WO2021190631A1 - 在哺乳动物细胞表面展示双特异性抗体的方法及载体 - Google Patents

在哺乳动物细胞表面展示双特异性抗体的方法及载体 Download PDF

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WO2021190631A1
WO2021190631A1 PCT/CN2021/083248 CN2021083248W WO2021190631A1 WO 2021190631 A1 WO2021190631 A1 WO 2021190631A1 CN 2021083248 W CN2021083248 W CN 2021083248W WO 2021190631 A1 WO2021190631 A1 WO 2021190631A1
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polynucleotide
cleaved
expression vector
component
restriction endonuclease
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French (fr)
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周辰
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DdbioCo Ltd Shang Hai
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DdbioCo Ltd Shang Hai
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Priority to BR112022019393A priority Critical patent/BR112022019393A2/pt
Priority to CN202180024152.3A priority patent/CN115315271A/zh
Priority to JP2022558475A priority patent/JP2023518898A/ja
Priority to US17/914,796 priority patent/US20230331871A1/en
Priority to KR1020227037513A priority patent/KR20220160130A/ko
Priority to MX2022012005A priority patent/MX2022012005A/es
Priority to EP21774814.4A priority patent/EP4129332A4/en
Publication of WO2021190631A1 publication Critical patent/WO2021190631A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • This application relates to the field of biomedicine, in particular to a method for constructing a bispecific antigen-binding polypeptide expression vector.
  • the conventional method of developing bispecific antibodies usually first screens specific antibodies against two targets separately, then pair each of a group of specific antibodies with each of the other group of antibodies, express and purify them, and perform physical and chemical analysis. Or biological activity and other analysis and testing.
  • There are problems such as many times of screening, high cost, long cycle, and serious deterioration of quality after long-term storage, which makes it difficult to meet the needs of industrialized mass production.
  • This application provides a method for displaying bispecific antigen-binding polypeptides (e.g., bispecific antibodies) on the surface of cells (e.g., mammalian cells).
  • a bacterial library capable of expressing different components (for example, antigen-binding polypeptides or fragments thereof, expression vector components for two or more different targets), and use restriction endonucleases to cut at specific restriction sites
  • the required components are used to obtain corresponding vector fragments, and these vector fragments can be connected to form the bispecific antigen-binding polypeptide expression vector.
  • the expression vector is transferred into a cell, and the bispecific antigen-binding polypeptide can be displayed on the cell surface, and the expression of the bispecific antigen-binding polypeptide and the binding affinity to each antigen can be directly analyzed.
  • the bispecific antigen binding protein can be successfully expressed on the surface of mammalian cells, and multiple peptide chains can be expressed simultaneously.
  • the carrier can be applied to the expression screening of bispecific antigen binding proteins in any structural form, thereby speeding up the screening and development of bispecific antibody drugs, and improving the drug-making rate.
  • the present application provides a method for constructing a bispecific antigen-binding polypeptide expression vector, the method comprising: a) providing a first polynucleotide, the first polynucleotide being 5'to The 3'direction includes S5-LC1-S6; b) provides a second polynucleotide that includes B4-VH1-B3 in the 5'to 3'direction; c) provides a third polynucleotide Acid, the third polynucleotide includes B2-LC2-B4 in a 5'to 3'direction; d) a fourth polynucleotide is provided, and the fourth polynucleotide includes a 5'to 3'direction B5-VH2-B6; e) providing a fifth polynucleotide, the fifth polynucleotide comprising S6-expression vector fragment I-B2 in a 5'to 3'direction; f) providing a sixth poly
  • the end produced by the specific cleavage of the B2 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B3, B4, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end of the B3 that is specifically cleaved by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the specific cleavage of the B4 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B3, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the specific cleavage of the B5 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B3, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end of the B6 that is specifically cleaved by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B3, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the specific cleavage of the S5 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, BB3, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the specific cleavage of the S6 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, S5, and B3. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the restriction endonuclease is selected from SfiI and BsmBI.
  • the B2, B3, B4, B5, and B6 can be specifically recognized and cleaved by BsmBI.
  • the S5 and S6 can be specifically recognized and cleaved by Sfil.
  • the B2 includes the nucleic acid sequence shown in SEQ ID NO:1.
  • the B3 includes the nucleic acid sequence shown in SEQ ID NO: 2.
  • the B4 includes the nucleic acid sequence shown in SEQ ID NO: 3.
  • the B5 includes the nucleic acid sequence shown in SEQ ID NO:4.
  • the B6 includes the nucleic acid sequence shown in SEQ ID NO:5.
  • the S5 includes the nucleic acid sequence shown in SEQ ID NO:6.
  • the S6 includes the nucleic acid sequence shown in SEQ ID NO:7.
  • the method further includes introducing the first polynucleotide into a first bacterium to obtain a bacterial library of LC1 light chain components.
  • the method includes inserting the first polynucleotide into a component vector to form an LC1 storage ligation product, and introducing the LC1 storage ligation product into the first bacteria to obtain LC1 light chain component bacteria library.
  • the method further includes obtaining a first light chain component plasmid containing the first polynucleotide from the LC1 light chain component bacterial library.
  • the method further includes obtaining the cleaved first polynucleotide from the first light chain component plasmid.
  • the method includes using a restriction endonuclease that specifically recognizes the S5 and S6 to digest the first light chain component plasmid to obtain the cleaved The first polynucleotide.
  • the method further includes introducing the second polynucleotide into a second bacterium to obtain a VH1 heavy chain component bacterial library. In some embodiments, the method includes inserting the second polynucleotide into a component vector to form a VH1 storage ligation product, and introducing the VH1 storage ligation product into the second bacteria to obtain the VH1 Bacterial library of heavy chain components. In some embodiments, the method further includes obtaining a first heavy chain component plasmid containing the second polynucleotide from the VH1 heavy chain component bacterial library. In some embodiments, the method further includes obtaining the cleaved second polynucleotide from the first heavy chain component plasmid.
  • the method further includes cutting the first heavy chain component plasmid with a restriction endonuclease that specifically recognizes the B4 and B3, so as to obtain the cut all the plasmids.
  • the second polynucleotide is a restriction endonuclease that specifically recognizes the B4 and B3, so as to obtain the cut all the plasmids.
  • the method further includes introducing the third polynucleotide into a third bacterium to obtain a bacterial library of LC2 light chain components.
  • the method includes inserting the third polynucleotide into a component vector to form an LC2 storage ligation product, and introducing the LC2 storage ligation product into the third bacteria to obtain the LC2 light Chain component bacterial library.
  • the method further includes obtaining a second light chain component plasmid containing the third polynucleotide from the LC2 light chain component bacterial library.
  • the method further includes obtaining the cleaved third polynucleotide from the second light chain component plasmid.
  • the method includes using a restriction endonuclease that specifically recognizes the B2 and B4 to digest the second light chain component plasmid, thereby obtaining the cleaved The third polynucleotide.
  • the method further includes introducing the fourth polynucleotide into a fourth bacterium to obtain a VH2 heavy chain component bacterial library.
  • the method includes inserting the fourth polynucleotide into a component vector to form a VH2 storage ligation product, and introducing the VH2 storage ligation product into the fourth bacteria to obtain the VH2 recombination product.
  • Chain component bacterial library In some embodiments, the method further includes obtaining a second heavy chain component plasmid containing the fourth polynucleotide from the VH2 heavy chain component bacterial library.
  • the method further includes obtaining the cleaved fourth polynucleotide from the second heavy chain component plasmid.
  • the method includes using a restriction endonuclease that specifically recognizes the B5 and B6 to digest the second heavy chain component plasmid, so as to obtain the cleaved The fourth polynucleotide.
  • the method further includes introducing the fifth polynucleotide into a fifth bacterium to obtain an expression vector component I bacterial library. In some embodiments, the method includes inserting the fifth polynucleotide into a component vector to form an expression vector fragment I storage ligation product, and introducing the storage ligation product into the fifth bacterium to obtain the Expression vector component I bacterial library. In some embodiments, the method further includes obtaining a display fragment component plasmid I containing the fifth polynucleotide from the expression vector component I bacterial library. In some embodiments, the method further includes obtaining the cleaved fifth polynucleotide from the display fragment component plasmid I. In some embodiments, the method includes using restriction endonucleases that specifically recognize the S6 and B2 to digest the display fragment component plasmid I, so as to obtain the cleaved first Five polynucleotides.
  • the method further includes introducing the sixth polynucleotide into a sixth bacterium to obtain an expression vector assembly II bacterial library.
  • the method includes inserting the sixth polynucleotide into a component vector to form an expression vector fragment II storage ligation product, and introducing the storage ligation product into the sixth bacteria to obtain the Expression vector component II bacterial library.
  • the method further includes obtaining a display fragment component plasmid II containing the sixth polynucleotide from the expression vector component II bacterial library.
  • the method further includes obtaining the cleaved sixth polynucleotide from the display fragment component plasmid II.
  • the method includes using restriction endonucleases that specifically recognize the B3 and B5 to digest the display fragment component plasmid II, so as to obtain the cleaved first Six polynucleotides.
  • the method further includes introducing the seventh polynucleotide into a seventh bacterium to obtain an expression vector assembly III bacterial library.
  • the method includes inserting the seventh polynucleotide into a component vector to form an expression vector fragment III storage ligation product, and introducing the storage ligation product into the seventh bacterium to obtain the Expression vector component III bacterial library.
  • the method further includes obtaining a display fragment assembly plasmid III containing the seventh polynucleotide from the expression vector assembly III bacterial library.
  • the method further includes obtaining the cleaved seventh polynucleotide from the display fragment component plasmid III.
  • the method includes using a restriction endonuclease that specifically recognizes the B6 and S5 to digest the display fragment component plasmid III, so as to obtain the cleaved first Seven polynucleotides.
  • the method includes cryopreserving the LC1 light chain component bacterial library, the LC2 light chain component bacterial library, the VH1 heavy chain component bacterial library, the VH2 heavy chain component bacterial library, The expression vector module I bacterial library, the expression vector module II bacterial library and the expression vector module III bacterial library.
  • the component vector is derived from a pUC vector.
  • the pUC vector is a pUC19 vector or is derived from a pUC19 vector.
  • the LC1 light chain component bacterial library contains at least 10 different clones.
  • the LC2 light chain component bacterial library contains at least 10 different clones.
  • the VH1 heavy chain component bacterial library contains at least 10 different clones.
  • the VH2 heavy chain component bacterial library contains at least 10 different clones.
  • the bispecific antigen-binding polypeptide expression vector contains at least 10 different clones.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and/or the fourth polynucleotide are obtained from a sample material.
  • the sample material includes antibodies or antigen-binding fragments thereof that target specific antigens.
  • the antibody or antigen-binding fragment thereof targets PD-1 and/or PD-L1.
  • the directed ligation includes the use of a ligase.
  • the ligase includes T4 DNA ligase.
  • the present application provides a bispecific antigen-binding polypeptide expression vector produced according to the described method.
  • this application provides a bispecific antigen-binding polypeptide display library constructed using the bispecific antigen-binding polypeptide expression vector.
  • the library is a mammalian cell display library.
  • the library can display at least 10 different bispecific antibodies or antibody fragments thereof.
  • this application provides a method for screening antibodies or antibody fragments, the method comprising using the library described in this application.
  • FIG 1 shows the structure of the bispecific antigen-binding polypeptide expression vector of the present application
  • Figure 2 shows the structure of the bispecific antigen-binding polypeptide expression vector as a specific example in this application
  • Figure 3 shows the expression of the bispecific antigen-binding polypeptide of the present application on the cell surface
  • Figures 4A-4J show that the bispecific antigen-binding polypeptide described in this application binds to the antigen in a dose-dependent manner.
  • the term "storage ligation product” generally refers to the product formed by ligating the polynucleotide after the digestion process and the nucleotide of the component carrier.
  • the polynucleotide and the component vector containing the recognition site of the same restriction endonuclease can be ligated by a ligase (e.g., DNA ligase) to obtain the storage ligation product.
  • a ligase e.g., DNA ligase
  • component vector generally refers to a nucleic acid that can contain a target nucleic acid (such as the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide described in this application).
  • a target nucleic acid such as the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide described in this application.
  • the fifth polynucleotide, the sixth polynucleotide and the seventh polynucleotide and can be used to introduce the nucleic acid of interest into a nucleic acid molecule inside a cell.
  • vector may include, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • an engineered vector can include an origin of replication, a restriction site, and a selectable marker.
  • the vector is usually a nucleotide sequence, usually a DNA sequence.
  • the component vector described in the present application may be derived from a plasmid vector, for example, a pUC series plasmid vector.
  • the pUC vector may be a pUC19 vector or be derived from a pUC19 vector.
  • introduction generally refers to the process of inserting an exogenous polynucleotide into a cell, and may include “transfection", “transformation” or “transduction”.
  • “Introduction” may include introduction into a eukaryotic cell or a prokaryotic cell, that is, the nucleotide can enter the cell and be converted into an autonomous replicon.
  • the cell may be a host cell.
  • the introduced cells include primary cells of the subject and their progeny.
  • the cell may be a prokaryotic cell, for example, a bacterial cell.
  • 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 heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region is usually composed of three domains, CH1, CH2, and CH3.
  • Each light chain includes a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region includes a domain, CL.
  • the VH and VL regions can be further subdivided into multiple highly variable regions, called complementarity determining regions (CDR), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL are composed of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • bispecific binding polypeptide generally refers to a polypeptide capable of specifically binding at least two different antigens (for example, a first target and a second target), which at least includes a first Fab that recognizes the first target And the second Fab that recognizes the second target.
  • Fab generally refers to the composition of a complete L chain (which may include VL and CL) together with the variable region domain (VH) of a heavy chain and the first constant domain (CH1) of a heavy chain Antigen-binding fragments. Each Fab can have a single antigen binding site.
  • the bispecific antigen binding polypeptide may include a first Fab and a second Fab.
  • the "first Fab” generally refers to an Fab that can recognize the first target of the bispecific antigen-binding polypeptide, and is usually formed by the combination of the first light chain and the first heavy chain variable region.
  • second Fab generally refers to a second Fab that can recognize the second target of the bispecific antigen-binding polypeptide, and is usually formed by the combination of the second light chain and the variable region of the second heavy chain.
  • the term "LC” generally refers to a polynucleotide comprising a nucleic acid sequence encoding the light chain or light chain fragment of the bispecific antigen-binding polypeptide.
  • the light chain or light chain fragment may have the ability to bind to the heavy chain of the same or similar antibody.
  • the light chain or light chain fragment may comprise a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region can be divided into ⁇ type and ⁇ type.
  • the light chain also includes a light chain having a ⁇ variable region (V- ⁇ ) connected to a ⁇ constant region (C- ⁇ ) or a ⁇ variable region (V- ⁇ ) connected to a ⁇ constant region (C- ⁇ ).
  • the light chain described in the present application may include a complete light chain and antigen-binding fragments thereof.
  • the polynucleotide encoding the light chain (ie, the first light chain) of the first Fab that recognizes the first target in the bispecific antigen-binding polypeptide may be referred to as LC1
  • the polynucleotide encoding the bispecific antigen-binding polypeptide recognizes
  • the polynucleotide of the light chain (ie, the second light chain) of the second Fab of the second target may be referred to as LC2.
  • VH generally refers to a polynucleotide comprising a nucleic acid sequence encoding a heavy chain variable region of a bispecific antigen-binding polypeptide.
  • the variable region of the heavy chain may have the ability to bind to the light chain of the same or similar antibody or its antigen-binding fragment.
  • the heavy chain variable region may include regions of heavy chain (H) CDR1, framework (FR)2, CDR2, FR3, CDR3, and FR4.
  • the heavy chain variable region described in the present application may include a complete heavy chain variable region and antigen-binding fragments thereof.
  • the polynucleotide encoding the heavy chain variable region of the first Fab that recognizes the first target in the bispecific antigen-binding polypeptide may be referred to as VH1, which encodes the The polynucleotide that recognizes the heavy chain variable region of the second Fab of the second target (ie, the "second heavy chain variable region") in the bispecific antigen-binding polypeptide may be referred to as VH2.
  • directed linking generally refers to the linking of different polynucleotides in one direction or one order.
  • a restriction endonuclease that specifically recognizes a cleavage site can be used to cut to form a sticky end that does not recognize or connect with a restriction endonuclease that specifically recognizes other cleavage sites.
  • the directed ligation may include the use of a ligase, for example, a DNA ligase.
  • polynucleotide generally refers to at least two nucleotides linked together.
  • the polynucleotide can be a polymer of any length, including, for example, 10, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, 100,000, and the like.
  • the polynucleotide may contain phosphodiester bonds.
  • a "polynucleotide” can be either ribonucleotide or deoxyribonucleotide or a modified form of either of the two nucleotides.
  • the polynucleotides described in this application may be linear.
  • the term "circularization” generally refers to the process of linking multiple polynucleotides end to end to form a circle.
  • the fifth polynucleotide, the cleaved sixth polynucleotide, and the cleaved seventh polynucleotide can be circularized to form the bispecific antigen-binding polypeptide expression vector.
  • the term "clone” generally refers to the number of colonies.
  • the clone may be a bacterial library (eg, the LC1 light chain component bacterial library, the LC2 light chain component bacterial library, the VH1 heavy chain component bacterial library, and/or the VH2 heavy chain component bacterial library) Or the number of colonies in the expression vector.
  • the clone may be the number of different colonies in the bacterial library.
  • the clone may be the number of progeny populations produced by a single clone.
  • cleaved polynucleotide generally refers to a polynucleotide that has been treated with a restriction endonuclease to produce sticky ends.
  • restriction endonuclease generally refers to an enzyme that cuts double-stranded DNA.
  • the restriction endonuclease can produce sticky ends with protruding single-stranded DNA, which can be bonded with DNA ligase.
  • the restriction endonuclease may have the functions of recognition and restriction cleavage.
  • the cutting site of the restriction endonuclease has a certain distance from its recognition site.
  • the restriction endonuclease can be selected from SfiI and BsmBI.
  • the term "restriction endonuclease that specifically recognizes it” generally refers to a polynucleotide that can only recognize a base sequence containing a certain recognition site, but cannot recognize a polynucleotide that contains a base sequence different from the recognition site. Restriction endonucleases based on polynucleotide sequences.
  • the term "specific cleavage” generally means that only a polynucleotide containing a base sequence at a certain recognition site can be cleaved, but not a polynucleotide containing a base sequence different from the recognition site.
  • corresponding restriction endonuclease generally refers to a restriction endonuclease capable of recognizing and cleaving the same nucleic acid sequence.
  • the ends generated after recognition and cleavage by the corresponding restriction endonucleases can generally recognize or connect to each other.
  • the term “display” generally refers to the expression of the bispecific antigen-binding polypeptide in a cell containing the bispecific antigen-binding polypeptide expression vector.
  • component plasmid generally refers to a bacterium obtained from the bacterial library and containing the polynucleotide (e.g., the first polynucleotide, the second polynucleotide, the third polynucleotide). Nucleotides, fourth polynucleotides, fifth polynucleotides, sixth polynucleotides, and/or, seventh polynucleotides).
  • the component plasmid may also include restriction endonuclease recognition sites.
  • 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 method for constructing a bispecific antigen-binding polypeptide expression vector.
  • the method may include providing polynucleotides, for example, a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide. Nucleotide, and/or, the seventh polynucleotide.
  • the polynucleotide may include a recognition site for a restriction endonuclease.
  • the recognition site sequence of the restriction endonuclease is designed not to be included in the polynucleotide encoding the antigen-binding polypeptide or a fragment thereof.
  • the restriction endonucleases of the present application can specifically recognize S5, S6, B4, B3, B2, B5, and B6, respectively. Wherein, the B2, B3, B4, B5, B6, S5 and S6 can each independently be a recognition site for restriction endonucleases.
  • restriction endonuclease recognition sites in this application can be specifically recognized by 1, 2, 3, 4, 5, 6, 7 or more restriction endonucleases, respectively .
  • the restriction endonuclease can be selected from SfiI and BsmBI. In other cases, other feasible restriction endonucleases can also be selected.
  • the recognition site of the restriction endonuclease may be a site specifically recognized and cleaved by SfiI, for example, it may be referred to as S5 and S6, respectively.
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO:6.
  • the S6 may include the nucleic acid sequence shown in SEQ ID NO:7.
  • the recognition site of the restriction endonuclease may be a site specifically recognized and cleaved by BsmBI, for example, may be referred to as B2, B3, B4, B5, and B6, respectively.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO:1.
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 2.
  • the B4 may include the nucleic acid sequence shown in SEQ ID NO: 3.
  • the B5 may include the nucleic acid sequence shown in SEQ ID NO:4.
  • the B6 may include the nucleic acid sequence shown in SEQ ID NO: 5.
  • the recognition sites of restriction endonucleases in this application include, but are not limited to, the recognition sites listed in the text, and can also include the recognition sites of other restriction endonucleases not listed, and the Other recognition sites of restriction endonucleases, as long as they do not cause undesired recognition or cleavage of the target sequence (for example, the polynucleotide encoding the antigen-binding polypeptide or fragment thereof).
  • the polynucleotides described in the present application may also include polynucleotides LC1, VH1, LC2, VH2 encoding the bispecific antigen-binding polypeptide or fragments thereof.
  • the LC1 may encode the first light chain of the bispecific antigen-binding polypeptide
  • the VH1 may encode the first heavy chain variable region of the bispecific antigen-binding polypeptide
  • the first The light chain can be combined with the variable region of the first heavy chain to form a first Fab that recognizes the first target.
  • the LC2 may encode the second light chain of the bispecific antigen-binding polypeptide
  • the VH2 may encode the second heavy chain variable region of the bispecific antigen-binding polypeptide
  • the second The light chain can be combined with the variable region of the second heavy chain to form a second Fab that recognizes the second target.
  • the first target and the second target may be antigens.
  • the combination of the first Fab and the second Fab can constitute the bispecific antigen-binding polypeptide.
  • the polynucleotides described in the present application may include expression vector fragments, such as expression vector fragment I, expression vector fragment II, and expression vector fragment III.
  • expression vector fragment I, expression vector fragment II and expression vector fragment III of the desired length or type can be selected according to the length or properties of the bispecific antigen-binding polypeptide or fragments thereof to be expressed, and the length or properties of the restriction site.
  • the expression vector fragment I, expression vector fragment II and expression vector fragment III may be vector fragments from any vector capable of expressing the gene of interest.
  • the expression vector fragment I, expression vector fragment II, and expression vector fragment III may be fragments from the display vector pDGB4 (for pDGB4, please refer to Ivan Zhou, et al., "Four-way ligation for construction of a mammalian cell- based full-length antibody display library", Acta Biochim Biophys Sin 2011, 43:232–238).
  • the expression vector fragments of the present application may contain nucleotide sequences with specific functions, including but not limited to promoters, enhancers, signal peptides, screening Markers (for example, can include enzyme recognition sites, resistance genes, reporter genes, screening genes), and those skilled in the art can adjust the expression vector fragments according to the desired function (insertion/replacement and/or deletion, etc.). Functional nucleotide sequence). In some cases, the expression vector fragments can be adjusted under different circumstances to obtain different nucleotide sequences.
  • the first polynucleotide may include S5-LC1-S6 in a 5'to 3'direction, wherein S5 and S6 may each independently be a restriction endonuclease recognition site, and the LC1 can encode the first light chain of the bispecific antigen binding polypeptide.
  • the S5 and S6 can be specifically recognized and cleaved by Sfil, respectively.
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO: 6, and the S5 may include the nucleic acid sequence shown in SEQ ID NO: 7.
  • the second polynucleotide may comprise B4-VH1-B3 in a 5'to 3'direction, wherein B4 and B3 may each independently be a restriction endonuclease recognition site, and the VH1 may encode the The first heavy chain variable region of the bispecific antigen binding polypeptide.
  • the B4 and B3 can be specifically recognized and cleaved by BsmBI, respectively.
  • the B4 may include the nucleic acid sequence shown in SEQ ID NO: 3
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 2.
  • the third polynucleotide may comprise B2-LC2-B4 in a 5'to 3'direction, wherein B2 and B4 may each independently be a restriction endonuclease recognition site, and the LC2 may encode the The second light chain of the bispecific antigen binding polypeptide.
  • the B2 and B4 can be specifically recognized and cleaved by BsmBI, respectively.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO:1
  • the B4 may include the nucleic acid sequence shown in SEQ ID NO:3.
  • the fourth polynucleotide may comprise B5-VH2-B6 in a 5'to 3'direction, wherein B5 and B6 may each independently be a restriction endonuclease recognition site, and the VH2 may encode the The second heavy chain variable region of the bispecific antigen binding polypeptide.
  • the B5 and B6 can be specifically recognized and cleaved by BsmBI, respectively.
  • the B5 may include the nucleic acid sequence shown in SEQ ID NO: 4
  • the B6 may include the nucleic acid sequence shown in SEQ ID NO: 7.
  • the fifth polynucleotide may comprise S6-expression vector fragment I-B2 in a 5'to 3'direction, wherein S6 and B2 may each independently be a restriction endonuclease recognition site.
  • S6 can be specifically recognized and cleaved by Sfil
  • B3 can be specifically recognized and cleaved by BsmBI.
  • the S6 may include the nucleic acid sequence shown in SEQ ID NO: 7
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO:1.
  • the sixth polynucleotide may comprise a B3-expression vector fragment II-B5 in a 5'to 3'direction, wherein B3 and B5 may each independently be a restriction endonuclease recognition site.
  • B3 and B5 can be specifically recognized and cleaved by BsmBI, respectively.
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 2
  • the B5 may include the nucleic acid sequence shown in SEQ ID NO: 4.
  • the seventh polynucleotide may comprise B6-expression vector fragment III-S5 in a 5'to 3'direction, wherein B6 and S5 may each independently be a restriction endonuclease recognition site.
  • B6 can be specifically recognized and cleaved by BsmBI
  • the S5 can be specifically recognized and cleaved by Sfil.
  • the B6 may include the nucleic acid sequence shown in SEQ ID NO: 5
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO: 6.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and/or the fourth polynucleotide of the present application may be obtained from sample materials.
  • the sample material may include antibodies or antigen-binding fragments thereof that target specific antigens.
  • the antigen may be any immunogenic fragment or determinant, including but not limited to PD-1, PD-L1, LAG-3, CD47, and CD3.
  • the antibody or antigen-binding fragment thereof targets PD-1 and/or PD-L1.
  • the method described in the present application may include combining the polynucleotide (e.g., the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide Acid, the sixth polynucleotide and the seventh polynucleotide) are introduced into the bacteria.
  • the polynucleotide e.g., the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide Acid, the sixth polynucleotide and the seventh polynucleotide
  • the polynucleotide may further include a nucleic acid sequence encoding a signal peptide, for example, a signal peptide expressing a natural resistance gene.
  • a nucleic acid sequence encoding a signal peptide for example, a signal peptide expressing a natural resistance gene.
  • the 3'end of the nucleic acid sequence encoding the signal peptide can be combined with an enzyme cleavage site at the 5'end of the polynucleotide.
  • its base sequence can be changed by unintentional mutation, but the amino acid sequence of the signal peptide remains unchanged.
  • the nucleic acid sequence encoding the signal peptide may include a nucleic acid sequence selected from any one of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, or the signal peptide may include a From the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.
  • the polynucleotide may be obtained according to conventional methods in the art, and the method may include, but is not limited to: standard PCR, long PCR, hot-start PCR, qPCR, RT-PCR, and isothermal amplification.
  • primers can be designed according to the sequence of the target fragments (for example, LC1, VH1, LC2, VH2, expression vector fragment I, expression vector fragment II, and expression vector fragment III), and then use this as a template respectively. Amplification is performed to obtain the polynucleotide.
  • the primer for amplifying the LC1 may include a nucleotide sequence selected from any one of SEQ ID NO: 20 and SEQ ID NO: 21.
  • the primer for amplifying the LC2 may include a nucleotide sequence selected from any one of SEQ ID NO: 22 and SEQ ID NO: 23.
  • the primer for amplifying the VH1 may include a nucleotide sequence selected from any one of SEQ ID NO: 24 and SEQ ID NO: 25.
  • the primer for amplifying the VH2 may include a nucleotide sequence selected from any one of SEQ ID NO: 26 and SEQ ID NO: 27.
  • the primer for amplifying the expression vector fragment I may comprise a nucleotide sequence selected from any one of SEQ ID NO: 14 and SEQ ID NO: 15.
  • the primer for amplifying the expression vector fragment II may comprise a nucleotide sequence selected from any one of SEQ ID NO: 16 and SEQ ID NO: 17.
  • the primer for amplifying the expression vector fragment III may comprise a nucleotide sequence selected from any one of SEQ ID NO: 18 and SEQ ID NO: 19.
  • the method in this application may further include the following steps: introducing the first polynucleotide into the first bacterium to obtain the LC1 light chain component bacterial library; introducing the second polynucleotide into the second bacterium to Obtain a VH1 heavy chain component bacterial library; introduce the third polynucleotide into a third bacterium to obtain an LC2 light chain component bacterial library; introduce the fourth polynucleotide into a fourth bacterium to obtain a VH2 heavy chain component bacteria Library; the fifth polynucleotide is introduced into the fifth bacterium to obtain the expression vector assembly I bacterial library; the sixth polynucleotide is introduced into the sixth bacterium to obtain the expression vector assembly II bacterial library; The seven polynucleotide is introduced into the seventh bacterium to obtain the expression vector module III bacterial library.
  • the polynucleotide can be inserted into a modular vector to form a storage ligation product.
  • PCR cloning can be used to insert the polynucleotide into a modular vector.
  • the component vectors may include plasmid vectors (e.g., pBR322, pUC series vectors), phage vectors (e.g., M13 vector, lambda vector), phage-derived plasmids (e.g., phagemid, cosmid), and bacterial artificial chromosomes (BAC).
  • the component vector may be derived from a pUC vector, for example, the component vector may be a pUC19 vector or derived from a pUC19 vector.
  • the storage ligation product can then be introduced into the bacteria to obtain the bacterial library.
  • the method may include inserting the first polynucleotide into a component vector to form an LC1 storage ligation product, and introducing the LC1 storage ligation product into the first bacteria to obtain an LC1 light chain component bacterial library .
  • the LC1 light chain component bacterial library may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the third polynucleotide into a component vector to form an LC2 storage ligation product, and introducing the LC2 storage ligation product into the third bacteria to obtain the LC2 light chain component bacterial library.
  • the LC2 light chain component bacterial library may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the second polynucleotide into a component vector to form a VH1 storage ligation product, and introducing the VH1 storage ligation product into the second bacteria to obtain the VH1 heavy chain component bacterial library.
  • the VH1 heavy chain component bacterial library may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the fourth polynucleotide into the component vector to form a VH2 storage ligation product, and introducing the VH2 storage ligation product into the fourth bacteria to obtain the VH2 heavy chain component bacterial library.
  • the VH2 heavy chain component bacterial library may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the fifth polynucleotide into a component vector to form an expression vector fragment I storage ligation product, and introducing the storage ligation product into the fifth bacteria to obtain the expression vector component I bacterial library .
  • the expression vector component I bacterial library may include at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the sixth polynucleotide into a component vector to form an expression vector fragment II storage ligation product, and introducing the storage ligation product into the sixth bacteria to obtain the expression vector component II bacterial library .
  • the expression vector component II bacterial library may include at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method may include inserting the seventh polynucleotide into a component vector to form an expression vector fragment III storage ligation product, and introducing the storage ligation product into the seventh bacteria to obtain the expression vector component III bacterial library .
  • the expression vector component III bacterial library may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, At least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the method described in the present application may also include cryopreserving the LC1 light chain component bacterial library, the LC2 light chain component bacterial library, the VH1 heavy chain component bacterial library, the VH2 heavy chain component bacterial library, and the expression
  • the vector component I bacterial library, the expression vector component II bacterial library, and the expression vector component III bacterial library may also include cryopreserving the LC1 light chain component bacterial library, the LC2 light chain component bacterial library, the VH1 heavy chain component bacterial library, the VH2 heavy chain component bacterial library, and the expression The vector component I bacterial library, the expression vector component II bacterial library, and the expression vector component III bacterial library.
  • the method may include obtaining a bacterial library and obtaining a component plasmid containing the polynucleotide from the bacterial library.
  • the method may include obtaining a first light chain component plasmid containing the first polynucleotide from the LC1 light chain component bacterial library, and the first light chain component plasmid may further include all Said S5 and said S6. In some cases, the method may include obtaining a second light chain component plasmid containing the third polynucleotide from the LC2 light chain component bacterial library, and the second light chain component plasmid may also include all Said B2 and said B4.
  • the method may include obtaining a first light chain component plasmid containing the second polynucleotide from the VH1 heavy chain component bacterial library, and the first heavy chain component plasmid may also include all Said B4 and said B3.
  • the method may include obtaining a second heavy chain component plasmid containing the fourth polynucleotide from the VH2 heavy chain component bacterial library, and the second heavy chain component plasmid may further include all Said B5 and said B6.
  • the method may include obtaining a display fragment assembly plasmid I containing the fifth polynucleotide from the expression vector assembly I bacterial library, and the display fragment assembly plasmid I may further include the S6 And the B2.
  • the method may include obtaining a display fragment assembly plasmid II containing the sixth polynucleotide from the expression vector assembly II bacterial library, and the display fragment assembly plasmid II may further include the B3 And the B5.
  • the method may include obtaining a display fragment assembly plasmid III containing the seventh polynucleotide from the expression vector assembly III bacterial library, and the display fragment assembly plasmid III may further include the B6 And the S5.
  • the method described in the present application may further include h) using a restriction endonuclease to specifically cleave the first polynucleotide, the second polynucleotide, the third polynucleotide, and the first polynucleotide.
  • the method may further include obtaining the cleaved polynucleotide from the component plasmid.
  • restriction endonucleases can be used to digest the plasmid to obtain the cleaved polynucleotide.
  • the method may include the following steps: obtaining the cleaved first polynucleotide from the first light chain component plasmid; obtaining the cleaved second polynucleotide from the second light chain component plasmid Acid; from the first heavy chain component plasmid to obtain the cleaved third polynucleotide; from the second heavy chain component plasmid to obtain the cleaved fourth polynucleotide; from the display
  • the fragment component plasmid I obtains the cleaved fifth polynucleotide; the display fragment component plasmid II obtains the cleaved sixth polynucleotide; the display fragment component plasmid III obtains the cleaved polynucleotide The seventh polynucleotide.
  • the restriction endonuclease that specifically recognizes the S5 and S6 can be used to digest the first light chain component plasmid, so as to obtain the cleaved first polynuclease. Glycidic acid.
  • the restriction endonuclease that specifically recognizes the B4 and B3 can be used to digest the first heavy chain component plasmid to obtain the cleaved second polynuclease. Glycidic acid.
  • the restriction endonuclease that specifically recognizes the B2 and B4 can be used to digest the second light chain component plasmid to obtain the cleaved third polynuclease. Glycidic acid.
  • the restriction endonuclease that specifically recognizes the S6 and B2 can be used to digest the display fragment component plasmid I, so as to obtain the cleaved fifth polynucleoside acid.
  • the restriction endonuclease that specifically recognizes the B3 and B5 can be used to digest the display fragment component plasmid II to obtain the cleaved sixth polynucleoside acid.
  • the restriction endonuclease that specifically recognizes the B6 and S5 can be used to digest the display fragment component plasmid III to obtain the cleaved seventh polynucleoside acid.
  • the modular plasmid can produce sticky ends at the 3'end and/or 5'end after being digested with restriction endonuclease.
  • the end produced by the specific cleavage of the B2 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B3, B4, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the restriction endonuclease that can specifically recognize B2 can only recognize or be connected to the end produced by the restriction endonuclease that can specifically recognize B2.
  • the end produced by the specific cleavage of the B3 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the restriction endonuclease that can specifically recognize B3 can only recognize or connect with the end produced by the restriction endonuclease that can specifically recognize B3.
  • the end produced by the specific cleavage of the B4 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B3, B5, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the restriction endonuclease that can specifically recognize B4 can only recognize or connect with the end produced by the restriction endonuclease that can specifically recognize B4.
  • the end produced by the specific cutting of the B5 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B3, B6, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the restriction endonuclease that can specifically recognize B5 can only recognize or connect with the end produced by the restriction endonuclease that can specifically recognize B5.
  • the end produced by the specific cleavage of the B6 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B3, S5, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other. Alternatively, the end produced by the restriction endonuclease that can specifically recognize B6 can only recognize or connect with the end produced by the restriction endonuclease that can specifically recognize B6.
  • the end produced by the specific cleavage of the S5 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, B3, and S6. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other. Alternatively, the end produced by the restriction endonuclease that can specifically recognize S5 can only recognize or connect with the end produced by the restriction endonuclease that can specifically recognize S5.
  • the end produced by the specific cleavage of the S6 by the restriction endonuclease that specifically recognizes it does not correspond to any one of the B2, B4, B5, B6, B3, and S5. Restriction endonucleases specifically cleave the resulting ends to recognize or connect to each other.
  • the end produced by the restriction endonuclease that can specifically recognize S6 can only recognize or be connected to the end produced by the restriction endonuclease that can specifically recognize S6.
  • the method may further include i) mixing the cleaved first polynucleotide, the cleaved second polynucleotide, and the cleaved third polynucleotide , The cleaved fourth polynucleotide, the cleaved fifth polynucleotide, the cleaved sixth polynucleotide, and the cleaved seventh polynucleotide, thereby So that it can be connected in a targeted manner and circularized to form the bispecific antigen-binding polypeptide expression vector.
  • the cleaved polynucleotides can be mixed in equal proportions, introduced into cells (for example, mammalian cells), colonies are picked and sequenced, so as to determine the expression vector containing the desired bispecific antigen-binding polypeptide sequence.
  • the structure of the expression vector may be as shown in Figure 1, which is formed by the directional connection and circularization of seven cut polynucleotides.
  • the 3'end of the first polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes S6 can be specifically cleaved by the restriction endonuclease that specifically recognizes S6.
  • the 5'-end ends of the fifth polynucleotides recognize and connect to each other.
  • the 3'end of the fifth polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes B2 can be specifically cleaved by the restriction endonuclease that specifically recognizes B2.
  • the ends of the third polynucleotides recognize and connect to each other.
  • the 3'end of the third polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes B4 can be specifically cleaved by the restriction endonuclease that specifically recognizes B4.
  • the 5'ends of the second polynucleotide of the second polynucleotide recognize and connect to each other.
  • the 3'end of the second polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes B3 can be specifically cleaved by the restriction endonuclease that specifically recognizes B3.
  • the 5'-end ends of the sixth polynucleotide of recognize and connect to each other.
  • the 3'end of the sixth polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes B5 can be specifically cleaved by the restriction endonuclease that specifically recognizes B5.
  • the ends of the 5'end of the fourth polynucleotide of are recognized and connected to each other.
  • the 3'end of the fourth polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes B6 can be specifically cleaved by the restriction endonuclease that specifically recognizes B6.
  • the 3'end of the seventh polynucleotide that is specifically cleaved by the restriction endonuclease that specifically recognizes S5 can be specifically cleaved by the restriction endonuclease that specifically recognizes S5.
  • the ends of the 5'ends of the first polynucleotides recognize and are connected to each other.
  • the directed ligation may include the use of a ligase.
  • the ligase may include T4 DNA ligase.
  • the bispecific antigen-binding polypeptide expression vector may comprise at least 10 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, At least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different clones.
  • the present application provides a bispecific antigen-binding polypeptide expression vector produced according to the method.
  • the bispecific antigen-binding polypeptide expression vector may include at least 10 (for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40 , At least 45, at least 50, or more) different clones.
  • the present application provides a bispecific antigen-binding polypeptide display library constructed using the bispecific antigen-binding polypeptide expression vector.
  • the display library may be a mammalian cell display library.
  • the library can display at least 10 species (e.g., at least 10 species, at least 11 species, at least 12 species, at least 13 species, at least 14 species, at least 15 species, at least 16 species, at least 17 species, At least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different bispecific antibodies or antibodies thereof Fragment.
  • the present application provides a method for screening antibodies or antibody fragments, and the method may include using the library described in the present application.
  • the display library may be a mammalian cell display library.
  • the library can display at least 10 species (e.g., at least 10 species, at least 11 species, at least 12 species, at least 13 species, at least 14 species, at least 15 species, at least 16 species, at least 17 species, At least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more) different bispecific antibodies or antibodies thereof Fragment.
  • the antibody or antibody fragment may be a bispecific antigen binding polypeptide.
  • the method may include selecting mammalian cells in the library, establishing a cell line stably expressing the bispecific antigen-binding polypeptide, and then performing screening.
  • FACS can be used to analyze the expression of the bispecific antigen-binding polypeptide on the cell surface and its specific affinity for at least two antigens.
  • the PD-1 antibody Pembrolizumab (Pembrolizumab) and the PD-L1 antibody Atezolizumab (Atezolizumab), and the pDGB4 vector were selected as the vector Example.
  • atezolizumab light chain nucleotide sequence SEQ ID NO: 30
  • Atezolizumab heavy chain variable region nucleotide sequence SEQ ID NO: 31
  • sp1 is at the 5'end of LC2
  • sp2 is at the 5'end of VH2
  • sp3 is at the 5'end of LC1.
  • the three base sequences encoding the signal peptide have been changed by unintentional mutations, but the amino acid sequence of the signal peptide remains unchanged.
  • the sequence is shown in Table 2 below:
  • the LC1, LC2-VH1, and LC2 expression frameworks are all driven by the CMV promoter. Synthetic primers, the sequence is shown in Table 3.
  • PCR Seven kinds of polynucleotides were amplified using PCR (LA Taq, Takara company, according to the company's product instructions). The templates and primer sequences used are shown in Table 3. After purification and recovery by gel electrophoresis (according to the instructions in the "Molecular Cloning Experiment Guide"), PCR products were obtained. Using the method of TA cloning (TA cloning kit, purchased from Takara), the PCR product was inserted into the pUC19 plasmid vector to obtain the storage ligation product.
  • TA cloning kit purchased from Takara
  • the storage carrier product was used to transform DH5a competent bacteria (Takara), cultured overnight at 37°C, and sent to the colony for sequencing to obtain the polynucleotide containing the desired sequence of the bacteria—the first polynucleotide containing LC1, containing The second polynucleotide of VH1, the third polynucleotide containing LC2, the fourth polynucleotide containing VH2, the fifth polynucleotide containing expression vector fragment I, the sixth polynucleotide containing expression vector fragment II The polynucleotide and the seventh polynucleotide containing the expression vector fragment III.
  • the bacteria can be frozen and stored as a bacterial library for later use.
  • the bacterial plasmids in the bacterial library in Example 1.4 were respectively extracted. Then use restriction endonucleases BsmBI and SfiI to digest the plasmid vector, use restriction endonucleases that specifically recognize S5 and S6 to cut the first polynucleotide, and use restriction endonucleases that specifically recognize B4 and B3
  • the enzyme cuts the second polynucleotide, uses the restriction endonuclease that specifically recognizes B2 and B4 to cut the third polynucleotide, and uses the restriction endonuclease that specifically recognizes B5 and B6 to cut the fourth polynuclease Utilize acid, use restriction endonuclease that specifically recognizes S6 and B2 to cut the fifth polynucleotide, use restriction endonuclease that specifically recognizes B3 and B5 to cut the sixth polynucleotide, use specificity
  • the seven cleaved polynucleotides obtained in Example 1.5 were mixed in equal molecular proportions, and ligase was added to make them ligated and circularized to form an expression vector, and then transferred to DH5a competent bacteria (Takara, Cat#, follow the manufacturer’s instructions) In the 2YT culture medium without antibiotics, incubate at 37°C, shaking at 250rpm for 60 minutes, spread ampicillin-resistant plates (Thermo, Cat#240845), and grow overnight at 37°C. The colonies are selected for sequencing, and an expression vector containing the correct sequence is obtained. The expression vector is transferred into FCHO cells to establish a cell line stably expressing the bispecific antigen-binding polypeptide to obtain a cell display library.
  • PEK PE-labeled mouse anti-human Kappa light chain antibody, to detect whether there is Kappa light chain on the cell surface
  • FITC-G FITC-labeled mouse anti-human IgG heavy chain antibody, to detect whether there is an IgG heavy chain on the cell surface
  • FITC-Ag1 FITC-labeled PD1 antigen, to detect whether the antibody displayed on the cell surface can bind to the PD1 antigen
  • FITC-Ag2 FITC-labeled PDL1 antigen, to detect whether the antibody displayed on the cell surface can bind to the PDL1 antigen.
  • FITC-G single staining more than 50% of cells have FITC signal, which means IgG heavy chain expression
  • PEK+FITC-Ag1 double staining, more than 50% of the cells have dual signals of PE and PFITC, indicating that the antibody expressed on the cell surface can bind to the PD1 antigen;
  • PEK+FITC-Ag2 double staining, more than 26% of cells have dual signals of PE and PFITC, indicating that the antibody expressed on the cell surface can bind to the PDL1 antigen;
  • PEK+FITC-Ag1+FITC-Ag2 triple staining more than 54% of cells have dual signals of PE and PFITC, although the ratio of double-positive cells only increased by 4%, the group of double-positive cells moved to the right, indicating FITC fluorescence signal Increased, it should be the result of the superimposed fluorescent signal of the antibody displayed on the cell surface that simultaneously binds to the PD1 and PDL1 antigens.
  • Example 3 The binding of the bispecific antigen-binding polypeptide to the antigen is dose-dependent
  • Example 2 using different concentrations of FITC-labeled PD1 antigen (FAg1, 0.3 ⁇ l, 1 ⁇ l, 3.3 ⁇ l) and different concentrations of FITC-labeled PDL1 antigen (FAg2, 0.3 ⁇ l, 1 ⁇ l, 3.3 ⁇ l) and expression double Cells with specific antigen-binding polypeptides are incubated together for flow cytometric analysis.
  • FIG. 4 A, non-staining negative control; B, PEK single staining; C, FAg1 single staining; D, FAg2 single staining; E, PEK+0.3 ⁇ l FAg1; F, PEK+1 ⁇ l FAg1; G, PEK+3.3 ⁇ l FAg1; H, PEK+0.3 ⁇ l FAg2; I, PEK+1 ⁇ l FAg2; J, PEK+3.3 ⁇ l Ag2.

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Abstract

提供了一种构建双特异性抗原结合多肽表达载体的方法以及根据该方法产生的双特异性抗原结合多肽表达载体，所述方法包括使用特异性识别酶切位点的限制性核酸内切酶处理得到七个具有特定粘性末端的核酸片段，使所述核酸片段定向连接。还提供了利用所述表达载体建立多肽展示文库的方法，该展示文库能够用于有效筛选抗体或抗体片段。

Description

在哺乳动物细胞表面展示双特异性抗体的方法及载体 技术领域
本申请涉及生物医药领域，具体的涉及一种用于构建双特异性抗原结合多肽表达载体的方法。
背景技术
目前，研发双特异性抗体的常规方法通常先分别筛选针对两个靶点的特异性抗体，然后将一组特异性抗体的每一个与另一组抗体的每一个配对，表达纯化后，进行理化或生物学活性等分析和检测。存在筛选次数多、成本高、周期长的问题，且长期保存后质量下降严重，难以满足工业化量产的需求。
因此，亟需能够满足工业化量产需求、质量可控且操作简便的方法来筛选双特异性抗体的方法。
发明内容
本申请提供了一种在细胞(例如，哺乳动物细胞)表面展示双特异性抗原结合多肽(例如，双特异性抗体)的方法。首先构建能够表达不同组件(例如，针对两个或两个以上不同靶点的抗原结合多肽或其片段、表达载体组件)的细菌文库，使用限制性内切核酸酶在特定的酶切位点切割所需组件得到相应的载体片段，将这些载体片段定向连接可形成所述双特异性抗原结合多肽表达载体。将该表达载体转入细胞，该细胞表面可展示双特异性抗原结合多肽，并可直接分析双特异性抗原结合多肽的表达及与每种抗原的结合亲和力。使用本申请的方法，可在哺乳动物细胞表面成功表达双特异性抗原结合蛋白，可同时表达多条肽链。所述载体可以应用于任何结构形式的双特异性抗原结合蛋白的表达筛选，从而加快双特异性抗体药物的筛选和开发速度，提高成药率。
一方面，本申请提供了一种用于构建双特异性抗原结合多肽表达载体的方法，所述方法包括：a)提供第一多核苷酸，所述第一多核苷酸以5’至3’方向包含S5-LC1-S6；b)提供第二多核苷酸，所述第二多核苷酸以5’至3’方向包含B4-VH1-B3；c)提供第三多核苷酸，所述第三多核苷酸以5’至3’方向包含B2-LC2-B4；d)提供第四多核苷酸，所述第四多核苷酸以5’至3’方向包含B5-VH2-B6；e)提供第五多核苷酸，所述第五多核苷酸以5’至 3’方向包含S6-表达载体片段I-B2；f)提供第六多核苷酸，所述第六多核苷酸以5’至3’方向包含B3-表达载体片段II-B5；g)提供第七多核苷酸，所述第七多核苷酸以5’至3’方向包含B6-表达载体片段III-S5；h)利用限制性内切核酸酶特异性切割所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸、所述第四多核苷酸、所述第五多核苷酸、所述第六多核苷酸和所述第七多核苷酸，得到切割后的所述第一多核苷酸、切割后的所述第二多核苷酸、切割后的所述第三多核苷酸、切割后的所述第四多核苷酸、切割后的所述第五多核苷酸、切割后的所述第六多核苷酸和切割后的所述第七多核苷酸；其中所述限制性内切核酸酶分别特异性识别S5、S6、B4、B3、B2、B5和B6；i)混合经所述切割后的第一多核苷酸、所述切割后的第二多核苷酸、所述切割后的第三多核苷酸、所述切割后的第四多核苷酸、所述切割后的第五多核苷酸、所述切割后的第六多核苷酸和所述切割后的第七多核苷酸，从而使得其能够定向连接而环化形成所述表达载体；其中所述LC1编码所述双特异性抗原结合多肽的第一轻链，所述VH1编码所述双特异性抗原结合多肽的第一重链可变区，且所述第一轻链可与所述第一重链可变区结合后形成识别第一靶标的第一Fab；所述LC2编码所述双特异性抗原结合多肽的第二轻链，所述VH2编码所述双特异性抗原结合多肽的第二重链可变区，且所述第二轻链可与所述第二重链可变区结合后形成识别第二靶标的第二Fab；其中所述B2、B3、B4、B5、B6、S5和S6各自独立地为限制性内切核酸酶识别位点。
在某些实施方式中，所述B2经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B3、B4、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述B3经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述B4经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B3、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述B5经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B3、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述B6经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B3、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后 产生的末端彼此识别或连接。
在某些实施方式中，所述S5经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、BB3和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述S6经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、S5和B3中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些实施方式中，所述限制性内切核酸酶选自SfiI和BsmBI。
在某些实施方式中，所述B2、B3、B4、B5和B6能够被BsmBI特异性识别及切割。
在某些实施方式中，所述S5和S6能够被Sfil特异性识别及切割。
在某些实施方式中，所述B2包含SEQ ID NO:1所示的核酸序列。
在某些实施方式中，所述B3包含SEQ ID NO:2所示的核酸序列。
在某些实施方式中，所述B4包含SEQ ID NO:3所示的核酸序列。
在某些实施方式中，所述B5包含SEQ ID NO:4所示的核酸序列。
在某些实施方式中，所述B6包含SEQ ID NO:5所示的核酸序列。
在某些实施方式中，所述S5包含SEQ ID NO:6所示的核酸序列。
在某些实施方式中，所述S6包含SEQ ID NO:7所示的核酸序列。
在某些实施方式中，所述方法还包括将所述第一多核苷酸导入第一细菌以获得LC1轻链组件细菌文库。在某些实施方式中，所述方法包括将所述第一多核苷酸插入组件载体形成LC1存储连接产物，并且将所述LC1存储连接产物导入所述第一细菌以获得LC1轻链组件细菌文库。在某些实施方式中，所述方法还包括由所述LC1轻链组件细菌文库获得包含所述第一多核苷酸的第一轻链组件质粒。在某些实施方式中，所述方法还包括由所述第一轻链组件质粒获得切割后的所述第一多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述S5和S6的限制性内切核酸酶对所述第一轻链组件质粒进行酶切处理，从而获得所述切割后的所述第一多核苷酸。
在某些实施方式中，所述方法还包括将所述第二多核苷酸导入第二细菌以获得VH1重链组件细菌文库。在某些实施方式中，所述方法包括将所述第二多核苷酸插入组件载体，形成VH1存储连接产物，并将所述VH1存储连接产物导入所述第二细菌中以获得所述VH1重链组件细菌文库。在某些实施方式中，所述方法还包括由所述VH1重链组件细菌文库获得包含所述第二多核苷酸的第一重链组件质粒。在某些实施方式中，所述方法还包括由所述第一重 链组件质粒获得切割后的所述第二多核苷酸。在某些实施方式中，所述方法还包括用特异性识别所述B4和B3的限制性内切核酸酶对所述第一重链组件质粒进行酶切处理，从而获得所述切割后的所述第二多核苷酸。
在某些实施方式中，所述方法还包括将所述第三多核苷酸导入第三细菌以获得LC2轻链组件细菌文库。在某些实施方式中，所述方法包括将所述第三多核苷酸插入组件载体形成LC2存储连接产物，并将所述LC2存储连接产物导入所述第三细菌中以获得所述LC2轻链组件细菌文库。在某些实施方式中，所述方法还包括由所述LC2轻链组件细菌文库获得包含所述第三多核苷酸的第二轻链组件质粒。在某些实施方式中，所述方法还包括由所述第二轻链组件质粒获得切割后的所述第三多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述B2和B4的限制性内切核酸酶对所述第二轻链组件质粒进行酶切处理，从而获得所述切割后的所述第三多核苷酸。
在某些实施方式中，所述方法还包括将所述第四多核苷酸导入第四细菌以获得VH2重链组件细菌文库。在某些实施方式中，所述方法包括将所述第四多核苷酸插入组件载体形成VH2存储连接产物，并将所述VH2存储连接产物导入所述第四细菌中以获得所述VH2重链组件细菌文库。在某些实施方式中，所述方法还包括由所述VH2重链组件细菌文库获得包含所述第四多核苷酸的第二重链组件质粒。在某些实施方式中，所述方法还包括由所述第二重链组件质粒获得切割后的所述第四多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述B5和B6的限制性内切核酸酶对所述第二重链组件质粒进行酶切处理，从而获得所述切割后的所述第四多核苷酸。
在某些实施方式中，所述方法还包括将所述第五多核苷酸导入第五细菌以获得表达载体组件I细菌文库。在某些实施方式中，所述方法包括将所述第五多核苷酸插入组件载体形成表达载体片段I存储连接产物，并将所述存储连接产物导入所述第五细菌中以获得所述表达载体组件I细菌文库。在某些实施方式中，所述方法还包括由所述表达载体组件I细菌文库获得包含所述第五多核苷酸的展示片段组件质粒I。在某些实施方式中，所述方法还包括由所述展示片段组件质粒I获得切割后的所述第五多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述S6和B2的限制性内切核酸酶对所述展示片段组件质粒I进行酶切处理，从而获得所述切割后的所述第五多核苷酸。
在某些实施方式中，所述方法还包括将所述第六多核苷酸导入第六细菌以获得表达载体组件II细菌文库。在某些实施方式中，所述方法包括将所述第六多核苷酸插入组件载体形成表达载体片段II存储连接产物，并将所述存储连接产物导入所述第六细菌中以获得所述表达 载体组件II细菌文库。在某些实施方式中，所述方法还包括由所述表达载体组件II细菌文库获得包含所述第六多核苷酸的展示片段组件质粒II。在某些实施方式中，所述方法还包括由所述展示片段组件质粒II获得切割后的所述第六多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述B3和B5的限制性内切核酸酶对所述展示片段组件质粒II进行酶切处理，从而获得所述切割后的所述第六多核苷酸。
在某些实施方式中，所述方法还包括将所述第七多核苷酸导入第七细菌以获得表达载体组件III细菌文库。在某些实施方式中，所述方法包括将所述第七多核苷酸插入组件载体形成表达载体片段III存储连接产物，并将所述存储连接产物导入所述第七细菌中以获得所述表达载体组件III细菌文库。在某些实施方式中，所述方法还包括由所述表达载体组件III细菌文库获得包含所述第七多核苷酸的展示片段组件质粒III。在某些实施方式中，所述方法还包括由所述展示片段组件质粒III获得切割后的所述第七多核苷酸。在某些实施方式中，所述方法包括使用特异性识别所述B6和S5的限制性内切核酸酶对所述展示片段组件质粒III进行酶切处理，从而获得所述切割后的所述第七多核苷酸。
在某些实施方式中，所述方法包括冷冻保存所述LC1轻链组件细菌文库、所述LC2轻链组件细菌文库、所述VH1重链组件细菌文库、所述VH2重链组件细菌文库、所述表达载体组件I细菌文库、所述表达载体组件II细菌文库和所述表达载体组件III细菌文库。
在某些实施方式中，所述组件载体源自pUC载体。
在某些实施方式中，所述pUC载体为pUC19载体或源自pUC19载体。
在某些实施方式中，所述LC1轻链组件细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述LC2轻链组件细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述VH1重链组件细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述VH2重链组件细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述双特异性抗原结合多肽表达载体包含至少10个不同的克隆。
在某些实施方式中，由样品材料获得所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸和/或所述第四多核苷酸。
在某些实施方式中，所述样品材料包括靶向特异性抗原的抗体或其抗原结合片段。
在某些实施方式中，所述抗体或其抗原结合片段靶向PD-1和/或PD-L1。
在某些实施方式中，所述定向连接包括使用连接酶。
在某些实施方式中，所述连接酶包括T4DNA连接酶。
另一方面，本申请提供了根据所述的方法所产生的双特异性抗原结合多肽表达载体。
另一方面，本申请提供了利用所述的双特异性抗原结合多肽表达载体建立的双特异性抗原结合多肽展示文库。
在某实施方式中，所述文库为哺乳动物细胞展示文库。
在某实施方式中，所述文库能够展示至少10种不同的双特异性抗体或其抗体片段。
另一方面，本申请提供了筛选抗体或抗体片段的方法，所述方法包括使用本申请所述的文库。
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双特异性抗原结合多肽表达载体的结构；
图2显示的是本申请中作为具体示例的双特异性抗原结合多肽表达载体的结构；
图3显示的是本申请所述双特异性抗原结合多肽在细胞表面表达；
图4A-4J显示的是本申请所述双特异性抗原结合多肽与抗原结合呈剂量依赖性。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“存储连接产物”通常是指经所述经酶切处理的多核苷酸和所述组件载体的核苷酸连接形成的产物。在本申请中，所述多核苷酸与包含相同限制性内切核酸酶的识 别位点的所述组件载体可以被连接酶(例如DNA连接酶)连接，而得到所述存储连接产物。
在本申请中，术语“组件载体”通常是指能包含目的核酸(如本申请所述第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸和第七多核苷酸)并且可以用于将该目的核酸导入细胞内部的核酸分子。术语“载体”可包括(但不限于)质粒、病毒、粘粒和人工染色体。一般而言，工程改造的载体可包含复制起点、酶切位点和可选择标记。载体通常为核苷酸序列，一般为DNA序列。在某些情形中，本申请所述组件载体可以源自质粒载体，例如，pUC系列质粒载体，又例如，所述pUC载体可以为pUC19载体或源自pUC19载体。
在本申请中，术语“导入”通常是指将外源多核苷酸插入细胞中的过程，可包括“转染”、“转化”或“转导”。“导入”可包括导入真核细胞或原核细胞中，即该核苷酸可进入该细胞中转变成自主复制子。所述细胞可以为宿主细胞。所述导入的细胞包括对象的初级细胞及其后代。所述细胞可以为原核细胞，例如，可以为细菌细胞。
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。每一条重链均包括一个重链可变区(VH)以及一个重链恒定区。重链恒定区通常由三个结构域CH1、CH2以及CH3构成。每一条轻链均包括一个轻链可变区(VL)以及一个轻链恒定区。轻链恒定区包括一个结构域，CL。VH以及VL区可以进一步被细分为多个高可变性区，被称为互补决定区(CDR)，其间散布有更为保守的被称为框架区(FR)的多个区。每个VH以及VL均由三个CDR及四个FR构成，按照以下顺序从氨基端至羧基端排布：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链及轻链的可变区包含与抗原相互作用的结合结构域。
在本申请中，术语“双特异性结合多肽”通常是指能够特异性结合至少两种不同抗原(例如，第一靶标和第二靶标)的多肽，其至少包含识别第一靶标的第一Fab和识别第二靶标的第二Fab。
在本申请中，术语“Fab”通常是指由完整L链(可包含VL和CL)连同一条重链的可变区结构域(VH)和一条重链的第一恒定域(CH1)组成的抗原结合片段。每个Fab可以具有单一的抗原结合位点。所述双特异性抗原结合多肽可以包括第一Fab和第二Fab。所述“第一Fab”通常是指可以识别所述双特异性抗原结合多肽的第一靶标的Fab，通常由所述第一轻链与所述第一重链可变区结合形成。所述“第二Fab”通常是指可以识别所述双特异 性抗原结合多肽的第二靶标的第二Fab，通常由所述第二轻链与所述第二重链可变区结合形成。
在本申请中，术语“LC”通常是指包含编码所述双特异性抗原结合多肽的轻链或轻链片段的核酸序列的多核苷酸。在本申请中，所述轻链或轻链片段可具备与同一或类似抗体的重链相结合的能力。在本申请中，所述轻链或轻链片段可包含轻链可变区(VL)和轻链恒定区(CL)。所述轻链恒定区可以分为κ型和λ型。所述轻链还包括具有与κ恒定区(C-κ)相连的λ可变区(V-λ)或与λ恒定区(C-λ)相连的κ可变区(V-κ)的轻链。本申请所述轻链可包括完整的轻链及其抗原结合片段。其中，编码所述双特异性抗原结合多肽中识别第一靶标的第一Fab的轻链(即第一轻链)的多核苷酸可以称为LC1，编码所述双特异性抗原结合多肽中识别第二靶标的第二Fab的轻链(即第二轻链)的多核苷酸可以称为LC2。
在本申请中，术语“VH”通常是指包含编码双特异性抗原结合多肽的重链可变区的核酸序列的多核苷酸。在本申请中，所述重链可变区可具备与同一或类似抗体的轻链或其抗原结合片段相结合的能力。所述重链可变区可包含重链(H)CDR1、框架(FR)2、CDR2、FR3、CDR3和FR4的区域。本申请所述重链可变区可包括完整的重链可变区及其抗原结合片段。其中，编码所述双特异性抗原结合多肽中识别第一靶标的第一Fab的重链可变区(即“第一重链可变区”)的多核苷酸可以称为VH1，编码所述双特异性抗原结合多肽中识别第二靶标的第二Fab的重链可变区(即“第二重链可变区”)的多核苷酸可以称为VH2。
在本申请中，术语“定向连接”通常是指不同多核苷酸按照一个方向或一个顺序依次连接。在本申请中，可以通过使用特异性识别一切割位点的限制性内切核酸酶，切割形成不与特异性识别其他的切割位点的限制性内切核酸酶彼此识别或连接的粘性末端，来保证多核苷酸的定向连接。所述定向连接可包括使用连接酶，例如，DNA连接酶。
在本申请中，术语“多核苷酸”通常是指连接在一起的至少两个核苷酸。所述多核苷酸可以是任何长度的聚合物，包括例如10、100、200、300、500、1000、2000、3000、5000、7000、10,000、100,000等。所述多核苷酸可以含有磷酸二酯键。“多核苷酸”可以是核糖核苷酸或脱氧核糖核苷酸或修饰形式的所述两种核苷酸中的任意一种。本申请所述多核苷酸可以是线性的。
在本申请中，术语“环化”通常是指多个多核苷酸首尾相连形成环形的过程。例如，本申请中的所述切割后的第一多核苷酸、切割后的第二多核苷酸、切割后的第三多核苷酸、切割后的第四多核苷酸、切割后的第五多核苷酸、切割后的第六多核苷酸和切割后的第七多核苷酸可环化形成所述双特异性抗原结合多肽表达载体。
在本申请中，术语“克隆”通常是指菌落的数量。例如，所述克隆可以为细菌文库(例如，所述LC1轻链组件细菌文库、所述LC2轻链组件细菌文库、所述VH1重链组件细菌文库和/或所述VH2重链组件细菌文库)或表达载体中菌落的数量。在某些情形下，所述克隆可以为所述细菌文库中不同的菌落的数量。在某些情形下，所述克隆可以为某一单一克隆所产生的子代群体的数量。
在本申请中，术语“切割后的”多核苷酸通常是指经过限制性内切核酸酶处理后产生粘性末端的多核苷酸。
在本申请中，术语“限制性内切核酸酶”通常是指一种将双股DNA切开的酶。所述限制性内切核酸酶可以产生具有突出单股DNA的黏性末端，从而可以与DNA连接酶黏合。在本申请中，所述限制性内切核酸酶可以具备识别和限制性切割的作用。例如，所述限制性内切核酸酶的切割位点距离其识别位点存在一定的距离。例如，所述限制性内切核酸酶可以选自SfiI和BsmBI。
在本申请中，术语“特异识别其的限制性内切核酸酶”通常是指只能识别含有某一识别位点的碱基序列的多核苷酸而不能识别含有与该识别位点不同的碱基序列的多核苷酸的限制性内切核酸酶。
在本申请中，术语“特异性切割”通常是指只能切割含有某一识别位点的碱基序列的多核苷酸而不能切割含有与该识别位点不同的碱基序列的多核苷酸。
在本申请中，术语“相应限制性内切核酸酶”通常是指能够识别和切割相同核酸序列的限制内切核酸酶。所述相应限制性内切核酸酶识别和切割后产生的末端通常能够彼此识别或连接。
在本申请中，术语“展示”通常是指在使包含所述双特异性抗原结合多肽表达载体的细胞表达所述双特异性抗原结合多肽。
在本申请中，术语“组件质粒”通常是指从所述细菌文库的细菌中获得的、包含所述多核苷酸(如，第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸，和/或，第七多核苷酸)的质粒。所述组件质粒还可以包含限制性内切核酸酶的识别位点。
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。
发明详述
一方面，本申请提供了一种用于构建双特异性抗原结合多肽表达载体的方法。
获得多核苷酸
所述方法可包括提供多核苷酸，例如，第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸，和/或，第七多核苷酸。
1)酶切位点
所述多核苷酸可包含限制性内切核酸酶的识别位点。本申请中，所述限制性内切核酸酶的识别位点序列被设计为不包含在编码所述抗原结合多肽或其片段的多核苷酸中。本申请的所述限制性内切核酸酶可分别特异性识别S5、S6、B4、B3、B2、B5和B6。其中，所述B2、B3、B4、B5、B6、S5和S6可以各自独立地为限制性内切核酸酶的识别位点。
本申请中的限制性内切核酸酶识别位点可以分别被1种、2种、3种、4种、5种、6种、7种或7种以上的限制性内切核酸酶特异性识别。在某些情形中，所述限制性内切核酸酶可选自SfiI和BsmBI。在其他情形中，也可选择其他可行的限制性内切核酸酶。
在某些情形中，所述限制性核酸内切酶的识别位点可以是被SfiI特异性识别并切割的位点，例如，可分别被称为S5和S6。例如，所述S5可包含SEQ ID NO:6所示的核酸序列。又例如，所述S6可包含SEQ ID NO:7所示的核酸序列。
所述限制性核酸内切酶的识别位点可以是被BsmBI特异性识别并切割的位点，例如，可分别被称为B2、B3、B4、B5和B6。例如，所述B2可包含SEQ ID NO:1所示的核酸序列。再例如，所述B3可包含SEQ ID NO:2所示的核酸序列。又例如，所述B4可包含SEQ ID NO:3所示的核酸序列。又例如，所述B5可包含SEQ ID NO:4所示的核酸序列。又例如，所述B6可包含SEQ ID NO:5所示的核酸序列。
应注意的是，本申请限制性核酸内切酶的识别位点包括但不限于文中列举出的识别位点，还可包含没有列举出的其他限制性内切酶的识别位点，以及所述限制性内切酶的其他识别位点，只要其不会对目标序列(如，编码所述抗原结合多肽或其片段的多核苷酸)造成不期望的识别或切割即可。
2)双特异性抗原结合多肽
本申请所述的多核苷酸(例如，第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸和第七多核苷酸)还可包括编码所述双特异性抗原结合多肽或其片段的多核苷酸LC1、VH1、LC2、VH2。
在本申请中，所述LC1可编码所述双特异性抗原结合多肽的第一轻链，所述VH1可编 码所述双特异性抗原结合多肽的第一重链可变区，所述第一轻链可与所述第一重链可变区结合后形成识别第一靶标的第一Fab。在本申请中，所述LC2可编码所述双特异性抗原结合多肽的第二轻链，所述VH2可编码所述双特异性抗原结合多肽的第二重链可变区，所述第二轻链可与所述第二重链可变区结合后形成识别第二靶标的第二Fab。在某些情形中，所述第一靶标和第二靶标可以是抗原。
在本申请中，所述第一Fab和第二Fab结合后可以构成所述双特异性抗原结合多肽。
本申请所述的多核苷酸(例如，第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸和第七多核苷酸)可包括表达载体片段，如，表达载体片段I、表达载体片段II和表达载体片段III。可以根据要表达的双特异性抗原结合多肽或其片段的长度或性质、酶切位点的长度或性质分别选择所需长度或种类的表达载体片段I、表达载体片段II和表达载体片段III。
在某些情形中，所述表达载体片段I、表达载体片段II和表达载体片段III可以是来自任何一个能够表达目的基因的载体的载体片段。例如，所述表达载体片段I、表达载体片段II和表达载体片段III可以是来自展示载体pDGB4的片段(关于pDGB4请参见Ivan Zhou,et al.,“Four-way ligation for construction of a mammalian cell-based full-length antibody display library”,Acta Biochim Biophys Sin 2011,43:232–238)。
本申请的表达载体片段(例如，表达载体片段I、表达载体片段II和表达载体片段III)可包含具有特定功能的核苷酸序列，包括但不限于，启动子、增强子、信号肽、筛选标记(例如，可包括酶的识别位点、抗性基因、报告基因、筛选基因)，本领域技术人员可根据所期望功能在表达载体片段中调整(插入/替换和/或删除等上述具有特定功能的核苷酸序列)。在某些情况下，所述的表达载体片段可以在不同的情况下被调整而得到不同的核苷酸序列。
在本申请中，所述第一多核苷酸以5’至3’方向可包含S5-LC1-S6，其中，S5和S6可以各自独立地为限制性内切核酸酶识别位点，所述LC1可以编码所述双特异性抗原结合多肽的第一轻链。在某些情形中，所述S5和S6可以分别被Sfil特异性识别及切割。例如，所述S5可包含SEQ ID NO:6所示的核酸序列，所述S5可包含SEQ ID NO:7所示的核酸序列。
所述第二多核苷酸以5’至3’方向可包含B4-VH1-B3，其中，B4和B3可以各自独立地为限制性内切核酸酶识别位点，所述VH1可以编码所述双特异性抗原结合多肽的第一重链可变区。在某些情形中，所述B4和B3可以分别被BsmBI特异性识别及切割。例如，所述B4可包含SEQ ID NO:3所示的核酸序列，所述B3可包含SEQ ID NO:2所示的核酸序列。
所述第三多核苷酸以5’至3’方向可包含B2-LC2-B4，其中，B2和B4可以各自独立地为 限制性内切核酸酶识别位点，所述LC2可以编码所述双特异性抗原结合多肽的第二轻链。在某些情形中，所述B2和B4可以分别被BsmBI特异性识别及切割。例如，所述B2可包含SEQ ID NO:1所示的核酸序列，所述B4可包含SEQ ID NO:3所示的核酸序列。
所述第四多核苷酸以5’至3’方向可包含B5-VH2-B6，其中，B5和B6可以各自独立地为限制性内切核酸酶识别位点，所述VH2可以编码所述双特异性抗原结合多肽的第二重链可变区。在某些情形中，所述B5和B6可以分别被BsmBI特异性识别及切割。例如，所述B5可包含SEQ ID NO:4所示的核酸序列，所述B6可包含SEQ ID NO:7所示的核酸序列。
所述第五多核苷酸以5’至3’方向可包含S6-表达载体片段I-B2，其中，S6和B2可以各自独立地为限制性内切核酸酶识别位点。在某些情形中，所述S6可以被Sfil特异性识别及切割，所述B3可以被BsmBI特异性识别及切割。例如，所述S6可包含SEQ ID NO:7所示的核酸序列，所述B2可包含SEQ ID NO:1所示的核酸序列。
所述第六多核苷酸以5’至3’方向可包含B3-表达载体片段II-B5，其中，B3和B5可以各自独立地为限制性内切核酸酶识别位点。在某些情形中，所述B3和B5可以分别被BsmBI特异性识别及切割。例如，所述B3可包含SEQ ID NO:2所示的核酸序列，所述B5可包含SEQ ID NO:4所示的核酸序列。
所述第七多核苷酸以5’至3’方向可包含B6-表达载体片段III-S5，其中，B6和S5可以各自独立地为限制性内切核酸酶识别位点。在某些情形中，所述B6可以被BsmBI特异性识别及切割，所述S5可以被Sfil特异性识别及切割。例如，所述B6可包含SEQ ID NO:5所示的核酸序列，所述S5可包含SEQ ID NO:6所示的核酸序列。
本申请所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸和/或所述第四多核苷酸可以由样品材料获得。在某些情形中，所述样品材料可以包括靶向特异性抗原的抗体或其抗原结合片段。所述抗原可以是任何免疫原性片段或决定簇，包括但不限于，PD-1、PD-L1、LAG-3、CD47、CD3。例如，所述抗体或其抗原结合片段靶向PD-1和/或PD-L1。
本申请所述的方法可包括将所述多核苷酸(例如，第一多核苷酸、第二多核苷酸、第三多核苷酸、第四多核苷酸、第五多核苷酸、第六多核苷酸和第七多核苷酸)导入细菌。
为了筛选导入了所述多核苷酸的阳性细菌，所述多核苷酸(例如，)还可包含编码信号肽的核酸序列，例如，表达天然抗性基因的信号肽。在一个实施例中，所述编码信号肽的核酸序列的3’端可以与所述多核苷酸5’端的酶切位点结合。在某些情形中，为了在编码信号肽的核酸序列的3’端部分引入合适的酶切位点，其碱基序列可通过无意突变而改变，但信号肽的氨基酸序列保持不变。例如，所述编码信号肽的核酸序列可以包含选自SEQ ID NO:8、SEQ  ID NO:10和SEQ ID NO:12中任一项所示的核酸序列，或者，所述信号肽可以包含选自SEQ ID NO:9、SEQ ID NO:11和SEQ ID NO:13中任一项所示的氨基酸序列。
可以根据本领域常规方法获得所述多核苷酸，所述方法可包括，但不限于：标准PCR、长PCR、热启动PCR、qPCR、RT-PCR和等温扩增。在某些情形中，可以根据所述目标片段(例如，LC1、VH1、LC2、VH2、表达载体片段I、表达载体片段II和表达载体片段III)的序列分别设计引物，再以此为模板分别进行扩增，以得到所述的多核苷酸。例如，扩增所述LC1的引物可包含选自SEQ ID NO:20和SEQ ID NO:21中任一项所示的核苷酸序列。例如，扩增所述LC2的引物可包含选自SEQ ID NO:22和SEQ ID NO:23中任一项所示的核苷酸序列。例如，扩增所述VH1的引物可包含选自SEQ ID NO:24和SEQ ID NO:25中任一项所示的核苷酸序列。例如，扩增所述VH2的引物可包含选自SEQ ID NO:26和SEQ ID NO:27中任一项所示的核苷酸序列。例如，扩增所述表达载体片段I的引物可包含选自SEQ ID NO:14和SEQ ID NO:15中任一项所示的核苷酸序列。例如，扩增所述表达载体片段II的引物可包含选自SEQ ID NO:16和SEQ ID NO:17中任一项所示的核苷酸序列。例如，扩增所述表达载体片段III的引物可包含选自SEQ ID NO:18和SEQ ID NO:19中任一项所示的核苷酸序列。
建立细菌文库
获得所述多核苷酸后，可以将其分别导入细菌以获得细菌文库。所以本申请中的所述方法还可包括以下步骤：将所述第一多核苷酸导入第一细菌以获得LC1轻链组件细菌文库；将所述第二多核苷酸导入第二细菌以获得VH1重链组件细菌文库；将所述第三多核苷酸导入第三细菌以获得LC2轻链组件细菌文库；将所述第四多核苷酸导入第四细菌以获得VH2重链组件细菌文库；将所述第五多核苷酸导入第五细菌以获得表达载体组件I细菌文库；将所述第六多核苷酸导入第六细菌以获得表达载体组件II细菌文库；将所述第七多核苷酸导入第七细菌以获得表达载体组件III细菌文库。
在某些情形中，可以将所述多核苷酸插入组件载体以形成存储连接产物。在某些情形中,可以使用PCR克隆将所述多核苷酸插入组件载体。所述组件载体可以包括质粒载体(如，pBR322，pUC系列载体)，噬菌体载体(如，M13载体，λ载体)，噬菌体衍生质粒(如，phagemid，cosmid)，细菌人工染色体(BAC)。在某些实施方式中，所述组件载体可以源自pUC载体，例如，所述组件载体可以为pUC19载体或源自pUC19载体。
然后可以将所述存储连接产物导入所述细菌，以获得所述细菌文库。
在本申请中，所述方法可包括将所述第一多核苷酸插入组件载体形成LC1存储连接产物， 并且将所述LC1存储连接产物导入所述第一细菌以获得LC1轻链组件细菌文库。在某些情形中，所述LC1轻链组件细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第三多核苷酸插入组件载体形成LC2存储连接产物，并将所述LC2存储连接产物导入所述第三细菌中以获得所述LC2轻链组件细菌文库。在某些情形中，所述LC2轻链组件细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第二多核苷酸插入组件载体，形成VH1存储连接产物，并将所述VH1存储连接产物导入所述第二细菌中以获得所述VH1重链组件细菌文库。在某些情形中，所述VH1重链组件细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第四多核苷酸插入组件载体形成VH2存储连接产物，并将所述VH2存储连接产物导入所述第四细菌中以获得所述VH2重链组件细菌文库。在某些情形中，所述VH2重链组件细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第五多核苷酸插入组件载体形成表达载体片段I存储连接产物，并将所述存储连接产物导入所述第五细菌中以获得所述表达载体组件I细菌文库。在某些情形中，所述表达载体组件I细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第六多核苷酸插入组件载体形成表达载体片段II存储连接产物， 并将所述存储连接产物导入所述第六细菌中以获得所述表达载体组件II细菌文库。在某些情形中，所述表达载体组件II细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
所述方法可包括将所述第七多核苷酸插入组件载体形成表达载体片段III存储连接产物，并将所述存储连接产物导入所述第七细菌中以获得所述表达载体组件III细菌文库。在某些情形中，所述表达载体组件III细菌文库可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
本申请所述的方法还可包括冷冻保存所述LC1轻链组件细菌文库、所述LC2轻链组件细菌文库、所述VH1重链组件细菌文库、所述VH2重链组件细菌文库、所述表达载体组件I细菌文库、所述表达载体组件II细菌文库和所述表达载体组件III细菌文库。
在本申请中，所述方法可包括获得细菌文库后，由细菌文库获得包含所述多核苷酸的组件质粒。
在某些情形中，所述方法可包括由所述LC1轻链组件细菌文库获得包含所述第一多核苷酸的第一轻链组件质粒，所述第一轻链组件质粒还可包括所述S5和所述S6。在某些情形中，所述方法可包括由所述LC2轻链组件细菌文库获得包含所述第三多核苷酸的第二轻链组件质粒，所述第二轻链组件质粒还可包括所述B2和所述B4。在某些情形中，所述方法可包括由所述VH1重链组件细菌文库获得包含所述第二多核苷酸的第一轻链组件质粒，所述第一重链组件质粒还可包括所述B4和所述B3。在某些情形中，所述方法可包括由所述VH2重链组件细菌文库获得包含所述第四多核苷酸的第二重链组件质粒，所述第二重链组件质粒还可包括所述B5和所述B6。在某些情形中，所述方法可包括由所述表达载体组件I细菌文库获得包含所述第五多核苷酸的展示片段组件质粒I，所述展示片段组件质粒I还可包括所述S6和所述B2。在某些情形中，所述方法可包括由所述表达载体组件II细菌文库获得包含所述第六多核苷酸的展示片段组件质粒II，所述展示片段组件质粒II还可包括所述B3和所述B5。在某些情形中，所述方法可包括由所述表达载体组件III细菌文库获得包含所述第七多核苷酸的展示片段组件质粒III，所述展示片段组件质粒III还可包括所述B6和所述S5。
获得切割后的多核苷酸
本申请所述方法还可包括h)利用限制性内切核酸酶特异性切割所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸、所述第四多核苷酸、所述第五多核苷酸、所述第六多核苷酸和所述第七多核苷酸，得到切割后的所述第一多核苷酸、切割后的所述第二多核苷酸、切割后的所述第三多核苷酸、切割后的所述第四多核苷酸、切割后的所述第五多核苷酸、切割后的所述第六多核苷酸和切割后的所述第七多核苷酸。
在某些情形中，获得包含所述多核苷酸的所述组件质粒后，所述方法还可包括由所述组件质粒获得切割后的所述多核苷酸。在某些情形中，可以使用限制性内切核酸酶对质粒进行酶切处理，从而获得所述切割后的多核苷酸。所述方法可包括以下步骤：由所述第一轻链组件质粒获得切割后的所述第一多核苷酸；由所述第二轻链组件质粒获得切割后的所述第二多核苷酸；由所述第一重链组件质粒获得切割后的所述第三多核苷酸；由所述第二重链组件质粒获得切割后的所述第四多核苷酸；由所述展示片段组件质粒I获得切割后的所述第五多核苷酸；由所述展示片段组件质粒II获得切割后的所述第六多核苷酸；由所述展示片段组件质粒III获得切割后的所述第七多核苷酸。
在某些情形中，可使用特异性识别所述S5和S6的限制性内切核酸酶对所述第一轻链组件质粒进行酶切处理，从而获得所述切割后的所述第一多核苷酸。
在某些情形中，可使用特异性识别所述B4和B3的限制性内切核酸酶对所述第一重链组件质粒进行酶切处理，从而获得所述切割后的所述第二多核苷酸。
在某些情形中，可使用特异性识别所述B2和B4的限制性内切核酸酶对所述第二轻链组件质粒进行酶切处理，从而获得所述切割后的所述第三多核苷酸。
在某些情形中，可使用特异性识别所述S6和B2的限制性内切核酸酶对所述展示片段组件质粒I进行酶切处理，从而获得所述切割后的所述第五多核苷酸。
在某些情形中，可使用特异性识别所述B3和B5的限制性内切核酸酶对所述展示片段组件质粒II进行酶切处理，从而获得所述切割后的所述第六多核苷酸。
在某些情形中，可使用特异性识别所述B6和S5的限制性内切核酸酶对所述展示片段组件质粒III进行酶切处理，从而获得所述切割后的所述第七多核苷酸。
所述组件质粒经限制性内切核酸酶酶切处理后可在3’端和/或5’端产生粘性末端。
在某些情形中，所述B2经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B3、B4、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别B2的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别B2的限制性内切核酸酶特异性切割后产生的末端彼此识别或 连接。
在某些情形中，所述B3经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别B3的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别B3的限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些情形中，所述B4经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B3、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别B4的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别B4的限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些情形中，所述B5经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B3、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别B5的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别B5的限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些情形中，所述B6经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B3、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别B6的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别B6的限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些情形中，所述S5经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、B3和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别S5的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别S5的限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
在某些情形中，所述S6经特异性识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、B3和S5中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。或者，能够特异性识别S6的限制性内切核酸酶特异性切割后产生的末端只能与能够特异性识别S6的限制性内切核酸酶特异性切割后产生的末端彼此识别或连 接。
连接得到双特异性抗原结合多肽表达载体
在本申请中，所述方法还可包括i)混合经所述切割后的第一多核苷酸、所述切割后的第二多核苷酸、所述切割后的第三多核苷酸、所述切割后的第四多核苷酸、所述切割后的第五多核苷酸、所述切割后的第六多核苷酸和所述切割后的第七多核苷酸，从而使得其能够定向连接而环化形成所述双特异性抗原结合多肽表达载体。例如，可以将所述切割后的多核苷酸等比例混合，导入细胞(例如，哺乳动物细胞)后，挑取菌落进行测序，从而确定含有所需双特异性抗原结合多肽序列的表达载体。
例如，所述表达载体的结构可以如图1所示，其由七个经切割后的多核苷酸定向连接环化形成。经所述特异性识别S6的限制性内切核酸酶特异性切割后的第一多核苷酸的3’端的末端可以与经所述特异性识别S6的限制性内切核酸酶特异性切割后的第五多核苷酸的5’端的末端彼此识别并连接。经所述特异性识别B2的限制性内切核酸酶特异性切割后的第五多核苷酸的3’端的末端可以与经所述特异性识别B2的限制性内切核酸酶特异性切割后的第三多核苷酸的末端彼此识别并连接。经所述特异性识别B4的限制性内切核酸酶特异性切割后的第三多核苷酸的3’端的末端可以与经所述特异性识别B4的限制性内切核酸酶特异性切割后的第二多核苷酸的5’端的末端彼此识别并连接。经所述特异性识别B3的限制性内切核酸酶特异性切割后的第二多核苷酸的3’端的末端可以与经所述特异性识别B3的限制性内切核酸酶特异性切割后的第六多核苷酸的5’端的末端彼此识别并连接。经所述特异性识别B5的限制性内切核酸酶特异性切割后的第六多核苷酸的3’端的末端可以与经所述特异性识别B5的限制性内切核酸酶特异性切割后的第四多核苷酸的5’端的末端彼此识别并连接。经所述特异性识别B6的限制性内切核酸酶特异性切割后的第四多核苷酸的3’端的末端可以与经所述特异性识别B6的限制性内切核酸酶特异性切割后的第七多核苷酸的5’端的末端彼此识别并连接。经所述特异性识别S5的限制性内切核酸酶特异性切割后的第七多核苷酸的3’端的末端可以与经所述特异性识别S5的限制性内切核酸酶特异性切割后的第一多核苷酸的5’端的末端彼此识别并连接。
在某些情形中，所述定向连接可包括使用连接酶。例如，所述连接酶可包括T4DNA连接酶。
所述双特异性抗原结合多肽表达载体可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50 个，或更多个)不同的克隆。
另一方面，本申请提供了根据所述方法产生的双特异性抗原结合多肽表达载体，所述双特异性抗原结合多肽表达载体可包含至少10个(例如，至少10个、至少11个、至少12个、至少13个、至少14个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个，或更多个)不同的克隆。
另一方面，本申请提供了利用所述双特异性抗原结合多肽表达载体建立的双特异性抗原结合多肽展示文库。在某些实施方式中，所述展示文库可以为哺乳动物细胞展示文库。在某些实施方式中，所述文库能够展示至少10种(例如，至少10种、至少11种、至少12种、至少13种、至少14种、至少15种、至少16种、至少17种、至少18种、至少19种、至少20种、至少25种、至少30种、至少35种、至少40种、至少45种、至少50种，或更多种)不同的双特异性抗体或其抗体片段。
另一方面，本申请提供了筛选抗体或抗体片段的方法，所述方法可包括使用本申请所述的文库。在某些实施方式中，所述展示文库可以为哺乳动物细胞展示文库。在某些实施方式中，所述文库能够展示至少10种(例如，至少10种、至少11种、至少12种、至少13种、至少14种、至少15种、至少16种、至少17种、至少18种、至少19种、至少20种、至少25种、至少30种、至少35种、至少40种、至少45种、至少50种，或更多种)不同的双特异性抗体或其抗体片段。在某些情形中。所述抗体或抗体片段可以是双特异性抗原结合多肽。例如，所述方法可包括，选择文库中的哺乳动物细胞，建立稳定表达双特异性抗原结合多肽的细胞株，然后进行筛选。例如，可以使用FACS分析所述双特异性抗原结合多肽在细胞表面的表达及其对至少两种抗原的特异性亲和力。
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 构建双特异性抗原结合多肽表达载体
1.1获得样品材料
为构建如图2所示的双特异性抗原结合多肽表达载体，选择靶向PD-1抗体帕博利珠单抗(Pembrolizumab)和PD-L1抗体阿特珠单抗(Atezolizumab)，以及pDGB4载体作为示例。 帕博利珠单抗轻链核苷酸序列：SEQ ID NO：28，帕博利珠单抗重链可变区核苷酸序列：SEQ ID NO：29，阿特珠单抗轻链核苷酸序列：SEQ ID NO：30，阿特珠单抗重链可变区核苷酸序列：SEQ ID NO：31，pDGB4载体核苷酸序列：SEQ ID NO：32。
1.2设计酶切位点
根据限制性内切核酸酶BsmBI和SfiI，设计5个BsmBI识别位点的序列(B2、B3、B4、B5和B6)和2个SfiI识别位点的序列(S5和S6)，序列如下表1所示：
表1
酶切位点 核酸序列
B2 SEQ ID NO:1
B3 SEQ ID NO:2
B4 SEQ ID NO:3
B5 SEQ ID NO:4
B6 SEQ ID NO:5
S5 SEQ ID NO:6
S6 SEQ ID NO:7
1.3选择信号肽
选择三个表达天然抗体基因的信号肽，其中，sp1在LC2的5’端，sp2在VH2的5’端，sp3在LC1的5’端。为了在编码信号肽的核酸序列的3’端部分引入合适的酶切位点，三个编码信号肽的碱基序列已通过无意突变而改变，但信号肽的氨基酸序列保持不变。序列如下表2所示：
表2
信号肽 序列
sp1核酸序列 SEQ ID NO:8
sp1氨基酸序列 SEQ ID NO:9
sp2核酸序列 SEQ ID NO:10
sp2氨基酸序列 SEQ ID NO:11
sp3核酸序列 SEQ ID NO:12
sp3氨基酸序列 SEQ ID NO:13
1.4获得多核苷酸
分别设计针对编码PD-1的轻链(LC1)和重链可变区序列(VH1)、PD-L1的轻链(LC2)和重链可变区(VH2)、以及三段载体pDGB4核酸序列(分别为表达载体片段I、表达载体片段II和表达载体片段III)的引物，引物的5-端均包含酶切位点。此外，LC1、LC2-VH1、LC2表达框架均用CMV启动子驱动表达。合成引物，序列见表3。
表3
多核苷酸 模板 正向引物 反向引物
LC1 PD1-LC(SEQ ID NO:28) P7(SEQ ID NO:20) P8(SEQ ID NO:21)
VH1 PD1-VH(SEQ ID NO:29) P11(SEQ ID NO:24) P12(SEQ ID NO:25)
LC2 PDL1-LC(SEQ ID NO:30) P9(SEQ ID NO:22) P10(SEQ ID NO:23)
VH2 PDL1-VH(SEQ ID NO:31) P13(SEQ ID NO:26) P14(SEQ ID NO:26)
表达载体片段I pDGB4(SEQ ID NO:32) P1(SEQ ID NO:14) P2(SEQ ID NO:15)
表达载体片段II pDGB4(SEQ ID NO:32) P3(SEQ ID NO:16) P4(SEQ ID NO:17)
表达载体片段III pDGB4(SEQ ID NO:32) P5(SEQ ID NO:18) P6(SEQ ID NO:19)
使用PCR(LA Taq，Takara公司,按照公司产品的说明书进行)扩增七种多核苷酸，所使用的模板和引物序列见表3所示。凝胶电泳纯化回收后(按照《分子克隆实验指南》中的记载操作)分别获得PCR产物。使用TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将PCR产物插入pUC19质粒载体，得到所述存储连接产物。用所述存储载体产物转化DH5a感受态细菌(Takara公司)，铺皿37℃培养过夜，送菌落测序，得到细菌含有所需序列的多核苷酸——含有LC1的第一多核苷酸、含有VH1的第二多核苷酸、含有LC2的第三多核苷酸、含有VH2的第四多核苷酸、含有表达载体片段I的第五多核苷酸、含有表达载体片段II的第六多核苷酸和含有表达载体片段III的第七多核苷酸。可将该细菌作为细菌文库冻存备用。
1.5酶切
使用质粒提取试剂盒(购自Axygen)，分别提取实施例1.4中的细菌文库中的细菌的质粒。然后使用限制性内切核酸酶BsmBI和SfiI消化质粒载体，使用特异性识别S5和S6的限制性内切核酸酶切割第一多核苷酸，使用特异性识别B4和B3的限制性内切核酸酶切割第二多核苷酸，使用特异性识别B2和B4的限制性内切核酸酶切割第三多核苷酸，使用特异性识别B5和B6的限制性内切核酸酶切割第四多核苷酸，使用特异性识别S6和B2的限制性内切核酸酶切割第五多核苷酸，使用特异性识别B3和B5的限制性内切核酸酶切割第六多核苷酸，使用特异性识别B6和S5的限制性内切核酸酶切割第七多核苷酸。电泳分离纯化，得到 七种所述切割后的多核苷酸。
1.6获得表达载体
等分子比例混合实施例1.5得到的七种切割后的多核苷酸，加入连接酶，使其定向连接环化形成表达载体，转入DH5a感受态细菌(Takara，Cat#，按照厂家的说明书操作)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃生长过夜。挑选菌落测序，得到含有正确序列的表达载体。将表达载体转入FCHO细胞，建立稳定表达双特异性抗原结合多肽的细胞株，获得细胞展示文库。
实施例2 双特异性抗原结合多肽在细胞表面表达
贴壁培养来自实施例1.6的细胞文库中的细胞，达到一定浓度后，用0.5mM EDTA-PBS消化分散细胞，离心收集细胞，用染色缓冲液(2％FBS-PBS)悬起细胞，分于96孔板，每孔5e4细胞/50ul，按实验要求加入1-3种以下所列荧光标记的抗体或者抗原，混合均匀，冰上孵育30分钟后，加染色缓冲液，200ul/孔，然后进行流式分析。不加荧光标记的抗原/抗体的孔设为阴性对照。
PEK：PE标记的鼠抗人Kappa轻链抗体，检测细胞表面是否有Kappa轻链；FITC-G：FITC标记的鼠抗人IgG重链抗体，检测细胞表面是否有IgG重链；FITC-Ag1：FITC标记的PD1抗原，检测细胞表面展示的抗体是否能与PD1抗原结合；FITC-Ag2：FITC标记的PDL1抗原，检测细胞表面展示的抗体是否能与PDL1抗原结合。
结果显示细胞表面有双特异性抗原结合多肽表达(图3)。图3中：
1，无染色阴性对照，既无PE信号，也无FITC信号；
2，PEK单染，50％以上细胞有PE信号，表示有Kappa轻链表达；
3，FITC-G单染，50％以上细胞有FITC信号，表示有IgG重链表达；
4，PEK+FITC-G双染，50％以上细胞有PE和PFITC双重信号，表示同时有Kappa轻链和IgG重链表达；
5，PEK+FITC-Ag1双染，50％以上细胞有PE和PFITC双重信号，表示细胞表面表达的抗体可以与PD1抗原结合；
6，PEK+FITC-Ag2双染，26％以上细胞有PE和PFITC双重信号，表示细胞表面表达的抗体可以与PDL1抗原结合；
7，PEK+FITC-Ag1+FITC-Ag2三染，54％以上细胞有PE和PFITC双重信号，虽然双阳性的细胞比率只增加了4％，但双阳性细胞群向右移动，表示FITC荧光信号增强了，应该是细胞表面展示的抗体同时结合PD1和PDL1抗原，荧光信号叠加的结果。
实施例3 双特异性抗原结合多肽与抗原结合呈剂量依赖性
按照实施例2的方法，使用不同浓度的FITC标记的PD1抗原(FAg1，0.3μl、1μl、3.3μl)和不同浓度的FITC标记的PDL1抗原(FAg2，0.3μl、1μl、3.3μl)与表达双特异性抗原结合多肽的细胞共同孵育，进行流式分析。
结果显示，随着抗原浓度增加，细胞群在图中位置向右移动，荧光信号随着FAg的增加而增加，表明双特异性抗体对两种抗原均显示剂量依赖性(图4)。图4中：A，无染色阴性对照；B，PEK单染；C，FAg1单染；D，FAg2单染；E，PEK+0.3μl FAg1；F，PEK+1μl FAg1；G，PEK+3.3μl FAg1；H，PEK+0.3μl FAg2；I，PEK+1μl FAg2；J，PEK+3.3μl Ag2。

Claims (64)

  1. 一种用于构建双特异性抗原结合多肽表达载体的方法，所述方法包括：
    a)提供第一多核苷酸，所述第一多核苷酸以5’至3’方向包含S5-LC1-S6；
    b)提供第二多核苷酸，所述第二多核苷酸以5’至3’方向包含B4-VH1-B3；
    c)提供第三多核苷酸，所述第三多核苷酸以5’至3’方向包含B2-LC2-B4；
    d)提供第四多核苷酸，所述第四多核苷酸以5’至3’方向包含B5-VH2-B6；
    e)提供第五多核苷酸，所述第五多核苷酸以5’至3’方向包含S6-表达载体片段I-B2；
    f)提供第六多核苷酸，所述第六多核苷酸以5’至3’方向包含B3-表达载体片段II-B5；
    g)提供第七多核苷酸，所述第七多核苷酸以5’至3’方向包含B6-表达载体片段III-S5；
    h)利用限制性内切核酸酶特异性切割所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸、所述第四多核苷酸、所述第五多核苷酸、所述第六多核苷酸和所述第七多核苷酸，得到切割后的所述第一多核苷酸、切割后的所述第二多核苷酸、切割后的所述第三多核苷酸、切割后的所述第四多核苷酸、切割后的所述第五多核苷酸、切割后的所述第六多核苷酸和切割后的所述第七多核苷酸；其中所述限制性内切核酸酶分别特异性识别S5、S6、B4、B3、B2、B5和B6；
    i)混合经所述切割后的第一多核苷酸、所述切割后的第二多核苷酸、所述切割后的第三多核苷酸、所述切割后的第四多核苷酸、所述切割后的第五多核苷酸、所述切割后的第六多核苷酸和所述切割后的第七多核苷酸，从而使得其能够定向连接而环化形成所述表达载体；
    其中所述LC1编码所述双特异性抗原结合多肽的第一轻链，所述VH1编码所述双特异性抗原结合多肽的第一重链可变区，且所述第一轻链可与所述第一重链可变区结合后形成识别第一靶标的第一Fab；
    所述LC2编码所述双特异性抗原结合多肽的第二轻链，所述VH2编码所述双特异性抗原结合多肽的第二重链可变区，且所述第二轻链可与所述第二重链可变区结合后形成识别第二靶标的第二Fab；
    其中所述B2、B3、B4、B5、B6、S5和S6各自独立地为限制性内切核酸酶识别位点。
  2. 根据权利要求1所述的方法，其中所述B2经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B3、B4、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  3. 根据权利要求1-2中任一项所述的方法，其中所述B3经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、S5和S6中的任一项经相应限制性 内切核酸酶特异性切割后产生的末端彼此识别或连接。
  4. 根据权利要求1-3中任一项所述的方法，其中所述B4经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B3、B5、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  5. 根据权利要求1-4中任一项所述的方法，其中所述B5经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B3、B6、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  6. 根据权利要求1-5中任一项所述的方法，其中所述B6经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B3、S5和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  7. 根据权利要求1-6中任一项所述的方法，其中所述S5经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、B3和S6中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  8. 根据权利要求1-7中任一项所述的方法，其中所述S6经特异识别其的限制性内切核酸酶特异性切割后产生的末端不与所述B2、B4、B5、B6、S5和B3中的任一项经相应限制性内切核酸酶特异性切割后产生的末端彼此识别或连接。
  9. 根据权利要求1-8中任一项所述的方法，其中所述限制性内切核酸酶选自SfiI和BsmBI。
  10. 根据权利要求1-9中任一项所述的方法，其中所述B2、B3、B4、B5和B6能够被BsmBI特异性识别及切割。
  11. 根据权利要求1-10中任一项所述的方法，其中所述S5和S6能够被Sfil特异性识别及切割。
  12. 根据权利要求1-11中任一项所述的方法，其中所述B2包含SEQ ID NO:1所示的核酸序列。
  13. 根据权利要求1-12中任一项所述的方法，其中所述B3包含SEQ ID NO:2所示的核酸序列。
  14. 根据权利要求1-13中任一项所述的方法，其中所述B4包含SEQ ID NO:3所示的核酸序列。
  15. 根据权利要求1-14中任一项所述的方法，其中所述B5包含SEQ ID NO:4所示的核酸序列。
  16. 根据权利要求1-15中任一项所述的方法，其中所述B6包含SEQ ID NO:5所示的核酸序 列。
  17. 根据权利要求1-16中任一项所述的方法，其中所述S5包含SEQ ID NO:6所示的核酸序列。
  18. 根据权利要求1-17中任一项所述的方法，其中所述S6包含SEQ ID NO:7所示的核酸序列。
  19. 根据权利要求1-18中任一项所述的方法，其还包括将所述第一多核苷酸导入第一细菌以获得LC1轻链组件细菌文库。
  20. 根据权利要求19所述的方法，其包括将所述第一多核苷酸插入组件载体形成LC1存储连接产物，并且将所述LC1存储连接产物导入所述第一细菌以获得LC1轻链组件细菌文库。
  21. 根据权利要求20所述的方法，其还包括由所述LC1轻链组件细菌文库获得包含所述第一多核苷酸的第一轻链组件质粒，由所述第一轻链组件质粒获得切割后的所述第一多核苷酸。
  22. 根据权利要求21所述的方法，其包括使用特异性识别所述S5和S6的限制性内切核酸酶对所述第一轻链组件质粒进行酶切处理，从而获得所述切割后的所述第一多核苷酸。
  23. 根据权利要求1-22中任一项所述的方法，其还包括将所述第二多核苷酸导入第二细菌以获得VH1重链组件细菌文库。
  24. 根据权利要求23所述的方法，其包括将所述第二多核苷酸插入组件载体，形成VH1存储连接产物，并将所述VH1存储连接产物导入所述第二细菌中以获得所述VH1重链组件细菌文库。
  25. 根据权利要求24所述的方法，其还包括由所述VH1重链组件细菌文库获得包含所述第二多核苷酸的第一重链组件质粒，由所述第一重链组件质粒获得切割后的所述第二多核苷酸。
  26. 根据权利要求25所述的方法，其包括使用特异性识别所述B4和B3的限制性内切核酸酶对所述第一重链组件质粒进行酶切处理，从而获得所述切割后的所述第二多核苷酸。
  27. 根据权利要求1-26中任一项所述的方法，其还包括将所述第三多核苷酸导入第三细菌以获得LC2轻链组件细菌文库。
  28. 根据权利要求27所述的方法，其包括将所述第三多核苷酸插入组件载体形成LC2存储连接产物，并将所述LC2存储连接产物导入所述第三细菌中以获得所述LC2轻链组件细菌文库。
  29. 根据权利要求28所述的方法，其还包括由所述LC2轻链组件细菌文库获得包含所述第三 多核苷酸的第二轻链组件质粒，由所述第二轻链组件质粒获得切割后的所述第三多核苷酸。
  30. 根据权利要求29所述的方法，其包括使用特异性识别所述B2和B4的限制性内切核酸酶对所述第二轻链组件质粒进行酶切处理，从而获得所述切割后的所述第三多核苷酸。
  31. 根据权利要求1-30中任一项所述的方法，其还包括将所述第四多核苷酸导入第四细菌以获得VH2重链组件细菌文库。
  32. 根据权利要求31所述的方法，其包括将所述第四多核苷酸插入组件载体形成VH2存储连接产物，并将所述VH2存储连接产物导入所述第四细菌中以获得所述VH2重链组件细菌文库。
  33. 根据权利要求32所述的方法，其还包括由所述VH2重链组件细菌文库获得包含所述第四多核苷酸的第二重链组件质粒，由所述第二重链组件质粒获得切割后的所述第四多核苷酸。
  34. 根据权利要求33所述的方法，其包括使用特异性识别所述B5和B6的限制性内切核酸酶对所述第二重链组件质粒进行酶切处理，从而获得所述切割后的所述第四多核苷酸。
  35. 根据权利要求1-34中任一项所述的方法，其还包括将所述第五多核苷酸导入第五细菌以获得表达载体组件I细菌文库。
  36. 根据权利要求35所述的方法，其包括将所述第五多核苷酸插入组件载体形成表达载体片段I存储连接产物，并将所述存储连接产物导入所述第五细菌中以获得所述表达载体组件I细菌文库。
  37. 根据权利要求36所述的方法，其还包括由所述表达载体组件I细菌文库获得包含所述第五多核苷酸的展示片段组件质粒I，由所述展示片段组件质粒I获得切割后的所述第五多核苷酸。
  38. 根据权利要求37所述的方法，其包括使用特异性识别所述S6和B2的限制性内切核酸酶对所述展示片段组件质粒I进行酶切处理，从而获得所述切割后的所述第五多核苷酸。
  39. 根据权利要求1-38中任一项所述的方法，其还包括将所述第六多核苷酸导入第六细菌以获得表达载体组件II细菌文库。
  40. 根据权利要求39所述的方法，其包括将所述第六多核苷酸插入组件载体形成表达载体片段II存储连接产物，并将所述存储连接产物导入所述第六细菌中以获得所述表达载体组件II细菌文库。
  41. 根据权利要求40所述的方法，其还包括由所述表达载体组件II细菌文库获得包含所述第 六多核苷酸的展示片段组件质粒II，由所述展示片段组件质粒II获得切割后的所述第六多核苷酸。
  42. 根据权利要求41所述的方法，其包括使用特异性识别所述B3和B5的限制性内切核酸酶对所述展示片段组件质粒II进行酶切处理，从而获得所述切割后的所述第六多核苷酸。
  43. 根据权利要求1-42中任一项所述的方法，其还包括将所述第七多核苷酸导入第七细菌以获得表达载体组件III细菌文库。
  44. 根据权利要求43所述的方法，其包括将所述第七多核苷酸插入组件载体形成表达载体片段III存储连接产物，并将所述存储连接产物导入所述第七细菌中以获得所述表达载体组件III细菌文库。
  45. 根据权利要求44所述的方法，其还包括由所述表达载体组件III细菌文库获得包含所述第七多核苷酸的展示片段组件质粒III，由所述展示片段组件质粒III获得切割后的所述第七多核苷酸。
  46. 根据权利要求45所述的方法，其包括使用特异性识别所述B6和S5的限制性内切核酸酶对所述展示片段组件质粒III进行酶切处理，从而获得所述切割后的所述第七多核苷酸。
  47. 根据权利要求43-46所述的方法，其包括冷冻保存所述LC1轻链组件细菌文库、所述LC2轻链组件细菌文库、所述VH1重链组件细菌文库、所述VH2重链组件细菌文库、所述表达载体组件I细菌文库、所述表达载体组件II细菌文库和/或所述表达载体组件III细菌文库。
  48. 根据权利要求20-47中任一项所述的方法，其中所述组件载体源自pUC载体。
  49. 根据权利要求48所述的方法，其中所述pUC载体为pUC19载体或源自pUC19载体。
  50. 根据权利要求19-49中任一项所述的方法，其中所述LC1轻链组件细菌文库包含至少10个不同的克隆。
  51. 根据权利要求23-50中任一项所述的方法，其中所述VH1重链组件细菌文库包含至少10个不同的克隆。
  52. 根据权利要求27-51中任一项所述的方法，其中所述LC2轻链组件细菌文库包含至少10个不同的克隆。
  53. 根据权利要求31-52中任一项所述的方法，其中所述VH2重链组件细菌文库包含至少10个不同的克隆。
  54. 根据权利要求1-53中任一项所述的方法，其中所述双特异性抗原结合多肽表达载体包含至少10个不同的克隆。
  55. 根据权利要求1-54中任一项所述的方法，其中由样品材料获得所述第一多核苷酸、所述第二多核苷酸、所述第三多核苷酸和/或所述第四多核苷酸。
  56. 根据权利要求55所述的方法，其中所述样品材料包括靶向特异性抗原的抗体或其抗原结合片段。
  57. 根据权利要求56所述的方法，其中所述抗体或其抗原结合片段靶向PD-1和/或PD-L1。
  58. 根据权利要求1-57中任一项所述的方法，其中所述定向连接包括使用连接酶。
  59. 根据权利要求58所述的方法，其中所述连接酶包括T4 DNA连接酶。
  60. 根据权利要求1-59中任一项所述的方法所产生的双特异性抗原结合多肽表达载体。
  61. 利用权利要求60所述的双特异性抗原结合多肽表达载体建立的双特异性抗原结合多肽展示文库。
  62. 根据权利要求61所述的文库，其为哺乳动物细胞展示文库。
  63. 根据权利要求60-61中任一项所述的文库，其能够展示至少10种不同的双特异性抗体或其抗体片段。
  64. 筛选抗体或抗体片段的方法，其包括使用根据权利要求61-63中任一项所述的文库。
PCT/CN2021/083248 2020-03-27 2021-03-26 在哺乳动物细胞表面展示双特异性抗体的方法及载体 Ceased WO2021190631A1 (zh)

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CN202180024152.3A CN115315271A (zh) 2020-03-27 2021-03-26 在哺乳动物细胞表面展示双特异性抗体的方法及载体
JP2022558475A JP2023518898A (ja) 2020-03-27 2021-03-26 哺乳動物細胞の表面に二重特異性抗体を提示する方法およびベクター
US17/914,796 US20230331871A1 (en) 2020-03-27 2021-03-26 Method for displaying bispecific antibody on surface of mammalian cell and vector
KR1020227037513A KR20220160130A (ko) 2020-03-27 2021-03-26 포유동물 세포 및 벡터의 표면에 이중특이항체를 디스플레이하는 방법
MX2022012005A MX2022012005A (es) 2020-03-27 2021-03-26 Metodo para el despliegue de anticuerpo biespecifico en la superficie de celula de mamifero y vector.
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