WO2020216194A1 - 一种用于肿瘤免疫治疗的多肽组合及其制备方法 - Google Patents

一种用于肿瘤免疫治疗的多肽组合及其制备方法 Download PDF

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WO2020216194A1
WO2020216194A1 PCT/CN2020/085736 CN2020085736W WO2020216194A1 WO 2020216194 A1 WO2020216194 A1 WO 2020216194A1 CN 2020085736 W CN2020085736 W CN 2020085736W WO 2020216194 A1 WO2020216194 A1 WO 2020216194A1
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Prior art keywords
antigen
polypeptide
toxin
terminus
intein
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PCT/CN2020/085736
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English (en)
French (fr)
Inventor
朱建伟
王静
陈俊升
谢跃庆
江华
宗会芳
韩雷
Original Assignee
上海交通大学
美国杰科实验室有限公司
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Application filed by 上海交通大学, 美国杰科实验室有限公司 filed Critical 上海交通大学
Priority to CN202080030426.5A priority Critical patent/CN113825773B/zh
Priority to EP20796067.5A priority patent/EP3960770A4/en
Priority to US17/605,472 priority patent/US20220325286A1/en
Publication of WO2020216194A1 publication Critical patent/WO2020216194A1/zh

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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/55—Fab or Fab'
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/385—Pseudomonas aeruginosa

Definitions

  • This application relates to the field of biomedicine, in particular to a polypeptide combination for tumor immunotherapy and a preparation method thereof.
  • Recombinant immunotoxin refers to a therapeutic chimeric protein composed of a cell targeting part and a toxin part. It has the function of specifically targeting and killing diseased cells. It can be prepared by chemical coupling or genetic recombination. The recombinant immunotoxin must bind to the target cell and be taken up by the target cell to function, and the enzymatically active fragment of the toxin must be transferred to the cytoplasm.
  • the shielding peptide can shield the antigen binding site of the antibody or its antigen-binding fragment, and reduce the non-specific binding of the antibody or its antigen-binding fragment during the circulation process.
  • the protease rich in the tumor microenvironment selectively cleaves the connecting peptide, removes the shielding peptide connected to the antibody or its antigen-binding fragment through the connecting peptide, and restores the antibody or
  • the antigen-binding ability of its antigen-binding fragments can selectively kill tumor cells and improve the specificity of tumor killing.
  • Trans-splicing can be a protein splicing reaction mediated by a fragmented intein.
  • the N-terminal fragment and the C-terminal fragment of the broken protein intein recognize each other and bind with non-covalent bonds. After the two are combined, their structure will be folded correctly, and the protein exon peptides on both sides of the broken protein intein will be connected .
  • This application provides a polypeptide combination for tumor immunotherapy and a preparation method thereof.
  • the polypeptide combination and its preparation method have one or more of the following properties: 1) improve the safety of medication and reduce non-specific toxicity to normal cells; 2) when located in normal tissues, the combination of polypeptides can form immunity The conjugate cannot bind to cells of normal tissues and exerts biological toxicity; and when located in the tumor microenvironment, the immunoconjugate formed by the combination of polypeptides specifically binds to tumor-specific antigens on the surface of tumor cells to kill tumor cells.
  • the preparation method has universal applicability and can be applied to various types of immunotoxins or protein drugs; 6) It can be used in eukaryotic cells separately It can be produced in expression system and prokaryotic cell expression system to achieve industrialized high yield.
  • the present application provides a polypeptide combination comprising a targeting component and a toxin component, wherein: the targeting component comprises a shielding peptide, a cleavable portion, an antigen binding portion and a first intein fragment , The shielding peptide and the antigen binding portion are connected through the cleavable portion, and the antigen binding portion is directly or indirectly connected to the first intein fragment; the toxin component includes a second intein Peptide fragments and toxins, and the second intein fragment is directly or indirectly connected to the toxin; the targeting component and the toxin component can be connected to the second intein fragment via the first intein fragment The interaction between the intein fragments forms an immunoconjugate; the immunoconjugate comprises the shielding peptide, the antigen-binding portion and the toxin, wherein the shielding peptide and the antigen-binding portion pass through the The cleavable portion is connected, and the antigen-binding portion is directly
  • the targeting component includes a first polypeptide chain and a second polypeptide chain; the first polypeptide chain includes the shielding peptide and a first antigen-binding unit, and the shielding peptide and The first antigen-binding unit is connected by the cleavable portion; the second polypeptide chain includes a second antigen-binding unit and the first intein fragment, and the second antigen-binding unit is connected to the first An intein fragment is directly or indirectly connected; and the first antigen binding unit and the second antigen binding unit can bind to each other to form the antigen binding portion.
  • the second polypeptide chain and the toxin component may be formed by the interaction between the first intein fragment and the second intein fragment.
  • the immunoconjugate does not include the first intein fragment and does not include the second intein fragment.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the antigen binding portion.
  • the C-terminus of the antigen-binding portion is directly or indirectly connected to the N-terminus of the first intein fragment.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the first antigen binding unit.
  • the C-terminus of the second antigen binding unit is directly or indirectly connected to the N-terminus of the first intein fragment.
  • the C-terminus of the second intein is directly or indirectly connected to the N-terminus of the toxin.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the antigen-binding portion.
  • the C-terminus of the antigen-binding portion is directly or indirectly connected to the N-terminus of the toxin.
  • the first polypeptide chain and the fusion polypeptide constitute the first antigen-binding unit and the second antigen-binding unit through mutual binding.
  • Immunoconjugates in the immunoconjugate, the first polypeptide chain and the fusion polypeptide constitute the first antigen-binding unit and the second antigen-binding unit through mutual binding.
  • the C-terminus of the second antigen binding unit is directly or indirectly connected to the N-terminus of the toxin.
  • the indirect connection comprises connection through a linker.
  • the linker comprises a peptide linker
  • the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the shielding peptide is selected from the following group: HER2 antigen shielding peptide, EGFR antigen shielding peptide, EpCAM antigen shielding peptide and Mesothelin antigen shielding peptide.
  • the shielding peptide comprises the amino acid sequence shown in SEQ ID NO:1.
  • the cleavable portion is selected from the following group: matrix metalloproteinase-sensitive linker, serine protease-sensitive linker, and urokinase plasminogen activator-sensitive linker. In some embodiments, the cleavable portion comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the targeting component targets tumor-specific antigens.
  • the tumor-specific antigen comprises HER2.
  • the antigen-binding portion comprises an antibody or antigen-binding fragment thereof.
  • the first antigen-binding unit and the second antigen-binding unit are derived from the same antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • the antigen binding fragment is Fab.
  • the first antigen binding unit comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the second antigen binding unit comprises the amino acid sequence shown in SEQ ID NO: 5.
  • the toxin is selected from the group consisting of bacterial toxins, human toxins, and phytotoxins. In certain embodiments, the toxin is selected from the group consisting of Pseudomonas aeruginosa exotoxin and diphtheria toxin. In certain embodiments, the toxin is selected from the group consisting of ricin, saponin, and gelonin. In some embodiments, the toxin comprises the amino acid sequence shown in SEQ ID NO:9.
  • the first intein fragment and/or the second intein fragment comprises a fragmented intein.
  • the fragmented intein is selected from the following group: SsP DnaB, Ssp DnaE, and Npu DnaE.
  • the first intein fragment is different from the second intein fragment.
  • the first intein fragment and the second intein fragment are derived from the same intein.
  • the first intein fragment comprises the amino acid sequence shown in SEQ ID NO:6.
  • the second intein fragment comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:7.
  • the toxin component comprises the amino acid sequence shown in SEQ ID NO:10.
  • the immunoconjugate comprises the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 12.
  • the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12.
  • the present application provides a method for preparing immunoconjugates, which includes the following steps: 1) providing a targeting component, the targeting component comprising a shielding peptide, a cleavable portion, an antigen binding portion and The first intein fragment, the shielding peptide and the antigen binding portion are connected by the cleavable portion, and the antigen binding portion is directly or indirectly connected to the first intein fragment; 2) providing toxin Component, the toxin component comprises a second intein fragment and a toxin, and the second intein fragment is directly or indirectly connected to the toxin; 3) the targeting component is combined with the toxin Contacting the first intein fragment and the second intein fragment under conditions that enable the interaction of the targeting component and the toxin component through the first intein fragment The interaction with the second intein fragment forms the immunoconjugate.
  • the providing of the targeting component includes: providing a first polypeptide chain, the first polypeptide chain comprising the shielding peptide and the first antigen binding unit, the shielding peptide and the first polypeptide chain An antigen binding unit is connected by the cleavable portion; a second polypeptide chain is provided, the second polypeptide chain includes a second antigen binding unit and the first intein fragment, and the second antigen binding unit Directly or indirectly connected to the first intein fragment; and combining the first polypeptide chain with the second polypeptide chain to form the targeting component, wherein all of the first polypeptide chain The first antigen binding unit and the second antigen binding unit of the second polypeptide chain are combined with each other to form the antigen binding portion in the targeting component.
  • wherein 3) includes allowing the second polypeptide chain and the toxin component to form an interaction between the first intein fragment and the second intein fragment.
  • a fusion polypeptide of the second antigen binding unit and the toxin, and the fusion polypeptide and the first polypeptide chain constitute the immunoconjugate.
  • the immunoconjugate does not include the first intein fragment and does not include the second intein fragment.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the antigen binding portion.
  • the C-terminus of the antigen-binding portion is directly or indirectly connected to the N-terminus of the first intein fragment.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the first antigen binding unit.
  • the C-terminus of the second antigen binding unit is directly or indirectly connected to the N-terminus of the first intein fragment.
  • the C-terminus of the second intein is directly or indirectly connected to the N-terminus of the toxin.
  • the C-terminus of the shielding peptide is directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion is directly or indirectly connected to the N-terminus of the antigen-binding portion.
  • the C-terminus of the antigen-binding portion is directly or indirectly connected to the N-terminus of the toxin.
  • the first polypeptide chain and the fusion polypeptide constitute the first antigen-binding unit and the second antigen-binding unit through mutual binding.
  • Immunoconjugates in the immunoconjugate, the first polypeptide chain and the fusion polypeptide constitute the first antigen-binding unit and the second antigen-binding unit through mutual binding.
  • the C-terminus of the second antigen binding unit is directly or indirectly connected to the N-terminus of the toxin.
  • the indirect connection comprises connection through a linker.
  • the linker comprises a peptide linker, and the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the shielding peptide is selected from the following group: HER2 antigen shielding peptide, EGFR antigen shielding peptide, EpCAM antigen shielding peptide and Mesothelin antigen shielding peptide.
  • the shielding peptide comprises the amino acid sequence shown in SEQ ID NO:1.
  • the cleavable portion is selected from the following group: matrix metalloproteinase-sensitive linker, serine protease-sensitive linker, and urokinase plasminogen activator-sensitive linker.
  • the cleavable portion comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the targeting component targets tumor-specific antigens.
  • the tumor-specific antigen comprises HER2.
  • the antigen-binding portion comprises an antibody or antigen-binding fragment thereof.
  • the first antigen-binding unit and the second antigen-binding unit are derived from the same antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • the antigen binding fragment is Fab.
  • the first antigen binding unit comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the second antigen binding unit comprises the amino acid sequence shown in SEQ ID NO: 5.
  • the toxin is selected from the group consisting of bacterial toxins, human toxins, and phytotoxins. In certain embodiments, the toxin is selected from the group consisting of Pseudomonas aeruginosa exotoxin and diphtheria toxin. In certain embodiments, the toxin is selected from the group consisting of ricin, saponin, and gelonin. In some embodiments, the toxin comprises the amino acid sequence shown in SEQ ID NO:9.
  • the first intein fragment and/or the second intein fragment comprises a fragmented intein.
  • the fragmented intein is selected from the following group: SsP DnaB, Ssp DnaE, and Npu DnaE.
  • the first intein fragment is different from the second intein fragment. In some embodiments, the first intein fragment and the second intein fragment are derived from the same intein.
  • the first intein fragment comprises the amino acid sequence shown in SEQ ID NO:6.
  • the second intein fragment comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:7.
  • the toxin component comprises the amino acid sequence shown in SEQ ID NO:10.
  • the immunoconjugate comprises the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 12.
  • the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12.
  • the interaction between the first intein fragment and the second intein fragment comprises trans-splicing.
  • a reducing agent is used to initiate the interaction between the first intein fragment and the second intein fragment.
  • the reducing agent is selected from the group consisting of DTT and ⁇ -mercaptoethanol.
  • the concentration of the reducing agent is 0.1-100 nM.
  • the reducing agent is in contact with the targeting component and the toxin component.
  • the method of preparing an immunoconjugate further comprises incubating after adding the reducing agent.
  • the temperature of the incubation is 1-50°C.
  • the incubation time is 60-300 minutes.
  • the application provides a vector comprising a nucleic acid encoding the first polypeptide chain in the polypeptide combination, a nucleic acid encoding the second polypeptide chain in the polypeptide combination, and a nucleic acid encoding the polypeptide combination
  • the nucleic acid of the second antigen-binding unit, the nucleic acid encoding the first intein fragment in the polypeptide combination, the nucleic acid that encodes the second intein fragment in the polypeptide combination, and the nucleic acid encoding the polypeptide combination The nucleic acid of the toxin described in and/or the nucleic acid encoding the shielding peptide described in the polypeptide combination.
  • This application provides a cell expressing the vector.
  • the application provides the use of the polypeptide combination, the carrier or the cell in the preparation of a medicine for treating diseases, and the diseases include tumors.
  • the tumor includes ovarian cancer and breast cancer.
  • the application provides the polypeptide combination, the vector or the cell, which treats tumors.
  • This application provides a method for treating tumors, which comprises administering the polypeptide combination, the vector or the cell.
  • FIG 1 shows the results of SDS-PAGE detection of the targeting components and toxin components described in this application.
  • FIG. 2 shows the results of SDS-PAGE detection of the immunoconjugates described in this application.
  • FIG. 3 shows the results of SDS-PAGE detection of the immunoconjugates described in the present application activated after protease treatment.
  • Figure 4 shows the results of the binding affinity analysis of the immunoconjugate described in this application to tumor cells expressing the HER2/erbB2/neu antigen.
  • Figure 5 shows the results of the cytotoxicity analysis of the immunoconjugates described in this application on tumor cells expressing HER2/erbB2/neu antigen.
  • Figure 6 shows the results of the in vivo pharmacodynamic study of the immunoconjugates described in this application.
  • Figure 7 shows the tumor volume in SKOV-3 tumor-bearing mice after administration of the immunoconjugate described in this application.
  • Figure 8 shows the results of the injury test of the immunoconjugate described in this application on mouse liver tissue.
  • polypeptide combination generally refers to a collection of two or more proteins or polypeptides.
  • the polypeptide combination may be a composition comprising two or more proteins or polypeptides.
  • different proteins or polypeptides can be located in different containers or physical spaces, respectively.
  • two or more different proteins or polypeptides can also be located in the same container or in the same physical space.
  • the polypeptide combination may not be a protein molecule or a protein complex.
  • none of the two or more proteins or polypeptides are located in the same protein molecule or the same protein complex.
  • the term "targeting component” generally refers to a protein or polypeptide comprising a shielding peptide, a cleavable portion, an antigen binding portion, and a first intein fragment.
  • the targeting component can target tumor cells.
  • the antigen binding portion can specifically bind to tumor cells.
  • the shielding peptide and the antigen-binding portion may be connected through the cleavable portion, and the antigen-binding portion may be directly or indirectly connected to the first intein fragment.
  • the term "shielding peptide” generally refers to a polypeptide or a fragment thereof that is attached to the antigen-binding portion and prevents the antigen-binding portion from binding to the antigen.
  • the shielding peptide may be located at the N-terminus of the antigen binding portion.
  • the shielding peptide may be connected to the antigen binding portion through a cleavable portion. In this application, when the shielding peptide is located in the environment of normal tissues, the shielding peptide cannot be separated from the antigen-binding portion connected to it, so that the antigen-binding portion cannot bind to the antigen.
  • the shielding peptide When the shielding peptide is located in the tumor environment, because the tumor cells release a specific protease, the shielding peptide can be separated from the antibody binding portion connected to it (for example, the cleavable portion can be cut off) , So that the antigen binding part can bind to the antigen of tumor cells.
  • the shielding peptide may be a shielding peptide used in Probody technology (see Polu KR, Lowman HB. Probody therapeutics for targeting antibodies to diseased tissue. Expert Opinion on Biological Therapy. 2014; 14:1049-1053) .
  • the term "cleavable part” generally refers to a polypeptide or fragment thereof that has cleavable properties and connects the shielding peptide and the antigen binding part.
  • the cleavable part can be cleaved by the action of protease.
  • the cleavable portion can be cleaved by proteases in the tumor environment (for example, proteases enriched around tumor cells), thereby separating the shielding peptide from the antigen binding portion.
  • the isolated antigen-binding portion can be bound to a tumor-specific antigen.
  • antigen-binding portion generally refers to an antibody or antigen-binding fragment thereof that can bind to a tumor-specific antigen.
  • tumor-specific antigen generally refers to an antigen that is specifically expressed on the surface of one or more tumor cells and is almost absent on normal cells.
  • HER2 generally refers to human epidermal growth factor receptor 2, and can also be referred to as Neu, ErbB-2 or CD340.
  • the HER2 may be a protein encoded by the ERBB2 gene, which belongs to a member of the epidermal growth factor receptor (EGFR/ErbB) family.
  • Recombinant humanized trastuzumab Trastuzumab It can specifically bind to the extracellular fourth subdomain of HER2.
  • the HER2 may belong to the tumor-specific antigen, for example, it may be a target of breast cancer or ovarian cancer.
  • the accession number of human HER2 in GenBank can be NP_001276866.1.
  • the term "antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein, which is composed of two identical light chains and two identical heavy chains.
  • each L chain is connected to the H chain through a covalent disulfide bond, while the two H chains are connected to each other through one or more disulfide bonds.
  • the number of disulfide bonds depends on the same species of the H chain. type.
  • Each H and L chain also has regularly spaced intrachain disulfide bonds.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three (for each ⁇ and ⁇ chain) or four (for ⁇ and ⁇ isotypes) constant domain (CH).
  • the term "antigen-binding fragment” generally refers to a part of a complete antibody, for example, the antigen-binding fragment may be the antigen-binding region and/or antibody variable region of a complete antibody.
  • the antigen-binding fragment can be obtained by chemical methods and/or genetic engineering methods. For example, proteases, including pepsin and papain, can be used to digest antibodies to produce antigen-binding fragments.
  • Fab generally refers to the production of two identical antigen-binding fragments after papain digests an antibody with a complete structure (for example, the Fc region and the hinge region are removed).
  • the Fab can be composed of a complete light chain, the variable region of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain.
  • VH variable region of the heavy chain
  • CH1 first constant domain
  • Fab' generally refers to an antigen-binding fragment with several additional residues at the carboxyl terminal of the CH1 domain compared to the Fab.
  • Fab' includes one or more residues from the hinge region of an antibody. Cysteine.
  • F(ab) 2 generally refers to an antigen-binding fragment obtained from a pair of Fabs linked by cysteine.
  • Fv generally refers to an antigen-binding fragment composed of the VL and VH domains of a single arm of an antibody.
  • single chain antibody generally refers to a molecule formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide linker.
  • the term "intein” generally refers to an inserted sequence located in the host protein.
  • the intein needs to be inserted into the exonin gene to replicate and transcribe, and can be excised from the precursor protein and connect the exonin on both sides to form a mature protein.
  • the nucleotide sequence corresponding to the intein can be chimeric in the nucleic acid sequence corresponding to the host protein, exist in the same open reading frame as the host protein gene, and perform simultaneous transcription and translation with the host protein gene.
  • the intein is excised from the host protein to form a mature active protein.
  • it can be divided into whole intein and fragmented intein.
  • the two splicing regions of the whole intein may co-exist in the same polypeptide fragment; and the two splicing regions of the fragmented intein may be split into two or more fragments and exist in different polypeptide fragments.
  • fragmented intein generally refers to an intein in which two cleavage regions can be located in more than two polypeptide fragments.
  • the fragmented intein can include an N-terminal protein splicing region (N fragment of intein, In) and a C-terminal protein splicing region (C fragment of intein, Ic).
  • N-terminus of the N-terminal protein splicing region can be connected to an extein (for example, N-extein); the C-terminus of the C-terminal protein splicing region can be connected to another extein (for example, C-extein) .
  • the extein N-extein and the extein C-extein may be located in two different open reading frames.
  • the N-terminal protein splicing region and the C-terminal protein splicing region belonging to the same fragmented intein the N-terminal protein splicing region and the C
  • the terminal protein cleavage region can be excised, and the extein N-extein and the extein C-extein can be connected to each other to form a connected protein or polypeptide.
  • first intein fragment usually refers to a partial fragment of intein, which may not include a complete intein fragment.
  • the first intein fragment can interact with other intein fragments, so that the protein or polypeptide fragment (such as exon) connected to the first intein fragment can interact with other proteins or polypeptides. Fragments (e.g. exopeptides) are linked to form linked proteins or polypeptides.
  • the first intein fragment can be linked to a second antigen binding unit.
  • the term "second intein fragment” generally refers to a partial fragment of the intein, which may not include a complete intein fragment.
  • the second intein fragment may be different from the first intein fragment.
  • the first intein fragment may be derived from the same intein as the second intein fragment.
  • the second intein fragment can interact with the first intein fragment.
  • the second intein fragment can interact with other intein fragments, so that the protein or polypeptide fragment (for example, exo peptide, for example, toxin) connected to the second intein fragment and other Protein or polypeptide fragments (for example, exo peptide) are connected to form a connected protein or polypeptide.
  • the second intein fragment can be linked to a toxin.
  • the first intein fragment can interact with the second intein fragment so that the second antigen binding unit and the toxin form a fusion polypeptide.
  • the term "toxin” generally refers to a poison produced by an organism.
  • the toxins are usually proteins that interfere with the action of other macromolecules in the organism, and the toxins can usually function by inhibiting protein synthesis through enzymatic hydrolysis.
  • Toxins can include bacterial toxins, plant toxins and human toxins according to their sources.
  • the bacterial toxins may include Pseudomonas aeruginosa exotoxin (PE) and diphtheria toxin (DT).
  • the plant toxins may include ricin, saponin and gelonin.
  • toxins can include the following functional areas: cell binding area, translocation area and toxic activity area.
  • the cell binding zone can enrich toxins on the surface of target cells.
  • PE24 generally refers to the fragment of Pseudomonas aeruginosa exotoxin (PE) after truncating the cell binding region.
  • the complete PE contains three functional areas, namely the cell binding area (Ia area, 1-252aa), the translocation area (II area, 253-364aa) and the toxic activity area (III area, 400-613aa).
  • the PE truncated body PE24 cuts off the cell binding region (Ia region, 1-252aa), and further shortens it to remove the T/B cell epitope, which greatly reduces the immunogenicity of the toxin (see Mazor R, Onda M,Park D,et al.Dual B-and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity.Oncotarget.2016; 7:29916-29926.).
  • the amino acid sequence of PE24 may be as shown in SEQ ID NO.9.
  • the term "toxin component” generally refers to a protein or polypeptide comprising the second intein fragment and the toxin.
  • the toxin component may have biological toxicity, and the biological toxicity may be produced by the toxin.
  • the second intein fragment in the toxin component can interact with the first intein fragment in the targeting component to produce the shielding peptide, The immunoconjugate of the cleavable portion, the antigen binding portion and the toxin.
  • the term "interaction” generally refers to the interaction between the first intein fragment and the second intein fragment.
  • the protein or polypeptide multi-linked by the first intein fragment can be linked to the protein or polypeptide linked to the second intein fragment to form a connected protein Or polypeptide.
  • the interaction may be splicing.
  • the interaction can be protein trans-splicing.
  • the first intein fragment and the second intein fragment may undergo protein trans-splicing to make the second antigen binding unit and the toxin form the fusion polypeptide.
  • protein trans-splicing refers to a protein splicing reaction mediated by a fragmented protein intein.
  • the N-terminal fragment (In) of the fragmented intein (In) and the C-terminal fragment (Ic) of the fragmented intein recognize each other and are bound by a non-covalent bond. After the two are combined, the structure is correctly folded, and It is removed by trans-cutting, and the N-terminal fragment of the fragmented intein and the exon on both sides of the C-terminal fragment of the fragmented intein are connected to obtain the exon formed by both sides protein.
  • first polypeptide chain generally refers to a single polypeptide chain comprising the shielding peptide and the first antigen binding unit.
  • the shielding peptide and the first antigen binding unit may be connected through the cleavable portion.
  • first antigen-binding unit generally refers to a part of the antigen-binding portion.
  • the first antigen binding unit has the ability to specifically bind to the tumor-specific antigen.
  • the first antigen-binding unit may be an antibody part or an antigen-binding fragment thereof that specifically binds to the tumor-specific antigen.
  • the first antigen binding unit may be a light chain (LC) of an antibody that specifically binds to the tumor-specific antigen.
  • second polypeptide chain generally refers to a single polypeptide chain comprising a second antigen binding unit and the first intein fragment.
  • the second antigen binding unit and the first intein fragment may be directly or indirectly connected.
  • the term "second antigen-binding unit” generally refers to a part of the antigen-binding portion.
  • the second antigen binding unit has the ability to specifically bind to the tumor-specific antigen.
  • the second antigen-binding unit may be an antibody part or an antigen-binding fragment thereof that specifically binds to the tumor-specific antigen.
  • the second antigen binding unit may be different from the first antigen binding unit.
  • the second antigen binding unit can be combined with the first antigen binding unit to form the antigen binding portion.
  • the second antigen binding unit may be the CH1 portion of the variable region and the constant region of the heavy chain of an antibody that specifically binds to the tumor-specific antigen.
  • the second antigen binding unit may be the variable region of the heavy chain of the antibody that specifically binds to the tumor-specific antigen, the CH1 portion of the constant region, and the hinge region.
  • the second antigen-binding unit can bind to the first antigen-binding unit to form a Fab that specifically binds to the tumor-specific antigen.
  • fusion polypeptide generally refers to a protein or polypeptide comprising the second antigen binding unit and the toxin.
  • the fusion polypeptide may be formed by the interaction of the first intein fragment in the second polypeptide chain and the second intein fragment in the toxin component.
  • the interaction may be protein trans-splicing of the first intein fragment and the second intein fragment.
  • the term "immunoconjugate” generally refers to a protein or polypeptide containing the shielding peptide, the antigen binding portion, and the toxin.
  • the shielding peptide and the antigen binding portion are connected via the cleavable portion, and the antigen binding portion is directly or indirectly connected to the toxin.
  • the indirect connection may be a connection through a linker.
  • the immunoconjugate may include the shielding peptide, the antigen-binding portion and the toxin in sequence from N-terminal to C-terminal.
  • the immunoconjugate may sequentially include the shielding peptide, the cleavable portion, the antigen binding portion and the toxin from N-terminal to C-terminal.
  • the immunoconjugate can have both biological toxicity and specific tumor-targeting properties.
  • linker generally refers to a molecule used to link two or more proteins or polypeptides.
  • the indirect connection between different proteins or polypeptides can be achieved through the linker.
  • the linker may be a peptide linker.
  • the second antigen binding unit may be connected to the toxin through the linker.
  • N-terminal generally refers to the end of a polypeptide chain, which may have an ⁇ -amino amino acid residue.
  • C-terminal generally refers to the end of a polypeptide chain, which may have an ⁇ -carboxyl amino acid residue.
  • N-terminus is the end of one end, and the C-terminus may be the end on the other side that is different from the N-terminus.
  • the term "reducing agent” generally refers to an agent capable of inducing the interaction between the first intein fragment and the second intein fragment.
  • the reducing agent can initiate protein trans-splicing between the first intein fragment and the second intein fragment.
  • the reducing agent may be a reagent that loses electrons in a redox reaction.
  • the reducing agent may be DTT (dithiothreitol, molecular formula is C 4 H 10 O 2 S 2 ).
  • 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%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • This application provides a polypeptide combination comprising a targeting component and a toxin component, wherein: the targeting component comprises a shielding peptide, a cleavable portion, an antigen binding portion and a first intein fragment, the shielding The peptide and the antigen-binding portion are connected through the cleavable portion, and the antigen-binding portion is directly or indirectly connected to the first intein fragment; the toxin component includes a second intein fragment and a toxin , And the second intein fragment is directly or indirectly connected to the toxin; the targeting component and the toxin component can pass through the first intein fragment and the second intein fragment Interaction between the two to form an immunoconjugate; the immunoconjugate comprises the shielding peptide, the antigen-binding portion and the toxin, wherein the shielding peptide and the antigen-binding portion are cleavable through the Part is connected, and the antigen binding part is directly or indirectly connected to the tox
  • This application also provides a method for preparing immunoconjugates, which includes the following steps: 1) providing a targeting component, the targeting component comprising a shielding peptide, a cleavable portion, an antigen binding portion and a first internal A peptide-containing fragment, the shielding peptide and the antigen binding portion are connected through the cleavable portion, and the antigen binding portion is directly or indirectly connected to the first intein fragment; 2) providing a toxin component, The toxin component includes a second intein fragment and a toxin, and the second intein fragment is directly or indirectly connected to the toxin; 3) making the targeting component and the toxin component in The first intein fragment and the second intein fragment are in contact with each other under conditions where the targeting component and the toxin component can interact with the toxin component through the first intein fragment and the The interaction between the second intein fragments forms the immunoconjugate.
  • the targeting component may include a shielding peptide, a cleavable portion, an antigen-binding portion, and a first intein fragment, wherein the shielding peptide can interact with the antigen-binding portion through the cleavable Part is connected, and the antigen binding part may be directly or indirectly connected to the first intein fragment.
  • the C-terminus of the shielding peptide can be directly or indirectly connected to the N-terminus of the cleavable portion.
  • the shielding peptide may be selected from the following group: HER2 antigen shielding peptide, EGFR antigen shielding peptide, EpCAM antigen shielding peptide and Mesothelin antigen shielding peptide.
  • the shielding peptide may include the amino acid sequence shown in SEQ ID NO:1.
  • the C-terminus of the cleavable portion can be directly or indirectly connected to the N-terminus of the antigen binding portion.
  • the cleavable part can be selected from the following groups: matrix metalloproteinase-sensitive linker, serine protease-sensitive linker, and urokinase plasminogen activator-sensitive linker.
  • the cleavable portion may include the amino acid sequence shown in SEQ ID NO: 2.
  • the C-terminus of the antigen-binding portion can be directly or indirectly connected to the N-terminus of the first intein fragment.
  • the first intein fragment may include the amino acid sequence shown in SEQ ID NO:6.
  • the targeting component can target tumor-specific antigens.
  • the tumor-specific antigen may comprise HER2.
  • the antigen-binding portion may comprise an antibody or an antigen-binding fragment thereof.
  • the antibody may be selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab') 2 , F(ab) 2 , dAb, isolated complementarity determining region CDR, Fv and scFv.
  • the antigen binding fragment may be Fab.
  • the targeting component may include a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may include the shielding peptide and the first antigen binding unit, the shielding peptide It can be connected to the first antigen binding unit through the cleavable portion.
  • the C-terminus of the shielding peptide can be directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion may be directly or indirectly connected to the N-terminus of the first antigen binding unit.
  • the first antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 3.
  • the first polypeptide chain of the present application from the N-terminus to the C-terminus, it may include the shielding peptide, the cleavable portion and the first antigen binding unit in sequence.
  • the first polypeptide chain may include the shielding peptide (which may include the amino acid sequence shown in SEQ ID NO:1), and the cleavable portion (which may include SEQ ID NO:1) in sequence from N-terminal to C-terminal.
  • the amino acid sequence shown in NO: 2 and the first antigen binding unit which may be the light chain of trastuzumab, which may include the amino acid sequence shown in SEQ ID NO: 3).
  • the first polypeptide chain may be PM-MLC, which may include the amino acid sequence shown in SEQ ID NO: 4.
  • the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO:4.
  • the second polypeptide chain may include a second antigen binding unit and the first intein fragment, and the second antigen binding unit may be directly or indirectly connected to the first intein fragment .
  • the C-terminus of the second antigen binding unit may be directly or indirectly connected to the N-terminus of the first intein fragment.
  • the second antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 5.
  • the second polypeptide chain of the present application may sequentially include the second antigen binding unit and the first intein fragment from N-terminus to C-terminus.
  • the second polypeptide chain may include the second antigen binding unit (which may be the variable region and CH1 of the heavy chain of trastuzumab from N-terminal to C-terminal, which may include SEQ ID NO: 5) and the first intein fragment (which may include the amino acid sequence shown in SEQ ID NO: 6).
  • the second polypeptide chain may be PM-HC-In, which may include the amino acid sequence shown in SEQ ID NO:7.
  • the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO:7.
  • the first antigen binding unit in the first polypeptide chain can be combined with the second antigen binding unit in the second polypeptide chain to form the antigen binding portion.
  • the first antigen-binding unit and the second antigen-binding unit may be derived from the same antibody or antigen-binding fragment thereof.
  • the first antigen binding unit and the second antigen binding unit may be derived from trastuzumab.
  • the first antigen binding unit may be the light chain of trastuzumab
  • the second antigen binding unit may be the variable region and the constant region CH1 region of the heavy chain of trastuzumab.
  • the first antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 3
  • the second antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 5.
  • the first antigen-binding unit may form a Fab with the second antigen-binding unit.
  • the first polypeptide chain and the second polypeptide chain can be expressed in an expression system of eukaryotic cells.
  • the toxin component may comprise a second intein fragment and a toxin, wherein the second intein fragment may be directly or indirectly linked to the toxin.
  • the second intein fragment may include the amino acid sequence shown in SEQ ID NO: 8.
  • the toxin may be selected from the following group: bacterial toxins, human toxins and phytotoxins.
  • the toxin may be selected from the group of Pseudomonas aeruginosa exotoxin and diphtheria toxin.
  • the toxin may be selected from the group consisting of ricin, saponin and gelonin.
  • the toxin may include PE24.
  • the toxin may include the amino acid sequence shown in SEQ ID NO:9.
  • the C-terminus of the second intein is directly or indirectly connected to the N-terminus of the toxin.
  • the indirect connection may include connection through a linker.
  • the linker may comprise a peptide linker.
  • the peptide linker may include the amino acid sequence shown in SEQ ID NO: 11.
  • the toxin component of the present application may sequentially include the second intein fragment and the toxin from N-terminal to C-terminal.
  • it may sequentially include the second intein fragment, the linker and the toxin from N-terminus to C-terminus.
  • it may sequentially include the second intein fragment (which may include the amino acid sequence shown in SEQ ID NO: 8), the linker (which It may include the amino acid sequence shown in SEQ ID NO: 11) and the toxin PE24 (which may include the amino acid sequence shown in SEQ ID NO: 9).
  • the toxin component may be Ic-PE24, which may include the amino acid sequence shown in SEQ ID NO: 10.
  • the toxin component may include the amino acid sequence shown in SEQ ID NO:10.
  • the toxin component can be expressed in the expression system of prokaryotic cells.
  • the targeting component and the toxin component may form an immunoconjugate through the interaction between the first intein fragment and the second intein fragment.
  • the first intein fragment may be different from the second intein fragment.
  • the first intein fragment and the second intein fragment may be derived from the same intein.
  • the first intein fragment and/or the second intein fragment may comprise a fragmented intein.
  • the fragmented intein may be selected from the following group: SsP DnaB, Ssp DnaE, and Npu DnaE.
  • the first intein fragment may be the N-terminal fragment of Npu DnaE
  • the second intein fragment may be the C-terminal part of Npu DnaE.
  • the first intein fragment and the second intein fragment can form a complete fragmented intein Npu DnaE.
  • the first intein fragment may include the amino acid sequence shown in SEQ ID NO: 6, and the second intein fragment may include the amino acid sequence shown in SEQ ID NO: 8.
  • the second polypeptide chain and the toxin component may be formed by the interaction between the first intein fragment and the second intein fragment to include the second antigen binding A fusion polypeptide of the unit and the toxin.
  • the interaction may be trans-shearing.
  • it may be a fragmented intein (for example, Npu DnaE) mediated trans-cleavage of a protein.
  • the first intein fragment and the second intein fragment are removed after being connected, thereby
  • the second antigen-binding unit connected to the first intein fragment and the toxin connected to the second intein fragment are connected to each other to form the second antigen-binding unit and the Fusion polypeptide of toxin.
  • the second antigen binding unit may be directly or indirectly connected to the toxin.
  • the C-terminus of the second antigen binding unit can be directly or indirectly connected to the N-terminus of the toxin.
  • the indirect connection may include connection through a linker.
  • the linker may comprise a peptide linker.
  • the peptide linker may include the amino acid sequence shown in SEQ ID NO: 11.
  • the fusion polypeptide described in the present application may sequentially include the second antigen binding unit and the toxin from N-terminus to C-terminus.
  • it may include the second antigen binding unit, the linker and the toxin in sequence from N-terminal to C-terminal.
  • the fusion polypeptide may sequentially include the second antigen binding unit (which may be the variable region of the heavy chain of trastuzumab and CH1 from the N-terminus to the C-terminus, which may include SEQ ID NO: 5
  • the amino acid sequence of) which may include the amino acid sequence shown in SEQ ID NO: 11
  • the toxin PE24 which may include the amino acid sequence of SEQ ID NO: 9).
  • the fusion polypeptide may include the amino acid sequence shown in SEQ ID NO: 12.
  • the immunoconjugate may include the shielding peptide, the antigen-binding portion, and the toxin, wherein the shielding peptide and the antigen-binding portion may be connected through the cleavable portion, and The antigen binding portion may be directly or indirectly linked to the toxin.
  • the first polypeptide chain can form an immunoconjugate with the fusion polypeptide.
  • the immunoconjugate may not include the first intein fragment and may not include the second intein fragment.
  • the first polypeptide chain and the fusion polypeptide may be combined with each other through the first antigen binding unit and the second antigen binding unit to form the immunoconjugate Things.
  • the first antigen binding unit and the second antigen binding unit may be connected by a disulfide bond between the variable region of the antibody light chain and the variable region of the antibody heavy chain.
  • the first antigen binding unit and the second antigen binding unit may be derived from trastuzumab.
  • the first antigen binding unit may be the light chain of trastuzumab
  • the second antigen binding unit may be the variable region and the constant region CH1 region of the heavy chain of trastuzumab.
  • the first antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 3, and the second antigen binding unit may include the amino acid sequence shown in SEQ ID NO: 5.
  • the antigen binding portion may be the Fab of trastuzumab.
  • the C-terminus of the shielding peptide may be directly or indirectly connected to the N-terminus of the cleavable portion.
  • the C-terminus of the cleavable portion may be directly or indirectly connected to the N-terminus of the antigen-binding portion.
  • the C-terminus of the antigen-binding portion may be directly or indirectly connected to the N-terminus of the toxin.
  • the indirect connection may include connection through a linker.
  • the linker may comprise a peptide linker.
  • the peptide linker may include the amino acid sequence shown in SEQ ID NO: 11.
  • the immunoconjugate of the present application may include the shielding peptide, the cleavable portion, the antigen binding portion, and the toxin in sequence from the N-terminus to the C-terminus.
  • it may include the shielding peptide, the cleavable portion, the antigen binding portion, the linker and the toxin in sequence from the N-terminus to the C-terminus.
  • the toxin component may include the shielding peptide (which may include the amino acid sequence shown in SEQ ID NO: 1) and the cleavable portion (which may include SEQ ID NO: 2), the antigen binding portion (trastuzumab Fab, which may include the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 5), the linker (which may include SEQ ID The amino acid sequence shown in NO: 11) and the toxin (PE24, which may include the amino acid sequence shown in SEQ ID NO: 9).
  • the shielding peptide which may include the amino acid sequence shown in SEQ ID NO: 1
  • the cleavable portion which may include SEQ ID NO: 2
  • the antigen binding portion which may include the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 5
  • the linker which may include SEQ ID The amino acid sequence shown in NO: 11
  • PE24 which may include the amino acid sequence shown in SEQ ID NO: 9).
  • the immunoconjugate may be M-Fab-PE24, which may include the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 12.
  • the immunoconjugate may include the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 12.
  • the method may include the step of providing the targeting component, and the step may include: providing a first polypeptide chain, the first polypeptide chain comprising the shielding peptide and the first antigen binding unit , The shielding peptide and the first antigen binding unit are connected through the cleavable portion; a second polypeptide chain is provided, and the second polypeptide chain comprises a second antigen binding unit and the first intein Fragment, the second antigen binding unit is directly or indirectly connected to the first intein fragment; and the first polypeptide chain and the second polypeptide chain are combined to form the targeting component, wherein The first antigen binding unit of the first polypeptide chain and the second antigen binding unit of the second polypeptide chain bind to each other to form the antigen binding portion in the targeting component.
  • step 3) of the method may further include allowing the second polypeptide chain and the toxin component to pass between the first intein fragment and the second intein fragment.
  • the fusion polypeptide comprising the second antigen binding unit and the toxin is formed by interaction, and the fusion polypeptide and the first polypeptide chain constitute the immunoconjugate.
  • the method may use a reducing agent to initiate the interaction between the first intein fragment and the second intein fragment.
  • the reducing agent may be selected from the following group: DTT and ⁇ -mercaptoethanol.
  • the concentration of the reducing agent may be 0.1-100 nM (for example, it may be about 0.1-80 nM, about 0.1-60 nM, about 0.1-40 nM, about 0.1-20 nM, about 0.1-10 nM, or about 1-10 nM. ).
  • the reducing agent may be in contact with the targeting component and the toxin component.
  • the step of adding the reducing agent may occur at the same time as the steps 1), 2) and 3) in the method or at different times.
  • the polypeptide combination after adding the reducing agent, may contain toxins with biological toxicity, and/or with biological toxicity.
  • the method for preparing the immunoconjugate may further include incubating after adding the reducing agent.
  • the temperature of the incubation may be about 1 to about 50°C (for example, it may be about 1°C to about 50°C, about 4°C to about 50°C, about 4°C to about 45°C, about 4°C. -About 40°C, about 4°C to about 37°C, about 8°C to about 37°C, about 13°C to about 37°C, about 17°C to about 37°C, about 17°C to about 35°C, about 17°C to about 30°C, about 17°C to about 25°C, about 17°C to about 23°C, or about 20°C to about 23°C).
  • the incubation time is about 60 to about 300 minutes (for example, it can be about 60 to about 300 minutes, about 60 to about 240 minutes, about 60 to about 180 minutes, about 60 to about 120 minutes. , About 90-about 120 minutes).
  • the application provides a vector comprising a nucleic acid encoding the first polypeptide chain in the polypeptide combination, a nucleic acid encoding the second polypeptide chain in the polypeptide combination, and a nucleic acid encoding the polypeptide combination
  • the nucleic acid of the second antigen-binding unit, the nucleic acid encoding the first intein fragment in the polypeptide combination, the nucleic acid that encodes the second intein fragment in the polypeptide combination, and the nucleic acid encoding the polypeptide combination The nucleic acid of the toxin described in and/or the nucleic acid encoding the shielding peptide described in the polypeptide combination.
  • This application provides a cell expressing the vector.
  • the application also provides a kit, which may include the first polypeptide chain, the second polypeptide chain, the toxin component, the cleavable portion, and the first polypeptide chain in the polypeptide combination.
  • a kit which may include the first polypeptide chain, the second polypeptide chain, the toxin component, the cleavable portion, and the first polypeptide chain in the polypeptide combination.
  • the components in the kit may not be mixed with each other.
  • the components in the kit can be placed in different containers and/or different physical spaces.
  • the kit may also include a reducing agent.
  • the reducing agent may be selected from the following group: DTT and ⁇ -mercaptoethanol.
  • the reducing agent may be contained in a separate container.
  • the application provides the use of the polypeptide combination, the carrier or the cell in the preparation of a medicine for treating diseases, and the diseases include tumors.
  • the treatment may include inhibiting or delaying the development or progression of tumors.
  • the treatment can reduce the size of the tumor (or even substantially eliminate the tumor), and/or can reduce and/or stabilize the disease state.
  • the tumor may include ovarian cancer and breast cancer.
  • the application provides the polypeptide combination, the vector or the cell, which treats tumors.
  • This application provides a method for treating tumors, which comprises administering the polypeptide combination, the vector or the cell.
  • the method can be implemented under in vivo conditions.
  • the first polypeptide chain, the second polypeptide chain, and the toxin component in the polypeptide combination can form the biologically toxic immunoconjugate under the conditions of the tumor cell microenvironment.
  • the immunoconjugate may possess biological toxicity, for example, it may have a killing effect on tumor cells, or promote tumor cell apoptosis.
  • the modes of administration may include oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration and / Or through subcutaneous depot administration.
  • This application also provides a method for administering the polypeptide combination to cells, which may include the following steps: mixing and administering the first polypeptide chain, the second polypeptide chain, the toxin component, and the reducing agent .
  • the first polypeptide chain, the second polypeptide chain, and the toxin component form the immunoconjugate under reducing conditions.
  • the immunoconjugate may possess biological toxicity, for example, it may have a killing effect on tumor cells, or promote tumor cell apoptosis.
  • This application also provides a method for administering the immunoconjugate to cells, which may include the following steps, administering the immunoconjugate to cells under in vitro conditions.
  • trastuzumab light chain and heavy chain design PCR amplification primers, and add restriction endonuclease sites at the 5'end of the inserted vector primers And protective bases for inserting into expression vectors.
  • the PCR synthesis fragment and the PM09 plasmid after restriction double enzyme digestion were ligated by DNA ligase to form PM-LC, PM-MLC, PM-HC-In eukaryotic vectors, respectively.
  • the PM-LC eukaryotic vector expresses the light chain of trastuzumab (LC, its amino acid sequence is shown in SEQ ID NO. 3); the PM-MLC eukaryotic vector expresses the trastuzumab linked to the shielding peptide Anti-light chain (MLC, whose amino acid sequence is shown in SEQ ID NO.4); PM-HC-In eukaryotic vector expresses trastuzumab heavy chain (HC- In, its amino acid sequence is shown in SEQ ID NO. 7).
  • LC light chain of trastuzumab
  • MLC shielding peptide Anti-light chain
  • PM-HC-In eukaryotic vector expresses trastuzumab heavy chain (HC- In, its amino acid sequence is shown in SEQ ID NO. 7).
  • the PCR-synthesized fragment and the pET32a plasmid after restriction double digestion were ligated by DNA ligase to form the pET-Ic-PE24 prokaryotic expression vector.
  • the pET-Ic-PE24 prokaryotic expression vector expresses the PE24 toxin (Ic-PE24, whose amino acid sequence is shown in SEQ ID NO. 10) connected with a second intein fragment.
  • the vector PM-LC and PM-HC-In prepared in Example 1.1, or PM-MLC and PM-HC-In were co-transfected into mammalian cells HEK293E, so that the transfected cells expressed and connected the first inner Trastuzumab Fab-Fab-In containing peptide fragments and Trastuzumab Fab-MFab-In connected with shielding peptide and first intein fragment.
  • the cell culture supernatant was collected by centrifugation, filtered with a 0.45 micron filter membrane and purified on a Capto L column.
  • the vector pET-Ic-PE24 prepared in Example 1.1 was transformed into E. coli competent BL21 (DE3), and the PE24 toxin connected with the second intein fragment—Ic-PE24 was induced by IPTG.
  • the expression cells were collected, after high-pressure homogenization, the supernatant was collected by centrifugation, and purified by nickel column.
  • SDS-PAGE detects purified antibody fragments and toxin fragments. The results are shown in Figure 1, where M represents marker, lane 1 is MFab-In, lane 2 is Ic-PE24, lane 3 is a mixture of M-Fab-In and IcPE24, and lane 4 is a combination of MFab-In and Ic-PE24. Mix and add 1mM DTT.
  • Example 1.2 The protein Fab-In or MFab-In prepared in Example 1.2 was reacted with Ic-PE24 prepared in Example 1.2, and 1 mM DTT reducing agent was added to induce the protein mediated by the first intein fragment and the second intein fragment
  • oxidized glutathione was added to terminate the reaction after 2 hours of reaction at 37°C, and the disulfide bond was formed by oxidation, thereby obtaining immunoconjugates Fab-PE24 and M-Fab-PE24, respectively.
  • the immunoconjugate Fab-PE24 comprises trastuzumab Fab and the toxin PE24 linked thereto, and its amino acid sequence is shown in SEQ ID NO. 3 and SEQ ID NO. 12;
  • the immuno conjugate M-Fab-PE24 includes a shielding peptide, a cleavable part, trastuzumab Fab and a toxin PE24 linked to it, and its amino acid sequence is shown in SEQ ID NO. 4 and SEQ ID NO. 12.
  • uPA freeze-dried powder (as a protease, which can cleavage the cleavable part of the shielding peptide) was dissolved in sterile PBS to prepare a 140 ⁇ g/ml protease solution.
  • the M-Fab-PE24 prepared in Example 1.3 was treated with uPA, 150 ⁇ g of M-Fab-PE24 was mixed with 8 ⁇ L of protease solution, and reacted overnight at 37°C in a water bath.
  • Digest cells in logarithmic growth phase with trypsin BT-474 human breast cancer cells, purchased from HTB-20 TM
  • SKOV-3 human ovarian cells, purchased from HTB-77 TM
  • MCF-7 human breast cancer cells, purchased from HTB-22 TM
  • Each group of cells were added with the immunoconjugate Fab-PE24 or M-Fab-PE24 prepared in Example 1, and incubated at 37°C for 72 hours.
  • Example 1 Injection of prepared Fab-PE24 (1.0 mg/kg), M-Fab-PE24 (1.0 mg/kg) and NCM-Fab-PE24 (1.0 mg/kg) prepared in Example 1.
  • NCM-Fab-PE24 differs from M-Fab-PE24 in that the cleavable part of the former connected to the shielding peptide has no restriction site, so the shielding peptide will not be removed.
  • the amino acid sequence of NCM-Fab-PE24 is shown in SEQ ID NO.13 and SEQ ID NO.12.
  • Intravenous injection once every other day, a total of six times.
  • the body weight and tumor volume of the mice were measured before each administration. After the administration, the body weight and tumor volume of the mice were measured twice a week. When the maximum tumor volume reached 1000 mm 3 , the experiment was terminated and the mice were euthanized. Peel off the nodules.
  • mice During the treatment of tumor-bearing Balb/c nude mice receiving the Fab-PE24 and M-Fab-PE24 prepared in Example 1, after the third administration (among them, the dosage, mode of administration and administration during the treatment See Example 4 for drug frequency) The next day, the mice were euthanized, the tumor tissue was taken out and embedded in OCT and then frozen, frozen section was performed using a cryostat, and the sections were subjected to immunofluorescence staining analysis. The specific steps are as follows:
  • Tissue sections were fixed with pre-chilled 4% (v/v) paraformaldehyde and fixed at 4°C for 10 minutes; the sections were washed with PBS for 10 minutes, and the PBS was blotted dry with absorbent paper; blocking solution (PBS containing 1% (v/v) Horse serum) incubate the slices at room temperature for 30 minutes; the primary antibody (rabbit anti-ETA antibody) is diluted with diluent (PBS containing 1% bovine serum albumin, 0.3% TritonX-100), and then the slices are incubated overnight at 4°C; washed with PBS Section 3 times, 15min each time; the secondary antibody (PE conjugated goat anti rabbit antibody) diluted with diluent, dripped onto the surface of the slice, and incubated the slice at room temperature for 1 hour; washed the slice 3 times with PBS, 15min each time; use DAPI The sections were counterstained at room temperature for 5 minutes; the sections were washed with PBS for 10 minutes;
  • mice During the treatment of tumor-bearing Balb/c nude mice receiving the Fab-PE24 and M-Fab-PE24 prepared in Example 1, after the third administration (among them, the dosage, mode of administration and administration during the treatment For the drug frequency, see Example 4).
  • the mice On the second day, the mice were subjected to orbital blood sampling, and the mice were allowed to stand at room temperature for 2 hours, and centrifuged at 4000 rpm for 10 minutes to obtain the supernatant.
  • a kit (purchased from Nanjing Jiancheng Biological Co., Ltd.) was used to detect the activities of alanine aminotransferase (AST) and aspartate aminotransferase (ALT) in the serum of mice.
  • the AST and ALT levels of each group of mice reflected the immunoconjugate prepared in Example 1.

Abstract

提供一种多肽组合，其中靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，屏蔽肽与抗原结合部分通过可剪切部分连接，且抗原结合部分与第一内含肽片段直接或间接连接；毒素组分包含第二内含肽片段和毒素，且第二内含肽片段与毒素直接或间接连接；靶向组分与毒素组分可以通过第一内含肽片段与第二内含肽片段间的相互作用而形成免疫缀合物；免疫缀合物中屏蔽肽与抗原结合部分通过可剪切部分连接，且抗原结合部分与毒素连接。还提供了多肽组合的制备方法以及制药用途。

Description

一种用于肿瘤免疫治疗的多肽组合及其制备方法 技术领域
本申请涉及生物医药领域，具体的涉及一种用于肿瘤免疫治疗的多肽组合及其制备方法。
背景技术
重组免疫毒素是指由细胞靶向部分和毒素部分组成的治疗性嵌合蛋白，具有特异性靶向病变细胞并杀伤病变细胞的功能，可以通过化学偶联或者基因重组的方法制得。重组免疫毒素发挥作用时必须结合靶细胞并被靶细胞摄取，并且毒素的酶活性片段必须转移至胞质中。
屏蔽肽可以屏蔽抗体或其抗原结合片段的抗原结合部位，降低抗体或其抗原结合片段在循环过程中发生的非特异性结合。当抗体或其抗原结合片段到达肿瘤微环境时，肿瘤微环境富含的蛋白酶选择性切割连接肽，移除通过该连接肽连接在所述抗体或其抗原结合片段上的屏蔽肽，恢复抗体或其抗原结合片段的抗原结合能力，从而选择性地杀伤肿瘤细胞，提高肿瘤的杀伤特异性。
反式剪接可以为由断裂型蛋白质内含肽介导的蛋白质剪接反应。例如，断裂蛋白质内含肽的N端片段和C端片段相互识别并以非共价键结合，两者结合后正确折叠其结构，并且将断裂蛋白质内含肽两侧的蛋白质外显肽的连接。
发明内容
本申请提供了一种用于肿瘤免疫治疗的多肽组合及其制备方法。所述的多肽组合及其制备方法具有以下性质中的一种或多种：1)提高用药安全性，降低对正常细胞的非特异性毒性；2)位于正常组织时，所述多肽组合形成的免疫缀合物无法与正常组织的细胞结合而发挥生物毒性；而位于肿瘤微环境时，所述多肽组合形成的免疫缀合物与肿瘤细胞表面的肿瘤特异性抗原特异性结合，发挥杀伤肿瘤细胞，和/或诱导肿瘤细胞凋亡的能力；3)具有较高的灵活性和可调节性，例如可以采用不同的给药方式、给药剂量和不同的给药组合方法，以达到更好的治疗效果；4)可以针对不同的肿瘤相关抗原，提供不同的靶向组分；5)制备方法具有普遍适用性，可以应用于多种类型的免疫毒素或者蛋白药物；6)可以分别在真核细胞表达系统和原核细胞表达系统中生产，实现工业化高产量。
一方面，本申请提供了一种多肽组合，其包含靶向组分和毒素组分，其中：所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一内含肽片段直接或间接连接； 所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；所述靶向组分与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成免疫缀合物；所述免疫缀合物包含所述屏蔽肽、所述抗原结合部分及所述毒素，其中所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述毒素直接或间接连接。
在某些实施方式中，所述靶向组分包含第一多肽链及第二多肽链；所述第一多肽链包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽与所述第一抗原结合单元通过所述可剪切部分连接；所述第二多肽链包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元与所述第一内含肽片段直接或间接连接；且所述第一抗原结合单元与所述第二抗原结合单元能够相互结合形成所述抗原结合部分。
在某些实施方式中，所述第二多肽链与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽；在所述融合多肽中，所述第二抗原结合单元与所述毒素直接或间接连接；且所述第一多肽链与所述融合多肽构成所述免疫缀合物。
在某些实施方式中，所述免疫缀合物不包含所述第一内含肽片段且不包含所述第二内含肽片段。
在某些实施方式中，在所述靶向组分中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
在某些实施方式中，在所述靶向组分中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
在某些实施方式中，在所述靶向组分中，所述抗原结合部分的C端与所述第一内含肽片段的N端直接或间接连接。
在某些实施方式中，在所述第一多肽链中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
在某些实施方式中，在所述第一多肽链中，所述可剪切部分的C端与所述第一抗原结合单元的N端直接或间接连接。
在某些实施方式中，在所述第二多肽链中，所述第二抗原结合单元的C端与所述第一内含肽片段的N端直接或间接连接。
在某些实施方式中，在所述毒素部分中，所述第二内含肽的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述屏蔽肽的C端与所述可剪切部分的N端 直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述抗原结合部分的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述第一多肽链与所述融合多肽通过所述第一抗原结合单元与所述第二抗原结合单元的相互结合构成所述免疫缀合物。
在某些实施方式中，在所述融合多肽中，所述第二抗原结合单元的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，所述间接连接包含通过连接子连接。
在某些实施方式中，所述连接子包含肽连接子，所述肽连接子包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述屏蔽肽选自以下组：HER2抗原屏蔽肽、EGFR抗原屏蔽肽，EpCAM抗原屏蔽肽和Mesothelin抗原屏蔽肽。在某些实施方式中，所述屏蔽肽包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述可剪切部分选择以下组：基质金属蛋白酶敏感型连接子、丝氨酸蛋白酶敏感型连接子和尿激酶纤溶酶原激活物敏感型连接子。在某些实施方式中，所述可剪切部分包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向组分靶向肿瘤特异性抗原。在某些实施方式中，所述肿瘤特异性抗原包含HER2。在某些实施方式中，所述抗原结合部分包含抗体或其抗原结合片段。在某些实施方式中，所述第一抗原结合单元和所述第二抗原结合单元源自相同的所述抗体或其抗原结合片段。在某些实施方式中，所述抗体选自以下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab') 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。在某些实施方式中，所述抗原结合片段为Fab。
在某些实施方式中，所述第一抗原结合单元包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合单元包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。在某些实施方式中，所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。在某些实施方式中，所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。在某些实施方式中，所述毒素包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述第一内含肽片段和/或所述第二内含肽片段包含断裂型内含肽。在某些实施方式中，所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
在某些实施方式中，所述第一内含肽片段与所述第二内含肽片段不同。
在某些实施方式中，所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。
在某些实施方式中，所述第一内含肽片段包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述第二内含肽片段包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述第一多肽链包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述第二多肽链包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述毒素组分包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述免疫缀合物包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述融合多肽包含SEQ ID NO:12所示的氨基酸序列。
另一方面，本申请提供了一种制备免疫缀合物的方法，其包括以下步骤：1)提供靶向组分，所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一内含肽片段直接或间接连接；2)提供毒素组分，所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；3)使所述靶向组分与所述毒素组分在使得所述第一内含肽片段与所述第二内含肽片段能够相互作用的条件下接触，其中所述靶向组分与所述毒素组分通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成所述免疫缀合物。
在某些实施方式中，所述提供靶向组分包括：提供第一多肽链，所述第一多肽链包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽与所述第一抗原结合单元通过所述可剪切部分连接；提供第二多肽链，所述第二多肽链包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元与所述第一内含肽片段直接或间接连接；且使所述第一多肽链与所述第二多肽链结合形成所述靶向组分，其中所述第一多肽链的所述第一抗原结合单元与所述第二多肽链的所述第二抗原结合单元相互结合而形成所述靶向组分中的所述抗原结合部分。
在某些实施方式中，其中3)包括使所述第二多肽链与所述毒素组分经所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽，所述融合多肽与所述第一多肽链构成所述免疫缀合物。
在某些实施方式中，所述免疫缀合物不包含所述第一内含肽片段且不包含所述第二内含肽片段。
在某些实施方式中，在所述靶向组分中，所述屏蔽肽的C端与所述可剪切部分的N端直 接或间接连接。
在某些实施方式中，在所述靶向组分中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
在某些实施方式中，在所述靶向组分中，所述抗原结合部分的C端与所述第一内含肽片段的N端直接或间接连接。
在某些实施方式中，在所述第一多肽链中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
在某些实施方式中，在所述第一多肽链中，所述可剪切部分的C端与所述第一抗原结合单元的N端直接或间接连接。
在某些实施方式中，在所述第二多肽链中，所述第二抗原结合单元的C端与所述第一内含肽片段的N端直接或间接连接。
在某些实施方式中，所述毒素部分中，所述第二内含肽的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述抗原结合部分的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，在所述免疫缀合物中，所述第一多肽链与所述融合多肽通过所述第一抗原结合单元与所述第二抗原结合单元的相互结合构成所述免疫缀合物。
在某些实施方式中，在所述融合多肽中，所述第二抗原结合单元的C端与所述毒素的N端直接或间接连接。
在某些实施方式中，所述间接连接包含通过连接子连接。在某些实施方式中，所述连接子包含肽连接子，所述肽连接子包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述屏蔽肽选自以下组：HER2抗原屏蔽肽、EGFR抗原屏蔽肽，EpCAM抗原屏蔽肽和Mesothelin抗原屏蔽肽。
在某些实施方式中，所述屏蔽肽包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述可剪切部分选择以下组：基质金属蛋白酶敏感型连接子、丝氨酸蛋白酶敏感型连接子和尿激酶纤溶酶原激活物敏感型连接子。
在某些实施方式中，所述可剪切部分包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述靶向组分靶向肿瘤特异性抗原。在某些实施方式中，所述肿瘤特异性抗原包含HER2。在某些实施方式中，所述抗原结合部分包含抗体或其抗原结合片段。在某些实施方式中，所述第一抗原结合单元和所述第二抗原结合单元源自相同的所述抗体或其抗原结合片段。在某些实施方式中，所述抗体选自以下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab') 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。在某些实施方式中，所述抗原结合片段为Fab。
在某些实施方式中，所述第一抗原结合单元包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述第二抗原结合单元包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。在某些实施方式中，所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。在某些实施方式中，所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。在某些实施方式中，所述毒素包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述第一内含肽片段和/或所述第二内含肽片段包含断裂型内含肽。在某些实施方式中，所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
在某些实施方式中，所述第一内含肽片段与所述第二内含肽片段不同。在某些实施方式中，所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。
在某些实施方式中，所述第一内含肽片段包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述第二内含肽片段包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述第一多肽链包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述第二多肽链包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述毒素组分包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述免疫缀合物包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述融合多肽包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述第一内含肽片段与所述第二内含肽片段间的相互作用包含反式剪接作用。
在某些实施方式中，其中使用还原剂引发所述第一内含肽片段与所述第二内含肽片段间的相互作用。
在某些实施方式中，其中所述还原剂选自以下组：DTT和β巯基乙醇。在某些实施方式中，所述还原剂的浓度为0.1-100nM。在某些实施方式中，所述还原剂与所述靶向组分和所 述毒素组分相接触。
在某些实施方式中，所述制备免疫缀合物的方法还包括在加入所述还原剂后孵育。在某些实施方式中，其中所述孵育的温度为1-50℃。在某些实施方式中，其中所述孵育的时间为60-300分钟。
本申请提供一种载体，其包含编码所述多肽组合中的所述的第一多肽链的核酸，编码所述多肽组合中所述的第二多肽链的核酸，编码所述多肽组合中所述的毒素组分的核酸，编码所述多肽组合中所述的可剪切部分的核酸，编码所述多肽组合中所述的第一抗原结合单元的核酸，编码所述多肽组合中所述的第二抗原结合单元的核酸，编码所述多肽组合中所述的第一内含肽片段的核酸，编码所述多肽组合中所述的第二内含肽片段的核酸，编码所述多肽组合中所述的毒素的核酸和/或编码所述多肽组合中所述的屏蔽肽的核酸。
本申请提供一种细胞，其表达所述的载体。
本申请提供所述的多肽组合、所述的载体或所述的细胞在制备治疗疾病的药物中的用途，所述疾病包含肿瘤。
在某些实施方式中，所述肿瘤包括卵巢癌和乳腺癌。
本申请提供所述多肽组合、所述载体或所述细胞，其治疗肿瘤。
本申请提供一种治疗肿瘤的方法，其包括施用所述的多肽组合、所述的载体或所述的细胞。
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。对附图简要说明书如下：
图1显示的是本申请所述靶向组分和毒素组分的SDS-PAGE检测的结果。
图2显示的是本申请所述免疫缀合物的SDS-PAGE检测的结果。
图3显示的是经蛋白酶处理后活化的本申请所述免疫缀合物的SDS-PAGE检测的结果。
图4显示的是本申请所述免疫缀合物对表达HER2/erbB2/neu抗原的肿瘤细胞的结合亲 和力分析的结果。
图5显示的是本申请所述免疫缀合物对表达HER2/erbB2/neu抗原的肿瘤细胞的细胞毒性分析的结果。
图6显示的是本申请所述免疫缀合物的体内药效学研究的结果。
图7显示的是施用本申请所述免疫缀合物后，SKOV-3荷瘤小鼠体内的肿瘤体积情况。
图8显示的是本申请所述免疫缀合物对小鼠肝组织的损伤试验的结果。
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。
在本申请中，术语“多肽组合”通常是指两种以上蛋白质或多肽的集合。在本申请中，所述多肽组合可以是包含两种以上蛋白质或多肽的组合物。在本申请的所述多肽组合中，不同的蛋白质或多肽可以分别位于不同的容器或物理空间中。又例如，不同的两种以上蛋白质或多肽也可以位于同一容器，或同一物理空间中。在本申请中，所述多肽组合可以不为一个蛋白质分子，或者一个蛋白质复合物。例如，其中两种以上蛋白质或多肽，均不位于相同的蛋白质分子，或相同的蛋白质复合物中。
在本申请中，术语“靶向组分”通常是指包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段的蛋白质或多肽。所述靶向组分可以靶向肿瘤细胞。例如，所述抗原结合部分可以特异性结合肿瘤细胞。在所述靶向部分中，所述屏蔽肽与所述抗原结合部分可以通过所述可剪切部分连接，且所述抗原结合部分可以与所述第一内含肽片段直接或间接连接。
在本申请中，术语“屏蔽肽”通常是指连接在抗原结合部分，阻碍抗原结合部分与抗原相结合的多肽或其片段。例如，所述屏蔽肽可以位于所述抗原结合部分的N端。例如，所述屏蔽肽可以通过可剪切部分与所述抗原结合部分连接。在本申请中，当所述屏蔽肽位于正常组织的环境中时，所述屏蔽肽无法和与其连接的所述抗原结合部分所分离，使得所述抗原结合部分无法与抗原相结合。而当所述屏蔽肽位于肿瘤环境中时，由于肿瘤细胞释放了特异的蛋白酶，可以使得所述屏蔽肽和与其连接的所述抗体结合部分分离(例如，可以通过切断所述可剪切部分)，从而使得抗原结合部分可以与肿瘤细胞的抗原相结合。在本申请中，所述屏蔽肽可以为Probody技术中所采用的屏蔽肽(参见Polu KR,Lowman HB.Probody therapeutics for targeting antibodies to diseased tissue.Expert Opinion on Biological Therapy.2014；14:1049-1053)。
在本申请中，术语“可剪切部分”通常是指连接所述屏蔽肽和所述抗原结合部分的，具备 可剪切特性的多肽或其片段。在本申请中，所述可剪切部分可以通过蛋白酶的作用而切断。例如，所述可剪切部分可以被肿瘤环境中的蛋白酶(例如，肿瘤细胞周围富集的蛋白酶)而切断，从而使所述屏蔽肽与所述抗原结合部分分离。例如，分离的抗原结合部分可以与肿瘤特异性抗原所结合。
在本申请中，术语“抗原结合部分”通常是指可以与肿瘤特异性抗原结合的抗体或其抗原结合片段。
在本申请中，术语“肿瘤特异性抗原”通常是指特异性表达于某种或多种肿瘤细胞表面而几乎不存在于正常细胞上的抗原。
在本申请中，术语“HER2”通常是指人类表皮生长因子受体2，human epidermal growth factor receptor 2，亦可称为Neu、ErbB-2或CD340。所述HER2可以为由ERBB2基因编码的蛋白质，其属于表皮生长因子受体(EGFR/ErbB)家族中的成员。重组型人源化曲妥珠单抗Trastuzumab
Figure PCTCN2020085736-appb-000001
可以特异性结合所述HER2的胞外第四亚结构域。所述HER2可以属于所述肿瘤特异性抗原，例如，可以作为乳腺癌或卵巢癌的靶标。人HER2在GenBank中的登录号可以为NP_001276866.1。
在本申请中，术语“抗体”通常是指能够特异性识别和/或中和特定抗原的多肽分子。基本的四链抗体单元是异四聚体糖蛋白，其由两条相同的轻链和两条相同的重链构成。在IgG的情况中，每个L链通过一个共价二硫键连接到H链，而两条H链通过一个或多个二硫键彼此相连，二硫键的数目取决于H链的同种型。每个H和L链还具有规律间隔的链内二硫键。每条H链在N-末端具有可变结构域(VH)，接着是三个(对于每条α和γ链)或四个(对于μ和ε同种型)恒定结构域(CH)。
在本申请中，术语“抗原结合片段”通常是指完整抗体的一部分，例如，所述抗原结合片段可以是完整抗体的抗原结合区和/或抗体可变区。所述抗原结合片段可以通过化学方法和/或基因工程的方法获得。例如，可以采用蛋白酶，包括胃蛋白酶和木瓜蛋白酶，消化抗体后产生抗原结合片段。
在本申请中，术语“Fab”通常是指是指由木瓜蛋白酶消化具有完整结构的抗体后(例如，去除了Fc区和铰链区)而产生两个相同的抗原结合片段。Fab可以由完整的轻链、重链可变区(VH)和重链的第一恒定结构域(CH1)组成。每个Fab可以具有单一的抗原结合位点。
在本申请中，术语“Fab'”通常是指与所述Fab相比，在CH1结构域的羧基末端具有几个额外的残基的抗原结合片段，Fab'包括来自抗体铰链区的一个或多个半胱氨酸。
在本申请中，术语“F(ab) 2”通常是指由半胱氨酸相连接的成对的所述Fab所得到的抗原结合片段。
在本申请中，术语“Fv”通常是指由抗体单臂的VL和VH结构域组成的抗原结合片段。
在本申请中，术语“单链抗体(scFv)”通常是指由抗体重链可变区和轻链可变区通过短肽连接子(linker)连接而成的分子。
在本申请中，术语“内含肽”通常是指位于宿主蛋白质中的一段插入序列。所述内含肽需要插入于外显肽基因才能复制转录，可从前体蛋白中被切除并将两侧外显肽连接起来成为成熟蛋白质。例如，内含肽对应的核苷酸序列可以嵌合在宿主蛋白对应的核酸序列之中，与宿主蛋白基因存在于同一开放阅读框架内，并与宿主蛋白质基因进行同步转录和翻译，当翻译形成蛋白质前体后，内含肽从宿主蛋白质中切除，从而形成成熟的具有活性的蛋白。根据内含肽的存在形式，可以将其分为整体内含肽和断裂型内含肽。例如，所述整体内含肽的两个剪接区域可以共同存在于同一多肽片段；而所述断裂型内含肽的两个剪接区域可以分裂成两份以上片段，并存在于不同的多肽片段。
在本申请中，术语“断裂型内含肽”通常是指其中两个剪切区域可位于两个以上多肽片段的内含肽。例如，所述断裂型内含肽可以包含N端蛋白质剪接区域(N fragment of intein，In)和C端蛋白质剪接区域(C fragment of intein，Ic)。其中，所述N端蛋白质剪接区域的N端可以连接一个外显肽(例如N-extein)；所述C端蛋白质剪切区域的C端可以连接另一个外显肽(例如，C-extein)。例如，所述外显肽N-extein和所述外显肽C-extein可以位于两个不同的开放的阅读框中。例如，在适宜的环境下，经过属于同一所述断裂型内含肽的所述N端蛋白质剪接区域和所述C端蛋白质剪切区域的相互作用，所述N端蛋白质剪接区域和所述C端蛋白质剪切区域可以被切除，同时所述外显肽N-extein和所述外显肽C-extein可以相互连接而形成连接的蛋白质或多肽。
在本申请中，术语“第一内含肽片段”通常是指内含肽的部分片段，它可以不包含完整的内含肽片段。在一定条件下，第一内含肽片段可以和其他内含肽片段发生相互作用，使得所述第一内含肽片段上所连接的蛋白质或多肽片段(例如外显肽)与其他蛋白质或多肽片段(例如外显肽)相连接而形成连接的蛋白质或多肽。例如，所述第一内含肽片段可以连接第二抗原结合单元。
在本申请中，术语“第二内含肽片段”通常是指通常是指内含肽的部分片段，它可以不包含完整的内含肽片段。在本申请中，所述第二内含肽片段可以不同于所述第一内含肽片段。例如，所述第一内含肽片段可以与所述第二内含肽片段来源于相同的所述内含肽。在本申请中，在一定条件下，第二内含肽片段可以和第一内含肽片段发生相互作用。例如，所述第二内含肽片段可以和其他内含肽片段发生相互作用，使得所述第二内含肽片段上所连接的蛋白质或多肽片段(例如外显肽，例如，毒素)与其他蛋白质或多肽片段(例如外显肽，)相连接 而形成连接的蛋白质或多肽。在本申请中，所述第二内含肽片段可以连接毒素。例如，所述第一内含肽片段可以与所述第二内含肽片段相互作用，从而使所述第二抗原结合单元与所述毒素形成融合多肽。
在本申请中，术语“毒素”通常是指生物体所生产出来的毒物。所述毒素通常是一些会干扰生物体中其他大分子作用的蛋白质，所述毒素通常可以通过酶解作用抑制蛋白合成来发挥功能。毒素按照其来源，可以包括细菌毒素、植物毒素以及人源毒素。例如，所述细菌毒素可包括绿脓杆菌外毒素(PE)和白喉毒素(DT)。所述植物毒素可包括蓖麻毒素、皂草素和白树毒素。通常，毒素可以包括以下功能区：细胞结合区、转位区和毒性活性区。例如，所述细胞结合区可将毒素富集于靶细胞的表面。
在本申请中，术语“PE24”通常是指绿脓杆菌外毒素(PE)截去细胞结合区之后的片段。完整的PE含有三个功能区，即细胞结合区(Ia区，1-252aa)、转位区(II区，253-364aa)和毒性活性区(III区，400-613aa)。PE的截短体PE24截去了细胞结合区(Ia区，1-252aa)，并且进一步切短去除了T/B细胞抗原表位，极大地降低了毒素的免疫原性(参见Mazor R,Onda M,Park D,et al.Dual B-and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity.Oncotarget.2016；7:29916-29926.)。所述PE24的氨基酸序列可以如SEQ ID NO.9所示。
在本申请中，术语“毒素组分”通常是指包含所述第二内含肽片段和所述毒素的蛋白质或多肽。在本申请中，所述毒素组分可以具备生物毒性，所述生物毒性可以由所述毒素而产生。在本申请中，所述毒素组分中的所述第二内含肽片段，可与所述靶向组分中的所述第一内含肽片段相互作用，从而产生包含所述屏蔽肽、所述可剪切部分、所述抗原结合部分和所述毒素的所述免疫缀合物。
在本申请中，术语“相互作用”通常是指所述第一内含肽片段和所述第二内含肽片段之间的相互作用。在本申请中，经过所述相互作用，所述第一内含肽片段多连接的蛋白质或多肽，可以与所述第二内含肽片段所连接的蛋白质或多肽能够相互连接而形成连接的蛋白质或多肽。例如，所述相互作用可以为剪接作用。例如，所述相互作用可以为蛋白质反式剪接作用。例如，所述第一内含肽片段可以与所述第二内含肽片段经蛋白质反式剪接作用使所述第二抗原结合单元与所述毒素形成所述融合多肽。
在本申请中，术语“蛋白质反式剪接”是指由断裂型蛋白质内含肽介导的蛋白质剪接反应。例如，所述断裂型内含肽的N端片段(In)和所述断裂型内含肽的C端片段(Ic)相互识别并以非共价键结合，两者结合后正确折叠结构，并被反式剪切去除，将所述断裂型内含肽的N端片段和所述断裂型内含肽的C端片段两侧的外显肽进行连接，从而得到由两侧外显肽形 成的蛋白质。
在本申请中，术语“第一条多肽链”通常是指包含所述屏蔽肽和第一抗原结合单元的单一多肽链。在所述第一多肽链中，所述屏蔽肽与所述第一抗原结合单元可以通过所述可剪切部分连接。
在本申请中，术语“第一抗原结合单元”通常是指所述抗原结合部分的一部分。所述第一抗原结合单元具备与所述肿瘤特异性抗原特异性结合的能力。在本申请中，所述第一抗原结合单元可以为特异性结合所述肿瘤特异性抗原的抗体部分或其抗原结合片段。例如，所述第一抗原结合单元可以为特异性结合所述肿瘤特异性抗原的抗体的轻链(LC)。
在本申请中，术语“第二多肽链”通常是指包含第二抗原结合单元和所述第一内含肽片段的单一多肽链。在所述第二多肽链中，所述第二抗原结合单元与所述第一内含肽片段可以直接或间接连接。
在本申请中，术语“第二抗原结合单元”通常是指所述抗原结合部分的一部分。所述第二抗原结合单元具备与所述肿瘤特异性抗原特异性结合的能力。在本申请中，所述第二抗原结合单元可以为特异性结合所述肿瘤特异性抗原的抗体部分或其抗原结合片段。在本申请中，所述第二抗原结合单元可以不同于所述第一抗原结合单元。在本申请中，所述第二抗原结合单元可以与所述第一抗原结合单元相互结合形成所述抗原结合部分。例如，所述第二抗原结合单元可以为特异性结合所述肿瘤特异性抗原的抗体的重链的可变区和恒定区的CH1部分。又例如，所述第二抗原结合单元可以为特异性结合所述肿瘤特异性抗原的抗体的重链的可变区、恒定区的CH1部分和铰链区。在本申请中，所述第二抗原结合单元可以与所述第一抗原结合单元相互结合形成特异性结合所述肿瘤特异性抗原的Fab。
在本申请中，术语“融合多肽”通常是指包含所述第二抗原结合单元和所述毒素的蛋白质或多肽。在本申请中，所述融合多肽可以通过所述第二多肽链中的所述第一内含肽片段与所述毒素组分中所述第二内含肽片段的相互作用而形成。例如，所述相互作用可以为所述第一内含肽片段与所述第二内含肽片段的蛋白质反式剪接作用。
在本申请中，术语“免疫缀合物”通常是指含所述屏蔽肽、所述抗原结合部分及所述毒素的蛋白质或多肽。在本申请中，所述免疫缀合物中，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述毒素直接或间接连接。例如，所述间接连接可以为通过连接子连接。在本申请中，所述免疫缀合物可以从N端至C端，依次包含所述屏蔽肽、所述抗原结合部分和所述毒素。例如，所述免疫缀合物可以从N端至C端，依次包含所述屏蔽肽、所述可剪切部分、所述抗原结合部分和所述毒素。在本申请中，所述免疫缀合物可以既具备生物毒性，又具备特异性靶向肿瘤的性质。
在本申请中，术语“连接子”通常是指用于连接两种以上蛋白质或多肽的分子。在本申请中，不同的蛋白质或多肽之间的间接连接可以通过所述连接子实现。在本申请中，所述连接子可以为肽连接子。例如，所述第二抗原结合单元可以通过所述连接子与所述毒素连接。
在本申请中，术语“N端”通常是指多肽链的末端，可以具备α-氨基的氨基酸残基。
在本申请中，术语“C端”通常是指多肽链的末端，可以具备α-羧基的氨基酸残基。对一条多肽链而言，所述N端为其一端的末端，而所述C端可以为其不同与所述N端的另一侧的末端。
在本申请中，术语“还原剂”通常是指能够引发所述第一内含肽片段与所述第二内含肽片段间的相互作用的试剂。在本申请中，所述还原剂可以引发所述第一内含肽片段与所述第二内含肽片段之间发生的蛋白质反式剪接。例如，所述还原剂可以为在氧化还原反应中失去电子的试剂。例如，所述还原剂可以为DTT(二硫苏糖醇,分子式为C 4H 10O 2S 2)。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
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。
多肽组合及其制备方法
本申请提供一种多肽组合，其包含靶向组分和毒素组分，其中：所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一内含肽片段直接或间接连接；所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；所述靶向组分与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成免疫缀合物；所述免疫缀合物包含所述屏蔽肽、所述抗原结合部分及所述毒素，其中所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述毒素直接或间接连接。
本申请还提供了一种制备免疫缀合物的方法，其包括以下步骤：1)提供靶向组分，所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一内含肽片段直接或间接连接；2)提供毒素组分，所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；3)使所述靶向组分与所述毒素组分在使得所述第一内含肽片段与所述第二内含肽片段能够相互作用的条件下接触，其中所述靶向组分与所述毒素组分通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成所述免疫缀合物。
靶向组分
在本申请中，所述靶向组分可包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，其中所述屏蔽肽可以与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分可以与所述第一内含肽片段直接或间接连接。
在本申请的所述靶向组分中，所述屏蔽肽的C端可以与所述可剪切部分的N端直接或间接连接。
在本申请中，所述屏蔽肽可以选自以下组：HER2抗原屏蔽肽、EGFR抗原屏蔽肽，EpCAM抗原屏蔽肽和Mesothelin抗原屏蔽肽。在本申请中，所述屏蔽肽可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请的所述靶向组分中，所述可剪切部分的C端可以与所述抗原结合部分的N端直接或间接连接。
在本申请中，所述可剪切部分可以选择以下组：基质金属蛋白酶敏感型连接子、丝氨酸蛋白酶敏感型连接子和尿激酶纤溶酶原激活物敏感型连接子。在本申请中，所述可剪切部分可以包含SEQ ID NO:2所示的氨基酸序列。
在本申请的所述靶向组分中，所述抗原结合部分的C端可以与所述第一内含肽片段的N端直接或间接连接。
在本申请中，所述第一内含肽片段可以包含SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述靶向组分可以靶向肿瘤特异性抗原。例如，所述肿瘤特异性抗原可以包含HER2。
在本申请中，所述抗原结合部分可以包含抗体或其抗原结合片段。例如，所述抗体可以选自以下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。又例如，所述抗原结合片段可以选自下组：Fab，Fab’，F(ab') 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。在本申请中，所述抗原结合片段可以为Fab。
第一多肽链
在本申请中，所述靶向组分可包含第一多肽链及第二多肽链，其中所述第一多肽链可包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽可与所述第一抗原结合单元通过所述可剪切部分连接。
在本申请的所述第一多肽链中，所述屏蔽肽的C端可以与所述可剪切部分的N端直接或间接连接。
在本申请的所述第一多肽链中，所述可剪切部分的C端可以与所述第一抗原结合单元的N端直接或间接连接。
在本申请中，所述第一抗原结合单元可以包含SEQ ID NO:3所示的氨基酸序列。
在本申请的所述第一多肽链中，其从N端到C端可以依次包含所述屏蔽肽、所述可剪切部分和所述第一抗原结合单元。例如，所述第一多肽链可以从N端到C端依次包含所述屏蔽肽(其可包含SEQ ID NO:1所示的氨基酸序列)、所述可剪切部分(其可包含SEQ ID NO:2所示的氨基酸序列)和所述第一抗原结合单元(可以为曲妥珠单抗的轻链，其可包含SEQ ID NO:3所示的氨基酸序列)。
例如，所述第一多肽链可以为PM-MLC，其可包含SEQ ID NO:4所示的氨基酸序列。在本申请中，所述第一多肽链可包含SEQ ID NO:4所示的氨基酸序列。
第二多肽链
在本申请中，所述第二多肽链可包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元可与所述第一内含肽片段直接或间接连接。
在本申请的所述第二多肽链中，所述第二抗原结合单元的C端可以与所述第一内含肽片段的N端直接或间接连接。
在本申请中，所述第二抗原结合单元可包含SEQ ID NO:5所示的氨基酸序列。
在本申请的所述第二多肽链中，其从N端到C端可以依次包含所述第二抗原结合单元和所述第一内含肽片段。例如，所述第二多肽链可以从N端到C端依次包含所述第二抗原结合单元(可以为曲妥珠单抗的重链的可变区和CH1，其可包含SEQ ID NO:5所示的氨基酸序列)和所述第一内含肽片段(其可包含SEQ ID NO:6所示的氨基酸序列)。
例如，所述第二多肽链可以为PM-HC-In，其可包含SEQ ID NO:7所示的氨基酸序列。在本申请中，所述第二多肽链可包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述第一多肽链中的所述第一抗原结合单元可与所述第二多肽链中的所述第二抗原结合单元相互结合形成所述抗原结合部分。例如，所述第一抗原结合单元和所述第二抗原结合单元可以源自相同的所述抗体或其抗原结合片段。
例如，所述第一抗原结合单元和所述第二抗原结合单元可以源自曲妥珠单抗。例如，所述第一抗原结合单元可以为曲妥珠单抗的轻链，且所述第二抗原结合单元可以为曲妥珠单抗的重链的可变区以及恒定区CH1区域。例如，所述第一抗原结合单元可包含SEQ ID NO:3所示的氨基酸序列，且所述第二抗原结合单元可包含SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述第一抗原结合单元可以与所述第二抗原结合单元形成Fab。
在本申请中，所述第一多肽链和所述第二多肽链可以在真核细胞的表达系统中表达。
毒素组分
在本申请中，所述毒素组分可以包含第二内含肽片段和毒素，其中所述第二内含肽片段 可以与所述毒素直接或间接连接。
在本申请中，所述第二内含肽片段可包含SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述毒素可以选自以下组：细菌毒素、人源毒素和植物毒素。例如，所述毒素可以选自以下组：绿脓杆菌外毒素和白喉毒素。例如，所述毒素可以选自以下组：蓖麻毒素、皂草素和白树毒素。在本申请中，所述毒素可以包含PE24。例如，所述毒素可以包含SEQ ID NO:9所示的氨基酸序列。
在本申请的所述毒素组分中，所述第二内含肽的C端与所述毒素的N端直接或间接连接。
在本申请中，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，所述肽连接子可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请的所述毒素组分中，其从N端到C端可以依次包含所述第二内含肽片段和所述毒素。例如，在所述毒素组分中，其从N端到C端可以依次包含所述第二内含肽片段、所述连接子和所述毒素。例如，在所述毒素组分中，其从N端到C端可以依次包含所述第二内含肽片段(其可包含SEQ ID NO:8所示的氨基酸序列)、所述连接子(其可包含SEQ ID NO:11所示的氨基酸序列)和所述毒素PE24(其可包含SEQ ID NO:9所示的氨基酸序列)。
例如，所述毒素组分可以为Ic-PE24，其可包含SEQ ID NO:10所示的氨基酸序列。在本申请中，所述毒素组分可包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述毒素组分可以在原核细胞的表达系统中表达。
在本申请中，所述靶向组分与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成免疫缀合物。
在本申请中，所述第一内含肽片段可以与所述第二内含肽片段不同。
在本申请中，所述第一内含肽片段可以和所述第二内含肽片段源自相同的内含肽。在本申请中，所述第一内含肽片段和/或所述第二内含肽片段可以包含断裂型内含肽。在本申请中，所述断裂型内含肽可以选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
在本申请中，所述第一内含肽片段可以为Npu DnaE的N端片段，且所述第二内含肽片段可以为Npu DnaE的C端部分。例如，所述第一内含肽片段和所述第二内含肽片段可以形成完整的断裂型内含肽Npu DnaE。例如，所述第一内含肽片段可包含SEQ ID NO:6所示的氨基酸序列，且所述第二内含肽片段可包含SEQ ID NO:8所示的氨基酸序列。
融合多肽
在本申请中，所述第二多肽链与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽。
在本申请中，所述相互作用可以为反式剪切作用。例如，可以为断裂型内含肽(例如，Npu DnaE)介导的蛋白质反式剪切作用。在本申请中，由于所述第一内含肽片段与所述第二内含肽片段间相互作用，所述第一内含肽片段与所述第二内含肽片段连接后被去除，从而使连接于所述第一内含肽片段的所述第二抗原结合单元与连接于所述第二内含肽片段的所述毒素相互连接，而形成包含所述第二抗原结合单元及所述毒素的融合多肽。
在本申请的所述融合多肽中，所述第二抗原结合单元可以与所述毒素直接或间接连接。在本申请的所述融合多肽中，所述第二抗原结合单元的C端可以与所述毒素的N端直接或间接连接。
在本申请中，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，所述肽连接子可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请所述的融合多肽中，其从N端到C端可以依次包含所述第二抗原结合单元和所述毒素。例如，其从N端到C端可以依次包含所述第二抗原结合单元、所述连接子和所述毒素。例如，所述融合多肽从N端到C端可以依次包含所述第二抗原结合单元(可以为曲妥珠单抗的重链的可变区和CH1，其可包含SEQ ID NO:5所示的氨基酸序列)、所述连接子(其可包含SEQ ID NO:11所示的氨基酸序列)和所述毒素PE24(其可包含SEQ ID NO:9所示的氨基酸序列)。
在本申请中，所述融合多肽可以包含SEQ ID NO:12所示的氨基酸序列。
免疫缀合物
在本申请中，所述免疫缀合物可以包含所述屏蔽肽、所述抗原结合部分及所述毒素，其中所述屏蔽肽与所述抗原结合部分可以通过所述可剪切部分连接，且所述抗原结合部分可以与所述毒素直接或间接连接。
在本申请中，所述第一多肽链可以与所述融合多肽构成免疫缀合物。
在本申请中，所述免疫缀合物可以不包含所述第一内含肽片段且可以不包含所述第二内含肽片段。
在本申请的所述免疫缀合物中，所述第一多肽链可以与所述融合多肽通过所述第一抗原结合单元与所述第二抗原结合单元的相互结合构成所述免疫缀合物。例如，所述第一抗原结合单元可以与所述第二抗原结合单元通过抗体轻链可变区与抗体重链可变区之间的二硫键而连接。例如，所述第一抗原结合单元和所述第二抗原结合单元可以源自曲妥珠单抗。例如，所述第一抗原结合单元可以为曲妥珠单抗的轻链，且所述第二抗原结合单元可以为曲妥珠单抗的重链的可变区以及恒定区CH1区域。例如，所述第一抗原结合单元可包含SEQ ID NO:3所示的氨基酸序列，且所述第二抗原结合单元可包含SEQ ID NO:5所示的氨基酸序列。在本 申请中，所述抗原结合部分可以为曲妥珠单抗的Fab。
在本申请的所述免疫缀合物中，所述屏蔽肽的C端可以与所述可剪切部分的N端直接或间接连接。
在本申请的所述免疫缀合物中，所述可剪切部分的C端可以与所述抗原结合部分的N端直接或间接连接。
在本申请的所述免疫缀合物中，所述抗原结合部分的C端可以与所述毒素的N端直接或间接连接。在本申请中，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，所述肽连接子可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请的所述免疫缀合物中，其从N端到C端可以依次包含所述屏蔽肽、所述可剪切部分、所述抗原结合部分和所述毒素。例如，其从N端到C端可以依次包含所述屏蔽肽、所述可剪切部分、所述抗原结合部分、所述连接子和所述毒素。例如，所述毒素组分从N端到C端可以依次包含所述屏蔽肽(其可包含SEQ ID NO:1所示的氨基酸序列)、所述可剪切部分(其可包含SEQ ID NO:2所示的氨基酸序列)、所述抗原结合部分(曲妥珠Fab，其可包含SEQ ID NO:3和SEQ ID NO:5所示的氨基酸序列)、所述连接子(其可包含SEQ ID NO:11所示的氨基酸序列)和所述毒素(PE24，其可包含SEQ ID NO:9所示的氨基酸序列)。
例如，所述免疫缀合物可以为M-Fab-PE24，其可包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。在本申请中，所述免疫缀合物可包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。
制备方法
在本申请中，所述方法可以包括提供所述靶向组分的步骤，该步骤可以包括：提供第一多肽链，所述第一多肽链包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽与所述第一抗原结合单元通过所述可剪切部分连接；提供第二多肽链，所述第二多肽链包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元与所述第一内含肽片段直接或间接连接；且使所述第一多肽链与所述第二多肽链结合形成所述靶向组分，其中所述第一多肽链的所述第一抗原结合单元与所述第二多肽链的所述第二抗原结合单元相互结合而形成所述靶向组分中的所述抗原结合部分。
在本申请中，所述方法的步骤3)中还可以包括使所述第二多肽链与所述毒素组分经所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽，所述融合多肽与所述第一多肽链构成所述免疫缀合物。
在本申请中，所述方法可以使用还原剂引发所述第一内含肽片段与所述第二内含肽片段间的相互作用。
例如，所述还原剂可以选自以下组：DTT和β巯基乙醇。
在本申请中，所述还原剂的浓度可以为0.1-100nM(例如，可以为约0.1-80nM、约0.1-60nM、约0.1-40nM、约0.1-20nM、约0.1-10nM或约1-10nM)。
在本申请中，所述还原剂可以与所述靶向组分和所述毒素组分相接触。在本申请中，加入还原剂的步骤可以与所述方法中的步骤1)、2)和3)同时发生或者不同时发生。在本申请中，加入所述还原剂后，所述多肽组合中可以包含具备生物毒性的毒素，和/或具备生物毒性。
在本申请中，所述制备免疫缀合物的方法还可以包括在加入所述还原剂后孵育。在本申请中，所述孵育的温度可以为约1-约50℃(例如，可以为约1℃-约50℃、约4℃-约50℃、约4℃-约45℃、约4℃-约40℃、约4℃-约37℃、约8℃-约37℃、约13℃-约37℃、约17℃-约37℃、约17℃-约35℃、约17℃-约30℃、约17℃-约25℃、约17℃-约23℃或约20℃-约23℃)。在本申请中，其中所述孵育的时间为约60-约300分钟(例如，可以为约60-约300分钟、约60-约240分钟、约60-约180分钟、约60-约120分钟、约90-约120分钟)。
载体、细胞以及制药用途
本申请提供一种载体，其包含编码所述多肽组合中的所述的第一多肽链的核酸，编码所述多肽组合中所述的第二多肽链的核酸，编码所述多肽组合中所述的毒素组分的核酸，编码所述多肽组合中所述的可剪切部分的核酸，编码所述多肽组合中所述的第一抗原结合单元的核酸，编码所述多肽组合中所述的第二抗原结合单元的核酸，编码所述多肽组合中所述的第一内含肽片段的核酸，编码所述多肽组合中所述的第二内含肽片段的核酸，编码所述多肽组合中所述的毒素的核酸和/或编码所述多肽组合中所述的屏蔽肽的核酸。
本申请提供一种细胞，其表达所述的载体。
本申请还提供一种试剂盒，其可包含所述多肽组合中的所述第一多肽链、所述第二多肽链、所述毒素组分、所述可剪切部分、所述第一抗原结合单元、所述第二抗原结合单元、所述第一内含肽片段、所述第二内含肽片段、所述毒素和所述屏蔽肽中的一种或多种。在本申请中，所述试剂盒中的组分可以相互之间不混合。例如，所述试剂盒中的组分可以放置在不同的容器和/或不同的物理空间内。
在本申请中，所述试剂盒还可以包含还原剂。所述还原剂可以选自以下组：DTT和β巯基乙醇。在本申请中，所述还原剂可以包含于独立的容器中。
本申请提供所述的多肽组合、所述的载体或所述的细胞在制备治疗疾病的药物中的用途，所述疾病包含肿瘤。
例如，所述治疗可以包含抑制或延缓包含肿瘤的发展或进展。例如，所述治疗可以减小 肿瘤的大小(甚至基本消除肿瘤)，和/或，可以减轻和/或稳定疾病状态。
在本申请中，所述肿瘤可以包括卵巢癌和乳腺癌。
本申请提供所述的多肽组合、所述的载体或所述的细胞，其治疗肿瘤。
本申请提供一种治疗肿瘤的方法，其包括施用所述的多肽组合、所述的载体或所述的细胞。
在本申请中，所述方法可以在体内条件下实现。例如，所述多肽组合中的所述第一多肽链、所述第二多肽链和所述毒素组分可以在肿瘤细胞微环境条件下形成具备生物毒性的所述免疫缀合物。在本申请中，所述免疫缀合物可以具备生物毒性，例如，可以对肿瘤细胞产生杀伤作用，或者促进肿瘤细胞的凋亡。
在本申请中，所述施用的方式可以包括口服给药、静脉内给药、肌肉内给药、在肿瘤部位的原位给药、吸入、直肠给药、阴道给药、经皮给药和/或通过皮下储存库给药。
本申请还提供向细胞施用所述多肽组合的方法，其可以包括以下的步骤：将所述第一多肽链、所述第二多肽链、所述毒素组分和所述还原剂混合施用。在体外条件下，所述第一多肽链、所述第二多肽链和所述毒素组分在还原性条件下形成所述免疫缀合物。在本申请中，所述免疫缀合物可以具备生物毒性，例如，可以对肿瘤细胞产生杀伤作用，或者促进肿瘤细胞的凋亡。
本申请还他提供向细胞施用所述免疫缀合物的方法，其可以包括以下的步骤，在体外条件下向细胞施用所述免疫缀合物。
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融合蛋白、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 抗原屏蔽型免疫毒素的制备及活化
1.1 表达载体的构建
分别以曲妥珠单抗的轻链和重链、Npu DnaE内含肽、合成的PE24基因序列为模板，设计PCR扩增引物，在插入载体引物的5’端加入限制性内切酶位点和保护碱基，用于插入表达载体使用。
将PCR合成片段与限制性双酶切后的PM09质粒通过DNA连接酶进行连接，分别形成PM-LC，PM-MLC，PM-HC-In真核载体。
其中，PM-LC真核载体表达曲妥珠单抗的轻链(LC，其氨基酸酸序列如SEQ ID NO.3所示)；PM-MLC真核载体表达连接有屏蔽肽的曲妥珠单抗的轻链(MLC，其氨基酸酸序列如 SEQ ID NO.4所示)；PM-HC-In真核载体表达连接有第一内含肽片段的曲妥珠单抗的重链(HC-In，其氨基酸酸序列如SEQ ID NO.7所示)。
将PCR合成片段与限制性双酶切后的pET32a质粒通过DNA连接酶进行连接，形成pET-Ic-PE24原核表达载体。
其中，pET-Ic-PE24原核表达载体表达连接有第二内含肽片段的PE24毒素(Ic-PE24，其氨基酸酸序列如SEQ ID NO.10所示)。
1.2 目的蛋白表达与纯化
将实施例1.1制备的载体PM-LC与PM-HC-In，或者，PM-MLC与PM-HC-In共同转染哺乳动物细胞HEK293E，从而使被转染的细胞分别表达连接有第一内含肽片段的曲妥珠Fab——Fab-In和连接有屏蔽肽以及第一内含肽片段的曲妥珠Fab——MFab-In。
转染7天后，离心收集细胞培养上清，0.45微米滤膜过滤后上Capto L柱纯化。
将实施例1.1制备的载体pET-Ic-PE24转化大肠杆菌感受态BL21(DE3)，经过IPTG诱导表达连接有第二内含肽片段的PE24毒素——Ic-PE24。收集表达菌体，高压匀质机裂菌后，离心收集上清，进行镍柱纯化。
SDS-PAGE检测纯化后的抗体片段和毒素片段。结果如图1所示，其中M代表marker，泳道1为MFab-In，泳道2为Ic-PE24，泳道3为M-Fab-In与IcPE24的混合，泳道4为MFab-In与Ic-PE24的混合，并加入1mM DTT。
1.3 抗原屏蔽型免疫毒素的制备
将实施例1.2制备的蛋白质Fab-In或MFab-In分别与实施例1.2制备的Ic-PE24反应，加入1mM DTT还原剂，诱导第一内含肽片段和第二内含肽片段介导的蛋白质反式剪接反应，37℃反应2小时后加入氧化型谷胱甘肽终止反应，氧化形成二硫键，从而分别得到免疫缀合物Fab-PE24以及M-Fab-PE24。
其中，所述免疫缀合物Fab-PE24包含曲妥珠单抗Fab和与其连接的毒素PE24，其氨基酸酸序列如SEQ ID NO.3和SEQ ID NO.12所示；所述免疫缀合物M-Fab-PE24包含屏蔽肽、可剪切部分、曲妥珠单抗Fab和与其连接的毒素PE24，其氨基酸酸序列如SEQ ID NO.4和SEQ ID NO.12所示。
SDS-PAGE检测蛋白质反式剪接反应的生成物。结果如图2所示，其中M代表marker，泳道1为M-Fab-PE24，泳道2为加入uPA(尿激酶型纤溶酶原激活物，Urokinase type plasminogen activator)进行处理的M-Fab-PE24。
1.4 抗原屏蔽型免疫毒素的活化
uPA冻干粉(作为蛋白酶，可断裂连接屏蔽肽的可剪切部分)用无菌PBS溶解，配成140 μg/ml的蛋白酶溶液。
将实施例1.3制备的M-Fab-PE24接受uPA处理，150μg M-Fab-PE24与8μL蛋白酶溶液混合，37℃水浴反应过夜。
SDS-PAGE检测反应生成物。结果如图3所示，其中泳道1为M-Fab-PE24，泳道2为加入uPA进行处理的M-Fab-PE24(又称活化的M-Fab-PE24)。
实施例2 抗原亲和力分析
用胰蛋白酶消化处于对数生长期的细胞BT-474(人乳腺癌细胞，购自
Figure PCTCN2020085736-appb-000002
HTB-20 TM)、SKOV-3(人卵巢细胞，购自
Figure PCTCN2020085736-appb-000003
HTB-77 TM)和MCF-7(人乳腺癌细胞，购自
Figure PCTCN2020085736-appb-000004
HTB-22 TM)并计数。
每个样品取1×10 6个细胞进入后续实验；4℃，1,500rpm离心5min，弃上清，重悬细胞至100μL流式工作液中；分别加入1μL曲妥珠单抗(本课题组成员表达纯化)或者实施例1制备的Fab-PE24或者M-Fab-PE24。
细胞样品4℃孵育30min；4℃，1,500rpm离心5min，弃上清，重悬于1mL PBS+2％FBS洗涤三次；4℃，1,500rpm离心5min，弃上清，重悬细胞至100μL流式工作液中；加入1μL FITC荧光标记的识别人IgG的荧光抗体。
细胞样品4℃孵育30min；4℃，1,500rpm离心5min，弃上清，重悬于1mL PBS+2％FBS洗涤三次；4℃，1,500rpm离心5min，去上清，重悬于0.5mL PBS+2％FBS并于4小时内流式细胞仪以FITC通道检测。
结果如图4所示，随着对数荧光强度的增强，M-Fab-PE24会首先脱离与肿瘤细胞BT-474、SKOV-3和MCF-7的特异性结合，进一步增强对数荧光强度，Fab-PE24和活化的M-Fab-PE24随即脱离与肿瘤细胞BT-474、SKOV-3和MCF-7的特异性结合。结果表明，相对于Fab-PE24，M-Fab-PE24的抗原亲和力明显下降，而经过蛋白酶处理后活化的M-Fab-PE24的抗原亲和力恢复至与Fab-PE24相当的水平。
实施例3 细胞毒性分析
0.25％胰酶消化处于对数生长期的肿瘤细胞BT-474、SKOV-3和MCF-7，移液器吹吸使其混匀后用移液管转移至50mL离心管中，测定细胞活率及密度。其中，用于细胞杀伤的肿瘤细胞细胞的活率需达到95％以上。
记录细胞悬液体积并将细胞收集于50mL离心管中，于台式离心机室温下1,000rpm离心5分钟；根据不同肿瘤细胞所需的铺板密度稀释细胞悬液，充分混匀细胞，加至96孔细胞 培养板中(5.0×10 3个细胞/孔)，将细胞培养板轻轻摇晃使细胞均匀分布，然后置于37℃，5％CO 2细胞培养箱中培养过夜(16-20小时)。
每组细胞分别加入实施例1制备的免疫缀合物Fab-PE24或M-Fab-PE24，37℃孵育72小时。
移除原有培养液，加入100μL新鲜培养液，10μL CCK8溶液；置于37℃培养箱孵育1小时。通过酶标仪检测450nm波长下各组细胞的吸光度值，从而计算各组细胞活力，结果参见图5。
如图5所示，随着Fab-PE24或M-Fab-PE24浓度的增加，肿瘤细胞BT-474、SKOV-3和MCF-7的细胞活率均降低。其中，与Fab-PE24相比，M-Fab-PE24所引起的细胞毒性显著下降，而经过蛋白酶处理后活化的M-Fab-PE24所引起的细胞毒性显示出了恢复。各组免疫毒素的IC 50具体参见表1。
表1
Figure PCTCN2020085736-appb-000005
实施例4 体内药效学评价
Balb/c裸鼠(购自上海斯莱克实验动物有限公司)接种SK-OV3肿瘤细胞后约10天，肿瘤体积达到平均100mm 3，进行随机分组，分为四组，分别接受PBS、实施例1制备的Fab-PE24(1.0mg/kg)、实施例1制备的M-Fab-PE24(1.0mg/kg)和NCM-Fab-PE24(1.0mg/kg)的注射。
其中，NCM-Fab-PE24与M-Fab-PE24的区别在于前者连接屏蔽肽的可剪切部分没有酶切作用位点，因此屏蔽肽不会被移除。NCM-Fab-PE24氨基酸酸序列如SEQ ID NO.13和SEQ ID NO.12所示。
静脉注射给药，每隔一天给药一次，共六次。每次给药前测量小鼠体重和肿瘤体积，给药结束后，每周测量两次小鼠体重和肿瘤体积，当最大瘤体积到达1000mm 3时，终止实验，小鼠安接受乐死处理，剥除瘤块。
各组小鼠肿瘤体积变化见图6。如图6所示，给药期间PBS对照组肿瘤生长较快，NCM- Fab-PE24也没有明显的治疗作用，Fab-PE24和M-Fab-PE24对肿瘤的抑制效果比较明显。其中，M-Fab-PE24的抑瘤效果相当于Fab-PE24。
实施例5 肿瘤分布
荷瘤Balb/c裸鼠接受实施例1制备的Fab-PE24和M-Fab-PE24的治疗过程中，第三次给药结束后(其中，治疗过程中的给药剂量、给药方式和给药频次参见实施例4)第二天对小鼠进行安乐死处理，取出肿瘤组织用OCT包埋后冷冻，使用冷冻切片机进行冷冻切片，对切片进行免疫荧光染色分析，具体步骤如下：
组织切片用预冷的4％(v/v)多聚甲醛进行固定，4℃条件下固定10min；PBS洗涤切片10min，使用吸水纸吸干PBS；封闭液(PBS含1％(v/v)马血清)孵育切片，室温30min；一抗(rabbit anti-ETA antibody)用稀释液(PBS含1％牛血清白蛋白，0.3％TritonX-100)稀释后，4℃条件下孵育切片过夜；PBS洗涤切片3次，每次15min；二抗(PE conjugated goat anti rabbit antibody)用稀释液稀释后，滴加至切片表面，室温条件下孵育切片1小时；PBS洗涤切片3次，每次15min；使用DAPI对切片进行复染，室温5min；PBS洗涤切片10min；使用荧光显微镜观察切片。
各组处理后的肿瘤分布参见图7。图7的结果显示，Fab-PE24组和M-Fab-PE24组均可观察到荧光，表明免疫缀合物Fab-PE24和M-Fab-PE24均能够渗透进入肿瘤组织，而NCM-Fab-PE24在肿瘤组织几乎没有分布。这就说明带有带有屏蔽肽的免疫缀合物M-Fab-PE24在肿瘤微环境蛋白酶作用下恢复抗原特异性结合。而接受相同抗体处理的PBS组肿瘤切片仅可观察到染核的DAPI荧光，这一结果说明图7的结果可以排除抗体本身对组织的非特异性结合的干扰。
实施例6 肝毒性分析
荷瘤Balb/c裸鼠接受实施例1制备的Fab-PE24和M-Fab-PE24的治疗过程中，第三次给药结束后(其中，治疗过程中的给药剂量、给药方式和给药频次参见实施例4)的第二天对小鼠进行眼眶采血，室温静置2小时，4000rpm离心10min取上清。
使用试剂盒(购自南京建成生物有限公司)分别检测小鼠血清中谷丙转氨酶(AST)和谷草转氨酶(ALT)的活性，通过各组小鼠的AST和ALT水平反映实施例1制备的免疫缀合物对小鼠肝脏的毒性影响。因此，AST和ALT可以作为评价所述免疫缀合物非特异性毒性的指标。
实验结果如图8所示，相对于PBS对照组，免疫缀合物Fab-PE24和M-Fab-PE24均导致 血清转氨酶活力升高，且三组之间存在显著性差异；但Fab-PE24组的AST和ALT水平显著高于M-Fab-PE24组。这就说明，Fab-PE24对肝组织的损伤效果大于M-Fab-PE24。
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 (106)

  1. 多肽组合，其包含靶向组分和毒素组分，其中：
    所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一内含肽片段直接或间接连接；
    所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；
    所述靶向组分与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成免疫缀合物；
    所述免疫缀合物包含所述屏蔽肽、所述抗原结合部分及所述毒素，其中所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述毒素直接或间接连接。
  2. 根据权利要求1所述的多肽组合，其中所述靶向组分包含第一多肽链及第二多肽链；所述第一多肽链包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽与所述第一抗原结合单元通过所述可剪切部分连接；所述第二多肽链包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元与所述第一内含肽片段直接或间接连接；且所述第一抗原结合单元与所述第二抗原结合单元能够相互结合形成所述抗原结合部分。
  3. 根据权利要求2所述的多肽组合，其中所述第二多肽链与所述毒素组分可以通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽；在所述融合多肽中，所述第二抗原结合单元与所述毒素直接或间接连接；且所述第一多肽链与所述融合多肽构成所述免疫缀合物。
  4. 根据权利要求1-3中任一项所述的多肽组合，其中所述免疫缀合物不包含所述第一内含肽片段且不包含所述第二内含肽片段。
  5. 根据权利要求1-4中任一项所述的多肽组合，其中在所述靶向组分中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
  6. 根据权利要求1-5中任一项所述的多肽组合，其中在所述靶向组分中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
  7. 根据权利要求1-6中任一项所述的多肽组合，其中在所述靶向组分中，所述抗原结合部分的C端与所述第一内含肽片段的N端直接或间接连接。
  8. 根据权利要求2-7中任一项所述的多肽组合，其中在所述第一多肽链中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
  9. 根据权利要求2-8中任一项所述的多肽组合，其中在所述第一多肽链中，所述可剪切部分的C端与所述第一抗原结合单元的N端直接或间接连接。
  10. 根据权利要求2-9中任一项所述的多肽组合，在所述第二多肽链中，所述第二抗原结合单元的C端与所述第一内含肽片段的N端直接或间接连接。
  11. 根据权利要求1-10中任一项所述的多肽组合，其中在所述毒素部分中，所述第二内含肽的C端与所述毒素的N端直接或间接连接。
  12. 根据权利要求1-11中任一项所述的多肽组合，其中在所述免疫缀合物中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
  13. 根据权利要求1-12中任一项所述的多肽组合，其中在所述免疫缀合物中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
  14. 根据权利要求1-13中任一项所述的多肽组合，其中在所述免疫缀合物中，所述抗原结合部分的C端与所述毒素的N端直接或间接连接。
  15. 根据权利要求3-14中任一项所述的多肽组合，其中在所述免疫缀合物中，所述第一多肽链与所述融合多肽通过所述第一抗原结合单元与所述第二抗原结合单元的相互结合构成所述免疫缀合物。
  16. 根据权利要求3-15中任一项所述的多肽组合，在所述融合多肽中，所述第二抗原结合单元的C端与所述毒素的N端直接或间接连接。
  17. 根据权利要求1-16中任一项所述的多肽组合，其中所述间接连接包含通过连接子连接。
  18. 根据权利要求17所述的多肽组合，其中所述连接子包含肽连接子，所述肽连接子包含SEQ ID NO:11所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的多肽组合，其中所述屏蔽肽选自以下组：HER2抗原屏蔽肽、EGFR抗原屏蔽肽，EpCAM抗原屏蔽肽和Mesothelin抗原屏蔽肽等。
  20. 根据权利要求1-18中任一项所述的多肽组合，其中所述屏蔽肽包含SEQ ID NO:1所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的多肽组合，其中所述可剪切部分选择以下组：基质金属蛋白酶敏感型连接子、丝氨酸蛋白酶敏感型连接子和尿激酶纤溶酶原激活物敏感型连接子。
  22. 根据权利要求1-21中任一项所述的多肽组合，其中所述可剪切部分包含SEQ ID NO:2所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的多肽组合，其中所述靶向组分靶向肿瘤特异性抗原。
  24. 根据权利要求23所述的多肽组合，其中所述肿瘤特异性抗原包含HER2。
  25. 根据权利要求1-24中任一项所述的多肽组合，其中所述抗原结合部分包含抗体或其抗原结合片段。
  26. 根据权利要求25所述的多肽组合，其中所述第一抗原结合单元和所述第二抗原结合单元源自相同的所述抗体或其抗原结合片段。
  27. 根据权利要求25-26中任一项所述的多肽组合，其中所述抗体选自以下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
  28. 根据权利要求25-27中任一项所述的多肽组合，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab') 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。
  29. 根据权利要求25-28中任一项所述的多肽组合，其中所述抗原结合片段为Fab。
  30. 根据权利要求2-29中任一项所述的多肽组合，其中所述第一抗原结合单元包含SEQ ID NO:3所示的氨基酸序列。
  31. 根据权利要求2-30中任一项所述的多肽组合，其中所述第二抗原结合单元包含SEQ ID NO:5所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的多肽组合，其中所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。
  33. 根据权利要求1-32中任一项所述的多肽组合，其中所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。
  34. 根据权利要求1-33中任一项所述的多肽组合，其中所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。
  35. 根据权利要求1-34中任一项所述的多肽组合，其中所述毒素包含SEQ ID NO:9所示的氨 基酸序列。
  36. 根据权利要求1-35中任一项所述的多肽组合，其中所述第一内含肽片段和/或所述第二内含肽片段包含断裂型内含肽。
  37. 根据权利要求36所述的多肽组合，其中所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
  38. 根据权利要求1-37中任一项所述的多肽组合，其中所述第一内含肽片段与所述第二内含肽片段不同。
  39. 根据权利要求1-38中任一项所述的多肽组合，其中所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。
  40. 根据权利要求1-39中任一项所述的多肽组合，其中所述第一内含肽片段包含SEQ ID NO:6所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的多肽组合，其中所述第二内含肽片段包含SEQ ID NO:8所示的氨基酸序列。
  42. 根据权利要求2-41中任一项所述的多肽组合，其中所述第一多肽链包含SEQ ID NO:4所示的氨基酸序列。
  43. 根据权利要求2-42中任一项所述的多肽组合，其中所述第二多肽链包含SEQ ID NO:7所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的多肽组合，其中所述毒素组分包含SEQ ID NO:10所示的氨基酸序列。
  45. 根据权利要求1-44中任一项所述的多肽组合，其中所述免疫缀合物包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。
  46. 根据权利要求3-45中任一项所述的多肽组合，其中所述融合多肽包含SEQ ID NO:12所示的氨基酸序列。
  47. 一种制备免疫缀合物的方法，其包括以下步骤：
    1)提供靶向组分，所述靶向组分包含屏蔽肽、可剪切部分、抗原结合部分和第一内含肽片段，所述屏蔽肽与所述抗原结合部分通过所述可剪切部分连接，且所述抗原结合部分与所述第一 内含肽片段直接或间接连接；
    2)提供毒素组分，所述毒素组分包含第二内含肽片段和毒素，且所述第二内含肽片段与所述毒素直接或间接连接；
    3)使所述靶向组分与所述毒素组分在使得所述第一内含肽片段与所述第二内含肽片段能够相互作用的条件下接触，其中所述靶向组分与所述毒素组分通过所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成所述免疫缀合物。
  48. 根据权利要求47所述的方法，其中所述提供靶向组分包括：
    提供第一多肽链，所述第一多肽链包含所述屏蔽肽和第一抗原结合单元，所述屏蔽肽与所述第一抗原结合单元通过所述可剪切部分连接；
    提供第二多肽链，所述第二多肽链包含第二抗原结合单元和所述第一内含肽片段，所述第二抗原结合单元与所述第一内含肽片段直接或间接连接；且
    使所述第一多肽链与所述第二多肽链结合形成所述靶向组分，其中所述第一多肽链的所述第一抗原结合单元与所述第二多肽链的所述第二抗原结合单元相互结合而形成所述靶向组分中的所述抗原结合部分。
  49. 根据权利要求48所述的方法，其中3)包括使所述第二多肽链与所述毒素组分经所述第一内含肽片段与所述第二内含肽片段间的相互作用而形成包含所述第二抗原结合单元及所述毒素的融合多肽，所述融合多肽与所述第一多肽链构成所述免疫缀合物。
  50. 根据权利要求47-49中任一项所述的方法，其中所述免疫缀合物不包含所述第一内含肽片段且不包含所述第二内含肽片段。
  51. 根据权利要求47-50中任一项所述的方法，其中在所述靶向组分中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
  52. 根据权利要求47-51中任一项所述的方法，其中在所述靶向组分中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
  53. 根据权利要求47-52中任一项所述的方法，其中在所述靶向组分中，所述抗原结合部分的C端与所述第一内含肽片段的N端直接或间接连接。
  54. 根据权利要求48-53中任一项所述的方法，其中在所述第一多肽链中，所述屏蔽肽的C端 与所述可剪切部分的N端直接或间接连接。
  55. 根据权利要求48-54中任一项所述的方法，其中在所述第一多肽链中，所述可剪切部分的C端与所述第一抗原结合单元的N端直接或间接连接。
  56. 根据权利要求48-55中任一项所述的方法，在所述第二多肽链中，所述第二抗原结合单元的C端与所述第一内含肽片段的N端直接或间接连接。
  57. 根据权利要求47-56中任一项所述的方法，其中在所述毒素部分中，所述第二内含肽的C端与所述毒素的N端直接或间接连接。
  58. 根据权利要求47-57中任一项所述的方法，其中在所述免疫缀合物中，所述屏蔽肽的C端与所述可剪切部分的N端直接或间接连接。
  59. 根据权利要求47-58中任一项所述的方法，其中在所述免疫缀合物中，所述可剪切部分的C端与所述抗原结合部分的N端直接或间接连接。
  60. 根据权利要求47-59中任一项所述的方法，其中在所述免疫缀合物中，所述抗原结合部分的C端与所述毒素的N端直接或间接连接。
  61. 根据权利要求49-60中任一项所述的方法，其中在所述免疫缀合物中，所述第一多肽链与所述融合多肽通过所述第一抗原结合单元与所述第二抗原结合单元的相互结合构成所述免疫缀合物。
  62. 根据权利要求49-61中任一项所述的方法，在所述融合多肽中，所述第二抗原结合单元的C端与所述毒素的N端直接或间接连接。
  63. 根据权利要求47-62中任一项所述的方法，其中所述间接连接包含通过连接子连接。
  64. 根据权利要求63所述的方法，其中所述连接子包含肽连接子，所述肽连接子包含SEQ ID NO:11所示的氨基酸序列。
  65. 据权利要求47-64中任一项所述的方法，其中所述屏蔽肽选自以下组：HER2抗原屏蔽肽、EGFR抗原屏蔽肽，EpCAM抗原屏蔽肽和Mesothelin抗原屏蔽肽。
  66. 根据权利要求47-65中任一项所述的方法，其中所述屏蔽肽包含SEQ ID NO:1所示的氨基酸序列。
  67. 根据权利要求47-66中任一项所述的方法，其中所述可剪切部分选择以下组：基质金属蛋 白酶敏感型连接子、丝氨酸蛋白酶敏感型连接子和尿激酶纤溶酶原激活物敏感型连接子。
  68. 根据权利要求47-67中任一项所述的方法，其中所述可剪切部分包含SEQ ID NO:2所示的氨基酸序列。
  69. 根据权利要求47-68中任一项所述的方法，其中所述靶向组分靶向肿瘤特异性抗原。
  70. 根据权利要求69所述的方法，其中所述肿瘤特异性抗原包含HER2。
  71. 根据权利要求47-70中任一项所述的方法，其中所述抗原结合部分包含抗体或其抗原结合片段。
  72. 根据权利要求71所述的方法，其中所述第一抗原结合单元和所述第二抗原结合单元源自相同的所述抗体或其抗原结合片段。
  73. 根据权利要求71-72中任一项所述的方法，其中所述抗体选自以下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
  74. 根据权利要求71-73中任一项所述的方法，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab') 2，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。
  75. 根据权利要求71-74中任一项所述的方法，其中所述抗原结合片段为Fab。
  76. 根据权利要求48-75中任一项所述的方法，其中所述第一抗原结合单元包含SEQ ID NO:3所示的氨基酸序列。
  77. 根据权利要求48-76中任一项所述的方法，其中所述第二抗原结合单元包含SEQ ID NO:5所示的氨基酸序列。
  78. 根据权利要求47-77中任一项所述的方法，其中所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。
  79. 根据权利要求47-78中任一项所述的方法，其中所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。
  80. 根据权利要求47-79中任一项所述的方法，其中所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。
  81. 根据权利要求47-80中任一项所述的方法，其中所述毒素包含SEQ ID NO:9所示的氨基酸序列。
  82. 根据权利要求47-81中任一项所述的方法，其中所述第一内含肽片段和/或所述第二内含肽片段包含断裂型内含肽。
  83. 根据权利要求82所述的方法，其中所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
  84. 根据权利要求47-83中任一项所述的方法，其中所述第一内含肽片段与所述第二内含肽片段不同。
  85. 根据权利要求47-84中任一项所述的方法，其中所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。
  86. 根据权利要求47-85中任一项所述的方法，其中所述第一内含肽片段包含SEQ ID NO:6所示的氨基酸序列。
  87. 根据权利要求47-86中任一项所述的方法，其中所述第二内含肽片段包含SEQ ID NO:8所示的氨基酸序列。
  88. 根据权利要求48-87中任一项所述的方法，其中所述第一多肽链包含SEQ ID NO:4所示的氨基酸序列。
  89. 根据权利要求48-88中任一项所述的方法，其中所述第二多肽链包含SEQ ID NO:7所示的氨基酸序列。
  90. 根据权利要求47-89中任一项所述的方法，其中所述毒素组分包含SEQ ID NO:10所示的氨基酸序列。
  91. 根据权利要求47-90中任一项所述的方法，其中所述免疫缀合物包含SEQ ID NO:4和SEQ ID NO:12所示的氨基酸序列。
  92. 根据权利要求49-91中任一项所述的方法，其中所述融合多肽包含SEQ ID NO:12所示的氨基酸序列。
  93. 根据权利要求47-92中任一项所述的方法，其中所述第一内含肽片段与所述第二内含肽片段间的相互作用包含反式剪接作用。
  94. 根据权利要求47-93中任一项所述的方法，其中使用还原剂引发所述第一内含肽片段与所述第二内含肽片段间的相互作用。
  95. 根据权利要求94所述的方法，其中所述还原剂选自以下组：DTT和β巯基乙醇。
  96. 根据权利要求94-95中任一项所述的方法，其中所述还原剂的浓度为0.1-100nM。
  97. 根据权利要求94-96中任一项所述的方法，其中所述还原剂与所述靶向组分和所述毒素组分相接触。
  98. 根据权利要求94-97中任一项所述的方法，其还包括在加入所述还原剂后孵育。
  99. 根据权利要求98所述的方法，其中所述孵育的温度为1-50℃。
  100. 根据权利要求47-99中任一项所述的方法，其中所述孵育的时间为60-300分钟。
  101. 载体，其包含编码根据权利要求2-46中任一项所述的第一多肽链的核酸，编码根据权利要求2-46中任一项所述的第二多肽链的核酸，编码根据权利要求1-46中任一项所述的毒素组分的核酸，编码根据权利要求1-46中任一项所述的可剪切部分的核酸，编码根据权利要求1-46中任一项所述的第一抗原结合单元的核酸，编码根据权利要求2-46中任一项所述的第二抗原结合单元的核酸，编码根据权利要求1-46中任一项所述的第一内含肽片段的核酸，编码根据权利要求1-46中任一项所述的第二内含肽片段的核酸，编码根据权利要求1-46中任一项所述的毒素的核酸，和/或，编码根据权利要求1-46中任一项所述的屏蔽肽的核酸。
  102. 细胞，其表达根据权利要求101所述的载体。
  103. 权利要求1-46中任一项所述的多肽组合、权利要求101所述的载体，或权利要求102所述的细胞在制备治疗疾病的药物中的用途，所述疾病包含肿瘤。
  104. 根据权利要求103所述的用途，所述的肿瘤包括卵巢癌和乳腺癌。
  105. 权利要求1-46中任一项所述的多肽组合、权利要求101所述的载体，或权利要求102所述的细胞，其治疗肿瘤。
  106. 治疗肿瘤的方法，其包括施用权利要求1-46中任一项所述的多肽组合、权利要求101所述的载体，或权利要求102所述的细胞。
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