WO2020125120A1 - 一种抗体文库的构建方法及其应用 - Google Patents

一种抗体文库的构建方法及其应用 Download PDF

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WO2020125120A1
WO2020125120A1 PCT/CN2019/108128 CN2019108128W WO2020125120A1 WO 2020125120 A1 WO2020125120 A1 WO 2020125120A1 CN 2019108128 W CN2019108128 W CN 2019108128W WO 2020125120 A1 WO2020125120 A1 WO 2020125120A1
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antibody
gene
screening
cells
domain
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French (fr)
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姚永超
李友佳
殷莎
刘广杰
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深圳市爱思迪生物科技有限公司
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Priority to EP19901269.1A priority Critical patent/EP3789521B1/en
Priority to US16/973,722 priority patent/US20210340523A1/en
Priority to JP2021517102A priority patent/JP7141154B2/ja
Publication of WO2020125120A1 publication Critical patent/WO2020125120A1/zh

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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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
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—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
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Antibodies are immunoglobulins expressed by B cells that bind to specific antigens. In most animals, antibodies are composed of pairs of heavy and light chains. Each chain is composed of two different regions, a variable region (Fv) and a constant region (Fc). Among them, the Fv region of the heavy chain and the light chain is responsible for binding to the target antigen, which is called the antigen binding determinant.
  • An antibody composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide of 15-20 amino acids (linker) is called a single chain antibody (scFv). Antibody drugs dominate the tumor and autoimmune diseases.
  • monoclonal antibodies and monoclonal antibody-based antibody drug conjugates, bispecific antibodies, chimeric antigen receptor T (CAR-T) cells and other immunotherapy have become the most popular tumor treatment methods .
  • various screening technologies for antibodies or antibody fragments are mainly developed based on B-cell monoclonal technology and protein display technology.
  • the hybridoma technology is the earliest technology used to establish the monoclonality of mouse B cells.
  • the spleen cells of the animal immunized with a predetermined antigen are fused with myeloma cells cultured in vitro and can grow indefinitely to obtain B hybridoma cells.
  • This hybridoma cell can not only be cultured in vitro like myeloma cells, but also be able to synthesize and secrete specific antibodies like B lymphocytes.
  • a single hybridoma cell line is obtained through monoclonalization, and the antibodies it produces are directed against the same epitope. This antibody is called a monoclonal antibody.
  • Phage display technology has become the most widely used display technology to date due to its relatively simple, robust, and convenient construction of large human antibody libraries. Phage display technology is to insert the gene encoding the polypeptide or protein into the appropriate position of the structural gene of the phage coat protein, and to fused and express the foreign polypeptide or protein and the coat protein without affecting the normal function of other coat proteins, so that the foreign polypeptide Or protein and coat protein are displayed on the surface of phage.
  • the polypeptide or protein displayed on the surface of the phage maintains a relatively independent spatial structure and biological activity, and can recognize and bind the target molecule.
  • the peptide library or protein library displayed by the phage is combined with the immobilized target molecule, and the unbound phage is washed away, and then the bound phage is eluted with an acid or alkali or a competitive molecule.
  • the neutralized phage infects E. coli for amplification, after After 3-5 rounds of enrichment, gradually increase the proportion of phages that can specifically recognize the target molecule, and finally obtain the peptide or protein that recognizes the target molecule.
  • the genes of the variable region of the antibody are inserted into the bacteriophage genome, the expressed antibodies are displayed on the surface of the bacteriophage, and a phage display antibody library is constructed to screen antibodies against various antigens.
  • Phage display antibody library technology is not limited by species, and can construct antibody libraries of various species. Antibodies selected from human natural libraries can be directly used for antibody drug research without going through the humanization process. Phage display technology and bacterial display technology are limited by the small capacity of the display system and are more suitable for displaying small peptides. Therefore, it can only be used to display antibody libraries for display of antibody libraries, and cannot display complete antibodies. In addition, antibodies are eukaryotic cell proteins, and bacteriophages and bacteria cannot guarantee complete and effective expression of eukaryotic proteins.
  • yeast display technology has become one of the most powerful tools for screening human antibody libraries and maturing antibody affinity.
  • the most widely used system for yeast display is the Saccharomyces cerevisiae Aga1p/2p ⁇ -lectin system, which relies on disulfide bonds to link the GPI-anchored Aga1p protein to the displayed antibody.
  • yeast display technology Compared with phage display technology, yeast display technology has many advantages. This includes the use of multicolor flow cytometry to quantify the intensity of antibody expression on the yeast surface and the intensity of binding to fluorescently labeled antigens. Yeast can express secreted antibodies, which helps to screen clones with higher expression, better folding, and proper secretion.
  • Yeast display can accommodate all forms of antibodies and antibody fragments, including domain antibodies (dAbs), scFv, Fabs, and even IgG.
  • dAbs domain antibodies
  • scFv domain antibodies
  • Fabs fragment antigen-binding protein
  • IgG immunoglobulin G
  • yeast electroporation transformation technology has improved the transformation efficiency to 1.5 ⁇ 10 8 transformants/ ⁇ g DNA, which is enough to construct an antibody library of up to 10 10 independent clones.
  • Mammalian cell display technology has become the most important technology for the development of human antibodies.
  • the folding, secretion and post-translational modification of proteins in mammalian cells are closest to the human body.
  • the mammalian cell display system is the most natural system for expressing and secreting human antibodies, which is conducive to the natural folding, stabilization and reduction of aggregation of antibodies.
  • Mammalian cell display technology has similar advantages as yeast display technology. Flow cytometry can be used to analyze the antibody on the surface of mammalian cells and sort out the cells with the best antibody expression signal.
  • Mammalian cell display technology can display full-length human antibodies, which can be used to construct an antibody library containing intact antibodies of Fc segments.
  • mammalian cell display technology has always had the problems of low transfection efficiency and difficulty in increasing library capacity. Because of these limitations, the initial use of mammalian cell display technology is for antibody optimization of relatively small libraries. In recent years, with the advancement of technology, a large number of mammalian cell-based libraries have been established for antibody screening.
  • Antibody drugs have achieved good results in tumor treatment, but there is no monoclonal antibody that can completely solve the problem of tumor treatment.
  • the main reasons are: (1) there is no one of the currently proven tumor targets Can completely cover a certain tumor.
  • a tumor antigen may be highly expressed in tumor cells, but it does not mean that this tumor antigen can be highly expressed in all tumor cells, and some tumor cells that do not express or lowly express this tumor antigen will become resistant to antibody drugs, resulting in During the treatment of the tumor, the tumor rebounded rapidly.
  • Few targets are only expressed in tumor cells, but not in normal cells. Almost all tumor antigens are also expressed in some normal tissues of humans, resulting in damage to normal tissues by antibody drugs, which has great side effects.
  • the antigen of tumor cells has extremely high mutation load, that is, the antigen has diversity and the mutation rate is high.
  • Tumor cells have a large number of different neoplastic antigens. These neoantigens are different even in the same tumor. Even if there is a definite tumor antigen, mutations may occur, resulting in the ineffectiveness of antibody drugs.
  • the proven targets are limited. For many tumors, there are no effective targets or target combinations available, and a large number of unknown tumor targets are not proven.
  • Existing antibody screening technologies are basically used for screening monoclonal antibodies. Monoclonal antibodies only recognize one epitope of a single tumor antigen target.
  • this application provides an antibody library construction method and application.
  • the method is based on the principle design of the synNotch system to control intracellular gene expression, and the extracellular recognition domain of the synNotch system is changed to In the coding domain of the extracellular antibody library, the regulated target gene is changed to a screening marker gene, an antibody-activated antibody screening system is obtained, and a polyclonal antibody technology for screening complex antigens is obtained, thereby solving the complex, diverse and variable tumor antigens and
  • the problem of limited available targets has broad application prospects and huge market value.
  • the present application provides a method for constructing an antibody library.
  • the method is based on the principle that the synNotch system controls gene expression in a cell, the first element and the second element are inserted into the same vector or different vectors, and transfected into the cell.
  • an antibody expression cell library that is, the antibody library;
  • the two vectors of steps (1) and (2) can be combined on one vector. It is also feasible to use this vector to construct an antibody library, and two-step transfection divided into two vectors can improve transfection efficiency and make it easier to construct large A large antibody library, the vector includes an expression vector.
  • the synNotch system contains the core regulatory domain of the natural intercellular signaling receptor Notch, and also contains a synthetic extracellular recognition domain and a synthetic intracellular Transcriptional domain; the synthetic extracellular recognition domain is a single-chain antibody.
  • the screening marker genes that can be positively screened by drugs in this application may be, but not limited to, puromycin resistance gene, neomycin resistance gene, blasticidin resistance gene, hygromycin B resistance gene, etc., respectively Drugs: screening with drugs puromycin, G418, blasticidin, hygromycin B; screening marker genes that can be negatively screened by drugs may be, but not limited to, herpes simplex virus thymidine kinase (HSV-TK) gene, Cytosine deaminase (CD) gene and iCasp9 suicide system gene were screened with drugs: ganciclovir or FIAU, 5-fluorocytosine, AP1903 or AP20187, respectively.
  • HSV-TK herpes simplex virus thymidine kinase
  • CD Cytosine deaminase
  • iCasp9 suicide system gene were screened with drugs: ganciclovir or FIAU, 5-fluorocytosine
  • the drug resistance gene includes any one or a combination of at least two of the puromycin resistance gene, neomycin resistance gene, blasticidin resistance gene and hygromycin B resistance gene .
  • the suicide gene includes any one or a combination of at least two of herpes simplex virus thymidine kinase (HSV-TK) gene, cytosine deaminase (CD) gene and iCasp9 suicide system gene.
  • HSV-TK herpes simplex virus thymidine kinase
  • CD cytosine deaminase
  • iCasp9 suicide system gene iCasp9
  • the fluorescent protein gene includes any one of EGFP, YFP, mCherry, DsRed and BFP or a combination of at least two.
  • the molecular tag includes any one or a combination of at least two of His-tag, Flag-tag, HA-tag, Myc-tag and Strep-tag.
  • the screening method for screening marker genes includes any one or a combination of at least two of drug screening, flow cytometry detection and sorting, and magnetic bead sorting.
  • the fluorescent protein gene detection and sorting method includes flow cytometry detection and sorting.
  • the coding domain of the extracellular antibody library includes an antibody sequence, an antibody heavy chain sequence, an antibody light chain sequence, an antibody variable region sequence, a single chain antibody sequence, a single domain Any one or a combination of at least two of the antibody sequence and the Fab fragment sequence.
  • the coding domain portion of the extracellular antibody library in this application includes, but is not limited to, intact antibodies, the chains (heavy or light chains) that make up the antibodies, and fragments of the antibodies (antibody variable regions, single chain antibodies, single domain antibodies, Fab segments) The constructed library.
  • the source of the coding domain of the extracellular antibody library may be, but not limited to, prepared for immunized animals, from diseased people, from healthy people, vaccinated people, artificially synthesized.
  • the Notch core domain includes human P1391-R1763 fragments, mouse P1390-R1752 fragments, fragments before or after, plus or minus 200 amino acids before or after these fragments, or the similarity with these fragments is not low In 85% of the sequence.
  • the Notch core domain of the present application may be from Notch not limited to humans, Notch from mice, and sequences with a similarity of not less than 85%; the Notch core domain may be P1391-R1763 fragment (human), P1390-R1752 Fragments (mouse), fragments before or after, plus or minus 200 amino acids, and sequences with a similarity of not less than 85% to these fragments.
  • the cis activator includes pTet and/or UAS-pSV40.
  • the method of screening antibodies is to contact a library of antibody-expressing cells with an antigen. Only the antibody-expressing cells that can recognize the antigen will activate the expression of the screening marker gene in the cell, and the expression target will be screened according to the expression of the screening marker gene Antibody cells.
  • the present application provides an antibody, which is selected by the method described in the third aspect.
  • the present invention also provides a system for screening antibodies, which includes:
  • a first element including a cis activator and a selection marker gene
  • the second element includes the gene encoding the coding domain of the extracellular antibody library, the gene encoding the Notch core domain, and the gene encoding the intracellular transcription domain.
  • the cis activator, selection marker gene, extracellular antibody library coding domain, Notch core domain, and intracellular transcription domain are defined as described above.
  • the present invention also provides a method for screening antibodies using the above system, which includes:
  • the first element includes a cis activator and a selection marker gene
  • the second element includes a gene encoding the coding domain of the extracellular antibody library, a gene encoding the Notch core domain and encoding Genes of intracellular transcription domains
  • cells expressing the target antibody are screened to screen the target antibody.
  • the cis activator, selection marker gene, extracellular antibody library coding domain, Notch core domain, and intracellular transcription domain are defined as described above.
  • the method provided in this application can screen both monoclonal antibodies and polyclonal antibodies: the previous antibody screening technology is suitable for screening monoclonal antibodies, even if it is used for screening polyclonal antibodies, the diversity and specificity of the antibodies screened Sex and stability are also difficult to guarantee; the antibody screening technology of this application has greater diversity in polyclonal antibodies screened against antigens, and can obtain antibodies with better specificity and higher affinity by changing the screening conditions. A large number of monoclonal antibody coding sequences are available, which is simpler than previous monoclonal screening techniques;
  • the method provided in this application does not require the expression and purification of antigens: in the past antibody screening technology, it is necessary to express and purify the peptide fragments of the antigen protein or antigen epitope when performing antibody screening, which is used to immunize animals and plate for ELISA screening of antibodies and binding Antibodies are screened for antibodies by flow cytometry, and some of these antigen proteins or peptides are not easy to purify, or they are different from the original antigen protein modification, or are not the original spatial structure. These will affect the specificity and affinity of the selected antibody; this application The antibody screening technology only needs to display the target antigen on the surface of the cell, and then the antibody can be screened. To obtain the cells displaying the target antigen, only the gene of the target antigen needs to be transfected into the cell, or directly to determine the expression target The wild-type cells of the antigen are very convenient;
  • the method provided in this application does not require preset antigens: the previous antibody screening technology needs to detect the antigen in advance and then screen the antibody against the antigen.
  • the tumor cells are extremely complex, there are a large number of undetected antigens, and the clear tumor antigens
  • the target is very limited, and the antibodies screened against the limited antigen target cannot meet the needs of tumor treatment; this application does not require a preset antigen, and can use the entire tumor cell as an antigen, and polyclonal antibodies selected from the antibody library can be Identify various antigen targets on the surface of tumor cells, thus avoiding the problem of limited antigen targets;
  • the method provided in this application can facilitate negative screening: the purpose of negative antibody screening is to remove unwanted antibodies, such as antibodies that may recognize normal cells, because tumor cells and normal cells have a large number of identical antigens, so they are targeted at tumor cells In addition to identifying the tumor cells, the selected antibodies may also identify normal cells, so that the developed antibody drugs have the side effect of harming normal tissues.
  • antibody screening technology screened monoclonal antibodies, and the screened antibodies can be removed one by one. To verify the effect on normal cells and remove unwanted antibodies, this method is inefficient and not suitable for polyclonal antibody screening; the antibody screening technology of this application can use negative screening marker genes, negative screening drugs or flow cytometry Instrument negative sorting and other methods are convenient for negative screening;
  • the methods provided in this application are diverse in screening methods: the antibody screening technology provided in this application can be used for positive and negative screening with drugs, and can also be screened with flow cytometry, or in combination with drug screening and flow cytometry screening. Free choice and matching according to your own research conditions and research experience is more conducive to obtaining target antibodies.
  • 1 is a schematic diagram of the synNotch system of the present application for controlling gene expression in cells
  • FIG. 3 is a schematic diagram of a vector of a cis-activator plus a selection marker gene of the present application
  • FIG. 4 is a schematic diagram of a vector of the pTet cis activator of the present application for regulating a fluorescent protein fusion puromycin resistance gene;
  • FIG. 6 is a picture of green fluorescent protein expressed by tTA activated pTet cis activator taken by a fluorescent microscope of the present application;
  • FIG. 7 is a picture of the expression of red fluorescent protein expressed by the tTA activated pTet cis activator taken by a fluorescent microscope of the present application;
  • FIG. 8 is a schematic diagram of a universal expression vector of the antibody library of the present application.
  • FIG. 9 is a schematic diagram of the expression vector of the anti-CD19 single chain antibody of the present application.
  • FIG. 10 is a schematic diagram of the expression vector of the anti-GPC3 single chain antibody of the present application.
  • FIG. 11 is a schematic diagram of the CD19 and GFPC3 antigen expression vectors of this application.
  • 12 is a flow cytometer for detecting the expression of CD19 and GFPC3 antigen in K562 cells
  • FIG. 13 is a graph showing the results of screening CD19 antibody expressing cells from the mixed antibody expressing cell library using the antibody screening system of the present application;
  • FIG. 14 is a graph showing the results of screening GPC3 antibody expressing cells from the mixed antibody expressing cell library using the antibody screening system of the present application;
  • 15 is a schematic diagram of a vector of pTet cis activator of the present application to regulate green fluorescent protein
  • 16 is a schematic diagram of the expression vector of the anti-Raji single chain antibody gene library of the present application.
  • 17 is a schematic diagram of an antigen expression vector used for negative screening in the present application.
  • FIG. 18 is a graph showing the expression results of CD19 antigen expression vector and negative selection antigen expression vector in K562 cells detected by flow cytometry of the present application;
  • FIG. 19 is a diagram of the induced activation of a library of antibody-expressing cells before screening by flow cytometry of the present application.
  • FIG. 20 is a diagram of induced activation of a library of antibody-expressing cells after screening and screening by flow cytometry of the present application;
  • 21 is a schematic diagram of a vector of pTet cis activator of the present application to regulate fluorescent protein fusion positive and negative selection genes;
  • 22 is a schematic diagram of an expression vector of a single-chain antibody gene library that can be subjected to preliminary drug screening in this application;
  • FIG. 23 is a schematic diagram of positive drug screening using the antibody screening system of the present application.
  • FIG. 25 is a schematic diagram of negative drug screening using the antibody screening system of this application.
  • FIG. 26 is a graph showing the results of negative expression of antibody-expressing cells detected by flow cytometry in this application.
  • FIG. 27 is a schematic diagram of a single-chain antibody and antibody constant region fusion expression vector of the present application.
  • FIG. 28 is a graph showing the results of antibody-antigen binding efficiency screened by the flow cytometer of the present application by the antibody screening method of the present application.
  • the schematic diagram of the synNotch system controlling gene expression in cells is shown in Figure 1.
  • the synNotch system undergoes induced transmembrane region cleavage, thereby releasing the intracellular transcription domain into the nucleus and binding Upstream cis activator to activate the expression of regulated target genes;
  • the schematic diagram of the synNotch-based antibody screening system is shown in Figure 2.
  • the extracellular recognition domain is changed to the coding domain of the extracellular antibody library, and the regulated target gene is changed to the screening marker gene, thereby obtaining Antigen-activated antibody screening system;
  • the cis activator uses pTet (SEQ ID NO. 3). After receiving the tTA signal, pTet can start the expression of the screening gene, and the fluorescent protein fusion puromycin resistance gene (SEQ ID NO) .4), the two are connected by a 2A (SEQ ID NO.5) sequence that can be automatically broken, and the vector diagram of the cis activator plus the selection marker gene is shown in Figure 3;
  • two fluorescent proteins EGFP SEQ ID NO. 6
  • mCherry SEQ ID NO. 7
  • the terminator was SV40 poly A (SEQ ID NO.8), directly synthesize the coding gene of the entire sequence ( Figure 4), construct it into a lentiviral vector, package the lentivirus, and transfect into 293T cells respectively, and monoclonalize the transfected cells after 3 days;
  • the tTA expression vector consists of the ef1- ⁇ promoter (SEQ ID NO. 9) and the tTA gene (SEQ ID NO .10), consisting of terminator, constructing tTA expression vector into lentiviral vector and packaging into lentivirus;
  • the vector inserted with the anti-human GPC3 single-chain antibody gene ( Figure 10) was packaged into a lentivirus, and transfected into a monoclonal cell line with mCherry selection markers to obtain GPC3 single-chain antibody-expressing cells.
  • CD19 and GFPC3 antigen-expressing cells synthesis of CD19 (SEQ ID NO. 15) and GPC3 (SEQ ID NO. 16) antigen coding sequences, which were constructed on expression vectors respectively. Fusion of CD19 or GPC3, blue fluorescent protein (SEQ ID NO.17), and puromycin resistance gene (Figure 11);
  • the constructed expression vector was packaged into a lentivirus, and the virus was transfected into K562 cells. After 2 days of transfection, 1 ⁇ g/mL puromycin was added to the culture medium to obtain K562 cells stably expressing CD19 or GPC3. The expression of blue fluorescent protein detected by flow cytometry was almost 100% ( Figure 12), indicating that the antigen has been stably expressed in K562 cells.
  • the cis-activator pTet is used. After receiving the tTA signal, pTet starts the expression of the screening marker green fluorescent protein gene, and constructs the lentiviral vector by artificially synthesizing the entire sequence (FIG. 15). Lentivirus was packaged and transfected into 293T cells. After 3 days, the transfected cells were monoclonalized. A portion of the monoclonal cells to be screened were taken out and transfected with lTA-expressing lentivirus. After 2-3 days, they were observed with a fluorescence microscope. Cell lines with green fluorescence expression and no fluorescence expression before transfection were selected as monoclonal cell lines for stable transfection with cis activator and selection marker genes.
  • mice were immunized with CD19 positive Raji cells, boosted once every two weeks, and immunized once again in the fourth week, and the mice were sacrificed 3 days later to isolate the splenic lymphocytes of the mice; Lymphocyte RNA;
  • the antibody gene library expression vector is packaged into a lentivirus and transfected into the monoclonal cell line that has been stably transfected with the cis activator and the selection marker gene in step (1) to obtain an antibody expression cell library.
  • Example 1 For the preparation of antigen-expressing cells expressing CD19, see Example 1. This antigen-expressing cell is used for the positive screening of anti-CD19 antibodies, synthesizing a transmembrane domain (SEQ ID NO.20) encoding gene, replacing the CD19 antigen in the positive screening vector Gene to obtain an antigen expression vector for negative selection ( Figure 17);
  • the constructed antigen expression vectors were packaged into lentiviruses and transfected into K562 cells by virus; 2 days after transfection, 1 ⁇ g/mL puromycin was added to the culture medium to obtain positive screening antigen expression cell lines and negative screening, respectively. Antigen-expressing cell line.
  • the expression of blue fluorescent protein detected by flow cytometry was almost 100% ( Figure 18), indicating that the antigen has been stably expressed in K562 cells.
  • the antibody-expressing cell library and the CD19 antigen-expressing positive screening cells were mixed 1:1. After the mixed cells were cultured for 2 days, a small number of cells expressing green fluorescent protein appeared in the antibody-expressing cell library ( Figure 19). After two days of mixed culture of antigen-selecting cells, a small number of cells expressing green fluorescent protein appeared in the antibody-expressing cell library. Using a sorted flow cytometer, cells expressing green fluorescent protein were sorted and cultured until most The green fluorescence disappears;
  • the sorted antibody-expressing cell library and negative-selecting antigen-expressing cells were mixed 1:1, and after 3 days, cells with double negative for green fluorescent protein and blue fluorescent protein were sorted by sorting flow cytometry, which is Cells expressing CD19 antibody, at this time, the antibody-expressing cells had no expression of green fluorescent protein when co-cultured with negative selection antigen-expressing cells, and had green fluorescent protein expression when co-cultured with CD19 antigen-expressing cells ( Figure 20), after positive and negative screening The antibody-expressing cells had no green fluorescent protein expression when co-cultured with negative selection antigen-expressing cells, but had green fluorescent protein expression when co-cultured with CD19 antigen-expressing cells.
  • the cis-activator pTet, iCasp9 negative selection system (SEQ ID NO.21), green fluorescent protein, and puromycin resistance gene are used as the selection marker, and the three are connected by a 2A sequence that can be automatically broken;
  • an intermediate vector other than the extracellular antibody library coding domain gene in the expression vector of the antibody gene library to facilitate subsequent insertion of the extracellular antibody library gene ( Figure 22), and artificially synthesize the sequence of each component of the intermediate vector, from N-terminus to C-terminus
  • the serially connected parts are the mPGK1 promoter, the CD8 ⁇ signal peptide coding sequence, the coding domain of the antibody gene library (the coding region sequence with the ⁇ -galactosidase (lacZ) N-terminal ⁇ fragment, and the front Asc I restriction enzyme.
  • Not I restriction enzyme site which facilitates the insertion of the antibody library sequence, and can display the fragment insertion status by blue and white spots
  • NOTCH1 fragment coding sequence tTA coding sequence, terminator, EFS promoter (SEQ ID NO.22), blasticidin resistance (SEQ ID NO.23) gene, terminator;
  • the synthesized sequence was constructed into a lentiviral vector for further insertion into the antibody gene library sequence.
  • the DNA fragments of the scFv library of mice immunized with Raji cells were digested by Asc I and Not I, and inserted between the Asc I and Not I restriction enzyme sites of the intermediate vector of the antibody gene library expression vector to obtain the antibody gene library Expression vector, the antibody gene library expression vector is packaged into a lentivirus, transfected into the monoclonal cell line that has been stably transfected with cis activator and selection marker gene in step (1), and 10 ⁇ g/mL is added to the culture medium 2 days later The blasticidin was screened to obtain a library of antibody-expressing cells;
  • the antibody-expressing cell library and CD19 antigen-expressing positive screening cells were mixed 1:1. After the mixed cells were cultured for 2 days, 1 ⁇ g/mL of puromycin was added to the medium for screening. After 6-8 days of positive screening, (positive The screening schematic diagram is shown in Figure 23).
  • the antibody library expressing cells are almost all cells expressing green fluorescent protein ( Figure 24).
  • the mixed cells of the antibody library expressing cells and the positive screening cells expressing CD19 antigen are positively screened by puromycin.
  • the antibody expressing cells are almost all cells expressing green fluorescent protein;

Abstract

本申请提供了一种抗体文库的构建方法及其应用，所述方法其包括将第一元件和第二元件插入同一载体或不同载体，将所述载体转染至细胞内，得到抗体表达细胞文库，即所述抗体文库；其中，所述第一元件包括顺式激活子和筛选标记基因，所述第二元件包括胞外抗体文库编码域、Notch核心结构域和胞内转录结构域。

Description

一种抗体文库的构建方法及其应用 技术领域
本申请属于生物技术领域，涉及一种抗体文库的构建方法及其应用。
背景技术
抗体是由B细胞表达、结合特定抗原的免疫球蛋白。大多数动物中，抗体由成对的重链和轻链构成。各链由可变区(Fv)和恒定区(Fc)两种不同的区域组成。其中，重链和轻链的Fv区负责与靶抗原的结合，称为抗原结合决定簇。由抗体重链可变区和轻链可变区通过15-20个氨基酸的短肽(linker)连接而成的抗体称为单链抗体(scFv)。抗体药物在肿瘤，自身免疫病两大领域占主导地位。特别是在肿瘤治疗领域，单克隆抗体以及基于单克隆抗体的抗体药物偶联物、双特异性抗体、嵌合抗原受体T(CAR-T)细胞等免疫治疗成为目前最热门的肿瘤治疗手段。当前，抗体或抗体片段的各种筛选技术主要是基于B细胞单克隆技术、蛋白展示技术发展而来。
杂交瘤技术是最早用于建立小鼠B细胞单克隆化的技术。将预定抗原免疫后的动物脾细胞与体外培养、可无限生长的骨髓瘤细胞融合，得到B杂交瘤细胞。这种杂交瘤细胞既能像骨髓瘤细胞一样在体外培养无限增殖，又能像B淋巴细胞那样合成和分泌特异性抗体。通过单克隆化得到单个杂交瘤细胞的细胞系，它所产生的抗体只针对同一抗原表位，这种抗体即所谓单克隆抗体。但由于鼠源抗体对于人来说属于“异种”蛋白，所以当这些单克隆抗体进入人体后，会诱使人体产生针对这些抗体的抗体(即人抗鼠抗体)。人抗鼠抗体会中和鼠源抗体，使得鼠源抗体药物失效。将鼠源抗体的Fc段替换为人源抗体的Fc段得到人鼠嵌合抗体，进一步将嵌合抗体Fv区的FR片段人源化得到人源化抗体，从而降低鼠源单克隆抗的免疫原性。而全人源的单抗更是完全用人类的遗传信息来编码抗体，避免了抗异种蛋白反应。将人B细胞与表达mIL-6和hTERT的鼠骨髓瘤细胞融合而成的人鼠杂交瘤细胞以及用EBV转化人B细胞使其永生化，均是开发全人源单抗的有效手段。
B细胞的单克隆化技术耗时耗力，效率低，无法进行抗体的高通量筛选。蛋白展示技术克服了杂交瘤技术无法进行高通量筛选的缺点，可以从一个巨大的文库中(超过10 10个独立克隆)筛选出目的克隆。常用的蛋白展示技术包括噬菌体展示技术、细菌展示技术、核糖体展示技术、酵母展示技术以及哺乳动物细胞展示技术，己经被广泛的应用于新抗体的筛选和抗体亲和力的改进。
噬菌体展示技术由于其相对简单、稳健、方便构建大的人类抗体库等优点，成为迄今为止应用最广泛的展示技术。噬菌体展示技术是将多肽或蛋白质的编码基因插入噬菌体外壳蛋 白结构基因的适当位置，在不影响其他外壳蛋白正常功能的情况下，使外源多肽或蛋白与外壳蛋白融合表达，从而使外源多肽或蛋白与外壳蛋白展示在噬菌体表面。展示到噬菌体表面的多肽或蛋白保持相对独立的空间结构和生物活性，可以识别和结合靶分子。噬菌体展示的肽库或蛋白库与固定后的靶分子结合，洗去未结合的噬菌体，然后用酸碱或者竞争的分子洗脱下结合的噬菌体，中和后的噬菌体感染大肠杆菌扩增，经过3-5轮的富集，逐步提高可以特异性识别靶分子的噬菌体比例，最终获得识别靶分子的多肽或者蛋白。将抗体可变区的基因插入噬菌体基因组中，表达的抗体展示到噬菌体的表面，构建噬菌体展示抗体库，可以筛选针对各种抗原的抗体。相对于杂交瘤技术，通过噬菌体展示抗体库技术筛选抗体，可以不经过免疫，缩短抗体生产的周期。也可以筛选在体内免疫原性弱，或者有毒性的抗原的抗体，适用范围广。噬菌体展示抗体库技术不受种属的限制，可以构建各种物种的抗体库。从人天然库中筛选到的抗体，可以不经过人源化过程，直接用于抗体药物研究。噬菌体展示技术以及细菌展示技术受限于展示系统的小容量，更适合展示小肽。因而将其用于抗体文库的展示方面，只能展示抗体的片段，无法进行完整抗体的展示。另外，抗体为真核细胞蛋白，噬菌体和细菌不能保证完全有效地表达真核蛋白。
酵母展示技术已成为筛选人抗体文库以及抗体亲和力成熟的最有力工具之一。最广泛用于酵母展示的系统是酿酒酵母Aga1p/2pα-凝集素系统，它依靠二硫键将GPI锚定的Aga1p蛋白与展示的抗体连接。与噬菌体展示技术相比，酵母展示技术有许多优点。包括使用多色流式细胞仪来定量酵母表面的抗体表达强度以及与荧光标记的抗原结合强度。酵母可表达分泌型的抗体，有助于筛选更高表达，更好的折叠，和适当分泌的克隆。酵母展示可以容纳所有形式的抗体和抗体片段，包括域抗体(dAbs)、scFv、Fabs、甚至IgG。但是，由于转化效率和流式分析技术的限制，酵母展示的人抗体库容大小是有限的(10 7-10 9个独立克隆)。然而，最近改进的酵母电穿孔转化技术已经将转化效率提高到1.5×10 8个转化子/μg DNA，这足以构建多达10 10个独立克隆的抗体库。
哺乳动物细胞展示技术成为开发人源抗体最重要的技术。哺乳动物细胞中蛋白质的折叠、分泌和翻译后修饰与人体最为接近。哺乳动物细胞展示系统是表达和分泌人类抗体最自然的系统，有利于抗体的自然折叠、稳定、减少聚集。哺乳动物细胞展示技术与酵母展示技术有相似的优点，可以通过流式细胞仪技术分析哺乳动物细胞表面的抗体情况，并分选出最佳抗体表达信号的细胞。哺乳动物细胞展示技术可以展示全长人源抗体，用于构建包含Fc段完整抗体的抗体库。然而，哺乳动物细胞展示技术一直存在转染效率低、文库容量难以提高的问题。由于这些限制，哺乳动物细胞展示技术最初用途是用于相对较小文库的抗体优化。而最 近几年中，随着技术的进步，已经建立了大量基于哺乳动物细胞的文库，用于抗体的筛选。
除了这些主流的抗体筛选技术外，还有一些有鲜明特点的抗体筛选技术，比如：基于生长信号体的抗体筛选技术(Growth Signalobody)、基于嵌合抗原受体的抗体筛选技术(CARbodies)、深度测序抗体发现技术，都展示着巨大的潜力。
得益于多种抗体筛选技术的进步以及人们对疾病的进一步认识，众多抗原靶点的发现，使得抗体药物的开发飞速发展。抗体以及基于抗体的药物对肿瘤的治疗效果与所选择的抗原靶点息息相关，目前FDA批准的抗体药物靶点数量有限，针对这些靶点的抗体均为单克隆抗体或多个单克隆抗体混合而成多样性有限的多克隆抗体。
抗体药物在肿瘤治疗上取得了很好的疗效，但目前还没有任何单克隆抗体能完全解决肿瘤治疗的问题，主要原因在于：(1)目前探明的肿瘤靶点中，没有哪一个靶点可完全覆盖某种肿瘤。肿瘤细胞中可能高表达某个肿瘤抗原，但不等于此肿瘤抗原能在所有肿瘤细胞中都高表达，部分不表达或低表达此肿瘤抗原的肿瘤细胞会对抗体药物产生抗药性，从而导致在肿瘤的治疗过程中，肿瘤的迅速反弹。(2)很少有靶点只表达在肿瘤细胞中，而在正常细胞中不表达。几乎所有的肿瘤抗原，在人的某些正常组织有也有表达，导致抗体药物对正常组织的损伤，具有很大的药副作用。(3)肿瘤细胞的抗原具有极高的突变负荷，即抗原具有多样性且突变率高。肿瘤细胞具有大量各不相同的肿瘤新抗原，这些新抗原即使在同一种肿瘤里，也是不相同的，即便有确定的肿瘤抗原，也可能发生突变，导致抗体药物无效。(4)已探明的靶点有限，针对许多肿瘤，无有效的靶点或靶点组合可用，大量未知的肿瘤靶点并未探明。现有的抗体筛选技术，基本都用于筛选单克隆抗体。单克隆抗体只识别单一肿瘤抗原靶点的一个抗原表位，即便是由多个单克隆抗体混合而成的多克隆抗体，其识别的抗原表位也有限，远远不能覆盖肿瘤细胞复杂的抗原表位。很多抗体筛选技术也可用于筛选多克隆抗体，但其筛选操作便利性、筛选出抗体的成功率、得到的抗体特异性等方面都面临不小的挑战。
因此，开发一种抗体文库的构建方法，用于筛选针对复杂抗原的多克隆抗体技术，从而解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
发明内容
针对现有技术的不足及实际的需求，本申请提供一种抗体文库的构建方法及应用，所述方法基于synNotch系统控制细胞内基因表达的原理设计，将synNotch系统的胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，得到受抗原激活的 抗体筛选系统，得到了筛选针对复杂抗原的多克隆抗体技术，从而解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种抗体文库的构建方法，所述方法以synNotch系统控制细胞内基因表达为原理，将第一元件和第二元件插入同一载体或不同载体，转染至细胞内，得到抗体表达细胞文库，即所述抗体文库；
其中，所述第一元件包括顺式激活子和筛选标记基因，所述第二元件包括胞外抗体文库编码域、Notch核心结构域和胞内转录结构域。
具体地，包括如下步骤：
(1)向细胞中转染带有顺式激活子和筛选标记基因的载体；
(2)向步骤(1)的细胞内转染携带从N端到C端依次为胞外抗体文库编码域、Notch核心结构域、胞内转录结构域的载体，得到抗体表达细胞文库，即所述抗体文库。
步骤(1)和(2)的两个载体可以合在一个载体上，用这个载体来构建抗体文库也是可行的，而分成两个载体进行两步骤转染能够提高转染效率，更容易构建大容量的抗体文库，所述载体包括表达载体。
本申请是基于synNotch系统控制细胞内基因表达的原理设计，synNotch系统包含了天然的细胞间信号传导受体Notch的核心调节结构域，同时含有合成性的胞外识别结构域和合成性的胞内转录结构域；合成性的胞外识别结构域为一单链抗体，当单链抗体识别并结合抗原时，synNotch系统发生诱导性跨膜区域剪切，从而释放胞内转录结构域进入细胞核，结合上游顺式激活子来激活受调控目标基因的表达；因此，synNotch系统改造的细胞，可以通过特定的抗原识别结合来驱动某一特定的基因表达，用来改造T细胞，使得T细胞可以受抗原调控表达细胞因子而杀伤靶细胞，或者T细胞可以受抗原调控表达CAR，从而提高CAR-T细胞对靶细胞的识别精度。
本申请中，申请人为解决现有技术筛选抗体的缺点，提供一种简洁高效的抗体文库构建方法，以synNotch系统控制细胞内基因表达为基本原理，通过大量实验摸索，优化整体方案流程，反复设计验证，将synNotch系统的胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，从而得到受抗原激活的抗体筛选系统；抗体文库的构建方法为先向细胞中转染带有顺式激活子加筛选标记基因的载体，再向这个细胞中转染携带从N端到C端分别为胞外抗体文库编码域、Notch核心结构域、胞内转录结构域的载体，从而得到抗体表达细胞文库。
优选地，所述筛选标记基因包括药物抗性基因、自杀基因、荧光蛋白基因和分子标签中的任意一种或至少两种的组合。
若要进行抗体的正筛选，将筛选标记基因设计为药物抗性基因，则激活的细胞可以在有筛选药物的培养基中存活，其余的细胞死亡，从而筛选出针对目标抗原的抗体表达细胞；若要进行抗体的负筛选，将筛选标记基因设计为自杀基因，则激活的细胞在有筛选药物的培养基中凋亡，其余的细胞存活，从而除去针对目标抗原的抗体表达细胞。
本申请可通过药物进行正筛选的筛选标记基因可以是但不限于嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、潮霉素B抗性基因等，分别用药物：用药物嘌呤霉素、G418、杀稻瘟素、潮霉素B进行筛选；可通过药物进行负筛选的筛选标记基因可以是但不限于单纯疱疹病毒胸苷激酶(HSV-TK)基因、胞嘧啶脱氨酶(CD)基因、iCasp9自杀系统基因，分别用药物：更昔洛韦或FIAU、5-氟胞嘧啶、AP1903或AP20187进行筛选。
优选地，所述药物抗性基因包括嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因和潮霉素B抗性基因中的任意一种或至少两种的组合。
优选地，所述自杀基因包括单纯疱疹病毒胸苷激酶(HSV-TK)基因、胞嘧啶脱氨酶(CD)基因和iCasp9自杀系统基因中的任意一种或至少两种的组合。
优选地，所述荧光蛋白基因包括EGFP、YFP、mCherry、DsRed和BFP中的任意一种或至少两种的组合。
优选地，所述分子标签包括His-tag、Flag-tag、HA-tag、Myc-tag和Strep-tag中的任意一种或至少两种的组合。
本申请的筛选标记基因可以但不限于荧光蛋白基因：EGFP、YFP、mCherry、DsRed、BFP，分子标签，即蛋白/多肽标签包括但不限于：His-tag、Flag-tag、HA-tag、Myc-tag、Strep-tag，通过流式细胞仪检测荧光或抗标签抗体，即可进行正筛选，也可进行负筛选。
所述筛选标记基因的筛选方法包括药物筛选、流式细胞仪检测并分选和磁珠分选中的任意一种或至少两种的组合。
优选地，所述药物抗性基因的筛选药物包括嘌呤霉素、G418、杀稻瘟素和潮霉素B中的任意一种。
优选地，所述自杀基因的筛选药物包括更昔洛韦或FIAU、5-氟胞嘧啶、AP1903和AP20187中的任意一种或至少两种的组合。
优选地，所述荧光蛋白基因的检测和分选方法包括流式细胞仪检测和分选。
优选地，所述分子标签的检测和分选方法包括流式细胞仪检测和分选。
优选地，所述标记基因的分选包括磁珠分选。
优选地，所述胞外抗体文库编码域包括(完整抗体、组成抗体的链或抗体片段)抗体序列、抗体重链序列、抗体轻链序列、抗体可变区序列、单链抗体序列、单域抗体序列和Fab片段序列中的任意一种或至少两种的组合。
本申请中胞外抗体文库编码域部分包括但不限于完整的抗体、组成抗体的链(重链或轻链)、抗体的片段(抗体可变区、单链抗体、单域抗体、Fab段)所构成的文库。胞外抗体文库编码域的来源，可以但不限于为免疫动物制备、来自疾病人群、来自健康人群、接种过疫苗的人群、人工合成。
优选地，所述胞外抗体文库编码域的来源包括免疫动物、疾病人群、健康人群、接种过疫苗的人群和人工合成中的任意一种或至少两种的组合。
优选地，所述Notch核心结构域包括人的Notch、小鼠的Notch、或与人的Notch或小鼠的Notch相似性不低于85％的序列。
优选地，所述Notch核心结构域包括人的P1391-R1763片段，小鼠的P1390-R1752片段，这些片段的前或后、加或减200个氨基酸以内的片段，或者与这些片段相似性不低于85％的序列。
优选地，所述人的Notch的氨基酸序列如SEQ ID NO.1所示。
优选地，所述小鼠的Notch的氨基酸序列如SEQ ID NO.2所示。
本申请的Notch核心结构域可以来自不限于人的Notch、小鼠的Notch，以及与它们相似性不低于85％的序列；Notch核心结构域可为P1391-R1763片段(人)、P1390-R1752片段(小鼠)，这些片段的前或后、加或减200个氨基酸以内的片段，以及与这些片段相似性不低于85％的序列。
优选地，所述转录结构域包括但不局限于tTA和/或Gal4-VP64。
优选地，所述顺式激活子包括pTet和/或UAS-pSV40。
优选地，所述转录结构域包括tTA和/或Gal4-VP64。
优选地，所述转染的方法包括病毒转染、化学转染试剂转染和电击转染中的任意一种或至少两种的组合。
优选地，所述编码域、结构域或基因包含编码蛋白质的氨基酸序列、编码蛋白的DNA序列和编码蛋白的RNA序列中的任意一种或至少两种的组合。
第二方面，本申请提供一种抗体文库，所述文库由第一方面所述方法构建得到。
第三方面，本申请提供一种筛选抗体的方法，采用第二方面所述抗体文库进行筛选，包 括如下步骤：
(1)将抗体文库与抗原接触；以及
(2)根据筛选标记基因的表达情况，筛选表达目标抗体的细胞；
其中，所述抗体包括单克隆抗体或多克隆抗体；
所述抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原和包被在培养支架上的抗原中的任意一种或至少两种的组合。
本申请中，筛选抗体的方法是将抗体表达细胞文库与抗原接触，只有能识别抗原的抗体表达细胞，才会激活细胞中筛选标记基因的表达，根据筛选标记基因的表达情况，筛选出表达目标抗体的细胞。
第四方面，本申请提供一种抗体，所述抗体由第三方面所述方法筛选得到。
在另一方面，本发明还提供了一种用于筛选抗体的系统，其包括：
第一元件，所述第一元件包括顺式激活子和筛选标记基因；和
第二元件，所述第二元件包括编码胞外抗体文库编码域的基因、编码Notch核心结构域的基因和编码胞内转录结构域的基因。
所述顺式激活子、筛选标记基因、胞外抗体文库编码域、Notch核心结构域和胞内转录结构域如上所述定义。
在另一方面，本发明还提供了一种使用上述系统来筛选抗体的方法，其包括：
将第一元件和第二元件转染至细胞，其中第一元件包括顺式激活子和筛选标记基因，第二元件包括编码胞外抗体文库编码域的基因、编码Notch核心结构域的基因和编码胞内转录结构域的基因；
使所述细胞表达所述胞外抗体文库编码域；
使所述细胞与抗原接触；以及
根据所述筛选标记基因的表达情况，筛选表达目标抗体的细胞，从而筛选目标抗体。
所述顺式激活子、筛选标记基因、胞外抗体文库编码域、Notch核心结构域和胞内转录结构域如上所述定义。
与现有技术相比，本申请具有如下有益效果：
1、本申请提供的方法既可筛选单克隆抗体，又可筛选多克隆抗体：以往的抗体筛选技术适合于筛选单克隆抗体，即使用于筛选多克隆抗体，所筛选出抗体的多样性、特异性及稳定性也难以保证；本申请的抗体筛选技术，针对抗原筛选出的多克隆抗体具有更大多样性，并 且能通过改变筛选条件获得特异性更好、亲和力更高的抗体，通过测序即可获得大量单克隆抗体的编码序列，比以往的单克隆筛选技术更简单；
2，本申请提供的方法不需要表达纯化抗原：以往的抗体筛选技术在进行抗体筛选时，需要表达纯化出抗原蛋白或者抗原表位的肽段，用于免疫动物、铺板进行ELISA筛选抗体、结合抗体进行流式筛选抗体，而这些抗原蛋白或肽段有的不容易纯化、或者和原来抗原蛋白修饰不同、或者不是原来的空间结构，这些都会影响所筛选出抗体的特异性，亲和力；本申请的抗体筛选技术，只需要将目标抗原展示在细胞的表面，即可进行抗体的筛选，要得到展示目标抗原的细胞，只需要将目标抗原的基因转染到细胞中，或者直接用确定表达目标抗原的野生型细胞，十分方便；
3，本申请提供的方法不需要预设抗原：以往的抗体筛选技术需要预先探明抗原，再针对抗原筛选抗体，但是，肿瘤细胞极其复杂，有大量未探明的抗原，而明确的肿瘤抗原靶点十分有限，针对有限抗原靶点筛选出的抗体，无法满足肿瘤治疗的需要；本申请不需要预设抗原，可以以整个肿瘤细胞为抗原，从抗体库中筛选出的多克隆抗体，可以识别肿瘤细胞表面的各种抗原靶点，从而避免了已知抗原靶点有限的问题；
4、本申请提供的方法可方便进行负筛选：抗体负筛选的目的是去除不需要的抗体，比如去除可能识别正常细胞的抗体，因为肿瘤细胞和正常细胞有大量相同的抗原，因此针对肿瘤细胞筛选出的抗体除了可以识别肿瘤细胞外，还可能识别正常的细胞，从而使得开发的抗体药物具有伤害正常组织的副作用，以往的抗体筛选技术筛选单克隆抗体，可以将筛出的抗体一个一个去验证对正常细胞的作用，去除不需要的抗体，这种方法效率低，不适合多克隆抗体的筛选；本申请的抗体筛选技术，可以通过用负筛选标记基因，用负筛选药物或流式细胞仪阴性分选等方式，很方便进行负筛选；
5、本申请提供的方法筛选方式多样：本申请提供的抗体筛选技术，可以用药物进行正负筛选，也可用流式细胞仪来进行筛选，还可以药物筛选和流式细胞仪筛选配合进行，根据自己的研究条件和研究经验自由选择和搭配，更有利于获得目标抗体。
附图说明
图1为本申请的synNotch系统控制细胞内基因表达的原理图；
图2为本申请的基于synNotch的抗体筛选系统；
图3为本申请的顺式激活子加筛选标记基因的载体示意图；
图4为本申请的pTet顺式激活子调控荧光蛋白融合嘌呤霉素抗性基因的载体示意图；
图5为本申请的tTA表达载体示意图；
图6为本申请的荧光显微镜拍摄tTA激活pTet顺式激活子表达绿色荧光蛋白图；
图7为本申请的荧光显微镜拍摄tTA激活pTet顺式激活子表达红色荧光蛋白图；
图8为本申请的抗体文库的通用表达载体示意图；
图9为本申请的抗CD19单链抗体的表达载体示意图；
图10为本申请的抗GPC3单链抗体的表达载体示意图；
图11为本申请的CD19和GFPC3抗原表达载体示意图；
图12为本申请的流式细胞仪检测CD19和GFPC3抗原在K562细胞中的表达图；
图13为本申请的用抗体筛选系统从混合的抗体表达细胞文库中筛选CD19抗体表达细胞结果图；
图14为本申请的用抗体筛选系统从混合的抗体表达细胞文库中筛选GPC3抗体表达细胞结果图；
图15为本申请的pTet顺式激活子调控绿色荧光蛋白的载体示意图；
图16为本申请的抗Raji单链抗体基因文库表达载体示意图；
图17为本申请的用于负筛选的抗原表达载体示意图；
图18为本申请的流式细胞仪检测CD19抗原表达载体和负筛选抗原表达载体在K562细胞中的表达结果图；
图19为本申请的流式细胞仪检测筛选前抗体表达细胞文库诱导激活情况图；
图20为本申请的流式细胞仪检测筛选后抗体表达细胞文库诱导激活情况图；
图21为本申请的pTet顺式激活子调控荧光蛋白融合正负筛选基因的载体示意图；
图22为本申请的可进行药物初筛选的单链抗体基因文库表达载体示意图；
图23为本申请的用抗体筛选系统进行药物正筛选原理图；
图24为本申请的流式细胞仪检测进行正筛选的抗体表达细胞结果图；
图25为本申请的用抗体筛选系统进行药物负筛选原理图；
图26为本申请的流式细胞仪检测进行负筛选的抗体表达细胞结果图；
图27为本申请的单链抗体与抗体恒定区融合表达载体示意图；
图28为本申请的流式细胞仪通过本申请抗体筛选方法筛选出的抗体与抗原结合效率结果图。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1筛选已知的CD19和GPC3抗体
synNotch系统控制细胞内基因表达的原理图见图1，synNotch系统细胞外的单链抗体识别并结合抗原时，synNotch系统发生诱导性跨膜区域剪切，从而释放胞内转录结构域进入细胞核，结合上游顺式激活子来激活受调控目标基因的表达；
基于synNotch的抗体筛选系统的原理图见图2，在synNotch系统的的基础上，将胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，从而得到受抗原激活的抗体筛选系统；
从已知的CD19和GPC3混合抗体库中分别筛选特异性针对CD19和GPC3的抗体；
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系
本实施例中，顺式激活子采用pTet(SEQ ID NO.3)，pTet接收到tTA信号后，即可启动筛选基因的表达，筛选标记用荧光蛋白融合嘌呤霉素抗性基因(SEQ ID NO.4)，二者之间用可自动断裂的2A(SEQ ID NO.5)序列连接，顺式激活子加筛选标记基因的载体示意图见图3；
本实施例分别用两种荧光蛋白EGFP(SEQ ID NO.6)和mCherry(SEQ ID NO.7)与嘌呤霉素抗性基因融合构建两种筛选标记，终止子采用SV40多聚A(SEQ ID NO.8)，通过人工合成直接合成整个序列(图4)的编码基因，构建到慢病毒载体中，包装慢病毒，分别转染到293T细胞中，3天后将转染的细胞单克隆化；
要对单克隆的细胞系进行筛选，获得正确的单克隆细胞，需要构建tTA表达载体(图5)，tTA表达载体由ef1-α启动子(SEQ ID NO.9)，tTA基因(SEQ ID NO.10)，终止子构成，将tTA表达载体构建到慢病毒载体中，包装成慢病毒；
将待筛选的单克隆细胞分别取出一部分细胞转染tTA表达慢病毒，2-3天后，荧光显微镜观察，有EGFP(图6，筛选稳定转染pTet顺式激活子调控绿色荧光蛋白融合嘌呤霉素抗性基因的单克隆细胞时，显微镜观察绿色荧光表达情况。)或mCherry(图7，筛选稳定转染pTet顺式激活子调控红色荧光蛋白融合嘌呤霉素抗性基因的单克隆细胞时，显微镜观察红色荧光表达情况。)表达而转染之前无荧光表达的细胞系，均为稳定转染pTet顺式激活子和筛选标记基因的单克隆细胞系；
(2)构建抗体表达细胞文库：首先构建抗体基因文库表达载体中除胞外抗体文库编码域基因外的中间载体，方便后续插入胞外抗体文库编码域基因(图8，抗体文库的通用表达载体示意图)，人工合成中间载体各组成部分的序列，从N端到C端依次串联的部分分别为mPGK1启动子(SEQ ID NO.11)，CD8α信号肽(SEQ ID NO.12)编码序列，抗体基因文库 编码域(带有β-半乳糖苷酶(lacZ)N端α片段的编码区序列，前面Asc I限制性内切酶位点，后面为Not I限制性内切酶位点，方便抗体库序列的插入，并能通过蓝白斑显示片段插入情况)，NOTCH1片段(SEQ ID NO.2中含跨膜区的P1390-R1752片段)编码序列，tTA编码序列，终止子，将合成的序列构建到慢病毒载体中，以备进一步插入抗体基因序列；
合成已知的抗人CD19单链抗体(SEQ ID NO.13)和抗人GPC3单链抗体(SEQ ID NO.14)的编码序列，两端带有Asc I和Not I限制性内切酶位点，分别插入到中间载体的Asc I和Not I限制性内切酶位点之间；将插入有抗人CD19单链抗体基因的载体(图9)包装成慢病毒，转染到带EGFP筛选标记的单克隆细胞系中，得到CD19单链抗体表达细胞；
将插入有抗人GPC3单链抗体基因的载体(图10)包装成慢病毒，转染到带mCherry筛选标记的单克隆细胞系中，得到GPC3单链抗体表达细胞。
(3)构建CD19和GFPC3抗原表达细胞：合成CD19(SEQ ID NO.15)和GPC3(SEQ ID NO.16)抗原的编码序列，分别构建到表达载体上，表达载体中通过两个2A序列将CD19或GPC3、蓝色荧光蛋白(SEQ ID NO.17)、嘌呤霉素抗性基因三者融合(图11)；
将构建好的表达载体包装成慢病毒，通过病毒转染到K562细胞中，转染2天后，培养基中加入1μg/mL的嘌呤霉素筛选得到稳定表达CD19或GPC3的K562细胞。流式细胞仪检测蓝色荧光蛋白的表达几乎达到100％(图12)，表明抗原在K562细胞中已稳定表达。
(4)从混合的抗体表达细胞文库中筛选CD19抗体表达细胞：将CD19单链抗体表达细胞和GPC3单链抗体表达细胞1:1混合，再将混合细胞与CD19抗原表达细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过8-10天的筛选，抗体表达细胞已几乎全为表达CD19抗体带有绿色荧光的细胞(图13)，将CD19单链抗体和绿色荧光共同表达的细胞与GPC3单链抗体和红色荧光共同表达的细胞1:1混合，经过与CD19抗原表达细胞混合并加嘌呤霉素进行筛选，得到几乎全为表达CD19抗体带有绿色荧光的细胞；
(5)从混合的细胞中筛选表达抗GPC3抗体的细胞：将CD19抗体表达细胞和GPC3抗体表达细胞1:1混合，再将混合细胞与GPC3抗原表达细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过8-10天的筛选，抗体表达细胞已几乎全为表达GPC3抗体带有红色荧光的细胞(图14)，将CD19单链抗体和绿色荧光共同表达的细胞与GPC3单链抗体和红色荧光共同表达的细胞1:1混合，经过与GPC3抗原表达细胞混合并加嘌呤霉素进行筛选，得到几乎全为表达GPC3抗体带有红色荧光的细胞；
上述实验结果显示，可以通过本抗体筛选方法从已知的抗体库中，筛选出针对目标抗原 的单链抗体。
实施例2.制备抗体文库并用流式细胞仪筛选CD19抗体表达细胞
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系
本实施例采用顺式激活子pTet，pTet接收到tTA信号后，启动筛选标记绿色荧光蛋白基因的表达，通过人工合成整个序列(图15)，构建到慢病毒载体中。包装慢病毒，转染到293T细胞中，3天后将转染的细胞单克隆化，将待筛选的单克隆细胞分别取出一部分细胞转染表达tTA的慢病毒，2-3天后，荧光显微镜观察，挑选有绿色荧光表达而转染之前无荧光表达的细胞系，为稳定转染转染顺式激活子和筛选标记基因的单克隆细胞系。
(2)构建抗体表达细胞文库
用CD19阳性的Raji细胞免疫C57BL/6J小鼠，两周时加强免疫一次，第四周再免疫一次，3天后处死小鼠，分离出小鼠的脾脏淋巴细胞；用RNA提取纯化试剂盒提取脾脏淋巴细胞的RNA；
将提取的RNA用逆转录试剂盒分别通过轻链的反转录引物(SEQ ID NO.18)和重链的反转录引物(SEQ ID NO.19)进行反转录，用简并引物分别扩增重链和轻链，再通过重叠PCR的方法将轻链和重链连接起来，形成轻链-Linker-重链的scFv文库，将scFv文库的DNA片段通过Asc I和Not I双酶切，插入到抗体基因文库表达载体中间载体的Asc I和Not I限制性内切酶位点之间，得到抗体基因文库表达载体(图16)，Raji细胞免疫小鼠制备的单链抗体克隆到抗体基因文库表达载体上；
将抗体基因文库表达载体包装成慢病毒，转染到步骤(1)已稳定转染顺式激活子和筛选标记基因的单克隆细胞系中，得到抗体表达细胞文库。
(3)构建抗原表达细胞；
表达CD19的抗原表达细胞制备见实施例1，此抗原表达细胞用于抗CD19抗体的正筛选，合成一个穿膜结构域(SEQ ID NO.20)的编码基因，替换正筛选载体中的CD19抗原基因，得到用于负筛选的抗原表达载体(图17)；
将构建好的抗原表达载体分别包装成慢病毒，通过病毒转染到K562细胞中；转染2天后，培养基中加入1μg/mL的嘌呤霉素筛选分别得到正筛选抗原表达细胞系和负筛选抗原表达细胞系。流式细胞仪检测蓝色荧光蛋白的表达几乎达到100％(图18)，表明抗原在K562细胞中已稳定表达。
(4)流式细胞仪筛选表达CD19抗体的细胞；
将抗体表达细胞文库和表达CD19抗原的正筛选细胞1:1混合，混合细胞培养2天后， 抗体表达细胞文库有少量表达绿色荧光蛋白的细胞出现(图19)，将抗体表达细胞文库和表达CD19抗原的正筛选细胞混合培养2天后，抗体表达细胞文库有少量表达绿色荧光蛋白的细胞出现，用分选型的流式细胞仪，分选出表达绿色荧光蛋白的细胞，继续培养，直到大部分绿色荧光消失；
再将分选出的抗体表达细胞文库和负筛选抗原表达细胞1:1混合，3天后，用分选型流式细胞仪分选出绿色荧光蛋白和蓝色荧光蛋白双阴性的细胞，即为表达CD19抗体的细胞，此时的抗体表达细胞与负筛选抗原表达细胞共培养时无绿色荧光蛋白表达，与CD19抗原表达细胞共培养时有绿色荧光蛋白表达(图20)，经过正负筛选后的抗体表达细胞与负筛选抗原表达细胞共培养时无绿色荧光蛋白表达，与CD19抗原表达细胞共培养时有绿色荧光蛋白表达。
实施例3.制备抗体文库并用药物筛选CD19抗体表达细胞
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系；
本实施例中，顺式激活子pTet，筛选标记用iCasp9负筛选系统(SEQ ID NO.21)、绿色荧光蛋白、嘌呤霉素抗性基因，三者之间用可自动断裂的2A序列连接；
通过人工合成整个编码序列(图21)，构建到慢病毒载体中，包装慢病毒，转染到293T细胞中，3天后将转染的细胞单克隆化；将待筛选的单克隆细胞分别取出一部分细胞转染实施例1中表达tTA的慢病毒，2-3天后，荧光显微镜观察，挑选有绿色荧光表达而转染之前无荧光表达的细胞系，为稳定转染有顺式激活子和筛选标记基因的单克隆细胞系。
(2)构建抗体表达细胞文库；
首先构建抗体基因文库表达载体中除胞外抗体文库编码域基因外的中间载体，方便后续插入胞外抗体文库基因(图22)，人工合成中间载体各组成部分的序列，从N端到C端依次串联的部分分别为mPGK1启动子，CD8α信号肽编码序列，抗体基因文库编码域(带有β-半乳糖苷酶(lacZ)N端α片段的编码区序列，前面Asc I限制性内切酶位点，后面为Not I限制性内切酶位点，方便抗体库序列的插入，并能通过蓝白斑显示片段插入情况)，NOTCH1片段编码序列，tTA编码序列，终止子，EFS启动子(SEQ ID NO.22)，杀稻瘟素抗性(SEQ ID NO.23)基因，终止子；
将合成的序列构建到慢病毒载体中，以备进一步插入抗体基因文库序列。将Raji细胞免疫小鼠的scFv文库DNA片段通过Asc I和Not I双酶切，插入到抗体基因文库表达载体中间载体的Asc I和Not I限制性内切酶位点之间，得到抗体基因文库表达载体,将抗体基因文库表达载体包装成慢病毒，转染到步骤(1)已稳定转染顺式激活子和筛选标记基因的单克隆细胞 系中，2天后在培养基中加入10μg/mL的杀稻瘟菌素进行初筛，得到抗体表达细胞文库；
(3)药物筛选表达CD19抗体的细胞；
将抗体表达细胞文库和表达CD19抗原的正筛选细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过6-8天的正筛选，(正筛选原理图见图23)，抗体文库表达细胞已几乎全为表达绿色荧光蛋白的细胞(图24)，将抗体文库表达细胞和表达CD19抗原的正筛选细胞混合细胞经过嘌呤霉素进行正筛选，抗体表达细胞已几乎全为表达绿色荧光蛋白的细胞；
再在混合细胞的培养基中加入10μg/mL的杀稻瘟菌素，直到蓝色荧光的正筛选抗原细胞完全消失，继续培养经过正筛选后的抗体文库表达细胞直到绿色荧光蛋白几乎不表达时，将抗体文库表达细胞和负筛选抗原表达细胞1:1混合，并在培养基中加入10nM的AP1903进行负筛选(负筛选原理图见图25)，直到培养的抗体表达细胞中不再表达绿色荧光蛋白(图26)，将抗体文库表达细胞和负筛选细胞混合细胞经过AP1903负筛选，抗体表达细胞已几乎全都为不表达绿色荧光蛋白的细胞；
最后在混合细胞的培养基中加入10μg/mL的杀稻瘟菌素，直到蓝色荧光的负筛选抗原细胞完全消失，此时，抗体文库表达细胞即为表达CD19抗体的细胞。
实施例4.抗体结合性质的鉴定
(1)克隆筛选出的CD19抗体表达细胞的抗体编码序列；
将经过筛选的CD19抗体表达细胞用DNeasy Blood&Tissue Kit基组提取试剂盒(Qiagen)按照试剂盒的操作指南提出细胞的基因组，以提取的基因组为模板，用引物对(SEQ ID NO.24，SEQ ID NO.25)扩增scFv的编码基因，用Asc I和Not I双酶切后，插入到经Asc I和Not I双酶切的表达载体上，与人的IgG1恒定区(SEQ ID NO.26)融合(图27)；
将连接好的载体转染到感受态细胞中，涂在含100mg/L氨苄青霉素的LB固体培养基的培养皿上，第二天，从培养皿上挑出20个左右的单克隆分别接种于含100mg/L氨苄青霉素的LB液体培养基中摇瓶培养，培养皿上的剩余菌落用LB培养基洗脱下来，用质粒提取试剂盒按照试剂盒说明书提取多克隆的scFv表达质粒，摇瓶培养的单克隆菌过夜培养后也分别提取单克隆的scFv表达质粒。
(2)表达纯化抗体；
分别将多克隆的scFv表达质粒和单克隆的scFv表达质粒用PEI转染到293T细胞中，转染72小时后，收集培养上清；将培养上清与结合缓冲液1:1混合、过滤后待用，先用5-10倍体积的结合缓冲液平衡Pteoein A柱，再将准备好的培养上清样品上样，然后用结合缓冲液 冲洗柱子，直到结合液中不含蛋白，最后用洗脱液过柱，收集洗出液，直至洗出液中不含蛋白，透析并浓缩所收集的抗体。
(3)流式细胞仪检测抗体的结合；
100μL表达CD19和不表达CD19的K562细胞悬液，铺到V型细胞板，500g，离心3min，沉积细胞，弃去上清加入100μL纯化的抗体溶液(定量IgG浓度为0.5μg/mL)，4℃孵育30min，500g，离心3min，用200μL PBS洗三遍；
按照实验设计孔，每孔加入80μl FITC标记的羊抗人二抗(1:150稀释)，4℃孵育30min，500g，离心3min，弃去上清，200μL PBS洗涤3次，最后一次洗完加入200μL PBS悬浮细胞，悬浮细胞用过滤网过滤后上流式细胞仪检测，结果显示，无论多克隆的scFv或单克隆的scFv都能不同程度特异性结合表达CD19抗原的K562细胞(图28)，筛选得到多克隆的scFv或单克隆的scFv都能不同程度特异性结合表达CD19抗原的K562细胞而不结合无CD19抗原表达的K562细胞。
综上所述，本申请提供了一种抗体文库的构建方法及其应用，基于synNotch系统控制细胞内基因表达的原理设计，通过大量实验摸索，优化整体方案流程，反复设计验证，将synNotch系统的胞外识别结构域改变为抗体文库，受调控的目标基因改变为筛选标记基因，从而得到了筛选针对复杂抗原的多克隆抗体技术，解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (17)

  1. 一种抗体文库的构建方法，其包括将第一元件和第二元件插入同一载体或不同载体，将所述载体转染至细胞内，得到抗体表达细胞文库，即所述抗体文库；
    其中，所述第一元件包括顺式激活子和筛选标记基因，所述第二元件包括胞外抗体文库编码域、Notch核心结构域和胞内转录结构域。
  2. 根据权利要求1所述的方法，其中，所述筛选标记基因包括药物抗性基因、自杀基因、荧光蛋白基因和分子标签中的任意一种或至少两种的组合。
  3. 根据权利要求2所述的方法，其中，所述药物抗性基因包括嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因和潮霉素B抗性基因中的任意一种或至少两种的组合；
    所述自杀基因包括单纯疱疹病毒胸苷激酶基因、胞嘧啶脱氨酶基因和iCasp9自杀系统基因中的任意一种或至少两种的组合；
    所述荧光蛋白基因包括EGFP、YFP、mCherry、DsRed和BFP中的任意一种或至少两种的组合；
    所述分子标签包括His-tag、Flag-tag、HA-tag、Myc-tag和Strep-tag中的任意一种或至少两种的组合。
  4. 根据权利要求1-3中任一项所述的方法，其中，所述筛选标记基因的筛选方法包括药物筛选、流式细胞仪检测并分选和磁珠分选中的任意一种或至少两种的组合。
  5. 根据权利要求3所述的方法，其中，所述药物抗性基因的筛选药物包括嘌呤霉素、G418、杀稻瘟素和潮霉素B中的任意一种或至少两种的组合；
    所述自杀基因的筛选药物包括更昔洛韦或FIAU、5-氟胞嘧啶、AP1903和AP20187中的任意一种或至少两种的组合。
  6. 根据权利要求1-5中任一项所述的方法，其中，所述胞外抗体文库编码域包括抗体序列、抗体重链序列、抗体轻链序列、抗体可变区序列、单链抗体序列、单域抗体序列和Fab片段序列中的任意一种或至少两种的组合。
  7. 根据权利要求1-6中任一项所述的方法，其中，所述胞外抗体文库编码域的来源包括免疫动物、疾病人群、健康人群、接种过疫苗的人群和人工合成中的任意一种或至少两种的组合。
  8. 根据权利要求1-7中任一项所述的方法，其中，所述Notch核心结构域包括人的Notch、小鼠的Notch或与人的Notch或小鼠的Notch相似性不低于85％的序列；
    其中，所述人的Notch的氨基酸序列如SEQ ID NO.1所示；
    所述小鼠的Notch的氨基酸序列如SEQ ID NO.2所示。
  9. 根据权利要求1-8中任一项所述的方法，其中，所述顺式激活子包括pTet和/或UAS-pSV40。
  10. 根据权利要求1-9中任一项所述的方法，其中，所述胞内转录结构域包括tTA和/或Gal4-VP64。
  11. 根据权利要求1-10中任一项所述的方法，其中，所述转染的方法包括病毒转染、化学转染试剂转染和电击转染中的任意一种或至少两种的组合。
  12. 根据权利要求1-11中任一项所述的方法，其中，所述编码域、结构域或基因包含编码蛋白质的氨基酸序列、编码蛋白的DNA序列或编码蛋白的RNA序列中的任意一种或至少两种的组合。
  13. 由权利要求1-12中任一项所述方法构建得到的抗体文库。
  14. 一种通过采用权利要求13所述的抗体文库来筛选抗体的方法，包括如下步骤：
    (1)将所述抗体文库与抗原接触；以及
    (2)根据筛选标记基因的表达情况，筛选表达目标抗体的细胞；
    其中，所述抗体包括单克隆抗体或多克隆抗体；
    所述抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原和包被在培养支架上的抗原中的任意一种或至少两种的组合。
  15. 由权利要求14所述方法筛选得到的抗体。
  16. 一种用于筛选抗体的系统，其包括：
    第一元件，所述第一元件包括顺式激活子和筛选标记基因；和
    第二元件，所述第二元件包括编码胞外抗体文库编码域的基因、编码Notch核心结构域的基因和编码胞内转录结构域的基因。
  17. 一种使用权利要求16所述的系统来筛选抗体的方法，其包括：
    将第一元件和第二元件转染至细胞，其中所述第一元件包括顺式激活子和筛选标记基因，并且所述第二元件包括编码胞外抗体文库编码域的基因、编码Notch核心结构域的基因和编码胞内转录结构域的基因；
    使所述细胞表达所述胞外抗体文库编码域；
    使所述细胞与抗原接触；以及
    根据所述筛选标记基因的表达情况，筛选表达目标抗体的细胞，从而筛选目标抗体。
PCT/CN2019/108128 2018-12-21 2019-09-26 一种抗体文库的构建方法及其应用 WO2020125120A1 (zh)

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Families Citing this family (8)

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Publication number Priority date Publication date Assignee Title
AU2019365135A1 (en) * 2018-10-22 2021-05-13 Fusion Antibodies Plc Antibody library and method
CN109576292B (zh) * 2018-12-21 2022-11-29 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用
CN111304244A (zh) * 2019-11-13 2020-06-19 沣潮医药科技（上海）有限公司 携带基因元件组合的载体组件、受体细胞文库、制备和筛选方法及用途
CN110964750A (zh) * 2019-11-13 2020-04-07 沣潮医药科技（上海）有限公司 携带基因元件组合的嵌合抗原受体细胞文库、制备和筛选方法及用途
CN114437232B (zh) * 2020-11-05 2023-11-24 中国科学院分子细胞科学卓越创新中心 一种细胞表面大分子定量展示系统及其制备方法和用途
CN115197968A (zh) * 2021-04-09 2022-10-18 新乡医学院 抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用
WO2023025208A1 (zh) * 2021-08-24 2023-03-02 赛斯尔擎生物技术（上海）有限公司 一种修饰细胞的方法
CN114621929B (zh) * 2021-12-21 2023-05-30 河南省肿瘤医院 一种抗肿瘤树突状细胞及其制备方法、表达载体及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108064283A (zh) * 2015-02-24 2018-05-22 加利福尼亚大学董事会 结合触发的转录开关及其使用方法
CN108369230A (zh) * 2015-09-25 2018-08-03 阿布维特罗有限责任公司 用于对天然配对t细胞受体序列进行t细胞受体靶向鉴别的高通量方法
CN109576292A (zh) * 2018-12-21 2019-04-05 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3789487A1 (en) * 2013-04-03 2021-03-10 Memorial Sloan Kettering Cancer Center Effective generation of tumor-targeted t-cells derived from pluripotent stem cells
JP6878408B2 (ja) * 2015-09-01 2021-05-26 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニアＴｈｅ Ｒｅｇｅｎｔｓ Ｏｆ Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃａｌｉｆｏｒｎｉａ モジュールポリペプチドライブラリならびにその作製方法及びその使用
WO2018205936A1 (zh) * 2017-05-08 2018-11-15 上海津曼特生物科技有限公司 双特异性重组蛋白及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108064283A (zh) * 2015-02-24 2018-05-22 加利福尼亚大学董事会 结合触发的转录开关及其使用方法
CN108369230A (zh) * 2015-09-25 2018-08-03 阿布维特罗有限责任公司 用于对天然配对t细胞受体序列进行t细胞受体靶向鉴别的高通量方法
CN109576292A (zh) * 2018-12-21 2019-04-05 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3789521A4 *

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