrader can achieve about 80% higher degradation efficiency of the target molecule. It is believed that the conjugates of the present application may have the ability to promote the degradation of target molecules.
  • Blank control group add buffer, such as Hank's Balanced Salt Solution (HBSS); test group A: add wild-type EGFR single-domain antibody ( ⁇ EGFR, 7D12); test group B: add wild-type EGFR single-domain antibody
  • HBSS Hank's Balanced Salt Solution
  • test group A add wild-type EGFR single-domain antibody ( ⁇ EGFR, 7D12); test group B: add wild-type EGFR single-domain antibody
  • HBSS Hank's Balanced Salt Solution
  • Figure 14 shows the results of immunoblotting assays to characterize the degradation of target molecules by the conjugates of the present application. The results show that the content of the target molecule EGFR can be significantly reduced after co-incubating with HeLa cells for 24 hours.
  • ammonium chloride can inhibit the functions of lysosomes and proteasomes, and the test group can add protein degrader inhibitor ammonium chloride (Sigma, Cat.No. .V900222-500G) to verify the degradation pathway of the conjugates of the present application.
  • Figure 15 shows the results of application of conjugates to promote degradation of target molecules in the presence of ammonium chloride.
  • the results show that in the presence of a protein degrader inhibitor, the degradation efficiency of the conjugate obtained by connecting the targeting moiety of the target molecule of the present application to the targeting moiety of the protein degrader is significantly reduced. It is believed that the conjugation of the present application can degrade the target molecule by a protein degrader.
  • PD-L1/MDA (5x10 5 ) cells were suspended in Hank's Balanced Salt Solution (HBSS) containing 50% high-concentration Matrigel, in a total volume of 100 ⁇ L, on the right side of 6-week-old female BALB/c Nude mice. Subcutaneous injection over the legs (approved by the Ethics Committee of Peking University Animal Experiments). After about 1 week, the bulge can be clearly observed, and there is a foreign body sensation to the touch. The volume is about 50-70mm 3 when measured with a vernier caliper in about 2 weeks.
  • HBSS Hank's Balanced Salt Solution
  • test group A injection buffer, such as Hank's Balanced Salt Solution (HBSS);
  • test group B injection of wild-type PD-L1 single domain antibody ( ⁇ PD-L1 );
  • test group B the conjugate obtained by injecting the targeting moiety of the wild-type PD-L1 single-domain antibody connexin degrader (NbTAC-PD-L1);
  • test group C injecting the targeting moiety of PD-L1 target molecule ⁇ PD- L1 L108FSY-linked protein degrader targeting moiety of the resulting conjugate (GlueTAC-PD-L1).
  • Take 20 mg of shredded tumor tissue add 200 ⁇ L of RIPA lysis solution, ice bath, disperse the tumor with a homogenizer, remove the fat layer by centrifugation, and react at room temperature for 1 hour to fully lyse the cancer cells.
  • 40 ⁇ L of samples were added with 5x loading buffer, analyzed by Western blotting, and labeled with PD-L1 antibody (CST, Cat. No. 13684) and GADPH antibody (Abcam, Cat. No. ab128915), respectively, and then developed.
  • CST Cat. No. 13684
  • GADPH antibody Abcam, Cat. No. ab128915
  • Figure 16 shows the result that the conjugate of the present application promotes the degradation of target molecules in tumor tissue in vivo.
  • the results show that the conjugate of the present application can significantly reduce the content of the target molecule in vivo.

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Abstract

一种用于靶分子修饰的缀合物及其制备方法。还涉及一种缀合物，其包含靶分子靶向部分以及蛋白降解器靶向部分，其中，所述靶分子靶向部分能够特异性识别所述靶分子并与该靶分子共价结合，所述蛋白降解器靶向部分与所述靶分子靶向部分直接或间接连接。所述的用于靶分子修饰的缀合物可以具有促进靶分子降解的效果。

Description

用于靶分子修饰的缀合物及其制备方法 技术领域
本申请涉及生物医药领域，具体的涉及一种用于靶分子修饰的缀合物及其制备方法。
背景技术
特异性降解靶分子成为疾病治疗中十分有前景的一种治疗手段。蛋白降解靶向嵌合物(Proteolysis Targeting Chimera，PROTAC)的化合物进入细胞后可以同时靶向胞内肿瘤相关蛋白和E3连接酶实现胞内蛋白的降解。然而，此类化合物难以实现细胞表面膜蛋白的降解，而且筛选靶向膜蛋白的高亲和力的小分子物质较为困难。溶酶体靶向嵌合体(lysosome targeting chimera,LYTAC)技术将靶向细胞表面受体CI-M6PR的寡糖通过化学反应偶联到靶蛋白的抗体上，然而LYTAC化合物的制备中寡糖的合成路线非常复杂，而采用的偶联技术也无法得到均一的产物，LYTAC化合物与靶分子的结合能力和降解效率也有待提高。AbTAC则开发了双特异性抗体，其中一个结构域靶向内吞受体RNF43，另一结构域靶向靶蛋白，然而制备均一的双特异性抗体具有较大的挑战性，同时该技术受限于靶细胞的内吞受体RNF43的表达。因此，开发一种不依赖于特定受体的、高效的膜蛋白降解技术迫在眉睫。
发明内容
本申请提供一种用于靶分子修饰的缀合物及其制备方法。本申请的用于靶分子修饰的缀合物可以具有合成简单，产物单一，与靶分子结合能力高和/或对靶分子降解效率高的特点，可以用于体内和/或体外对于靶分子的特异性降解，可以用于疾病的预防和治疗。
一方面，本申请提供一种缀合物，其包含：靶分子靶向部分以及蛋白降解器靶向部分；其中，所述靶分子靶向部分能够特异性识别靶分子并与该靶分子共价结合，所述蛋白降解器靶向部分与所述靶分子靶向部分直接或间接连接。
在一种实施方式中，所述靶分子靶向部分包含第一官能团，所述靶分子包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团能够与所述第二官能团反应而形成共价键。
在一种实施方式中，在所述第一官能团与所述第二官能团之间的距离小于约5E-10米时， 所述第一官能团能够与所述第二官能团反应而形成共价键。
在一种实施方式中，所述第二官能团包含亲电基团。
在一种实施方式中，所述第二官能团包含天然氨基酸的侧链基团。
在一种实施方式中，所述第二官能团选自以下组：氨基、咪唑基、巯基、羟基、羧基、甲硫基，以及前述的衍生物。
在一种实施方式中，所述靶分子包含细胞表面的分子、细胞内的分子和/或细胞外的分子。
在一种实施方式中，所述靶分子选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
在一种实施方式中，所述靶分子选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。
在一种实施方式中，所述靶分子选自以下组：PD-L1(细胞程序死亡-配体1)、EGFR(表皮生长因子受体)、HER-2(人类表皮生长因子受体-2)、Ras蛋白、CD38(淋巴细胞分化抗原CD38)，以及前述的功能性片段。
在一种实施方式中，所述第一官能团选自以下组：
Figure PCTCN2022080594-appb-000001
以及前述的衍生物，其中，R 1为O、N或不存在，R 2为卤素、烯基或炔基，R 3为卤素、烯基或炔基，R 4为卤素，n1和n2各自独立地至少为0。
在一种实施方式中，所述第一官能团选自以下组：
Figure PCTCN2022080594-appb-000002
Figure PCTCN2022080594-appb-000003
以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：
Figure PCTCN2022080594-appb-000004
Figure PCTCN2022080594-appb-000005
Figure PCTCN2022080594-appb-000006
以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
在一种实施方式中，所述靶分子靶向部分包含抗体或其抗原结合片段。
在一种实施方式中，所述抗体选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。
在一种实施方式中，所述抗原结合片段选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。
在一种实施方式中，所述靶分子靶向部分包含单域抗体或其抗原结合片段。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。
在一种实施方式中，所述靶分子靶向部分能够特异性识别所述PD-L1。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸被所述非天然氨基酸替换。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸被所述6-溴-己酰赖氨酸替换、第110位氨基酸被所述6-溴-己酰赖氨酸替换、第113位氨基酸被所述氟代硫酸盐-L-酪氨酸替换和/或第108位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
在一种实施方式中，所述靶分子靶向部分包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，在第116位氨基酸包含所述第一官能团。
在一种实施方式中，所述靶分子靶向部分能够特异性识别所述EGFR。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述非天然氨基酸替换。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
在一种实施方式中，所述靶分子靶向部分包含SEQ ID NO：12所示的氨基酸序列。
在一种实施方式中，所述蛋白降解器包含溶酶体和/或蛋白酶体。
在一种实施方式中，所述蛋白降解器靶向部分选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
在一种实施方式中，所述蛋白降解器靶向部分包含能够靶向溶酶体或蛋白酶体的分子或其功能活性片段。
在一种实施方式中，所述蛋白降解器靶向部分选自以下组：溶酶体靶向肽(LTP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。
在一种实施方式中，所述蛋白降解器靶向部分包含所述溶酶体靶向肽，所述溶酶体靶向肽的氨基酸序列如SEQ ID NO：63-77中任一项所示。
在一种实施方式中，所述缀合物还包含穿膜部分，所述穿膜部分能够促进所述靶分子进 入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
在一种实施方式中，所述穿膜部分选自以下组：穿膜肽(CPP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。
在一种实施方式中，所述穿膜部分包含聚精氨酸，所述聚精氨酸的重复单元数量至少为2。
在一种实施方式中，所述聚精氨酸的聚合单体包含D构型的精氨酸和/或L构型的精氨酸。
在一种实施方式中，所述缀合物包含所述穿膜肽和所述溶酶体靶向肽，所述穿膜肽和所述溶酶体靶向肽直接或间接连接。
在一种实施方式中，所述蛋白降解器靶向部分包含能够靶向溶酶体或蛋白酶体和/或能够结合细胞表面促进内吞的受体的分子或其功能活性片段。
在一种实施方式中，所述蛋白降解器靶向部分包含能够结合6-磷酸甘露糖受体(M6PR)的分子。
在一种实施方式中，所述蛋白降解器靶向部分包含一个或多个甘露糖6-磷酸酯(M6P)或其功能活性片段。
在一种实施方式中，所述蛋白降解器靶向部分包含约1个至约500个所述甘露糖6-磷酸酯或其功能活性片段。
在一种实施方式中，所述蛋白降解器靶向部分包含支架分子，所述支架分子直接或间接连接所述一个或多个甘露糖6-磷酸酯或其功能活性片段。
在一种实施方式中，所述支架分子包含聚糖结构。
在一种实施方式中，所述支架分子包含糖蛋白，所述糖蛋白包含被所述一个或多个甘露糖6-磷酸酯或其功能活性片段修饰的一个或多个氨基酸。
在一种实施方式中，所述糖蛋白包含N-羧基酸酐(NCA)或其衍生物的基团。
在一种实施方式中，所述蛋白降解器靶向部分包含能够结合去唾液酸糖蛋白受体(ASGPR)的分子或其功能活性片段。
在一种实施方式中，所述蛋白降解器靶向部分包含所述去唾液酸糖蛋白受体的一个或多个配体。
在一种实施方式中，所述蛋白降解器靶向部分包含一个或多个N-乙酰半乳糖胺(GalNAc)。
在一种实施方式中，所述蛋白降解器靶向部分包含一个或多个半乳糖。
在一种实施方式中，所述蛋白降解器靶向部分包含一个或多个葡萄糖。
在一种实施方式中，所述蛋白降解器靶向部分包含所述去唾液酸糖蛋白受体的约1个至约500个配体。
在一种实施方式中，所述蛋白降解器靶向部分包含支架分子，所述支架分子直接或间接连接所述去唾液酸糖蛋白受体的所述一个或多个配体。
在一种实施方式中，所述蛋白降解器靶向部分包含所述一个或多个N-乙酰半乳糖胺修饰的一个或多个丙氨酸。
在一种实施方式中，所述支架分子包含一个、两个或三个所述N-乙酰半乳糖胺。
在一种实施方式中，所述支架分子包含一个、两个或三个所述半乳糖。
在一种实施方式中，所述缀合物包含融合蛋白。
在一种实施方式中，所述缀合物包含所述靶分子靶向部分、所述蛋白降解器靶向部分和所述穿膜部分，所述靶分子靶向部分、所述蛋白降解器靶向部分与所述穿膜部分之间相互独立地直接或间接连接。
在一种实施方式中，所述间接连接包含通过连接子连接。
在一种实施方式中，所述连接子包含肽连接子。
在一种实施方式中，所述连接子包含可酶切或不可酶切的连接子。
在一种实施方式中，所述可酶切的连接子包含可通过蛋白酶酶切的连接子。
在一种实施方式中，所述缀合物包含所述靶分子靶向部分、所述穿膜肽和所述溶酶体靶向肽。
在另一方面，本申请提供一种多肽，其包含靶分子靶向部分，所述靶分子靶向部分能够特异性识别靶分子并与该靶分子共价结合。
在一种实施方式中，所述靶分子靶向部分包含第一官能团，所述靶分子包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团能够与所述第二官能团反应而形成共价键。
在一种实施方式中，在所述第一官能团与所述第二官能团之间的距离小于约5E-10米时，所述第一官能团能够与所述第二官能团反应而形成共价键。
在一种实施方式中，所述第二官能团包含亲电基团。
在一种实施方式中，所述第二官能团包含天然氨基酸的侧链基团。
在一种实施方式中，所述第二官能团选自以下组：氨基、咪唑基、巯基、羟基、羧基、甲 硫基，以及前述的衍生物。
在一种实施方式中，所述靶分子包含细胞表面的分子、细胞内的分子和/或细胞外的分子。
在一种实施方式中，所述靶分子选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
在一种实施方式中，所述靶分子选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。
在一种实施方式中，所述靶分子选自以下组：PD-L1(细胞程序死亡-配体1)、EGFR(表皮生长因子受体)、HER-2(人类表皮生长因子受体-2)、Ras蛋白、CD38(淋巴细胞分化抗原CD38)，以及前述的功能性片段。
在一种实施方式中，所述第一官能团选自以下组：
Figure PCTCN2022080594-appb-000007
以及前述的衍生物，其中，R 1为O、N或不存在，R 2为卤素、烯基或炔基，R 3为卤素、烯基或炔基，R 4为卤素，n1和n2各自独立地至少为0。
在一种实施方式中，所述第一官能团选自以下组：
Figure PCTCN2022080594-appb-000008
Figure PCTCN2022080594-appb-000009
以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：
Figure PCTCN2022080594-appb-000010
Figure PCTCN2022080594-appb-000011
Figure PCTCN2022080594-appb-000012
以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
在一种实施方式中，所述靶分子靶向部分选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
在一种实施方式中，所述靶分子靶向部分包含抗体或其抗原结合片段。
在一种实施方式中，所述抗体选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。
在一种实施方式中，所述抗原结合片段选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。
在一种实施方式中，所述靶分子靶向部分包含单域抗体或其抗原结合片段。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。
在一种实施方式中，所述靶分子靶向部分能够特异性识别所述PD-L1。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸被所述非天然氨基酸替换。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸被所述6-溴-己酰赖氨酸替换、第110位氨基酸被所述6-溴-己酰赖氨酸替换、第113位氨基酸被所述氟代硫酸盐-L-酪氨酸替换和/或第108位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
在一种实施方式中，所述靶分子靶向部分包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，在第116位氨基酸包含所述第一官能团。
在一种实施方式中，所述靶分子靶向部分能够特异性识别所述EGFR。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述非天然氨基酸替换。
在一种实施方式中，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
在一种实施方式中，所述靶分子靶向部分包含SEQ ID NO：12所示的氨基酸序列。
在另一方面，本申请提供一种核酸分子，其编码本申请的缀合物和/或本申请的多肽。
在另一方面，本申请提供一种载体，其包含本申请的核酸分子。
在另一方面，本申请提供一种免疫缀合物，其包含本申请的缀合物和/或本申请的多肽。
在另一方面，本申请提供一种细胞，其包含本申请的缀合物、本申请的核酸分子、本申请的载体和/或本申请的免疫缀合物。
在另一方面，本申请提供一种组合物，其包含本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物和/或本申请的细胞，以及任选地药学上可接受的载剂。
在另一方面，本申请提供一种试剂盒，其包含本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、和/或本申请的组合物。
在另一方面，本申请提供本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
在一种实施方式中，其中所述疾病和/或病症包含肿瘤。
在一种实施方式中，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性 肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
在一种实施方式中，其中所述疾病和/或病症包含实体瘤。
在一种实施方式中，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
在另一方面，本申请提供本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒，其用于预防和/或治疗疾病和/或病症。
在一种实施方式中，其中所述疾病和/或病症包含肿瘤。
在一种实施方式中，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
在一种实施方式中，其中所述疾病和/或病症包含实体瘤。
在一种实施方式中，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
在另一方面，本申请提供一种预防和/或治疗疾病和/或病症的方法，其包含向有需要的对象施用本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。
在一种实施方式中，其中所述疾病和/或病症包含肿瘤。
在一种实施方式中，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
在一种实施方式中，其中所述疾病和/或病症包含实体瘤和/或血液瘤。
在一种实施方式中，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
在另一方面，本申请提供一种影响靶分子含量的方法，施用本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物和/或本申请的试剂盒。
在一种实施方式中，所述方法包含体外方法、离体方法和/或体内方法。
在一种实施方式中，所述靶分子为细胞表面的分子和/或细胞内的分子。
在一种实施方式中，所述靶分子为皮肤细胞、骨髓细胞、宫颈细胞、肺细胞和/或乳腺细胞表面的分子。
在一种实施方式中，所述靶分子为皮肤癌细胞、骨髓瘤细胞、宫颈癌细胞、肺癌细胞和/ 或乳腺癌细胞表面的分子。
在一种实施方式中，所述细胞选自以下组：PD-L1阳性细胞、EGFR阳性细胞、HER-2阳性细胞、CD38阳性细胞和Ras功能和/或活性异常的细胞。
在另一方面，本申请提供一种制备本申请缀合物和/或本申请多肽的方法，包含在使得所述缀合物和/或所述多肽表达的条件下，培养本申请的细胞。
在一种实施方式中，通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
在另一方面，本申请提供一种制备本申请缀合物的方法，包含将所述缀合物的所述靶分 子靶向部分与所述蛋白降解器靶向部分直接或间接连接。
在一种实施方式中，通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
在一种实施方式中，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
在一种实施方式中，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是，本申请的缀合物降解靶分子可以实现的一种降解途径的示意图。
图2A和图2B显示的是，可以作为靶分子靶向部分前体的野生型单域抗体的一级质谱图。
图3A和图3B显示的是，PD-L1单域抗体的不同位点被BrC6K或FSY替换后的所得化合物库的质谱图。
图4A和图4B显示的是，SDS-PAGE分析PD-L1单域抗体的不同位点被BrC6K或FSY替换后的所得化合物库同靶分子交联结果。
图5A、图5B和图5C显示的是，具有高邻近交联反应活性的靶分子靶向部分与靶分子交联后的交联肽段的串联质谱。
图6A、图6B、图6C和图6D显示的是，PD-L1单域抗体或EGFR单域抗体中不同位点被BrC6K或FSY替换后的所得靶分子靶向部分的质谱图。
图7A、图7B、和图7C显示的是，PD-L1单域抗体或EGFR单域抗体中不同位点被BrC6K或FSY替换后的所得靶分子靶向部分与靶分子体外交联检测结果。
图8A、图8B、图8C和图8D显示的是，靶分子靶向部分结合细胞表面靶分子的检测结果。
图9A和图9B显示的是，转肽酶介导蛋白降解器靶向部分和靶分子靶向部分连接制备本申请缀合物的示意图。
图10显示的是，PD-L1靶分子靶向部分αPD-L1 L108FSY，以及PD-L1靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物GlueTAC FSY/PD-L1的一级质谱图。
图11显示的是，野生型EGFR单域抗体连接蛋白降解器靶向部分所得的偶联物NbTAC-EGFR，以及EGFR靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物GlueTAC-EGFR的一级质谱图。
图12显示的是，对照组和GlueTAC试验组的活细胞共聚焦荧光成像。
图13A、图13B、图13C和图13D显示的是，免疫印迹试验表征本申请缀合物促进靶分子降解的结果。
图14显示的是，免疫印迹试验表征本申请缀合物促进靶分子降解的结果。
图15显示的是，氯化铵存在情况下，申请缀合物促进靶分子降解的结果。
图16显示的是，本申请缀合物在体内促进肿瘤组织的靶分子降解的结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“药学上可接受的载剂”通常是指药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物可以是无毒的。生理学可接受的载体可以包括合适的物质。药学上可接受的载体(carrier)与基因工程中用于插入核酸的载体(vector)通常并不是同一物质。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。肿瘤细胞可以局部地或通过血流和淋巴系统扩散到身体其他部分。在本申请中，所述肿瘤可以包括良性肿瘤和恶性肿瘤。在本申请中，所述肿瘤可以包括实体瘤。在本申请中，所述肿瘤可以包括癌症。在本申请中，所述肿瘤的实例包括但不限于乳腺癌。
在本申请中，术语“实体瘤”通常是指可以通过临床检查(例如X线照射、CT扫描、B超或触诊等)手段检测到的有形的肿瘤。所述肿瘤可以包括异常细胞生长形成的赘生物或实体病变。
在本申请中，术语“缀合物”通常是指由两个或更多独立的部分接合在一起形成的任何物质。例如，缀合物可以包含由一段多肽与另一段或多段多肽连接而成的物质。例如，缀合物可以由共价键连接而成。例如，缀合物可以是通过转肽酶将单域抗体与穿膜肽以及溶酶体靶向肽连接形成的物质。
在本申请中，术语“靶分子靶向部分”通常是指能够特异性识别和/或特异性结合靶分子的部分。例如，靶分子靶向部分可以是抗原结合蛋白、抗体或抗体的抗原结合片段。例如，靶分子靶向部分还可以具有与靶分子形成共价键的能力。
在本申请中，术语“特异性识别”通常是指识别特异性靶分子，但是基本不识别或结合样品中其它分子的物质或部分。在某些情形中，特异性结合至抗原的抗体还可以结合至靶分子的不同等位形式。
在本申请中，术语“蛋白降解器”通常是指蛋白质降解相关的细胞内特定结构。例如，蛋白降解器可以包含细胞内具有降解蛋白能力的特定形态结构和功能的微器官。例如，蛋白降解器可以包含胞内的溶酶体。
在本申请中，术语“蛋白降解器靶向部分”通常是指能够靶向蛋白降解器的部分。例如，具有蛋白降解器靶向部分的物质，相比于不具有蛋白降解器靶向部分的该物质，可以以更高的数量比例进入蛋白降解器。例如，当一种物质连接了蛋白降解器靶向部分，该物质进入蛋白降解器的比例可以提高。例如，蛋白降解器靶向部分可以为溶酶体靶向肽或者可以为能够结合6-磷酸甘露糖受体(M6PR)的分子。
在本申请中，术语“穿膜肽”通常是指是一类能够穿过细胞膜或组织屏障的短肽。例如，穿膜肽可以通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。
在本申请中，术语“溶酶体靶向肽”通常是指一类能够靶向溶酶体的肽段。例如，溶酶体靶向肽可以运载蛋白质、RNA、DNA等靶分子进入溶酶体。
在本申请中，术语“官能团”通常是指物质中执行一些功能或活性并与其他分子反应的部分。例如，一种官能团可以与另一种官能团发生化学反应以形成共价键连接。
在本申请中，术语“亲电基团”通常是指电子不足或电子缺乏的分子或其部分。例如，亲电基团可以选自以下组：氨基、咪唑基、巯基、羟基、羧基、甲硫基，以及前述的衍生物。
在本申请中，术语“氨基”通常是指-NH 2。例如，氨基可以被任意取代。
在本申请中，术语“咪唑基”通常是指1，3二氮唑基(Imidazolyl)。例如，咪唑基可以被任意取代。
在本申请中，术语“巯基”通常是指-SH。例如，巯基可以被任意取代。
在本申请中，术语“羟基”通常是指-OH。例如，羟基可以被任意取代。
在本申请中，术语“羧基”通常是指-COOH。例如，羧基可以被任意取代。
在本申请中，术语“甲硫基”通常是指-SCH 3。例如，甲硫基可以被任意取代。
在本申请中，术语“卤素”通常是指氟、氯、溴和/或碘。
在本申请中，术语“烷基”、“烯基”、“环烷基”通常可以在名称前加一个标识表示在特定情况下基团中存在的原子数，例如，C 1-C 4烷基，C 3-C 7环烷氧基，C 1-C 4烷基羰基氨基等，“C”后所跟下标数字表示在基团中存在的碳原子数。例如，C 3烷基是指具有三个碳原子的烷基(例如，正丙基，异丙基)；C 1-4中基团的成员可具有落入1-4范围内的任何数目的碳原子。在本申请中，语“烷基”、“烯基”、“环烷基”通常可以任意地被取代。
在本申请中，术语“包含”或“包括”通常是指包括明确指定的特征，但不排除其他要素。在某些情形中，“包含”或者“包括”也涵盖“为”或者“由…组成”的情况。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本申请中，术语“PD-L1”通常是指PD-1的两种细胞表面糖蛋白配体之一(另一种是PD-L2)。例如，PD-L1可以在结合PD-1后下调T细胞活化和细胞因子分泌。例如，术语“PD-L1”可以包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型和物种同系物，以及包含PD-L1功能活性片段的物质。完整hPD-L1序列可见于GenBank登录号Q9NZQ7下。
在本申请中，术语“EGFR”通常是指人表皮生长因子受体1(又称为HER-1)。例如，EGFR的GenBank登录号可以为AF125253。例如，术语“EGFR”可以包括人EGFR，EGFR的变体、同种型和物种同系物，以及包含EGFR功能活性片段的物质。
在本申请中，术语“HER-2”通常是指人表皮生长因子受体2。例如，HER-2的GenBank登录号可以为P04626。例如，术语“HER-2”可以包括人HER-2，HER-2的变体、同种型和物种同系物，以及包含HER-2功能活性片段的物质。
在本申请中，术语“Ras”通常是指一种GTP结合蛋白。例如，Ras的GenBank登录号可以为P01112。例如，术语“Ras”可以包括人Ras，Ras的变体、同种型和物种同系物、失去GTP水解能力的Ras，以及包含Ras功能活性片段的物质。
在本申请中，术语“CD38”通常是指淋巴细胞表面抗原CD38。例如，CD38的GenBank登录号可以为P28907。例如，术语“CD38”可以包括人CD38，CD38的变体、同种型和物种同系物，以及包含CD38功能活性片段的物质。
在本申请中，术语“免疫检查点分子”通常是指一种调节免疫系统的分子。例如，免疫检查点分子可以防止免疫系统攻击细胞。例如，免疫检查点分子可以是PD-L1。
在本申请中，术语“肿瘤相关分子”通常是指与肿瘤的发生和/或发展相关的分子。例如，肿瘤相关分子可以是与肿瘤的生长和/或转移相关的分子。例如，肿瘤相关分子可以是肿瘤细胞高表达的分子。
在本申请中，术语“信号通路蛋白”通常是指细胞信号通路相关的蛋白。例如，信号通路蛋白可以激活MAPK信号通路的蛋白。
在本申请中，术语“CDR”也称“互补决定区”通常是指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、 HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。例如，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知。例如，靶分子靶向部分的氨基酸序列编号可以按照IMGT编号方案。例如，靶分子靶向部分的CDR可以根据Kabat编号系统确定。
在本申请中，术语“抗体”通常是指免疫球蛋白或其片段或其衍生物，可以涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语可以包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本申请的目的，术语“抗体”也可以包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、亲和抗体(affibody)、dAb和/或保持抗原结合功能的其它抗体片段。通常，这样的片段可以包括抗原结合结构域。基本的4链抗体单元可以是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体可以由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体可以包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元可以一般为约150,000道尔顿。每个L链可以通过一个共价二硫键与H链连接，而两个H链可以通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还可以具有规则间隔的链内二硫化桥键。每个H链在N末端可以具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端可以具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)可以相对应。特定的氨基酸残基可以被认为在轻链和重链可变结构域之间形成界面。VH和VL配对可以一起形成单个抗原结合位点。对于不同类别抗体的结构和性质。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列可以被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，可以具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“多肽”通常是指氨基酸的聚合物。例如，多肽可以不是指特定长度的产物；因此，多肽的定义中可以包括了肽、寡肽和蛋白质。这个术语也可以不是指或排除多肽的表达后修饰，例如，糖基化、乙酰化作用、磷酸化作用等等。例如，多肽可以包含一 个或多个氨基酸(包括，例如，非天然氨基酸，等)类似物的多肽、有取代键的多肽、以及本领域已知的修饰，既可以包括自然发生的也可以包括非自然发生的修饰。
在本申请中，术语“抗原结合片段”通常是指具有特异结合抗原能力的一个或多个多肽片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv，亲和抗体(affibody)和/或dAb。
在本申请中，术语“可变区”通常是指在抗体之间可变结构域的某些区段在序列上可能存在较大差异的区域。轻链中的“可变区”可以包含轻链可变区VL；重链中的“可变区”可以包含重链可变区VH。可变结构域可以介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性可以并非在整个可变结构域范围内均匀分布。它可以通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分可以称为框架区(FR)。天然重链和轻链的可变结构域各自可以包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下可以形成β-折叠结构的一部分。每条链中的CDR可以通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成。
在本申请中，术语“Fab”通常是指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(VH)的第一恒定区(CH1)组成。
在本申请中，术语“Fab′”或“Fab′片段”通常是指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“(Fab’)2”通常是指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段可以含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段可以具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv”或“Fv片段”通常是指人单克隆抗体的单价抗原结合片段，可以包括所有或部分重链可变区和轻链可变区，并且可以缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区可以包括例如CDR。例如，Fv片段可以包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区可以是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv可以保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“di-scFv”通常是指两个scFv可以以任何顺序(例如相对于多肽的N末端和C末端)进行连接得到的物质。
在本申请中，术语“亲和抗体”通常是指一种亲和蛋白。例如，亲和抗体(Affibody)分子可以是基于58个氨基酸残基蛋白结构域的亲和蛋白，所述结构域可以源自葡萄球菌A蛋白的三螺旋束IgG结合结构域。例如使用噬菌体展示技术可以从文库中选择靶向期望分子的亲和抗体变体。亲和抗体分子可以以简单稳健的结构和低的分子量(6kDa)的特点从而用于多种应用，例如，可以用作受体相互作用的检测试剂和抑制剂。有关亲和抗体及其生产的进一步描述发现可以记载在US 5,831,012中。标记的亲和抗体也可以用于成像应用中，以测定同种型的丰度。
在本申请中，术语“dAb”通常是指具有VH域或VL域的抗原结合片段，可以参考例如WO2006030220A1、WO2006003388A2或其它公布的专利申请。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可以称为纳米抗体(Nanobody)(Nb)和/或单域抗体。
在本申请中，术语“单克隆抗体”通常是指单分子组成的抗体分子制备物。单克隆抗体可以通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体可以是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”可以表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区可以源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性可以降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”可以保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可以包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术和其它技术，如使用文库，是本领域已知的。
在本申请中，术语“6-溴-己酰赖氨酸”或通常是指包含BrC6K基团的赖氨酸，例如可以是
Figure PCTCN2022080594-appb-000013
例如，6-溴-己酰赖氨酸可以包含其衍生物。
在本申请中，术语“氟代硫酸盐-L-酪氨酸”通常是指指包含FSY基团的酪氨酸，例如可以是
Figure PCTCN2022080594-appb-000014
例如，氟代硫酸盐-L-酪氨酸可以包含其衍生物。
发明详述
一方面，本申请提供一种缀合物。
图1显示的是，本申请的缀合物降解靶分子可以实现的降解途径之一的示意图。例如，本申请的缀合物可以包含：靶分子靶向部分以及蛋白降解器靶向部分，所述靶分子靶向部分可以特异性识别所述靶分子并与可以该靶分子共价结合；其中，所述蛋白降解器靶向部分可以与所述靶分子靶向部分直接或间接连接。
例如，所述靶分子靶向部分可以包含第一官能团，所述靶分子可以包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，第二官能团可以包含亲电基团，第一官能团可以和第二官能团发生交联反应，例如可以是取代反应和/或加成反应。例如，靶分子靶向部分和靶分子可以通过取代反应和/或加成反应，形成共价键连接。例如，当所述靶分子靶向部分与所述靶分子通过抗体-抗原非共价作用接近时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，当所述靶分子靶向部分与所述靶分子接近时，靶分子靶向部分的骨架到靶分子的骨架之间的距离可以约等于带有第一官能团的侧链长度加上带有第二官能团的侧链长度之和。例如，第一官能团和第二官能团的取向可以是头对头，也可以是带有第一官能团的侧链和带有第二官能团的侧链可以通过旋转以实现头对头的取向。例如，在某些状态下，第一官能团和第二官能团之间的距离小于约5埃(5E-10米)时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，第一官能团和第二官能团之间的距离可以通过调节所述靶分子靶向部分的带有第一官能团的侧链长度来实现。例如，可以通过检测50％以上的第一官能团和第二官能团之间发生交联的时间，判断交联效率或邻近反应活性。例如，50％以上的第一官能团和第二官能团之间发生交联的时间小于约72小时、小于约48小时、小于约24小时、小于约12小时、小于约8小时、或小于约4小时，可以认为第一官能团具有与第二官能团发生邻近反应的活性。例如，靶分子靶向部分与所述靶分子通过非共价作用，例如抗体-抗原作用接近时，可以给予第一官能团具有与第二官能团发生邻近反应的条件和/或时间。
例如，第二官能团包含天然氨基酸的侧链基团。例如，天然氨基酸可以包含甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartate)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamate)、赖氨酸(Lysine)、谷胺酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、和/或脯氨酸(Proline)。例如，第二官能团也可以包含非天然氨基酸的侧链基团，例如一些靶分子可以包含硒半胱氨酸、吡咯赖氨酸和/或非天然氨基酸。例如，第二官能团也可以是任意的侧链基团，例如靶分子可以具有任意结构。
例如，第二官能团可以是亲电基团。例如，可以是包含电负性较大的原子，例如卤素原子、氧原子、氮原子、硫原子的基团。例如，第二官能团可以包含氨基、咪唑基、巯基、羟基、羧基和/或甲硫基。例如，第二官能团可以包含
Figure PCTCN2022080594-appb-000015
和/或
Figure PCTCN2022080594-appb-000016
例如，包含第二官能团的侧链可以选自以下组：-CH 2SH、-CH 2CH 2COOH、-CH 2CH 2CONH 2、-CH 2CH 2SCH 3、-(CH 2) 3NH-C(NH)NH 2、-(CH 2) 4NH 2、-CH 2-C 6H 4OH、-CH 2OH、-CH(OH)CH 3、-CH 2COOH、-CH 2CONH 2、和-CH 2-C 3H 3N 2。例如，包含第二官能团的氨基酸可以选自以下组：半胱氨酸、谷氨酸、谷胺酰胺、甲硫氨酸、精氨酸、赖氨酸、酪氨酸、丝氨酸、苏氨酸、天冬氨酸、天冬酰胺和组氨酸。例如，包含第二官能团的氨基酸可以选自以下组：组氨酸、赖氨酸、酪氨酸、半胱氨酸、丝氨酸、苏氨酸、谷氨酸和甲硫氨酸。
例如，靶分子可以包含细胞表面的分子、细胞内的分子和/或细胞外的分子。例如，靶分子可以是细胞表面的受体分子。例如，靶分子可以选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。例如，靶分子可以包含多肽。例如，靶分子可以选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。例如，靶分子可以包含表皮生长因子受体(HER)家族的成员。例如，靶分子可以包含丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号通路的成员。例如，所述靶分子可以选自以下组：PD-L1、EGFR、HER-2、Ras、CD38，以及前述的功能性片段。
例如，靶分子可以包含SEQ ID NO：98所示的氨基酸序列。例如，PD-L1可以在第69位氨基酸包含所述第二官能团，例如组氨酸。
例如，第一官能团可以选自以下组：硫酰氟、卤代烃、羰基卤代烃、α羰基不饱和烃及上述基团的衍生物。例如，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000017
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000018
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000019
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000020
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6。
例如，所述第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000021
Figure PCTCN2022080594-appb-000022
以及前述的衍生物。在本申请中，衍生物可以是指所述官能团或化合物的可被一个或多个额外基团取代或未被取代，所述额外基团可以单独且独立选自烷基、烯基、炔基、环烷基、芳基、杂芳基、杂环烷基、羟基、烷氧基、巯基、氰基、卤素、羰基、硫代羰基、异氰酸基、硫氰酸基、异硫氰酸基、硝基、全卤代烷基、全氟代烷基和包括单取代及双取代氨基基团的氨基，及其受保护的衍生物。任选取代基的非限制性示例可以包括卤素、-CN、＝O、＝N-OH、＝N-OR、＝N-R、OR、-C(O)R、-C(O)OR、-OC(O)R、-OC(O)OR、-C(O)NHR、-C(O)NR 2、-OC(O)NHR、-OC(O)NR 2、-SR-、-S(O)R、-S(O) 2R、-NHR、-N(R) 2、-NHC(O)R、-NRC(O)R、-NHC(O)OR、-NRC(O)OR、S(O) 2NHR、-S(O) 2N(R) 2、-NHS(O) 2NR 2、-NRS(O) 2NR 2、-NHS(O) 2R、-NRS(O) 2R、C 1-C 8烷基、C 1-C 8烷氧基、芳基、杂芳基、环烷基、杂环烷基、卤素取代的C 1-C 8烷基、和卤素取代的C 1-C 8烷氧基，其中各R可以独立选自H、卤素、C 1-C 8烷基、C 1-C 8烷氧基、芳基、杂芳基、环烷基、杂环烷基、卤素取代的C 1-C 8烷基、和卤素取代的C 1-C 8烷氧基。此类取代基团的位置和数量可以按各基团熟知的价态限制确定。
例如，靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以包含所述第一官能团，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000023
Figure PCTCN2022080594-appb-000024
Figure PCTCN2022080594-appb-000025
以及前述的衍生物。
例如，所述靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以包含所述第一官能团，所述非天然氨基酸可以选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
例如，靶分子靶向部分可以选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。例如，靶分子靶向部分可以包含多肽。例如，靶分子靶向部分可以包含抗体或其抗原结合片段。例如，抗体可以选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。例如，抗原结合片段可以选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。例如，所述靶分子靶向部分可以包含单域抗体或其抗原结合片段。
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、 综合考虑Kabat/Chothia等。这些编码系统为本领域内已知。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖可以根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，所述CDR可以由Kabat编号方案来确定。
此外，需要说明的是，本申请所述靶分子靶向部分可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述靶分子靶向部分中。保守氨基酸替换可以是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组可以包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请靶分子靶向部分的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改可以不会使抗原结合性消失，
例如，靶分子靶向部分可以为抗体。例如，靶分子靶向部分可以在抗体可变区包含所述第一官能团。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分可以在抗体可变区的氨基酸包含第一官能团。例如，靶分子靶向部分可以在CDR区包含所述第一官能团。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分可以在CDR区的氨基酸包含第一官能团。例如，靶分子靶向部分可以在CDR区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸包含所述第一官能团。例如，靶分子靶向部分可以在CDR3区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸包含所述第一官能团。例如，CDR区可以是根据任意一种CDR区划分方式划分的，例如Kabat划 分方法。例如，本申请靶分子靶向部分的CDR1、CDR2和CDR3可以分别如SEQ ID NO：1、2和3所示。例如，本申请靶分子靶向部分可以特异性结合PD-L1。例如，本申请靶分子靶向部分的CDR1、CDR2和CDR3可以分别如SEQ ID NO：4、5和6所示。例如，本申请靶分子靶向部分可以特异性结合EGFR。
例如，靶分子靶向部分可以为抗体。例如，靶分子靶向部分可以在抗体可变区包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分在抗体可变区的氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分可以在CDR区包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分在CDR区的氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分在CDR区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分在CDR3区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，可以在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以为包含本申请第一官能团的物质，例如包含本申请第一官能团的非天然氨基酸。例如，所述靶分子靶向部分在第108位氨基酸、第110位氨基酸和/或第113位氨基酸的侧链可以包含本申请第一官能团。例如，所述靶分子靶向部分在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含的第一官能团可以与靶分子上的第二官能团发生邻近反应形成共价键连接。例如，所述靶分子靶向部分可以特异性识别所述PD-L1。所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以被所述非天然氨基酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以各自独立地被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换。例如，所述靶分子靶向部 分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位可以被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分可以包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，可以在第116位氨基酸包含所述第一官能团。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以为包含本申请第一官能团的物质，例如包含本申请第一官能团的非天然氨基酸。例如，所述靶分子靶向部分在第116位氨基酸的侧链可以包含本申请第一官能团。例如，所述靶分子靶向部分在第116位氨基酸包含的第一官能团可以与靶分子上的第二官能团发生邻近反应形成共价键连接。例如，所述靶分子靶向部分可以特异性识别所述EGFR。所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述非天然氨基酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分可以包含SEQ ID NO：12所示的氨基酸序列。
例如，蛋白降解器可以包含溶酶体。例如，蛋白降解器靶向部分可以选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。例如，蛋白降解器靶向部分可以包含能够靶向溶酶体的分子或其功能活性片段。例如，蛋白降解器靶向部分还可以包含穿膜部分，所述穿膜部分能够促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，蛋白降解器靶向部分可以包含以下组：溶酶体靶向肽(LTP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。例如，缀合物还可以包含穿膜部分，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分可以与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分可以与所述蛋白降解器靶向部分直接或间接连接。例如，穿膜部分可以选自以下组：穿膜肽(CPP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。例如，穿 膜肽可以包含聚精氨酸，所述聚精氨酸的重复单元数量可以至少为2。
例如，蛋白降解器靶向部分可以包含前蛋白转化酶枯草溶菌素9(PCSK9)或其功能活性片段，前蛋白转化酶枯草溶菌素9(PCSK9)可以与细胞表面的受体结合，可以促进PCSK9以及包含PCSK9的复合物的降解。例如，蛋白降解器靶向部分可以包含酪氨酸内吞基序(Tyrosine containing cytoplasmic motif)或其功能活性片段，例如可以是NPX 1Y(如SEQ ID NO：63所示)或FX 2NPX 3Y(SEQ ID NO：65所示)，其中X 1、X 2或X 3可以是任意氨基酸，例如，X 1、X 2或X 3可以各自独立地为甘氨酸G和/或丙氨酸A。例如，蛋白降解器靶向部分可以包含聚乳酸-羟基乙酸共聚物和/或聚乳酸，聚乳酸-羟基乙酸共聚物的聚合单体可以包含乳酸和羟基乙酸，乳酸和羟基乙酸的比例可以是任意的。例如，蛋白降解器靶向部分可以包含表皮生长因子EGF或其功能活性片段，序列可以如SEQ ID NO：67所示。例如，蛋白降解器靶向部分可以包含RGD序列，可以是L-精氨酸、甘氨酸和L-天冬氨酸组成的三肽序列，序列可以如RGD所示。
例如，蛋白降解器靶向部分可以包含以下组：溶酶体靶向十八烷基罗丹明B(Lysosomotropic octadecyl-rhodamine-B)、联吡啶缩氨基硫脲(dipyridyl-thio-semi-carbazones)、铁螯合剂Dp44mT(di-2-pyridylketone 4,4,-di-methyl-3-thiosemicarbazone)、铁螯合剂Bp4eT(2-benzoylpyridine-4-ethyl-3thiosemicarbazone)、铁螯合剂PIH(Pyridoxal isonicotinoyl hydrazone)，以及它们的衍生物。例如，蛋白降解器靶向部分可以包含聚精氨酸，例如可以是聚L-精氨酸、可以是聚D-精氨酸，也可以是聚L-精氨酸-D-精氨酸共聚物，例如聚合精氨酸的重复单元数可以至少为2。例如，蛋白降解器靶向部分可以选自以下组：八聚L-精氨酸、八聚D-精氨酸、九聚L-精氨酸、九聚D-精氨酸、十二聚L-精氨酸、和十二聚D-精氨酸。例如，蛋白降解器靶向部分可以选自以下组：物质-P(Substance P，SP，序列可以如SEQ ID NO：68所示)、神经肽Y(Neuropeptide Y，序列可以如SEQ ID NO：69所示)、TP穿膜肽(TP transportan，序列可以如SEQ ID NO：70所示)、穿膜肽TAT(序列可以如SEQ ID NO：71所示)、穿透肽(pAntp,Penetratin，序列可以如SEQ ID NO：72所示)、低pH插入肽(pH low-insertion peptide，pHLIP，序列可以如SEQ ID NO：73所示)、跨膜序列ATRAM(acidity-triggered rational membrane，序列可以如SEQ ID NO：74所示)、可激活细胞穿透肽(ACPP，序列可以如SEQ ID NO：75所示)、加速穿透序列(pHK-PAS，序列可以如SEQ ID NO：76所示)，以及上述的功能活性片段。
例如，蛋白降解器靶向部分可以靶向选自以下组的物质：离子非依赖性甘露糖6磷酸受体(CI-M6PR，也称为M6PR，uniprot登录号可以是P20645)、小窝蛋白-1(Caveolin-1，uniprot 登录号可以是Q03135)，网格蛋白(Clathrin，uniprot登录号可以是Q00610)，催乳素受体(Prolactin receptor，PRLR，uniprot登录号可以是P16471)，淀粉样前体样蛋白2(Amyloid Precursor Like Protein 2，APLP2，uniprot登录号可以是Q06481)，溶酶体相关膜糖蛋白3(Lysosome-associated membrane glycoprotein 3，LAMP3，CD63，uniprot登录号可以是Q9UQV4)，甘丙肽受体-1(GALR-1receptor，uniprot登录号可以是P47211)，网格蛋白相关蛋白(Clathrin-associated proteins，uniprot登录号可以是P63010)，Disabled-2蛋白(Dab2，uniprot登录号可以是Q5VWQ8)，ARH蛋白(Low density lipoprotein receptor adapter protein 1，uniprot登录号可以是Q5SW96)，潜伏膜蛋白(latent membrane protein，LMP，uniprot登录号可以是P13285)，MDM2蛋白(E3泛素蛋白连接酶，uniprot登录号可以是Q00987)，指环蛋白43(RNF43，Ring Finger Protein 43，uniprot登录号可以是Q68DV7)，指环蛋白126(RNF126，Ring Finger Protein 126，uniprot登录号可以是Q9BV68)，锌指蛋白(zinc and ring finger 3，ZNRF3，uniprot登录号可以是Q9ULT6)，以及上述的功能活性片段。
例如，蛋白降解器靶向部分可以包含能够靶向溶酶体或蛋白酶体和/或能够结合细胞表面促进内吞的受体的分子或其功能活性片段。例如，所述蛋白降解器靶向部分包含能够结合6-磷酸甘露糖受体(M6PR)的分子。例如，所述蛋白降解器靶向部分可以包含一个或多个甘露糖6-磷酸酯(M6P)或其功能活性片段。例如，所述蛋白降解器靶向部分可以包含约1个至约500个所述甘露糖6-磷酸酯或其功能活性片段。例如，所述蛋白降解器靶向部分可以包含支架分子，所述支架分子直接或间接连接所述一个或多个甘露糖6-磷酸酯或其功能活性片段。例如，所述支架分子可以包含聚糖结构。例如，所述支架分子可以包含糖蛋白，所述糖蛋白可以包含被所述一个或多个甘露糖6-磷酸酯或其功能活性片段修饰的一个或多个氨基酸。例如，所述糖蛋白可以包含N-羧基酸酐(NCA)或其衍生物的基团。例如，所述蛋白降解器靶向部分可以包含能够结合去唾液酸糖蛋白受体(ASGPR)的分子或其功能活性片段。例如，所述蛋白降解器靶向部分可以包含所述去唾液酸糖蛋白受体的一个或多个配体。例如，所述蛋白降解器靶向部分可以包含一个或多个N-乙酰半乳糖胺(GalNAc)。例如，所述蛋白降解器靶向部分可以包含一个或多个半乳糖。例如，所述蛋白降解器靶向部分可以包含一个或多个葡萄糖。例如，所述蛋白降解器靶向部分可以包含所述去唾液酸糖蛋白受体的约1个至约500个配体。例如，所述蛋白降解器靶向部分可以包含支架分子，所述支架分子可以直接或间接连接所述去唾液酸糖蛋白受体的所述一个或多个配体。例如，所述蛋白降解器靶向部分可以包含所述一个或多个N-乙酰半乳糖胺修饰的一个或多个丙氨酸。例如，所述支架分子可以包含一个、两个或三个所述N-乙酰半乳糖胺。例如，所述支架分子可以包含一个、两个或三 个所述半乳糖。例如，所述缀合物可以包含融合蛋白。
例如，所述缀合物可以包含所述靶分子靶向部分、所述蛋白降解器靶向部分和所述穿膜部分，所述靶分子靶向部分、所述蛋白降解器靶向部分与所述穿膜部分之间可以相互独立地直接或间接连接。例如，所述间接连接可以包含通过连接子连接。例如，所述连接子可以包含肽连接子。例如，所述连接子可以包含可酶切或不可酶切的连接子。例如，所述可酶切的连接子可以包含可通过蛋白酶酶切的连接子。
例如，所述缀合物可以包含所述靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，其中所述靶分子靶向部分可以包含第一官能团，所述靶分子可以包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团可以与所述第二官能团反应而形成共价键，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，其中所述靶分子靶向部分可以包含第一官能团，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000026
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000027
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000028
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000029
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6；所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，其中所述靶分子靶向部分可以包含第一官能团，所述第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000030
Figure PCTCN2022080594-appb-000031
以及前述的衍生物，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000032
Figure PCTCN2022080594-appb-000033
以及前述的衍生物，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR区的N端前5个氨基酸中和/或CDR区的C端后5个氨基酸中的包含第一官能团，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000034
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000035
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000036
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000037
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6；所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR区的N端前5个氨基酸中和/或CDR区的C端后5个氨基酸中的包含第一官能团，所述第一官能 团可以选自以下组：
Figure PCTCN2022080594-appb-000038
Figure PCTCN2022080594-appb-000039
以及前述的衍生物，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区、CDR区的N端前5个氨基酸和/或CDR区的C端后5个氨基酸包含非天然氨基酸的残基，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000040
Figure PCTCN2022080594-appb-000041
以及前述的衍生物，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区、CDR区的N端前5个氨基酸和/或CDR区的C端后5个氨基酸包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR3区的N端前5个氨基酸中和/或CDR3区的C端后5个氨基酸中包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为PD-L1单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以被氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽， 靶分子靶向部分可以为PD-L1单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸可以被氟代硫酸盐-L-酪氨酸替换，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为EGFR单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以被氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，靶分子靶向部分可以为EGFR单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以被氟代硫酸盐-L-酪氨酸替换，所述穿膜部分可以促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
例如，本申请的缀合物可以包含靶分子靶向部分、所述穿膜部分和所述溶酶体靶向肽，蛋白降解器靶向部分的氨基酸序列可以如SEQ ID NO：63-91中任一项所示，所述穿膜部分的氨基酸序列可以如SEQ ID NO：78-79中任一项所示，所述溶酶体靶向肽的氨基酸序列可以如SEQ ID NO：63-77所示，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
多肽
在另一方面，本申请提供一种多肽。例如，多肽可以包含靶分子靶向部分，所述靶分子靶向部分可以特异性识别所述靶分子并与该靶分子共价结合。
例如，所述靶分子靶向部分可以包含第一官能团，所述靶分子可以包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，第二官能团可以包含亲电基团，第一官能团可以和第二官能团发生交联反应，例如可以是取代反应和/或加成反应。例如，靶分子靶向部分和靶分子可以通过取代反应和/或加成反应，形成共价键连接。例如，当所述靶分子靶向部分与所述靶分子通过抗体-抗原非共价作用接近时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，当所述靶分子靶向部分与所述靶分子接近时，靶分子靶向部分的骨架到靶分子的骨架之间的距离 可以约等于带有第一官能团的侧链长度加上带有第二官能团的侧链长度之和。例如，第一官能团和第二官能团的取向可以是头对头，也可以是带有第一官能团的侧链和带有第二官能团的侧链可以通过旋转以实现头对头的取向。例如，在某些状态下，第一官能团和第二官能团之间的距离小于约5埃(5E-10米)时，所述第一官能团可以与所述第二官能团反应而形成共价键。例如，第一官能团和第二官能团之间的距离可以通过调节所述靶分子靶向部分的带有第一官能团的侧链长度来实现。例如，可以通过检测50％以上的第一官能团和第二官能团之间发生交联的时间，判断交联效率或邻近反应活性。例如，50％以上的第一官能团和第二官能团之间发生交联的时间小于约72小时、小于约48小时、小于约24小时、小于约12小时、小于约8小时、或小于约4小时，可以认为第一官能团具有与第二官能团发生邻近反应的活性。例如，靶分子靶向部分与所述靶分子通过非共价作用，例如抗体-抗原作用接近时，可以给予第一官能团具有与第二官能团发生邻近反应的条件和/或时间。
例如，第二官能团包含天然氨基酸的侧链基团。例如，天然氨基酸可以包含甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartate)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamate)、赖氨酸(Lysine)、谷胺酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、和/或脯氨酸(Proline)。例如，第二官能团也可以包含非天然氨基酸的侧链基团，例如一些靶分子可以包含硒半胱氨酸、吡咯赖氨酸和/或非天然氨基酸。例如，第二官能团也可以是任意的侧链基团，例如靶分子可以具有任意结构。
例如，第二官能团可以是亲电基团。例如，可以是包含电负性较大的原子，例如卤素原子、氧原子、氮原子、硫原子的基团。例如，第二官能团可以包含氨基、咪唑基、巯基、羟基、羧基和/或甲硫基。例如，第二官能团可以包含
Figure PCTCN2022080594-appb-000042
和/或
Figure PCTCN2022080594-appb-000043
例如，包含第二官能团的侧链可以选自以下组：-CH 2SH、-CH 2CH 2COOH、-CH 2CH 2CONH 2、-CH 2CH 2SCH 3、-(CH 2) 3NH-C(NH)NH 2、-(CH 2) 4NH 2、-CH 2-C 6H 4OH、-CH 2OH、-CH(OH)CH 3、-CH 2COOH、-CH 2CONH 2、和-CH 2-C 3H 3N 2。例如，包含第二官能团的氨基酸可以选自以下组：半胱氨酸、谷氨酸、谷胺酰胺、甲硫氨酸、精氨酸、赖氨酸、酪氨酸、丝氨酸、苏氨酸、天冬氨酸、天冬酰胺和组氨酸。例如，包含第二官能团的氨基酸可以选自以下组：组氨酸、赖氨酸、酪氨酸、半胱氨酸、丝氨酸、苏氨酸、谷氨酸和甲硫氨酸。
例如，靶分子可以包含细胞表面的分子、细胞内的分子和/或细胞外的分子。例如，靶分子可以是细胞表面的受体分子。例如，靶分子可以选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。例如，靶分子可以包含多肽。例如，靶分子可以选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。例如，靶分子可以包含表皮生长因子受体(HER)家族的成员。例如，靶分子可以包含丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号通路的成员。例如，所述靶分子可以选自以下组：PD-L1、EGFR、HER-2、Ras、CD38，以及前述的功能性片段。
例如，靶分子可以包含SEQ ID NO：98所示的氨基酸序列。例如，PD-L1可以在第69位氨基酸包含所述第二官能团，例如组氨酸。
例如，第一官能团可以选自以下组：硫酰氟、卤代烃、羰基卤代烃、α羰基不饱和烃及上述基团的衍生物。例如，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000044
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000045
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000046
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6。
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000047
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6。
例如，所述第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000048
Figure PCTCN2022080594-appb-000049
以及前述的衍生物。在本申请中，衍生物可以是指所述官能团或化合物的可被一个或多个额外基团取代或未被取代，所述额外基团可以单独且独立选自烷基、烯基、炔基、环烷基、芳基、杂芳基、杂环烷基、羟基、烷氧基、巯基、氰基、卤素、羰基、硫代羰基、异氰酸基、硫氰酸基、异硫氰酸基、硝基、全卤代烷基、全氟代烷基和包括单取代及双取代氨基基团的氨基，及其受保护的衍生物。任选取代基的非限制性示例可以包括卤素、-CN、＝O、＝N-OH、＝N-OR、＝N-R、OR、-C(O)R、-C(O)OR、-OC(O)R、-OC(O)OR、-C(O)NHR、-C(O)NR 2、-OC(O)NHR、-OC(O)NR 2、-SR-、-S(O)R、-S(O) 2R、-NHR、-N(R) 2、-NHC(O)R、-NRC(O)R、-NHC(O)OR、-NRC(O)OR、S(O) 2NHR、-S(O) 2N(R) 2、-NHS(O) 2NR 2、-NRS(O) 2NR 2、-NHS(O) 2R、-NRS(O) 2R、C 1-C 8烷基、C 1-C 8烷氧基、芳基、杂芳基、环烷基、杂环烷基、卤素取代的C 1-C 8烷基、和卤素取代的C 1-C 8烷氧基，其中各R可以独立选自H、卤素、C 1-C 8烷基、C 1-C 8烷氧基、芳基、杂芳基、环烷基、杂环烷基、卤素取代的C 1-C 8烷基、和卤素取代的C 1-C 8烷氧基。此类取代基团的位置和数量可以按各基团熟知的价态限制确定。
例如，靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以包含所述第一官能团，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000050
Figure PCTCN2022080594-appb-000051
Figure PCTCN2022080594-appb-000052
以及前述的衍生物。
例如，所述靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以包含所述第一官能团，所述非天然氨基酸可以选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
例如，靶分子靶向部分可以选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。例如，靶分子靶向部分可以包含多肽。例如，靶分子靶向部分可以包含抗体或其抗原结合片段。例如，抗体可以选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。例如，抗原结合片段可以选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。例如，所述靶分子靶向部分可以包含单域抗体或其抗原结合片段。
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖可以根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，所述CDR可以由Kabat编号方案来确定。
此外，需要说明的是，本申请所述靶分子靶向部分可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性 的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述靶分子靶向部分中。保守氨基酸替换可以是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组可以包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请靶分子靶向部分的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改可以不会使抗原结合性消失，
例如，靶分子靶向部分可以为抗体。例如，靶分子靶向部分可以在抗体可变区包含所述第一官能团。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分可以在抗体可变区的氨基酸包含第一官能团。例如，靶分子靶向部分可以在CDR区包含所述第一官能团。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分可以在CDR区的氨基酸包含第一官能团。例如，靶分子靶向部分可以在CDR区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸包含所述第一官能团。例如，靶分子靶向部分可以在CDR3区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸包含所述第一官能团。例如，CDR区可以是根据任意一种CDR区划分方式划分的，例如Kabat划分方法。例如，本申请靶分子靶向部分的CDR1、CDR2和CDR3可以分别如SEQ ID NO：1、2和3所示。例如，本申请靶分子靶向部分可以特异性结合PD-L1。例如，本申请靶分子靶向部分的CDR1、CDR2和CDR3可以分别如SEQ ID NO：4、5和6所示。例如，本申请靶分子靶向部分可以特异性结合EGFR。
例如，靶分子靶向部分可以为抗体。例如，靶分子靶向部分可以在抗体可变区包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分在抗体可变区的氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分可以在CDR区包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。例如，与野生型的靶分子靶向部分相比，本申请靶分子靶向部分在CDR区的氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分在CDR区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意 氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，靶分子靶向部分在CDR3区的N端前5个氨基酸中的任意氨基酸和/或C端后5个氨基酸中的任意氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，可以在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以为包含本申请第一官能团的物质，例如包含本申请第一官能团的非天然氨基酸。例如，所述靶分子靶向部分在第108位氨基酸、第110位氨基酸和/或第113位氨基酸的侧链可以包含本申请第一官能团。例如，所述靶分子靶向部分在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含的第一官能团可以与靶分子上的第二官能团发生邻近反应形成共价键连接。例如，所述靶分子靶向部分可以特异性识别所述PD-L1。所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以被所述非天然氨基酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以各自独立地被所述氟代硫酸盐-L-酪氨酸替换和/或被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位可以被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分可以包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，可以在第116位氨基酸包含所述第一官能团。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以为包含本申请第一官能团的物质，例如包含本申请第一官能团的非天然氨基酸。例如，所述靶分子靶向部分在第116位氨基酸的侧链可以包含本申请第一官能团。例如，所述靶分子靶向部分在第116位氨基酸包含的第一官能团可以与靶分子上的第二官能团发生邻近反应形成共价键连接。例如，所述靶分子靶向部分可以特异性识别所述EGFR。所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可 以被所述非天然氨基酸替换。
例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述氟代硫酸盐-L-酪氨酸替换。例如，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸可以被所述6-溴-己酰赖氨酸替换。例如，所述靶分子靶向部分可以包含SEQ ID NO：12所示的氨基酸序列。
例如，本申请的多肽可以包含靶分子靶向部分，其中所述靶分子靶向部分可以包含第一官能团，所述靶分子可以包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团可以与所述第二官能团反应而形成共价键。
例如，本申请的多肽可以包含靶分子靶向部分，其中所述靶分子靶向部分可以包含第一官能团，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000053
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000054
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000055
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000056
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6。
例如，本申请的多肽可以包含靶分子靶向部分，其中所述靶分子靶向部分可以包含第一官能团，所述第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000057
Figure PCTCN2022080594-appb-000058
以及前述的衍生物。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以包含非天然氨基酸的残基，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000059
Figure PCTCN2022080594-appb-000060
以及前述的衍生物。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR区的N端前5个氨基酸中和/或CDR区的C端后5个氨基酸中的包含第一官能团，第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000061
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基，R 3可以为卤素、烯基或炔基，R 4可以为卤素，n1和n2可以各自独立地至少为0；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000062
其中，R 1可以为O、N或不存在，R 2可以为卤素、烯基或炔基。例如，R 2可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000063
其中，R 3可以为卤素、烯基或炔基，n1可以至少为0。例如，R 3可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n1可以为0至6；
例如，第一官能团可以为
Figure PCTCN2022080594-appb-000064
其中，R 4可以为卤素、烯基或炔基，n2可以至少为0。例如，R 4可以为F、Cl、Br或I、C 2-C 6烯基或C 2-C 6炔基。其中，n2可以为0至6。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR区的N端前5个氨基酸中和/或CDR区的C端后5个氨基酸中的包含第一官能团，所述第一官能团可以选自以下组：
Figure PCTCN2022080594-appb-000065
Figure PCTCN2022080594-appb-000066
以及前述的衍生物。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区、CDR区的N端前5个氨基酸和/或CDR区的C端后5个氨基酸包含非天然氨基酸的残基，所述非天然氨基酸可以选自以下组：
Figure PCTCN2022080594-appb-000067
Figure PCTCN2022080594-appb-000068
以及前述的衍生物。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区、CDR区的N端前5个氨基酸和/或CDR区的C端后5个氨基酸包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区氨基酸中、CDR3区的N端前5个氨基酸中和/或CDR3区的C端后5个氨基酸中包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪 氨酸和/或6-溴-己酰赖氨酸。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为抗体，其中所述靶分子靶向部分可以在CDR区包含非天然氨基酸的残基，所述非天然氨基酸可以包含氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为PD-L1单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸、第110位氨基酸和/或第113位氨基酸可以被氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为PD-L1单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，第108位氨基酸可以被氟代硫酸盐-L-酪氨酸替换。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为EGFR单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以被氟代硫酸盐-L-酪氨酸和/或6-溴-己酰赖氨酸替换。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分可以为EGFR单域抗体，其中所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，第116位氨基酸可以被氟代硫酸盐-L-酪氨酸替换。
例如，本申请的多肽可以包含靶分子靶向部分，靶分子靶向部分的氨基酸序列可以如SEQ ID NO：9-12中任一项所示。
例如，多肽还可以包含小分子物质，所述小分子物质可以与所述靶分子靶向部分直接或间接连接。例如，所述小分子物质可以是荧光分子，例如荧光探针TAMRA(5-Carboxytetramethylrhodamine)。
核酸，免疫缀合物，细胞，组合物和试剂盒
另一方面，本申请提供了分离的核酸分子，其可以编码本申请所述的缀合物和/或多肽。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(ii)通过克隆重组产生的；(iii)纯化的，例如通过酶切和凝胶电泳分级分离；或者(iv)合成的，例如通过化学合成。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可以包含允许编码区在适当宿主中正确表达的表达控制元件。这样 的控制元件为本领域技术人员所熟知的，例如，可以包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可以通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体可以为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个宿主细胞可以包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可以包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可以将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、293T细胞、COS-1细胞、SP2/0细胞、NS0细胞或骨髓瘤细胞。可以通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如热转化、电穿孔、病毒侵染、Ca离子转化、lipofectine转染、lipofectamin转染或其他转染试剂等。
另一方面，本申请还提供了免疫缀合物，其可以包含本申请所述的缀合物和/或多肽。
在某些实施方式中，可以将本申请所述的缀合物和/或多肽或其片段与另一试剂，如化学治疗剂、毒素、免疫治疗剂、成像探针、分光镜探针等的连接。该连接可以通过一个或多个共价键，或非共价相互作用，并且可以包括螯合作用。可以使用多种接头(所述接头可以为本领域所知)以形成免疫缀合物。此外，可以以融合蛋白质的形式提供免疫缀合物，其可以由编码免疫缀合物的多核苷酸表达。所述免疫缀合物还可以包含例如抗体-药物缀合物(ADC)。在ADC中，抗体和治疗剂可以通过接头交联，该接头可以是可切割的接头，例如可以是体内和/或体外可酶切的接头，例如可以是肽类接头、二硫类接头或腙类接头。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的缀合物和/或多肽、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下可以对接受者无毒。本发明的药物组合物可以包括但不限于液体、 冷冻和冻干组合物。
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂。
另一方面，本申请还提供了一种试剂盒，可以包含本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、和/或本申请的组合物。例如，本申请的试剂盒可以用于预防和/或治疗疾病和/或病症、影响靶分子含量、和/或制备本申请缀合物和/或本申请多肽。
治疗方法
另一方面，本申请还提供了本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒在制备药物中的用途，所述药物可以用于预防和/或治疗疾病和/或病症。例如，其中所述疾病和/或病症可以包含肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1高表达肿瘤、EGFR高表达肿瘤、HER-2高表达肿瘤、和Ras高表达的肿瘤。例如，其中所述疾病和/或病症可以包含实体瘤。例如，其中所述疾病和/或病症可以包含乳腺癌、肺癌、皮肤肿瘤和/或宫颈癌。例如，其中所述疾病和/或病症可以包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
另一方面，本申请还提供了本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒，其可以用于预防和/或治疗疾病和/或病症。例如，其中所述疾病和/或病症可以包含肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳 性肿瘤、和Ras功能和/或活性异常的肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1高表达肿瘤、EGFR高表达肿瘤、HER-2高表达肿瘤、CD38高表达肿瘤和Ras高表达的肿瘤。例如，其中所述疾病和/或病症可以包含实体瘤。例如，其中所述疾病和/或病症可以包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
另一方面，本申请还提供了一种预防和/或治疗疾病和/或病症的方法，其可以包含向有需要的对象施用本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。例如，其中所述疾病和/或病症可以包含肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、和Ras功能和/或活性异常的肿瘤。例如，其中所述疾病和/或病症可以选自以下组：PD-L1高表达肿瘤、EGFR高表达肿瘤、HER-2高表达肿瘤、CD38高表达肿瘤和Ras高表达的肿瘤。例如，其中所述疾病和/或病症可以包含实体瘤。例如，其中所述疾病和/或病症可以包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
另一方面，本申请还提供了一种影响靶分子含量的方法，可以包含施用本申请的缀合物、本申请的多肽、本申请的核酸分子、本申请的载体、本申请的免疫缀合物、本申请的细胞、本申请的组合物和/或本申请的试剂盒。例如，所述方法可以包含降低靶分子的含量。例如，所述方法可以包含体外方法、离体方法和/或体内方法。例如，所述靶分子可以为细胞表面的分子和/或细胞内的分子。例如，所述靶分子可以为皮肤细胞、骨髓细胞、宫颈细胞、肺细胞和/或乳腺细胞表面的分子。例如，所述靶分子可以为皮肤癌细胞、骨髓瘤细胞、宫颈癌细胞、肺癌细胞和/或乳腺癌细胞表面的分子。例如，所述细胞可以选自以下组：PD-L1阳性细胞、EGFR阳性细胞、HER-2阳性细胞、CD38阳性细胞和Ras功能和/或活性异常的肿瘤。例如，所述细胞可以选自以下组：PD-L1高表达细胞、EGFR高表达细胞、HER-2高表达细胞、CD38高表达细胞和Ras高表达的细胞。
制备方法
另一方面，本申请还提供了一种制备本申请缀合物和/或本申请多肽的方法，可以包含在使得所述缀合物和/或所述多肽表达的条件下，培养本申请的细胞。
例如，可以通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
例如，所述靶分子靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，和/或所述靶分子靶向部分的N末端可以包含寡聚甘氨酸序列。
例如，所述蛋白降解器靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基 酸序列LPETG或LPXTG，其中X可以为任意氨基酸，和/或所述蛋白降解器靶向部分的N末端可以包含寡聚甘氨酸序列。
例如，所述靶分子靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，所述蛋白降解器靶向部分的N末端可以包含寡聚甘氨酸序列，可以通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
例如，所述蛋白降解器靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，所述靶分子靶向部分的N末端可以包含寡聚甘氨酸序列，可以通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
另一方面，本申请还提供了一种筛选高邻近反应活性的靶分子靶向部分的方法。例如，为了解决单域抗体的亲和力不足和血清半衰期短的问题，可以通过向单域抗体中引入具有邻近反应活性的非天然氨基酸制备共价抗体。例如，引入具有邻近反应活性的非天然氨基酸的位点可以是单域抗体的CDR区、CDR区N端前5个氨基酸位点和/或CDR区C端前5个氨基酸位点。例如，为了筛选具有更高的邻近反应活性的靶分子靶向部分，可以采用以下筛选方法：可以向IgG、scFv、Fab或者单域抗体的CDR区、CDR区N端前5个氨基酸位点和/或CDR区C端前5个氨基酸位点插入具有邻近反应活性的非天然氨基酸，或可以根据抗体和抗原复合物的晶体结构，在抗原的界面氨基酸，的5埃周围搜寻抗体上可以用于插入具有邻近反应活性的非天然氨基酸的候选位点，例如抗原的界面氨基酸可以是抗原上距离抗体最接近的氨基酸，例如可以是具有亲核基团的界面氨基酸，抗体的候选位点可以是距离该界面氨基酸5埃的所有位点；将候选位点突变为非天然氨基酸后，可以获得具有邻近反应活性的共价抗体库用于筛选具有更高的邻近反应活性的靶分子靶向部分。例如，可以通过遗传密码子拓展技术将具有邻近反应活性的非天然氨基酸替换候选位点的氨基酸。例如，可以通过将共价抗体库与少量的抗原孵育后，通过变性凝胶电泳分离得到具有更高的邻近反应活性的靶分子靶向部分与抗原的交联条带，可以通过胶内酶解，串联质谱分析，以及可以通过交联肽段的分析得到具有更高的邻近反应活性的共价抗体中邻近反应性非天然氨基酸的插入位置和抗原上反应的亲核残基。
例如，验证本申请的靶分子靶向部分是否可以与靶分子共价结合的方法可以是，将靶分子靶向部分与靶分子共同孵育，用变性剂洗涤后，检测靶分子靶向部分与靶分子是否仍可以结合。例如，可以通过荧光标记的αPD-L1共价抗体与另一种荧光标记的靶分子孵育，可以通 过共聚焦荧光成像表征靶分子靶向部分与靶分子是否仍可以结合。例如，可以通过western blot检验蛋白交联产生分子量更高的交联条带的情况。
例如，可以通过转肽酶在共价抗体的C末端或N末端连接功能性多肽得到缀合物。例如，可以通过荧光标记缀合物、靶分子白串联的绿色荧光蛋白和溶酶体追踪剂三者的共定位成像表征降解子的降解过程。例如，可以将不同结构形式的缀合物同肿瘤细胞共同孵育，并可选地在部分培养基中加入溶酶体抑制剂，随后，收集细胞，提取蛋白质，经高温裂解和变性，western blot半定量检验缀合物对靶分子的降解程度。
本申请所述的靶分子靶向部分可以通过共价键连接无限降低了抗体对抗原的解离值，可以解决片段抗体半衰期短和易脱靶的缺点。通过转肽酶偶联功能分子的方式制备得到的缀合物则可以在结合靶分子后快速内吞并最终进入溶酶体中降解。利用靶分子靶向部分与靶分子不可逆结合的特点，可以实现缀合物-靶分子复合物在转运的过程中不能发生解离，避免靶分子在到达溶酶体前发生解离，而无法降解。同PROTAC技术相比，本申请可以实现对膜蛋白的高效降解，同LYTAC和AbTAC相比，本申请对膜蛋白的降解可以不依赖特定的肿瘤表面受体而能达到相同的降解效率，本申请的缀合物可以在基础科学研究和抗肿瘤治疗等领域具有更为广阔的应用前景。
另一方面，本申请还提供了一种制备本申请缀合物的方法，可以包含将所述缀合物的所述靶分子靶向部分与所述蛋白降解器靶向部分直接或间接连接。
例如，可以通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
例如，所述靶分子靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，和/或所述靶分子靶向部分的N末端可以包含寡聚甘氨酸序列。
例如，所述蛋白降解器靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，和/或所述蛋白降解器靶向部分的N末端可以包含寡聚甘氨酸序列。
例如，所述靶分子靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，所述蛋白降解器靶向部分的N末端可以包含寡聚甘氨酸序列，可以通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
例如，所述蛋白降解器靶向部分的C末端可以包含如SEQ ID NO：92或93所示的氨基酸序列LPETG或LPXTG，其中X可以为任意氨基酸，所述靶分子靶向部分的N末端可以包 含寡聚甘氨酸序列，可以通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的缀合物、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1靶分子靶向部分的制备与检测
1.1靶分子靶向部分前体的表达载体的构建
为了制备能够特异性识别靶分子并与该靶分子共价结合的靶分子靶向部分，首先可以制备能够结合靶分子的靶分子靶向部分前体。例如，野生型单域抗体可以作为靶分子靶向部分前体。
利用重组酶(诺唯赞，Cat.No.C112-01)通过同源重组的方式改造表达载体pET 20b(+)(Novagen，Cat.No.69739-3)，向其中引入酶切位点Not I，得到野生型单域抗体专用表达载体Nb vector，其中线性化pET 20b(+)所用引物对如下所示：
上游引物：GAAGCTGAGTGCGGCCGCAGGAAGCTGAGTTGGCTGC(如SEQ ID NO:7所示)；
下游引物：CTGCGGCCGCACTCAGCTTCCTTTCGGGCTTTGTTAGC(如SEQ ID NO:8所示)；
合成包含PD-L1单域抗体表达序列的双链核酸(PD-L1单域抗体氨基酸序列可以如SEQ ID NO:14或16所示，核酸序列可以如SEQ ID NO:15或17所示)，和包含EGFR单域抗体表达序列的双链核酸(EGFR单域抗体氨基酸序列可以如SEQ ID NO:19所示，核酸序列可以如SEQ ID NO:20所示)。任选地，为了后续的荧光染料标记和酶催化偶联多肽，单域抗体的C末端可以添加了一段转肽酶识别序列，如SEQ ID NO：92所示的氨基酸序列LPETG，随后通过限制性内切酶Nco I和Not I将单域抗体的编码序列亚克隆至Nb载体(Nb vector)中。
靶分子靶向部分前体的表达和纯化
将包含有单域抗体的Nb vector质粒通过热激法转化到表达型宿主菌BL 21(DE3)(TIANGEN，Cat.No.CB105-02)中，其涂布在含有氨苄青霉素(100μg/mL)的固体培养基的平板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种以1:100的比例转接至新鲜的自诱导培养基中(每升含6g Na 2HPO 4，3g KH 2PO 4，20g蛋白胨(typtone)，5g酵母 提取物(yeast extract)，5g NaCl，200mg MgCl 2,200mg CaCl 2,0.5g葡萄糖，2.5g乳糖，6mL甘油)，37℃摇床培养至OD值达到0.6-0.8时，转移到27℃摇床培养12-16小时。4000rpm转速离心，弃去上清的培养基，收集菌体。按照每1g菌泥加入15mL高渗溶液(20％Sucrose，25mM Tris,150mM NaCl，1mM EDTA,1mg/mL溶菌酶，pH 7.4)的比例重悬，室温孵育30min。4℃条件下，17000rpm高速离心40min，去除沉淀，收集上清。然后使用镍离子亲和层析柱(生工，Cat.No.C600792-0505)纯化单域抗体蛋白，并使用FPLC(GE Healthcare)对单域抗体进一步纯化，纯化好的蛋白质浓缩后保存在含有甘油的PBS(pH 7.4)缓冲溶液中，纯化效果通过UPLC/MS进行表征，结果表明单域抗体的分子量正确，纯度在95％以上。
图2A和图2B显示的是，可以作为靶分子靶向部分前体的野生型单域抗体的一级质谱图。图2A为野生型单域抗体αPD-L1的质谱图，理论值为16016Da，测定值为16015Da，图2B为野生型单域抗体αEGFR的质谱图，理论值为15603Da，测定值为15601Da。
1.2靶分子靶向部分的表达载体的构建
为了制备能够特异性识别靶分子并与该靶分子共价结合的靶分子靶向部分，之后可以在靶分子靶向部分前体的氨基酸位点引入可以靶分子形成共价键连接的第一官能团，例如，可以将单域抗体的界面氨基酸使用非天然氨基酸替换。例如，界面氨基酸位点的确定方法可以是先将单域抗体按照本领域常用的CDR划分方式划分CDR区，界面氨基酸可以是CDR区内的任意氨基酸，界面氨基酸也可以是CDR的N端前5个氨基酸中的任意氨基酸，界面氨基酸也可以是CDR的C端后5个氨基酸中的任意氨基酸。例如，可以通过琥珀密码子TAG突变的方式，将靶分子靶向部分前体的氨基酸位点替换为可以与靶分子形成共价连接的非天然氨基酸。
靶分子靶向部分的表达载体的构建
合成包含PD-L1单域抗体表达序列的双链核酸，和包含EGFR单域抗体表达序列的双链核酸，并利用重组酶(诺唯赞，Cat.No.C112-01)通过同源重组的方式克隆到pBAD/His(Thermo，Cat.No.V43001)中，其中线性化pBAD/His载体的PCR反应中所用引物对如下所示：
上游引物:AGTTGGCTGCTGCCACCAGAATTTGCCTGGCGGCA(如SEQ ID NO:21所示)；
下游引物:GCAGGTATTTCATATGGTTAATTCCTCCTGTTAGCCC(如SEQ ID NO:22所示)；
使用PCR聚合酶(诺唯赞，Cat.No.P505-d1)通过覆盖线性扩增PCR向单域抗体的表达序列中引入琥珀密码子TAG突变用于非天然氨基酸的插入。
表1显示的是，在不同位点引入TAG突变的覆盖线性扩增PCR所用引物。
表1在不同位点引入TAG突变的覆盖线性扩增PCR所用引物
Figure PCTCN2022080594-appb-000069
Figure PCTCN2022080594-appb-000070
Figure PCTCN2022080594-appb-000071
吸取3μL扩增产物加入到DH5α大肠杆菌(TIANGEN，Cat.No.CB101-02)化学感受态中，42℃热激1min后，冰浴，复苏，最后涂布在含有氨苄青霉素(100μg/mL)的固体培养基的平板上，37℃过夜。随机挑取至少3个单克隆菌落，以线性化pBAD/His载体的上游引物作为测序引物测序，选择正确的载体用于非天然氨基酸插入单域抗体的表达和纯化。
靶分子靶向部分的表达和纯化
将带有琥珀密码子TAG突变的单域抗体表达载体质粒分别同tRNA合成酶(tRNA Synthetase)/tRNA载体质粒通过热激法共转化到宿主菌DH10B(庄盟生物，Cat.No.ZC112-2)中，涂布在含有氨苄青霉素(100μg/mL)和氯霉素(34μg/mL)的固体培养基的平板上， 37℃过夜。每个平板各挑取一个单菌落接种于含有抗生素的LB培养基中，37℃，220rpm培养12小时。随后，从处于平台期的菌液中吸取相同体积的液体，并在混合后，以1:100的比例转接至新鲜的培养基中(每升含6g Na 2HPO 4，3g KH 2PO 4，20g蛋白胨(typtone)，5g酵母提取物(yeast extract)，5g NaCl，200mg MgCl 2，200mg CaCl 2,0.5g葡萄糖，6mL甘油)，37℃摇床培养至OD值达到0.6-0.8时，加入终浓度为1mM的非天然氨基酸，37℃继续培养30min，加入终浓度为0.2％的阿拉伯糖诱导蛋白表达，随后转移到27℃摇床培养12-16小时。4000rpm转速离心，弃去上清的培养基，收集菌体。加入裂解液(25mM Tris,150mM NaCl，1mg/mL溶菌酶，1x蛋白酶抑制剂混合物(Protease inhibitor cocktail)，pH 7.4)重悬，湿冰上超声破碎，4℃条件下，17000rpm高速离心40min，去除沉淀，收集上清。然后使用镍离子亲和层析柱(生工，Cat.No.C600792-0505)纯化单域抗体蛋白，并使用FPLC(GE Healthcare)对单域抗体进一步纯化，纯化好的蛋白质保存在含有甘油的PBS(pH 7.4)缓冲溶液中，纯化效果通过UPLC/MS进行表征。例如，非天然氨基酸的合成可以是本领域已知的，例如，6-溴-己酰赖氨酸(BrC6K)的合成酶可以记载于Angew.Chem.Int.Ed.Engl.56,14521-14525(2017).之中，氟代硫酸盐-L-酪氨酸(FSY)的合成酶可以记载于J.Am.Chem.Soc.140,4995-4999(2018).之中。
例如，可以分别向单域抗体中引入了BrC6K和FSY两种邻近交联反应活性非天然氨基酸。结果表明，不同位点引入非天然氨基酸的分子量均有在质谱结果中体现，此法可以得到不同位点插入非天然氨基酸的靶分子靶向部分化合物库。
表2显示的是，PD-L1单域抗体中不同位点被BrC6K或FSY替换后分子量。
表2 PD-L1单域抗体中不同位点被BrC6K或FSY替换后分子量
不同位点 被BrC6K替换后分子量 被FSY替换代后分子量
R32 16165 16105
T54 16220 16160
D99 16206 16146
S100 16234 16174
F101 16174 16114
E102 16192 16132
D103 16206 16146
P104 16224 16164
T105 16220 16160
T107 16220 16160
L108 16208 16148
V109 16222 16162
T110 16220 16160
S111 16234 16174
S112 16234 16174
G113 16264 16204
A114 16250 16190
F115 16174 16114
Q116 16193 16133
Y117 16158 16098
图3A和图3B显示的是，PD-L1单域抗体的不同位点被BrC6K或FSY替换后的所得化合物库的质谱图。图3A显示的是PD-L1单域抗体的不同位点被BrC6K后的所得化合物库的质谱图，图3B显示的是PD-L1单域抗体的不同位点被FSY替换后的所得化合物库的质谱图。
1.3靶分子靶向部分与靶分子形成共价键连接的检测
取少量复溶的人PD-L1蛋白(义翘神州，Cat.No.10084-HNAH)2μg置于1.5mL EP管中，加入30μg本申请中纯化出的PD-L1单域抗体中不同位点被BrC6K或FSY替换后的所得化合物库(化合物库中包含不同位点被BrC6K或FSY替换后的多个单域抗体的复合物)和70μL PBS缓冲液(pH 8.0)，37℃反应一定时间后，冻干除去溶剂。加入40μL 1x上样缓冲液(康为世纪，Cat.No.CW0027S)，重悬，室温静置30min，SDS-PAGE(金斯瑞，Cat.No.M00659)凝胶电泳分离分析反应体系。电泳完成后，拆开胶板，取出凝胶，蒸馏水洗涤后，考马斯亮蓝染色，脱色液脱色以鉴定交联。
图4A和图4B显示的是，SDS-PAGE分析PD-L1单域抗体的不同位点被BrC6K或FSY替换后的所得化合物库同靶分子交联结果。图4A显示的是，PD-L1单域抗体的不同位点被BrC6K替换后的所得化合物库与靶分子孵育0小时以及孵育72小时后的交联结果；图4B显示的是，PD-L1单域抗体的不同位点被FSY替换后的所得化合物库与靶分子孵育后的交联结果。结果显示，插入非天然氨基酸的单域抗体在和抗原蛋白孵育后，体系中产生了新的大分子量条带，分子量之和等于单域抗体加上抗原，表明靶分子靶向部分化合物库中的靶分子靶向部分都可以与抗原蛋白形成了新的共价相互作用，这种共价结合不受变性剂SDS的影响。所得化合物库中的靶分子靶向部分都具有邻近交联反应活性。
1.4高邻近交联反应活性的靶分子靶向部分的筛选
在干净的保鲜膜上，采用新的刀片将上述凝胶上的化合物库同抗原交联条带切下，分割为边长约1mm的小块。50％乙腈(Fisher,Cat.No.955-4)水溶液处理三次脱去残留的考马斯亮蓝染色液，纯乙腈对胶粒脱水。随后DTT(二硫苏糖醇)还原打开蛋白中的二硫键到游离态，碘乙酰修饰还原出的半胱氨酸的巯基。再次用纯乙腈脱去胶粒中的水份。随后用胰蛋白酶(Trypsin K/R，Promega,Cat.No.V511B)和胰凝乳蛋白酶(Chymotrypsin，Promega,Cat.No.V1061)两种酶同时作用将胶粒中的蛋白酶解为肽段，0.1％甲酸(Fluka)提取肽段，脱盐处理后提交串联质谱分析。串联质谱为Thermo的QE PLUS或LUMOS三合一质谱，流动相为0.1％甲酸水溶液和0.1％甲酸乙腈，梯度时间为90min。与此同时从胶上分离出PD-L1单域抗体条带和PD-L1条带，类似的处理后使用多级质谱鉴定切出来的肽段，p-Find软件搜索肽段，得到的结果作为对照分析。对于凝胶上的化合物库同抗原交联条带，采用p-link软件搜索鉴定到的交联肽段，绘制相互作用界面，可以确定具有高邻近交联反应活性的靶分子靶向部分上的非天然氨基酸插入位点，以及可以确定靶分子上发生交联的位点。
图5A、图5B和图5C显示的是，具有高邻近交联反应活性的靶分子靶向部分与靶分子交联后的交联肽段的串联质谱。图5A显示的是，PD-L1单域抗体中110位被BrC6K替换时，可以与靶分子PD-L1的第69位组氨酸具有更高的邻近交联反应活性；图5B显示的是，PD-L1单域抗体中113位被BrC6K替换时，可以与靶分子PD-L1的第69位组氨酸具有更高的邻近交联反应活性；图5C显示的是，PD-L1单域抗体中108位被FSY替换时，可以与靶分子PD-L1的第69位组氨酸具有更高的邻近交联反应活性。结果表明，SDS-PAGE结合胶内酶解交联蛋白并用多级质谱分析交联肽段的方法可以用于从靶分子靶向部分化合物库中筛选出具有更高的邻近交联反应活性的靶分子靶向部分；PD-L1单域抗体中110位、113位和108位被具有邻近交联反应活性的非天然氨基酸替换后，所得靶分子靶向部分具有更高的邻近交联反应活性。
将靶分子靶向部分表达和纯化：第110位被BrC6K替换后的PD-L1单域抗体(αPD-L1 T110BrC6K)、第113位被BrC6K替换后的PD-L1单域抗体(αPD-L1 G113BrC6K)、第108位被FSY替换后的PD-L1单域抗体(αPD-L1 L108FSY)和第116位被FSY替换后的EGFR单域抗体(αEGFR Q116FSY)。
图6A、图6B、图6C和图6D显示的是，PD-L1单域抗体或EGFR单域抗体中不同位点被BrC6K或FSY替换后的所得靶分子靶向部分的质谱图。图6A显示的是，第110位被BrC6K替换后的PD-L1单域抗体(αPD-L1 T110BrC6K)，图6B显示的是，第113位被BrC6K替换 后的PD-L1单域抗体(αPD-L1 G113BrC6K)，图6C显示的是，第108位被FSY替换后的PD-L1单域抗体(αPD-L1 L108FSY)，图6D显示的是，第116位被FSY替换后的EGFR单域抗体(αEGFR Q116FSY)。
取复溶的人PD-L1蛋白(义翘神州，Cat.No.10084-HNAH)2μg置于1.5mL EP管中，加入5μg不同位点被非天然氨基酸替换的所得靶分子靶向部分或野生型PD-L1单域抗体和70μL PBS缓冲液(pH 8.0)，37℃反应一定时间后，SDS-PAGE分析可以再以两种方式测试：方式一，加入50μg的GGG-TAMRA羧基罗丹明荧光底物(北京中科亚光生物科技有限公司合成)和0.5μg的转肽酶Sortase A(表达纯化得到，序列如SEQ ID NO：99所示)，37℃孵育1小时使荧光底物连接到靶分子靶向部分上，加入5x上样缓冲液(康为世纪，Cat.No.CW0027S)，室温静置30min，SDS-PAGE(金斯瑞，Cat.No.M00659)凝胶电泳分离分析反应体系。电泳完成后，蒸馏水清洗胶三次后，TYPHOON多功能激光扫描成像系统(GE healthhcare)扫描胶内荧光；方式二，冻干除去溶剂。加入40μL稀释为1x的5x上样缓冲液(康为世纪，Cat.No.CW0027S)，重悬，室温静置30min，SDS-PAGE(金斯瑞，Cat.No.M00659)凝胶电泳分离，考马斯亮蓝染色，分析交联反应。
图7A、图7B、和图7C显示的是，PD-L1单域抗体或EGFR单域抗体中不同位点被BrC6K或FSY替换后的所得靶分子靶向部分与靶分子体外交联检测结果。图7A显示的是SDS-PAGE分析野生型PD-L1单域抗体，靶分子靶向部分αPD-L1 T110BrC6K，靶分子靶向部分αPD-L1 G113BrC6K分别同PD-L1蛋白共同孵育24小时的混合液，本申请的靶分子靶向部分都可以与靶分子形成交联条带；图7B显示的是，SDS-PAGE分析αPD-L1 L108FSY同PD-L1蛋白共同孵育24小时的混合液，本申请的靶分子靶向部分都可以与靶分子形成交联条带；图7C显示的是，SDS-PAGE分析野生型EGFR单域抗体，αEGFR Q116FSY分别同靶分子EGFR蛋白共同孵育12小时的混合液，下图为荧光扫描胶结果显示本申请的靶分子靶向部分都可以与靶分子形成交联条带。结果表明，本申请得到的靶分子靶向部分都能够具有与靶分子邻近交联反应活性，本申请得到的靶分子靶向部分αPD-L1 L108FSY能够具有更高的与PD-L1邻近交联反应活性，αEGFR Q116FSY能够具有更高的与EGFR邻近交联反应活性。
1.5靶分子靶向部分结合细胞表面靶分子的检测
为了验证靶分子靶向部分可以共价结合在细胞膜表面的靶分子，采用转肽酶Sortase A在靶分子靶向部分的C末端标记荧光染料，例如，羧基罗丹明荧光底物。将带有LPETG转肽酶连接序列的靶分子靶向部分用超滤的方法更换到反应液中(25mM Tris,150mM NaCl，pH 7.4)，调整抗体浓度为100μM，加入10倍质量当量的GGG-TAMRA底物和0.1倍质量当量 的转肽酶Sortase A，37℃孵育1小时使荧光底物连接到靶分子靶向部分上，加入终浓度为5mM的MTSET试剂(Biorigin,Cat#BN15003)，Bio-rad脱盐柱离心除去底物的同时更换到PBS(pH 7.4)溶液中，加入10％的甘油于-80℃保存或使用。
表达PD-L1的细胞的构建
构建表达PD-L1或表达PD-L1-GFP细胞的方法可以是本领域常用的构建方法。例如，可以将3种质粒pLV-PD-L1-C-GFP Spark(义翘神州，Cat.No.HG10084-ACGLN)、psPAX2(淼灵生物，Cat.No.P0261)和pMD2.G(淼灵生物，Cat.No.P0262)共同转染进入生长密度为80％的HEK 293T细胞中，使用含有10％胎牛血清的DMEM培养基，放于恒温为37℃并含有5％的CO 2的培养箱中培养24小时后更换新鲜的DMEM培养基，转染48小时后开始收取第一次病毒并更换新鲜培养基，72小时和96小时后收取第二次和第三次病毒颗粒。在24孔板中种植MDA-MB-231 Luc乳腺癌细胞(诺和生物，MDA-MB-231+Luciferase)，待生长密度为70％的时候，向4个孔中分别加入100μL，200μL，400μL和500μL病毒原液并加入终浓度为8μg/mL的聚凝胺，培养24小时后传代，扩大培养至直径10厘米培养皿中后用0.25％的胰酶消化悬浮为单细胞状态，随后送至细胞分选仪完成分选工作，分选通道选择GFP。得到多克隆后采用有限稀释法进一步得到不同PD-L1表达量的单克隆稳转系PD-L1/MDA或PD-L1-GFP/MDA，扩大培养后用快速冻存液冻存于-80℃冰箱或继续培养，可以通过共聚焦荧光显微镜拍摄荧光抗体标记的细胞表征PD-L1的转入。
消化吹散培养皿中的表达PD-L1的人乳腺癌MDA细胞(PD-L1/MDA)，以1:5的比例铺被到8孔板中，37℃培养12小时。加入终浓度为200nM荧光标记的靶分子靶向部分或野生型单域抗体，继续培养5小时。随后普通清洗组使用PBS溶液(pH 7.4)洗三次，每次15min；强变性清洗组则采用强变性溶液(Stringent溶液，500mM NaCl,3％吐温20,100mM甘氨酸,pH 3.0)，洗三次，每次15min，该溶液为强烈的变性条件，因此通过非共价相互作用结合在细胞表面的物质(例如野生型单域抗体)将从细胞上解离。使用蔡司共聚焦荧光显微镜对TAMRA通道进行荧光成像。
图8A、图8B、图8C和图8D显示的是，靶分子靶向部分结合细胞表面靶分子的检测结果。图8A显示，PD-L1单域抗体被BrC6K替换后的靶分子靶向部分αPD-L1 BrC6K在强变性溶液洗涤后依然与靶分子结合，图8B显示，免疫印迹试验表征αPD-L1 BrC6K与靶分子PD-L1发生交联反应；图8C显示，EGFR单域抗体被FSY替换后的靶分子靶向部分αEGFR FSY在强变性溶液洗涤后依然与靶分子结合，图8D显示，免疫印迹试验表征αEGFR FSY与靶分子PD-L1发生交联反应。结果显示，强变性溶液处理后，依然可以在细胞表面检测到靶分子靶向部 分(αPD-L1 BrC6K或αEGFR FSY)，而野生型单域抗体(αPD-L1或αEGFR)基本消失。这表明靶分子靶向部分在结合细胞膜表面靶分子后，在邻近效应的促进下，靶分子靶向部分上的具有邻近交联反应活性的非天然氨基酸将同靶分子上的亲核残基发生反应，二者的结合方式由非共价结合转变为共价交联连接。
实施例2缀合物的制备与检测
本申请提供一种将蛋白降解器靶向部分连接在靶分子靶向部分制备具有降解能力的缀合物的方法。例如，可以通过酶催化偶联反应将穿膜肽(Cell penetrating peptide)和溶酶体靶向肽(Lysosomal targeting peptide)连接于靶分子靶向部分的N端或C端，同时保持靶分子靶向部分对靶分子的靶向结合和邻近交联反应的能力。
2.1蛋白降解器靶向部分的合成
本申请中所述穿膜肽指可以帮助胞外物质穿过细胞膜的多肽，例如，多个带正电的精氨酸串联在一起得到的多肽具有良好的穿膜能力，穿膜肽可以是多聚精氨酸；又例如，NPXY(如SEQ ID NO:64所示，其中X可以为甘氨酸G、或丙氨酸A)，ALAPYIP(如SEQ ID NO:77所示)可以很好的靶向溶酶体。靶向溶酶体或蛋白酶体的其它多肽同样可以在本申请中发挥类似的作用，由二者组成的用于偶联反应的转肽反应底物序列包括但不限于表3显示的蛋白降解器靶向部分。
表3本申请的蛋白降解器靶向部分序列信息
Figure PCTCN2022080594-appb-000072
Figure PCTCN2022080594-appb-000073
上述多肽底物交由多肽合成公司(北京中科亚光生物科技有限公司)，可以采用标准的固相多肽合成法合成，经由HPLC纯化后，纯度>95％。其中，G或G’、L或L’表示转肽酶Sortase A识别并连接的序列，CPP可以表示多聚精氨酸，例如9个D构型的精氨酸连接而成的多肽，LTP可以表示NPXY靶向溶酶体序列(如SEQ ID NO:64所示，其中X可以为甘氨酸G、或丙氨酸A)，LTS可以表示ALAPYIP靶向溶酶体序列(如SEQ ID NO:77所示)，MMP-B或B-MMP可以表示屏蔽多聚精氨酸的负电序列，其中MMP序列可以被细胞表面的蛋白酶酶切，从而使B序列的带负电序列被剪切，使得多聚精氨酸发挥穿膜能力。
2.2缀合物的合成
图9A和图9B显示的是，转肽酶介导蛋白降解器靶向部分和靶分子靶向部分连接制备本申请缀合物的示意图。图9A显示的是靶分子靶向部分的C端连接蛋白降解器靶向部分制备本申请缀合物；图9B显示的是靶分子靶向部分的N端连接蛋白降解器靶向部分制备本申请 缀合物。
靶分子靶向部分的C端连接蛋白降解器靶向部分
将带有LPETG转肽酶连接序列的靶分子靶向部分用超滤或脱盐的方法更换为反应液(25mM Tris,150mM NaCl，5mM CaCl 2，pH 7.4)，调整抗体终浓度为100μM，加入终浓度为1mM的蛋白降解器靶向部分(例如G n-CPP+LTS，其中n可以为1到10)和终浓度为10μM的转肽酶Sortase A，30℃孵育1小时，LC-MS监测反应进程。随后，加入终浓度为5mM的MTSET试剂，混匀后室温孵育10min。使用脱盐柱(Bio-rad,Cat.No.7326221)或PD-10(GE,Cat.No.52-1308-00)除去底物的同时更换到PBS(pH 7.4)溶液中。His标签(His-Tag)可以由6-10个连续的组氨酸残基组成，His标签可被镍柱吸附；由于参与连接反应的靶分子靶向部分的C端的His标签在反应中被切除，而转肽酶中C末端带有His标签，因此可以采用镍磁珠或树脂除去体系中残留未反应的靶分子靶向部分和转肽酶。因为是位点特异性连接并经过纯化后得到的产品，每一个靶分子靶向部分上可以均连接一分子的蛋白降解器靶向部分。得到的缀合物用液质联用质谱(LC-MS表征)。
图10显示的是，PD-L1靶分子靶向部分αPD-L1 L108FSY，以及PD-L1靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物GlueTAC FSY/PD-L1的一级质谱图。
图11显示的是，野生型EGFR单域抗体连接蛋白降解器靶向部分所得的偶联物NbTAC-EGFR，以及EGFR靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物GlueTAC-EGFR的一级质谱图。
靶分子靶向部分的N端连接蛋白降解器靶向部分
为了可以在靶分子靶向部分的N端偶联蛋白降解器靶向部分或其它功能肽，本申请在靶分子靶向部分的N末端添加了寡聚甘氨酸序列(例如采用三聚甘氨酸)。合成N末端添加了寡聚甘氨酸序列的PD-L1单域抗体表达序列的双链核酸(氨基酸序列可以如SEQ ID NO:16所示，核酸序列可以如SEQ ID NO:17所示)，随后通过Nco I和Not I将单域抗体的编码序列亚克隆至Nb vector。
采用本申请的方法在L108位和G113位引入琥珀密码子突变，所用引物如下：
在L108引入TAG突变的覆盖线性扩增PCR所用引物：
上游引物：GATCCGACCTGTACCTAGGTTACCAGCAGCGGTG(如SEQ ID NO:94所示)；
下游引物：ACCGCTGCTGGTAACCTAGGTACAGGTCGGATCTTC(如SEQ ID NO:95所示)。
在G113引入TAG突变的覆盖线性扩增PCR所用引物：
上游引物：CCTGGTTACCAGCAGCTAGGCATTTCAGTATTGGG(如SEQ ID NO:96所示)；
下游引物：CCAATACTGAAATGCCTAGCTGCTGGTAACCAGGG(如SEQ ID NO:97所示)。
将测序正确的突变质粒后，分别采用采用本申请表达和纯化的方法纯化得到N端带有三聚甘氨酸的野生型单域抗体NG 3-αPD-L1，靶分子靶向部分NG 3-αPD-L1 L108FSY和靶分子靶向部分NG 3-αPD-L1 G113BrC6K。
将N端为三聚甘氨酸的单域抗体蛋白用超滤或脱盐的方法更换为反应液(25mM Tris,150mM NaCl，5mM CaCl 2，pH 7.4)，调整抗体终浓度为100μM，加入终浓度为200μM的蛋白降解器靶向部分(例如带有LPETG转肽酶连接序列的CPP+LTS序列)和终浓度为10μM的转肽酶Sortase A，30℃孵育1小时，LC-MS监测反应进程。随后，加入终浓度为5mM的MTSET试剂，混匀后室温孵育10min。使用阳离子交换树脂从反应体系中纯化出目标产物，浓缩后使用脱盐柱(Bio-rad,Cat.No.7326221)或PD-10(GE,Cat.No.52-1308-00)除去底物的同时更换到PBS(pH 7.4)溶液中，因为是位点特异性连接并经过纯化后得到的产品，每一个单域抗体上可以均连接一分子的蛋白降解器靶向部分。得到的蛋白用液质联用质谱(LC-MS表征)。
实施例3缀合物介导的靶分子降解
3.1采用荧光成像的方法检测缀合物对靶分子的结合和促进靶分子降解
为了观察到本申请的缀合物在靶向靶分子后发生邻近交联反应，产生共价连接的复合物，并进入细胞在蛋白降解器内一同降解，本申请在PD-L1蛋白的C末端融合了绿色荧光蛋白，通过监测靶分子、缀合物和溶酶体定位探针的荧光信号，可以观测缀合物促进靶分子降解的过程。
消化吹散培养皿中表达PD-L1和GFP融合蛋白的人乳腺癌MDA细胞(PD-L1-GFP/MDA，通过本申请的方法构建)，以1:5的比例铺被到8孔板中，37℃培养12小时。分别加入终浓度为200nM的TAMRA荧光标记的野生型PD-L1单域抗体(对照组)、终浓度为200nM的TAMRA荧光标记的PD-L1靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物GlueTAC-PD-L1(GlueTAC试验组)，并加入溶酶体荧光探针(Lysotracker Deepred，Thermo,Cat.No.L12492)，继续培养5小时。随后使用PBS缓冲液(pH 7.4)洗3次，使用蔡司共聚焦荧光显微镜对GFP、TAMRA和Deepred三个通道分别进行荧光成像。
图12显示的是，对照组和GlueTAC试验组的活细胞共聚焦荧光成像。结果显示，对照组中荧光标记的单域抗体和靶分子在空间分布上没有明显的一致性；荧光标记的缀合物和靶分子在空间分布上有一定程度的一致性，其荧光呈现出聚集的效果，可以认为缀合物和靶分子以结合成为复合物的形式存在。进一步地，荧光合并图显示红色荧光的溶酶体定位探针的空间分布和缀合物-靶分子复合物也有一定程度的一致性，可以认为靶分子在本申请缀合物的介导下被送入溶酶体中降解。
3.2采用免疫印迹定量分析缀合物促进靶分子降解的能力
为了量化不同结构形式的缀合物在降解靶分子上的能力，消化吹散培养皿中的表达PD-L1和GFP融合蛋白的人乳腺癌MDA-MB-231细胞(PD-L1-GFP/MDA，通过本申请的方法构建)或人非小细胞肺癌HCC827细胞(Procell,Cat.No.CL0094)，以1:3的比例铺被到12孔板中，37℃培养12小时。空白对照组：加入缓冲液，例如Hank's平衡盐溶液(Hank's Balanced Salt Solution,HBSS)；试验组A：加入终浓度为200nM的野生型PD-L1单域抗体(αPD-L1)；试验组B：加入终浓度为200nM的野生型PD-L1单域抗体的N端连接蛋白降解器靶向部分所得的偶联物(NbTAC-N/PD-L1)；试验组C：加入终浓度为200nM的野生型PD-L1单域抗体的C端连接蛋白降解器靶向部分所得的偶联物(NbTAC-C/PD-L1)；试验组D：加入终浓度为200nM的PD-L1单域抗体第108位被FSY替换后的靶分子靶向部分(αPD-L1 L108FSY)；试验组E：加入终浓度为200nM的PD-L1靶分子靶向部分αPD-L1 G113BrC6K连接蛋白降解器靶向部分所得的缀合物GlueTAC BrC6K/PD-L1；试验组F：加入终浓度为200nM的PD-L1靶分子靶向部分αPD-L1 L108FSY连接蛋白降解器靶向部分所得的缀合物GlueTAC FSY/PD-L1。细胞继续培养一段时间后取出，弃去培养基，每孔中加入60μL RIPA裂解液(Thermo,Cat.No.89901)，冰上孵育30min。吸取细胞裂解液于1.5mL EP管中，加入15μL 5x上样缓冲液，95℃加热30min。12000rpm高速离心后，SDS-PAGE电泳。依次经过转膜，封闭，一抗标记和二抗标记后在成像仪(Biorad)上显影，其中一抗分别为鼠抗人PD-L1(CST)，鼠抗人Tubulin(Cell Signaling Technologies,Cat#2125S)和鼠抗His(Cell Signaling Technologies,Cat#12698S)，二抗为兔抗鼠IgG(Abcam,Cat.No.ab6728)，最后显影得到的图使用Image Lab软件计算每各条带的灰度值，以定量计算PD-L1的相对含量。
图13A、图13B、图13C和图13D显示的是，免疫印迹试验表征本申请缀合物促进靶分子降解的结果。图13A、图13B和图13D显示的是，本申请缀合物与人非小细胞肺癌HCC827细胞孵育24小时后促进靶分子PD-L1降解结果，图13C显示的是，本申请缀合物与人乳腺癌MDA-MB-231细胞孵育24小时后促进靶分子PD-L1降解结果。结果显示，与野生型PD- L1单域抗体相比，野生型PD-L1单域抗体的N端或C端连接蛋白降解器靶向部分所得的偶联物，可以促进靶分子降解。进一步地，具有邻近交联反应活性的靶分子靶向部分连接蛋白降解器靶向部分所得的本申请缀合物，可以实现约80％的更高的靶分子降解效率。可以认为，本申请的缀合物可以具有促进靶分子降解的能力。
根据本申请中免疫印迹定量分析缀合物促进靶分子降解的方法，检测本申请缀合物降解宫颈癌HeLa细胞的靶分子EGFR的能力。空白对照组：加入缓冲液，例如Hank's平衡盐溶液(Hank's Balanced Salt Solution,HBSS)；试验组A：加入野生型EGFR单域抗体(αEGFR，7D12)；试验组B：加入野生型EGFR单域抗体连接蛋白降解器靶向部分所得的偶联物(NbTAC-EGFR)；试验组C：加入EGFR单域抗体第116位被FSY替换后的靶分子靶向部分(αEGFR Q116FSY)；试验组D：加入EGFR靶分子靶向部分αEGFR Q116FSY连接蛋白降解器靶向部分所得的缀合物GlueTAC-EGFR。
图14显示的是，免疫印迹试验表征本申请缀合物促进靶分子降解的结果。结果显示，本申请缀合物GlueTAC-EGFR与HeLa细胞共同孵育24小时后，可以显著降低靶分子EGFR的含量。
3.3缀合物促进靶分子降解的降解途径的验证
根据本申请中免疫印迹定量分析缀合物促进靶分子降解的方法，氯化铵可以抑制溶酶体和蛋白酶体的功能，试验组可以加入蛋白降解器抑制剂氯化铵(Sigma，Cat.No.V900222-500G)验证本申请缀合物的降解途径。
图15显示的是，氯化铵存在情况下，申请缀合物促进靶分子降解的结果。结果显示，在蛋白降解器抑制剂的存在下，本申请靶分子靶向部分连接蛋白降解器靶向部分所得的缀合物的降解效率显著降低。可以认为本申请的缀合可以通过蛋白降解器降解靶分子。
实施例4缀合物介导的体内靶分子降解
用含有50％高浓度基质胶的Hank's平衡盐溶液(Hank's Balanced Salt Solution,HBSS)悬浮PD-L1/MDA(5x10 5)细胞，总体积100μL，在6周龄的雌性BALB/c Nude鼠的右腿上方皮下注射(经过北京大学动物实验伦理委员会的批准)。约1周后可以明显观察到凸起，触摸有异物感，2周左右使用游标卡尺测量，体积约为50-70mm 3。此时在肿瘤部位原位注射50μg样品，空白对照组：注射缓冲液，例如Hank's平衡盐溶液(Hank's Balanced Salt Solution,HBSS)；试验组A：注射野生型PD-L1单域抗体(αPD-L1)；试验组B：注射野生型PD-L1单域抗体连接蛋白降解器靶向部分所得的偶联物(NbTAC-PD-L1)；试验组C：注射PD-L1靶分子靶向部分αPD-L1 L108FSY连接蛋白降解器靶向部分所得的缀合物(GlueTAC-PD-L1)。
每组3只，并在给药24小时后取出肿瘤组织，肿瘤体积＝(长x宽x宽)/2，剪碎。取20mg剪碎后的肿瘤组织，加入200μL RIPA裂解液，冰浴，匀浆仪分散肿瘤，离心去除脂肪层，室温反应1小时，充分裂解癌细胞。取40μL加入5x上样缓冲液(loading buffer)处理样品，Western blotting分析，分别用PD-L1抗体(CST,Cat.No.13684)和GADPH抗体(Abcam，Cat.No.ab128915)标记后显影。
图16显示的是，本申请缀合物在体内促进肿瘤组织的靶分子降解的结果。结果显示，本申请缀合物在体内可以显著降低靶分子的含量。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (141)

  1. 一种缀合物，其包含：靶分子靶向部分以及蛋白降解器靶向部分；其中，所述靶分子靶向部分能够特异性识别靶分子并与该靶分子共价结合，所述蛋白降解器靶向部分与所述靶分子靶向部分直接或间接连接。
  2. 根据权利要求1所述的缀合物，所述靶分子靶向部分包含第一官能团，所述靶分子包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团能够与所述第二官能团反应而形成共价键。
  3. 根据权利要求2所述的缀合物，在所述第一官能团与所述第二官能团之间的距离小于约5E-10米时，所述第一官能团能够与所述第二官能团反应而形成共价键。
  4. 根据权利要求2-3中任一项所述的缀合物，所述第二官能团包含亲电基团。
  5. 根据权利要求2-4中任一项所述的缀合物，所述第二官能团包含天然氨基酸的侧链基团。
  6. 根据权利要求2-5中任一项所述的缀合物，所述第二官能团选自以下组：氨基、咪唑基、巯基、羟基、羧基、甲硫基，以及前述的衍生物。
  7. 根据权利要求1-6中任一项所述的缀合物，所述靶分子包含细胞表面的分子、细胞内的分子和/或细胞外的分子。
  8. 根据权利要求1-7中任一项所述的缀合物，所述靶分子选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
  9. 根据权利要求1-8中任一项所述的缀合物，所述靶分子选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。
  10. 根据权利要求1-9中任一项所述的缀合物，所述靶分子选自以下组：PD-L1(细胞程序死亡-配体1)、EGFR(表皮生长因子受体)、HER-2(人类表皮生长因子受体-2)、Ras蛋白、CD38(淋巴细胞分化抗原CD38)，以及前述的功能性片段。
  11. 根据权利要求2-10中任一项所述的缀合物，所述第一官能团选自以下组：
    Figure PCTCN2022080594-appb-100001
    以及前述的衍生物，其中，R 1为O、N或不存在，R 2为卤素、烯基或炔基，R 3为卤素、烯基或炔基，R 4为卤素，n1和n2各自独立地至少为0。
  12. 根据权利要求2-11中任一项所述的缀合物，所述第一官能团选自以下组：
    Figure PCTCN2022080594-appb-100002
    Figure PCTCN2022080594-appb-100003
    以及前述的衍生物。
  13. 根据权利要求2-12中任一项所述的缀合物，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：
    Figure PCTCN2022080594-appb-100004
    Figure PCTCN2022080594-appb-100005
    以及前述的衍生物。
  14. 根据权利要求2-13中任一项所述的缀合物，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
  15. 根据权利要求1-14中任一项所述的缀合物，所述靶分子靶向部分选自以下组：多肽、核 酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
  16. 根据权利要求1-15中任一项所述的缀合物，所述靶分子靶向部分包含抗体或其抗原结合片段。
  17. 根据权利要求16所述的缀合物，所述抗体选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。
  18. 根据权利要求16-17中任一项所述的缀合物，所述抗原结合片段选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。
  19. 根据权利要求1-18中任一项所述的缀合物，所述靶分子靶向部分包含单域抗体或其抗原结合片段。
  20. 根据权利要求2-19中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。
  21. 根据权利要求10-20中任一项所述的缀合物，所述靶分子靶向部分能够特异性识别所述PD-L1。
  22. 根据权利要求13-21中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸被所述非天然氨基酸替换。
  23. 根据权利要求14-22中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸被所述6-溴-己酰赖氨酸替换、第110位氨基酸被所述6-溴-己酰赖氨酸替换、第113位氨基酸被所述氟代硫酸盐-L-酪氨酸替换和/或第108位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
  24. 根据权利要求1-23中任一项所述的缀合物，所述靶分子靶向部分包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
  25. 根据权利要求2-24中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，在第116位氨基酸包含所述第一官能团。
  26. 根据权利要求10-25中任一项所述的缀合物，所述靶分子靶向部分能够特异性识别所述EGFR。
  27. 根据权利要求13-26中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所 述非天然氨基酸替换。
  28. 根据权利要求14-27中任一项所述的缀合物，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
  29. 根据权利要求1-28中任一项所述的缀合物，所述靶分子靶向部分包含SEQ ID NO：12所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的缀合物，所述蛋白降解器包含溶酶体和/或蛋白酶体。
  31. 根据权利要求1-30中任一项所述的缀合物，所述蛋白降解器靶向部分选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
  32. 根据权利要求30-31中任一项所述的缀合物，所述蛋白降解器靶向部分包含能够靶向所述溶酶体和/或所述蛋白酶体的分子或其功能活性片段。
  33. 根据权利要求1-32中任一项所述的缀合物，所述蛋白降解器靶向部分选自以下组：溶酶体靶向肽(LTP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。
  34. 根据权利要求1-33中任一项所述的缀合物，所述蛋白降解器靶向部分包含所述溶酶体靶向肽，所述溶酶体靶向肽的氨基酸序列如SEQ ID NO：63-77中任一项所示。
  35. 根据权利要求1-34中任一项所述的缀合物，所述缀合物还包含穿膜部分，所述穿膜部分能够促进所述靶分子进入细胞内，其中，所述穿膜部分与所述靶分子靶向部分直接或间接连接，和/或所述穿膜部分与所述蛋白降解器靶向部分直接或间接连接。
  36. 根据权利要求35所述的缀合物，所述穿膜部分选自以下组：穿膜肽(CPP)、低聚糖肽、甘露糖6-磷酸酯(M6P)、N-乙酰半乳糖胺(GalNAc)、半乳糖，以及前述的任意组合。
  37. 根据权利要求35-36中任一项所述的缀合物，所述穿膜部分包含聚精氨酸，所述聚精氨酸的重复单元数量至少为2。
  38. 根据权利要求37所述的缀合物，所述聚精氨酸的聚合单体包含D构型的精氨酸和/或L构型的精氨酸。
  39. 根据权利要求35-38中任一项所述的缀合物，所述缀合物包含所述穿膜肽和所述溶酶体靶向肽，所述穿膜肽和所述溶酶体靶向肽直接或间接连接。
  40. 根据权利要求30-39中任一项所述的缀合物，所述蛋白降解器靶向部分包含能够靶向所述溶酶体和/或所述蛋白酶体，和/或能够结合细胞表面促进内吞的受体的分子或其功能活性片段。
  41. 根据权利要求1-40中任一项所述的缀合物，所述蛋白降解器靶向部分包含能够结合6-磷酸甘露糖受体(M6PR)的分子。
  42. 根据权利要求1-41中任一项所述的缀合物，所述蛋白降解器靶向部分包含一个或多个甘露糖6-磷酸酯(M6P)或其功能活性片段。
  43. 根据权利要求42所述的缀合物，所述蛋白降解器靶向部分包含约1个至约500个所述甘露糖6-磷酸酯或其功能活性片段。
  44. 根据权利要求42-43中任一项所述的缀合物，所述蛋白降解器靶向部分包含支架分子，所述支架分子直接或间接连接一个或多个所述甘露糖6-磷酸酯或其功能活性片段。
  45. 根据权利要求44所述的缀合物，所述支架分子包含聚糖结构。
  46. 根据权利要求44-45中任一项所述的缀合物，所述支架分子包含糖蛋白，所述糖蛋白包含被所述一个或多个所述甘露糖6-磷酸酯或其功能活性片段修饰的一个或多个氨基酸。
  47. 根据权利要求46所述的缀合物，所述糖蛋白包含N-羧基酸酐(NCA)或其衍生物的基团。
  48. 根据权利要求1-47中任一项所述的缀合物，所述蛋白降解器靶向部分包含能够结合去唾液酸糖蛋白受体(ASGPR)的分子或其功能活性片段。
  49. 根据权利要求48所述的缀合物，所述蛋白降解器靶向部分包含所述去唾液酸糖蛋白受体的一个或多个配体。
  50. 根据权利要求1-49中任一项所述的缀合物，所述蛋白降解器靶向部分包含一个或多个N-乙酰半乳糖胺(GalNAc)。
  51. 根据权利要求1-50中任一项所述的缀合物，所述蛋白降解器靶向部分包含一个或多个半乳糖。
  52. 根据权利要求1-51中任一项所述的缀合物，所述蛋白降解器靶向部分包含一个或多个葡萄糖。
  53. 根据权利要求48-52中任一项所述的缀合物，所述蛋白降解器靶向部分包含所述去唾液酸糖蛋白受体的约1个至约500个配体。
  54. 根据权利要求48-53中任一项所述的缀合物，所述蛋白降解器靶向部分包含支架分子，所述支架分子直接或间接连接所述去唾液酸糖蛋白受体的一个或多个配体。
  55. 根据权利要求50-54中任一项所述的缀合物，所述蛋白降解器靶向部分包含一个或多个所述N-乙酰半乳糖胺修饰的一个或多个丙氨酸。
  56. 根据权利要求54-55中任一项所述的缀合物，所述支架分子包含一个、两个或三个所述N-乙酰半乳糖胺。
  57. 根据权利要求54-56中任一项所述的缀合物，所述支架分子包含一个、两个或三个所述半乳糖。
  58. 根据权利要求1-57中任一项所述的缀合物，所述缀合物包含融合蛋白。
  59. 根据权利要求35-58中任一项所述的缀合物，所述缀合物包含所述靶分子靶向部分、所述蛋白降解器靶向部分和所述穿膜部分，所述靶分子靶向部分、所述蛋白降解器靶向部分与所述穿膜部分之间相互独立地直接或间接连接。
  60. 根据权利要求59所述的缀合物，所述间接连接包含通过连接子连接。
  61. 根据权利要求60所述的缀合物，所述连接子包含肽连接子。
  62. 根据权利要求60-61中任一项所述的缀合物，所述连接子包含可酶切和/或不可酶切的连接子。
  63. 根据权利要求62所述的缀合物，所述可酶切的连接子包含可通过蛋白酶酶切的连接子。
  64. 根据权利要求36-63中任一项所述的缀合物，所述缀合物包含所述靶分子靶向部分、所述穿膜肽和所述溶酶体靶向肽。
  65. 一种多肽，其包含靶分子靶向部分，所述靶分子靶向部分能够特异性识别靶分子并与该靶分子共价结合。
  66. 根据权利要求65所述的多肽，所述靶分子靶向部分包含第一官能团，所述靶分子包含第二官能团，当所述靶分子靶向部分与所述靶分子接近时，所述第一官能团能够与所述第二官能团反应而形成共价键。
  67. 根据权利要求66所述的多肽，在所述第一官能团与所述第二官能团之间的距离小于约5E-10米时，所述第一官能团能够与所述第二官能团反应而形成共价键。
  68. 根据权利要求66-67中任一项所述的多肽，所述第二官能团包含亲电基团。
  69. 根据权利要求66-68中任一项所述的多肽，所述第二官能团包含天然氨基酸的侧链基团。
  70. 根据权利要求66-69中任一项所述的多肽，所述第二官能团选自以下组：氨基、咪唑基、巯基、羟基、羧基、甲硫基，以及前述的衍生物。
  71. 根据权利要求65-70中任一项所述的多肽，所述靶分子包含细胞表面的分子、细胞内的分子和/或细胞外的分子。
  72. 根据权利要求65-71中任一项所述的多肽，所述靶分子选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
  73. 根据权利要求65-72中任一项所述的多肽，所述靶分子选自以下组：免疫检查点分子、肿瘤相关分子和信号通路蛋白。
  74. 根据权利要求65-73中任一项所述的多肽，所述靶分子选自以下组：PD-L1(细胞程序死亡-配体1)、EGFR(表皮生长因子受体)、HER-2(人类表皮生长因子受体-2)、Ras蛋白、
    CD38(淋巴细胞分化抗原CD38)，以及前述的功能性片段。
  75. 根据权利要求66-74中任一项所述的多肽，所述第一官能团选自以下组：
    Figure PCTCN2022080594-appb-100006
    以及前述的衍生物，其中，R 1为O、N或不存在，R 2为卤素、烯基或炔基，R 3为卤素、烯基或炔基，R 4为卤素，n1和n2各自独立地至少为0。
  76. 根据权利要求66-75中任一项所述的多肽，所述第一官能团选自以下组：
    Figure PCTCN2022080594-appb-100007
    以及前述的衍生物。
  77. 根据权利要求66-76中任一项所述的多肽，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：
    Figure PCTCN2022080594-appb-100008
    Figure PCTCN2022080594-appb-100009
    Figure PCTCN2022080594-appb-100010
    以及前述的衍生物。
  78. 根据权利要求66-77中任一项所述的多肽，所述靶分子靶向部分包含非天然氨基酸的残基，所述非天然氨基酸包含所述第一官能团，所述非天然氨基酸选自以下组：氟代硫酸盐-L-酪氨酸、6-溴-己酰赖氨酸，以及前述的衍生物。
  79. 根据权利要求65-78中任一项所述的多肽，所述靶分子靶向部分选自以下组：多肽、核酸、小分子、多糖、脂质、纳米颗粒，以及前述的任意组合。
  80. 根据权利要求65-79中任一项所述的多肽，所述靶分子靶向部分包含抗体或其抗原结合片段。
  81. 根据权利要求80所述的多肽，所述抗体选自以下组：鼠源抗体、骆驼源抗体、嵌合抗体、人源化抗体和全人源抗体。
  82. 根据权利要求80-81中任一项所述的多肽，所述抗原结合片段选自以下组：VHH，Fab，Fab′，Fv片段，(Fab') 2，(Fab) 2，scFv，di-scFv，亲和抗体(affibody)和dAb。
  83. 根据权利要求65-82中任一项所述的多肽，所述靶分子靶向部分包含单域抗体或其抗原结合片段。
  84. 根据权利要求66-83中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，在第108位氨基酸、第110位氨基酸和/或第113位氨基酸包含所述第一官能团。
  85. 根据权利要求74-84中任一项所述的多肽，所述靶分子靶向部分能够特异性识别所述PD-L1。
  86. 根据权利要求77-85中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸、第110位氨基酸和/或第113位氨基酸被所述非天然氨基酸替换。
  87. 根据权利要求78-86中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：13所示的相应氨基酸序列相比，所述靶分子靶向部分的第108位氨基酸被所述6-溴-己酰赖氨酸替换、第110位氨基酸被所述6-溴-己酰赖氨酸替换、第113位氨基酸被所述氟代硫酸盐-L-酪氨酸替换和/或第108位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
  88. 根据权利要求65-87中任一项所述的多肽，所述靶分子靶向部分包含SEQ ID NO：9-11中任一项所示的氨基酸序列。
  89. 根据权利要求66-88中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，在第116位氨基酸包含所述第一官能团。
  90. 根据权利要求74-89中任一项所述的多肽，所述靶分子靶向部分能够特异性识别所述EGFR。
  91. 根据权利要求77-90中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述非天然氨基酸替换。
  92. 根据权利要求78-91中任一项所述的多肽，所述靶分子靶向部分包含的氨基酸序列与SEQ ID NO：18所示的相应氨基酸序列相比，所述靶分子靶向部分的第116位氨基酸被所述氟代硫酸盐-L-酪氨酸替换。
  93. 根据权利要求65-92中任一项所述的多肽，所述靶分子靶向部分包含SEQ ID NO：12所示的氨基酸序列。
  94. 一种核酸分子，其编码权利要求1-64中任一项所述的缀合物和/或权利要求65-93中任一项所述的多肽。
  95. 一种载体，其包含权利要求94所述的核酸分子。
  96. 一种免疫缀合物，其包含权利要求1-64中任一项所述的缀合物和/或权利要求65-93中任一项所述的多肽。
  97. 一种细胞，其包含权利要求1-64中任一项所述的缀合物、权利要求94所述的核酸分子、权利要求95所述的载体和/或权利要求96所述的免疫缀合物。
  98. 一种组合物，其包含权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所述的多肽、权利要求94所述的核酸分子、权利要求95所述的载体、权利要求96所述的免疫缀合物和/或权利要求97所述的细胞，以及任选地药学上可接受的载剂。
  99. 一种试剂盒，其包含权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所 述的多肽、权利要求94所述的核酸分子、权利要求95所述的载体、权利要求96所述的免疫缀合物、权利要求97所述的细胞、和/或权利要求98所述的组合物。
  100. 权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所述的多肽、权利要求94所述的核酸分子、权利要求95所述的载体、权利要求96所述的免疫缀合物、权利要求97所述的细胞、权利要求98所述的组合物、和/或权利要求99所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  101. 根据权利要求100所述的用途，其中所述疾病和/或病症包含肿瘤。
  102. 根据权利要求100-101中任一项所述的用途，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
  103. 根据权利要求100-102中任一项所述的用途，其中所述疾病和/或病症包含实体瘤。
  104. 根据权利要求100-103中任一项所述的用途，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
  105. 权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所述的多肽、权利要求94所述的核酸分子、权利要求95所述的载体、权利要求96所述的免疫缀合物、权利要求97所述的细胞、权利要求98所述的组合物、和/或权利要求99所述的试剂盒，其用于预防和/或治疗疾病和/或病症。
  106. 用于根据权利要求105所述的用途的缀合物、多肽、核酸分子、载体、免疫缀合物、细胞、组合物、和/或试剂盒，其中所述疾病和/或病症包含肿瘤。
  107. 用于根据权利要求105-106中任一项所述的用途的缀合物、多肽、核酸分子、载体、免疫缀合物、细胞、组合物、和/或试剂盒，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
  108. 用于根据权利要求105-107中任一项所述的用途的缀合物、多肽、核酸分子、载体、免疫缀合物、细胞、组合物、和/或试剂盒，其中所述疾病和/或病症包含实体瘤。
  109. 用于根据权利要求105-108中任一项所述的用途的缀合物、多肽、核酸分子、载体、免疫缀合物、细胞、组合物、和/或试剂盒，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
  110. 一种预防和/或治疗疾病和/或病症的方法，其包含向有需要的对象施用权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所述的多肽、权利要求94所述的核酸 分子、权利要求95所述的载体、权利要求96所述的免疫缀合物、权利要求97所述的细胞、权利要求98所述的组合物、和/或权利要求99所述的试剂盒。
  111. 根据权利要求110所述的方法，其中所述疾病和/或病症包含肿瘤。
  112. 根据权利要求110-111中任一项所述的方法，其中所述疾病和/或病症选自以下组：PD-L1阳性肿瘤、EGFR阳性肿瘤、HER-2阳性肿瘤、CD38阳性肿瘤和Ras功能和/或活性异常的肿瘤。
  113. 根据权利要求110-112中任一项所述的方法，其中所述疾病和/或病症包含实体瘤和/或血液瘤。
  114. 根据权利要求110-113中任一项所述的方法，其中所述疾病和/或病症包含乳腺癌、肺癌、皮肤癌、骨髓瘤和/或宫颈癌。
  115. 一种影响靶分子含量的方法，施用权利要求1-64中任一项所述的缀合物、权利要求65-93中任一项所述的多肽、权利要求94所述的核酸分子、权利要求95所述的载体、权利要求96所述的免疫缀合物、权利要求97所述的细胞、权利要求98所述的组合物和/或权利要求99所述的试剂盒。
  116. 根据权利要求115所述的方法，所述方法包含体外方法、离体方法和/或体内方法。
  117. 根据权利要求115-116中任一项所述的方法，所述靶分子为细胞表面的分子和/或细胞内的分子。
  118. 根据权利要求115-117中任一项所述的方法，所述靶分子为皮肤细胞、骨髓细胞、宫颈细胞、肺细胞和/或乳腺细胞表面的分子。
  119. 根据权利要求115-118中任一项所述的方法，所述靶分子为皮肤癌细胞、骨髓瘤细胞、宫颈癌细胞、肺癌细胞和/或乳腺癌细胞表面的分子。
  120. 根据权利要求115-119中任一项所述的方法，所述细胞选自以下组：PD-L1阳性细胞、EGFR阳性细胞、HER-2阳性细胞、CD38阳性细胞和Ras功能和/或活性异常的细胞。
  121. 一种制备权利要求1-64中任一项所述缀合物和/或权利要求65-93中任一项所述多肽的方法，包含在使得所述缀合物和/或所述多肽表达的条件下，培养权利要求97所述的细胞。
  122. 根据权利要求121所述的方法，所述缀合物和/或所述多肽包含靶分子靶向部分与蛋白降解器靶向部分。
  123. 根据权利要求122所述的方法，通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
  124. 根据权利要求122-123中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
  125. 根据权利要求122-124中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列。
  126. 根据权利要求122-125中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
  127. 根据权利要求122-126中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列。
  128. 根据权利要求123-127中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
  129. 根据权利要求123-128中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
  130. 根据权利要求123-129中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
  131. 根据权利要求123-130中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
  132. 一种制备权利要求1-64中任一项所述缀合物的方法，包含将所述缀合物的所述靶分子靶向部分与所述蛋白降解器靶向部分直接或间接连接。
  133. 根据权利要求132所述的方法，通过转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
  134. 根据权利要求132-133中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
  135. 根据权利要求132-134中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述靶分子靶向部分的N末端包含寡聚甘氨酸序列。
  136. 根据权利要求132-135中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，其中X为任意氨基酸。
  137. 根据权利要求132-136中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG和/或所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列。
  138. 根据权利要求133-137中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
  139. 根据权利要求133-138中任一项所述的方法，所述靶分子靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述蛋白降解器靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
  140. 根据权利要求133-139中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：93所示的氨基酸序列LPXTG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分，其中X为任意氨基酸。
  141. 根据权利要求133-140中任一项所述的方法，所述蛋白降解器靶向部分的C末端包含如SEQ ID NO：92所示的氨基酸序列LPETG，所述靶分子靶向部分的N末端包含寡聚甘氨酸序列，通过所述转肽酶A(Srt A)连接所述靶分子靶向部分与所述蛋白降解器靶向部分。
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