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WO2021190629A1 - 抗原特异性结合多肽基因展示载体的构建方法与应用 - Google Patents

抗原特异性结合多肽基因展示载体的构建方法与应用 Download PDF

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WO2021190629A1
WO2021190629A1 PCT/CN2021/083246 CN2021083246W WO2021190629A1 WO 2021190629 A1 WO2021190629 A1 WO 2021190629A1 CN 2021083246 W CN2021083246 W CN 2021083246W WO 2021190629 A1 WO2021190629 A1 WO 2021190629A1
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polynucleotide
display
vector
antigen
component
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PCT/CN2021/083246
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English (en)
French (fr)
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周辰
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泷搌（上海）生物科技有限公司
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Priority to KR1020227037512A priority Critical patent/KR20220162151A/ko
Priority to EP21776379.6A priority patent/EP4130260A4/en
Priority to MX2022012004A priority patent/MX2022012004A/es
Priority to BR112022019392A priority patent/BR112022019392A2/pt
Priority to JP2022558484A priority patent/JP2023518900A/ja
Priority to US17/914,650 priority patent/US20230124855A1/en
Priority to CN202180024143.4A priority patent/CN115362254A/zh
Publication of WO2021190629A1 publication Critical patent/WO2021190629A1/zh

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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This application relates to the field of biomedicine, in particular to a method for constructing an antigen-specific binding polypeptide gene display vector, which can be used to screen antigen-specific binding polypeptides.
  • hybridoma technology there are two main types of antibody discovery methods commonly used in the prior art: hybridoma technology and antibody display technology.
  • hybridoma technology murine hybridoma and transgenic mouse hybridoma.
  • antibody display technologies phage display, yeast display and mammalian cell display.
  • Each antibody discovery technology has very clear advantages, but also has very big shortcomings and limitations. For example, the process of constructing antibody drug seed library lacks quality control, resulting in poor quality of antibody library, small storage capacity, less diversity, and effective cloning ratio Low, it is difficult to screen high-quality lead antibodies, or the antibody library screening technology does not have quantitative screening, and the screening throughput is small, the screening effect is not good, and the time is consuming.
  • This application provides a method for constructing an antigen-specific binding polypeptide gene display vector.
  • the antigen-specific binding polypeptide gene display vector is composed of four fragments. By constructing a component library and a display vector, 5 of the four fragments The'end and the 3'end have sticky ends with specific sequences, thereby directional circularization forms the antigen-specific binding polypeptide gene display vector.
  • the method for constructing an antigen-specific binding polypeptide gene display vector in this application and the method for screening an antigen-specific binding polypeptide using the antigen-specific binding polypeptide gene display vector of this application have at least one of the following properties: 1) Constructing the antigen specificity described in this application
  • the binding polypeptide gene display vector can use special restriction endonuclease recognition sites to ensure directional connection and prevent wrong connection.
  • the number of molecules of each component fragment during connection can be controlled to 1:1, thereby improving the connection and transformation Efficiency;
  • strategies such as constructing the VH component library and LC component library are used to improve the connection and transformation efficiency of each fragment;
  • the combined PCR strategy used in the conventional antibody library construction methods in this field is not used, which effectively reduces the introduction caused by PCR The probability of mutation;
  • the quality control can be carried out relatively easily, which can meet the needs of industrialized mass production;
  • the display vector can be introduced into the cell and directly screened by the biological activity analysis experiment, which effectively shortens the construction of the antigen-specific binding polypeptide gene The time from the display of the vector to the screening of the antigen-specific polypeptides with unique sequences.
  • the time from constructing the display vector to screening for positive clones with unique sequences can be at least about 1 week (at least about 10 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks); 5)
  • the bacterial library of the display vector has large clonal diversity and high screening efficiency.
  • the library containing the antigen-specific binding polypeptide gene display vector can reach about 50% or more (for example, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more or more), the conversion efficiency is high, and the success rate of database construction is high.
  • the present application provides a method for constructing an antigen-specific binding polypeptide gene display vector, the method comprising: a) providing a first display vector polynucleotide, the first display vector polynucleotide being 5 The'to 3'direction includes B2-display VH-B3; b) a second display vector polynucleotide is provided, and the second display vector polynucleotide includes S5-display LC-S6 in the 5'to 3'direction; c) Provide a third display vector polynucleotide, the third display vector polynucleotide comprising B3-display vector fragment I-S5 in a 5'to 3'direction; d) provide a fourth display vector polynucleotide, the fourth The display vector polynucleotide comprises S6-display vector fragment II-B2 in the 5'to 3'direction; e) using restriction endonuclease to specifically cut the first display vector polyn
  • the end produced by the specific cleavage of the B2 by the restriction endonuclease that specifically recognizes it is different from any one of the B3, S5, and S6 by the corresponding restriction endonuclease.
  • the ends generated after specific cleavage recognize or connect to each other.
  • the end of the B3 that is specifically cleaved by the restriction endonuclease that specifically recognizes it is not the same as any one of the B2, S5, and S6 through the corresponding restriction endonuclease.
  • the ends generated after specific cleavage recognize or connect to each other.
  • the end of the S5 that is specifically cleaved by the restriction endonuclease that specifically recognizes it is not the same as any one of the B2, B3, and S6 through the corresponding restriction endonuclease.
  • the ends generated after specific cleavage recognize or connect to each other.
  • the end of the S6 that is specifically cleaved by the restriction endonuclease that specifically recognizes it is not the same as any one of the B2, B3, and S5 through the corresponding restriction endonuclease.
  • the ends generated after specific cleavage recognize or connect to each other.
  • the restriction endonuclease is selected from SfiI, Esp3I and BsmBI.
  • the B2 and B3 can be specifically recognized and cleaved by an enzyme selected from the group consisting of BsmBI and Esp3I.
  • the S5 and S6 can be specifically recognized and cleaved by Sfil.
  • the B2 includes the nucleic acid sequence shown in SEQ ID NO: 8.
  • the B3 includes the nucleic acid sequence shown in SEQ ID NO:9.
  • the S5 includes the nucleic acid sequence shown in SEQ ID NO: 10.
  • the S6 includes the nucleic acid sequence shown in SEQ ID NO: 11.
  • the method further comprises introducing the first display vector polynucleotide into the first display bacteria to obtain a bacterial library displaying VH components.
  • the method includes inserting the first display vector polynucleotide into a display component vector to form a display VH storage ligation product, and introducing the display VH storage ligation product into the first display bacterium to Obtain the bacterial library displaying the VH components.
  • the method further includes introducing the second display vector polynucleotide into a second display bacteria to obtain a bacterial library displaying LC components.
  • the method includes inserting the second display vector polynucleotide into a display component vector to form a display LC storage ligation product, and introducing the display LC storage ligation product into the second display bacteria To obtain the bacterial library displaying LC components.
  • the method further includes introducing the third display vector polynucleotide into a third display bacteria to obtain a display vector component I bacterial library.
  • the method includes inserting the third display vector polynucleotide into a display component vector to form a display vector fragment I storage ligation product, and introducing the storage ligation product into the third display bacterium to Obtain the display vector component I bacterial library.
  • the method further includes introducing the fourth display vector polynucleotide into a fourth display bacteria to obtain a display vector assembly II bacterial library.
  • the method includes inserting the fourth display vector polynucleotide into a display component vector to form a display vector fragment II storage ligation product, and introducing the storage ligation product into the fourth display bacterium to Obtain the display vector component II bacterial library.
  • the display vector component vector is derived from a pUC vector.
  • the pUC vector is a pUC19 vector or is derived from a pUC19 vector.
  • the method further includes obtaining a display VH component plasmid containing the first display vector polynucleotide from the display VH component bacterial library, and obtaining the cleaved VH component plasmid from the display VH component plasmid The first display vector polynucleotide.
  • the method includes using a restriction endonuclease that specifically recognizes the B2 and B3 to digest the VH component plasmid to obtain the cut first Display vector polynucleotide.
  • the method further includes obtaining a display LC component plasmid containing the second display vector polynucleotide from the display LC component bacterial library; obtaining the cleaved LC component plasmid from the display LC component plasmid The second display vector polynucleotide.
  • the method includes using restriction endonucleases that specifically recognize the S5 and S6 to digest the LC component plasmid to obtain the cut second Display vector polynucleotide.
  • the method further includes obtaining a display fragment assembly plasmid I containing the third display vector polynucleotide from the expression vector assembly I bacterial library; Of said third display vector polynucleotide.
  • the method includes using restriction endonucleases that specifically recognize the B3 and S5 to digest the display fragment component plasmid I, so as to obtain the cleaved first Three display vector polynucleotides.
  • the method further includes obtaining a display fragment assembly plasmid II containing the fourth display vector polynucleotide from the expression vector assembly II bacterial library, and obtaining the fragment assembly plasmid II from the display fragment assembly plasmid II. Said fourth display vector polynucleotide.
  • the method includes using a restriction endonuclease that specifically recognizes the S6 and B2 to digest the display fragment component plasmid II, so as to obtain the cleaved first Four display vector polynucleotides.
  • the method includes:
  • VH component vector comprising a sixth polynucleotide, the sixth polynucleotide comprising the B3-VH component vector linking fragment-B2 in a 5'to 3'direction;
  • B is a recognition site of a restriction endonuclease capable of specifically recognizing B2 and/or B3, and the antigen-specific VH encodes the heavy chain variable region of the antigen-specific binding polypeptide.
  • the method includes:
  • LC component carrier comprising an eighth polynucleotide, the eighth polynucleotide comprising the S6-LC component carrier connecting fragment-S5 in a 5'to 3'direction;
  • the S is a recognition site of a restriction endonuclease capable of specifically recognizing S5 and/or S5, and the antigen-specific LC encodes the light chain of the antigen-specific binding polypeptide.
  • the method includes,
  • a) Provide a ninth polynucleotide, which comprises the B2-VH component vector tool fragment-B3 in a 5'to 3'direction;
  • the method includes,
  • a tenth polynucleotide which comprises the S5-LC component carrier tool fragment-S6 in a 5'to 3'direction;
  • the expression module vector is derived from a pMD vector.
  • the pMD vector is a pMD19 vector or is derived from a pMD19 vector.
  • the method includes the following steps:
  • the method includes using a restriction endonuclease that specifically recognizes the B2 and B3 to digest the VH component vector storage plasmid, thereby obtaining the released first Six polynucleotides.
  • the method includes the following steps:
  • the method includes using a restriction endonuclease that specifically recognizes the S5 and S6 to digest the LC component vector storage plasmid, thereby obtaining the released first Eight polynucleotides.
  • the method includes:
  • the antigen-specific VH component library comprising a first polynucleotide, the first polynucleotide comprising B2-antigen-specific VH in a 5'to 3'direction -B3;
  • the antigen-specific LC component library includes a second polynucleotide, and the second polynucleotide includes S5-antigen-specific LC in a 5'to 3'direction -S6;
  • the display vector comprising a third polynucleotide and a fourth polynucleotide, the third polynucleotide comprising B3-display vector fragment I- in a 5'to 3'direction S5, the fourth polynucleotide comprises S6-display vector fragment II-B2 in a 5'to 3'direction;
  • restriction endonucleases to specifically cleave the antigen-specific VH component library, the antigen-specific LC component library, and the display vector to obtain the released first polynucleotide.
  • the antigen-specific LC encodes the light chain of the antigen-specific binding polypeptide
  • the antigen-specific VH encodes the heavy chain variable region of the antigen-specific binding polypeptide
  • B2, B3, S5 and S6 are each independently a restriction endonuclease recognition site.
  • the method includes using restriction endonucleases that specifically recognize B2 and B3 to digest the antigen-specific VH component library, thereby obtaining the released first Polynucleotide.
  • the method includes using restriction endonucleases that specifically recognize S5 and S6 to digest the antigen-specific LC component library, thereby obtaining the released second Polynucleotide.
  • the method includes using a restriction endonuclease that specifically recognizes B3 and a restriction endonuclease that specifically recognizes S5 to digest the display vector to obtain the The released third polynucleotide.
  • the method includes using a restriction endonuclease that specifically recognizes S6 and a restriction endonuclease that specifically recognizes B2 to digest the display vector to obtain the The released fourth polynucleotide.
  • the fifth polynucleotide, the seventh polynucleotide, the ninth polynucleotide, the tenth polynucleotide, the first display vector polynucleotide, the The second display vector polynucleotide, the third display vector polynucleotide and/or the fourth display vector polynucleotide are examples of the fifth polynucleotide, the seventh polynucleotide, the ninth polynucleotide, the tenth polynucleotide, the first display vector polynucleotide, the The second display vector polynucleotide, the third display vector polynucleotide and/or the fourth display vector polynucleotide.
  • the sample material includes antibodies or antigen-binding fragments thereof that target specific antigens and/or IgG.
  • the antibody or antigen-binding fragment thereof targets ROR1, PD-1 and/or PD-L1.
  • the IgG is human IgG.
  • the human IgG is human IgG1 or human IgG2.
  • the directed ligation includes the use of a ligase.
  • the ligase includes T4 DNA ligase.
  • the method includes introducing the antigen-specific binding polypeptide gene display vector into a cell, and the antigen-specific binding polypeptide is obtained from the cell.
  • the method includes:
  • the method includes cryopreserving the antigen-specific binding polypeptide gene display bacterial library, the VH module vector storage bacterial library, the LC module vector storage bacterial library, and the VH module display bacterial library , The display LC module bacterial library, the display vector module I bacterial library, and the display vector module II bacterial library.
  • the VH component vector storage bacterial library contains at least 10 different clones.
  • the LC component vector storage bacterial library contains at least 10 different clones.
  • the library of bacteria displaying VH components contains at least 10 different clones.
  • the library of bacteria displaying LC components contains at least 10 different clones.
  • the display vector component I bacterial library contains at least 10 identical clones.
  • the display vector component II bacterial library contains at least 10 identical clones.
  • the ratio of effective clones in the antigen-specific binding polypeptide gene display bacterial library is at least about 10%.
  • the cell is a mammalian cell.
  • this application provides a method for screening antigen-specific binding polypeptides or fragments thereof, which includes using the antigen-specific binding polypeptide gene display vector.
  • this application provides an antigen-specific binding polypeptide gene display vector produced according to the method.
  • the present application provides an antigen-specific binding polypeptide gene display bacterial library produced according to the method.
  • Figure 1 shows the structure of the display carrier described in this application
  • Figure 2 shows an example of the display carrier described in this application
  • FIG. 3 shows the structure of the VH component carrier described in the present application
  • FIG. 4 shows the structure of the LC component carrier described in this application
  • Figure 5A shows the amino acid sequence of the antigen-specific LC described in this application
  • Figure 5B shows the amino acid sequence of the antigen-specific VH described in this application;
  • Figure 6 shows the FACS analysis of the expression of ROR1 antigen-specific binding polypeptide on the surface of CHO cells
  • Figure 7 shows the SDS-PAGE denaturing reduction gel electrophoresis analysis of 8 exemplary antibodies
  • Figure 8 shows the FACS analysis results of the positive antibodies screened using the method of this application.
  • Figure 9 shows a schematic diagram of a light chain storage vector in the process of constructing a phage library
  • Figure 10 shows a schematic diagram of a heavy chain storage vector in the process of constructing a phage library
  • Figure 11 shows a schematic diagram of a linker storage vector in the process of constructing a phage library
  • Figure 12 shows a schematic diagram of the pCom3x vector in the process of constructing a phage library
  • Figure 13 shows a schematic diagram of a phage display vector in the process of constructing a phage library
  • Fig. 14 shows a schematic diagram of the plasmid displaying the ligation product in the process of constructing the phage library.
  • the term "antigen-binding polypeptide” generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • the term may include antibodies or antigen-binding portions thereof, or the antigen-binding regions and/or antibody variable regions of intact antibodies.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein, which is composed of two identical light chains and two identical heavy chains. In the case of IgG, each L chain is connected to the H chain through a covalent disulfide bond, and the two H chains are connected to each other through one or more disulfide bonds. The number of disulfide bonds depends on the same species of the H chain. type. Each H and L chain also has regularly spaced intrachain disulfide bonds.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain) or four (for mu and epsilon isotypes) constant domains (CH).
  • VH variable domain
  • CH constant domains
  • the antigen-binding polypeptide can be obtained by chemical methods and/or genetic engineering methods. For example, proteases, including pepsin and papain, can be used to generate the antigen-binding fragment after digesting the antibody. In this application, the antibody fragment may be Fab.
  • Fab generally refers to the production of two identical antigen-binding fragments after papain digests an antibody with a complete structure (for example, the Fc region and the hinge region are removed).
  • the Fab can be composed of a complete light chain, the variable region of the heavy chain (VH), and the first constant domain (CH1) of the heavy chain.
  • VH variable region of the heavy chain
  • CH1 first constant domain
  • first polynucleotide generally refers to a polynucleotide containing antigen-specific VH, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Nuclease) recognition site.
  • endonucleases e.g., restriction endonucleases
  • the first polynucleotide may comprise B2-antigen-specific VH-B3 in a 5'to 3'direction, wherein the B2 and B3 may be recognition sites of restriction endonucleases.
  • the released The first polynucleotide may include the antigen-specific VH, and the antigen-specific VH may also have sticky ends with specific sequences after digestion.
  • an endonuclease that recognizes the endonuclease recognition site in the first polynucleotide for example, a restriction endonuclease that recognizes B2 and B3, such as BsmBI
  • the released The first polynucleotide may include the antigen-specific VH, and the antigen-specific VH may also have sticky ends with specific sequences after digestion.
  • second polynucleotide generally refers to a polynucleotide containing antigen-specific LC, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Nuclease) recognition site.
  • the first polynucleotide may comprise S5-antigen-specific LC-S6 in a 5'to 3'direction, wherein the S5 and S6 may be recognition sites of restriction endonucleases.
  • the released The first polynucleotide may include the antigen-specific LC, and the antigen-specific LC may also have sticky ends of a specific sequence after digestion.
  • an endonuclease that recognizes the endonuclease recognition site in the first polynucleotide for example, a restriction endonuclease that recognizes S5 and S6, such as SfiI
  • the released The first polynucleotide may include the antigen-specific LC, and the antigen-specific LC may also have sticky ends of a specific sequence after digestion.
  • antigen-specific VH generally refers to the nucleotides that contain the variable region of the antibody heavy chain that can specifically bind to the antigen
  • antigen-specific LC generally refers to the coding containing the The nucleotides of the light chain of the antibody to which the antigen specifically binds.
  • the sequences of antigen-specific VH and antigen-specific LC can be obtained by any method in the prior art, including but not limited to phage display technology, yeast surface display technology, ribosome display technology, mRNA display technology and/or hybridoma technology For example, it can be obtained by the method of phage library display.
  • third polynucleotide generally refers to a polynucleotide containing fragment I of the display vector, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Nuclease) recognition site.
  • the third polynucleotide may comprise a B3-display vector fragment I-S5 in a 5'to 3'direction, wherein the B3 and S5 may be recognition sites of restriction endonucleases.
  • the third polynucleotide may be included in a display vector, and an endonuclease that recognizes the endonuclease recognition site in the third polynucleotide (for example, a restriction endonuclease that recognizes B3 and S5) Enzymes, such as SfiI, BsmBI and/or Esp3I)
  • an endonuclease that recognizes the endonuclease recognition site in the third polynucleotide for example, a restriction endonuclease that recognizes B3 and S5 Enzymes, such as SfiI, BsmBI and/or Esp3I
  • the third polynucleotide may be released, and the released third polynucleotide may include the display vector fragment I, and
  • the display vector fragment I can also have sticky ends with specific sequences after digestion.
  • the term "released third polynucleotide” generally refers to a fragment of the third polynucleotide released after the display vector is processed.
  • the treatment may be restriction endonuclease digestion treatment.
  • a suitable restriction endonuclease for example, SfiI, BsmBI and/or Esp3I
  • SfiI, BsmBI and/or Esp3I can be selected for the recognition site of the restriction endonuclease on the display vector, so that the released first The three polynucleotides are released and isolated from the display vector.
  • the term "fourth polynucleotide” generally refers to a polynucleotide containing fragment II of the display vector, which may have an endonuclease (e.g., restriction endonuclease) at the 5'end and/or 3'end Nuclease) recognition site.
  • the fourth polynucleotide may comprise the S6-display vector fragment II-B2 in a 5'to 3'direction, wherein the S6 and B2 may be recognition sites for restriction endonucleases.
  • the fourth polynucleotide may be included in a display vector, and an endonuclease that recognizes the endonuclease recognition site in the fourth polynucleotide (for example, a restriction endonuclease that recognizes S6 and B2) Enzymes, such as SfiI, BsmBI and/or Esp3I) after the digestion treatment, the fourth polynucleotide may be released, and the released fourth polynucleotide may include the display vector fragment II, the The display vector fragment II can also have sticky ends with specific sequences after digestion.
  • an endonuclease that recognizes the endonuclease recognition site in the fourth polynucleotide for example, a restriction endonuclease that recognizes S6 and B2
  • Enzymes such as SfiI, BsmBI and/or Esp3I
  • the term "released fourth polynucleotide” generally refers to a fragment of the fourth polynucleotide released after the display vector is processed.
  • the treatment may be restriction endonuclease digestion treatment.
  • a suitable restriction endonuclease for example, SfiI, BsmBI and/or Esp3I
  • SfiI, BsmBI and/or Esp3I can be selected for the recognition site of the restriction endonuclease on the display vector, so that the released first The tetrapolynucleotide is released and isolated from the display vector.
  • the term "fifth polynucleotide” generally refers to a polynucleotide containing the antigen-specific VH, which may have endonucleases (e.g., restriction enzymes) at the 5'end and/or 3'end Endonuclease) recognition site.
  • the fifth polynucleotide may comprise B-the antigen-specific VH-B in the 5'to 3'direction, wherein the B may be a recognition site of a restriction endonuclease, and the restriction The endonuclease may be a restriction endonuclease capable of recognizing B2 and/or B3.
  • the fifth polynucleotide may comprise the antigen-specific VH.
  • the term "seventh polynucleotide” generally refers to a polynucleotide containing the antigen-specific LC, which may have endonucleases (e.g., restriction enzymes) at the 5'end and/or 3'end. Endonuclease) recognition site.
  • the fifth polynucleotide may include S-the antigen-specific LC-S in a 5'to 3'direction, wherein the S may be a recognition site of a restriction endonuclease, and the restriction The endonuclease may be a restriction endonuclease capable of recognizing S5 and/or S6.
  • the polynucleotide may comprise the antigen-specific LC.
  • VH module vector generally refers to a circular polynucleotide comprising a sixth polynucleotide and/or a VH module vector tool fragment.
  • VH component vector generally refers to a circular polynucleotide comprising the eighth polynucleotide and/or LC component vector tool fragment.
  • the term "display vector” generally refers to a circular polynucleotide comprising a display vector fragment I and a display vector fragment II, and it may also include a display VH and a display LC. After being processed, the display vector can release the third display vector polynucleotide, the fourth display vector polynucleotide, the third display vector polynucleotide and/or the fourth display vector polynucleotide.
  • first display vector polynucleotide generally refers to a polynucleotide containing a display VH, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Enzyme) recognition site.
  • the first display vector polynucleotide may include B2-display VH-B3 in a 5'to 3'direction, wherein the B2 and B3 may be recognition sites for restriction endonucleases.
  • the first display vector polynucleotide of may include the display VH, and the two ends of the display VH may also have sticky ends of a specific sequence after digestion.
  • second display vector polynucleotide generally refers to a polynucleotide containing a display LC, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Enzyme) recognition site.
  • the first display vector polynucleotide may include S5-display LC-S6 in a 5'to 3'direction, wherein the S5 and S6 may be recognition sites for restriction endonucleases.
  • the second display vector polynucleotide of may include the display LC, and the two ends of the display LC may also have cohesive ends of a specific sequence after digestion.
  • the term “display VH” generally refers to nucleotides encoding the heavy chain variable region of an antigen-binding polypeptide
  • the term “display LC” generally refers to nucleotides encoding the light chain of an antigen-binding polypeptide.
  • the "display VH” and “antigen-specific VH” in the present application can be nucleotides that encode the heavy chain variable region derived from a binding polypeptide against the same antigen, or can be a nucleotide that encodes a heavy chain variable region derived from a binding polypeptide against a different antigen.
  • nucleotides of the variable region of the chain can be nucleotides that encode the light chain derived from the binding polypeptide against the same antigen, or they can be encoded from the light chain The nucleotides of the light chain of the antigen-binding polypeptide.
  • display vector fragment generally refers to a fragment obtained by cutting the display vector with restriction endonucleases (such as BsmBI and/or SfiI), for example, display vector fragment I and display vector fragment II.
  • restriction endonucleases such as BsmBI and/or SfiI
  • the 5'end and 3'end of the display vector fragment may contain recognition sites for restriction endonucleases.
  • third display vector polynucleotide generally refers to a polynucleotide comprising display vector fragment I, which may have endonucleases (e.g., restriction endonucleases) at the 5'end and/or 3'end Dicer) recognition site.
  • the first display vector polynucleotide may comprise a B3-display vector fragment I-S5 in a 5'to 3'direction, wherein the B3 and S5 may be recognition sites of restriction endonucleases.
  • an endonuclease that recognizes the endonuclease recognition site in the second display vector polynucleotide for example, a restriction endonuclease that recognizes S5, such as SfiI, or a restriction endonuclease that recognizes B3
  • Nuclease, for example, BsmBI and/or Esp3I after the digestion treatment, the cleaved third display vector polynucleotide may include the display vector fragment I, and the two ends of the display vector fragment I may also be digested by restriction enzymes. After the sticky end of the specific sequence.
  • the term "fourth display vector polynucleotide” generally refers to a polynucleotide comprising display vector fragment II, which may have an endonuclease (e.g., restriction endonuclease) at the 5'end and/or 3'end Dicer) recognition site.
  • the first display vector polynucleotide may comprise S6-display vector fragment II-B2 in a 5'to 3'direction, wherein the S6 and B2 may be recognition sites of restriction endonucleases.
  • an endonuclease that recognizes the endonuclease recognition site in the second display vector polynucleotide for example, a restriction endonuclease that recognizes S6, such as SfiI, or a restriction endonuclease that recognizes B2
  • the fourth display vector polynucleotide after cutting may include the display vector fragment II, and the two ends of the display vector fragment II may also be digested by restriction enzymes. After the sticky end of the specific sequence.
  • VH module vector generally refers to a circular polynucleotide formed by inserting the ninth polynucleotide into an expression module vector.
  • LC module vector generally refers to a circular polynucleotide formed by inserting the tenth polynucleotide into an expression module vector.
  • the term "expression module vector” generally refers to a vector into which a polynucleotide (for example, the ninth polynucleotide and/or the tenth polynucleotide) can be inserted.
  • the expression module vector may be derived from a pMD vector.
  • the expression component vector can be a pMD19 vector or derived from a pMD19 vector.
  • the term "ninth polynucleotide” generally refers to a polynucleotide containing a VH component vector tool fragment, which may have endonucleases (for example, restriction endonucleases) at the 5'end and/or 3'end Dicer) recognition site.
  • the ninth polynucleotide may comprise the B2-VH component vector tool fragment-B3 in a 5'to 3'direction, wherein the B2 and B3 may be recognition sites of restriction endonucleases.
  • the term "tenth polynucleotide” generally refers to a polynucleotide comprising a LC component vector tool fragment, which may have endonucleases (for example, restriction endonucleases) at the 5'end and/or 3'end Dicer) recognition site.
  • the tenth polynucleotide may include the S5-LC component vector tool fragment-S6 in a 5'to 3'direction, wherein the S5 and S6 may be recognition sites for restriction endonucleases.
  • component vector tool fragment generally means that it can have recognition sites for endonucleases (for example, restriction endonucleases) at the 5'end and/or 3'end, but not in its interior. Any polynucleotide that has a recognition site for an endonuclease (e.g., restriction endonuclease).
  • the length of the component carrier tool fragment is usually different from the antigen-specific VH and antigen-specific LC. In some cases, the length of the component vector tool fragment can be about 1 kb. In some cases, the component vector tool fragment can be derived from the Fc region of IgG.
  • the component vector tool fragment may be derived from an Fc region selected from the group consisting of human IgG1 and human IgG2.
  • the recognition sites of endonucleases e.g., restriction endonucleases
  • the recognition sites of endonucleases can be S5 and S6. .
  • module vector generally refers to a circular polynucleotide formed by inserting a ninth polynucleotide and/or a tenth polynucleotide into an expression module vector.
  • the term "ninth bacterium” generally refers to the bacterium used to introduce or contain the ninth nucleotide.
  • the ninth bacterium may include the VH component vector.
  • the ninth bacterium can express, replicate and/or store (for example, cryopreservation) the ninth nucleotide and/or the VH component vector.
  • a VH component vector storage plasmid containing the VH component vector can be obtained from the ninth bacterium.
  • the term "tenth bacterium” generally refers to the bacterium used to introduce or contain the tenth nucleotide.
  • the ninth bacterium may include the LC component carrier.
  • the tenth bacterium can express, replicate and/or store (for example, cryopreserve) the tenth nucleotide and/or the LC component vector.
  • an LC component vector storage plasmid containing the LC component vector can be obtained from the tenth bacterium.
  • the term "sixth polynucleotide” generally refers to a polynucleotide comprising a VH component vector linking fragment, which may have an endonuclease (e.g., restriction endonuclease) at the 5'end and/or 3'end Dicer) recognition site.
  • the fourth polynucleotide may include the B3-VH component vector linkage fragment-B2 in a 5'to 3'direction, wherein the B3 and B2 may be recognition sites of restriction endonucleases.
  • the sixth polynucleotide may be contained in a VH component vector, and the endonuclease that recognizes the endonuclease recognition site in the sixth polynucleotide (for example, the restriction endonuclease that recognizes B3 and B2) After nuclease, such as BsmBI and/or Esp3I) digestion treatment, the sixth polynucleotide may be released, and the released fourth polynucleotide may include the antigen-specific VH, the antigen There may be sticky ends of specific sequences after digestion at both ends of the specific VH.
  • the endonuclease that recognizes the endonuclease recognition site in the sixth polynucleotide for example, the restriction endonuclease that recognizes B3 and B2
  • the sixth polynucleotide may be released, and the released fourth polynucleotide may include the antigen-specific VH, the antigen There may be sticky ends of specific sequence
  • the term "released sixth polynucleotide” generally refers to a fragment of the sixth polynucleotide released after the VH component carrier is processed.
  • the treatment may be restriction endonuclease digestion treatment.
  • a suitable restriction endonuclease for example, BsmBI and/or Esp3I
  • BsmBI and/or Esp3I can be selected for the recognition site of the restriction endonuclease on the display vector, so that the released sixth number
  • the nucleotides are released from the VH module carrier and separated.
  • the term "eighth polynucleotide” generally refers to a polynucleotide comprising a linking fragment of an LC component vector, which may have an endonuclease (e.g., restriction endonuclease) at the 5'end and/or 3'end Dicer) recognition site.
  • the fourth polynucleotide may comprise the S6-LC component vector linking fragment-S5 in a 5'to 3'direction, wherein the S6 and S5 may be recognition sites of restriction endonucleases.
  • the eighth polynucleotide may be included in the LC component vector, and the endonuclease that recognizes the endonuclease recognition site in the eighth polynucleotide (for example, the restriction endonuclease that recognizes S6 and S5) After nuclease, such as SfiI) digestion treatment, the eighth polynucleotide can be released, and the released eighth polynucleotide can include the antigen-specific LC, and the antigen-specific LC is two There may also be a sticky end with a specific sequence after digestion.
  • the endonuclease that recognizes the endonuclease recognition site in the eighth polynucleotide for example, the restriction endonuclease that recognizes S6 and S5
  • the eighth polynucleotide can be released, and the released eighth polynucleotide can include the antigen-specific LC, and the antigen-specific LC is two There may also be a
  • the term "released eighth polynucleotide” generally refers to a fragment of the eighth polynucleotide released after the LC component carrier is processed.
  • the treatment may be restriction endonuclease digestion treatment.
  • a suitable restriction endonuclease for example, SfiI
  • SfiI a suitable restriction endonuclease
  • 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, BsmBI and Esp3I.
  • the term "first bacterium” generally refers to the bacterium used to introduce or contain the antigen-specific binding polypeptide gene display vector.
  • the first bacterium may include the antigen-specific VH, the antigen-specific LC, the display vector fragment I, and the display vector fragment II.
  • the first bacterium can express, replicate and/or store (for example, cryopreservation) antigen-specific VH, said antigen-specific LC, said display vector fragment I and said display vector fragment II, Or the antigen specifically binds to the DNA of the polypeptide expression vector.
  • the term "antigen-specific binding polypeptide gene display bacterial library” generally refers to a bacterial library obtained by introducing the antigen-specific binding polypeptide gene display vector into the first bacterium.
  • the antigen-specific binding polypeptide gene display bacterial library may be a bacterial library comprising nucleic acid sequences encoding the light chain of the antigen-specific binding polypeptide or the heavy chain variable region of the antigen-specific polypeptide.
  • the antigen binding polypeptide that specifically show a bacterial gene library may comprise from about 105-- about 109 (e.g., may comprise from about 105-- 8 to about 10, from about 105-- 7 to about 10, from about 10 6 to about 10 7 ) nucleic acid sequences encoding the antigen-specific binding polypeptides.
  • the antigen-specific binding polypeptide gene display bacterial library may include about 10 7 to about 10 12 (for example, it may include about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 107-- about 109, from about 107-- 8 to about 10) of the first bacterium.
  • first display bacteria generally refers to the bacteria used to introduce or contain the first display vector polynucleotide.
  • the first bacterium may comprise the display VH.
  • the first display bacteria can express, replicate, and/or store (for example, cryopreservation) to display VH, and/or the first display vector polynucleotide.
  • the term "display VH component bacterial library” generally refers to a bacterial library obtained by introducing the first display vector polynucleotide into the first display bacteria.
  • the bacterial library displaying the VH module may be a bacterial library containing a nucleic acid sequence encoding a heavy chain variable region of an antigen-specific polypeptide.
  • the assembly of bacterial VH display libraries may comprise from about 105-- about 109 (e.g., may comprise from about 105-- 8 to about 10, from about 105-- 7 to about 10, from about 106-- about 10 7 ) the nucleic acid sequence encoding the display VH.
  • the bacterial library displaying VH components may include about 10 7 to about 10 12 (for example, it may include about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 7 to about 10 109, about 107-- 8 to about 10) of the first display bacteria.
  • second display bacteria generally refers to bacteria used to introduce or contain the second display vector polynucleotide.
  • the second bacterium may comprise the display LC.
  • the second display bacteria can express, replicate, and/or store (for example, cryopreservation) the display LC, and/or the second display vector polynucleotide.
  • the term "display LC component bacterial library” generally refers to a bacterial library obtained by introducing the second display vector polynucleotide into the second display bacteria.
  • the bacterial library displaying LC components may be a bacterial library containing a nucleic acid sequence encoding a light chain of an antigen-specific polypeptide.
  • the LC component of bacterial display libraries may comprise from about 105-- about 109 (e.g., may comprise from about 105-- 8 to about 10, from about 105-- 7 to about 10, from about 106-- about 10 7 ) nucleic acid sequences encoding the display LC.
  • the bacterial library displaying LC components may include about 10 7 to about 10 12 (for example, about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 7 to about 10 109, about 107-- 8 to about 10) of the second display bacteria.
  • third display bacteria generally refers to bacteria used to introduce or contain the third display vector polynucleotide.
  • the third bacterium may include the display vector fragment I.
  • the third display bacteria can express, replicate and/or store (e.g., cryopreserve) the display vector fragment I, and/or the third display vector polynucleotide.
  • the term "display vector component I bacterial library” generally refers to a bacterial library obtained by introducing the third display vector polynucleotide into the third display bacteria.
  • the display vector component I bacterial library may be a bacterial library containing a nucleic acid sequence encoding the display vector component I.
  • the display vector component I bacterial library may contain about 10 5 to about 10 9 (for example, it may contain about 10 5 to about 10 8 , about 10 5 to about 10 7 or about 10 6- About 10 7 ) nucleic acid sequences encoding the display LC.
  • the display vector component I bacterial library may include about 10 7 -about 10 12 (for example, it may include about 10 7 -about 10 11 , about 10 7 -about 10 10 , about 10 7- about 109, about 107-- about 108) show the third bacterium.
  • the term "fourth display bacterium” generally refers to a bacterium used to introduce or contain the fourth display vector polynucleotide.
  • the fourth bacterium may include the display vector fragment II.
  • the fourth display bacteria can express, replicate and/or store (for example, cryopreservation) the display vector fragment II, and/or the fourth display vector polynucleotide.
  • the term "display vector component II bacterial library” generally refers to a bacterial library obtained by introducing the third display vector polynucleotide into the fourth display bacteria.
  • the display vector component II bacterial library may be a bacterial library containing a nucleic acid sequence encoding the display vector component II.
  • the display vector component II bacterial library may contain about 10 5 to about 10 9 (for example, it may contain about 10 5 to about 10 8 , about 10 5 to about 10 7 , about 10 6- About 10 7 ) nucleic acid sequences encoding the display LC.
  • the display vector component II bacterial library may include about 10 7 to about 10 12 (for example, it may include about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 7- about 109, about 107-- about 108) show the fourth bacteria.
  • the term "introduction” generally refers to the process of transferring or introducing exogenous polynucleotides into cells.
  • 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 "linked” generally refers to linking two or more polynucleotide molecules together.
  • the ligation can be achieved by a ligase (e.g., DNA ligase).
  • a ligase e.g., DNA ligase
  • the 3'end of one polynucleotide is connected to the 5'end of another polynucleotide to form a complete polynucleotide molecule.
  • the term "clone” generally refers to the number of colonies.
  • the clone may be the number of colonies in a bacterial library (eg, the light chain component bacterial library, the heavy chain component bacterial library, the display bacterial library, and/or the phage library).
  • 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.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length.
  • Polymeric forms of acids such as deoxyribonucleotides or ribonucleotides, or their analogs, include, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, 100,000, and the like.
  • the polynucleotide may contain phosphodiester bonds.
  • 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 an antigen-specific binding polypeptide gene display vector.
  • the antigen-specific binding polypeptide gene display vector described in the present application can be formed by directional circularization of four fragments, which can respectively include antigen-specific VH, antigen-specific LC, display vector fragment I and display vector fragment II.
  • the method described in the present application may include constructing a component vector, which may include providing polynucleotides, for example, a ninth polynucleotide and a tenth polynucleotide.
  • the ninth polynucleotide may include the B2-VH component vector tool fragment-B3 in the 5'to 3'direction
  • the tenth polynucleotide may include the S5-LC component vector tool fragment in the 5'to 3'direction -S6.
  • the vector tool fragment can be derived from the Fc fragment of IgG.
  • the Fc fragment of human IgG For example, the Fc fragment of human IgG1. Another example is the Fc fragment of human IgG2.
  • DDB214 and DDB215 can be used as primers, and the Fc fragment of human IgG1 can be used as a template to amplify the ninth nucleotide.
  • the DDB214 may include the amino acid sequence shown in SEQ ID NO:1
  • the DDB215 may include the amino acid sequence shown in SEQ ID NO:2.
  • the tenth nucleotide can be amplified by using DDB216 and DDB217 as primers and the Fc fragment of human IgG1 as a template.
  • the DDB216 may include the amino acid sequence shown in SEQ ID NO:3
  • the DDB217 may include the amino acid sequence shown in SEQ ID NO:4.
  • Both ends of the ninth nucleotide may include restriction endonuclease recognition sites B2 and B3, and both ends of the tenth nucleotide may include restriction endonuclease recognition sites S5 and B3.
  • the method may include inserting the polynucleotides (for example, a ninth polynucleotide and a tenth polynucleotide) into an expression module vector to obtain a module vector (for example, a VH module vector and an LC module vector).
  • a module vector for example, a VH module vector and an LC module vector.
  • the expression component vector can be derived from any vector, for example, any vector that can be amplified and/or easily stored.
  • the vector used as an expression component vector may have properties such as high copy number and low molecular weight.
  • the expression module vector may be derived from a pMD vector, for example, the expression module vector may be a pMD19 vector or be derived from a pMD19 vector.
  • the pMD vector or pMD-derived vector in order to construct the expression component vector, can be engineered/modified.
  • one or more endonuclease recognition sites in the vector can be removed by site-directed mutagenesis (for example, one or more recognition sites of BsmBI and/or SfiI are removed).
  • one or more endonuclease recognition sites can also be added to the vector through site-directed mutagenesis (for example, one or more recognition sites for BsmBI and/or SfiI can be added at selected positions. ).
  • one or more BsmBI recognition sites originally contained in the vector can be removed by site-directed mutagenesis, and then one or more additional BsmBI recognition sites can be added to another position in the vector to obtain An engineered vector (e.g., an engineered pMD vector).
  • An engineered vector e.g., an engineered pMD vector
  • the expression component vector (for example, the expression component vector of the VH component vector) may contain the recognition site of BsmBI. In some cases, the expression component vector may include two recognition sites for BsmBI.
  • one or more SfiI recognition sites originally contained in the vector can be removed by site-directed mutagenesis, and then one or more additional SfiI recognition sites can be added to another position in the vector to Obtain an engineered vector (e.g., an engineered pMD vector).
  • an engineered vector e.g., an engineered pMD vector
  • the expression component vector (for example, the expression component vector of the LC component vector) may include the recognition site of SfiI. In some cases, the expression component vector may include two SfiI recognition sites.
  • the method may further include inserting the polynucleotides (for example, the ninth polynucleotide and the tenth polynucleotide) into an expression component vector to obtain a component vector storage plasmid (for example, a VH component The vector storage plasmid and the LC component vector storage plasmid), and then the component vector storage plasmid is introduced into the bacteria (for example, the ninth bacteria and the tenth bacteria) to obtain the component vector storage bacterial library (for example, the VH component vector storage bacterial library and the LC The component carrier stores the bacterial library).
  • a component vector storage plasmid for example, a VH component
  • the component vector storage plasmid for example, a VH component
  • the component vector storage bacterial library for example, the VH component vector storage bacterial library and the LC
  • the component carrier stores the bacterial library).
  • the VH component vector described in the present application is shown in Figure 3, which can be obtained by inserting the ninth polynucleotide into the expression component vector and ligating it.
  • the VH component vector includes a sixth polynucleotide, and the sixth polynucleotide may include a B3-VH component vector linking fragment-B2 in a 5'to 3'direction, wherein B2 and B3 can be BsmBI and/ Or Esp3I specific recognition and cleavage.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO: 8
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 9.
  • the VH component vector After processing (for example, restriction enzyme digestion), the VH component vector can produce a released sixth polynucleotide, the 5'end and 3'end of the released sixth polynucleotide can have The sticky end of the specific sequence produced by restriction digestion.
  • the LC component vector described in the present application is shown in FIG. 4, which can be obtained by inserting the tenth polynucleotide into the expression component vector and ligating.
  • the LC component vector includes an eighth polynucleotide, and the eighth polynucleotide may include the S6-LC component vector linking fragment-S5 in a 5'to 3'direction, wherein S6 and S5 can be specific for SfiI, respectively. Identify and cut.
  • the S6 may include the nucleic acid sequence shown in SEQ ID NO: 11
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO: 10.
  • the enzyme After being processed (for example, digestion), it can produce a released eighth polynucleotide, and the 5'end and 3'end of the released eighth polynucleotide can carry the product produced by digestion.
  • the sticky end of a specific sequence After being processed (for example, digestion), it can produce a released eighth polynucleotide, and the 5'end and 3'end of the released eighth polynucleotide can carry the product produced by digestion.
  • the sticky end of a specific sequence After being processed (for example, digestion), it can produce a released eighth polynucleotide, and the 5'end and 3'end of the released eighth polynucleotide can carry the product produced by digestion.
  • the sticky end of a specific sequence After being processed (for example, digestion), it can produce a released eighth polynucleotide, and the 5'end and 3'end of the released eighth polynucleotide can carry the product produced by digestion.
  • the sticky end of a specific sequence After being processed (
  • the method described in the present application includes providing a fifth polynucleotide, the fifth polynucleotide comprising B-antigen-specific VH-B in a 5'to 3'direction, wherein the B is capable of specifically recognizing B2 And/or the recognition site of the restriction endonuclease of B3.
  • an antigen-specific VH fragment can be used as a template to amplify the antigen-specific VH so that the 5'end and 3'end of the antigen-specific VH are connected with restriction endonucleases (for example, BsmBI and/or Esp3I) recognition site.
  • the method described in the present application includes providing a seventh polynucleotide comprising S-antigen-specific LC-S in a 5'to 3'direction, wherein the S is capable of specifically recognizing S5 And/or S6 restriction endonuclease recognition site.
  • an antigen-specific LC fragment can be used as a template to amplify the antigen-specific LC, so that the 5'end and 3'end of the antigen-specific LC are connected to the recognition by restriction endonuclease (for example, SfiI) Site.
  • methods of the prior art can be used to obtain antigen-specific VH fragments and antigen-specific LC fragments.
  • it can be obtained from an animal immunized with an antigen, or it can be obtained from an antibody library, including combinatorial antibody library, phage display library, yeast surface display library, ribosome display library, and mRNA display library.
  • the method described in the present application may include cutting the fifth polynucleotide and the VH component vector with a restriction endonuclease to obtain the cut fifth polynucleotide and the released sixth polynucleotide Acid, and then mix the cleaved fifth polynucleotide and the released sixth polynucleotide, so that it can be directionally connected and circularized to form an antigen-specific VH component library.
  • the antigen-specific VH component library may comprise the antigen-specific VH.
  • a restriction endonuclease for example, a restriction endonuclease that recognizes B2 and B3
  • the released antigen-specific VH can be obtained, and the antigen-specific VH can be obtained.
  • the 5'end and 3'end of the VH can have sticky ends with specific sequences.
  • the method described in the present application may include cutting the seventh polynucleotide and the LC component vector with a restriction endonuclease to obtain the cleaved seventh polynucleotide and the released eighth polynucleotide Acid, and then mix the cleaved seventh polynucleotide and the released eighth polynucleotide, so that it can be directionally connected and circularized to form an antigen-specific LC component library.
  • the antigen-specific LC component library may include the antigen-specific LC. After cutting the antigen-specific LC component library with a restriction endonuclease (for example, a restriction endonuclease that recognizes S5 and S6), the released antigen-specific LC can be obtained, and the antigen-specific The 5'end and 3'end of the LC can have sticky ends with specific sequences.
  • a restriction endonuclease for example, a restriction endonuclease that recognizes S5 and S6
  • the method described in the present application may further include constructing a display vector, which may be composed of four display vector polynucleotides (for example, the first display vector polynucleotide, the second display vector polynucleotide, the third display vector polynucleotide Nucleotides and the fourth display vector polynucleotide).
  • a display vector which may be composed of four display vector polynucleotides (for example, the first display vector polynucleotide, the second display vector polynucleotide, the third display vector polynucleotide Nucleotides and the fourth display vector polynucleotide).
  • the display vector polynucleotides may include antigen-binding polypeptides or Fragments, for example, display LC and/or display VH.
  • the display LC can encode the light chain of the antigen-binding polypeptide
  • the display VH can encode the variable region of the heavy chain of the antigen-binding polypeptide
  • the light chain can bind to the variable region of the heavy chain.
  • the Fab that recognizes the target is formed.
  • the target may be an antigen.
  • the target is PD-1.
  • the display vector polynucleotides described in the present application may include display vector fragments For example, display vector fragment I and display vector fragment II.
  • the display vector fragment I and the display vector fragment II of the desired length or type can be selected according to the length or properties of the antigen-binding polypeptide or its fragments to be expressed, and the length or properties of the restriction site.
  • the display vector fragment I and the display vector fragment II can be derived from any vector fragment capable of expressing the target gene.
  • the expression vector fragment I and expression vector fragment II 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 display vector fragments of the present application may include nucleotide sequences with specific functions, including but not limited to promoters, enhancers, signal peptides, selection markers (for example, Including enzyme recognition sites, resistance genes, reporter genes, screening genes), those skilled in the art can adjust the display vector fragments according to the desired function (insertion/replacement and/or deletion, etc.) sequence). In some cases, the display vector fragments can be adjusted under different circumstances to obtain different nucleotide sequences.
  • the first display vector polynucleotide may include B2-display VH-B3 in the 5'to 3'direction, wherein B2 and B3 may each independently be restriction endonuclease recognition sites,
  • the display VH can encode the variable region of the heavy chain of the antigen binding polypeptide.
  • the B2 and B3 can be specifically recognized and cleaved by BsmBI, respectively.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO: 8
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 9.
  • the second display vector polynucleotide may include S5-display LC-S6 in the 5'to 3'direction, wherein S5 and S6 may be restriction endonuclease recognition sites independently, and the display LC may The light chain that encodes the antigen-binding polypeptide.
  • the S5 and S6 can be specifically recognized and cleaved by SfiI, respectively.
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO: 0, and the S6 may include the nucleic acid sequence shown in SEQ ID NO: 11.
  • the third display vector polynucleotide may comprise a B3-display vector fragment I-S5 in a 5'to 3'direction, wherein B3 and S5 may each independently be a restriction endonuclease recognition site.
  • the S5 can be specifically recognized and cleaved by Sfil
  • the B3 can be specifically recognized and cleaved by BsmBI and/or Esp3I.
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO: 9
  • the S5 may include the nucleic acid sequence shown in SEQ ID NO: 10.
  • the fourth display vector polynucleotide may comprise S6-display vector fragment II-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
  • B2 can be specifically recognized and cleaved by BsmBI and/or Esp3I.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO: 8
  • the S6 may include the nucleic acid sequence shown in SEQ ID NO: 11.
  • the first display vector polynucleotide, the second display vector polynucleotide, the third display vector polynucleotide, and/or the display vector fourth polynucleotide of the present application can be obtained from sample materials.
  • the sample material may include an antigen-targeting antibody or antigen-binding fragment thereof.
  • 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.
  • the display vector polynucleotide (for example, the first display vector polynucleotide, the second display vector polynucleotide, the third display vector polynucleotide, and the second display vector polynucleotide)
  • the four-display vector may also include a nucleic acid sequence encoding a signal peptide, for example, a signal peptide that expresses 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.
  • nucleic acid sequence encoding the signal peptide may comprise a nucleic acid sequence selected from any one of SEQ ID NO: 12 and SEQ ID NO: 14, or the signal peptide may comprise a nucleic acid sequence selected from SEQ ID NO: 13. And the amino acid sequence shown in any one of SEQ ID NO: 15.
  • 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, display LC, display VH, display vector fragment I, and display vector fragment II), and then use this as a template to amplify separately to obtain the target fragments.
  • the primers for amplifying the display LC may include the nucleotide sequences shown in SEQ ID NO: 20 and SEQ ID NO: 21.
  • the primers for amplifying the display VH may include the nucleotide sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23.
  • the primer for amplifying the display vector fragment I may include the nucleotide sequences shown in SEQ ID NO: 18 and SEQ ID NO: 19.
  • the primer for amplifying the display vector fragment II may include the nucleotide sequences shown in SEQ ID NO: 16 and SEQ ID NO: 17.
  • the method in this application may further include the following steps: introducing the first display vector polynucleotide into the first display bacteria to obtain a bacterial library displaying VH components; and introducing the second display vector polynucleotide into the second display vector Display bacteria to obtain a bacterial library displaying LC module; introduce the third display vector polynucleotide into a third display bacteria to obtain a display vector module I bacterial library; introduce the fourth display vector polynucleotide into a fourth display bacteria to Obtain the display vector component II bacterial library.
  • the first display vector polynucleotide, the second display vector polynucleotide, the third display vector polynucleotide and the fourth display vector polynucleotide may all be linear nucleic acid molecules .
  • the display vector polynucleotide can be inserted into the display component vector to form a storage connection 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 a library of displayed bacteria.
  • the display bacterial library (e.g., the VH module display bacterial library and the LC module display bacterial library) may include about at least 10 (e.g., about at least 100, at least 200, at least 300, at least 400 , At least 500, at least 600, at least 800, about at least 1,000, about at least 10,000 or more) different clones.
  • the display bacterial library (for example, the display vector module I bacterial library and the display vector module II bacterial library) may comprise about at least 10 (for example, about at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 800, about at least 1,000, about at least 10,000 or more) identical clones.
  • the ratio of effective clones in the display bacterial library may be at least about 50% (For example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%).
  • the bacteria in the display bacterial library (for example, the display VH module bacterial library, the display LC module bacterial library, the display carrier module I bacterial library, and the display carrier module II bacterial library) can be cultured in liquid.
  • the culture time of the liquid culture can be no more than about 8 hours, for example, can be no more than about 4 hours, no more than about 5 hours, no more than about 6 hours, or no more than about 7 hours.
  • the liquid culture operation is relatively simple.
  • the bacteria in the display bacteria library can be cultured with a small amount of bacterial solution, and then colonies can be selected.
  • the culture time of the plate culture may be about 12-18 hours, for example, it may be about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
  • the plate culture can select colonies (for example, select single clones) and then perform sequencing analysis.
  • Antigen-specific binding polypeptide gene display vector
  • the method described in the present application may further include using restriction endonucleases (for example, restriction endonucleases that specifically recognize the S5, S6, B2, and B3) to specifically cleave the VH component library, the The LC component library and the display vector obtain the released first polynucleotide, the released second polynucleotide, the released third polynucleotide, and the released all The fourth polynucleotide.
  • restriction endonucleases for example, restriction endonucleases that specifically recognize the S5, S6, B2, and B3
  • the 5'end of the released first polynucleotide may have a sticky end cut by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes B2, for example, BsmBI and/or Esp3I).
  • the 3'end may have a sticky end cut by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes B3, for example, BsmBI and/or Esp3I).
  • the 5'end of the released second polynucleotide has a sticky end cleaved by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes S5, for example, SfiI), and the 3'end has Restriction endonuclease (for example, a restriction endonuclease that specifically recognizes S6, for example, SfiI) cuts the sticky end.
  • a restriction endonuclease for example, a restriction endonuclease that specifically recognizes S5
  • SfiI Restriction endonuclease
  • the 5'end of the released third polynucleotide has a sticky end cut by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes B3, for example, BsmBI and/or Esp3I),
  • the 3'end has a sticky end cut by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes S5, for example, SfiI).
  • the 5'end of the released fourth polynucleotide has a sticky end cleaved by a restriction endonuclease (for example, a restriction endonuclease that specifically recognizes S6, for example, SfiI), and the 3'end has A restriction endonuclease (for example, a restriction endonuclease that specifically recognizes B2, for example, BsmBI and/or Esp3I) cleaves the sticky end.
  • a restriction endonuclease for example, a restriction endonuclease that specifically recognizes S6, for example, SfiI
  • a restriction endonuclease for example, a restriction endonuclease that specifically recognizes B2, for example, BsmBI and/or Esp3I
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide may all be linear nucleic acid molecules.
  • the method of the present application may further include mixing the released first polynucleotide, the released second polynucleotide, the released third polynucleotide, and the released The fourth polynucleotide, so that it can be directionally connected and circularized to form the antigen-specific binding polypeptide gene display vector.
  • the directed ligation may include the use of a ligase, for example, T4 DNA ligase.
  • the antigen-specific binding polypeptide gene display vector can be used for the first bacteria to obtain an antigen-specific binding polypeptide gene display bacterial library.
  • the antigen-specific binding polypeptide gene display bacterial library may comprise about at least 10 (for example, about at least 100, about at least 200, about at least 300, about at least 400, about at least 500, About at least 1,000, about at least 10,000 or more) clones.
  • the ratio of effective clones in the antigen-specific binding polypeptide gene display bacterial library may be at least about 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, At least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%).
  • the required time can be at least about 1 week (for example, at least about 10 days, at least about 2 weeks, at least about 3 weeks, At least about 4 weeks).
  • the bacteria in the antigen-specific binding polypeptide gene display bacterial library can be cultured in liquid.
  • the culture time of the liquid culture can be no more than about 24 hours, for example, can be no more than about 5 hours, can be no more than about 10 hours, can be no more than about 15 hours, no more than about 20 hours, no more than about 22 hours or no more than about 22 hours.
  • the liquid culture operation is relatively simple.
  • the bacteria in the bacterial library can be plated with a small amount of bacterial solution and then selected for colonies.
  • the culture time of the plate culture may be about 12-18 hours, for example, may be about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
  • the plate culture can select colonies (for example, select single clones) and then perform sequencing analysis.
  • the method may further include introducing the antigen-specific binding polypeptide gene display vector into a cell, and obtaining the antigen-specific binding polypeptide from the cell.
  • the cell may be a mammalian cell.
  • the antigen-specific binding polypeptide can then be obtained from the bacteria.
  • the recognition site sequence of the restriction endonuclease is designed not to be included in the polynucleotide encoding the antigen-binding polypeptide or fragment thereof.
  • the restriction endonucleases of this application can specifically recognize B2, B3, S5, and S6, respectively. Wherein, the B2, B3, 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, or more than 4 restriction endonucleases, respectively.
  • the restriction endonuclease may be selected from SfiI, BsmBI and/or Esp3I. In other cases, other feasible restriction endonucleases can also be selected.
  • the restriction endonuclease can be selected from SfiI, BsmBI and Esp3I.
  • the BsmBI and Esp3I can be isoenzymes, which can recognize the same recognition site of restriction endonucleases.
  • the S5 and S6 can be identified and cut by SfiI.
  • the B2 and B3 can be recognized and cut by BsmBI and/or Esp3I.
  • SfiI can recognize a sequence consisting of 13 bases (5' to 3') GGCCNNNN/NGGCC, which can form an overhang at the 3'end (overhang, for example, a single-stranded sequence containing 3 bases) after digestion , Where N can represent any of the four bases of GATC. Therefore, there are 4 ⁇ 5 different sequences that can be recognized by SfiI.
  • BsmBI and Esp3I can recognize a sequence consisting of 12 bases (5' to 3') CGTTCCN/NNNNN, which can form an overhang at the 5'end after digestion, such as a single-stranded sequence containing 4 bases. Sequence), where N can represent any of the four bases of GATC. Therefore, there are 4 ⁇ 6 different sequences that can be recognized by BsmBI and Esp3I.
  • 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: 10.
  • the S6 may include the nucleic acid sequence shown in SEQ ID NO: 11.
  • the recognition site of the restriction endonuclease may be a site specifically recognized and cleaved by BsmBI and/or Esp3I, for example, may be referred to as B2 and B3, respectively.
  • the B2 may include the nucleic acid sequence shown in SEQ ID NO: 8.
  • the B3 may include the nucleic acid sequence shown in SEQ ID NO:9.
  • 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).
  • this application also provides the antigen-specific binding polypeptide gene display bacterial library.
  • this application also provides a display vector produced according to the method.
  • Total RNA is extracted from human peripheral blood lymphocytes, and mRNA is further isolated from total RNA (Takara Cat#Z652N/636592, see product instructions for specific test procedures).
  • the 5'-end of the light chain forward primer contains the nucleotide sequence of R1 GCCCCAGGCGGCC (SEQ ID NO: 78), and the 5'-end of the reverse primer contains the nucleotide sequence of R2 GGCCACATAGGCC (SEQ ID NO: 79);
  • the 5'-end of the heavy chain variable region forward primer contains the nucleotide sequence of R5 GGCCCAACCGGCC (SEQ ID NO: 80), and the 5'-end of the reverse primer contains the nucleotide sequence of R6 GGCCCTCAGCGGCC (SEQ ID NO: 81) .
  • the primers were synthesized by Jin Weizhi Company.
  • the 5'-end of the forward primer contains the nucleotide sequence of R3 GGCCACATAGGCC (SEQ ID NO: 79), and the 5'-end of the reverse primer contains the nucleotide sequence of R4 GGCCCAACCGGCC (SEQ ID NO: 80).
  • the specific primer sequence can refer to the following table 1-1:
  • the first step is to use the mRNA obtained in Example 1.1 as a template, and use Promega's MMLV to perform reverse transcription to synthesize cDNA (according to the Promega product instructions, where the primer is ThermoCat#N8080127, and the reverse transcriptase is Promega Cat#M1701) .
  • the second step is to use the cDNA obtained in the first step as a template and use the primers obtained in Example 1.2 to amplify the KLC, LLC and VH gene libraries of the component antibodies by PCR (Takara Cat#RR900A, carried out according to the company’s product instructions) . After purification and recovery by gel electrophoresis (using Axygen's gel recovery kit, follow the instructions in the "Molecular Cloning Experiment Guide”) to obtain PCR products-KLC fragments, LLC fragments and VH fragments.
  • the specific primer sequence can refer to the following table 1-2:
  • PCR was performed using the R1-1kb-R2 forward primer and R1-1kb-R2 reverse primer prepared in Example 1.4.1, After purification and recovery by gel electrophoresis (using Axygen gel recovery kit), the PCR product-R1-1kb-R2 (SEQ ID NO: 165) is obtained.
  • the R3-linker-R4 forward primer (SEQ ID NO: 160) and the R3-linker-R4 reverse primer (SEQ ID NO: 161) were used for PCR, purified and recovered by gel electrophoresis Then (using the Axygen gel recovery kit), the PCR product-R3-linker-R4 (SEQ ID NO: 167) is obtained.
  • the length of the linker may be 72 bp, and its nucleotide sequence is shown in SEQ ID NO: 166.
  • PCR was performed using the R5-1kb-R6 forward primer and R5-1kb-R6 reverse primer prepared in Example 1.4.1, After purification and recovery by gel electrophoresis, the PCR product-R5-1kb-R6 (SEQ ID NO: 168) is obtained.
  • the specific primer sequence can refer to the following table 1-3:
  • the linker storage vector can be used to transform TG1 competent bacteria (Lucigen company), cultured overnight at 37°C, send the colonies for sequencing, and then collect the colonies to obtain the linker component bacteria.
  • the connecting sub-assembly bacteria can be frozen for later use.
  • the polynucleotide including KLC and LLC prepared in Example 1.3 was digested with restriction endonucleases R1 and R2 to obtain the desired light chain fragment (approximately 0.65 kb).
  • the light chain storage vector DDB-R1-1kb-R2 prepared in Example 1.4 was digested with restriction endonucleases R1 and R2. A light chain storage vector fragment (approximately 2.7 kb) was obtained.
  • the obtained target light chain fragment and the light chain storage vector fragment are mixed, and then ligated by ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a light chain storage ligation product, and then use the light chain storage ligation product to transform TG1 competent Bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer's instructions), spread ampicillin-resistant plates (Thermo, Cat#240845), cultivate overnight at 37°C, send colonies for sequencing, and then collect all colonies to obtain light chain component bacteria library.
  • the quality of the light chain component bacterial library can be tested and/or the light chain component bacterial library can be frozen for use.
  • the polynucleotide prepared in Example 1.3 was digested with restriction endonucleases R5 and R6 to obtain the target heavy chain variable region fragment (about 0.35 kb).
  • the heavy chain storage vector DDB-R5-1kb-R6 prepared in Example 1.4 was digested with restriction endonucleases R5 and R6. A fragment of the heavy chain storage vector (approximately 2.7 kb) was obtained.
  • the obtained target heavy chain variable region fragments and heavy chain storage vector fragments are mixed, and then ligated with ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a heavy chain storage ligation product, and then use the heavy chain storage ligation product to transform TG1 competent bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer’s instructions), spread ampicillin-resistant plates (Thermo, Cat#240845), culture overnight at 37°C, pick the colonies for sequencing, and then collect all colonies to obtain a heavy Chain component bacterial library.
  • the quality of the heavy chain component bacterial library can be tested and/or the heavy chain component bacterial library can be frozen for later use.
  • the light chain component plasmid prepared in Example 1.5.1 was digested with restriction endonucleases R1 and R2, and purified and recovered by gel electrophoresis to obtain the light chain insert LC.
  • the heavy chain component plasmid prepared in Example 1.5.2 was digested with restriction endonucleases R5 and R6, and after purification and recovery by gel electrophoresis, the heavy chain insert HC was obtained.
  • the linker assembly bacteria prepared in Example 1.4.4 was extracted to obtain the linker assembly plasmid.
  • the linker forward primer (SEQ ID NO: 156) and the linker reverse primer (SEQ ID NO: 157) were used for amplification. Increase the 0.8kb fragment containing the linker, then cut the 0.8kb PCR product with restriction endonucleases R3 and R4, and purify and recover by gel electrophoresis (using a small fragment gel recovery kit, purchased from Laifeng Bio, Cat#DK402) to obtain a 72pb linker fragment.
  • the pComb3x vector was purchased and the vector map is shown in Figure 12, and the map of the modified pComb3x-fab vector used for antibody Fab display is shown in Figure 13.
  • the SfiI restriction site at the 3'end of the Fab gene in the pComb3x-fab vector was removed by nonsense mutation.
  • restriction endonuclease R2 cleavage site was added downstream of the stop codon of the light chain in the nonsense mutated vector, and a restriction endonuclease was introduced at the end of the signal peptide of the heavy chain variable region through nonsense mutations.
  • the restriction site of nuclease R5 was cut to obtain the modified phage display vector DDB-R1R2R5R6, and its map is shown in Figure 14.
  • the display vector DDB-R1R2R5R6 prepared in Example 1.7 was digested with restriction endonuclease R7 and restriction endonuclease R8 to obtain a 3.6 kb display vector fragment.
  • the light chain insert LC (0.65kb), heavy chain insert HC (0.35kb), linker fragment (72bp) and the phage display vector fragment (3.6kb) obtained in Example 1.6 were 1:1:1: Mix with the molecular ratio of 1, and use T4 DNA ligase at 20°C to ligate for more than 20 hours to obtain the ligation product for display.
  • the ligation product was purified by PCR-Clean-up and transferred to TG1 competent bacteria (Lucigen, Cat#60502-2, operated according to the manufacturer’s instructions), and cultured in 2YT medium without antibiotics 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 all the colonies grown on the plate are collected, which is the phage display bacterial library.
  • the phage display bacterial library can be stored for later use.
  • Example 1.8 An appropriate amount of bacterial liquid was taken from the displayed bacterial library prepared in Example 1.8, and cultured in 2YT medium (containing 100 ⁇ g/ml of ampicillin and 2% glucose) at 37° C. until the OD 600 reached 0.5. Then, M13KO7 helper phage (purchased from NEB, Cat#N0315S, MOI is about 10-20) was added to the bacterial solution and mixed, then stood still at 37°C for 30 minutes, then shaken at 37°C, 250rpm for 30 minutes, and centrifuged The supernatant of the culture medium containing the M13KO7 helper phage was discarded, and the bacteria were resuspended in a culture medium (containing ampicillin and kanamycin) 4 times the original volume of the bacterial liquid, and shaken overnight at 30°C and 250 rpm. The next day, the phage was collected by PEG precipitation, the phage concentration was titrated, and stored in aliquots. The display antibody phage library
  • Pembrolizumab (Pembrolizumab) and the pDGB4 vector were selected as examples.
  • Pembrolizumab light chain nucleotide sequence SEQ ID NO: 5
  • pembrolizumab heavy chain variable region nucleotide sequence SEQ ID NO: 6
  • pDGB4 vector nucleotide sequence SEQ ID NO: 7.
  • BsmBI and SfiI design two BsmBI recognition site sequences (B2 and B3) and two SfiI recognition site sequences (S5 and S6), where the nucleotide sequence of B2 is as SEQ ID NO: 8, the nucleotide sequence of B3 is shown in SEQ ID NO: 9, the nucleotide sequence of S5 is shown in SEQ ID NO: 10, and the nucleotide sequence of S6 is shown in SEQ ID NO: 11. Show.
  • SP1 and SP2 Two signal peptides expressing natural antibody genes were selected: SP1 and SP2.
  • SP1 expression shows VH, the nucleotide sequence is shown in SEQ ID NO: 12, and the amino acid sequence is shown in SEQ ID NO: 13;
  • SP2 expression shows LC, the nucleotide sequence is shown in SEQ ID NO: 14, and the amino acid sequence is shown in SEQ ID NO: 15 is shown.
  • the primers for display VH, display LC, display vector fragment I and display vector fragment II were designed respectively, and all of them were expressed by the CMV promoter. Synthesize primers and use the sequence in 2.1 as a template for PCR amplification. The sequence is shown in Table 2.
  • PCR (LA Taq, Takara company, according to the company's product instructions) was used to amplify the four display vector polynucleotides.
  • the used templates and primer sequences are shown in Table 2.
  • PCR products were obtained.
  • TA cloning (TA cloning kit, purchased from Takara)
  • the PCR product was inserted into the pUC19 plasmid vector to obtain the storage ligation product.
  • Transform DH5a competent bacteria with the storage vector product, culture overnight at 37°C in the plate, send the colony for sequencing, and obtain the display vector polynucleotide containing the required sequence of the bacteria-the first display vector polynucleus containing the VH A nucleotide, a second display vector polynucleotide containing a display LC, a third display vector polynucleotide containing a display vector fragment I, and a fourth display vector polynucleotide containing an expression vector fragment II.
  • the bacteria can be frozen and stored as a bacterial library for later use.
  • Example 2.4 Using a plasmid extraction kit (purchased from Axygen), the bacterial plasmids in the bacterial library in Example 2.4 were respectively extracted. The plasmid vector was then digested with restriction endonucleases BsmBI and SfiI. Electrophoresis is separated and purified to obtain four cut display vector polynucleotides.
  • Example 2 Mix the four cleaved display vector polynucleotides obtained in Example 2.5 in equal molecular proportions, add ligase to ligate and circularize them to form an expression vector, and transfer them to DH5a competent bacteria (Takara, follow the manufacturer’s instructions).
  • DH5a competent bacteria Teakara, follow the manufacturer’s instructions.
  • culture was shaken at 250 rpm at 37°C for 60 minutes, and an ampicillin-resistant plate (Thermo, Cat#240845) was spread and grown overnight at 37°C. Select the colony for sequencing, and get the display vector containing the correct sequence.
  • the specific structure of the display carrier is shown in Figure 2.
  • PCR was performed with primers DDB214 and DDB215. After purification and recovery by gel electrophoresis, the PCR product B2-KB-B3 was obtained.
  • the nucleotide sequence of DDB214 is shown in SEQ ID NO:1
  • the nucleotide sequence of DDB215 is shown in SEQ ID NO: 2.
  • the structure of the VH component carrier is shown in Figure 3.
  • PCR was performed with primers DDB216 and DDB217. After purification and recovery by gel electrophoresis, the PCR product S5-KB-S6 was obtained.
  • the nucleotide sequence of DDB216 is shown in SEQ ID NO: 3.
  • the nucleotide sequence of DDB217 is shown in SEQ ID NO: 4.
  • the structure of the LC component carrier is shown in Figure 4.
  • Example 1 Take 500 ⁇ l of the phage library constructed in Example 1 (Fab library, original storage volume 4 ⁇ 10 10 , effective clones greater than 80%, prepared phage library 2 ⁇ 10 13 /ml), and biotin-labeled ROR1 antigen (Acro Biosystems, Cat#RO1-H82E6) is mixed with the phage library (antigen concentration 10 ⁇ g/ml), and shaken at room temperature for 2 hours to allow the phage displaying antigen-specific Fab to bind to the biotin-labeled antigen).
  • Fab library original storage volume 4 ⁇ 10 10 , effective clones greater than 80%, prepared phage library 2 ⁇ 10 13 /ml
  • biotin-labeled ROR1 antigen Acro Biosystems, Cat#RO1-H82E6
  • the phage displaying ROR1 antigen-specific Fab was eluted with 50 ⁇ l of glycine solution at pH 2.2, and neutralized to pH 7.0 with 20 ⁇ l of Tris buffer at pH 8.0, and finally 75 ⁇ l of phage solution was obtained.
  • Example 4.2 Take 75 ⁇ l of the phage solution obtained in the second round of screening in Example 4.2, mix with 500 ⁇ l of TG1 bacteria in the logarithmic growth phase, and stand still at 37°C for 30 minutes. Then take the infected TG1 bacterial solution, spread Amp resistant plates, and incubate overnight at 37°C.
  • each well contains 400 ⁇ l culture medium (2YT+Amp+0.2% glucose), shake culture at 37°C for 6 hours, add 400 ⁇ l culture medium containing IPTG to each well (2YT+Amp+2mM IPTG), the final concentration of IPTG is 1mM, shake culture overnight at 30°C at 250rpm.
  • Two 96-well ELISA plates were coated with ROR1 antigen without biotin label, 100ng/100 ⁇ l/well, and coated overnight at 4°C.
  • Design primers containing recognition sites for restriction endonucleases (Esp3I and SfiI). 29 primers used to amplify antigen-specific VH, including 24 forward primers and 5 reverse primers; 19 primers used to amplify antigen-specific KLC (kappa light chain), including 18 forward primers , 1 reverse primer; 26 primers used to amplify antigen-specific LLC (lambda light chain), including 25 forward primers and 1 reverse primer; each set of forward primers are mixed in equal proportions , VH reverse primers are also mixed in equal proportions, and then forward and reverse primers are mixed in equal proportions to form three sets of primers, which are used to amplify VH, KLC and LLC.
  • this embodiment takes KLC as an example, where the forward primer of VH is shown in SEQ ID NO: 30-53, the reverse primer of VH is shown in SEQ ID NO: 54-58, and the forward primer of KLC is shown in SEQ ID NO: 54-58.
  • the reverse primer of KLC is shown in SEQ ID NO: 59-76, and the reverse primer of KLC is shown in SEQ ID NO 77.
  • the small DNA extracted from the 35 positive clones screened in Example 4 were mixed in equal amounts and amplified using the above three sets of primers. Electrophoresis analysis and purification of the antigen-specific VH and antigen-specific LC with recognition sites obtained by PCR (take KLC as an example).
  • the antigen-specific VH obtained in Example 5.1 was digested with Esp3I, and the digested antigen-specific VH was analyzed and purified by electrophoresis.
  • the VH component vector obtained in Example 3.1 was digested with Esp3I, and the digested 2.8 kb component vector fragment was purified by electrophoresis analysis.
  • the purified antigen-specific VH and a 2.8 kb component vector fragment were ligated to obtain a ROR1 specific VH component library.
  • the antigen-specific LC obtained in Example 5.1 was digested with SfiI, and the digested antigen-specific LC was analyzed and purified by electrophoresis.
  • the LC component vector obtained in Example 3.2 was digested with SfiI, and the digested 2.8kb component vector fragment was purified by electrophoresis analysis.
  • the purified antigen-specific LC and the 2.8 kb component vector fragment were ligated to obtain the ROR1 specific LC component library.
  • the VH component library was digested with Esp3I, and the digested 0.35 kb antigen-specific VH with sticky ends (ie, the released first polynucleotide) was purified by electrophoresis.
  • the KLC component library was digested with SfiI, and the digested 0.65 kb antigen-specific LC with sticky ends (ie, the released second polynucleotide) was purified by electrophoresis.
  • the display vector obtained in Example 1 was double digested with Esp3I and SfiI, and a 3kb display vector fragment I (ie, the released third polynucleotide) with a sticky end was purified and a 5kb display vector fragment with a sticky end was obtained.
  • the four fragments after digestion with the above-mentioned enzymes were mixed in equal molecular proportions, and ligated at 20°C for 4 hours, the ligation system was 10 ⁇ l, and the total amount of fragments was 25 ng, to obtain an antigen-specific binding polypeptide gene display vector.
  • the ligation product was purified with PCR Cleanup kit, and the ligation product was collected by elution with 10 ⁇ l ddH 2 O. Take 4 ⁇ l of the purified ligation product for electrotransformation (Takara DH5a, electrocompetent bacteria), plate, culture overnight at 37°C, count the colonies, and the storage capacity reaches 2.3 ⁇ 10 5 to obtain an antigen-specific binding polypeptide gene display bacterial library. Collect all the colonies and extract the carrier DNA to obtain the antigen-specific binding polypeptide display library.
  • the ROR1 specific binding polypeptide expression vector DNA was obtained from the antigen-specific binding polypeptide display library obtained in Example 6, and 40 ⁇ g of DNA was transformed into FCHO cells. After 60 hours of transformation, FACS was used to analyze the expression of the full-length antibody on the cell surface and the ROR1 antigen specificity of the antibody. The results in Figure 6 indicate that the full-length ROR1 antibody is expressed on the cell surface, and the expressed antibody can specifically bind to FITC-labeled ROR1 (obtained by labeling with a FITC labeling kit).
  • the ROR1 antigen without biotin label is from Acro Biosystems (Cat#RO1-H5250-1mg).
  • Figure 6 shows cells double-stained with PE-labeled mouse anti-human Kappa light chain antibody and FITC-labeled ROR1 antigen, and FACS analysis of the fluorescence signal on the cell surface.
  • A negative control
  • B cell bank expressing ROR1 specific antibodies.
  • hygromycin hygromycin concentration 500 ⁇ g/ml
  • BD PE-labeled mouse anti-human kappa light chain antibody
  • FITC-labeled ROR1 antigen PE and FITC double-positive cells were sorted by FACS.
  • the single cell clones were added to a 96-well plate, one cell per well, and hygromycin pressure culture.
  • 92 stably transformed single cell clones were obtained.
  • the cells were digested with 0.5mM EDTA-PBS buffer, and 92 single cell clones were double-stained with PE-labeled mouse anti-human kappa light chain antibody and FITC-labeled ROR1 antigen (antigen concentration 0.15ng/50 ⁇ l).
  • a total of 30 cell clones located at different positions (representing different affinities) were selected for PCR amplification of antibody genes.
  • the cells of the positive clones were collected by centrifugation, the supernatant was discarded, and the cell genome DNA was extracted with 20 ⁇ l of cell genome extraction solution (Quick Extraction Buffer, Lucigen) according to the reagent instructions. Take 2 ⁇ l of cell genomic DNA extract from each clone, and amplify the VH and LC of each clone by PCR.
  • the forward primer used to amplify the VH fragment is TGGGCTCTGCTCCTCCTGACC (SEQ ID NO: 24), and the reverse primer used to amplify the VH fragment is AGTTCCACGACACCGTCACCGGTTC (SEQ ID NO: 25), the forward primer used to amplify the LC fragment It is GGACCTGAGGATCCTCTTCTTGG (SEQ ID NO: 26), and the forward primer used to amplify the LC fragment is TAAATTCCTCGGCCGTGCAGGCCTTATCAACACTCTCCCCTGTTGAAGCTCT (SEQ ID NO: 27).
  • VH and LC fragments amplified by PCR were separated and purified by electrophoresis. Sequencing analysis of the purified VH and LC fragments identified 14 unique VHs and 13 unique LCs, combined to obtain 17 unique sequence positive clones (the light and heavy chain 6 CDRs have at least one amino acid difference).
  • here is a pair of unique sequences of VH and LC.
  • the VH amino acid sequence of the unique sequence is shown in SEQ ID NO: 28, and the kappa LC amino acid sequence of the unique sequence is shown in SEQ ID NO: 29.
  • Example 8 The positive VH fragment with unique sequence obtained in Example 8 was digested with Esp3I, and the positive LC fragment with unique sequence in Example 8 was digested with SfiI. Use PCR cleanup to purify the digested VH and LC fragments. Insert the VH fragment and LC fragment into the soluble heavy chain expression vector, and send the colony sequencing for confirmation. Extract VH and LC expression vector DNA confirmed by sequencing.
  • 293EXP cells were expanded by suspension culture, and 17 antibody light and heavy chain expression vector pairs were formed according to the light and heavy chain pairing determined in Example 8. Each pair was mixed at a ratio of 18 ⁇ g of light chain expression vector and 12 ⁇ g of heavy chain expression vector to transform 30 ml of suspended 293EXP cells (1.2 ⁇ 10 6 /ml).
  • the culture medium supernatant was collected, and the antibody was purified with GenScript's magnetic beads (Cat#L00695) according to the product specification, dialysis equilibrated to PBS-antibody solution, and stored at -80°C. Analysis by SDS-PAGE denaturing gel electrophoresis (Figure 7) showed that the antibody purity reached more than 90%.
  • ELISA was used to analyze the binding affinity of the purified antibody to the antigen.
  • the EC 50 of 8 exemplary antibodies is listed as an illustration. The results are shown in Table 4 below.
  • the vector DNA was extracted according to the implementation.
  • 15 clones of antigen-specific VH and antigen-specific LC were respectively amplified by PCR, and purified by restriction enzyme digestion.
  • the antigen-specific binding polypeptide display library was constructed. Then 48 colonies were randomly selected and sent for sequencing. The sequencing results were analyzed, 36 clones with correct VH and LC were selected, the vector DNA was extracted, and the CHO cells were transiently transfected.
  • FIG. 8 shows cells double-stained with PE-labeled mouse anti-human Kappa light chain antibody and FITC-labeled ROR1 antigen, and FACS analysis of the fluorescence signal on the cell surface.
  • A negative control
  • B cell clones expressing non-ROR1 specific antibodies
  • C-H 6 exemplary positive cell clones expressing ROR1 specific antibodies.

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Abstract

本申请公开了一种构建抗原特异性结合多肽基因展示载体的方法，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四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。本申请还公开了根据该方法产生的抗原特异性结合多肽基因展示载体和细菌文库。本申请所述方法能够用于有效筛选抗原特异性抗原结合多肽或其片段。

Description

抗原特异性结合多肽基因展示载体的构建方法与应用 技术领域
本申请涉及生物医药领域，具体的涉及一种构建抗原特异性结合多肽基因展示载体的方法，该展示载体可用于筛选抗原特异性结合多肽。
背景技术
目前，现有技术中常用的抗体发现方法主要有两大类：杂交瘤技术和抗体展示技术。杂交瘤技术又有鼠源杂交瘤和转基因小鼠杂交瘤两类。抗体展示技术主要有三类：分别是噬菌体展示，酵母展示和哺乳动物细胞展示。每个抗体发现技术有很明确的优点，但也有非常大的缺点和局限，例如，构建抗体药种子库的过程缺少质量控制，导致抗体库的质量差、库容小，多样性少，有效克隆比例低，难以筛选到高质量的先导抗体，或者，抗体库筛选技术没有定量筛选，且筛选通量小，筛选效果不佳，时间耗费大。
因此，需要创新的抗体发现技术，以提高筛选创新抗体药先导抗体分子的质量、数量及多样性，加快抗体药开发速度，提高开发成功率。
发明内容
本申请提供了一种构建抗原特异性结合多肽基因展示载体的方法，所述抗原特异性结合多肽基因展示载体由四个片段构成，通过构建组件库和展示载体，使所述四个片段的5’端和3’端带有特定序列的粘性末端，从而定向环化形成所述抗原特异性结合多肽基因展示载体。本申请构建抗原特异性结合多肽基因展示载体的方法以及使用本申请抗原特异性结合多肽基因展示载体筛选抗原特异性结合多肽的方法具备至少一个以下性质：1)构建本申请所述的抗原特异性结合多肽基因展示载体可以采用特殊的限制性内切核酸酶的识别位点，既保证定向连接，又防止错误连接，连接时各个组件片段的分子个数可以控制为1∶1，从而提高连接转化效率；同时，采用构建VH组件库和LC组件库等策略，提高各个片段的连接和转化效率；2)没有采用本领域常规抗体库构建方法所使用的组合PCR策略，有效减少PCR所导致的引入突变的几率；3)能够较容易地进行质量控制，能够满足工业化量产的需求；4)可将展示载体导入细胞后直接通过生物活性分析实验筛选，有效缩短了从构建抗原特异性结合多肽基因展示载体到筛选得到独特序列的抗原特异性多肽的时间。例如，从构建展示载体到筛选出独特序列的阳性克隆的时间可以为至少约1周(至少约10天、至少约2周、至少约3周、至少约4周、至少约5周、至少约6周、至少约7周、至少约8周)；5)展示载体的细菌文库 中克隆多样性大，筛选效率高，在某些情形中，所述包含抗原特异性结合多肽基因展示载体的库中，有效克隆比例可达到约50％以上(例如，约55％以上、约60％以上、约65％以上、约70％以上、约75％以上、约80％以上、约85％以上、约90％以上、约95％以上或更高)，转化效率高，建库成功率高。
一方面，本申请提供了一种用于构建抗原特异性结合多肽基因展示载体的方法，所述方法包括：a)提供第一展示载体多核苷酸，所述第一展示载体多核苷酸以5’至3’方向包含B2-展示VH-B3；b)提供第二展示载体多核苷酸，所述第二展示载体多核苷酸以5’至3’方向包含S5-展示LC-S6；c)提供第三展示载体多核苷酸，所述第三展示载体多核苷酸以5’至3’方向包含B3-展示载体片段I-S5；d)提供第四展示载体多核苷酸，所述第四展示载体多核苷酸以5’至3’方向包含S6-展示载体片段II-B2；e)利用限制性内切核酸酶特异性切割所述第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和所述第四展示载体多核苷酸，得到切割后的所述第一展示载体多核苷酸、切割后的所述第二展示载体多核苷酸、切割后的所述第三展示载体多核苷酸和切割后的所述第四展示载体多核苷酸；其中所述限制性内切核酸酶分别特异性识别B2、B3、S5和S6；f)混合所述切割后的第一展示载体多核苷酸、所述切割后的第二展示载体多核苷酸、所述切割后的第三展示载体多核苷酸和所述切割后的第四展示载体多核苷酸，从而使得其能够定向连接而环化形成所述抗原特异性结合多肽基因展示载体；其中，所述展示VH编码抗原特异性结合多肽的重链可变区，所述展示LC编码抗原特异性结合多肽的轻链；其中所述B2、B3、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、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、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、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。
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、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。
在某些实施方式中，所述限制性内切核酸酶选自SfiI、Esp3I和BsmBI。
在某些实施方式中，所述B2和B3能够被选自下组的酶特异性识别及切割：BsmBI和Esp3I。
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:8所示的核酸序列。
在某些实施方式中，所述B3包含SEQ ID NO:9所示的核酸序列。
在某些实施方式中，所述S5包含SEQ ID NO:10所示的核酸序列。
在某些实施方式中，所述S6包含SEQ ID NO:11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展示载体多核苷酸导入第一展示细菌以获得展示VH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展示载体多核苷酸插入展示组件载体，形成展示VH存储连接产物，并且将所述展示VH存储连接产物导入所述第一展示细菌以获得所述展示VH组件细菌文库。
在某些实施方式中，所述方法还包括将所述第二展示载体多核苷酸导入第二展示细菌以获得展示LC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展示载体多核苷酸插入展示组件载体，形成展示LC存储连接产物，并将所述展示LC存储连接产物导入所述第二展示细菌中以获得所述展示LC组件细菌文库。
在某些实施方式中，所述方法还包括将所述第三展示载体多核苷酸导入第三展示细菌以获得展示载体组件I细菌文库。
在某些实施方式中，所述方法包括将所述第三展示载体多核苷酸插入展示组件载体形成展示载体片段I存储连接产物，并将所述存储连接产物导入所述第三展示细菌中以获得所述展示载体组件I细菌文库。
在某些实施方式中，所述方法还包括将所述第四展示载体多核苷酸导入第四展示细菌以获得展示载体组件II细菌文库。
在某些实施方式中，所述方法包括将所述第四展示载体多核苷酸插入展示组件载体形成展示载体片段II存储连接产物，并将所述存储连接产物导入所述第四展示细菌中以获得所述展示载体组件II细菌文库。
在某些实施方式中，所述展示载体组件载体源自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，所述方法还包括由所述展示VH组件细菌文库获得包含所述第一展示载体多核苷酸的展示VH组件质粒，由所述展示VH组件质粒获得切割后的所述第一展示载体多核苷酸。
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B3的限制性内切核酸酶对所述展示VH组件质粒进行酶切处理，从而获得所述切割后的所述第一展示载体多核苷酸。
在某些实施方式中，所述方法还包括由所述展示LC组件细菌文库获得包含所述第二展示载体多核苷酸的展示LC组件质粒；由所述展示LC组件质粒获得切割后的所述第二展示载体多核苷酸。
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展示LC组件质粒进行酶切处理，从而获得所述切割后的所述第二展示载体多核苷酸。
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三展示载体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三展示载体多核苷酸。
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S5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三展示载体多核苷酸。
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四展示载体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四展示载体多核苷酸。
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II进行酶切处理，从而获得所述切割后的所述第四展示载体多核苷酸。
在某些实施方式中，所述方法包括：
a)提供第五多核苷酸，所述第五多核苷酸以5’至3’方向包含B-抗原特异性VH-B；
b)提供VH组件载体，所述VH组件载体包含第六多核苷酸，所述第六多核苷酸以5’至3’方向包含B3-VH组件载体连接片段-B2；
c)利用限制性内切核酸酶切割所述第五多核苷酸和所述VH组件载体，得到切割后的第五多核苷酸和经释放的第六多核苷酸；
d)混合所述切割后的第五多核苷酸和所述经释放的第六多核苷酸，从而使得其能够定向连接而环化形成抗原特异性VH组件库；
其中所述B为能够特异性识别B2和/或B3的限制性内切核酸酶的识别位点，所述抗原 特异性VH编码所述抗原特异性结合多肽的重链可变区。
在某些实施方式中，所述方法包括：
a)提供第七多核苷酸，所述第七多核苷酸以5’至3’方向包含S-抗原特异性LC-S；
b)提供LC组件载体，所述LC组件载体包含第八多核苷酸，所述第八多核苷酸以5’至3’方向包含S6-LC组件载体连接片段-S5；
c)利用限制性内切核酸酶切割所述第七多核苷酸和所述LC组件载体，得到切割后的第七多核苷酸和经释放的第八多核苷酸；
d)混合所述切割后的第七多核苷酸和所述经释放的第八多核苷酸，从而使得其能够定向连接而环化形成抗原特异性LC组件库，
其中所述S为能够特异性识别S5和/或S5的限制性内切核酸酶的识别位点，所述抗原特异性LC编码所述抗原特异性结合多肽的轻链。
在某些实施方式中，所述方法包括，
a)提供第九多核苷酸，所述第九多核苷酸以5’至3’方向包含B2-VH组件载体工具片段-B3；
b)将所述第九多核苷酸插入表达组件载体，获得所述VH组件载体。
在某些实施方式中，所述方法包括，
a)提供第十多核苷酸，所述第十多核苷酸以5’至3’方向包含S5-LC组件载体工具片段-S6；
b)将所述第十多核苷酸插入表达组件载体，获得所述LC组件载体。
在某些实施方式中，所述表达组件载体源自pMD载体。
在某些实施方式中，所述pMD载体为pMD19载体或源自pMD19载体。
在某些实施方式中，所述方法包括以下步骤：
a)将所述VH组件载体导入第九细菌，获得VH组件载体存储细菌文库；
b)由所述VH组件载体存储细菌文库得到VH组件载体存储质粒；
c)由所述VH组件载体存储质粒获得经释放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B2和B3的限制性内切核酸酶对所述VH组件载体存储质粒进行酶切处理，从而获得所述经释放的所述第六多核苷酸。
在某些实施方式中，所述方法包括以下步骤：
a)将所述LC组件载体导入第十细菌，获得LC组件载体存储细菌文库；
b)由所述LC组件载体存储细菌文库得到LC组件载体存储质粒；
c)由所述LC组件载体存储质粒获得经释放的所述第八多核苷酸。
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LC组件载体存储质粒进行酶切处理，从而获得所述经释放的所述第八多核苷酸。
在某些实施方式中，所述方法包括：
a)提供所述抗原特异性VH组件库，所述抗原特异性VH组件库包括第一多核苷酸，所述第一多核苷酸以5’至3’方向包含B2-抗原特异性VH-B3；
b)提供所述抗原特异性LC组件库，所述抗原特异性LC组件库包括第二多核苷酸，所述第二多核苷酸以5’至3’方向包含S5-抗原特异性LC-S6；
c)提供所述展示载体，所述展示载体包含第三多核苷酸和第四多核苷酸，所述第三多核苷酸以5’至3’方向包含B3-展示载体片段I-S5，所述第四多核苷酸以5’至3’方向包含S6-展示载体片段II-B2；
d)利用限制性内切核酸酶特异性切割所述抗原特异性VH组件库、所述抗原特异性LC组件库和所述展示载体，得到经释放的所述第一多核苷酸、经释放的所述第二多核苷酸、经释放的所述第三多核苷酸和经释放的所述第四多核苷酸；其中所述限制性内切核酸酶分别特异性识别B2、B3、S5和S6；
e)混合所述经释放的第一多核苷酸、所述经释放的第二多核苷酸、所述经释放的第三多核苷酸和所述经释放的第四多核苷酸，从而使得其能够定向连接而环化形成抗原特异性结合多肽基因展示载体；
其中，所述抗原特异性LC编码所述抗原特异性结合多肽的轻链，所述抗原特异性VH编码所述抗原特异性结合多肽的重链可变区；
其中所述B2、B3、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，所述方法包括使用特异性识别B2和B3的限制性内切核酸酶对所述抗原特异性VH组件库进行酶切处理，从而获得所述经释放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S5和S6的限制性内切核酸酶对所述抗原特异性LC组件库进行酶切处理，从而获得所述经释放的所述第二多核苷酸。
在某些实施方式中，所述方法包括使用特异性识别B3的限制性内切核酸酶和特异性识别S5的限制性内切核酸酶对所述展示载体进行酶切处理，从而获得所述经释放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S6的限制性内切核酸酶和特异性识别B2的限制性内切核酸酶对所述展示载体进行酶切处理，从而获得所述经释放的所述第四多核 苷酸。
在某些实施方式中，由样品材料获得所述第五多核苷酸、第七多核苷酸、第九多核苷酸、第十多核苷酸、第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和/或所述第四展示载体多核苷酸。
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和/或IgG。
在某些实施方式中，所述抗体或其抗原结合片段靶向ROR1、PD-1和/或PD-L1。
在某些实施方式中，所述IgG为人IgG。
在某些实施方式中，所述人IgG为人IgG1或人IgG2。
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T4 DNA连接酶。
在某些实施方式中，所述方法包括将所述抗原特异性结合多肽基因展示载体导入细胞，由所述细胞得到所述抗原特异性结合多肽。
在某些实施方式中，所述方法包括：
a)将所述抗原特异性结合多肽基因展示载体导入第一细菌，获得抗原特异性结合多肽基因展示细菌文库；
b)从所述抗原特异性结合多肽基因展示细菌文库获得抗原特异性结合多肽展示基因库；
c)由所述抗原特异性结合多肽展示基因库得到抗原特异性结合多肽表达载体DNA；
d)将所述抗原特异性结合多肽表达载体DNA导入细胞；
e)由所述细胞得到所述抗原特异性结合多肽。
在某些实施方式中，所述方法包括冷冻保存所述抗原特异性结合多肽基因展示细菌文库、所述VH组件载体存储细菌文库、所述LC组件载体存储细菌文库、所述展示VH组件细菌文库、所述展示LC组件细菌文库、所述展示载体组件I细菌文库和所述展示载体组件II细菌文库。
在某些实施方式中，所述VH组件载体存储细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述LC组件载体存储细菌文库包含至少10个不同的克隆。
在某些实施方式中，所述展示VH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，所述展示LC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，所述展示载体组件I细菌文库包含至少10个相同的克隆。
在某些实施方式中，所述展示载体组件II细菌文库包含至少10个相同的克隆。
在某些实施方式中，所述抗原特异性结合多肽基因展示细菌文库中有效克隆的比例为至少约10％。
在某些实施方式中，所述细胞为哺乳动物细胞。
另一方面，本申请提供了筛选抗原特异性结合多肽或其片段的方法，其包括使用所述的抗原特异性结合多肽基因展示载体。
另一方面，本申请提供了根据所述的方法所产生的抗原特异性结合多肽基因展示载体。
另一方面，本申请提供了根据所述的方法所产生的抗原特异性结合多肽基因展示细菌文库。
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展示载体的结构；
图2显示的是本申请所述展示载体的示例；
图3显示的是本申请所述VH组件载体的结构；
图4显示的是本申请所述LC组件载体的结构；
图5A显示的是本申请所述抗原特异性LC的氨基酸序列，图5B显示的是本申请所述抗原特异性VH的氨基酸序列；
图6显示的是FACS分析ROR1抗原特异性结合多肽在CHO细胞表面的表达；
图7显示的是8个示例性抗体的SDS-PAGE变性还原凝胶电泳分析；
图8显示的是使用本申请的方法筛选得到的阳性抗体的FACS分析结果；
图9显示的是构建噬菌体库过程中的轻链存储载体的示意图；
图10显示的是构建噬菌体库过程中的重链存储载体的示意图；
图11显示的是构建噬菌体库过程中的连接子存储载体的示意图；
图12显示的是构建噬菌体库过程中的pCom3x载体的示意图；
图13显示的是构建噬菌体库过程中的噬菌体展示载体的示意图；
图14显示的是构建噬菌体库过程中的展示用连接产物的质粒的示意图。
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“抗原结合多肽”通常是指能够特异性识别和/或中和特定抗原的多肽分子。该术语可包括抗体或其抗原结合部分，或者完整抗体的抗原结合区和/或抗体可变区。基本的四链抗体单元是异四聚体糖蛋白，其由两条相同的轻链和两条相同的重链构成。在IgG的情况中，每个L链通过一个共价二硫键连接到H链，而两条H链通过一个或多个二硫键彼此相连，二硫键的数目取决于H链的同种型。每个H和L链还具有规律间隔的链内二硫键。每条H链在N-末端具有可变结构域(VH)，接着是三个(对于每条α和γ链)或四个(对于μ和ε同种型)恒定结构域(CH)。所述抗原结合多肽可以通过化学方法和/或基因工程的方法获得。例如，可以采用蛋白酶，包括胃蛋白酶和木瓜蛋白酶，消化抗体后产生所述抗原结合片段。在本申请中，所述抗体片段可以为Fab。
在本申请中，术语“Fab”通常是指由木瓜蛋白酶消化具有完整结构的抗体后(例如，去除了Fc区和铰链区)而产生两个相同的抗原结合片段。Fab可以由完整的轻链、重链可变区(VH)和重链的第一恒定结构域(CH1)组成。每个Fab可以具有单一的抗原结合位点。
在本申请中，术语“第一多核苷酸”通常是指包含抗原特异性VH的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一多核苷酸可以以5’至3’方向包含B2-抗原特异性VH-B3，其中所述B2、B3可以为限制性内切核酸酶的识别位点。例如，经识别所述第一多核苷酸中的内切酶识别位点的内切酶(例如，识别B2和B3的限制性内切核酸酶，例如BsmBI)酶切处理后，经释放的所述第一多核苷酸可包含所述抗原特异性VH，所述抗原特异性VH两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“第二多核苷酸”通常是指包含抗原特异性LC的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一多核苷酸可以以5’至3’方向包含S5-抗原特异性LC-S6，其中所述S5、S6可以为限制性内切核酸酶的识别位点。例如，经识别所述第一多核苷酸中的内切酶识别位点的内切酶(例如，识别S5和 S6的限制性内切核酸酶，例如SfiI)酶切处理后，经释放的所述第一多核苷酸可包含所述抗原特异性LC，所述抗原特异性LC两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“抗原特异性VH”通常是指包含编码能够与抗原进行特异性结合的抗体重链可变区的核苷酸，术语“抗原特异性LC”通常是指编码包含能够与抗原进行特异性结合的抗体轻链的核苷酸。可由现有技术中的任何方法得到抗原特异性VH和抗原特异性LC的序列，这些方法包括但不限于噬菌体展示技术、酵母表面展示技术、核糖体展示技术、mRNA展示技术和/或杂交瘤技术，例如，可通过噬菌体库展示的方法得到。
在本申请中，术语“第三多核苷酸”通常是指包含展示载体片段I的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第三多核苷酸可以以5’至3’方向包含B3-展示载体片段I-S5，其中所述B3、S5可以为限制性内切核酸酶的识别位点。所述第三多核苷酸可以包含在展示载体中，经识别所述第三多核苷酸中的内切酶识别位点的内切酶(例如，识别B3和S5的限制性内切核酸酶，例如SfiI、BsmBI和/或Esp3I)酶切处理后，所述第三多核苷酸可以被释放，经释放的所述第三多核苷酸可包含所述展示载体片段I，所述展示载体片段I两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“经释放的第三多核苷酸”通常是指展示载体经处理后释放出的第三多核苷酸的片段。在本申请中，所述处理可以为经限制性内切核酸酶酶切处理。例如，可以针对所述展示载体上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，SfiI、BsmBI和/或Esp3I)，从而使所述经释放的第三多核苷酸从所述展示载体中释放而分离。
在本申请中，术语“第四多核苷酸”通常是指包含展示载体片段II的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第四多核苷酸可以以5’至3’方向包含S6-展示载体片段II-B2，其中所述S6、B2可以为限制性内切核酸酶的识别位点。所述第四多核苷酸可以包含在展示载体中，经识别所述第四多核苷酸中的内切酶识别位点的内切酶(例如，识别S6和B2的限制性内切核酸酶，例如SfiI、BsmBI和/或Esp3I)酶切处理后，所述第四多核苷酸可以被释放，经释放的所述第四多核苷酸可包含所述展示载体片段II，所述展示载体片段II两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“经释放的第四多核苷酸”通常是指展示载体经处理后释放出的第四多核苷酸的片段。在本申请中，所述处理可以为经限制性内切核酸酶酶切处理。例如，可以针对所述展示载体上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，SfiI、BsmBI和/或Esp3I)，从而使所述经释放的第四多核苷酸从所述展示载体中释放而分离。
在本申请中，术语“第五多核苷酸”通常是指包含所述抗原特异性VH的多核苷酸，其可 以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第五多核苷酸可以以5’至3’方向包含B-所述抗原特异性VH-B，其中所述B可以为限制性内切核酸酶的识别位点，该限制性内切核酸酶可以是能够识别B2和/或B3的限制性内切核酸酶。经识别所述第五多核苷酸中的内切酶识别位点的内切酶(例如，识别B的限制性内切核酸酶，例如BsmBI和/或Esp3I)酶切处理后，切割后的所述第五多核苷酸可包含所述抗原特异性VH。
在本申请中，术语“第七多核苷酸”通常是指包含所述抗原特异性LC的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第五多核苷酸可以以5’至3’方向包含S-所述抗原特异性LC-S，其中所述S可以为限制性内切核酸酶的识别位点，该限制性内切核酸酶可以是能够识别S5和/或S6的限制性内切核酸酶。经识别所述第七多核苷酸中的内切酶识别位点的内切酶(例如，识别S的限制性内切核酸酶，例如SfiI)酶切处理后，切割后的所述第七多核苷酸可包含所述抗原特异性LC。
在本申请中，术语“VH组件载体”通常是指包含第六多核苷酸和/或VH组件载体工具片段的环状多核苷酸。
在本申请中，术语“VH组件载体”通常是指包含第八多核苷酸和/或LC组件载体工具片段的环状多核苷酸。
在本申请中，术语“展示载体”通常是指包含展示载体片段I和展示载体片段II的环状多核苷酸，其还可包含展示VH和展示LC。经处理后，所述展示载体可释放第三展示载体多核苷酸、第四展示载体多核苷酸、第三展示载体多核苷酸和/或第四展示载体多核苷酸。
在本申请中，术语“第一展示载体多核苷酸”通常是指包含展示VH的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一展示载体多核苷酸可以以5’至3’方向包含B2-展示VH-B3，其中所述B2、B3可以为限制性内切核酸酶的识别位点。例如，经识别所述第一展示载体多核苷酸中的内切酶识别位点的内切酶(例如，识别B2和B3的限制性内切核酸酶，例如BsmBI)酶切处理后，切割后的所述第一展示载体多核苷酸可包含所述展示VH，所述展示VH两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“第二展示载体多核苷酸”通常是指包含展示LC的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一展示载体多核苷酸可以以5’至3’方向包含S5-展示LC-S6，其中所述S5、S6可以为限制性内切核酸酶的识别位点。例如，经识别所述第二展示载体多核苷酸中的内切酶识别位点的内切酶(例如，识别S5和S6的限制性内切核酸酶，例如SfiI)酶切处理后，切割后的所述第二展示载 体多核苷酸可包含所述展示LC，所述展示LC两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“展示VH”通常是指编码抗原结合多肽的重链可变区的核苷酸，术语“展示LC”通常是指编码抗原结合多肽轻链的核苷酸。本申请的“展示VH”和“抗原特异性VH”可以是编码源自针对相同抗原的结合多肽的重链可变区的核苷酸，也可以是编码源自针对不同抗原的结合多肽的重链可变区的核苷酸，本申请的“展示LC”和“抗原特异性LC”可以是编码源自针对相同抗原的结合多肽的轻链的核苷酸，也可以是编码源自针对不同抗原的结合多肽的轻链的核苷酸。
在本申请中，术语“展示载体片段”通常是指利用限制性内切核酸酶(例如BsmBI和/或SfiI)切割展示载体得到的片段，例如，展示载体片段I和展示载体片段II。展示载体片段的5’端和3’端可包含限制性内切核酸酶的识别位点。
在本申请中，术语“第三展示载体多核苷酸”通常是指包含展示载体片段I的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一展示载体多核苷酸可以以5’至3’方向包含B3-展示载体片段I-S5，其中所述B3、S5可以为限制性内切核酸酶的识别位点。例如，经识别所述第二展示载体多核苷酸中的内切酶识别位点的内切酶(例如，识别S5的限制性内切核酸酶，例如SfiI，或者，识别B3的限制性内切核酸酶，例如BsmBI和/或Esp3I)酶切处理后，切割后的所述第三展示载体多核苷酸可包含所述展示载体片段I，所述展示载体片段I两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“第四展示载体多核苷酸”通常是指包含展示载体片段II的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一展示载体多核苷酸可以以5’至3’方向包含S6-展示载体片段II-B2，其中所述S6、B2可以为限制性内切核酸酶的识别位点。例如，经识别所述第二展示载体多核苷酸中的内切酶识别位点的内切酶(例如，识别S6的限制性内切核酸酶，例如SfiI，或者，识别B2的限制性内切核酸酶，例如BsmBI和/或Esp3I)酶切处理后，切割后的所述第四展示载体多核苷酸可包含所述展示载体片段II，所述展示载体片段II两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“VH组件载体”通常是指将第九多核苷酸插入表达组件载体后形成的环状多核苷酸。
在本申请中，术语“LC组件载体”通常是指将第十多核苷酸插入表达组件载体后形成的环状多核苷酸。
在本申请中，术语“表达组件载体”通常是指可以插入多核苷酸(例如，第九多核苷酸和/或第十多核苷酸)的载体。所述表达组件载体可以源自pMD载体。例如，所述表达组件载体 可以为pMD19载体或源自pMD19载体。
在本申请中，术语“第九多核苷酸”通常是指包含VH组件载体工具片段的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第九多核苷酸可以以5’至3’方向包含B2-VH组件载体工具片段-B3，其中所述B2、B3可以为限制性内切核酸酶的识别位点。
在本申请中，术语“第十多核苷酸”通常是指包含LC组件载体工具片段的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第十多核苷酸可以以5’至3’方向包含S5-LC组件载体工具片段-S6，其中所述S5、S6可以为限制性内切核酸酶的识别位点。
在本申请中，术语“组件载体工具片段”通常是指可在5’端和/或3’端具有内切酶(例如，限制性内切核酸酶)的识别位点，而在其内部不具有内切酶(例如，限制性内切核酸酶)的识别位点的任何多核苷酸。组件载体工具片段的长度通常与抗原特异性VH和抗原特异性LC不相同。在某些情形中，组件载体工具片段的长度可以为约1kb。在某些情形中，组件载体工具片段可以源自IgG的Fc区。例如，组件载体工具片段可以源自选自下组的Fc区：人IgG1和人IgG2。例如，内切酶(例如，限制性内切核酸酶)的识别位点可以是B2和B3，又例如，内切酶(例如，限制性内切核酸酶)的识别位点可以是S5和S6。
在本申请中，术语“组件载体”通常是指将第九多核苷酸和/或第十多核苷酸插入表达组件载体后形成的环状多核苷酸。
在本申请中，术语“第九细菌”通常是指用于引入或包含所述第九核苷酸的细菌。所述第九细菌可以包含所述VH组件载体。在本申请中，所述第九细菌可以表达、复制和/或存储(例如，冷冻保存)所述第九核苷酸和/或所述VH组件载体。在本申请中，可以从所述第九细菌中获得包含所述VH组件载体的VH组件载体存储质粒。
在本申请中，术语“第十细菌”通常是指用于引入或包含所述第十核苷酸的细菌。所述第九细菌可以包含所述LC组件载体。在本申请中，所述第十细菌可以表达、复制和/或存储(例如，冷冻保存)所述第十核苷酸和/或所述LC组件载体。在本申请中，可以从所述第十细菌中获得包含所述LC组件载体的LC组件载体存储质粒。
在本申请中，术语“第六多核苷酸”通常是指包含VH组件载体连接片段的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第四多核苷酸可以以5’至3’方向包含B3-VH组件载体连接片段-B2，其中所述B3、B2可以为限制性内切核酸酶的识别位点。所述第六多核苷酸可以包含在VH组件载体中，经识别所述第 六多核苷酸中的内切酶识别位点的内切酶(例如，识别B3和B2的限制性内切核酸酶，例如BsmBI和/或Esp3I)酶切处理后，所述第六多核苷酸可以被释放，经释放的所述第四多核苷酸可包含所述抗原特异性VH，所述抗原特异性VH两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“经释放的第六多核苷酸”通常是指VH组件载体经处理后释放出的第六多核苷酸的片段。在本申请中，所述处理可以为经限制性内切核酸酶酶切处理。例如，可以针对所述展示载体上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，BsmBI和/或Esp3I)，从而使所述经释放的第六多核苷酸从所述VH组件载体中释放而分离。
在本申请中，术语“第八多核苷酸”通常是指包含LC组件载体连接片段的多核苷酸，其可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第四多核苷酸可以以5’至3’方向包含S6-LC组件载体连接片段-S5，其中所述S6、S5可以为限制性内切核酸酶的识别位点。所述第八多核苷酸可以包含在LC组件载体中，经识别所述第八多核苷酸中的内切酶识别位点的内切酶(例如，识别S6和S5的限制性内切核酸酶，例如SfiI)酶切处理后，所述第八多核苷酸可以被释放，经释放的所述第八多核苷酸可包含所述抗原特异性LC，所述抗原特异性LC两端还可存在经酶切后的特定序列的粘性末端。
在本申请中，术语“经释放的第八多核苷酸”通常是指LC组件载体经处理后释放出的第八多核苷酸的片段。在本申请中，所述处理可以为经限制性内切核酸酶酶切处理。例如，可以针对所述展示载体上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，SfiI)，从而使所述经释放的第八多核苷酸从所述LC组件载体中释放而分离。
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，BsmBI和Esp3I。
在本申请中，术语“第一细菌”通常是指用于引入或包含所述抗原特异性结合多肽基因展示载体的细菌。所述第一细菌可以包含所述抗原特异性VH、所述抗原特异性LC、所述展示载体片段I和所述展示载体片段II。在本申请中，所述第一细菌可以表达、复制和/或存储(例如，冷冻保存)抗原特异性VH、所述抗原特异性LC、所述展示载体片段I和所述展示载体片段II，或所述抗原特异性结合多肽表达载体DNA。
在本申请中，术语“抗原特异性结合多肽基因展示细菌文库”通常是指通过将所述抗原特异性结合多肽基因展示载体导入所述第一细菌中获得的细菌文库。在本申请中，所述抗原特异性结合多肽基因展示细菌文库可以为包含编码抗原特异性结合多肽的轻链或抗原特异性多肽的重链可变区的核酸序列的细菌文库。在本申请中，所述抗原特异性结合多肽基因展示细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码所述抗原特异性结合多肽的核酸序列。在本申请中，所述抗原特异性结合多肽基因展示细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第一细菌。
在本申请中，术语“第一展示细菌”通常是指用于引入或包含所述第一展示载体多核苷酸的细菌。所述第一细菌可以包含所述展示VH。在本申请中，所述第一展示细菌可以表达、复制和/或存储(例如，冷冻保存)展示VH，和/或所述第一展示载体多核苷酸。
在本申请中，术语“展示VH组件细菌文库”通常是指通过将所述第一展示载体多核苷酸导入所述第一展示细菌中获得的细菌文库。在本申请中，所述展示VH组件细菌文库可以为包含编码抗原特异性多肽的重链可变区的核酸序列的细菌文库。在本申请中，所述展示VH组件细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码所述展示VH的核酸序列。在本申请中，所述展示VH组件细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第一展示细菌。
在本申请中，术语“第二展示细菌”通常是指用于引入或包含所述第二展示载体多核苷酸的细菌。所述第二细菌可以包含所述展示LC。在本申请中，所述第二展示细菌可以表达、复制和/或存储(例如，冷冻保存)展示LC，和/或所述第二展示载体多核苷酸。
在本申请中，术语“展示LC组件细菌文库”通常是指通过将所述第二展示载体多核苷酸导入所述第二展示细菌中获得的细菌文库。在本申请中，所述展示LC组件细菌文库可以为包含编码抗原特异性多肽的轻链的核酸序列的细菌文库。在本申请中，所述展示LC组件细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码所述展示LC的核酸序列。在本申请中，所述展示LC组件细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第二展示细菌。
在本申请中，术语“第三展示细菌”通常是指用于引入或包含所述第三展示载体多核苷酸的细菌。所述第三细菌可以包含所述展示载体片段I。在本申请中，所述第三展示细菌可以表 达、复制和/或存储(例如，冷冻保存)展示载体片段I，和/或所述第三展示载体多核苷酸。
在本申请中，术语“展示载体组件I细菌文库”通常是指通过将所述第三展示载体多核苷酸导入所述第三展示细菌中获得的细菌文库。在本申请中，所述展示载体组件I细菌文库可以为包含编码展示载体组件I的核酸序列的细菌文库。在本申请中，所述展示载体组件I细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码所述展示LC的核酸序列。在本申请中，所述展示载体组件I细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第三展示细菌。
在本申请中，术语“第四展示细菌”通常是指用于引入或包含所述第四展示载体多核苷酸的细菌。所述第四细菌可以包含所述展示载体片段II。在本申请中，所述第四展示细菌可以表达、复制和/或存储(例如，冷冻保存)展示载体片段II，和/或所述第四展示载体多核苷酸。
在本申请中，术语“展示载体组件II细菌文库”通常是指通过将所述第三展示载体多核苷酸导入所述第四展示细菌中获得的细菌文库。在本申请中，所述展示载体组件II细菌文库可以为包含编码展示载体组件II的核酸序列的细菌文库。在本申请中，所述展示载体组件II细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码所述展示LC的核酸序列。在本申请中，所述展示载体组件II细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第四展示细菌。
在本申请中，术语“导入”通常是指将外源多核苷酸转移或导入细胞中的过程。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“连接”通常是指将两个或者两个以上的多核苷酸分子连接在一起。例如，所述连接可以通过连接酶(例如，DNA连接酶)实现。例如，使一个多核苷酸的3’端与另一个多核苷酸的5’端连接，从而形成一个完整的多核苷酸分子。
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，所述轻链组件细菌文库、所述重链组件细菌文库、所述展示细菌文库和/或所述噬菌体文库)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“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其 类似物，包括例如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。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
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，本申请提供一种构建抗原特异性结合多肽基因展示载体的方法。
本申请所述的抗原特异性结合多肽基因展示载体可以由四个片段定向环化连接而成，分别可包含抗原特异性VH、抗原特异性LC、展示载体片段I和展示载体片段II。
组件载体
本申请所述的方法可包括构建组件载体，其可包括提供多核苷酸，例如，第九多核苷酸和第十多核苷酸。所述第九多核苷酸以5’至3’方向可包含B2-VH组件载体工具片段-B3，所述第十多核苷酸以5’至3’方向包含S5-LC组件载体工具片段-S6。所述载体工具片段可以来自IgG的Fc片段。例如，人IgG1的Fc片段。又例如，人IgG2的Fc片段。在某些情形中，可利用DDB214和DDB215作为引物，以人IgG1的Fc片段为模板，扩增得到所述第九核苷酸。其中，所述DDB214可包含SEQ ID NO:1所示的氨基酸序列，所述DDB215可包含SEQ ID NO:2所示的氨基酸序列。在某些情形中，可以利用利用DDB216和DDB217作为引物，以人IgG1的Fc片段为模板，扩增得到所述第十核苷酸。其中，所述DDB216可包含SEQ ID NO:3所示的氨基酸序列，所述DDB217可包含SEQ ID NO:4所示的氨基酸序列。所述第九核苷酸的两端可包含限制性核酸内切酶的识别位点B2和B3，所述第十核苷酸的两端可包含限制性核酸内切酶的识别位点S5和S6。
所述方法可包括将所述多核苷酸(例如，第九多核苷酸和第十多核苷酸)插入表达组件载体，获得组件载体(例如，VH组件载体和LC组件载体)。
所述表达组件载体可以源自任何载体，例如任何可扩增和/或容易保存的载体。在某些情形中，用于作为表达组件载体的所述载体可具有高拷贝数、分子量小等性质。在某些情形中，所述表达组件载体可以源自pMD载体，例如，所述表达组件载体可以为pMD19载体或源自pMD19载体。
在本申请中，为了构建所述表达组件载体，可以对所述pMD载体或源自pMD的载体进行工程化/修饰。例如，可以通过定点突变去除所述载体中的一个或多个内切核酸酶识别位点(例如，去除其中的一个或多个BsmBI和/或SfiI的识别位点)。在某些情形中，也可以通过定点突变在所述载体中增加一个或多个内切核酸酶识别位点(例如，在选定的位置增加一个或多个BsmBI和/或SfiI的识别位点)。
例如，可通过定点突变的方法去除所述载体中原本含有的一个或多个BsmBI识别位点，然后可在所述载体中的另一个位置添加一个或多个另外的BsmBI识别位点，以获得经改造的载体(例如，经改造的pMD载体)。
在本申请中，所述表达组件载体(例如，VH组件载体的表达组件载体)中可以包含BsmBI的识别位点。在某些情形中，所述表达组件载体可包含两个BsmBI的识别位点。
又例如，可通过定点突变的方法去除所述载体中原本含有的一个或多个SfiI识别位点，然后可在所述载体中的另一个位置添加一个或多个另外的SfiI识别位点，以获得经改造的载体(例如，经改造的pMD载体)。
在本申请中，所述表达组件载体(例如，LC组件载体的表达组件载体)中可以包含SfiI的识别位点。在某些情形中，所述表达组件载体可包含两个SfiI的识别位点。
在某些情形中，所述方法还可包括将所述多核苷酸(例如，第九多核苷酸和第十多核苷酸)插入表达组件载体，获得组件载体存储质粒(例如，VH组件载体存储质粒和LC组件载体存储质粒)，然后将所述组件载体存储质粒导入细菌(例如，第九细菌和第十细菌)，获得组件载体存储细菌文库(例如，VH组件载体存储细菌文库和LC组件载体存储细菌文库)。
例如，本申请所述的VH组件载体如图3所示，其可由将所述第九多核苷酸插入表达组件载体连接获得。所述VH组件载体包含第六多核苷酸，所述第六多核苷酸可以以5’至3’方向包含B3-VH组件载体连接片段-B2，其中B2和B3可以分别被BsmBI和/或Esp3I特异性识别及切割。例如，所述B2可包含SEQ ID NO:8所示的核酸序列，所述B3可包含SEQ ID NO:9所示的核酸序列。
经处理(例如，酶切)后，所述的VH组件载体可产生经释放的第六多核苷酸，所述经释放的第六多核苷酸的5’端和3’端可带有经酶切产生的特定序列的粘性末端。
例如，本申请所述的LC组件载体如图4所示，其可由将所述第十多核苷酸插入表达组件载体连接获得。所述LC组件载体包含第八多核苷酸，所述第八多核苷酸可以以5’至3’方向包含S6-LC组件载体连接片段-S5，其中S6和S5可以分别被SfiI特异性识别及切割。例如，所述S6可包含SEQ ID NO:11所示的核酸序列，所述S5可包含SEQ ID NO:10所示的 核酸序列。
经处理(例如，酶切)后，其可产生经释放的第八多核苷酸，所述经释放的第八多核苷酸的5’端和3’端可带有经酶切产生的特定序列的粘性末端。
抗原特异性VH和抗原特异性LC
本申请所述的方法包括提供第五多核苷酸，所述第五多核苷酸以5’至3’方向包含B-抗原特异性VH-B，其中所述B为能够特异性识别B2和/或B3的限制性内切核酸酶的识别位点。例如，可以以抗原特异性VH片段为模板，扩增所述抗原特异性VH，使得所述抗原特异性VH的5’端和3’端连接限制性内切核酸酶(例如，BsmBI和/或Esp3I)的识别位点。
本申请所述的方法包括提供第七多核苷酸，所述第七多核苷酸以5’至3’方向包含S-抗原特异性LC-S，其中所述S为能够特异性识别S5和/或S6的限制性内切核酸酶的识别位点。例如，可以以抗原特异性LC片段为模板，扩增所述抗原特异性LC，使得所述抗原特异性LC的5’端和3’端连接限制性内切核酸酶(例如，SfiI)的识别位点。
在某些情形中，可以使用现有技术的方法获得抗原特异性VH片段和抗原特异性LC片段。例如，可以从抗原免疫后的动物得到，还可以从抗体库中得到，包括组合抗体库、噬菌体展示库、酵母表面展示库、核糖体展示库、mRNA展示库。
组件库
本申请所述的方法可包括利用限制性内切核酸酶切割所述第五多核苷酸和所述VH组件载体，得到切割后的第五多核苷酸和经释放的第六多核苷酸，然后混合所述切割后的第五多核苷酸和所述经释放的第六多核苷酸，从而使得其能够定向连接而环化形成抗原特异性VH组件库。
所述抗原特异性VH组件库可包含所述抗原特异性VH。利用限制性内切核酸酶(例如，识别B2和B3的限制性内切核酸酶)切割所述抗原特异性VH组件库后，可以得到经释放的所述抗原特异性VH，所述抗原特异性VH的5’端和3’端可以带有特定序列的粘性末端。
本申请所述的方法可包括利用限制性内切核酸酶切割所述第七多核苷酸和所述LC组件载体，得到切割后的第七多核苷酸和经释放的第八多核苷酸，然后混合所述切割后的第七多核苷酸和所述经释放的第八多核苷酸，从而使得其能够定向连接而环化形成抗原特异性LC组件库。
所述抗原特异性LC组件库可包含所述抗原特异性LC。利用限制性内切核酸酶(例如，识别S5和S6的限制性内切核酸酶)切割所述抗原特异性LC组件库后，可以得到经释放的所述抗原特异性LC，所述抗原特异性LC的5’端和3’端可以带有特定序列的粘性末端。
展示载体
本申请所述方法还可包括构建展示载体，所述展示载体可以由四个展示载体多核苷酸组成(例如，第一展示载体多核苷酸、第二展示载体多核苷酸、第三展示载体多核苷酸和第四展示载体多核苷酸)。
本申请所述的展示载体多核苷酸(第一展示载体多核苷酸、第二展示载体多核苷酸、第三展示载体多核苷酸和第四展示载体多核苷酸)可包括抗原结合多肽或其片段，例如，展示LC和/或展示VH。在本申请中，所述展示LC可编码所述抗原结合多肽的轻链，所述展示VH可编码抗原结合多肽的重链可变区，所述轻链可与所述重链可变区结合后形成识别靶标的Fab。在某些情形中，所述靶标可以是抗原。例如，所述靶标为PD-1。
本申请所述的展示载体多核苷酸(例如，第一展示载体多核苷酸、第二展示载体多核苷酸、第三展示载体多核苷酸和第四展示载体多核苷酸)可包括展示载体片段，如，展示载体片段I和展示载体片段II。可以根据要表达的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展示载体片段I和展示载体片段II。
在某些情形中，所述展示载体片段I和展示载体片段II可以是来自任何一个能够表达目的基因的载体片段。例如，所述表达载体片段I和表达载体片段II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)。
本申请的展示载体片段(例如，展示载体片段I和展示载体片段II)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展示载体片段中调整(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，所述的展示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，所述第一展示载体多核苷酸以5’至3’方向可包含B2-展示VH-B3，其中，B2和B3可以各自独立地为限制性内切核酸酶识别位点，所述展示VH可以编码抗原结合多肽的重链可变区。在某些情形中，所述B2和B3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:8所示的核酸序列，所述B3可包含SEQ ID NO:9所示的核酸序列。
所述第二展示载体多核苷酸以5’至3’方向可包含S5-展示LC-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，所述展示LC可以编码抗原结合多肽的轻链。在某些情形中，所述S5和S6可以分别被SfiI特异性识别及切割。例如，所述S5可包含SEQ ID  NO:0所示的核酸序列，所述S6可包含SEQ ID NO:11所示的核酸序列。
所述第三展示载体多核苷酸以5’至3’方向可包含B3-展示载体片段I-S5，其中，B3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S5可以被Sfil特异性识别及切割，所述B3可以被BsmBI和/或Esp3I特异性识别及切割。例如，所述B3可包含SEQ ID NO:9所示的核酸序列，所述S5可包含SEQ ID NO:10所示的核酸序列。
所述第四展示载体多核苷酸以5’至3’方向可包含S6-展示载体片段II-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B2可以被BsmBI和/或Esp3I特异性识别及切割。例如，所述B2可包含SEQ ID NO:8所示的核酸序列，所述S6可包含SEQ ID NO:11所示的核酸序列。
本申请所述第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和/或所述展示载体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。
为了筛选导入了所述展示载体多核苷酸的阳性细菌，所述展示载体多核苷酸(例如，第一展示载体多核苷酸、第二展示载体多核苷酸、第三展示载体多核苷酸和第四展示载体多核苷酸)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:12和SEQ ID NO:14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:13和SEQ ID NO:15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，展示LC、展示VH、展示载体片段I和展示载体片段II)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，扩增所述展示LC的引物可包含SEQ ID NO:20和SEQ ID NO:21所示的核苷酸序列。例如，扩增所述展示VH的引物可包含SEQ ID NO:22和SEQ ID NO:23中所示的核苷酸序列。例如，扩增所述展示载体片段I的引物可包含SEQ ID NO:18和SEQ ID NO:19中所示的核苷酸序列。例如，扩增所述展示载体片段II的引物可包含SEQ ID NO:16和SEQ ID NO:17中所示的核苷酸序列。
获得所述展示载体多核苷酸后，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：将所述第一展示载体多核苷酸导入第一展示细菌以获得展示VH组件细菌文库；将所述第二展示载体多核苷酸导入第二展示细菌以获得展示LC组件细菌文库；将所述第三展示载体多核苷酸导入第三展示细菌以获得展示载体组件I细菌文库；将所述第四展示载体多核苷酸导入第四展示细菌以获得展示载体组件II细菌文库。
在本申请中，所述第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和所述第四展示载体多核苷酸可以均为线性核酸分子。
在某些情形中，可以将所述展示载体多核苷酸插入展示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，以获得展示细菌文库。
在本申请中，所述展示细菌文库(例如，展示VH组件细菌文库和展示LC组件细菌文库)可以包含约至少10个(例如，约至少100个、至少200个、至少300个、至少400个、至少500个、至少600个、至少800个、约至少1000个、约至少10000个或更多)不同的克隆。
在本申请中，所述展示细菌文库(例如，展示载体组件I细菌文库和展示载体组件II细菌文库)可以包含约至少10个(例如，约至少100个、至少200个、至少300个、至少400个、至少500个、至少600个、至少800个、约至少1000个、约至少10000个或更多)相同的克隆。
在本申请中，所述展示细菌文库(例如，展示VH组件细菌文库、展示LC组件细菌文库、展示载体组件I细菌文库和展示载体组件II细菌文库)中有效克隆的比例可以为至少约50％(例如，至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％)。
在本申请中，所述展示细菌文库(例如，展示VH组件细菌文库、展示LC组件细菌文库、展示载体组件I细菌文库和展示载体组件II细菌文库)中细菌可以进行液体培养。所述液体培养的培养时间可以为不大于约8小时，例如，可以为不大于约4小时、不大于约5小时、不大于约6小时或者不大于约7小时。在本申请中，所述液体培养操作较为简便。在某些情形下，所述展示细菌文库中的细菌可以取少量菌液铺皿培养，然后再挑选菌落。所述铺 皿培养的培养时间可以为约12-18小时，例如，可以为约12小时、13小时、14小时、15小时、16小时、17小时或18小时。在本申请中，所述铺皿培养可以挑选菌落(例如，挑选单克隆)再进行测序分析。
抗原特异性结合多肽基因展示载体
本申请所述的方法还可包括利用限制性内切核酸酶(例如，特异性识别所述S5、S6、B2和B3的限制性内切核酸酶)特异性切割所述VH组件库、所述LC组件库和所述展示载体，得到经释放的所述第一多核苷酸、经释放的所述第二多核苷酸、经释放的所述第三多核苷酸和经释放的所述第四多核苷酸。
经释放的所述第一多核苷酸5’端可具有限制性内切核酸酶(例如，特异性识别B2的限制性内切核酸酶，例如，BsmBI和/或Esp3I)切割后的粘性末端，3’端可具有限制性内切核酸酶(例如，特异性识别B3的限制性内切核酸酶，例如，BsmBI和/或Esp3I)切割后的粘性末端。
经释放的所述第二多核苷酸5’端具有限制性内切核酸酶(例如，特异性识别S5的限制性内切核酸酶，例如，SfiI)切割后的粘性末端，3’端具有限制性内切核酸酶(例如，特异性识别S6的限制性内切核酸酶，例如，SfiI)切割后的粘性末端。
经释放的所述第三多核苷酸5’端具有限制性内切核酸酶(例如，特异性识别B3的限制性内切核酸酶，例如，BsmBI和/或Esp3I)切割后的粘性末端，3’端具有限制性内切核酸酶(例如，特异性识别S5的限制性内切核酸酶，例如，SfiI)切割后的粘性末端。
经释放的所述第四多核苷酸5’端具有限制性内切核酸酶(例如，特异性识别S6的限制性内切核酸酶，例如，SfiI)切割后的粘性末端，3’端具有限制性内切核酸酶(例如，特异性识别B2的限制性内切核酸酶，例如，BsmBI和/或Esp3I)切割后的粘性末端。
在本申请中，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所述第四多核苷酸可以均为线性核酸分子。
本申请的方法还可包括混合经所述经释放的第一多核苷酸、所述经释放的第二多核苷酸、所述经释放的第三多核苷酸和所述经释放的第四多核苷酸，从而使得其能够定向连接而环化形成所述抗原特异性结合多肽基因展示载体。在某些情形中，所述定向连接可包括使用连接酶，例如，T4 DNA连接酶。
在某些情形中，可以将所述抗原特异性结合多肽基因展示载体第一细菌，以获得抗原特异性结合多肽基因展示细菌文库
在本申请中，所述抗原特异性结合多肽基因展示细菌文库可以包含约至少10个(例如， 约至少100个，约至少200个、约至少300个、约至少400个、约至少500个、约至少1000个、约至少10000个或更多)克隆。
在本申请中，所述抗原特异性结合多肽基因展示细菌文库中有效克隆的比例可以为至少约50％(例如，至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％)。
使用本申请所述的方法，从构建展示载体到筛选出独特序列的抗原特异性结合多肽，所需时间可以至少约1周(例如，至少约10天、至少约2周、至少约3周、至少约4周)。
在本申请中，所述抗原特异性结合多肽基因展示细菌文库中的细菌可以进行液体培养。所述液体培养的培养时间可以为不大于约24小时，例如，可以为不大于约5小时、可以为不大于约10小时、可以为不大于约15小时、不大于约20小时、不大于约22小时或者不大于约22小时。在本申请中，所述液体培养操作较为简便。在某些情形下，所述细菌文库中的细菌可以取少量菌液铺皿培养，然后再挑选菌落。所述铺皿培养的培养时间可以为约12-18小时，例如，可以为约12小时、13小时、14小时、15小时、16小时、17小时或18小时。在本申请中，所述铺皿培养可以挑选菌落(例如，挑选单克隆)再进行测序分析。
所述方法还可包括将所述抗原特异性结合多肽基因展示载体导入细胞，由所述细胞得到所述抗原特异性结合多肽。例如，所述细胞可以为哺乳动物细胞。然后可以由所述细菌得到所述抗原特异性结合多肽。
酶切位点
在本申请中，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B2、B3、S5和S6。其中，所述B2、B3、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种或4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、BsmBI和/或Esp3I。在其他情形中，也可选择其他可行的限制性内切核酸酶。
在本申请中，所述限制性内切核酸酶可以选自SfiI、BsmBI和Esp3I。在本申请中，所述BsmBI和Esp3I可以为同工酶，其可以识别相同的限制性内切核酸酶的识别位点。
在本申请中，例如，所述S5和S6可以被SfiI识别及切割。在本申请中，例如，所述B2和B3能够被BsmBI和/或Esp3I识别及切割。
例如，SfiI可以识别由13个碱基构成的序列(5’至3’)GGCCNNNN/NGGCC，其经过酶切后可形成3’端的突出序列(overhang，例如包含3个碱基的单链序列)，其中N可以代表 GATC四种碱基中的任何一种。因此，有4^5种不同的序列均能够被SfiI识别。
例如，BsmBI和Esp3I可以识别由12个碱基构成的序列(5’至3’)CGTCTCN/NNNNN，其经过酶切后可形成5’端的突出序列(overhang，例如包含4个碱基的单链序列)，其中N可以代表GATC四种碱基中的任何一种。因此，有4^6种不同的序列均能够被BsmBI和Esp3I识别。
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:10所示的核酸序列。又例如，所述S6可包含SEQ ID NO:11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和/或Esp3I特异性识别并切割的位点，例如，可分别被称为B2和B3。例如，所述B2可包含SEQ ID NO:8所示的核酸序列。再例如，所述B3可包含SEQ ID NO:9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。
另一方面，本申请还提供了所述的抗原特异性结合多肽基因展示细菌文库。
另一方面，本申请还提供了根据所述方法所产生的展示载体。
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 构建人PBMC的噬菌体表面抗体(Fab)展示库
1.1获取免疫材料总RNA/mRNA
从人的外周血淋巴细胞提取总RNA，并且进一步从总RNA分离mRNA(Takara Cat#Z652N/636592，具体试验步骤参见产品说明书)。
1.2设计合成引物
参照《Phage Display》(A Laboratory Mannual，ISBN 0-87969-546-3)，设计针对人重链可变区VH、轻链KLC(全长Kappa轻链)、轻链LLC(全长Lamda轻链)的引物。其中，轻链正向引物5’-端含有R1的核苷酸序列GGCCCAGGCGGCC(SEQ ID NO:78)，反向引物5’-端含有R2的核苷酸序列GGCCACATAGGCC(SEQ ID NO:79)；重链可变区正向引 物5’-端含有R5的核苷酸序列GGCCCAACCGGCC(SEQ ID NO:80)，反向引物5’-端含有R6的核苷酸序列GGCCCTCAGCGGCC(SEQ ID NO:81)。引物由金维智公司合成。
以pComb3x载体为模板，设计扩增连接子的正向和反向引物。其中，正向引物5’-端含有R3的核苷酸序列GGCCACATAGGCC(SEQ ID NO:79)，反向引物5’-端含有R4的核苷酸序列GGCCCAACCGGCC(SEQ ID NO:80)。
具体的引物序列可参照下表1-1：
表1-1引物序列-1
引物名称 SEQ ID NO:
轻链KLC的正向引物 82-99
轻链KLC的反向引物 100
轻链LLC的正向引物 101-125
轻链LLC的反向引物 126
VH的正向引物 127-150
VH的反向引物 151-155
连接子的正向引物 156
连接子的反向引物 157
1.3获得第一多核苷酸和第三多核苷酸
通过两步法扩增组件抗体基因库。
第一步，以实施例1.1中获得的mRNA为模板，用Promega的MMLV进行逆转录合成cDNA(按照Promega公司产品的说明书进行，其中引物为Thermo Cat#N8080127、逆转录酶为Promega Cat#M1701)。
第二步，以第一步获得的cDNA为模板，利用实施例1.2中获得的引物，通过PCR(Takara Cat#RR900A，按照公司产品的说明书进行)扩增组件抗体的KLC，LLC和VH基因库。凝胶电泳纯化回收后(利用Axygen的凝胶回收试剂盒，按照《分子克隆实验指南》中的记载操作)分别获得PCR产物——KLC片段、LLC片段和VH片段。
1.4构建存储载体
1.4.1设计引物
参照实施例1.2的内容设计引物。
设计合成用于获得存储载体的引物。具体的引物序列可参照下表1-2：
表1-2引物序列-2
引物名称 SEQ ID NO:
R1-1kb-R2的正向引物 158
R1-1kb-R2的反向引物 159
R5-1kb-R6的正向引物 162
R5-1kb-R6的反向引物 163
1.4.2 PCR扩增
以1kb长的人IgG1的Fc(SEQ ID NO:164)为模板，利用实施例1.4.1制备的引物R1-1kb-R2的正向引物和R1-1kb-R2的反向引物，进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R1-1kb-R2(SEQ ID NO:165)。
以pComb3x载体为模板，利用R3-连接子-R4的正向引物(SEQ ID NO:160)和R3-连接子-R4的反向引物(SEQ ID NO:161)进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R3-连接子-R4(SEQ ID NO:167)。其中所述连接子的长度可以为72bp，其核苷酸序列如SEQ ID NO:166所示。
以1kb长的人IgG1的Fc(SEQ ID NO:164)为模板，利用实施例1.4.1制备的引物R5-1kb-R6的正向引物和R5-1kb-R6的反向引物，进行PCR，凝胶电泳纯化回收后，得到PCR产物——R5-1kb-R6(SEQ ID NO:168)。
1.4.3构建轻链存储载体和重链存储载体
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将1.4.2制备的R1-1kb-R2片段插入pMD19-T载体，得到用于插入全长轻链基因库的轻链存储载体DDB-R1-1kb-R2，其载体图谱如图9所示。
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将1.4.2制备的R5-1kb-R6片段插入pMD19-T载体得到含有R5-1kb-R6片段的载体，然后以这个载体为模板，利用引物突变去除这个载体中原有的BsmBI酶切位点，得到用于插入VH基因库的重链存储载体DDB-R5-1kb-R6，其载体图谱如图10所示。
具体的引物序列可参照下表1-3：
表1-3引物序列-3
引物名称 SEQ ID NO:
载体突变的正向引物 169
载体突变的反向引物 170
1.4.4构建连接子存储载体并获得连接子组件细菌
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将实施例1.4.2制备的R3-连接子-R4插入pMD19-T载体，得到连接子存储载体DDB-R3-连接子-R4，其载体图谱如图11所示。可用所述连接子存储载体转化TG1感受态细菌(Lucigen公司)，铺皿37℃培养过夜，送菌落测序，然后收集菌落得到连接子组件细菌。可将该连接子组件细菌冻存备用。
1.5获得组件细菌文库
1.5.1获得轻链组件细菌文库
利用限制性内切核酸酶R1和R2酶切实施例1.3所制得的包括KLC和LLC的多核苷酸获得目的轻链片段(约为0.65kb)。
利用限制性内切核酸酶R1和R2酶切实施例1.4所制得的轻链存储载体DDB-R1-1kb-R2。获得轻链存储载体片段(约为2.7kb)。
将所得的目的轻链片段和轻链存储载体片段混合，再利用连接酶T4 DNA ligase(购自NEB，Thermo)连接得到轻链存储连接产物，然后用所述轻链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，送菌落测序，然后收集全部菌落得到轻链组件细菌文库。可检测该轻链组件细菌文库的质量和/或将该轻链组件细菌文库冻存备用。
1.5.2获得重链组件细菌文库
利用限制性内切核酸酶R5和R6酶切实施例1.3所制得的多核苷酸获得目的重链可变区片段(约为0.35kb)。
利用限制性内切核酸酶R5和R6酶切实施例1.4所制得的重链存储载体DDB-R5-1kb-R6。获得重链存储载体片段(约为2.7kb)。
将所得的目的重链可变区片段和重链存储载体片段混合，再利用连接酶T4 DNA ligase(购自NEB，Thermo)连接得到重链存储连接产物，然后用所述重链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，挑菌落测序，然后收集全部菌落得到重链组件细菌文库。可检测该重链组件细菌文库的质量和/或将该重链组件细菌文库冻存备用。
1.6获得轻链组件质粒、重链组件质粒和连接子片段
使用质粒提取试剂盒(购自Axygen)，分别提取实施例1.5.1制备的轻链组件细菌文库、实施例1.5.2制备的重链组件细菌文库中的质粒，分别得到轻链组件质粒和重链组件质粒。
利用限制性内切核酸酶R1和R2酶切实施例1.5.1制备的轻链组件质粒，经凝胶电泳纯化回收后，得到轻链插入片段LC。
利用限制性内切核酸酶R5和R6酶切实施例1.5.2制备的重链组件质粒，经凝胶电泳纯化回收后，得到重链插入片段HC。
使用质粒提取试剂盒(购自Axygen)，提取实施例1.4.4制备的连接子组件细菌中的质粒，得到连接子组件质粒。以该连接子组件质粒或实施例1.4.4中的连接子存储载体为模板， 用连接子的正向引物(SEQ ID NO:156)和连接子的反向引物(SEQ ID NO:157)扩增含有连接子的0.8kb的片段，然后用限制性内切核酸酶R3和R4酶切该0.8kb的PCR产物，经凝胶电泳纯化回收(利用小片段凝胶回收试剂盒，购自莱枫生物，Cat#DK402)得到72pb的连接子片段。
1.7获得展示载体
购买获得pComb3x载体，其载体图谱如图12所示，其中用于抗体Fab展示的经改造的pComb3x-fab载体图谱如图13所示。
通过无义突变除去pComb3x-fab载体中在Fab基因3’端的SfiI酶切位点。
然后在该无义突变后的载体中轻链终止密码子的下游加入限制性内切核酸酶R2的酶切位点，在重链可变区信号肽的末端通过无义突变引入限制性内切核酸酶R5的酶切位点，获得经改造后的噬菌体展示载体DDB-R1R2R5R6，其图谱如图14所示。
1.8制备展示细菌文库
利用限制性内切核酸酶R7和限制性内切核酸酶R8酶切实施例1.7制备的展示载体DDB-R1R2R5R6，获得3.6kb展示载体片段。
将实施例1.6获得的轻链插入片段LC(0.65kb)、重链插入片段HC(0.35kb)、连接子片段(72bp)和所述噬菌体展示载体片段(3.6kb)按照1:1:1:1的分子比例混合，用T4 DNA连接酶20℃连接20小时以上，得到展示用连接产物。
利用PCR-Clean-up纯化连接产物，转入TG1感受态细菌(Lucigen，Cat#60502-2，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，收集平皿上生长的全部菌落，即为噬菌体展示细菌文库。可将该噬菌体展示细菌文库保存备用。
1.9制备展示型抗体噬菌体文库
从实施例1.8所制备的展示细菌文库中取适量的菌液，在2YT培养液(含有氨苄青霉素100μg/ml和2％葡萄糖)中37℃培养至OD 600达到0.5。然后向该菌液中加入M13KO7辅助噬菌体(购自NEB，Cat#N0315S，MOI约为10-20)混匀后，37℃静止30分钟，接着在37℃，250rpm条件下震摇30分钟，离心弃去含有M13KO7辅助噬菌体的培养液上清部分，以该菌液原始体积4倍的培养液(含有氨苄青霉素和卡那霉素)重悬细菌，30℃，250rpm条件下振摇过夜。第二天用PEG沉淀收集噬菌体，滴定噬菌体浓度，分装保存。即得到展示型抗体噬菌体文库。
实施例2 构建展示载体
2.1获得样品材料
为构建如图1所示的展示载体，选择靶向PD-1抗体帕博利珠单抗(Pembrolizumab)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:5，帕博利珠单抗重链可变区核苷酸序列：SEQ ID NO:6，pDGB4载体核苷酸序列：SEQ ID NO:7。
2.2设计酶切位点
选择限制性内切核酸酶BsmBI和SfiI，设计2个BsmBI识别位点的序列(B2和B3)和2个SfiI识别位点的序列(S5和S6)，其中，B2的核苷酸序列如SEQ ID NO:8所示，B3的核苷酸序列如SEQ ID NO:9所示，S5的核苷酸序列如SEQ ID NO:10所示，S6的核苷酸序列如SEQ ID NO:11所示。
2.3选择信号肽
选择两个表达天然抗体基因的信号肽：SP1和SP2。为了在信号肽的3’-端部分引入合适的酶切位点，二个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。SP1表达展示VH，核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:13所示；SP2表达展示LC，核苷酸序列如SEQ ID NO:14所示，氨基酸序列如SEQ ID NO:15所示。
2.4获得展示载体多核苷酸
分别设计针对展示VH、展示LC以及展示载体片段I和展示载体片段II的引物，且均用CMV启动子驱动表达。合成引物，以2.1中的序列为模板，进行PCR扩增。序列见表2。
表2各片段序列
Figure PCTCN2021083246-appb-000001
使用PCR(LA Taq，Takara公司，按照公司产品的说明书进行)扩增四个展示载体多核苷酸，所使用的模板和引物序列见表2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展示载体多核苷酸——含有展示VH的第一展示载体多核苷酸、含有展示LC的第二展示载体多核苷酸、含有展示载体片段I的第三展示载体多核苷酸和含有表达载体片段II的第四展示载体多核苷酸。可将该细菌作为细菌文库冻存备用。
2.5酶切
使用质粒提取试剂盒(购自Axygen)，分别提取实施例2.4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。电泳分离纯化，得到四种所述切割后的展示载体多核苷酸。
2.6连接获得展示载体
等分子比例混合实施例2.5得到的四种切割后的展示载体多核苷酸，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，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展示载体。展示载体具体结构如图2所示。
实施例3 构建VH组件载体和LC组件载体
3.1 VH组件载体
以1kb长的人IgG1的Fc为模板，用引物DDB214和DDB215进行PCR，凝胶电泳纯化回收后，得到PCR产物B2-KB-B3，其中，DDB214的核苷酸序列如SEQ ID NO:1所示，DDB215的核苷酸序列如SEQ ID NO:2所示。将B2-KB-B3插入表达组件载体pMD19载体，得到VH组件载体存储质粒，将其转化TG1感受态细菌(Lucigen公司)，铺皿37℃培养过夜，挑克隆测序，确定含有正确序列的VH组件载体。VH组件载体的结构如图3所示。
3.2 LC组件载体
以1kb长的人IgG1的Fc为模板，用引物DDB216和DDB217进行PCR，凝胶电泳纯化回收后，得到PCR产物S5-KB-S6，其中，DDB216的核苷酸序列如SEQ ID NO:3所示，DDB217的核苷酸序列如SEQ ID NO:4所示。将S5-KB-S6插入表达组件载体pMD19载体，得到LC组件载体存储质粒，将其转化TG1感受态细菌(Lucigen公司)，铺皿37℃培养过 夜，挑克隆测序，确定含有正确序列的LCH组件载体。LC组件载体的结构如图4所示。
实施例4 获得抗原特异性VH和抗原特异性LC
4.1第一轮筛选
取500μl实施例1中构建的噬菌体库(Fab库，原始库容4×10 10，有效克隆大于80％，制备的噬菌体库2×10 13/ml)，将生物素标记的ROR1抗原(Acro Biosystems，Cat#RO1-H82E6)与噬菌体库混合(抗原浓度10μg/ml)，室温转摇2小时，使展示抗原特异性Fab的噬菌体与生物素标记的抗原结合)。接下来取80μl磁珠(购自英俊)与噬菌体库-抗原混合，室温转摇20分钟，通过磁珠表面的亲和素与生物素的结合捕获抗原特异性噬菌体，形成磁珠-亲和素-生物素-抗原-Fab抗体片段交联体。然后通过磁力架收集形成的携带ROR1抗原特异性Fab的交联体，用pH2.2的甘氨酸溶液洗脱展示ROR1抗原特异性Fab的噬菌体，用pH8.0的Tris缓冲液中和至pH7.0，最终得到550μl噬菌体溶液。
4.2第二轮筛选
取250μl第一轮筛选得到的噬菌体溶液，与等量的4％Milk-PBS混合，最终体积0.5ml。接下来将4μg生物素标记的抗原与噬菌体溶液混合，最终抗原浓度8μg/ml，室温转摇3小时，使展示抗原特异性Fab的噬菌体与生物素标记的抗原结合。然后取40μl磁珠与噬菌体溶液-抗原混合，室温转摇20分钟，通过磁珠表面的亲和素与生物素的结合捕获抗原特异性噬菌体，形成磁珠-亲和素-生物素-抗原-Fab抗体片段交联体。通过磁力架收集形成的携带ROR1抗原特异性Fab的交联体。然后用1×PBST洗磁珠4遍，再用1×PBS洗4遍。最后用50μl pH2.2的甘氨酸溶液洗脱展示ROR1抗原特异性Fab的噬菌体，用20μl pH8.0的Tris缓冲液中和至pH7.0，最终得到75μl噬菌体溶液。
4.3侵染TG1细菌
取实施例4.2中第二轮筛选得到的75μl噬菌体溶液，与500μl对数生长期的TG1细菌混合，37度静止30分钟。然后取侵染的TG1菌液，铺Amp抗性平皿，37度培养过夜。
4.4 ELISA筛选阳性克隆
计数铺皿生长的菌落，接种2块96孔深孔板，每孔含有400μl培养液(2YT+Amp+0.2％葡萄糖)，37度振摇培养6个小时，每孔加400μl含IPTG的培养液(2YT+Amp+2mM IPTG)，IPTG终浓度1mM，在30℃下250rpm振摇培养过夜。用没有生物素标记的ROR1抗原包被2块96孔ELISA板，100ng/100μl/孔，4℃包被过夜。
将抗原包被过夜的96孔ELISA板洗涤封闭后，每孔加100μl过夜培养的菌液，37℃孵孵育1个小时，再次洗涤，加二抗(HRP标记的抗人IgG-Fab的抗体)，37℃孵育40分钟。 洗涤后加显色液，避光保存30分钟，酶标仪读取OD600数值。结果如下表3-1和3-2所示。
表3-1阳性克隆初筛结果-1
#1 1 2 3 4 5 6 7 8 9 10 11 12
A 0.365 0.143 1.138 0.080 0.333 0.235 0.714 0.819 0.073 0.101 0.074 0.079
B 0.118 0.673 0.089 0.101 0.076 1.049 0.065 0.402 0.408 0.263 0.065 0.073
C 1.827 0.106 0.102 0.096 0.070 0.070 0.074 0.066 0.168 0.067 0.185 1.480
D 0.119 0.118 0.078 0.185 0.080 0.127 0.072 0.068 0.067 0.079 0.110 0.091
E 0.151 1.598 0.163 1.526 0.102 0.090 0.079 0.065 0.058 0.281 0.083 0.085
F 0.104 0.102 0.107 0.116 0.114 0.093 0.115 2.056 0.063 0.07 0.066 0.196
G 0.095 0.103 0.091 0.161 0.199 0.106 1.637 0.075 0.559 0.069 0.284 0.130
H 0.137 0.131 0.227 0.150 0.516 0.241 0.096 0.090 0.302 0.094 0.103 0.290
表3-2阳性克隆初筛结果-2
Figure PCTCN2021083246-appb-000002
取读数大于0.25的克隆，共35个，送样测序。序列分析表明，独特VH有34个，独特 LC有28个。轻链氨基酸序列比对结果见图5A，重链可变区氨基酸序列比对结果见图5B。
实施例5 构建VH组件库和LC组件库
5.1扩增抗原特异性VH和抗原特异性LC
设计包含限制性内切核酸酶(Esp3I和SfiI)识别位点的引物。用于扩增抗原特异性VH的引物29个，其中正向引物24个，反向引物5个；用于扩增抗原特异性KLC(kappa轻链)的引物19个，其中正向引物18个，反向引物1个；用于扩增抗原特异性LLC(lambda轻链)的引物26个，其中正向引物25个，反向引物1个；每组正向引物的每个引物均等比例混合，VH的反向引物也等比例混合，然后正反向引物再等比例混合，形成三组引物，分别用于扩增VH、KLC和LLC。作为实例性说明，本实施例以KLC为例，其中，VH的正向引物如SEQ ID NO:30-53所示，VH的反向引物如SEQ ID NO:54-58所示，KLC的正向引物如SEQ ID NO:59-76所示，KLC的反向引物如SEQ ID NO 77所示。
将实施例4中筛选得到的35个阳性克隆的小提DNA等量混合，使用上述三组引物分别扩增。电泳分析纯化PCR得到的带有识别位点的抗原特异性VH和抗原特异性LC(以KLC为例)。
5.2酶切和连接
用Esp3I酶切实施例5.1得到的抗原特异性VH，电泳分析纯化酶切后的抗原特异性VH。用Esp3I酶切实施例3.1得到的VH组件载体，电泳分析纯化酶切后的2.8kb的组件载体片段。将纯化的抗原特异性VH和2.8kb的组件载体片段连接，得到ROR1特异性的VH组件库。
用SfiI酶切实施例5.1得到的抗原特异性LC，电泳分析纯化酶切后的抗原特异性LC。用SfiI酶切实施例3.2得到的LC组件载体，电泳分析纯化酶切后的2.8kb的组件载体片段。将纯化的抗原特异性LC和2.8kb的组件载体片段连接，得到ROR1特异性的LC组件库。
实施例6 构建抗原特异性结合多肽展示库
用Esp3I酶切VH组件库，电泳分析纯化酶切后的0.35kb的带有粘性末端的抗原特异性VH(即，经释放的第一多核苷酸)。用SfiI酶切KLC组件库，电泳分析纯化酶切后的0.65kb的带有粘性末端的抗原特异性LC(即，经释放的第二多核苷酸)。用Esp3I和SfiI双酶切实施例1得到的展示载体，纯化得到具有粘性末端的3kb的展示载体片段I(即，经释放的第三多核苷酸)和具有粘性末端的5kb的展示载体片段II(即，经释放的第四多核苷酸)。将上述四个酶切后的片段等分子比例混合，20℃连接4小时，连接体系10μl，含有片段总量25ng， 得到抗原特异性结合多肽基因展示载体。
用PCR Cleanup试剂盒纯化连接产物，用10μl ddH 2O洗脱收集连接产物。取4μl纯化的连接产物做电转(Takara DH5a，电转感受态细菌)，铺皿，37℃培养过夜，计数菌落，库容达到2.3×10 5，获得抗原特异性结合多肽基因展示细菌文库。收集全部菌落，提取载体DNA得到抗原特异性结合多肽展示库。
实施例7 筛选单克隆抗体
从实施例6得到的抗原特异性结合多肽展示库得到ROR1特异性结合多肽表达载体DNA，将40μg DNA转化入FCHO细胞。转化60小时后，使用FACS分析细胞表面全长抗体的表达和抗体的ROR1抗原特异性。图6的结果表明细胞表面有ROR1全长抗体表达，表达的抗体可以与FITC标记的ROR1(使用FITC标记试剂盒进行标记得到)特异性结合。没有生物素标记的ROR1抗原来自Acro Biosystems(Cat#RO1-H5250-1mg)。图6显示的是用PE标记的鼠抗人Kappa轻链抗体和FITC标记的ROR1抗原双染细胞，FACS分析细胞表面的荧光信号。A，阴性对照；B，表达ROR1特异性抗体的细胞库。
用潮霉素(潮霉素浓度500μg/ml)加压筛选稳定转化的FCHO细胞库。潮霉素加压培养10天后得到稳定转化细胞库，用PE标记的鼠抗人kappa轻链抗体(BD)和FITC标记的ROR1抗原双染细胞库，使用FACS分选PE和FITC双阳性细胞，将单细胞克隆加入96孔板，每孔一个细胞，潮霉素加压培养。
潮霉素加压培养14天后得到稳定转化单细胞克隆92个。用0.5mM的EDTA-PBS缓冲液消化细胞，用PE标记的鼠抗人kappa轻链抗体和FITC标记的ROR1抗原双染92个单细胞克隆(抗原浓度0.15ng/50μl)。
进行FACS分析，得到PE和FITC荧光双阳性细胞克隆71个，阳性率为77％(71/92＝77％)。
实施例8 获得阳性克隆序列
根据阳性细胞群在FACS分析图中的定位，选择定位于不同位置(代表不同亲和力)的细胞克隆共30个做抗体基因PCR扩增。分别离心收集阳性克隆的细胞，弃上清，用20μl细胞基因组提取液(Quick Extraction Buffer，Lucigen)按照试剂说明书提取细胞基因组DNA。每个克隆各取2μl细胞基因组DNA提取液，PCR扩增每个克隆的VH和LC。用于扩增VH片段的正向引物为TGGGCTCTGCTCCTCCTGACC(SEQ ID NO:24)，用于扩增VH片段的反向引物为AGTTCCACGACACCGTCACCGGTTC(SEQ ID NO:25)，用于扩增LC片段的正向引物为GGACCTGGAGGATCCTCTTCTTGG(SEQ ID NO:26)，用于扩增LC片段的正 向引物为TAAATTCCTCGGCCGTGCAGGCCTTATCAACACTCTCCCCTGTTGAAGCTCT(SEQ ID NO:27)。
电泳分离纯化PCR扩增的VH和LC片段。测序分析纯化的VH和LC片段，确定14个独特VH和13个独特LC，组合得到17个独特序列阳性克隆(轻重链6个CDR至少有一个氨基酸不同)。作为示例，在此列举其中一对VH和LC的独特序列，独特序列的VH氨基酸序列如SEQ ID NO:28所示，独特序列的kappa LC氨基酸序列如SEQ ID NO:29所示。
实施例9 抗体亲和力分析
用Esp3I酶切实施例8中得到的具有独特序列阳性VH片段，用SfiI酶切实施例8中的具有独特序列阳性LC片段。使用PCR cleanup纯化酶切的VH和LC片段。将VH片段和LC片段分别插入可溶性重链表达载体，送菌落测序确定。提取测序确定的VH和LC表达载体DNA。
悬浮培养扩增293EXP细胞，按实施例8确定的轻重链配对共组成17个抗体轻重链表达载体配对。每个配对按轻链表达载体18μg，重链表达载体12μg的比例混合，转化30ml悬浮293EXP细胞(1.2×10 6/ml)。转化第六天收集培养液上清，用金斯瑞的磁珠(Cat#L00695)，按产品说明书纯化抗体，透析平衡为PBS-抗体溶液，-80℃保存。用SDS-PAGE变性凝胶电泳分析(图7)，结果显示抗体纯度达到90％以上。使用ELISA分析纯化抗体与抗原结合的亲和力，此处列举其中8个示例性抗体的EC 50作为说明，结果见下表4。
表4 8个示例性抗体的亲和力分析结果(EC50)
抗体编号 EC50(μg/ml) 抗体编号 EC50(μg/ml)
1 0.531 103 0.177
11 0.161 115 0.225
32 0.207 140 0.159
101 0.102 162 0.229
实施例10 快速筛选抗原特异性多肽
根据实施例4的噬菌体库筛选得到的阳性克隆的ELISA数据，从表3-1和3-2中选择ELISA读数0.8以上15个克隆，取等量菌液混合后，小提载体DNA，按照实施例5的流程，分别PCR扩增15个克隆的抗原特异性VH和抗原特异性LC，酶切纯化。按照实施例6的流程，构建抗原特异性结合多肽展示库。然后随机挑选48个菌落送样测序。分析测序结果，挑选VH和LC均正确的克隆36个，小提载体DNA，瞬转CHO细胞。60个小时后，用0.5mM  EDTA-PBS缓冲液消化细胞，用PE标记的鼠抗人kappa轻链抗体和FITC标记的ROR1抗原双染36个细胞群(抗原浓度0.15ng/50μl)，FACS分析，结果如图8所示，得到6个PE和FITC双阳性细胞克隆，阳性率17％(6/36)。图8显示的是用PE标记的鼠抗人Kappa轻链抗体和FITC标记的ROR1抗原双染细胞，FACS分析细胞表面的荧光信号。A，阴性对照；B，表达非ROR1特异性抗体的细胞克隆；C-H，表达ROR1特异性抗体的6个示例性阳性细胞克隆。

Claims (66)

  1. 一种用于构建抗原特异性结合多肽基因展示载体的方法，其包括：
    a)提供第一展示载体多核苷酸，所述第一展示载体多核苷酸以5’至3’方向包含B2-展示VH-B3；
    b)提供第二展示载体多核苷酸，所述第二展示载体多核苷酸以5’至3’方向包含S5-展示LC-S6；
    c)提供第三展示载体多核苷酸，所述第三展示载体多核苷酸以5’至3’方向包含B3-展示载体片段I-S5；
    d)提供第四展示载体多核苷酸，所述第四展示载体多核苷酸以5’至3’方向包含S6-展示载体片段II-B2；
    e)利用限制性内切核酸酶特异性切割所述第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和所述第四展示载体多核苷酸，得到切割后的所述第一展示载体多核苷酸、切割后的所述第二展示载体多核苷酸、切割后的所述第三展示载体多核苷酸和切割后的所述第四展示载体多核苷酸；其中所述限制性内切核酸酶分别特异性识别B2、B3、S5和S6；
    f)混合所述切割后的第一展示载体多核苷酸、所述切割后的第二展示载体多核苷酸、所述切割后的第三展示载体多核苷酸和所述切割后的第四展示载体多核苷酸，从而使得其能够定向连接而环化形成所述抗原特异性结合多肽基因展示载体；
    其中，所述展示VH编码抗原特异性结合多肽的重链可变区，所述展示LC编码抗原特异性结合多肽的轻链；
    其中所述B2、B3、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、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、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、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。
  5. 根据权利要求1-4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、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。
  6. 根据权利要求1-5中任一项所述的方法，其中所述限制性内切核酸酶选自SfiI、Esp3I和BsmBI。
  7. 根据权利要求1-6中任一项所述的方法，其中所述B2和B3能够被选自下组的酶特异性识别及切割：BsmBI和Esp3I。
  8. 根据权利要求1-7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。
  9. 根据权利要求1-8中任一项所述的方法，其中所述B2包含SEQ ID NO:8所示的核酸序列。
  10. 根据权利要求1-9中任一项所述的方法，其中所述B3包含SEQ ID NO:9所示的核酸序列。
  11. 根据权利要求1-10中任一项所述的方法，其中所述S5包含SEQ ID NO:10所示的核酸序列。
  12. 根据权利要求1-11中任一项所述的方法，其中所述S6包含SEQ ID NO:11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，其还包括将所述第一展示载体多核苷酸导入第一展示细菌以获得展示VH组件细菌文库。
  14. 根据权利要求13所述的方法，其包括将所述第一展示载体多核苷酸插入展示组件载体，形成展示VH存储连接产物，并且将所述展示VH存储连接产物导入所述第一展示细菌以获得所述展示VH组件细菌文库。
  15. 根据权利要求1-14中任一项所述的方法，其还包括将所述第二展示载体多核苷酸导入第二展示细菌以获得展示LC组件细菌文库。
  16. 根据权利要求15所述的方法，其包括将所述第二展示载体多核苷酸插入展示组件载体，形成展示LC存储连接产物，并将所述展示LC存储连接产物导入所述第二展示细菌中以获得所述展示LC组件细菌文库。
  17. 根据权利要求1-16中任一项所述的方法，其还包括所述第三展示载体多核苷酸导入第三展示细菌以获得展示载体组件I细菌文库。
  18. 根据权利要求17所述的方法，其包括将所述第三展示载体多核苷酸插入展示组件载体形成展示载体片段I存储连接产物，并将所述存储连接产物导入所述第三展示细菌中以获得所述展示载体组件I细菌文库。
  19. 根据权利要求1-18中任一项所述的方法，其还包括所述第四展示载体多核苷酸导入第四展示细菌以获得展示载体组件II细菌文库。
  20. 根据权利要求19所述的方法，其包括将所述第四展示载体多核苷酸插入展示组件载体形成展示载体片段II存储连接产物，并将所述存储连接产物导入所述第四展示细菌中以获得所述展示载体组件II细菌文库。
  21. 根据权利要求14-20中任一项所述的方法，其中所述展示载体组件载体源自pUC载体。
  22. 根据权利要求21所述的方法，其中所述pUC载体为pUC19载体或源自pUC19载体。
  23. 根据权利要求13-22中任一项所述的方法，其还包括由所述展示VH组件细菌文库获得包含所述第一展示载体多核苷酸的展示VH组件质粒，由所述展示VH组件质粒获得切割后的所述第一展示载体多核苷酸。
  24. 根据权利要求13-23所述的方法，其包括使用特异性识别所述B2和B3的限制性内切核酸酶对所述展示VH组件质粒进行酶切处理，从而获得所述切割后的所述第一展示载体多核苷酸。
  25. 根据权利要求15-24中任一项所述的方法，其还包括由所述展示LC组件细菌文库获得包含所述第二展示载体多核苷酸的展示LC组件质粒，由所述展示LC组件质粒获得切割后的所述第二展示载体多核苷酸。
  26. 根据权利要求15-25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展示LC组件质粒进行酶切处理，从而获得所述切割后的所述第二展示载体多核苷酸。
  27. 根据权利要求17-26中任一项所述的方法，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三展示载体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三展示载体多核苷酸。
  28. 根据权利要求17-27中任一项所述的方法，其包括使用特异性识别所述B3和S5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三展示载体多核苷酸。
  29. 根据权利要求19-28中任一项所述的方法，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四展示载体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四展示载体多核苷酸。
  30. 根据权利要求19-29中任一项所述的方法，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II进行酶切处理，从而获得所述切割后的所述第四展示载体多核苷酸。
  31. 根据权利要求1-30所述的方法，其包括：
    a)提供第五多核苷酸，所述第五多核苷酸以5’至3’方向包含B-抗原特异性VH-B；
    b)提供VH组件载体，所述VH组件载体包含第六多核苷酸，所述第六多核苷酸以5’至3’方向包含B3-VH组件载体连接片段-B2；
    c)利用限制性内切核酸酶切割所述第五多核苷酸和所述VH组件载体，得到切割后的第五多核苷酸和经释放的第六多核苷酸；
    d)混合所述切割后的第五多核苷酸和所述经释放的第六多核苷酸，从而使得其能够定向连接而环化形成抗原特异性VH组件库；其中所述B为能够特异性识别B2和/或B3的限制性内切核酸酶的识别位点，所述抗原特异性VH编码所述抗原特异性结合多肽的重链可变区。
  32. 根据权利要求1-31中任一项所述的方法，其包括：
    a)提供第七多核苷酸，所述第七多核苷酸以5’至3’方向包含S-抗原特异性LC-S；
    b)提供LC组件载体，所述LC组件载体包含第八多核苷酸，所述第八多核苷酸以5’至3’方向包含S6-LC组件载体连接片段-S5；
    c)利用限制性内切核酸酶切割所述第七多核苷酸和所述LC组件载体，得到切割后的第七多核苷酸和经释放的第八多核苷酸；
    d)混合所述切割后的第七多核苷酸和所述经释放的第八多核苷酸，从而使得其能够定向连接而环化形成抗原特异性LC组件库，
    其中所述S为能够特异性识别S5和/或S5的限制性内切核酸酶的识别位点，所述抗原特异性LC编码所述抗原特异性结合多肽的轻链。
  33. 根据权利要求1-32中任一项所述的方法，其包括，
    a)提供第九多核苷酸，所述第九多核苷酸以5’至3’方向包含B2-VH组件载体工具片段-B3；
    b)将所述第九多核苷酸插入表达组件载体，获得所述VH组件载体。
  34. 根据权利要求1-33中任一项所述的方法，其包括，
    a)提供第十多核苷酸，所述第十多核苷酸以5’至3’方向包含S5-LC组件载体工具片段-S6；
    b)将所述第十多核苷酸插入表达组件载体，获得所述LC组件载体。
  35. 根据权利要求33-34中任一项所述的方法，其中所述表达组件载体源自pMD载体。
  36. 根据权利要求35所述的方法，其中所述pMD载体为pMD19载体或源自pMD19载体。
  37. 根据权利要求31-36中任一项所述的方法，其包括以下步骤：
    a)将所述VH组件载体导入第九细菌，获得VH组件载体存储细菌文库；
    b)由所述VH组件载体存储细菌文库得到VH组件载体存储质粒；
    c)由所述VH组件载体存储质粒获得经释放的所述第六多核苷酸。
  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B2和B3的限制性内切核酸酶对所述VH组件载体存储质粒进行酶切处理，从而获得所述经释放的所述第六多核苷酸。
  39. 根据权利要求32-38所述的方法，其包括以下步骤：
    a)将所述LC组件载体导入第十细菌，获得LC组件载体存储细菌文库；
    b)由所述LC组件载体存储细菌文库得到LC组件载体存储质粒；
    c)由所述LC组件载体存储质粒获得经释放的所述第八多核苷酸。
  40. 根据权利要求39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LC组件载体存储质粒进行酶切处理，从而获得所述经释放的所述第八多核苷酸。
  41. 根据权利要求31-40中任一项所述的方法，其包括：
    a)提供所述抗原特异性VH组件库，所述抗原特异性VH组件库包括第一多核苷酸，所述第一多核苷酸以5’至3’方向包含B2-抗原特异性VH-B3；
    b)提供所述抗原特异性LC组件库，所述抗原特异性LC组件库包括第二多核苷酸，所述第二多核苷酸以5’至3’方向包含S5-抗原特异性LC-S6；
    c)提供所述展示载体，所述展示载体包含第三多核苷酸和第四多核苷酸，所述第三多核苷酸以5’至3’方向包含B3-展示载体片段I-S5，所述第四多核苷酸以5’至3’方向包含S6-展示载体片段II-B2；
    d)利用限制性内切核酸酶特异性切割所述抗原特异性VH组件库、所述抗原特异性LC组件库和所述展示载体，得到经释放的所述第一多核苷酸、经释放的所述第二多核苷酸、经释放的所述第三多核苷酸和经释放的所述第四多核苷酸；其中所述限制性内切核酸酶分别特异性识别B2、B3、S5和S6；
    e)混合所述经释放的第一多核苷酸、所述经释放的第二多核苷酸、所述经释放的第三多核苷酸和所述经释放的第四多核苷酸，从而使得其能够定向连接而环化形成抗原特异性结合多肽基因展示载体；
    其中，所述抗原特异性LC编码所述抗原特异性结合多肽的轻链，所述抗原特异性VH编码所述抗原特异性结合多肽的重链可变区；
    其中所述B2、B3、S5和S6各自独立地为限制性内切核酸酶识别位点。
  42. 根据权利要求41所述的方法，其包括使用特异性识别B2和B3的限制性内切核酸酶对所述抗原特异性VH组件库进行酶切处理，从而获得所述经释放的所述第一多核苷酸。
  43. 根据权利要求41-42中任一项所述的方法，其包括使用特异性识别S5和S6的限制性内切核酸酶对所述抗原特异性LC组件库进行酶切处理，从而获得所述经释放的所述第二多核苷酸。
  44. 根据权利要求41-43中任一项所述的方法，其包括使用特异性识别B3的限制性内切核酸酶和特异性识别S5的限制性内切核酸酶对所述展示载体进行酶切处理，从而获得所述经释放的所述第三多核苷酸。
  45. 根据权利要求41-44中任一项所述的方法，其包括使用特异性识别S6的限制性内切核酸酶和特异性识别B2的限制性内切核酸酶对所述展示载体进行酶切处理，从而获得所述经释放的所述第四多核苷酸。
  46. 根据权利要求41-45中任一项所述的方法，其中由样品材料获得所述第五多核苷酸、第七多核苷酸、第九多核苷酸、第十多核苷酸、第一展示载体多核苷酸、所述第二展示载体多核苷酸、所述第三展示载体多核苷酸和/或所述第四展示载体多核苷酸。
  47. 根据权利要求46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和/或IgG。
  48. 根据权利要求47所述的方法，其中所述抗体或其抗原结合片段靶向ROR1、PD-1和/或PD-L1。
  49. 根据权利要求47所述的方法，其中所述IgG为人IgG。
  50. 根据权利要求49所述的方法，其中所述人IgG为人IgG1或人IgG2。
  51. 根据权利要求1-50中任一项所述的方法，其中所述定向连接包括使用连接酶。
  52. 根据权利要求51所述的方法，其中所述连接酶包括T4 DNA连接酶。
  53. 根据权利要求1-52中任一项所示的方法，其包括将所述抗原特异性结合多肽基因展示载体导入细胞，由所述细胞得到所述抗原特异性结合多肽。
  54. 根据权利要求1-53中任一项所示的方法，其包括：
    a)将所述抗原特异性结合多肽基因展示载体导入第一细菌，获得抗原特异性结合多肽基因展示细菌文库；
    b)从所述抗原特异性结合多肽基因展示细菌文库获得抗原特异性结合多肽展示基因库；
    c)由所述抗原特异性结合多肽展示基因库得到抗原特异性结合多肽表达载体 DNA；
    d)将所述抗原特异性结合多肽表达载体DNA导入细胞；
    e)由所述细胞得到所述抗原特异性结合多肽。
  55. 根据权利要求54所述的方法，其包括冷冻保存所述抗原特异性结合多肽基因展示细菌文库、所述VH组件载体存储细菌文库、所述LC组件载体存储细菌文库、所述展示VH组件细菌文库、所述展示LC组件细菌文库、所述展示载体组件I细菌文库和所述展示载体组件II细菌文库。
  56. 根据权利要求54-55中任一项所述的方法，其中所述VH组件载体存储细菌文库包含至少10个不同的克隆。
  57. 根据权利要求54-56中任一项所述的方法，其中所述LC组件载体存储细菌文库包含至少10个不同的克隆。
  58. 根据权利要求54-57中任一项所述的方法，其中所述展示VH组件细菌文库包含至少10个不同的克隆。
  59. 根据权利要求54-58中任一项所述的方法，其中所述展示LC组件细菌文库包含至少10个不同的克隆。
  60. 根据权利要求54-59中任一项所述的方法，其中所述展示载体组件I细菌文库包含至少10个相同的克隆。
  61. 根据权利要求54-60中任一项所述的方法，其中所述展示载体组件II细菌文库包含至少10个相同的克隆。
  62. 根据权利要求54-61所述的方法，其中所述抗原特异性结合多肽基因展示细菌文库中有效克隆的比例为至少约10％。
  63. 根据权利要求54-62所述的方法，其中所述细胞为哺乳动物细胞。
  64. 筛选抗原特异性结合多肽或其片段的方法，其包括使用根据权利要求1-63中一项所述的抗原特异性结合多肽基因展示载体。
  65. 根据权利要求1-63中任一项所述的方法所产生的抗原特异性结合多肽基因展示载体。
  66. 根据权利要求1-63中任一项所述的方法所产生的抗原特异性结合多肽基因展示细菌文库。
PCT/CN2021/083246 2020-03-27 2021-03-26 抗原特异性结合多肽基因展示载体的构建方法与应用 WO2021190629A1 (zh)

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