WO2022270396A1 - Double-screening method - Google Patents

Double-screening method Download PDF

Info

Publication number
WO2022270396A1
WO2022270396A1 PCT/JP2022/024080 JP2022024080W WO2022270396A1 WO 2022270396 A1 WO2022270396 A1 WO 2022270396A1 JP 2022024080 W JP2022024080 W JP 2022024080W WO 2022270396 A1 WO2022270396 A1 WO 2022270396A1
Authority
WO
WIPO (PCT)
Prior art keywords
library
target molecule
linker
mrna
selection
Prior art date
Application number
PCT/JP2022/024080
Other languages
French (fr)
Japanese (ja)
Inventor
涼 米原
重文 熊地
泰平 村上
麻衣子 本橋
直人 根本
Original Assignee
株式会社Epsilon Molecular Engineering
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社Epsilon Molecular Engineering filed Critical 株式会社Epsilon Molecular Engineering
Publication of WO2022270396A1 publication Critical patent/WO2022270396A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • 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
    • C40B40/04—Libraries containing only organic compounds
    • 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

Definitions

  • the present invention relates to a screening method that combines a cell-free translation system and a prokaryotic expression system. More specifically, it relates to a method of preparing a temporary library from a large-scale library of 10 13 to 10 14 and screening a target molecule that can be expressed in prokaryotic cells from the library.
  • genotype-phenotype mapping techniques are useful for enabling screening of functional peptides from various peptide libraries. It has been known. Techniques for associating genotypes with phenotypes can be classified into so-called ribozyme types, virus types, cell types, external intelligence types, and the like.
  • virus type is a term indicating a form in which a genotype (DNA or RNA) and a phenotype (protein) are simply combined. ” does not indicate Specifically, a representative example is phage display in which a protein encoded by the genome (for example, coat protein) is simply bound to the genome.
  • phage display uses cells, so cells Produces protein.
  • mRNA display and cDNA display are cell-free translation systems, cells do not produce proteins.
  • the phage display method is most commonly used because it is easy to handle when considering production on an industrial scale (see Non-Patent Document 1, hereinafter referred to as "conventional example 1").
  • Non-Patent Document 6 hereinafter referred to as "Conventional Example 2”
  • the cDNA display method can handle large-scale libraries of 10 12 or more.
  • Example 1 is an excellent method in that it can ensure accurate correspondence between the genotype and the expressed peptide, and that a simple and rapid technique has already been established. For example, the entire process of selection and evolution using phage display is usually completed in about 2-3 weeks (see Non-Patent Documents 1 and 2). However, there is a problem that it can only handle libraries with a maximum library size of about 10 8 .
  • Example 3 can handle a library having a larger size of 10 12 or more as described above (hereinafter sometimes referred to as a "large-scale library"), so candidates with more molecular diversity This is an excellent method in that it allows you to select In addition, since it is a cell-free translation system, it is possible to incorporate non-natural residues into proteins/peptides, and it is an excellent reaction in that post-translational modifications such as disulfide shuffling reactions can be performed ( Non-Patent Documents 3-5).
  • Prior art 2 is an excellent invention in that a delicate nanobody can be produced using the ribosome display method and the phage display method.
  • peptides obtained by the ribosome display method are reverse transcribed in a separate reaction system using reverse transcriptase to obtain cDNA.
  • the cDNA obtained here is amplified by PCR, and then competent cells are transformed to produce a plasmid containing the amplified cDNA, and the desired peptide is obtained by phage display. , and there is the problem that it takes time and effort to finally obtain the target peptide.
  • a screening method comprising a step of determining whether or not candidate clones obtained by screening from the large-scale library can be expressed in cells before transferring to a cell-based expression system. there were.
  • DNAs of target molecules are selected using a cell-free translation system to construct a library, and DNAs of the target molecules are selected from DNA fragments contained in the library and concentrated.
  • a protein that can be produced in an organism among the proteins made from the target molecules contained in the primary library obtained in the selection/selection step; and an expression/selection step of expressing and selecting a DNA fragment encoding using a cell.
  • a cDNA display linker having a main chain and a side chain can be used.
  • the main chain of the linker comprises (m1) a solid phase binding site for fixing the linker to a solid phase; (m2) a cleavage site for separating the linker from the solid phase; a photocrosslinking base for binding the mRNA to the strand; (m4) a side chain binding site; (m5) a reverse transcription initiation site; wherein the side chain comprises (s1) a peptide binding site for binding a peptide synthesized corresponding to the mRNA bound to the main chain; and (s2) a label. (s3) a detectable fluorescent molecule that binds to said label binding site; and (s4) a backbone binding site that binds to said side chain binding site of said backbone.
  • the solid phase binding site is composed of a molecule selected from the group consisting of biotin, streptavidin, or analogues thereof; the cleavage site is composed of ribo-G or inosine;
  • the base is composed of cyanovinylcarbazole or an analogue thereof;
  • the peptide binding site is composed of puromycin or an analogue thereof;
  • the fluorescent molecules for detection are fluorescein, FITC (fluorescein isothiocyanate), TET (tetrachloro fluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568, Alexa647 and the like.
  • Said primary library can be constructed using said linkers from a cDNA library with a library size of 10 10 -10 14 .
  • the selection/selection step includes (a1) an mRNA preparation step of preparing mRNA of a target molecule; (a3) binding the linker-mRNA conjugate to a solid phase, followed by translation in a cell-free system; , a translation step of forming a linker-mRNA-peptide conjugate; (a4) a reverse transcription step of amplifying the linker-mRNA-peptide conjugate by PCR and reverse transcription to obtain an mRNA/cDNA-protein conjugate; a5) a primary library preparation step of cleaving the amplified cDNA display molecules from the solid phase at a solid phase cleavage site to obtain a primary library; and (a6) selecting DNAs of target molecules contained in the primary library.
  • a concentration step of concentrating can be magnetic particles selected from the group consisting of sepharose beads, silica beads and latex beads.
  • the selection/selection step includes (b1) a phagemid vector forming step of mixing DNA in the primary library with a phagemid to form a phagemid vector; (b2) incorporating a gene fragment of a target molecule into the phagemid vector; (b3) culturing the cells obtained in the transforming step, adding a helper phage to infect the cells, and culturing the phage to display the target molecule. and (b4) a biopanning step of biopanning phages contained in the phage library to select phages that bind to the target molecule.
  • the cells can be competent cells.
  • the competent cell can be Escherichia coli.
  • the number of times the steps (a1) to (a6) are repeated may be 5 times or less.
  • a primary library by screening cDNAs of target molecules from a large-scale library, to obtain a secondary library selected and enriched from the primary library at high speed and with high efficiency, and A screening method is provided that enables the selection of cDNA molecules of interest while removing molecules that are harmful to organisms from the next library.
  • phage ELISA can be used to easily evaluate the affinity of candidates that bind to target molecules. Therefore, purification in a short period of time becomes possible, and it becomes possible to make full use of the advantages of phage display, which are currently not fully utilized.
  • FIG. 1 shows a scheme of hit compound screening by the cDNA display method.
  • FIG. 2 shows the elution method for each screening round.
  • FIG. 3 shows a scheme of phage display.
  • FIG. 4 shows the CDR regions of VHH clone #1 and VHH clone #2.
  • FIG. 5 is an electropherogram showing the results of SDS-PAGE performed after purification of VHH.
  • FIG. 6 is a graph showing the results of affinity measurement for the target molecule FGFR1 of VHH clone #1.
  • FIG. 7 is a graph showing the results of affinity measurement for the target molecule FGFR1 of VHH clone #2.
  • the method of screening for a target molecule of the present invention comprises (a) a selection/selection step of selecting, concentrating and selecting DNA of the target molecule, and (b) an expression/selection step of expressing and selecting using a cell.
  • the selection/selection step (a) includes (a1) constructing a primary library, and then (a2) selecting the DNA of the target molecule from the primary library.
  • linker refers to a linker-mRNA conjugate, a linker-mRNA-protein conjugate, or a linker-mRNA/cDNA-protein conjugate (hereinafter referred to as "IVV") used in the cDNA display method described later. ), and means a linker for cDNA display having a main chain and a side chain.
  • the linker as a whole is preferably designed to be flexible and hydrophilic.
  • the main chain of the linker has, from the 3′ direction to the 5′ direction, (m1) a solid phase binding site, (m2) a solid phase cleavage site, (m3) a photocrosslinking base, and (m4) It contains (m4) a side chain linking site and (m5) a reverse transcription initiation site.
  • the solid phase binding site (m1) is a site that binds the cDNA display linker via a molecule bound to a solid phase.
  • molecules that form a bond with a solid phase include biotin or analogues thereof when avidin and streptavidin are bound to the solid phase, and maltose and G when maltose-binding protein is bound to the solid phase.
  • biotin or analogues thereof when avidin and streptavidin are bound to the solid phase and maltose and G when maltose-binding protein is bound to the solid phase.
  • the solid phase binding site (m1) is a site for binding the above-mentioned mRNA-protein conjugate or mRNA/cDNA-protein conjugate to a solid phase via a linker.
  • the solid phase-binding site (m1) is preferably composed of, for example, any of the molecules described above because it can maintain an appropriate distance from the solid phase and can be successfully separated from the solid phase, which will be described later.
  • the solid-phase cleavage site (m2) is preferably composed of modified amino acids that are site-specifically cleaved, and examples of such modified amino acids include ribo-G and inosine. This is because ribonuclease G and inosine are site-specifically cleaved by ribonuclease V and endonuclease V, respectively. A single solid-phase cleavage site for the linker used in this step is sufficient.
  • the main chain of the linker has a side chain binding site (m3) near its 3' end, and the side chain described later binds to the side chain binding site. Further, for example, when the side chain of the linker is composed of Amino-Modifier C6 dT, the 5' end of the side chain is 5'-Thiol-Modifier C6 and crosslinked using EMCS to and side chains.
  • the main chain of the linker is mainly composed of DNA and has a nucleotide sequence complementary to mRNA capable of binding to the target molecule. It is preferable that this sequence contains cyanovinylcarbazole, which forms a crosslink with a pyrimidine base, so that the above linker and mRNA can be crosslinked by irradiation with light.
  • the cyanovinylcarbazole is preferably 3-cyanovinylcarbazole (hereinafter sometimes abbreviated as "cnvK”), because photocrosslinking with mRNA can be formed in an extremely short time.
  • the cnvK is located between the solid cleavage site and the side chain binding site of the main chain.
  • the reverse transcription site is a region that is located on the 3' end side of the linker and functions as a primer for reverse transcription when reverse transcription is performed on the linker. If the reverse transcription site exceeds 15 bases, the binding efficiency as a linker becomes poor, so it is preferably composed of about 1 to 15 bases. From the viewpoint of binding efficiency with the linker and reaction efficiency as a primer, it preferably consists of 3 to 5 bases.
  • the side chain of the linker has a peptide binding site (s1), a label binding site (s2), a fluorescent molecule for detection (s3), and a main chain binding site (s4).
  • the side chain peptide-binding site (s1) is a site that binds a peptide synthesized corresponding to the mRNA bound to the main chain, and may be composed of puromycin or an analogue thereof.
  • the label-binding site may be composed of one or more spacer sequences, and is preferably bound with a fluorescent molecule because it facilitates detection of the formed cDNA display molecule.
  • the fluorescent molecule for detection is not particularly limited, it is preferably fluorescein or fluorescein isothiocyanate isomer (hereinafter sometimes abbreviated as "FITC”) or other fluorescein derivative from the viewpoint of ease of handling.
  • the side chain is linked to the side chain binding site of the main chain at the main chain binding site (s4), and is a linker for cDNA display method comprising the main chain and side chains having the structure as described above (hereinafter simply " Sometimes referred to as "linker”.) is formed.
  • a desired initial library (DNA library) with a library size of 10 10 to 10 14 is used. This is because a library of this size is too large to be handled by other display methods and can be handled only by the cDNA display method.
  • the above initial library is transcribed, and mRNA encoding each DNA contained in this library is prepared. Since the initial library contains a huge amount of DNA other than the target molecule, it goes without saying that correspondingly, the mRNA prepared here also contains a huge amount of mRNA other than the target molecule. .
  • this mRNA and the linker are mixed to form a mixture, and the mixture is irradiated with light of a desired wavelength for a desired time, for example, light of a wavelength of 360 to 400 nm for 30 seconds to 3 minutes, and the mRNA and The above linker is coupled by photocrosslinking to obtain an mRNA-linker conjugate.
  • the mRNA-linker conjugate is then subjected to a cDNA display device to generate IVV, a cDNA display molecule.
  • the mRNA of the mRNA-linker conjugate is translated in a cell-free translation system, and the peptide corresponding to the mRNA is bound to the peptide binding site of the linker to form the mRNA-peptide-linker conjugate. Thereafter, the peptide bound to the mRNA-peptide-linker conjugate is reverse transcribed to form an mRNA/cDNA-peptide-linker conjugate in which the cDNA is ligated to the reverse transcription site (see FIG. 1).
  • Peptides in the IVV are then mixed with detection molecules bound to a solid phase, and IVV containing peptides not bound to the detection molecules are removed by washing. Molecules bound to the solid phase are then eluted.
  • DNAs encoding molecules other than the target molecule are eliminated and the amount thereof decreases, so that the amount of DNAs encoding the target molecule increases relatively. This phenomenon is called "concentration" of the target molecule.
  • a series of processes from preparation of mRNA to elution from the solid phase is referred to as "round”, such as 1st round and 2nd round .
  • the amount of IVV containing peptides that do not bind to the detection molecule is very large, and these also cause non-specific adsorption.
  • mRNA is prepared from the eluate containing the DNA molecule encoding the target molecule, and IVV is produced and concentrated in the same manner as above.
  • the amount of DNA other than the target molecule can be greatly reduced.
  • the target molecule can be obtained as follows. 1. Screening for Molecules of Interest A primary library is generated by screening a library containing cDNAs for molecules of interest using the cDNA display method.
  • target molecules for example, human fibroblast growth factor receptors 1-4 (Fibroblast Growth Factor Receptors 1-4 may be abbreviated as "FGFR1-4" hereinafter) can be used.
  • a molecule for detecting the target molecule (hereinafter referred to as "detection molecule”) refers to a molecule for detecting the target molecule. It may be a part of the region as long as it is possible.
  • FGFR extracellular domain
  • extracellular domains include FGFR1/Fc (hereinafter sometimes abbreviated as "FGFR-Fc”), FGF receptor 1 ⁇ (IIIc)/Fc chimera (hereinafter sometimes abbreviated as "FGFR-Fc”). etc. can be mentioned.
  • FGFR-Fc FGFR1/Fc
  • FGF receptor 1 ⁇ (IIIc)/Fc chimera FGF receptor 1 ⁇ (IIIc)/Fc chimera
  • FGFR-Fc FGF receptor 1 ⁇ (IIIc)/Fc chimera
  • FGFR1-Fc as the detection molecule is placed in a container, about 15 to about 25 equivalents of the biotinylation reagent is added to the container, and reacted at about 20 to about 30° C. for about 20 to about 40 minutes to obtain the detection molecule. is biotinylated. Thereafter, a desalting column or the like is used to remove unreacted biotinylation reagent.
  • biotinylation reagent for example, EZ-link sulfo NHS-SS biotin can be used, and as a desalting column, for example, Zeba Spin Desalting Columns (both manufactured by Thermo Fisher) can be used.
  • naive libraries obtained from these animals include alpaca-derived naive VHH libraries (manufactured by RePHAGEN), which can be used to construct primary libraries as follows. They can be used to generate a primary library as follows. For example, specific genes of the above naive library, such as S-hinge and L-hinge genes, are used as templates, PCR is performed using the primers represented by SEQ ID NOS: 1 to 3 shown in Table 1 below, and the DNA contained in the above library is obtained. is preferable from the viewpoint of reaction efficiency.
  • overlap PCR is performed using the primers of the sequences represented by SEQ ID NOS: 4 and 5 above to prepare a full-length VHH-encoding DNA library (hereinafter referred to as "full-length library”).
  • Full-length library a full-length VHH-encoding DNA library
  • Overlap PCR conditions are preferably as follows from the viewpoint of reaction efficiency. PCR conditions: After 2 minutes at 98°C, 5 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 35 seconds were performed, followed by reaction at 72°C for 1 minute and cooling to 10°C.
  • the full-length library can contain, for example, a promoter, an enhancer, a sequence involved in translation initiation, a desired VHH gene, a purification tag, a linker hybridization region, etc.
  • a promoter for example, a promoter, an enhancer, a sequence involved in translation initiation, a desired VHH gene, a purification tag, a linker hybridization region, etc.
  • the T7 promoter examples include those containing omega ( ⁇ ) enhancers, Kozak sequences, desired VHH genes, His-tags, Y-tags, and the like.
  • a linker having a main chain and a side chain is used to combine the cDNA of the desired VHH, the corresponding mRNA and the protein (VHH) into one. It is preferable because it can be obtained as a set.
  • the main chain of this linker contains BioTEG as a solid-phase binding site at the 5' end, and ribo-G (hereinafter sometimes abbreviated as "rG") as a cleavage site from the solid phase in the vicinity thereof.
  • inosine hereinafter sometimes abbreviated as “I”
  • cyanovinylcarbazole an example of the main chain sequence is shown in SEQ ID NO: 6 below. Hereinafter, this main chain may be referred to as "biotin fragment”.
  • K represents 3-cyanovinylcarbazole (hereinafter sometimes referred to as "cnvK").
  • linker having the main chain represented by SEQ ID NO: 6 below is referred to as “cnvK rG Linker" in the present specification.
  • the side chain is sometimes referred to as a "puromycin segment” because it contains puromycin or an analog thereof as a peptide binding site ("P" in the above side chain sequence) at its free end.
  • the side chain preferably has a 5'(5S)TCTFZZCCP sequence from the viewpoint of efficiency of formation of the cDNA display molecule described below.
  • “(5S)” represents 5' Thiol C6,
  • “F” represents the fluorescent group FITC-dT, and "Z” represents Spacer 18, respectively.
  • the chemical synthesis of the main chain and side chains may be carried out according to a conventional method, or may be outsourced to Tsukuba Oligo Service Co., Ltd. or other companies.
  • biotin fragment and EMCS (manufactured by Dojindo Laboratories) are incubated in a predetermined solution to bind, and the biotin fragment-EMCS conjugate is precipitated with ethanol.
  • ethanol precipitation for example, Quick-Precip Plus Solution (manufactured by Edge BioSystems) or the like may be used.
  • the puromycin-segment is dissolved in a disodium hydrogen phosphate buffer, stirred using a shaker, and then subjected to reduction treatment to obtain the reduced puromycin-segment-containing solution.
  • the reduced puromycin-segment-containing solution is mixed with the EMCS-modified biotin fragment (ethanol precipitate) and left at the desired temperature for the desired time, eg, about 2-6° C. overnight to bind. to form the cnvK rG linker.
  • reduction treatment is performed to prevent the formation of intramolecular crosslinks of the linker.
  • dithiothreitol hereinafter sometimes abbreviated as "DTT”
  • DTT dithiothreitol
  • a conjugate of the above fragments can then be obtained as an ethanol precipitate in the same manner as above.
  • the ethanol precipitate obtained is preferably dissolved in the desired solution for purification.
  • it may be dissolved in about 50 to about 150 ⁇ L of nuclease-free water (manufactured by Nacalai Tesque, Inc.) to obtain a lysate.
  • the above-mentioned lysate was separated by subjecting it to polyacrylamide gel electrophoresis (hereinafter sometimes abbreviated as "PAGE"), the band containing the cnvK rG Linker was excised, and the excised gel was crushed to remove the cnvK rG linker. Extract. Next, the above extract is transferred to a centrifugal tube filter and centrifuged to separate the gel, followed by ethanol precipitation to obtain the cnvK rG linker.
  • a centrifugal tube filter include Costar (registered trademark) Spin-X (registered trademark) (cellulose acetate with a pore size of 0.22 ⁇ m (manufactured by Corning)) and the like.
  • cDNA display molecules for primary library preparation can be prepared as follows using buffers with compositions as shown in Table 3 below.
  • Transcription 1 The DNA for cDNA display molecule synthesis prepared in (2) is transcribed, and the resulting transcription product (mRNA) is purified and quantified.
  • mRNA transcription product
  • 2x binding buffer, binding (selection) buffer, His-tag binding/washing buffer, His-tag elution buffer (same as His-tag binding/washing buffer except imidazole concentration is 200-300 mM), selection A buffer or the like can be used.
  • the above transcription can be performed, for example, using the T7 RiboMAX Express Large Scale RNA Production System (manufactured by Promega) according to the attached protocol.
  • the content of molecules other than the target molecule decreases as the round progresses, so the amount of DNA used at the start of a new round can be reduced. For example, about 5-7 ⁇ g in the first round, and about 0.05 to about 2 ⁇ g in the second and subsequent rounds.
  • Transcripts obtained in each round are quantified after purification. may be used to quantify the concentration of purified transcripts.
  • the mRNA purified above is mixed with the above cnvK rG linker in equal amounts in a buffer, annealed, photocrosslinked, and ligated to obtain an mRNA-linker.
  • a procedure for hybridizing the 3' end side of mRNA to the cnvK rG linker for example, equal amounts of the above purified mRNA and cnvK rG linker are added to Tris buffer and incubated at about 88 to 92°C for 0.5 to 1.5 minutes. and then cooled to 65-75°C at a rate of about 0.05-0.15°C/sec and incubated at 65-75°C for 0.5-1.5 minutes.
  • the temperature lowering rate can be increased to 1.5 to 2.5°C/sec to lower the temperature to about 5 to 15°C.
  • a Handheld UV Lamp (6W, UVGL-56, 254/365 nm, 100V (Analytik jena US, An Endress + Hauser Company)
  • UV at 350-370 nm. may be irradiated for 2 to 8 minutes.
  • an mRNA display molecule is prepared by the following procedure. For example, about 3 to about 8 pmol of the above mRNA-linker is added to a 25 to 100 ⁇ L scale cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), manufactured by Promega), and the mixture is heated to about 25 to about 35°C for 15 to 15 minutes. Incubate for 45 minutes, to which salt is added at the desired concentration. For example, a final concentration of 50-100 mM MgCl 2 and a final concentration of 800-1,000 mM KCl can be added. The mixture can then be incubated at about 36° C.
  • a chelator such as EDTA was added to a final concentration of 50-100 mM and incubated at 2-6°C for 1-10 minutes. , can form an mRNA display molecule.
  • cDNA display molecule is formed as follows. The solid phase is placed in a tube and washed with a buffer. After that, the peptide displayed on the mRNA molecule is reverse transcribed to form an mRNA/cDNA-peptide conjugate (hereinafter referred to as "cDNA display molecule") on the solid phase. Prepare. This cDNA display molecule can be released from the solid phase by an enzyme that cleaves the enzymatic cleavage site.
  • Dynabeads Myone Streptavidin C1 can be used for protein low binding tubes. Take the Dynabeads in a tube, add an appropriate amount of binding buffer to wash, and then add the mRNA display molecule. This tube is stirred at about 20 to about 30° C. for 15 to 45 minutes to solidify and washed with the binding buffer in the same manner as above.
  • a reaction solution for reverse transcription (1-3 ⁇ L of 25 mM dNTP Mix and 0.5-2 ⁇ L of GeneAce Reverse Transcriptase (100-300 U/ ⁇ L) (both manufactured by NIPPON GENE)) is added here,
  • the reaction can be performed by incubating at about 40-45° C. for 15-45 minutes to obtain the cDNA display molecule.
  • the cDNA display molecules are washed with an appropriate amount of binding buffer, followed by the addition of His-tag binding/wash buffer and RNase T1 and incubation with agitation.
  • a primary library is prepared by binding the above cDNA molecules to a solid phase, washing with a desired buffer, and then eluting the washed cDNA display molecules from the solid phase.
  • Obtainable For example, put His Mag Sepharose Ni Beads (manufactured by GE Health Care) in an Eppendorf tube, wash the beads with a buffer in the same manner as in (4) above, and then wash the beads with a buffer in the same manner as in (4) above. Wash and purify. About 25-35 ⁇ L of His-tag elution buffer is then added and vortexed to elute the cDNA display molecules as described above.
  • a cDNA library can be obtained as a primary library (library indicated as Initial in FIG. 2).
  • Selection of Target Molecules (1) Selection of Target Molecules that Bind to Ligand Using the primary library described above, selection is performed in the following procedure to obtain target molecules that bind to ligands.
  • the scale of synthesis of the target molecule-displaying cDNA display library in each round of selection can be decreased as the round progresses (see, eg, Table 5 below), thereby enriching the target molecule.
  • the elution method for binding to the solid phase in the selection cycle is not particularly limited as long as it does not affect the activity of the target molecule, and a buffer containing competitive elution, TCEP (tris(2-carboxyethyl)phosphine), etc. is used. Elution can be performed. An example of such elution is shown in the flow chart of FIG.
  • R1 Selection Round 1
  • a detection molecule is added thereto, and incubated at a desired temperature for a desired time to immobilize the detection molecule.
  • the cDNA display molecule is added, bound to the detection molecule, and eluted after washing.
  • 16 ⁇ L of biotinylated FGFR1-Fc is added to the above tube and mixed by inversion at about 20 to about 30° C. for 15 to 45 minutes to immobilize FGFR-Fc and immobilize the detection molecule. Beads can be obtained.
  • the tube can then be washed several times with the desired buffer, eg, selection buffer, followed by the following elution. If an elution buffer is used, the cDNA display molecule bound to the detection molecule is eluted by cleaving the SS bond of the binding molecule. After that, an alkali elution operation of adding NaOH alkali to the tube containing the immobilized beads and incubating is performed a desired number of times to neutralize the obtained alkali solution. The resulting supernatant is mixed with the above-mentioned alkaline eluate to prepare a sample for purification, and a portion thereof is taken to prepare a sample for PCR.
  • the desired buffer eg, selection buffer
  • annealing temperature 65 PCR can be performed under conditions of ⁇ 70°C and extension reaction for 20-40 seconds.
  • the resulting PCR product can be subjected to 8M urea 3-5% denaturing PAGE.
  • the above PCR product can be purified, and the resulting purified product can be subjected to the second round of selection as an R1 library.
  • AMPure XP manufactured by Beckman Coulter
  • a desired number of rounds can be selected by repeating the same procedure.
  • a selection buffer is added to each tube to which the solid phase (beads) has been added, and the beads in each tube are washed. After that, the selection buffer is added again for washing, after which another detection molecule such as FGFR1-His is added, mixed by inversion, and then eluted.
  • the obtained eluate may be amplified by PCR and used as a library after purification.
  • the library used in R2 and later may be prepared by mixing the TCEP eluted solution.
  • enriched library The above DNA library (hereinafter sometimes referred to as "enriched library") is further enriched using phage display methods, and at the same time target molecules that can be expressed in E. coli can be obtained. can try.
  • the phage display method is shown schematically in FIG.
  • the phagemid and each DNA library are treated with restriction enzymes to obtain phagemid vectors.
  • restriction enzymes For example, add FastDigest BamHI and react at about 36 to about 38°C for 45 to 90 minutes, then add FastDigest SfiI (both restriction enzymes are manufactured by Thermo Fisher Scientific) and heat at 45 to 55°C for 45 minutes to Incubate for 90 minutes.
  • AMPure XP can be used to purify the resulting phagemid vector.
  • the resulting phagemid vector is purified by gel electrophoresis, and the enzyme is deactivated after dephosphorylation.
  • the above phagemid vector is loaded onto a 1% agarose gel containing 1xGel green (Fuji Film Wako Pure Chemical Industries, Ltd.) and electrophoresed at 100 V for 20 to 40 minutes. Thereafter, the phagemid vector of interest can be extracted from the gel and purified to obtain a purified phagemid vector.
  • FastGene Gel/PCR Extraction Kit Nippon Genetics Co., Ltd.
  • FastAP Thermosensitive Alkaline Phosphatase manufactured by Thermo Fisher Scientific
  • the phagemid vector and the restriction enzyme-treated library are mixed at a ratio of 1:5 to 10 (molar ratio) and reacted to obtain a phagemid into which the gene fragment of the target molecule is integrated.
  • vector can be obtained.
  • Ligation high Ver. 2 manufactured by TOYOBO
  • the reaction can be performed overnight at about 10 to about 20°C.
  • E. coli is transformed with the obtained phagemid vector. This transformation can be performed by electroporation or the like.
  • the E. coli is not particularly limited as long as it is a competent cell for phage display, and for example, E. coli TG-1 (manufactured by Lucigen) can be used.
  • Transformed TG-1 can be seeded onto agar plates and cultured overnight at about 28 to about 32° C. in a 5% CO 2 incubator to allow E. coli colonies to form on the medium. All these colonies may be collected by picking and cultured under the same conditions as above until the OD600 reaches the desired value. After completion of this culture, the number of E. coli was counted, and 20 times the amount of helper phage was added here. This is because a helper phage is required for phagemid propagation. After adding the helper phage, the cells are cultured under the same conditions as above, and the culture solution is centrifuged to collect the supernatant.
  • the collected supernatant is transferred to a new tube, a 15-25% PEG solution containing 2.5 M NaCl is added thereto, mixed by inversion, and cooled on ice.
  • PBS Phosphate Buffered Saline
  • FGFR1 immobilization solution is then dispensed into each well of a 96-well ELISA plate (Immuno Clear Standard Modules_C8_MaxiSorp: Cat # 445101, Thermo Scientific) or the like and incubated overnight for these immobilizations.
  • eluate is then added to each well to recover the phage display molecules bound to the detection molecule.
  • a 50-150 mM trimethylamine solution is added to each well and allowed to stand at room temperature for 5-15 minutes to elute bound phages.
  • the obtained eluate is preferably immediately neutralized with Tris-HCl (pH 6.5-7.5) or the like.
  • the eluate neutralized as described above is mixed with competent cells in the same manner as described above, and this mixture is seeded on an agar medium to form colonies. After colony picking and culturing, helper phage and perform biopanning again. Biopanning can also be performed using immunotubes, as described below. Whether or not VHHs that bind to FGFR1 are enriched in the second library obtained as described above can be confirmed by phage ELISA in a polyclonal state. Phagemids are extracted from Escherichia coli for clones that give responses, and DNA sequences are analyzed by the Sanger method or the like to identify clones.
  • Target Molecule Plasmids can be prepared from clones identified as described above and expressed in bacteria. For example, using Corynebacterium glutamicum (C. glutamicum) as a bacterium, the target molecule can be expressed as follows.
  • Corynebacterium glutamicum C. glutamicum
  • in vitro homologous recombinants can be obtained by placing a desired buffer in a new tube, adding the above-mentioned purified DNA, bacterial expression plasmid, and E. coli extraction reaction solution, and incubating. A competent cell is transformed with this homologous recombinant, and a plasmid is extracted from the resulting transformant.
  • the desired buffer include SLiCE buffer containing 25-75 mM Tris-HCl (pH 7.2-7.7), 5-15 mM MgCl 2 , 0.5-1.5 mM ATP, and 0.5-1.5 mM DTT.
  • the plasmid obtained as described above is transformed into a bacterium such as C. glutamicum by electroporation or the like, and the resulting transformant is placed in a desired medium. and pre-culture.
  • the pre-culture can be performed overnight at about 28 to about 32° C. using CM2G medium.
  • 1/20 of this preculture solution is transferred to PM1S medium and cultured at about 23 to about 27° C. for 60 to 80 hours to secrete the target molecule into the medium.
  • the culture solution is transferred to a centrifuge tube and centrifuged, and the collected culture supernatant is preferably treated with a 0.22 ⁇ m filter to remove the cells.
  • imidazole is added to the culture supernatant containing the target molecule obtained as described above, and then the carrier is added and mixed by inversion. Subsequently, this carrier is separated by a spin column, and target molecules are eluted with an elution buffer to be described later.
  • a carrier include Ni Sepharose 6 Fast Flow (manufactured by Cytiva, hereinafter sometimes simply referred to as "carrier"). etc. can be mentioned.
  • the elution buffer 25-75 mM Tris-HCl (pH 7.0-8.0) containing 250-350 mM NaCl and 400-600 mM imidazole can be used. Also, the purity of the eluted target molecule can be confirmed by SDS-PAGE or the like. As described above, the target molecule can be obtained.
  • Example 1 Screening of Target Molecules Using the cDNA display method, libraries containing cDNAs of target molecules were screened to prepare a primary library.
  • the target molecule was human fibroblast growth factor receptor 1 (hereinafter sometimes abbreviated as FGFR1), and a molecule for detecting the target molecule (hereinafter referred to as " as the extracellular domain of FGFR1, recombinant human FGFR1 protein (Active, Cat. No. ab168696, purchased from Abcam, hereinafter sometimes abbreviated as "FGFR-His”), and FGF receptor 1 ⁇ (IIIc)/Fc chimera (Cat. No.
  • FGFR-Fc human recombinant carrier-free, purchased from R&D System, hereinafter sometimes abbreviated as "FGFR-Fc"
  • FGFR-Fc human recombinant carrier-free
  • IgG1 (Fc) Cat. No. 110-HG, human recombinant carrier-free, purchased from R&D System, hereinafter sometimes abbreviated as "hFc” was used.
  • a cDNA display method used for screening is schematically shown in FIG.
  • the molecule of interest was biotinylated by the following procedure. 20 equivalents of a biotinylation reagent (EZ-link sulfo NHS-SS biotin; manufactured by Thermo Fisher) was added to each of FGFR1-Fc (detection molecule 1) and hFc (detection molecule 2), and incubated at 25°C for 30 minutes. reacted. Thereafter, desalting columns (Zeba Spin Desalting Columns; manufactured by Thermo Fisher) were used to remove unreacted biotinylation reagents, and the molecules to be detected were immobilized.
  • EZ-link sulfo NHS-SS biotin EZ-link sulfo NHS-SS biotin
  • FGFR1-Fc detection molecule 1
  • hFc detection molecule 2
  • desalting columns Zeba Spin Desalting Column
  • VHH variable region fragments of full-length alpaca-derived antibodies (single variable domains of heavy chain antibodies, hereinafter sometimes referred to as "VHH") as follows. . S-hinge and L-hinge genes from two types of alpaca-derived naive VHH libraries (both manufactured by RePHAGEN) were used as templates. Further, as the forward primer, using the primer of SEQ ID NO: 1 shown in Table 1 below, using the S hinge-specific reverse primer (SEQ ID NO: 2) and the L hinge-specific reverse primer (SEQ ID NO: 3), the following Amplification was carried out under PCR conditions to obtain S-hinge and L-hinge cDNA molecules (see Table 1 above). The PCR conditions were as follows: 98°C for 2 minutes, 5 cycles of 98°C for 10 seconds, 62°C for 5 seconds, and 72°C for 35 seconds, reaction at 72°C for 1 minute, and cooling to 10°C.
  • full-length library is a DNA fragment containing a T7 promoter, an omega ( ⁇ ) enhancer, a Kozak consensus sequence, a VHH gene, a His-tag, and a linker hybridization region (Y-tag) (see Table 1 above).
  • Overlap PCR was performed at 98°C for 2 minutes, followed by 5 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 35 seconds. It was cooled.
  • g guanosine
  • V represents Amino C6-dT
  • K represents 3-cyanovinylcarbazole (hereinafter sometimes referred to as "cnvK”) (hereinafter referred to as "cnvK rG Linker”). .).
  • the side chain puromycin segment has a 5' (5S) TCTFZZCCP sequence.
  • the free terminal "P" in the side chain sequence represents puromycin as a protein binding site.
  • “(5S)” represents 5' Thiol C6,
  • “F” represents the fluorescent group FITC-dT, and
  • “Z” represents Spacer 18, respectively.
  • the above reduced puromycin segment solution was mixed with the above ethanol precipitation product of the EMCS-modified biotin fragment and left overnight at 4°C. Subsequently, DTT was added to the reaction solution so that the final concentration was 50 mM, and the mixture was stirred at room temperature for 30 minutes. Thereafter, using Quick-Precip Plus Solution (manufactured by Edge BioSystems), ethanol precipitation was performed to obtain a conjugate of the above fragments as an ethanol precipitate. This ethanol precipitate was dissolved in 100 ⁇ L of Nuclease-free water (manufactured by Nacalai Tesque, Inc.) to obtain a lysate.
  • the above lysate was separated by 8M urea 12% denaturing polyacrylamide gel electrophoresis (electrophoresis conditions: 60°C, 200V, 30 minutes), and the cnvK rG Linker fraction was excised.
  • the excised gel was crushed using a Biomasher II set (manufactured by Nippi Co., Ltd.), 500 ⁇ L of nuclease-free water was added to the gel crushed product, and the mixture was stirred overnight at 4°C to extract the cnvK rG Linker.
  • the stirred solution was transferred to a Costar (registered trademark) Spin-X (registered trademark) centrifugal tube filter, 0.22 ⁇ m cellulose acetate (manufactured by Corning), and then centrifuged at 16,000 xg for 15 minutes to separate the gel from the extracted liquid. did.
  • ethanol precipitation was performed in the same manner as above using Quick-Precip Plus Solution to obtain cnvK poly rG Linker.
  • the resulting cnvK rG Linker was dissolved in nuclease-free water and stored at -20°C.
  • Example 2 Generation of cDNA Display
  • the buffers used are shown in Table 3 below.
  • a Handheld UV Lamp (6W, UVGL-56, 254/365 nm, 100V (Analytik jena US, An Endress + Hauser Company) was used to irradiate UV at 365 nm for 5 minutes to extract cnvK rG linkers and mRNA. Photocrosslinking was performed to obtain an mRNA-linker conjugate.
  • VHH conjugates were eluted from Dynabeads MyOne Streptavidin C1.
  • Example 3 Selection of VHHs (1) Selection of target VHHs that bind to ligands In order to obtain target VHHs that bind to ligands, selection was performed using the cDNA display library prepared in Example 2 above. The synthesis scale of the VHH-presenting cDNA display library used in each selection round is shown in Table 5 below. In addition, Fig. 2 shows a screening test flow chart showing cases classified by elution method (competitive elution, TCEP elution).
  • Selection Round 1 (hereinafter sometimes simply referred to as “R1”) 16.3 ⁇ L of biotinylated FGFR1-Fc (detection molecule 1) prepared in Example 1 above was placed in a protein low binding tube containing 100 ⁇ L of Dynabeads Myone Streptavidin C1 (hereinafter sometimes simply referred to as “streptavidin beads”). In addition, the mixture was mixed by inversion at 25°C for 30 minutes to immobilize FGFR-Fc. 100 pmol of FGFR-Fc-immobilized beads were placed in a protein low binding tube, and the entire amount of cDNA display prepared according to Example 2 using the DNA prepared in Example 1 (2-2) was added. In addition, they were mixed by inversion at 25°C for 30 minutes to bind with the detection molecules. Selection buffer was then added to the tube and washed four times.
  • R1 Selection Round 1
  • R4 Selection cycle 4 (1-4-1) Screening of R3-1 Library
  • a cDNA display method was performed using the R3 library, and a fraction eluted by competitive elution and a fraction eluted with TCEP were obtained.
  • 3 pmol of cDNA display using the R3-1 library and 1 pmol of FGFR1-Fc immobilized beads were placed in a protein low binding tube, and 1,000 ⁇ L of selection buffer, 50 pmol of hFc, and 140 pmol of streptavidin were added. , and 100 ng/ ⁇ L of Salmon sperm DNA were added, and mixed by inversion at 4° C. for 3 hours.
  • the competitively eluted solution obtained as described above was treated with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, and then at 98°C. 25 cycles of 10 seconds, 68°C for 5 seconds, and 72°C for 35 seconds were performed, followed by PCR amplification under the conditions of 72°C for 1 minute to obtain the R4-1-1 library.
  • the TCEP eluate was used with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 5 minutes. Seconds, 35 seconds at 72°C for 25 cycles, and amplification by PCR under the conditions of 72°C for 1 minute to obtain the R4-1-2 library.
  • R4-1-1, R4-1-2, R4-2-1, and R4-2- Each library of 2 was placed in a separate tube and bound in the same manner as above. 200 ⁇ L of selection buffer was added to these tubes, the beads in the tubes were washed three times, and 20 ⁇ L of TCEP was added to the beads for elution (TCEP elution).
  • the competitively eluted solution was subjected to 98° C. after 2 minutes at 98°C using cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers. 25 cycles of 10 seconds at °C, 5 seconds at 68°C, 35 seconds at 72°C, and 1 minute at 72°C for amplification.
  • the R5-2-2-1 library was obtained from the 2 libraries.
  • R4-1-1 library to R5-1-1-2 R4-1-2 library to R5-1-2-2 library
  • R4-2-1 library to R5 The R5-2-2-2 library was obtained from the -2-1-2 library and the R4-2-2 library.
  • R5 from the R4-1-1, R4-1-2, R4-2-1 and R4-2-2 libraries obtained in (1-4) above, a part was taken and FACS (Fluorescence-Activated Cell Sorting) was performed. ) was used for selection.
  • FACS Fluorescence-Activated Cell Sorting
  • biotin-fluorescein was added to a protein low binding tube containing hFc-immobilized beads to prepare hFc-immobilized fluorescent beads (hereinafter simply referred to as “hFcF” beads). 2 pmol of hFcF beads were taken in a new tube, and 0.5 pmol of FGFR1-Fc immobilized beads were added and mixed.
  • any of the above libraries 50 ⁇ L of selection buffer, 50 pmol streptavidin, and 500 ng/ ⁇ L of Salmon sperm DNA were mixed and allowed to react at 4°C for 1 hour. After completion of the reaction, 200 ⁇ L of selection buffer was added to each tube and washed three times. Subsequently, 500 ⁇ L of selection buffer was added to each tube to serve as a sorting solution.
  • the sorting solution obtained as described above was set in an FCM (Cell Sorter SH800: SONY), and the sample channel and droplet forming conditions during fractionation were set up, and sorting was performed.
  • the regions of the magnetic beads were identified based on the particle size fractionated by FACS, and the regions of the hFcF beads and the FGFR1-Fc immobilized beads were identified from the fluorescence intensity due to 488 nm laser irradiation. Sorting was performed under the optimum preparative conditions, and 500,000 particles were collected in protein low binding tubes for each. After that, centrifugation was performed at 13,000 xg for 10 minutes at 25°C. After centrifugation, the protein low binding tube was allowed to stand on a magnetic plate at room temperature for 10 minutes, after which the supernatant was removed and dissolved in 20 ⁇ L of water.
  • This lysate was treated with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, followed by 98°C for 10 seconds, 68°C for 5 seconds, 72 25 cycles of 35 seconds at ° C., amplified by PCR under the conditions of 72 ° C. for 1 minute, respectively, R5-1-1-FACS library, R5-1-2-FACS library, R5-2-1-FACS library and This was used as the R5-2-2-FACS library.
  • NGS next-generation sequencer
  • Amplicon PCR was performed to obtain amplification product 1.
  • Amplicon PCR conditions were (s1) 98°C for 1 minute, (s2) 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, 72°C for 35 seconds, and (s3) 72°C for 1 minute. did.
  • Index PCR manufactured by Illumina was performed according to the attached instructions to obtain amplification product 2.
  • the resulting amplification product 2 was purified according to the instructions of Agencourt AMPure XP and then quantified by NanoPad DS-11FX (DeNovix). Subsequently, sequence analysis was performed according to the instructions of MiSeq (manufactured by Illumina) and MiSeq Reagent Nano kit v2 (500 cycles), the obtained DNA sequences were translated into VHH amino acid sequences, and ranked by appearance frequency.
  • phagemid vector 1 An enzyme-treated phagemid (hereinafter sometimes referred to as "phagemid vector 1") was obtained. Then, using AMPure XP, the phagemid vector was crudely purified according to the attached instructions.
  • the above phagemid vector 1 and the library (VHH antibody gene fragment) treated with restriction enzymes as above were mixed at a ratio of 1:5 to 10 (molar ratio), Ligation high Ver. to obtain phagemid vector 2 into which the above VHH gene fragment was integrated.
  • the resulting phagemid vector 2 was concentrated by ethanol precipitation, and then electroporated to transform Escherichia coli TG-1 (phage display competent cells, manufactured by Lucigen).
  • the transformed TG-1 was seeded on 2YTAG agar plates and cultured overnight at 30°C in an incubator.
  • the tube was then chilled on ice for 1 hour and then centrifuged at 4,000 xg for 30 minutes at 4°C to remove the supernatant.
  • PBS Phosphate Buffered Saline
  • 10% glycerol was added to the obtained precipitates to dissolve them to obtain a phage display solution containing VHH-displaying phages.
  • the eluate neutralized in this way was mixed with 1,200 ⁇ L of E. coli TG-1 and allowed to stand at 30°C for 1 hour to obtain mixed solution 2.
  • 10 ⁇ L of this mixture 2 was plated on 2YTAG agar medium (1.5%, 10 cm dish). The rest of the mixture 2 was centrifuged at 4000 xg for 20 minutes at 25°C and the supernatant was removed. The resulting precipitate (Escherichia coli TG-1) was seeded on the same 2YTAG agar medium (15 cm dish) as above.
  • E. coli was infected with helper phage by adding 20 times the amount of E. coli and cultured overnight at 30°C.
  • the culture solution containing the grown E. coli was centrifuged in the same manner as in (3-1) above, and the supernatant was collected in a tube. Thereafter, the mixture was cooled on ice for 1 hour, centrifuged in the same manner as in (3-1) above, and the supernatant was removed to obtain a precipitate.
  • PBS containing 10% glycerol was added to this precipitate to dissolve it, phage display was prepared, and biopanning was performed again.
  • the resulting eluate was immediately neutralized with 1 mL of 0.5 M Tris-HCl (pH 6.8).
  • the eluate after neutralization was mixed with 10 mL of Escherichia coli TG-1 and allowed to stand at 30° C. for 1 hour to obtain mixed solution 4.
  • This mixture 4 was centrifuged in the same manner as above, and the resulting precipitate (Escherichia coli TG-1) was seeded on the above-described 2YTAG agar medium (15 cm dish) and placed in a CO 2 incubator at 30°C overnight. cultivated in the
  • This culture medium was treated in the same manner as in (3-2) above, the supernatant was collected in a new tube, a 20% PEG solution containing 25M NaCl was added, and the tube was covered and mixed by inversion. Thereafter, the mixture was cooled on ice for 1 hour, centrifuged in the same manner as above, and the supernatant was removed to obtain a precipitate. The resulting precipitate was dissolved in PBS containing 10% glycerol, phage display was prepared, and a second round of biopanning was performed. In the second biopanning, the same procedure as above was performed except that FGFR1-His was immobilized on the immunotube instead of FGFR1-Fc to obtain a second library.
  • Phage ELISA In order to confirm whether VHHs that bind to FGFR1 are enriched in the second library obtained as described above, phage ELISA was performed in a polyclonal state. First, FGFR1-Fc and FGFR1-His were diluted with PBS to 10 ⁇ g/mL to prepare an FGFR1-immobilized solution. 100 ⁇ L of the above FGFR1 immobilization solution was added to each well of a 96-well ELISA plate and incubated overnight at 4° C. to immobilize them. An operation of removing the immobilization solution from each well, adding PBS and washing was repeated three times, and then blocking was performed in the same manner as in (3-3) above. After blocking, the blocking solution was removed from each well, and PBS was added for washing, which was repeated three times.
  • 160 ⁇ L was taken from the phage display obtained in each round and mixed with 160 ⁇ L of 10% BSA-containing PBS to obtain Mixture 5. After that, 50 ⁇ L of the mixed solution 5 was added to each well and reacted at room temperature for 1 hour. The operation of removing the above mixture 5 from each well and then adding 200 ⁇ L of PBS-T to each well for washing was repeated five times.
  • Anti-M13-mAb-HRP manufactured by Sinobiological
  • VHH-displaying phages biopanned in (3-2) above were placed in an Eppendorf tube, E. coli TG-1 was added, and the mixture was allowed to stand at 30°C for 1 hour to form an F-E mixture.
  • This F-E mixture was centrifuged at 4000 ⁇ g for 20 minutes at 25° C., and the resulting precipitate (Escherichia coli TG-1) was dissolved in 2YTAG to obtain a lysate. Thereafter, this lysate was seeded on a 2YTAG agar medium in the same manner as described above, and colonies that appeared on the agar medium were single-picked and cloned to recover phage display.
  • Phage ELISA was performed in the same manner, except that FGFR1-His diluted with PBS to 2 ⁇ g/mL was used in the ELISA plate. After that, the phagemids of the responding clones were extracted from E. coli, and DNA sequences corresponding to the displayed VHHs were analyzed by the Sanger method. This sequence analysis was contracted to Eurofins Genomics. The above analysis identified two VHH clones, #1 and #2.
  • VHH monomeric VHH (hereinafter sometimes referred to as "sVHH") was expressed and purified as described above.
  • sVHH monomeric VHH (hereinafter sometimes referred to as "sVHH") was expressed and purified as described above.
  • pSLiCE_PK4_SMDPhage_F SEQ ID NO: 13
  • pSMDPhage_#1_R SEQ ID NO: 14
  • pSMDPhage_#2_R sequence No.
  • PCR was performed under the conditions of an annealing temperature of 55°C and an extension reaction of 5 seconds to obtain an sVHH expression plasmid as an amplified product.
  • the resulting amplified product was purified using a Gel/PCR Extraction Kit (manufactured by Nippon Genetics) according to the attached manual to obtain purified DNA.
  • the above purified DNA, C. glutamicum expression plasmid, and E. coli extraction were added to an 8-strip PCR tube containing SLiCE buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM ATP, and 1 mM DTT.
  • the SLiCE reaction solution was added, and in vitro homologous recombination was performed at 37°C for 15 minutes.
  • a competent cell JM109 manufactured by Takara Bio Inc.
  • the suspension was transferred to a spin column (EconoSpin; manufactured by Ajinomoto Bio-Pharma) and centrifuged at 100 xg for 30 seconds at 4°C to recover the carrier. Subsequently, add 600 ⁇ L of washing buffer (50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 30 mM imidazole), repeat the centrifugation operation twice under the same conditions as above, and then elute with 700 ⁇ L. Bound VHHs were eluted by adding buffer (50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 500 mM imidazole).
  • washing buffer 50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 500 mM imidazole.
  • SDS-PAGE The purity of the eluted sVHH was confirmed by SDS-PAGE (see Figure 5). SDS-PAGE was performed using a 4% stacking gel and a 15% separating gel. After applying 4 ⁇ L of sample to each well, electrophoresis was performed at 150 V for 1 hour. Precision Plus Protein TM Standards (BIO-RAD) were used as molecular weight markers. After electrophoresis, it was stained with Coomassie brilliant blue (CBB).
  • CBB Coomassie brilliant blue
  • the eluate buffer was replaced with PBS through a desalting column (Zeba Spin Desalting Columns; Thermo Fisher).
  • concentration of sVHH was quantified by the BCA method using bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard protein and Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher).
  • Example 5 Affinity measurement of sVHH Affinity measurement of sVHH was tested using surface plasmon resonance (SPR) resonance method.
  • SPR surface plasmon resonance
  • the binding activity to FGFR1-Fc of VHH clone #1 or #2 immobilized on Series S Sensor Chip CAP was measured using Biacore (trademark) T200 (manufactured by Cytiva), and CAP Single -Analyzed by cycle kinetics method.
  • the temperature was set at 25°C.
  • HBS-EP+ (10 mM HEPES (pH 7.4) containing 150 mM NaCl, 0.5 mM EDTA and 0.05% surfactant P20) (manufactured by Cytiva) was used.
  • the measurement order for each run was as follows, and all reagents were manufactured by Cytiva.
  • the present invention is useful in technical fields such as medicine and diagnostic agents.
  • SEQ ID NO: 1 Base sequence of target molecule primer
  • SEQ ID NO: 2 Target molecule primer base sequence
  • SEQ ID NO: 3 Target molecule primer base sequence
  • SEQ ID NO: 4 Overlap PCR primer base sequence
  • SEQ ID NO: 5 Overlap Nucleotide sequence of primer for wrap PCR
  • SEQ ID NO: 6 base sequence of main chain of cnvK linker
  • SEQ ID NO: 7 base sequence of primer (cnvK NewYtag)
  • SEQ ID NO: 8 base sequence of primer (T7 ⁇ New)
  • SEQ ID NO: 9 NGS Fw 1st PCR Base sequence of primer SEQ ID NO: 10: Base sequence of NGS Rv 1st PCR primer
  • SEQ ID NO: 11 Base sequence of primer for addition of restriction enzyme recognition sequence
  • SEQ ID NO: 12 Base sequence of primer for addition of restriction enzyme recognition sequence
  • SEQ ID NO: 13 Primer ( pSLiCE_PK4_SMDPhage_F) base sequence
  • SEQ ID NO: 14

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

[Problem] The purpose of the present invention is to provide a screening method equipped with a step which makes it possible to determine whether or not expression in a cell by a clone candidate, which is obtained by screening from a large-scale library before moving to a cell-based expression system, will be possible. [Solution] To provide a target molecule screening method equipped with: (a) a selection/screening step for constructing a library by selecting the DNA of a target molecule by using a non-cellular translation system, selecting the DNA of the target molecule from among the DNA fragments included in the library, and preparing a primary library by concentrating and screening the same; and (b) an expression/selection step for selecting a DNA fragment which codes a protein which can be produced in a living organism from among the proteins which can be produced from the target molecule included in the primary library obtained in the selection/screening step, and causing expression thereof using cells.

Description

ダブルスクリーニング方法Double screening method
　本発明は、無細胞翻訳系と、原核細胞による発現系とを組み合わせたスクリーニング方法に関する。より詳細には、1013～1014という大規模なサイズのライブラリから一時ライブラリを作製し、そのライブラリから原核細胞で発現できる目的分子をスクリーニングする方法に関する。 The present invention relates to a screening method that combines a cell-free translation system and a prokaryotic expression system. More specifically, it relates to a method of preparing a temporary library from a large-scale library of 10 13 to 10 14 and screening a target molecule that can be expressed in prokaryotic cells from the library.
　機能性ペプチドを開発する上で、種々の技術が知られているが、多様なペプチドライブラリから機能性ペプチドのスクリーニングを可能とするためには、遺伝子型－表現型対応付け技術が有用であることが知られている。遺伝子型と表現型との対応付け技術は、いわゆるリボザイム型、ウイルス型、細胞型、外部知性型等に分類することができる。 Various techniques are known for the development of functional peptides, but genotype-phenotype mapping techniques are useful for enabling screening of functional peptides from various peptide libraries. It has been known. Techniques for associating genotypes with phenotypes can be classified into so-called ribozyme types, virus types, cell types, external intelligence types, and the like.
　ここで、上記「ウイルス型」とは、遺伝子型（DNA又はRNA）と表現型（タンパク質）とが単純に結合している形態を示す用語であり、微生物学で用いられる「ウイルス」の「型」を示すものではない。具体的には、ゲノムに当該ゲノムがコードしているタンパク質（例えば、コートプロテイン）が単純に結合しているファージディスプレイが代表例として挙げられる。 Here, the above-mentioned "virus type" is a term indicating a form in which a genotype (DNA or RNA) and a phenotype (protein) are simply combined. ” does not indicate Specifically, a representative example is phage display in which a protein encoded by the genome (for example, coat protein) is simply bound to the genome.
　現在知られている技術としては、ファージディスプレイ、細胞表層ディスプレイ、リボソームディスプレイ、mRNAディスプレイ、cDNAディスプレイ等がある、これらのうち、ファージディスプレイと細胞表層ディスプレイとは細胞を用いることになるため、細胞がタンパク質を産生する。一方、mRNAディスプレイとcDNAディスプレイとは、無細胞翻訳系であるため、細胞がタンパク質を産生することはない。 Currently known techniques include phage display, cell surface display, ribosome display, mRNA display, cDNA display, etc. Among these, phage display and cell surface display use cells, so cells Produces protein. On the other hand, since mRNA display and cDNA display are cell-free translation systems, cells do not produce proteins.
　そして、現在のところ、工業規模での産生を考えたときに扱いやすいことからファージディスプレイ法が最も多く利用されている（非特許文献１参照、以下、「従来例１」という。）。また、最近、リボソームディスプレイ法でライブラリを作製し、次にファージディスプレイ法に乗せ換えて所望のペプチドを得るという技術が報告されている（非特許文献６参照。以下、「従来例２」という）。
　一方で、cDNAディスプレイ法(特許文献１参照、以下「従来例３」という。)は、1012以上という大規模なサイズのライブラリを扱うことができることが知られている。 At present, the phage display method is most commonly used because it is easy to handle when considering production on an industrial scale (see Non-Patent Document 1, hereinafter referred to as "conventional example 1"). In addition, recently, a technique has been reported in which a library is prepared by the ribosome display method and then transferred to the phage display method to obtain a desired peptide (see Non-Patent Document 6, hereinafter referred to as "Conventional Example 2"). .
On the other hand, it is known that the cDNA display method (see Patent Document 1, hereinafter referred to as "conventional example 3") can handle large-scale libraries of 10 12 or more.
ＷＯ２０１７/１７０７７６号公報WO2017/170776
　従来例１は、遺伝子型と発現ペプチドとの正確な対応性が確保できること、及び簡便で迅速な手法がすでに確立されているという点では優れた方法である。例えば、ファージディスプレイを用いるセレクション及び進化の全過程は、通常、約２～３週間で完了する（非特許文献１、２参照）。しかし、最大でも108程度のライブラリサイズを有するライブラリまでしか扱えないという問題があった。 Conventional Example 1 is an excellent method in that it can ensure accurate correspondence between the genotype and the expressed peptide, and that a simple and rapid technique has already been established. For example, the entire process of selection and evolution using phage display is usually completed in about 2-3 weeks (see Non-Patent Documents 1 and 2). However, there is a problem that it can only handle libraries with a maximum library size of about 10 8 .
　従来例３は、上述したように1012以上という、より大きなサイズを有するライブラリ（以下、「大規模ライブラリ」ということがある。）を扱うことができるため、より多くの分子多様性を有する候補をセレクションできるという点では優れた方法である。また、無細胞翻訳系であるため、非天然残基のタンパク質／ペプチド内への取り込みが可能であり、ジスルフィドシャッフリング反応等のような翻訳後修飾を行い得る点という点では優れた反応である（非特許文献３～５）。 Conventional Example 3 can handle a library having a larger size of 10 12 or more as described above (hereinafter sometimes referred to as a "large-scale library"), so candidates with more molecular diversity This is an excellent method in that it allows you to select In addition, since it is a cell-free translation system, it is possible to incorporate non-natural residues into proteins/peptides, and it is an excellent reaction in that post-translational modifications such as disulfide shuffling reactions can be performed ( Non-Patent Documents 3-5).
　しかし、無細胞翻訳系を使用した場合には、その後、細胞を用いた発現系で発現させることができる否を事前に評価できないという問題がある。すなわち、無細胞翻訳系で得られた候補クローンを上記細胞に組み込むためのプラスミドに組み込み、実際に上記細胞に導入してみない限り、その細胞にとって致死性のクローンであるかどうかが判定できないのである。このため、上記細胞が産生できるクローンを選別するための工程が不可欠となるという問題があった。 However, when using a cell-free translation system, there is a problem that it is not possible to evaluate in advance whether it can be expressed in an expression system using cells. That is, unless a candidate clone obtained by a cell-free translation system is incorporated into a plasmid for integration into the cell and actually introduced into the cell, it cannot be determined whether the clone is lethal to the cell. be. Therefore, there is a problem that a process for selecting clones that can be produced by the above cells is essential.
　従来技術２は、リボソームディスプレイ法とファージディスプレイ法とを用いて、デリケートなナノボディを作製できるという点では優れた発明である。しかしながら、従来技術２では、リボソームディスプレイ法で得られたペプチドを、逆転写酵素を用いた別の反応系で逆転写反応させてcDNAを取得する。そして、ここで得られたcDNAをPCRに供して増幅させ、その後に、増幅したcDNAを含むプラスミドを作製するために、コンピテント細胞を形質転換し、ファージディスプレイ法で所望のペプチドを得るという多くの工程が必要とされ、最終的に目的ペプチドを得るまでに時間と手間がかかるという問題があった。 Prior art 2 is an excellent invention in that a delicate nanobody can be produced using the ribosome display method and the phage display method. However, in prior art 2, peptides obtained by the ribosome display method are reverse transcribed in a separate reaction system using reverse transcriptase to obtain cDNA. Then, the cDNA obtained here is amplified by PCR, and then competent cells are transformed to produce a plasmid containing the amplified cDNA, and the desired peptide is obtained by phage display. , and there is the problem that it takes time and effort to finally obtain the target peptide.
　このため、細胞による発現系に移行する前に上記大規模ライブラリからスクリーニングして得た候補クローンが、細胞で発現可能か否かを判別することができる工程を備えるスクリーニング方法に対する強い社会的要請があった。 Therefore, there is a strong social demand for a screening method comprising a step of determining whether or not candidate clones obtained by screening from the large-scale library can be expressed in cells before transferring to a cell-based expression system. there were.
　以上のような状況の下で、本願発明の発明者等は鋭意研究を重ね、本願発明を完成したものである。 Under the circumstances described above, the inventors of the present invention conducted extensive research and completed the present invention.
　本発明は、(a)無細胞翻訳系を使用して目的分子のDNAを選択してライブラリを構成し、前記ライブラリに含まれるDNA断片の中から、前記目的分子のDNAを選択し、濃縮して淘汰することによって一次ライブラリを作製する、選択・淘汰工程と；(b)前記選択・淘汰工程で得られた一次ライブラリに含まれる前記目的分子から作られるタンパクのうち、生物において作製可能なタンパクをコードするDNA断片を、細胞を用いて発現させて選択する発現・選択工程と；を備える、目的分子のスクリーニング方法である。 In the present invention, (a) DNAs of target molecules are selected using a cell-free translation system to construct a library, and DNAs of the target molecules are selected from DNA fragments contained in the library and concentrated. (b) a protein that can be produced in an organism, among the proteins made from the target molecules contained in the primary library obtained in the selection/selection step; and an expression/selection step of expressing and selecting a DNA fragment encoding using a cell.
　前記選択・淘汰工程では、主鎖と側鎖とを有するcDNAディスプレイ用リンカーを使用することができる。ここで、前記リンカーの主鎖は、(m1)前記リンカーを固相へ固定するための固相結合部位と；(m2)固相から前記リンカーを切り離すための切断部位と；(m3)前記主鎖にmRNAを結合させるための光架橋塩基と；(m4)側鎖結合部位と；
　　(m5)逆転写開始部位と；を含み、前記側鎖は、(s1)前記主鎖に結合されたmRNAに対応して合成されたペプチドを結合するためのペプチド結合部位と；(s2)標識結合部位と；(s3)前記標識結合部位に結合する検出用蛍光分子と；(s4)前記主鎖の前記側鎖結合部位に結合する主鎖結合部位と；を備えることができる。 In the selection/selection step, a cDNA display linker having a main chain and a side chain can be used. Here, the main chain of the linker comprises (m1) a solid phase binding site for fixing the linker to a solid phase; (m2) a cleavage site for separating the linker from the solid phase; a photocrosslinking base for binding the mRNA to the strand; (m4) a side chain binding site;
(m5) a reverse transcription initiation site; wherein the side chain comprises (s1) a peptide binding site for binding a peptide synthesized corresponding to the mRNA bound to the main chain; and (s2) a label. (s3) a detectable fluorescent molecule that binds to said label binding site; and (s4) a backbone binding site that binds to said side chain binding site of said backbone.
　ここで、前記リンカーにおいて、前記固相結合部位は、ビオチン、ストレプトアビジン、又はその類縁体からなる群から選ばれる分子で構成され；前記切断部位は、リボＧ又はイノシンで構成され；前記光架橋塩基は、シアノビニルカルバゾール又はその類縁体で構成され；前記ペプチド結合部位は、ピューロマイシン又はその類縁体で構成され；前記検出用蛍光分子は、フルオレセイン、FITC（フルオレセインイソチオシアネート）、TET（テトラクロロフルオレセイン）、HEX（５'－ヘキサクロロ－フルオレセイン－ＣＥホスホロアミダイト）、Cy3、Cy5、Alexa568、Alexa647等からなる群から選ばれる。前記一次ライブラリは、1010～1014のライブラリサイズのcDNAライブラリから、前記リンカーを用いて構成することができる。 Here, in the linker, the solid phase binding site is composed of a molecule selected from the group consisting of biotin, streptavidin, or analogues thereof; the cleavage site is composed of ribo-G or inosine; The base is composed of cyanovinylcarbazole or an analogue thereof; The peptide binding site is composed of puromycin or an analogue thereof; The fluorescent molecules for detection are fluorescein, FITC (fluorescein isothiocyanate), TET (tetrachloro fluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568, Alexa647 and the like. Said primary library can be constructed using said linkers from a cDNA library with a library size of 10 10 -10 14 .
　前記選択・淘汰工程は、(a1)目的分子のmRNAを調製するmRNA調製工程と；(a2)300～365 nmの波長を15秒～60秒間照射して、前記(a1)工程で得られたmRNAと前記リンカーとを前記光架橋塩基で連結させて、リンカー－mRNA連結体を作製する連結工程と；(a3)前記リンカー－mRNA連結体を固相に結合させ、その後無細胞系で翻訳し、リンカー－mRNA－ペプチド連結体を形成する翻訳工程と；(a4)前記リンカー－mRNA－ペプチド連結体をPCRで増幅させ、逆転写してmRNA/cDNA－タンパク質連結体を得る逆転写工程と；(a5)前記増幅されたcDNAディスプレイ分子を前記固相から固相切断部位で切断し、一次ライブラリを得る、一次ライブラリ作製工程と；(a6)前記一次ライブラリに含まれる目的分子のDNAをセレクションして濃縮する濃縮工程と；を備えることができる。
　ここで、前記固相は、セファロースビーズ、シリカビーズ及びラテックスビーズからなる群から選ばれる磁性粒子とすることができる。 The selection/selection step includes (a1) an mRNA preparation step of preparing mRNA of a target molecule; (a3) binding the linker-mRNA conjugate to a solid phase, followed by translation in a cell-free system; , a translation step of forming a linker-mRNA-peptide conjugate; (a4) a reverse transcription step of amplifying the linker-mRNA-peptide conjugate by PCR and reverse transcription to obtain an mRNA/cDNA-protein conjugate; a5) a primary library preparation step of cleaving the amplified cDNA display molecules from the solid phase at a solid phase cleavage site to obtain a primary library; and (a6) selecting DNAs of target molecules contained in the primary library. a concentration step of concentrating;
Here, the solid phase can be magnetic particles selected from the group consisting of sepharose beads, silica beads and latex beads.
　前記前記選択・淘汰工程は、(b1)上記一次ライブラリ中のDNAをファージミドと混合してファージミドベクターを形成させるファージミドベクター形成工程と；(b2)前記ファージミドベクター中に目的分子の遺伝子断片を組み込み、前記細胞を形質転換させる形質転換工程と；(b3)前記形質転換工程で得られた細胞を培養し、ヘルパーファージを加えて上記細胞に感染させ、培養して上記目的分子を提示したファージを含むファージライブラリ作製工程と、(b4)前記ファージライブラリに含まれるファージをバイオパニングして上記目的分子と結合するファージを選択するバイオパニング工程と；を備えることができる。 The selection/selection step includes (b1) a phagemid vector forming step of mixing DNA in the primary library with a phagemid to form a phagemid vector; (b2) incorporating a gene fragment of a target molecule into the phagemid vector; (b3) culturing the cells obtained in the transforming step, adding a helper phage to infect the cells, and culturing the phage to display the target molecule. and (b4) a biopanning step of biopanning phages contained in the phage library to select phages that bind to the target molecule.
　前記細胞は、コンピテントセルとすることができる。また、前記コンピテントセルは、大腸菌とすることができる。さらに、前記（a1）～（a6）工程を繰り返す回数が５回以内とすることができる。 The cells can be competent cells. Also, the competent cell can be Escherichia coli. Furthermore, the number of times the steps (a1) to (a6) are repeated may be 5 times or less.
　本発明によれば、目的分子のcDNAを大規模ライブラリからスクリーニングして一次ライブラリを構築し、上記一次ライブラリ中で高速かつ高効率で選択・濃縮した二次ライブラリを得ることができ、かつ前記二次ライブラリから生物にとって有害な分子を除去しつつ、目的分子のcDNA分子を選択できるスクリーニング方法が提供される。 According to the present invention, it is possible to construct a primary library by screening cDNAs of target molecules from a large-scale library, to obtain a secondary library selected and enriched from the primary library at high speed and with high efficiency, and A screening method is provided that enables the selection of cDNA molecules of interest while removing molecules that are harmful to organisms from the next library.
　また、ファージELISAにより簡便に、標的分子と結合する候補の親和性を評価することができる。このため、短時間での精製が可能となり、現状では、生かし切れていないファージディスプレイの利点を十分に生かすことも可能となる。 In addition, phage ELISA can be used to easily evaluate the affinity of candidates that bind to target molecules. Therefore, purification in a short period of time becomes possible, and it becomes possible to make full use of the advantages of phage display, which are currently not fully utilized.
図１は、cDNAディスプレイ法によるヒット化合物スクリーニングのスキームを示す図である。FIG. 1 shows a scheme of hit compound screening by the cDNA display method. 図２は、スクリーニングラウンド毎の溶出法を示す図である。FIG. 2 shows the elution method for each screening round. 図３は、ファージディスプレイのスキームを示す図である。FIG. 3 shows a scheme of phage display. 図４は、VHHクローン＃１及びVHHクローン＃２のCDR領域を示す図である。FIG. 4 shows the CDR regions of VHH clone #1 and VHH clone #2. 図５は、VHHの精製の後に実施したSDS－PAGEの結果を示す電気泳動像である。FIG. 5 is an electropherogram showing the results of SDS-PAGE performed after purification of VHH. 図６は、VHHクローン＃１の標的分子FGFR1に対する親和性測定の結果を示すグラフである。FIG. 6 is a graph showing the results of affinity measurement for the target molecule FGFR1 of VHH clone #1. 図７は、VHHクローン＃２の標的分子FGFR1に対する親和性測定の結果を示すグラフである。FIG. 7 is a graph showing the results of affinity measurement for the target molecule FGFR1 of VHH clone #2.
　以下に、図面を参照しながら、本発明をさらに詳細に説明する。
　本発明の目的分子のスクリーニング方法は、(a)目的分子のDNAを選択し、濃縮して淘汰する、選択・淘汰工程と、(b)細胞を用いて発現させて選択する発現・選択工程とを備える。上記(a)の選択・淘汰工程では、(a1)一次ライブラリを構成し、次に、(a2)前記一次ライブラリから、前記目的分子のDNAを選択する工程と、を備える。 The present invention will be described in more detail below with reference to the drawings.
The method of screening for a target molecule of the present invention comprises (a) a selection/selection step of selecting, concentrating and selecting DNA of the target molecule, and (b) an expression/selection step of expressing and selecting using a cell. Prepare. The selection/selection step (a) includes (a1) constructing a primary library, and then (a2) selecting the DNA of the target molecule from the primary library.
　ここで、上記(a1)工程では、所定の構造のリンカーを使用する。本明細書において、「リンカー」とは、後述するcDNAディスプレイ法において用いられる、リンカー－mRNA連結体、リンカー－mRNA－タンパク質連結体、又はリンカー－mRNA/cDNA－タンパク質連結体（以下、「IVV」ということがある。）のいずれかを生成する際に使用するリンカーのことをいい、主鎖と側鎖とを有するcDNAディスプレイ用リンカーを意味する。前記リンカーは、全体として、柔軟性と親水性とを有するように、設計することが好ましい。 Here, in step (a1) above, a linker with a predetermined structure is used. As used herein, the term "linker" refers to a linker-mRNA conjugate, a linker-mRNA-protein conjugate, or a linker-mRNA/cDNA-protein conjugate (hereinafter referred to as "IVV") used in the cDNA display method described later. ), and means a linker for cDNA display having a main chain and a side chain. The linker as a whole is preferably designed to be flexible and hydrophilic.
　ここで、前記リンカーの主鎖は、3’方向から５’方向に向かって、(m1)固相結合部位と、（m2）固相切断部位と、（m3）光架橋塩基と、（m4）(m4)側鎖連結部位と、(m5)逆転写開始部位とを含む。前記固相結合部位(m1)は、前記cDNAディスプレイ用リンカーを固相に結合された分子を介して結合する部位である。 Here, the main chain of the linker has, from the 3′ direction to the 5′ direction, (m1) a solid phase binding site, (m2) a solid phase cleavage site, (m3) a photocrosslinking base, and (m4) It contains (m4) a side chain linking site and (m5) a reverse transcription initiation site. The solid phase binding site (m1) is a site that binds the cDNA display linker via a molecule bound to a solid phase.
　固相との結合を形成する分子としては、例えば、固相にアビジン及びストレプトアビジン等が結合されている場合にはビオチン又はその類縁体、マルトース結合タンパク質が結合されている場合にはマルトース、Ｇタンパク質が結合されている場合にはグアニンヌクレオチド、ポリヒスチジンペプチドが結合されている場合にはNi又はCo等の金属、グルタチオン－Ｓ－トランスフェラーゼが結合されている場合にはグルタチオンであることが好ましい。 Examples of molecules that form a bond with a solid phase include biotin or analogues thereof when avidin and streptavidin are bound to the solid phase, and maltose and G when maltose-binding protein is bound to the solid phase. Preferred are guanine nucleotides when proteins are bound, metals such as Ni or Co when polyhistidine peptides are bound, and glutathione when glutathione-S-transferase is bound.
　前記固相結合部位(m1)は、上述したmRNA－タンパク質連結体又はmRNA/cDNA－タンパク質連結体を、リンカーを介して固相に結合させるための部位である。前記固相結合部位(m1)は、例えば、上記のいずれか分子で構成されていることが固相と適度な間隔を維持することができ、後述する固相からの切り離しがうまくできることから好ましい。 The solid phase binding site (m1) is a site for binding the above-mentioned mRNA-protein conjugate or mRNA/cDNA-protein conjugate to a solid phase via a linker. The solid phase-binding site (m1) is preferably composed of, for example, any of the molecules described above because it can maintain an appropriate distance from the solid phase and can be successfully separated from the solid phase, which will be described later.
　前記固相切断部位(m2)は、部位特異的に切断される修飾アミノ酸で構成されていることが好ましく、こうした修飾アミノ酸としては、例えば、リボG、イノシン等を挙げることができる。リボGはリボヌクレアーゼで、またイノシンはエンドヌクレアーゼVでそれぞれ部位特的に切断されるからである。この工程で使用されるリンカーの固相切断部位は1箇所あれば十分である。 The solid-phase cleavage site (m2) is preferably composed of modified amino acids that are site-specifically cleaved, and examples of such modified amino acids include ribo-G and inosine. This is because ribonuclease G and inosine are site-specifically cleaved by ribonuclease V and endonuclease V, respectively. A single solid-phase cleavage site for the linker used in this step is sufficient.
　前記リンカーの主鎖は、その3’末端側近傍に側鎖結合部位(m3)を備えており、上記側鎖結合部位には、後述する側鎖が結合する。また、例えば、リンカーの側鎖が、Amino-Modifier C6 dTで構成されている場合には、前記側鎖の５'末端を5'-Thiol-Modifier C6として、EMCSを用いて架橋させ、主鎖と側鎖とを結合させることができる。 The main chain of the linker has a side chain binding site (m3) near its 3' end, and the side chain described later binds to the side chain binding site. Further, for example, when the side chain of the linker is composed of Amino-Modifier C6 dT, the 5' end of the side chain is 5'-Thiol-Modifier C6 and crosslinked using EMCS to and side chains.
　また、前記リンカーの主鎖は、主としてDNAで構成され、目的分子と結合し得るmRNAと相補的なヌクレオチド配列を有する。そして、この配列中に、ピリミジン塩基と架橋を形成するシアノビニルカルバゾールを含むことが、光の照射によって上記リンカーとmRNAとを架橋結合させることができるために好ましい。特に、上記シアノビニルカルバゾールが3-シアノビニルカルバゾール（以下、「cnvK」と略すことがある。）であることが、極めて短時間でmRNAとの光架橋を形成できることから好ましい。上記cnvKは、上記主鎖の固相断部位と側鎖結合部位との間に配置されている。 In addition, the main chain of the linker is mainly composed of DNA and has a nucleotide sequence complementary to mRNA capable of binding to the target molecule. It is preferable that this sequence contains cyanovinylcarbazole, which forms a crosslink with a pyrimidine base, so that the above linker and mRNA can be crosslinked by irradiation with light. In particular, the cyanovinylcarbazole is preferably 3-cyanovinylcarbazole (hereinafter sometimes abbreviated as "cnvK"), because photocrosslinking with mRNA can be formed in an extremely short time. The cnvK is located between the solid cleavage site and the side chain binding site of the main chain.
　さらに、逆転写部位は、上記リンカーの3’末端側に位置し、上記リンカー上で逆転写が行われる場合に逆転写用のプライマーとして機能する領域である。この逆転写部位は、15塩基を越えると、リンカーとしての結合効率が悪くなるため、約1～15塩基からなることが好ましい。リンカーとの結合効率及びプライマーとしての反応効率という面から、3～5塩基からなるものであることが好ましい。 Furthermore, the reverse transcription site is a region that is located on the 3' end side of the linker and functions as a primer for reverse transcription when reverse transcription is performed on the linker. If the reverse transcription site exceeds 15 bases, the binding efficiency as a linker becomes poor, so it is preferably composed of about 1 to 15 bases. From the viewpoint of binding efficiency with the linker and reaction efficiency as a primer, it preferably consists of 3 to 5 bases.
　上記リンカーの側鎖は、ペプチド結合部位(s1)と、標識結合部位(s2)と、検出用蛍光分子(s3)と、主鎖結合部位(s4)とを備えている。ここで、上記側鎖のペプチド結合部位(s1)は、主鎖に結合されたmRNAに対応して合成されたペプチドを結合する部位であり、ピューロマイシン又はその類縁体で構成されていてもよい。また、上記標識結合部位は、１以上のスペーサー配列で構成されていてもよく、蛍光分子が結合されていることが、形成されたcDNAディスプレイ分子の検出が容易になることから好ましい。 The side chain of the linker has a peptide binding site (s1), a label binding site (s2), a fluorescent molecule for detection (s3), and a main chain binding site (s4). Here, the side chain peptide-binding site (s1) is a site that binds a peptide synthesized corresponding to the mRNA bound to the main chain, and may be composed of puromycin or an analogue thereof. . In addition, the label-binding site may be composed of one or more spacer sequences, and is preferably bound with a fluorescent molecule because it facilitates detection of the formed cDNA display molecule.
　上記検出用蛍光分子（s3）は特に限定されないが、フルオレセイン又はフルオレセインイソチオシアネートアイソマー（以下、「FITC」と略すことがある。）その他のフルオレセイン誘導体であることが、取り扱いの容易さの点から好ましい。上記側鎖は、主鎖結合部位(s4)で、上記主鎖の側鎖結合部位と連結され、上述した通りの構造の主鎖と側鎖とを含むcDNAディスプレイ法用リンカー（以下、単に「リンカー」ということがある。）が形成される。 Although the fluorescent molecule for detection (s3) is not particularly limited, it is preferably fluorescein or fluorescein isothiocyanate isomer (hereinafter sometimes abbreviated as "FITC") or other fluorescein derivative from the viewpoint of ease of handling. . The side chain is linked to the side chain binding site of the main chain at the main chain binding site (s4), and is a linker for cDNA display method comprising the main chain and side chains having the structure as described above (hereinafter simply " Sometimes referred to as "linker".) is formed.
　本発明の(a)選択・淘汰工程では、ライブラリサイズが1010～1014である所望のイニシャルライブラリ（DNAライブラリ）を使用する。このサイズのライブラリは、他のディスプレイ法ではサイズが大きすぎて扱うことができず、cDNAディスプレイ法のみで扱うことができるからである。 In the (a) selection/selection step of the present invention, a desired initial library (DNA library) with a library size of 10 10 to 10 14 is used. This is because a library of this size is too large to be handled by other display methods and can be handled only by the cDNA display method.
　まず、常法に従って、上記イニシャルライブラリを転写し、このライブラリに含まれる各DNAをコードするmRNAを調製する。上記イニシャルライブラリには目的分子以外のDNAが膨大な量で含まれているため、それに対応して、ここで調製されたmRNAも目的分子以外のmRNAを膨大な量で含むことはいうまでもない。 First, according to a standard method, the above initial library is transcribed, and mRNA encoding each DNA contained in this library is prepared. Since the initial library contains a huge amount of DNA other than the target molecule, it goes without saying that correspondingly, the mRNA prepared here also contains a huge amount of mRNA other than the target molecule. .
　次いで、このmRNAと上記リンカーとを混合して混合物とし、所望の波長の光を所望の時間、例えば、360～400nmの波長の光を30秒～3分間、上記混合物に照射し、上記mRNAと上記リンカーとを光架橋によって結合させ、mRNA－リンカー結合体を得る。次いで、上記mRNA－リンカー結合体をcDNAディスプレイ装置に供して、cDNAディスプレイ分子であるIVVを作製する。 Next, this mRNA and the linker are mixed to form a mixture, and the mixture is irradiated with light of a desired wavelength for a desired time, for example, light of a wavelength of 360 to 400 nm for 30 seconds to 3 minutes, and the mRNA and The above linker is coupled by photocrosslinking to obtain an mRNA-linker conjugate. The mRNA-linker conjugate is then subjected to a cDNA display device to generate IVV, a cDNA display molecule.
　この装置内では、mRNA－リンカー結合体のmRNAを無細胞翻訳系で翻訳され、上記mRNAに対応するペプチドが上記リンカーのペプチド結合部位に結合され、mRNA－ペプチド－リンカー結合体が形成される。その後、mRNA－ペプチド－リンカー結合体に結合されたペプチドを逆転写して、上記逆転写部位にcDNAが連結されたmRNA/cDNA－ペプチド－リンカー連結体が形成される（図１参照）。 In this device, the mRNA of the mRNA-linker conjugate is translated in a cell-free translation system, and the peptide corresponding to the mRNA is bound to the peptide binding site of the linker to form the mRNA-peptide-linker conjugate. Thereafter, the peptide bound to the mRNA-peptide-linker conjugate is reverse transcribed to form an mRNA/cDNA-peptide-linker conjugate in which the cDNA is ligated to the reverse transcription site (see FIG. 1).
　その後、上記IVV中のペプチドを、固相に結合させた検出分子と混合し、上記検出分子と結合しないペプチドを含むIVVを、洗浄によって除去する。その後、固相に結合した分子を溶出させる。言い換えれば、IVVに含まれるDNAのうち、目的分子以外をコードするDNAが淘汰されて量が減少するため、目的分子をコードするDNAの量は相対的に増加することになる。この現象を目的分子の「濃縮」という。mRNAの調製から固相からの溶出までの一連の過程を、「ラウンド」といい、1stラウンド、2ndラウンドのようにいう。なお、上述したように、上記検出分子と結合しないペプチドを含むIVVの量は非常に多いため、これらは非特的吸着の原因ともなる。 Peptides in the IVV are then mixed with detection molecules bound to a solid phase, and IVV containing peptides not bound to the detection molecules are removed by washing. Molecules bound to the solid phase are then eluted. In other words, among DNAs contained in IVV, DNAs encoding molecules other than the target molecule are eliminated and the amount thereof decreases, so that the amount of DNAs encoding the target molecule increases relatively. This phenomenon is called "concentration" of the target molecule. A series of processes from preparation of mRNA to elution from the solid phase is referred to as "round", such as 1st round and 2nd round . In addition, as described above, the amount of IVV containing peptides that do not bind to the detection molecule is very large, and these also cause non-specific adsorption.
　引き続き、目的分子をコードするDNA分子を含む溶出液からmRNAを調製し、上記と同様にしてIVVを作製し濃縮を行う。以上のような操作を3～6回程度繰り返すことによって、目的分子以外のDNAの量を大幅に減少させることができるため、上記のようなラウンド中で形成されるペプチドに占める目的分子以外のペプチドの割合も、上記のDNAの量の減少に比例して減少させることができる。このことはまた、ペプチドをコードするDNAを濃縮することができることをも意味する。 Subsequently, mRNA is prepared from the eluate containing the DNA molecule encoding the target molecule, and IVV is produced and concentrated in the same manner as above. By repeating the above operation about 3 to 6 times, the amount of DNA other than the target molecule can be greatly reduced. can also be decreased in proportion to the decrease in the amount of DNA described above. This also means that the peptide-encoding DNA can be enriched.
　具体的には、本発明の方法を用いて、以下のようにして目的分子を得ることができる。
１．目的分子のスクリーニング
　cDNAディスプレイ法を用い、目的分子のcDNAを含むライブラリをスクリーニングして、一次ライブラリを作製する。目的分子として、例えば、ヒト線維芽細胞増殖因子受容体1～4（Fibroblast Growth Factor Receptor 1～4を、以下、「FGFR1～4」と略すことがある。）を使用することができる。
　また、上記目的分子を検出する分子（以下、「検出分子」という。）とは、上記の目的分子を検出するための分子をいうが、完全なタンパクのみならず、上記目的分子の検出に使用できるものである限り、その一部の領域であってもよい。例えば、上記目的分子としてFGFRを使用する場合には、その細胞外領域を検出分子として使用してもよい。こうした細胞外領域としては、FGFR1/Fc(以下、「FGFR-Fc」と略すことがある。）、FGF受容体1β(IIIc)/Fcキメラ（以下「FGFR-Fc」と略すことがある。）等を挙げることができる。これらを使用する場合には、上記細胞外領域は免疫グロブリン領域と呼ばれることから、陰性対照としては、例えば、IgG1 (Fc)(以下、「hFc」と略すことがある。）を使用することができる。 Specifically, using the method of the present invention, the target molecule can be obtained as follows.
1. Screening for Molecules of Interest A primary library is generated by screening a library containing cDNAs for molecules of interest using the cDNA display method. As target molecules, for example, human fibroblast growth factor receptors 1-4 (Fibroblast Growth Factor Receptors 1-4 may be abbreviated as "FGFR1-4" hereinafter) can be used.
A molecule for detecting the target molecule (hereinafter referred to as "detection molecule") refers to a molecule for detecting the target molecule. It may be a part of the region as long as it is possible. For example, when FGFR is used as the target molecule, its extracellular domain may be used as the detection molecule. Such extracellular domains include FGFR1/Fc (hereinafter sometimes abbreviated as "FGFR-Fc"), FGF receptor 1β(IIIc)/Fc chimera (hereinafter sometimes abbreviated as "FGFR-Fc"). etc. can be mentioned. When these are used, since the extracellular region is called an immunoglobulin region, for example, IgG1 (Fc) (hereinafter sometimes abbreviated as "hFc") can be used as a negative control. can.
（１）検出分子の固相化
　上記検出分子は、以下のようにして固定化して使用することが、操作効率の点から好ましい。例えば、検出分子としてのFGFR1－Fcを容器に入れ、その約15～約25当量のビオチン化試薬をその容器に加え、約20～約30℃にて約20～約40分間反応させて検出分子をビオチン化する。その後、脱塩カラム等を用いて、未反応のビオチン化試薬を除去する。こうしたビオチン化試薬としては、例えば、EZ－link sulfo NHS-SSビオチンを、また、脱塩カラムとしては、例えば、Zeba Spin Desalting Columns（いずれも、Thermo Fisher社製）を使用することができる (1) Immobilization of Detection Molecules From the viewpoint of operational efficiency, it is preferable to use the detection molecules after immobilization as follows. For example, FGFR1-Fc as the detection molecule is placed in a container, about 15 to about 25 equivalents of the biotinylation reagent is added to the container, and reacted at about 20 to about 30° C. for about 20 to about 40 minutes to obtain the detection molecule. is biotinylated. Thereafter, a desalting column or the like is used to remove unreacted biotinylation reagent. As such a biotinylation reagent, for example, EZ-link sulfo NHS-SS biotin can be used, and as a desalting column, for example, Zeba Spin Desalting Columns (both manufactured by Thermo Fisher) can be used.
（２）cDNAディスプレイ分子の合成
（２－１）重鎖抗体を用いた一次ライブラリの作製
　重鎖抗体を産生するラクダ科動物はとしては、ラクダ、アルパカ、ラマ等を挙げることができる。これらの動物から得られたナイーブライブラリとしては、アルパカ由来ナイーブVHHライブラリ（RePHAGEN社製）を挙げることができ、それらを用いて以下のようにして一次ライブラリを作製することができる。
それらを用いて以下のようにして一次ライブラリを作製することができる。
　例えば、上記ナイーブライブラリの特定の遺伝子、例えば、Sヒンジ及びLヒンジ遺伝子、を鋳型とし、下記表1に示す配列番号1～3で示すプライマーを使用してPCRを行い、上記ライブラリに含まれるDNAを増幅させることが、反応効率の点から好ましい。 (2) Synthesis of cDNA Display Molecules (2-1) Construction of Primary Library Using Heavy Chain Antibodies Examples of camelids that produce heavy chain antibodies include camels, alpacas, and llamas. Examples of naive libraries obtained from these animals include alpaca-derived naive VHH libraries (manufactured by RePHAGEN), which can be used to construct primary libraries as follows.
They can be used to generate a primary library as follows.
For example, specific genes of the above naive library, such as S-hinge and L-hinge genes, are used as templates, PCR is performed using the primers represented by SEQ ID NOS: 1 to 3 shown in Table 1 below, and the DNA contained in the above library is obtained. is preferable from the viewpoint of reaction efficiency.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　引き続き、例えば、上記配列番号4及び5で表される配列のプライマーを用いてオーバーラップPCRを行うことによって、全長VHHコード化DNAライブラリ（以下、「全長ライブラリ」という。）を作製することができる。オーバーラップPCR条件は、例えば、下記の通りとすることが、反応効率の点から好ましい。PCR条件：98℃で２分間の後、98℃で10秒、６5℃で５秒、72℃で35秒を５サイクル行い、72℃で１分反応させた後に10℃まで冷却した。
　ここで、上記全長ライブラリは、例えば、プロモーター、エンハンサー、翻訳開始に関与する配列、所望のVHH遺伝子、精製用タグ、リンカーハイブリダイゼーション領域等を含むものとすることができ、具体的には、T7プロモーター、オメガ（Ω）エンハンサー、コザック配列、所望のVHH遺伝子、Hisタグ、およびYタグ等を含むものを挙げることができる。 Subsequently, for example, overlap PCR is performed using the primers of the sequences represented by SEQ ID NOS: 4 and 5 above to prepare a full-length VHH-encoding DNA library (hereinafter referred to as "full-length library"). . Overlap PCR conditions, for example, are preferably as follows from the viewpoint of reaction efficiency. PCR conditions: After 2 minutes at 98°C, 5 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 35 seconds were performed, followed by reaction at 72°C for 1 minute and cooling to 10°C.
Here, the full-length library can contain, for example, a promoter, an enhancer, a sequence involved in translation initiation, a desired VHH gene, a purification tag, a linker hybridization region, etc. Specifically, the T7 promoter, Examples include those containing omega (Ω) enhancers, Kozak sequences, desired VHH genes, His-tags, Y-tags, and the like.
（２－２）cDNAディスプレイ用リンカーの調製
　cDNAディスプレイ法では、例えば、主鎖と側鎖とを有するリンカーを使用することが、所望のVHHのcDNA、対応するmRNA及びタンパク(VHH)を１つのセットとして取得できることから好ましい。具体的には、このリンカーの主鎖は、5’末端に固相結合部位としてのBioTEGを含み、その近傍に固相からの切断部位としてのリボG（以下、「rG」と略すことがある。）又はイノシン（以下、「I」と略すことがある。）を含み、mRNA連結部位としてのシアノビニルカルバゾールを備えていることが好ましい。上記主鎖の配列の一例を、下記配列番号6に示す。以下、この主鎖を「ビオチンフラグメント」ということがある。 (2-2) Preparation of linkers for cDNA display In the cDNA display method, for example, a linker having a main chain and a side chain is used to combine the cDNA of the desired VHH, the corresponding mRNA and the protein (VHH) into one. It is preferable because it can be obtained as a set. Specifically, the main chain of this linker contains BioTEG as a solid-phase binding site at the 5' end, and ribo-G (hereinafter sometimes abbreviated as "rG") as a cleavage site from the solid phase in the vicinity thereof. ) or inosine (hereinafter sometimes abbreviated as “I”), and preferably has cyanovinylcarbazole as an mRNA-ligating site. An example of the main chain sequence is shown in SEQ ID NO: 6 below. Hereinafter, this main chain may be referred to as "biotin fragment".
　なお、下記の配列中、5’末端から3番目のNはグアノシンを、また、24番目のNはAmino C6-dTをそれぞれ示す。Kは3-シアノビニルカルバゾール（以下、「cnvK」ということがある。）を表す。以下、下記配列番号6で表される主鎖を有するリンカーを、本明細書中では、「cnvK rG Linker」という。 In the sequence below, the 3rd N from the 5' end indicates guanosine, and the 24th N indicates Amino C6-dT. K represents 3-cyanovinylcarbazole (hereinafter sometimes referred to as "cnvK"). Hereinafter, the linker having the main chain represented by SEQ ID NO: 6 below is referred to as "cnvK rG Linker" in the present specification.
　5’－AANAATTTCCAKGCCGCCCCCCGNCCT －3’　　　　　　（配列番号6） 　5'-AANAATTTCCAKGCCGCCCCCCGNCCT -3' (sequence number 6)
　また、側鎖は、その遊離末端にペプチド結合部位（上記側鎖配列中の「P」）としてピューロマイシン又はその類縁体を含むことから、「ピューロマイシンセグメント」ということがある。上記側鎖は、5’(5S)TCTFZZCCPの配列を有していることが、後述するcDNAディスプレイ分子の形成効率の面から好ましい。また、「(5S)」は5' Thiol C6を、「F」は蛍光基であるFITC-dTを、そして「Z」は Spacer 18をそれぞれ表す。上記の主鎖及び側鎖の化学合成は、常法に従って行ってもよく、また、つくばオリゴサービス（株）その他の企業に製造を委託してもよい。 In addition, the side chain is sometimes referred to as a "puromycin segment" because it contains puromycin or an analog thereof as a peptide binding site ("P" in the above side chain sequence) at its free end. The side chain preferably has a 5'(5S)TCTFZZCCP sequence from the viewpoint of efficiency of formation of the cDNA display molecule described below. Also, "(5S)" represents 5' Thiol C6, "F" represents the fluorescent group FITC-dT, and "Z" represents Spacer 18, respectively. The chemical synthesis of the main chain and side chains may be carried out according to a conventional method, or may be outsourced to Tsukuba Oligo Service Co., Ltd. or other companies.
　所定の溶液中で上記ビオチンフラグメントとEMCS（（株）同仁化学研究所製)をインキュベートして結合させ、上記ビオチンフラグメント－EMCS結合体をエタノール沈殿させる。このエタノール沈殿には、例えば、Quick-Precip Plus Solution (Edge BioSystems社製)等を使用してもよい。 The biotin fragment and EMCS (manufactured by Dojindo Laboratories) are incubated in a predetermined solution to bind, and the biotin fragment-EMCS conjugate is precipitated with ethanol. For this ethanol precipitation, for example, Quick-Precip Plus Solution (manufactured by Edge BioSystems) or the like may be used.
　次にピューロマイシン－セグメントをリン酸水素二ナトリウムバッファーに溶解し、シェーカーを用いて撹拌し、次いで還元処理を行うことにより、上記還元ピューロマイシン－セグメント含有溶液を得ることができる。 Next, the puromycin-segment is dissolved in a disodium hydrogen phosphate buffer, stirred using a shaker, and then subjected to reduction treatment to obtain the reduced puromycin-segment-containing solution.
　例えば、上記還元ピューロマイシン－セグメント含有溶液を、上記のEMCS修飾済みビオチンフラグメント（エタノール沈殿物）と混合し、所望の温度で所望の時間、例えば、約2～6℃で一晩放置して結合させ、cnvK rG リンカーを形成させる。続いて、上記リンカーの分子内架橋の形成を防ぐために還元処理鵜を行う。例えば、ジチオスレイトール（以下、「DTT」と略すことがある。）を終濃度25～75 mMとなるように上記反応液に加え、室温で20～40分間撹拌して還元処理を行ってもよい。
　その後、上記フラグメントの結合体を、上記と同様にエタノール沈殿物として得ることができる。得られたエタノール沈殿物を、精製するために、所望の溶液に溶解することが好ましい。例えば、約50～約150μLのヌクレアーゼフリー水（ナカライテスク（株）製）等に溶解させて溶解産物としてもよい。 For example, the reduced puromycin-segment-containing solution is mixed with the EMCS-modified biotin fragment (ethanol precipitate) and left at the desired temperature for the desired time, eg, about 2-6° C. overnight to bind. to form the cnvK rG linker. Subsequently, reduction treatment is performed to prevent the formation of intramolecular crosslinks of the linker. For example, dithiothreitol (hereinafter sometimes abbreviated as "DTT") may be added to the above reaction solution to a final concentration of 25 to 75 mM and stirred at room temperature for 20 to 40 minutes to perform reduction treatment. good.
A conjugate of the above fragments can then be obtained as an ethanol precipitate in the same manner as above. The ethanol precipitate obtained is preferably dissolved in the desired solution for purification. For example, it may be dissolved in about 50 to about 150 μL of nuclease-free water (manufactured by Nacalai Tesque, Inc.) to obtain a lysate.
　上記溶解産物を、ポリアクリルアミドゲル電気泳動（以下、「PAGE」と略すことがある。）に供して分離し、cnvK rG Linkerを含むバンドを切り出し、切り出したゲルを破砕して、cnvK rGリンカーを抽出する。次いで、上記の抽出液を遠心チューブフィルターに移し遠心してゲルを分離し、その後エタノール沈殿を行ってcnvK rGリンカーを得ることができる。こうしたチューブフィルターとしては、Costar（登録商標） Spin-X（登録商標）（孔径0.22μmセルロースアセテート（Corning社製））等を挙げることができる。 The above-mentioned lysate was separated by subjecting it to polyacrylamide gel electrophoresis (hereinafter sometimes abbreviated as "PAGE"), the band containing the cnvK rG Linker was excised, and the excised gel was crushed to remove the cnvK rG linker. Extract. Next, the above extract is transferred to a centrifugal tube filter and centrifuged to separate the gel, followed by ethanol precipitation to obtain the cnvK rG linker. Examples of such a tube filter include Costar (registered trademark) Spin-X (registered trademark) (cellulose acetate with a pore size of 0.22 μm (manufactured by Corning)) and the like.
２．cDNAディスプレイ分子の作製
　一次ライブラリ作成用のcDNAディスプレイ分子は、下記表3に示すような組成のバッファーを用いて、以下のように調製することができる。
（１）転写
　上記１．（２）で作製したcDNAディスプレイ分子合成用DNAを転写し、得られた転写産物（mRNA）精製し、定量する。ここでは、例えば、2ｘ結合バッファー、結合（セレクション）バッファー、Hisタグ結合/洗浄バッファー、Hisタグ溶出バッファー（イミダゾール濃度が200～300mMである点を除き、Hisタグ結合/洗浄バッファーと同じ）、セレクションバッファー等を使用することができる。 2. Preparation of cDNA Display Molecules cDNA display molecules for primary library preparation can be prepared as follows using buffers with compositions as shown in Table 3 below.
(1) Transcription 1. The DNA for cDNA display molecule synthesis prepared in (2) is transcribed, and the resulting transcription product (mRNA) is purified and quantified. Here, for example, 2x binding buffer, binding (selection) buffer, His-tag binding/washing buffer, His-tag elution buffer (same as His-tag binding/washing buffer except imidazole concentration is 200-300 mM), selection A buffer or the like can be used.
　上記の転写は、例えば、T7 RiboMAX Express Large Scale RNA Production System(Promega社製)を使用し、添付のプロトコルに従って行うことができる。cDNAディスプレイ法では、目的分子以外の含有量が、ラウンドが進むにつれて減少するため、新たなラウンドに入るときに使用するDNAの量をより少量にすることができる。例えば、第1ラウンドで約5～7μｇ、第2ラウンド以降は約0.05～約2μｇとすることができる。各ラウンド中で得られた転写産物は精製後に定量するが、例えば、RNaClean XP(Beckman Coulter社製)等を使用し、それら添付されたプロトコル従って精製し、その後、NanoPad DS-11FX(DeNovix)等を用いて精製した転写産物の濃度を定量するようにしてもよい。 The above transcription can be performed, for example, using the T7 RiboMAX Express Large Scale RNA Production System (manufactured by Promega) according to the attached protocol. In the cDNA display method, the content of molecules other than the target molecule decreases as the round progresses, so the amount of DNA used at the start of a new round can be reduced. For example, about 5-7 μg in the first round, and about 0.05 to about 2 μg in the second and subsequent rounds. Transcripts obtained in each round are quantified after purification. may be used to quantify the concentration of purified transcripts.
（２）ライゲーション
　次いで、上記で精製したmRNAを上記cnvK rGリンカーとバッファー中で等量で混合し、アニールさせた後に光架橋を形成させ、連結してmRNA－リンカーを得る。mRNAの３’末端側をcnvK rGリンカーにハイブリダイズさせる手順としては、例えば、それぞれ等量の上記精製mRNA及びcnvK rGリンカーを、トリスバッファーに加えて、約88～92℃で0.5～1.5分間インキュベートし、次いで、約0.05～0.15℃／秒の速度で65～75℃まで降温させ、65～75℃で0.5～1.5分間インキュベートする。引き続き、上記同様の速度で約20～約30℃まで降温させた後に、降温速度を1.5～2.5℃／秒の速度に上げて、約5～15℃まで降温させることができる。引き続き、これらを光架橋するために、例えば、Handheld UV Lamp (6W, UVGL-56, 254/365 nm、100V (Analytik jena US, An Endress + Hauser Company))を使用して、350～370nmのUVを2～8分間照射するようにしてもよい。 (2) Ligation Next, the mRNA purified above is mixed with the above cnvK rG linker in equal amounts in a buffer, annealed, photocrosslinked, and ligated to obtain an mRNA-linker. As a procedure for hybridizing the 3' end side of mRNA to the cnvK rG linker, for example, equal amounts of the above purified mRNA and cnvK rG linker are added to Tris buffer and incubated at about 88 to 92°C for 0.5 to 1.5 minutes. and then cooled to 65-75°C at a rate of about 0.05-0.15°C/sec and incubated at 65-75°C for 0.5-1.5 minutes. Subsequently, after the temperature is lowered to about 20 to about 30°C at the same speed as above, the temperature lowering rate can be increased to 1.5 to 2.5°C/sec to lower the temperature to about 5 to 15°C. In order to subsequently photocrosslink these, for example, using a Handheld UV Lamp (6W, UVGL-56, 254/365 nm, 100V (Analytik jena US, An Endress + Hauser Company)) UV at 350-370 nm. may be irradiated for 2 to 8 minutes.
（３）mRNAディスプレイ分子の調製
　次に、所望のスケールの無細胞翻訳を用いて、以下の手順でmRNAディスプレイ分子を調製する。例えば、約3～約8 pmolの上記mRNA－リンカーを、25～100μLスケールの無細胞翻訳系(Rabbit reticulocyte Lysate (ヌクレアーゼ処理済)、Promega社製）に加え、約25～約35℃で15～45分間インキュベートし、ここに、塩を所望の濃度で加える。例えば、終濃度で50～100 mMのMgCl2、及び終濃度で800～1,000 mM のKClを加えることができる。次いで、この混合物を約36～約38℃にて0.5～1.5時間インキュベートし、上記mRNAに対応するペプチドを上記リンカーのペプチド結合部位に提示させることができる。次いで、リンカー中のmRNAに結合しているリボソームを除去するために、EDTA等のキレーターを、終濃度で50～100 mMとなるように加えて、2～6℃にて1～10分間インキュベートし、mRNAディスプレイ分子を形成させることができる。 (3) Preparation of mRNA display molecule Next, using a desired scale of cell-free translation, an mRNA display molecule is prepared by the following procedure. For example, about 3 to about 8 pmol of the above mRNA-linker is added to a 25 to 100 μL scale cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), manufactured by Promega), and the mixture is heated to about 25 to about 35°C for 15 to 15 minutes. Incubate for 45 minutes, to which salt is added at the desired concentration. For example, a final concentration of 50-100 mM MgCl 2 and a final concentration of 800-1,000 mM KCl can be added. The mixture can then be incubated at about 36° C. to about 38° C. for 0.5-1.5 hours to allow the peptide corresponding to the mRNA to be presented at the peptide binding site of the linker. Next, in order to remove ribosomes bound to the mRNA in the linker, a chelator such as EDTA was added to a final concentration of 50-100 mM and incubated at 2-6°C for 1-10 minutes. , can form an mRNA display molecule.
（４）cDNAディスプレイ分子の調製
　上記mRNAディスプレイ分子を用いて、以下のようにしてcDNAディスプレイ分子を形成させる。固相をチューブに取ってバッファーで洗浄し、その後、上記mRNA分子に提示されているペプチドを逆転写し、mRNA／cDNA―ペプチド連結体（以下、「cDNAディスプレイ分子」という。）を固相上で調製する。このcDNAディスプレイ分子は、酵素切断部位を切断する酵素によって、固相から切り離すことができる。 (4) Preparation of cDNA display molecule Using the above mRNA display molecule, a cDNA display molecule is formed as follows. The solid phase is placed in a tube and washed with a buffer. After that, the peptide displayed on the mRNA molecule is reverse transcribed to form an mRNA/cDNA-peptide conjugate (hereinafter referred to as "cDNA display molecule") on the solid phase. Prepare. This cDNA display molecule can be released from the solid phase by an enzyme that cleaves the enzymatic cleavage site.
　固相としては、例えば、プロテインローバインディングチューブに、Dynabeads Myone Streptavidin C1（ThermoFischer Scientific社製、以下、単に「Dynabeads」ということがある。）を使用することができる。このDynabeadsをチューブに取って、ここに適量の結合バッファーを加えて洗浄し、その後、上記mRNAディスプレイ分子を加える。このチューブを約20～約30℃にて15～45分間撹拌して固相化し、上記同様に結合バッファーで洗浄する。その後、逆転写のために、逆転写用反応液（1～3μLの25mM dNTP Mix 及び0.5～2μLのGeneAce Reverse Transcriptase (100～300U/μL) (いずれもNIPPON GENE社製)）をここに加え、約40～45℃にて15～45分間インキュベートして反応を行い、cDNAディスプレイ分子を得ることができる。この後、上記cDNAディスプレイ分子を固相から溶出させるために、適量の結合バッファーで上記cDNAディスプレイ分子を洗浄し、その後、Hisタグ結合／洗浄バッファー及びRNase T1を加えて撹拌しつつインキュベートする。 As the solid phase, for example, Dynabeads Myone Streptavidin C1 (manufactured by ThermoFischer Scientific, hereinafter sometimes simply referred to as "Dynabeads") can be used for protein low binding tubes. Take the Dynabeads in a tube, add an appropriate amount of binding buffer to wash, and then add the mRNA display molecule. This tube is stirred at about 20 to about 30° C. for 15 to 45 minutes to solidify and washed with the binding buffer in the same manner as above. After that, for reverse transcription, a reaction solution for reverse transcription (1-3 μL of 25 mM dNTP Mix and 0.5-2 μL of GeneAce Reverse Transcriptase (100-300 U/μL) (both manufactured by NIPPON GENE)) is added here, The reaction can be performed by incubating at about 40-45° C. for 15-45 minutes to obtain the cDNA display molecule. After this, in order to elute the cDNA display molecules from the solid phase, the cDNA display molecules are washed with an appropriate amount of binding buffer, followed by the addition of His-tag binding/wash buffer and RNase T1 and incubation with agitation.
（５）一次ライブラリの調製
　cDNA ライブラリを作製するために、固相に上記cDNA分子を結合させて所望のバッファーで洗浄し、その後固相から洗浄したcDNAディスプレイ分子を溶出させることによって、一次ライブラリを得ることができる。例えば、エッペンドルフチューブに、His Mag Sepharose Ni Beads (GE Health Care社製造)を入れ、このビーズを上記（４）と同様にバッファーで洗浄し、その後、上記（４）と同様にしてバッファーを用いて洗浄して精製する。その後、上記のようにcDNAディスプレイ分子を溶出させるために、約25～35μLのHisタグ溶出バッファーを加えて、撹拌する。以上の操作によって、一次ライブラリ（図2中、Initialと表示されているライブラリ）として、cDNAライブラリを得ることができる。 (5) Preparation of Primary Library To generate a cDNA library, a primary library is prepared by binding the above cDNA molecules to a solid phase, washing with a desired buffer, and then eluting the washed cDNA display molecules from the solid phase. Obtainable. For example, put His Mag Sepharose Ni Beads (manufactured by GE Health Care) in an Eppendorf tube, wash the beads with a buffer in the same manner as in (4) above, and then wash the beads with a buffer in the same manner as in (4) above. Wash and purify. About 25-35 μL of His-tag elution buffer is then added and vortexed to elute the cDNA display molecules as described above. Through the above operations, a cDNA library can be obtained as a primary library (library indicated as Initial in FIG. 2).
３．目的分子のセレクション
（１）リガンドと結合する目的分子のセレクション
　上記一次ライブラリを用いて、リガンドと結合する目的分子を取得するために、以下の手順でセレクションを行う。各セレクションラウンドにおける目的分子提示cDNAディスプレイライブラリの合成スケールは、ラウンドが進むにつれて減少させることができ（例えば、後述する表5参照）、これによって、目的分子を濃縮することができる。セレクションサイクルにおいて固相と結合させた溶出方法は、それら目的分子の活性に影響を与えない限りとくに限定されず、競合溶出、TCEP(トリス(2－カルボキシエチル)ホスフィン)）等を含むバッファーを用いる溶出を行うことができる。こうした溶出の一例を図2フローチャートして示す。 3. Selection of Target Molecules (1) Selection of Target Molecules that Bind to Ligand Using the primary library described above, selection is performed in the following procedure to obtain target molecules that bind to ligands. The scale of synthesis of the target molecule-displaying cDNA display library in each round of selection can be decreased as the round progresses (see, eg, Table 5 below), thereby enriching the target molecule. The elution method for binding to the solid phase in the selection cycle is not particularly limited as long as it does not affect the activity of the target molecule, and a buffer containing competitive elution, TCEP (tris(2-carboxyethyl)phosphine), etc. is used. Elution can be performed. An example of such elution is shown in the flow chart of FIG.
（１－１）セレクショラウンド1（以下、単に「R1」ということがある）
　所望量のDynabeadsをチューブに入れ、検出分子をここに加えて所望の温度で所望の時間インキュベートして上記検出分子を固相化する。ここに、上記cDNAディスプレイ分子を加えて上記検出分子と結合させ、洗浄後に溶出させる。
　例えば、検出分子として、ビオチン化FGFR1－Fcを16μL上記チューブに加え、約20～約30℃にて15～45分間転倒混和することにより、FGFR－Fcを固相化し、検出分子固相化ビーズを得ることができる。 (1-1) Selection Round 1 (hereinafter sometimes simply referred to as “R1”)
A desired amount of Dynabeads is placed in a tube, a detection molecule is added thereto, and incubated at a desired temperature for a desired time to immobilize the detection molecule. Here, the cDNA display molecule is added, bound to the detection molecule, and eluted after washing.
For example, as a detection molecule, 16 μL of biotinylated FGFR1-Fc is added to the above tube and mixed by inversion at about 20 to about 30° C. for 15 to 45 minutes to immobilize FGFR-Fc and immobilize the detection molecule. Beads can be obtained.
　その後、このチューブに所望のバッファー、例えば、セレクションバッファーをこの加えて数回洗浄し、次いで以下の溶出を行うことができる。溶出バッファーを使用する場合には、上記検出分子と結合したcDNAディスプレイ分子を、上記結合分子のSS結合を切断することにより溶出させる。
　この後、上記固相化ビーズを入れたチューブに、NaOHアルカリを加えてインキュベートするアルカリ溶出操作を所望の回数行い、得られたアルカリ溶液を中和する。得られた上清は、上記アルカリ溶出液と混合し、精製用試料とし、その一部を取ってPCR用試料とする。 The tube can then be washed several times with the desired buffer, eg, selection buffer, followed by the following elution. If an elution buffer is used, the cDNA display molecule bound to the detection molecule is eluted by cleaving the SS bond of the binding molecule.
After that, an alkali elution operation of adding NaOH alkali to the tube containing the immobilized beads and incubating is performed a desired number of times to neutralize the obtained alkali solution. The resulting supernatant is mixed with the above-mentioned alkaline eluate to prepare a sample for purification, and a portion thereof is taken to prepare a sample for PCR.
　例えば、上記のPCR用試料中に含まれるcDNAを鋳型とし、cnvK NewYtag for poly A（配列番号7）及びT7 omega new（配列番号8）をプライマーとして用いて（下記表2参照）、アニーリング温度65～70℃、伸長反応20～40秒の条件でPCRを行うことができる。得られたPCR産物を、8M尿素3～5％変性PAGEに供することができる。また、上記PCR産物を精製し、得られた精製物をR1ライブラリとしてセレクションの2ndラウンドに供することができる。こうした精製には、例えば、AMPure XP(Beckman Coulter社製)を使用することができる。以下、同様の手順を繰り返して、所望のラウンド数のセレクションを行うことができる。 For example, using the cDNA contained in the above PCR sample as a template, cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers (see Table 2 below), annealing temperature 65 PCR can be performed under conditions of ~70°C and extension reaction for 20-40 seconds. The resulting PCR product can be subjected to 8M urea 3-5% denaturing PAGE. Alternatively, the above PCR product can be purified, and the resulting purified product can be subjected to the second round of selection as an R1 library. For such purification, for example, AMPure XP (manufactured by Beckman Coulter) can be used. A desired number of rounds can be selected by repeating the same procedure.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　なお、R2以降では、必要に応じて、上記セレクションバッファーを用いた競合溶出を行い、次のラウンドに供する試料を調製するようにしてもよい。具体的には、上記固相（ビーズ）を加えたチューブに、セレクションバッファーをそれぞれ加え、各チューブ中のビーズを洗浄する。その後、再度セレクションバッファーをここに加えて洗浄し、その後、別の検出分子、例えば、FGFR1－His、を加えて転倒混和し、その後溶出する。得られた溶出液をPCRで増幅させ、精製後、ライブラリとして使用しても良い。なお、R2以降で使用するライブラリは、TCEP溶出した溶液を混合して作製してもよい。 From R2 onwards, if necessary, competitive elution using the above selection buffer may be performed to prepare samples for the next round. Specifically, a selection buffer is added to each tube to which the solid phase (beads) has been added, and the beads in each tube are washed. After that, the selection buffer is added again for washing, after which another detection molecule such as FGFR1-His is added, mixed by inversion, and then eluted. The obtained eluate may be amplified by PCR and used as a library after purification. The library used in R2 and later may be prepared by mixing the TCEP eluted solution.
４．ファージディスプレイ法を用いた目的分子のスクリーニング
　上記DNAライブラリ（以下、「濃縮ライブラリ」ということがある。）を、ファージディスプレイ法を用いてさらに濃縮し、同時に、大腸菌で発現可能な目的分子の取得を試みることができる。ファージディスプレイ法を図3に模式的に示す。 4. Screening of Target Molecules Using Phage Display Method The above DNA library (hereinafter sometimes referred to as "enriched library") is further enriched using phage display methods, and at the same time target molecules that can be expressed in E. coli can be obtained. can try. The phage display method is shown schematically in FIG.
（１）ファージミド及び各DNAライブラリの制限酵素処理
　まず、上記ライブラリに、ファージミドベクターとのライゲーション用の制限酵素認識配列を付加する。上記各ライブラリを鋳型とし、例えば、Alp-to-pPK4-Nco-Sfi-VHH-F（フォラードプライマー、配列番号11）及びAlp-GGGS-HisTag-to-pPK4-R（リバースプライマー）（配列番号12）を用いて、アニーリング温度50～60℃、伸長反応45～75秒の条件でPCRを行い、得られた増幅産物を精製する。この精製には、AMPure XP(Beckman社製)等を使用することができる。 (1) Restriction Enzyme Treatment of Phagemid and Each DNA Library First, a restriction enzyme recognition sequence for ligation with a phagemid vector is added to the library. Using each of the above libraries as a template, for example, Alp-to-pPK4-Nco-Sfi-VHH-F (Forard primer, SEQ ID NO: 11) and Alp-GGGS-HisTag-to-pPK4-R (reverse primer) (SEQ ID NO: 12), perform PCR under the conditions of annealing temperature of 50-60°C and elongation reaction of 45-75 seconds, and purify the resulting amplified product. AMPure XP (manufactured by Beckman) or the like can be used for this purification.
　引き続き、ファージミドベクターを得るために、ファージミド及び各DNAライブラリを制限酵素処理する。例えば、FastDigest BamHIを加え、約36～約38℃にて45分～90分間反応させ、その後FastDigest SfiI（いずれの制限酵素もThermo Fisher Scientific社製）を加え、45～55℃にて45分～90分間反応させる。得られたファージミドベクターの精製には、例えば、AMPure XPを使用することができる。 Subsequently, the phagemid and each DNA library are treated with restriction enzymes to obtain phagemid vectors. For example, add FastDigest BamHI and react at about 36 to about 38°C for 45 to 90 minutes, then add FastDigest SfiI (both restriction enzymes are manufactured by Thermo Fisher Scientific) and heat at 45 to 55°C for 45 minutes to Incubate for 90 minutes. For example, AMPure XP can be used to purify the resulting phagemid vector.
　得られたファージミドベクターをゲル電気泳動によって精製し、脱リン酸後に酵素を失活させる。例えば、1xGel green（富士フイルム和光純薬(株)）を含む1％アガロースゲルに上記ファージミドベクターをロードし、100Vで20～40分間泳動させる。その後、ゲルから目的のファージミドベクターを抽出し精製して、精製ファージミドベクターを得ることができる。この抽出及び精製には、例えば、FastGene Gel/PCR Extraction Kit（日本ジェネティクス(株)）を使用してもよい。この精製ファージミドベクターに、例えば、FastAP Thermosensitive Alkaline Phosphatase（Thermo Fisher Scientific社製）を加えて脱リン酸し、その後、酵素を失活させる。 The resulting phagemid vector is purified by gel electrophoresis, and the enzyme is deactivated after dephosphorylation. For example, the above phagemid vector is loaded onto a 1% agarose gel containing 1xGel green (Fuji Film Wako Pure Chemical Industries, Ltd.) and electrophoresed at 100 V for 20 to 40 minutes. Thereafter, the phagemid vector of interest can be extracted from the gel and purified to obtain a purified phagemid vector. For this extraction and purification, for example, FastGene Gel/PCR Extraction Kit (Nippon Genetics Co., Ltd.) may be used. For example, FastAP Thermosensitive Alkaline Phosphatase (manufactured by Thermo Fisher Scientific) is added to this purified phagemid vector to dephosphorylate and then deactivate the enzyme.
　次いで、上記ファージミドベクターと制限酵素処理したライブラリ（目的分子をコードする遺伝子断片）とを、１：5～10（モル比）で混合して反応させ、上記目的分子の遺伝子断片が組み込まれたファージミドベクターを得ることができる。この遺伝子の組み込みには、例えば、Ligation high Ver. 2 (TOYOBO社製)を用いることができ、約10～約20℃にて一晩反応させればよい。得られたファージミドベクターで大腸菌を形質転換する。この形質転換は、エレクトロポレーション等によって行うことができる。上記大腸菌は、ファージディスプレイ用コンピテントセルであれば特に限定されず、例えば、大腸菌TG－1（Lucigen社製）等を使用することができる。 Next, the phagemid vector and the restriction enzyme-treated library (gene fragment encoding the target molecule) are mixed at a ratio of 1:5 to 10 (molar ratio) and reacted to obtain a phagemid into which the gene fragment of the target molecule is integrated. vector can be obtained. For example, Ligation high Ver. 2 (manufactured by TOYOBO) can be used for this gene integration, and the reaction can be performed overnight at about 10 to about 20°C. E. coli is transformed with the obtained phagemid vector. This transformation can be performed by electroporation or the like. The E. coli is not particularly limited as long as it is a competent cell for phage display, and for example, E. coli TG-1 (manufactured by Lucigen) can be used.
　形質転換したTG－1を寒天培地プレートに播種し、約28～約32℃にて一晩、5％CO2インキュベータ中で培養し、培地上に大腸菌のコロニーを形成させることができる。これらのコロニーをすべてピッキングして回収し、O.D.600が所望の値になるまで上記度同様の条件で培養してもよい。この培養終了後に大腸菌数を計数し、その20倍量のヘルパーファージをここに加えた。ファージミドが増殖するためには、ヘルパーファージが必要だからである。ヘルパーファージを加えた後に、上記と同様の条件で培養し、培養液を遠心分離して上清を回収する。
　回収した上記上清を新たなチューブに取り、ここに2.5MのNaCl含有15～25％PEG溶液を添加し、転倒混和後に氷上にて冷却する。再度遠心することにより、上記目的分子を提示したファージが沈殿物として得られ、これを5～15％グリセロール含有PBS(Phosphate Buffered Saline)で溶解し、ファージディスプレイ溶液とする。 Transformed TG-1 can be seeded onto agar plates and cultured overnight at about 28 to about 32° C. in a 5% CO 2 incubator to allow E. coli colonies to form on the medium. All these colonies may be collected by picking and cultured under the same conditions as above until the OD600 reaches the desired value. After completion of this culture, the number of E. coli was counted, and 20 times the amount of helper phage was added here. This is because a helper phage is required for phagemid propagation. After adding the helper phage, the cells are cultured under the same conditions as above, and the culture solution is centrifuged to collect the supernatant.
The collected supernatant is transferred to a new tube, a 15-25% PEG solution containing 2.5 M NaCl is added thereto, mixed by inversion, and cooled on ice. By centrifugation again, phage displaying the target molecule is obtained as a precipitate, which is dissolved in PBS (Phosphate Buffered Saline) containing 5 to 15% glycerol to obtain a phage display solution.
（２）ELISAプレートを用いたバイオパニング
　複数の検出分子を用意し、ELISA用の96ウェルプレートの各ウェルに分注し、これらを固定する。ブロッキングを行い、上記ファージディスプレイ溶液を加えて反応させ、その後溶出する。得られた溶出液で大腸菌を形質転換し、上記と同様の操作を行って増殖させ、ファージディスプレイをバイオパニングすることができる。
　例えば、検出分子として、FGFR1－Fc及びFGFR1－Hisを、適宜PBSで希釈してFGFR1固定化溶液とする。次いで、上記FGFR1固定化溶液を96ウェルELISAプレート(Immuno Clear Standard Modules_C8_MaxiSorp: Cat # 445101、Thermo Scientific社製)等の各ウェルに分注し、これらの固定化のために一晩インキュベートする。 (2) Biopanning using ELISA plate A plurality of detection molecules are prepared, dispensed into each well of a 96-well plate for ELISA, and immobilized. Blocking is performed, the above phage display solution is added and allowed to react, and then elution is performed. The resulting eluate is used to transform Escherichia coli, and the same procedures as described above are performed to proliferate the phage display biopanning.
For example, as detection molecules, FGFR1-Fc and FGFR1-His are appropriately diluted with PBS to form an FGFR1-immobilized solution. The above FGFR1 immobilization solution is then dispensed into each well of a 96-well ELISA plate (Immuno Clear Standard Modules_C8_MaxiSorp: Cat # 445101, Thermo Scientific) or the like and incubated overnight for these immobilizations.
　インキュベート後、各ウェルから溶液をいて所望の回数、例えば3回洗浄し、ブロッキングのために3％スキムミルク含有PBS等のブロッキング溶液を加え、室温にて時間静置する。その後、ブロッキング溶液を除去し、上記不ロッキング溶液に対してさらにBSAを加えたPBSに、その約1/20量の上記ファージディスプレイ溶液を加え、上記ELISAプレートの各ウェルに分注し、室温にて反応させる。 After incubation, remove the solution from each well and wash the desired number of times, for example, 3 times, add a blocking solution such as PBS containing 3% skim milk for blocking, and allow to stand at room temperature for an hour. After that, the blocking solution was removed, and about 1/20 of the amount of the phage display solution was added to PBS to which BSA was further added to the anti-locking solution. to react.
　反応終了後、各ウェルから上記混合液を除き、各ウェルをPBS－Tで所望の回数、例えば4～5回、洗浄する。この操作は、必要に応じて何回か繰り返してもよい。その後、溶出液を各ウェルに加えて、上記検出分子と結合したファージディスプレイ分子を回収する。例えば、50～150 mMのトリメチルアミン溶液を各ウェルに加えて、室温で5～15分間静置し、結合しているファージを溶出させる。得られた溶出液は、直ちにTris-HCl(pH 6.5～7.5)等を用いて中和することが好ましい。 After the reaction is completed, remove the above mixed solution from each well, and wash each well with PBS-T the desired number of times, for example, 4-5 times. This operation may be repeated several times as required. An eluate is then added to each well to recover the phage display molecules bound to the detection molecule. For example, a 50-150 mM trimethylamine solution is added to each well and allowed to stand at room temperature for 5-15 minutes to elute bound phages. The obtained eluate is preferably immediately neutralized with Tris-HCl (pH 6.5-7.5) or the like.
　上記のように中和した溶出液を、上記と同様に、コンピテントセルと混合し、この混合液を寒天培地上に播種してコロニーを形成させ、コロニーピッキングを行って培養した後に、ヘルパーファージを加え、再度バイオパニングを行う。バイオパニングは、後述するように、イムノチューブを用いて行うこともできる。
　上記のようにして得られた第2ライブラリ中に、FGFR1に結合するVHHが濃縮されているか否かは、ポリクローナルの状態におけるファージELISAで確認することができる。レスポンスが得られたクローンについて、ファージミドを大腸菌から抽出し、サンガー法等によってDNAの配列解析を行い、クローンを同定する。 The eluate neutralized as described above is mixed with competent cells in the same manner as described above, and this mixture is seeded on an agar medium to form colonies. After colony picking and culturing, helper phage and perform biopanning again. Biopanning can also be performed using immunotubes, as described below.
Whether or not VHHs that bind to FGFR1 are enriched in the second library obtained as described above can be confirmed by phage ELISA in a polyclonal state. Phagemids are extracted from Escherichia coli for clones that give responses, and DNA sequences are analyzed by the Sanger method or the like to identify clones.
４．目的分子の産生
　以上のようにして同定したクローンからプラスミドを作製し、細菌で発現させることができる。例えば、細菌としてコリネバクテリウム・グルタミカム（C. glutamicum）を用いて、以下のようにして目的分子を発現させることができる。 4. Production of Target Molecule Plasmids can be prepared from clones identified as described above and expressed in bacteria. For example, using Corynebacterium glutamicum (C. glutamicum) as a bacterium, the target molecule can be expressed as follows.
（１）目的分子発現用プラスミドの構築
　上記クローンのファージミドを鋳型とし、pSLiCE_PK4_SMDPhage_F （配列番号13）、pSMDPhage_#1_R （配列番号14）又はpSMDPhage_#2_R （配列番号15）をプライマーとし、アニーリング温度を50～60℃、伸長反応2～10秒の条件でPCRを行って増幅産物を得ることができる。この増幅産物は必要に応じて精製するが、Gel/PCR Extraction Kit（日本ジェネティクス社製）等を使用してもよい。 (1) Construction of a plasmid for expressing the target molecule Using the phagemid of the above clone as a template, pSLiCE_PK4_SMDPhage_F (SEQ ID NO: 13), pSMDPhage_#1_R (SEQ ID NO: 14) or pSMDPhage_#2_R (SEQ ID NO: 15) as primers, annealing at 50 Amplification products can be obtained by performing PCR under the conditions of ~60°C and extension reaction for 2 to 10 seconds. This amplified product is purified as necessary, and a Gel/PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.) or the like may be used.
　例えば、新たなチューブに所望のバッファーを入れ、ここに、上記の精製DNA、細菌発現用プラスミド、及び大腸菌抽出用反応液を加えてインキュベートすることにより、In vitro相同組換体を得ることができる。この相同組換え体を用いてコンピテントセルを形質転換し、得られた形質転換体からプラスミドを抽出する。上記所望のバッファーとしては、25～75 mM Tris-HCl(pH 7.2～7.7)、5～15 mM MgCl２、0.5～1.5 mM ATP、及び0.5 ～1.5 mM DTT含むSLiCEバッファー等を挙げることができる。 For example, in vitro homologous recombinants can be obtained by placing a desired buffer in a new tube, adding the above-mentioned purified DNA, bacterial expression plasmid, and E. coli extraction reaction solution, and incubating. A competent cell is transformed with this homologous recombinant, and a plasmid is extracted from the resulting transformant. Examples of the desired buffer include SLiCE buffer containing 25-75 mM Tris-HCl (pH 7.2-7.7), 5-15 mM MgCl 2 , 0.5-1.5 mM ATP, and 0.5-1.5 mM DTT.
（２）細菌を用いた目的分子の発現
　上記のようにして得られたプラスミドを、エレクトロポレーション等によって細菌、例えば、C. glutamicumを形質転換し、得られた形質転換体を所望の培地、に播種し前培養を行う。上記前培養は、例えば、CM2G培地を使用し、約28～約32℃にて一晩とすることができる。その後、この前培養液をPM1S培地に1/20量移し、約23～約27℃にて60～80時間、培養することにより、目的分子が上記培地中に分泌される。培養終了後、培養液を遠沈管に移して遠心し、菌体を除去するために回収した培養上清を0.22μｍのフィルターで処理することが好ましい。 (2) Expression of target molecule using bacteria The plasmid obtained as described above is transformed into a bacterium such as C. glutamicum by electroporation or the like, and the resulting transformant is placed in a desired medium. and pre-culture. For example, the pre-culture can be performed overnight at about 28 to about 32° C. using CM2G medium. Thereafter, 1/20 of this preculture solution is transferred to PM1S medium and cultured at about 23 to about 27° C. for 60 to 80 hours to secrete the target molecule into the medium. After completion of the culture, the culture solution is transferred to a centrifuge tube and centrifuged, and the collected culture supernatant is preferably treated with a 0.22 μm filter to remove the cells.
　以上のようにして得られた目的分子を含む培養上清は、例えば、イミダゾールを添加し、その後、担体を加えて転倒混和する。引き続き、スピンカラムでこの担体を分離し、後述する溶出バッファーで目的分子を溶出する。こうした担体としては、例えば、Ni Sepharose 6 Fast Flow (Cytiva社製、以下、単に「担体」ということがある。）を挙げることができ、上記スピンカラムとしては、EconoSpin（Ajinomoto Bio-Pharma社製）等を挙げることができる。 For example, imidazole is added to the culture supernatant containing the target molecule obtained as described above, and then the carrier is added and mixed by inversion. Subsequently, this carrier is separated by a spin column, and target molecules are eluted with an elution buffer to be described later. Examples of such a carrier include Ni Sepharose 6 Fast Flow (manufactured by Cytiva, hereinafter sometimes simply referred to as "carrier"). etc. can be mentioned.
　上記溶出バッファーとしては、250～350 mMのNaCl及び400～600 mMのイミダゾールを含む25～75 mM Tris-HCl (pH 7.0～8.0）使用することができる。また、溶出された目的分子の純度はSDS－PAGE等で確認することができる。
　以上のようにして、目的分子を得ることができる。 As the elution buffer, 25-75 mM Tris-HCl (pH 7.0-8.0) containing 250-350 mM NaCl and 400-600 mM imidazole can be used. Also, the purity of the eluted target molecule can be confirmed by SDS-PAGE or the like.
As described above, the target molecule can be obtained.
　以下に、実施例によって本発明をより詳細に説明するが、本発明は後述する実施例に限定されるものではない。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described later.
（実施例１）目的分子のスクリーニング
　cDNAディスプレイ法を用い、目的分子のcDNAを含むライブラリをスクリーニングして、一次ライブラリを作製した。本実施例においては、目的分子としてヒト線維芽細胞増殖因子受容体（Fibroblast Growth Factor Receptor 1(以下、FGFR1と略すことがある。)）を、また、上記目的分子を検出する分子（以下、「検出分子」という。）として、FGFR1の細胞外領域である、組換えヒトFGFR1タンパク(Active、Cat. No. ab168696、Abcam社より購入、以下、「FGFR-His」と略すことがある。）、及びFGF受容体1β(IIIc)/Fcキメラ(Cat. No. 661-FR, ヒト組換えキャリアフリー、R&D System社より購入、以下「FGFR-Fc」と略すことがある。）を使用した。陰性対照としては、IgG1 (Fc)(Cat. No. 110-HG, ヒト組換えキャリアフリー, R&D System 社より購入、以下、「hFc」と略すことがある。）を使用した。 (Example 1) Screening of Target Molecules Using the cDNA display method, libraries containing cDNAs of target molecules were screened to prepare a primary library. In this example, the target molecule was human fibroblast growth factor receptor 1 (hereinafter sometimes abbreviated as FGFR1), and a molecule for detecting the target molecule (hereinafter referred to as " as the extracellular domain of FGFR1, recombinant human FGFR1 protein (Active, Cat. No. ab168696, purchased from Abcam, hereinafter sometimes abbreviated as "FGFR-His"), and FGF receptor 1β(IIIc)/Fc chimera (Cat. No. 661-FR, human recombinant carrier-free, purchased from R&D System, hereinafter sometimes abbreviated as "FGFR-Fc") were used. As a negative control, IgG1 (Fc) (Cat. No. 110-HG, human recombinant carrier-free, purchased from R&D System, hereinafter sometimes abbreviated as "hFc") was used.
（１）検出分子の固相化
　スクリーニングに使用するcDNAディスプレイ法を、図１に模式的に示す。
　目的分子を、以下の手順によりビオチン化した。FGFR1－Fc（検出分子1）及びhFc（検出分子2）に対し、それぞれ、20当量のビオチン化試薬（EZ－link sulfo NHS-SSビオチン；Thermo Fisher社製）を加え、25℃にて30分間反応させた。その後、脱塩カラム（Zeba Spin Desalting Columns；Thermo Fisher社製）を用いて、未反応のビオチン化試薬を除去し、検出分子を固相化した。 (1) Immobilization of Detection Molecules A cDNA display method used for screening is schematically shown in FIG.
The molecule of interest was biotinylated by the following procedure. 20 equivalents of a biotinylation reagent (EZ-link sulfo NHS-SS biotin; manufactured by Thermo Fisher) was added to each of FGFR1-Fc (detection molecule 1) and hFc (detection molecule 2), and incubated at 25°C for 30 minutes. reacted. Thereafter, desalting columns (Zeba Spin Desalting Columns; manufactured by Thermo Fisher) were used to remove unreacted biotinylation reagents, and the molecules to be detected were immobilized.
（２）cDNAディスプレイの合成
　完全長アルパカ由来抗体の可変領域断片（重鎖抗体の単一可変ドメイン、以下、「VHH」ということがある。）から、VHH一次ライブラリを以下のようにして作製した。
　2種類のアルパカ由来ナイーブVHHライブラリ（いずれもRePHAGEN社製）からSヒンジ及びLヒンジ遺伝子を鋳型として使用した。また、フォワードプライマーとしては、下記表1に示す配列番号1のプライマーを使用し、Sヒンジ特異的リバースプライマー（配列番号2）及びLヒンジ特異的リバースプライマー（配列番号3）を使用し、以下のPCR条件で増幅させ、Sヒンジ及びLヒンジのcDNA分子を得た（上記表１参照）。PCR条件は、98℃で2分間の後、98℃で10秒、62℃で5秒、72℃で35秒を5サイクル行い、72℃で1分反応させた後に10℃まで冷却とした。 (2) Synthesis of cDNA display A VHH primary library was prepared from variable region fragments of full-length alpaca-derived antibodies (single variable domains of heavy chain antibodies, hereinafter sometimes referred to as "VHH") as follows. .
S-hinge and L-hinge genes from two types of alpaca-derived naive VHH libraries (both manufactured by RePHAGEN) were used as templates. Further, as the forward primer, using the primer of SEQ ID NO: 1 shown in Table 1 below, using the S hinge-specific reverse primer (SEQ ID NO: 2) and the L hinge-specific reverse primer (SEQ ID NO: 3), the following Amplification was carried out under PCR conditions to obtain S-hinge and L-hinge cDNA molecules (see Table 1 above). The PCR conditions were as follows: 98°C for 2 minutes, 5 cycles of 98°C for 10 seconds, 62°C for 5 seconds, and 72°C for 35 seconds, reaction at 72°C for 1 minute, and cooling to 10°C.
　その後、オーバーラップPCR用プライマー（配列番号4、5）を用いてオーバーラップPCRを行い、全長VHHコード化DNAライブラリ（以下、「全長ライブラリ」という。）を作製した。この全長ライブラリは、T7プロモーター、オメガ（Ω）エンハンサー、コザックコンセンサス配列、VHH遺伝子、Hisタグ、およびリンカーハイブリダイゼーション領域（Ｙタグ）を含むDNA断片である（上記表1参照）。
　なお、オーバーラップPCR条件は、98℃で2分間の後、98℃で10秒、65℃で5秒、72℃で35秒を5サイクル行い、72℃で１分反応させた後に10℃まで冷却とした。 Overlap PCR was then performed using overlap PCR primers (SEQ ID NOS: 4 and 5) to prepare a full-length VHH-encoding DNA library (hereinafter referred to as "full-length library"). This full-length library is a DNA fragment containing a T7 promoter, an omega (Ω) enhancer, a Kozak consensus sequence, a VHH gene, a His-tag, and a linker hybridization region (Y-tag) (see Table 1 above).
Overlap PCR was performed at 98°C for 2 minutes, followed by 5 cycles of 98°C for 10 seconds, 65°C for 5 seconds, and 72°C for 35 seconds. It was cooled.
（２－２）cDNAディスプレイ用リンカーの調製
　cDNAディスプレイには、主鎖と側鎖とを有するリンカーを使用した。このリンカーの主鎖は、固相結合部位としてのBioTEGを５’末端に含み、その近傍に固相からの切断部位としてのrGとを含み、mRNA連結部位としての3－シアノビニルカルバゾールを備えている。上記主鎖の配列を下記配列番号6に示す（以下、この主鎖を「ビオチンフラグメント」ということがある。）。なお、下記の配列中、gはグアノシン、VはAmino C6-dTを、Kは3-シアノビニルカルバゾール（以下、「cnvK」ということがある。）をそれぞれ表す（以下、「cnvK rG Linker」という。）。 (2-2) Preparation of Linker for cDNA Display A linker having a main chain and a side chain was used for cDNA display. The main chain of this linker contains BioTEG as a solid phase binding site at the 5′ end, contains rG as a cleavage site from the solid phase in the vicinity thereof, and has 3-cyanovinylcarbazole as an mRNA ligation site. there is The sequence of the main chain is shown in SEQ ID NO: 6 below (hereinafter, this main chain may be referred to as "biotin fragment"). In the following sequences, g represents guanosine, V represents Amino C6-dT, and K represents 3-cyanovinylcarbazole (hereinafter sometimes referred to as "cnvK") (hereinafter referred to as "cnvK rG Linker"). .).
　5’－AAgAATTTCCAKGCCGCCCCCCGVCCT －3’　　　　　　（配列番号6） 　5'-AAgAATTTCCAKGCCGCCCCCCGVCCT -3' (sequence number 6)
　また、側鎖となるピューロマイシンセグメントは、5' (5S)TCTFZZCCPの配列を有している。ここで、上記側鎖配列中の遊離末端となる「P」は、タンパク質結合部位としてのピューロマイシンを表す。また、「(5S)」は5' Thiol C6を、「F」は蛍光基であるFITC-dTを、そして「Z」は Spacer 18をそれぞれ表す。上記の主鎖及び側鎖の化学合成は、つくばオリゴサービス（株）に委託した。 In addition, the side chain puromycin segment has a 5' (5S) TCTFZZCCP sequence. Here, the free terminal "P" in the side chain sequence represents puromycin as a protein binding site. Also, "(5S)" represents 5' Thiol C6, "F" represents the fluorescent group FITC-dT, and "Z" represents Spacer 18, respectively. The above chemical synthesis of the main chain and side chains was entrusted to Tsukuba Oligo Service Co., Ltd.
　まず、0.2Mのリン酸ナトリウム(pH 7.2)に、15 nmolのビオチンフラグメント(終濃度150μM)及びEMCS（（株）同仁化学研究所製、終濃度16.7 mM)を加え、37℃で30分間インキュベートした。その後、Quick-Precip Plus Solution (Edge BioSystems社製)を用いて、上記ビオチンフラグメント－EMCS結合体をエタノール沈殿させた。
　次に37.5 nmol分のピューロマイシン－セグメントを終濃度417μMとなるように、50 mMのDTTを含む１Ｍのリン酸水素二ナトリウム水溶液に溶解し、シェーカーを用いて室温で１時間撹拌した。次いで、NAP5カラム（GEヘルスケアバイオサイエンス社製）を用いて、0.03 MのNaClを含む0.02 Mのリン酸ナトリウム緩衝液(pH 7.0)にバッファーを交換し、還元ピューロマイシンセグメント溶液を得た。 First, 15 nmol of biotin fragment (final concentration: 150 µM) and EMCS (manufactured by Dojindo Laboratories, final concentration: 16.7 mM) were added to 0.2 M sodium phosphate (pH 7.2) and incubated at 37°C for 30 minutes. did. Then, using Quick-Precip Plus Solution (manufactured by Edge BioSystems), the biotin fragment-EMCS conjugate was precipitated with ethanol.
Next, 37.5 nmol of puromycin segment was dissolved in a 1 M aqueous solution of disodium hydrogen phosphate containing 50 mM DTT to a final concentration of 417 μM, and the mixture was stirred at room temperature for 1 hour using a shaker. Then, using a NAP5 column (manufactured by GE Healthcare Bioscience), the buffer was exchanged with 0.02 M sodium phosphate buffer (pH 7.0) containing 0.03 M NaCl to obtain a reduced puromycin segment solution.
　上記還元ピューロマイシンセグメント溶液を、上記のEMCS修飾済みビオチンフラグメントのエタノール沈殿産物と混合し、4℃で一晩放置した。続いて、DTTを終濃度50 mMとなるように上記反応液に投入し、室温で30分間撹拌した。その後、Quick-Precip Plus Solution (Edge BioSystems社製)を用いて、エタノール沈殿を行ない、上記フラグメントの結合体をエタノール沈殿物として得た。このエタノール沈殿産物を、100μLのNuclease-free water（ナカライテスク（株）製）に溶解し、溶解産物とした。 The above reduced puromycin segment solution was mixed with the above ethanol precipitation product of the EMCS-modified biotin fragment and left overnight at 4°C. Subsequently, DTT was added to the reaction solution so that the final concentration was 50 mM, and the mixture was stirred at room temperature for 30 minutes. Thereafter, using Quick-Precip Plus Solution (manufactured by Edge BioSystems), ethanol precipitation was performed to obtain a conjugate of the above fragments as an ethanol precipitate. This ethanol precipitate was dissolved in 100 μL of Nuclease-free water (manufactured by Nacalai Tesque, Inc.) to obtain a lysate.
　上記溶解産物を８M尿素12％変性ポリアクリルアミドゲル電気泳動（泳動条件：60℃、200V、30分）により分離し、cnvK rG Linker画分を切り出した。バイオマッシャーII セット（（株）ニッピ製）を用いて切り出したゲルを破砕し、500μLのヌクレアーゼフリー水をこのゲル破砕物に加え、cnvK rG Linkerを抽出するために4℃で一晩撹拌した。撹拌した溶液をCostar（登録商標） Spin-X（登録商標）遠心チューブフィルター, 0.22μmセルロースアセテート（Corning社製）に移し、その後、16,000xgで15分間遠心し、ゲルと抽出した液とを分離した。 The above lysate was separated by 8M urea 12% denaturing polyacrylamide gel electrophoresis (electrophoresis conditions: 60°C, 200V, 30 minutes), and the cnvK rG Linker fraction was excised. The excised gel was crushed using a Biomasher II set (manufactured by Nippi Co., Ltd.), 500 μL of nuclease-free water was added to the gel crushed product, and the mixture was stirred overnight at 4°C to extract the cnvK rG Linker. The stirred solution was transferred to a Costar (registered trademark) Spin-X (registered trademark) centrifugal tube filter, 0.22 μm cellulose acetate (manufactured by Corning), and then centrifuged at 16,000 xg for 15 minutes to separate the gel from the extracted liquid. did.
　引き続き、Quick-Precip Plus Solutionを用いて上記と同様にエタノール沈殿を行ない、cnvK poly rG Linkerを得た。得られたcnvK rG Linkerはヌクレアーゼフリー水に溶解させて－20℃で保存した。 Subsequently, ethanol precipitation was performed in the same manner as above using Quick-Precip Plus Solution to obtain cnvK poly rG Linker. The resulting cnvK rG Linker was dissolved in nuclease-free water and stored at -20°C.
（実施例２）cDNAディスプレイの作製
　VHH提示cDNAディスプレイを以下のように調製した。使用するバッファーは下記表3に示す。 Example 2 Generation of cDNA Display A VHH-presenting cDNA display was prepared as follows. The buffers used are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（１）転写
　上記実施例１（２－２）で作製したcDNAディスプレイ合成用DNAを、T7 RiboMAX Express Large Scale RNA Production System(Promega社製)を用いて、添付されたマニュアルに従って転写した。使用したDNA量は第1ラウンドで6.6μｇ、第2ラウンド以降は0.1～1μｇとした。得られた転写産物は、RNaClean XP(Beckman Coulter社製)を使用し、添付されたマニュアルに従って精製した。精製産物の濃度は、NanoPad DS-11FX(DeNovix)を用いて定量した。 (1) Transcription The DNA for cDNA display synthesis prepared in Example 1 (2-2) above was transcribed using the T7 RiboMAX Express Large Scale RNA Production System (manufactured by Promega) according to the attached manual. The amount of DNA used was 6.6 μg in the first round, and 0.1-1 μg in the second and subsequent rounds. The resulting transcript was purified using RNaClean XP (manufactured by Beckman Coulter) according to the attached manual. Concentrations of purified products were quantified using NanoPad DS-11FX (DeNovix).
（２）ライゲーション
　精製した20pmol mRNA及び上記実施例１（２－２）で作製した20pmolのcnvK rGリンカーに、NaCl（終濃度0.2 M）及びTris－HCl（pH 7.5、終濃度0.05M）を加え、90℃で１分間インキュベートした。その後、0.1℃／秒の速度で70℃まで降温させ、70℃で1分間インキュベートした。次いで、0.1℃／秒の速度で25℃まで降温させ、2℃／秒の速度で10℃まで降温させ、cnvK rGリンカーをmRNAの３’末端側にハイブリダイズさせた。引き続き、Handheld UV Lamp (6W, UVGL-56, 254/365 nm、100V (Analytik jena US, An Endress + Hauser Company))を使用して、365nmのUVを5分間照射し、cnvK rGリンカーとmRNAを光架橋させ、mRNA－リンカー連結体を得た。 (2) Ligation NaCl (final concentration 0.2 M) and Tris-HCl (pH 7.5, final concentration 0.05 M) were added to the purified 20 pmol mRNA and 20 pmol cnvK rG linker prepared in Example 1 (2-2) above. , and incubated at 90°C for 1 minute. After that, the temperature was lowered to 70°C at a rate of 0.1°C/sec and incubated at 70°C for 1 minute. Then, the temperature was lowered at a rate of 0.1°C/sec to 25°C and then lowered at a rate of 2°C/sec to 10°C to hybridize the cnvK rG linker to the 3' end of the mRNA. Subsequently, a Handheld UV Lamp (6W, UVGL-56, 254/365 nm, 100V (Analytik jena US, An Endress + Hauser Company)) was used to irradiate UV at 365 nm for 5 minutes to extract cnvK rG linkers and mRNA. Photocrosslinking was performed to obtain an mRNA-linker conjugate.
（３）mRNAディスプレイ分子の調製
　6 pmolのmRNA－リンカーを、50μLスケールの無細胞翻訳系(Rabbit reticulocyte Lysate (nuclease-treated)、Promega社製）に加えて、30℃で30分間インキュベートした。次いで、MgCl2（終濃度75 mM）びKCl（終濃度900 mM）を加え、37℃にて１時間インキュベートし、上記mRNAに対応するペプチドを上記リンカー側鎖のピューロマイシンにディスプレイした。次いで、EDTA (pH 8.0)を終濃度70 mMとなるように加えて、4℃にて5分間インキュベートし、mRNA－リンカーのmRNAに結合しているリボソームを除去し、mRNAディスプレイを形成させた。 (3) Preparation of mRNA display molecule 6 pmol of mRNA-linker was added to a 50 µL scale cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), Promega) and incubated at 30°C for 30 minutes. Then, MgCl 2 (final concentration 75 mM) and KCl (final concentration 900 mM) were added and incubated at 37° C. for 1 hour to display the peptide corresponding to the mRNA on the puromycin side chain of the linker. Then, EDTA (pH 8.0) was added to a final concentration of 70 mM and incubated at 4°C for 5 minutes to remove ribosomes bound to the mRNA of the mRNA-linker to form mRNA display.
（４）cDNAディスプレイ分子の調製
　Protein Lobind Tubeに、Dynabeads Myone Streptavidin C1（ThermoFischer Scientific社製）を60μL入れ、200μLの結合バッファーを加えて洗浄した。その後、上記（１）－（３）で調製したmRNAディスプレイをこのチューブ中に加え、25℃にて30分間撹拌した。200μLの結合バッファーで洗浄した。その後、下記表4に示す組成の反応液中で42℃にて30分間インキュベートして逆転写反応を行い、mRNA／cDNA―VHH連結体（以下、単に「VHH連結体」という。）を調製した。200μLの結合バッファーで上記VHH連結体を洗浄し、その後、39μLのHisタグ結合／洗浄バッファーと、1μLの1,000 U／μLのRNase T1とを加え、37℃にて15分撹拌してmRNA／cDNA―VHH連結体（以下、「cDNAディスプレイ」という。）をDynabeads MyOne Streptavidin C1から溶出させた。 (4) Preparation of cDNA Display Molecules 60 μL of Dynabeads Myone Streptavidin C1 (manufactured by ThermoFischer Scientific) was placed in a Protein Lobind Tube, and 200 μL of binding buffer was added for washing. Then, the mRNA display prepared in (1)-(3) above was added to this tube and stirred at 25° C. for 30 minutes. Washed with 200 μL binding buffer. After that, reverse transcription reaction was performed by incubating at 42°C for 30 minutes in a reaction solution having the composition shown in Table 4 below to prepare an mRNA/cDNA-VHH conjugate (hereinafter simply referred to as "VHH conjugate"). . Wash the VHH conjugate with 200 μL of binding buffer, then add 39 μL of His tag binding/washing buffer and 1 μL of 1,000 U/μL of RNase T1, stir at 37° C. for 15 minutes to mix mRNA/cDNA. -VHH conjugates (hereinafter referred to as "cDNA display") were eluted from Dynabeads MyOne Streptavidin C1.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（５）cDNAディスプレイ分子の精製
　エッペンドルフチューブに、30μL のHis Mag Sepharose Ni Beads (GE Health Care社製造)を入れ、このビーズを200μLのHisタグ結合／洗浄バッファーで洗浄した。その後、上記（１）－（４）の方法で調製したcDNAディスプレイをここに加え、25℃にて30分間撹拌した。200μLのHisタグ結合／洗浄バッファーをこのチューブに加えて洗浄し、その後、30μLのHisタグ溶出バッファーを加え、37℃にて15分撹拌してcDNAディスプレイライブラリを溶出させた。 (5) Purification of cDNA Display Molecules 30 μL of His Mag Sepharose Ni Beads (manufactured by GE Health Care) were placed in an Eppendorf tube, and the beads were washed with 200 μL of His tag binding/washing buffer. Thereafter, the cDNA display prepared by the method (1)-(4) above was added thereto and stirred at 25° C. for 30 minutes. 200 μL of His-tag binding/washing buffer was added to the tube to wash, then 30 μL of His-tag elution buffer was added and stirred at 37° C. for 15 minutes to elute the cDNA display library.
（実施例３）VHHのセレクション
（１）リガンドと結合する目的VHHのセレクション
　リガンドと結合する目的VHHを取得するため、上記実施例２により調製したcDNAディスプレイライブラリを用いて、セレクションを行った。各セレクションラウンドで使用したVHH提示cDNAディスプレイライブラリの合成スケールを下記表５に示す。また、溶出方法（競合溶出、TCEP溶出）による場合分けを記したスクリーニング試験フローチャートを図2に示す。 (Example 3) Selection of VHHs (1) Selection of target VHHs that bind to ligands In order to obtain target VHHs that bind to ligands, selection was performed using the cDNA display library prepared in Example 2 above. The synthesis scale of the VHH-presenting cDNA display library used in each selection round is shown in Table 5 below. In addition, Fig. 2 shows a screening test flow chart showing cases classified by elution method (competitive elution, TCEP elution).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（１－１）セレクショラウンド1（以下、単に「R1」ということがある）
　100μLのDynabeads Myone Streptavidin C1（以下、単に「ストレプトアビジンビーズ」ということがある。）を入れたプロテインローバインディングチューブに、上記実施例１で調製したビオチン化FGFR1－Fc（検出分子1）を16.3μL加え、25℃にて30分間転倒混和し、FGFR－Fcを固相化した。
　100pmol分のFGFR－Fcを固相化したビーズをプロテインローバインディングチューブに取り、ここに上記実施例１（２－２）で作製したDNAを用いて上記実施例２にしたがって調整したcDNAディスプレイを全量加え、25℃にて30分間転倒混和して上記検出分子と結合させた。その後、セレクションバッファーをこのチューブに加えて４回洗浄した。 (1-1) Selection Round 1 (hereinafter sometimes simply referred to as “R1”)
16.3 μL of biotinylated FGFR1-Fc (detection molecule 1) prepared in Example 1 above was placed in a protein low binding tube containing 100 μL of Dynabeads Myone Streptavidin C1 (hereinafter sometimes simply referred to as “streptavidin beads”). In addition, the mixture was mixed by inversion at 25°C for 30 minutes to immobilize FGFR-Fc.
100 pmol of FGFR-Fc-immobilized beads were placed in a protein low binding tube, and the entire amount of cDNA display prepared according to Example 2 using the DNA prepared in Example 1 (2-2) was added. In addition, they were mixed by inversion at 25°C for 30 minutes to bind with the detection molecules. Selection buffer was then added to the tube and washed four times.
　次いで、40μLの10mM TCEP(Tris(2-carboxyethyl)phosphine)をこのチューブに加え、25℃にて15分間静置し、検出分子のSS結合を切断して結合したcDNAディスプレイを溶出し、TCEP溶液とした。その後、上記固相化ビーズに10μLの10mM NaOHを加え、25℃にて10分間静置するというアルカリ溶出操作を２回連続して行い、アルカリ溶出液を得た。この溶出で得られたアルカリ溶出液は、4μLの1M Tris－HCl (pH 8.0)を加えて中和した。溶出後の上記ストレプトアビジンビーズに16μLのセレクションバッファーを加え、その後、室温にて1分間静置し、上清を上記アルカリ溶出液と混合した。 Next, add 40 μL of 10 mM TCEP (Tris(2-carboxyethyl)phosphine) to this tube, leave it at 25°C for 15 minutes, cleave the SS bond of the detection molecule and elute the bound cDNA display, and extract the TCEP solution. and After that, 10 μL of 10 mM NaOH was added to the immobilized beads, and the mixture was allowed to stand at 25° C. for 10 minutes. The alkaline eluate obtained by this elution was neutralized by adding 4 μL of 1M Tris-HCl (pH 8.0). 16 μL of selection buffer was added to the streptavidin beads after elution, and then allowed to stand at room temperature for 1 minute, and the supernatant was mixed with the alkaline eluate.
　別々のチューブに入れた各溶出液を、cnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）とをプライマーとして98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件でPCR反応を行った。その後、上記のように得られたPCR産物を８M尿素4％変性PAGEに供した。得られたPCR産物をAMPure XP(Beckman Coulter社製)を使用し、添付されたマニュアルに従って精製し、等量（v/v）で混合して、R１ライブラリとした。 Each eluate in a separate tube was incubated at 98°C for 2 minutes with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers, followed by 98°C for 10 seconds and 68°C. 5 seconds at 72°C for 35 seconds for 25 cycles, and the PCR reaction was carried out at 72°C for 1 minute. The PCR products obtained as described above were then subjected to 8M urea 4% denaturing PAGE. The obtained PCR products were purified using AMPure XP (manufactured by Beckman Coulter) according to the attached manual, and mixed in equal amounts (v/v) to form the R1 library.
（１－２）セレクションラウンド2（R2）
　R2では、溶出操作を、TCEP及びアルカリを用いる溶出（以下、「TCEP溶出」という。）で行った。
　まず、上記（実施例１）で調製した検出分子2（ビオチン化hFc）から2.7μLを取り、新たなストレプトアビジンビーズ50μLを加えたプロテインローバインディングチューブに加え、25℃にて30分間転倒混和し、hFc固相化ビーズを作製した。 (1-2) Selection Round 2 (R2)
In R2, elution was carried out using TCEP and alkali (hereinafter referred to as "TCEP elution").
First, 2.7 µL of the detection molecule 2 (biotinylated hFc) prepared in the above (Example 1) was taken, added to a protein low binding tube containing 50 µL of new streptavidin beads, and mixed by inversion for 30 minutes at 25°C. , hFc-immobilized beads were produced.
　10pmol分のhFcが固相化されているビーズと、R１ライブラリを用いて上記実施例２にしたがって調整したcDNAディスプレイライブラリ全量とを、100μLのセレクションバッファーを入れたプロテインローバインディングチューブ中に加え、25℃にて60分間転倒混和した。転倒混和後の上清を回収してプロテインローバインディングチューブ中に入れ、10pmol分のFGFR1－Fc固相化ストレプトアビジンビーズをここに加え、100pmolのhFcをさらに加え、25℃にて60分間転倒混和した。その後、チューブに200μLのセレクションバッファーを加えて上記固相化ビーズをそれぞれ4回洗浄した。 10 pmol of hFc-immobilized beads and the total amount of the cDNA display library prepared according to Example 2 using the R1 library were added to a protein low binding tube containing 100 μL of selection buffer. The mixture was mixed by inversion for 60 minutes at ℃. Collect the supernatant after mixing by inversion and put it in a protein low binding tube, add 10 pmol of FGFR1-Fc-immobilized streptavidin beads, add 100 pmol of hFc, and mix by inversion at 25°C for 60 minutes. did. Thereafter, 200 μL of selection buffer was added to the tube to wash the immobilized beads four times.
　その後、チューブに40μLの10mM TCEPを加え、室温にて15分間反応させて結合したcDNAディスプレイを溶出した。引き続き、R1と同様のアルカリ溶出を行い、その後16μLのセレクションバッファーを用いて、室温にて1分反応させて溶出した。この溶出後、直ちに1M Tris-HCl(pH8.0)を4μL加えて溶出液を中和した。
　別々のチューブに入れた各溶出液を、それぞれPCRに供し、98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分反応させた後に10℃の条件で増幅させた。得られた増幅産物を、AMPure XPを使用し、添付されたマニュアルに従って精製した後、等量（v/v）で混合して、R2ライブラリとした。 After that, 40 μL of 10 mM TCEP was added to the tube and reacted at room temperature for 15 minutes to elute the bound cDNA display. Subsequently, alkaline elution was performed in the same manner as in R1, and then 16 μL of selection buffer was used to react at room temperature for 1 minute for elution. Immediately after this elution, 4 μL of 1M Tris-HCl (pH 8.0) was added to neutralize the eluate.
Each eluate in a separate tube was subjected to PCR, and after 2 minutes at 98°C, 25 cycles of 98°C for 10 seconds, 68°C for 5 seconds, 72°C for 35 seconds, and 72°C for 1 cycle were performed. After reacting for 1 minute, amplification was carried out at 10°C. The obtained amplification products were purified using AMPure XP according to the attached manual, and then mixed in equal amounts (v/v) to form the R2 library.
（１－３）セレクションサイクル3（R3）のスクリーニング手順
　R3では、溶出操作を、TCEP及びアルカリを用いる溶出（以下、「TCEP溶出」という。）とTCEP及びアルカリを用いない溶出（以下、「競合溶出」という）とに分けて行った。
　10pmol分のhFcが固相化されている上記ストレプトアビジンビーズと、R3ライブラリを用いて上記実施例２にしたがって調製したcDNAディスプレイライブラリを全量プロテインローバインディングチューブ中に入れ、100μLのセレクションバッファーをここに加えて、25℃にて60分間転倒混和した。引き続き、この上清を回収し、半量を10pmol分のFGFR1－Fc固相化ビーズを入れたチューブに加えた。これらのチューブに、50μLのセレクションバッファー、50pmolのhFc、14pmolのストレプトアビジン、及び100ng/μLのSalmon sperm DNAを含有するPBS-Tを加え、25℃にて60分間転倒混和した。 (1-3) Screening procedure for selection cycle 3 (R3) In R3, the elution operation is divided into elution using TCEP and alkali (hereinafter referred to as "TCEP elution") and elution using no TCEP and alkali (hereinafter referred to as "competitive elution"). elution”).
The streptavidin beads on which 10 pmol of hFc are immobilized and the cDNA display library prepared according to Example 2 above using the R3 library were all placed in a protein low binding tube, and 100 μL of selection buffer was added here. In addition, the mixture was mixed by inversion for 60 minutes at 25°C. Subsequently, this supernatant was collected and half of it was added to a tube containing 10 pmol of FGFR1-Fc immobilized beads. PBS-T containing 50 μL of selection buffer, 50 pmol of hFc, 14 pmol of streptavidin, and 100 ng/μL of Salmon sperm DNA was added to these tubes and mixed by inversion for 60 minutes at 25°C.
（１－３－１）競合溶出
　上記チューブに200μLのセレクションバッファーを加えて、チューブ中のビーズを3回洗浄した。その後、40μLのセレクションバッファーを加えて、4℃にて30分間転倒混和してさらに洗浄した。引き続き、100 pmolのFGFR1－Hisをチューブに加え、25℃にて30分間、転倒混和した。この操作で得られた溶出液をPCRに供した。プライマーとして、cnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）を使用して98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件でPCR反応を行った。増幅産物を、AMPure XPを使用し、添付されたマニュアルに従って精製した後、等量（v/v）で混合し、R3－1ライブラリとした。 (1-3-1) Competitive Elution 200 μL of selection buffer was added to the above tube, and the beads in the tube were washed three times. After that, 40 μL of selection buffer was added, and the mixture was mixed by inversion at 4° C. for 30 minutes for further washing. Subsequently, 100 pmol of FGFR1-His was added to the tube and mixed by inversion for 30 minutes at 25°C. The eluate obtained by this operation was subjected to PCR. As primers, cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) were used at 98°C for 2 minutes, followed by 98°C for 10 seconds, 68°C for 5 seconds, and 72°C for 35 seconds. The PCR reaction was carried out under the conditions of 25 cycles of seconds and 72°C for 1 minute. The amplified products were purified using AMPure XP according to the attached manual, and then mixed in equal amounts (v/v) to form the R3-1 library.
（１－３－２）TCEP溶出
　上記（１－２）と同様の操作を行い、TCEP溶出液を得た。プライマーとしてcnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）とを使用して、98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件で、得られた溶出液中に含まれるcDNAディスプレイを増幅させ、増幅産物を得た。得られた増幅産物を、上記と同様に精製し、等量（v/v）で混合してR3―2ライブラリとした。 (1-3-2) TCEP Elution A TCEP eluate was obtained by performing the same operation as in (1-2) above. Using cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers, 98°C for 2 minutes, 98°C for 10 seconds, 68°C for 5 seconds, 72°C for Under the conditions of 25 cycles of 35 seconds at 72° C. for 1 minute, the cDNA display contained in the obtained eluate was amplified to obtain an amplified product. The resulting amplification products were purified in the same manner as above and mixed in equal amounts (v/v) to form the R3-2 library.
（１－４）セレクションサイクル4（R4）
（１－４－１）R3－1ライブラリのスクリーニング
　R3ライブラリを用いたcDNAディスプレイ法を行い、競合溶出させた画分とTCEPで溶出させた画分とを得た。3 pmolのR3-1ライブラリを用いたcDNAディスプレイと、1 pmolのFGFR1－Fc固相化ビーズとをプロテインローバインディングチューブ中に入れ、ここに1,000μLのセレクションバッファー、50pmolのhFc、140pmolのストレプトアビジン、及び100ng/μLのSalmon sperm DNAを加え、4℃にて3時間転倒混和した。 (1-4) Selection cycle 4 (R4)
(1-4-1) Screening of R3-1 Library A cDNA display method was performed using the R3 library, and a fraction eluted by competitive elution and a fraction eluted with TCEP were obtained. 3 pmol of cDNA display using the R3-1 library and 1 pmol of FGFR1-Fc immobilized beads were placed in a protein low binding tube, and 1,000 μL of selection buffer, 50 pmol of hFc, and 140 pmol of streptavidin were added. , and 100 ng/μL of Salmon sperm DNA were added, and mixed by inversion at 4° C. for 3 hours.
　引き続き、このチューブに200μLのセレクションバッファーを加えて、チューブ内のビーズを3回洗浄した。その後、上記と同様に2つに分けて別々のチューブに入れた。
　これらチューブの一方のみを使用し10pmolのFGFR1－Hisを添加した。その後、これら2本のチューブを、4℃にて16時間転倒混和し、上記ビーズに結合したcDNAディスプレイを競合溶出した。 Subsequently, 200 µL of selection buffer was added to this tube to wash the beads in the tube three times. It was then split into two and placed in separate tubes as above.
Only one of these tubes was used and 10 pmol of FGFR1-His was added. These two tubes were then mixed by inversion at 4° C. for 16 hours to competitively elute the cDNA display bound to the beads.
　10pmol分のhFcが固相化されているビーズと、R3-1及びR3-2ライブラリを別々に用いて上記実施例２にしたがって調整したcDNAディスプレイライブラリ全量とを、100μLのセレクションバッファーを入れたプロテインローバインディングチューブ中に加え、25℃にて60分間転倒混和した。転倒混和後の上清を回収してプロテインローバインディングチューブ中に入れ、10pmol分のFGFR1－Fc固相化ストレプトアビジンビーズをここに加え、100pmolのhFcをさらに加え、25℃にて60分間転倒混和した。その後、チューブに200μLのセレクションバッファーを加えて上記固相化ビーズをそれぞれ4回洗浄した。その後、40μLの10mM TCEPを加え、室温にて15分間反応させて溶出し、TCEP溶出液を得た。 10 pmol of hFc-immobilized beads and the total amount of the cDNA display library prepared according to Example 2 using the R3-1 and R3-2 libraries separately were combined with 100 μL of a protein buffer containing a selection buffer. It was added to a low binding tube and mixed by inversion at 25°C for 60 minutes. Collect the supernatant after mixing by inversion and put it in a protein low binding tube, add 10 pmol of FGFR1-Fc-immobilized streptavidin beads, add 100 pmol of hFc, and mix by inversion at 25°C for 60 minutes. did. Thereafter, 200 μL of selection buffer was added to the tube to wash the immobilized beads four times. After that, 40 μL of 10 mM TCEP was added, reacted at room temperature for 15 minutes, and eluted to obtain a TCEP eluate.
　以上のようにして得られた競合溶出した溶液を、cnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）をプライマーに使用して98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件でPCRによって増幅させ、R4－1－1ライブラリとした。また、TCEP溶出液をcnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）とをプライマーに使用して98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件でPCRによって増幅させ、R4－1－2ライブラリとした。 The competitively eluted solution obtained as described above was treated with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, and then at 98°C. 25 cycles of 10 seconds, 68°C for 5 seconds, and 72°C for 35 seconds were performed, followed by PCR amplification under the conditions of 72°C for 1 minute to obtain the R4-1-1 library. In addition, the TCEP eluate was used with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 5 minutes. Seconds, 35 seconds at 72°C for 25 cycles, and amplification by PCR under the conditions of 72°C for 1 minute to obtain the R4-1-2 library.
（１－４－２）R3－2ライブラリのスクリーニング
　R3－2ライブラリについても、上記（１－４－１）においてR3－1ライブラリについてしたと同じ処理を行い、競合溶出によってR4－2－1を、また、TCEP溶出によってR4－2－2を得た。 (1-4-2) Screening of R3-2 library For the R3-2 library, the same treatment as for the R3-1 library in (1-4-1) above was performed, and R4-2-1 was extracted by competitive elution. , and TCEP elution gave R4-2-2.
（１－５）セレクションサイクル5（R5）
　R5の競合溶出では、1pmolのFGFR1－Fcが固相化されているビーズに対して上記（１－４）より得たR4－1－1、R4－1－2、R4－2－1、及びR4－2－2の各ライブラリをそれぞれ別々のチューブに入れ、上記と同様に結合させた。これらのチューブに200μLのセレクションバッファーを加えて、チューブ内のビーズを3回洗浄した。各チューブに20μLの10pmolのFGFR1－Hisを加えてインキュベートした。インキュベート開始20時間後に、20μLの溶液を回収し（E1）、回収したのと同量の10pmolのFGFR1－Hisを加え、インキュベート開始40時間後に再び20μLの溶液を回収した（E2）。別途、1pmolのFGFR1－Fcが固相化されているビーズに対して上記（１－４）より得たR4－1－1、R4－1－2、R4－2－1、及びR4－2－2の各ライブラリをそれぞれ別々のチューブに入れ、上記と同様に結合させた。これらのチューブに200μLのセレクションバッファーを加えて、チューブ内のビーズを3回洗浄し、ここに20μLのTCEPを加えて溶出した（TCEP溶出）。 (1-5) Selection cycle 5 (R5)
In competitive elution of R5, R4-1-1, R4-1-2, R4-2-1, and R4-1-1, R4-1-2, R4-2-1, and Each R4-2-2 library was placed in a separate tube and bound in the same manner as above. 200 μL of selection buffer was added to these tubes to wash the beads in the tubes three times. 20 μL of 10 pmol FGFR1-His was added to each tube and incubated. Twenty hours after the initiation of incubation, 20 μL of solution was collected (E1), the same amount of 10 pmol of FGFR1-His as that recovered was added, and 40 hours after initiation of incubation, 20 μL of the solution was again recovered (E2). Separately, R4-1-1, R4-1-2, R4-2-1, and R4-2- Each library of 2 was placed in a separate tube and bound in the same manner as above. 200 µL of selection buffer was added to these tubes, the beads in the tubes were washed three times, and 20 µL of TCEP was added to the beads for elution (TCEP elution).
　以上のように溶出した溶液のうち、競合溶出した溶液を、cnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）をプライマーに使用して98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、72℃で1分の条件で、別々にPCRを行って増幅させ、増幅産物を等量で混合して、R4－1－1ライブラリからR5－1－1―1及びR4－1－2ライブラリからR5－1－2―1ライブラリ、R4－2－1ライブラリからR5－2－1―1ライブラリ及びR4－2－2ライブラリからR5－2－2―1ライブラリを得た。
とした。TCEP溶出液についても同様の操作を行い、R4－1－1ライブラリからR5－1－1―2及びR4－1－2ライブラリからR5－1－2―2ライブラリ、R4－2－1ライブラリからR5－2－1―2ライブラリ及びR4－2－2ライブラリからR5－2－2―2ライブラリを得た。 Among the solutions eluted as described above, the competitively eluted solution was subjected to 98° C. after 2 minutes at 98°C using cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers. 25 cycles of 10 seconds at ℃, 5 seconds at 68℃, 35 seconds at 72℃, and 1 minute at 72℃ for amplification. -1-1 library to R5-1-1-1 and R4-1-2 library to R5-1-2-1 library, R4-2-1 library to R5-2-1-1 library and R4-2- The R5-2-2-1 library was obtained from the 2 libraries.
and Perform the same operation for the TCEP eluate, R4-1-1 library to R5-1-1-2, R4-1-2 library to R5-1-2-2 library, R4-2-1 library to R5 The R5-2-2-2 library was obtained from the -2-1-2 library and the R4-2-2 library.
　R5では、上記（１－４）で得たR4－1－1、R4－1―2、R4－2－1及びR4－2－2ライブラリから、一部を取ってFACS(Fluorescence-Activated Cell Sorting)を用いてセレクションを行った。まず、hFcが固相化されているビーズを入れたプロテインローバインディングチューブにビオチン－フルオレセインを加えてhFc固相化蛍光ビーズ（以下、単に「hFcF」ビーズという。）を作製した。2pmol分のhFcFビーズを新たなチューブに取って、ここに0.5pmol分のFGFR1－Fc固相化ビーズと加えて混合した。 In R5, from the R4-1-1, R4-1-2, R4-2-1 and R4-2-2 libraries obtained in (1-4) above, a part was taken and FACS (Fluorescence-Activated Cell Sorting) was performed. ) was used for selection. First, biotin-fluorescein was added to a protein low binding tube containing hFc-immobilized beads to prepare hFc-immobilized fluorescent beads (hereinafter simply referred to as “hFcF” beads). 2 pmol of hFcF beads were taken in a new tube, and 0.5 pmol of FGFR1-Fc immobilized beads were added and mixed.
　その後、上記のいずれかのライブラリ、50μLのセレクションバッファー、50pmolストレプトアビジン、及び500ng/μLのSalmon sperm DNAを混合し、4℃にて1時間反応させた。反応終了後に、200μLのセレクションバッファーを各チューブに加えて、3回洗浄した。引き続き、500μLのセレクションバッファーを各チューブに加え、ソーティング用溶液とした。以上のようにして得られたソーティング用溶液を、FCM(Cell Sorter SH800:SONY)にセットし、サンプル流路、分取時のドロップレット形成条件のセットアップを行い、ソーティングを行った。 After that, any of the above libraries, 50 μL of selection buffer, 50 pmol streptavidin, and 500 ng/μL of Salmon sperm DNA were mixed and allowed to react at 4°C for 1 hour. After completion of the reaction, 200 μL of selection buffer was added to each tube and washed three times. Subsequently, 500 μL of selection buffer was added to each tube to serve as a sorting solution. The sorting solution obtained as described above was set in an FCM (Cell Sorter SH800: SONY), and the sample channel and droplet forming conditions during fractionation were set up, and sorting was performed.
　FACSで分取された粒子径に基づいて磁性体ビーズの領域を同定し、488nmのレーザー照射による蛍光強度からhFcFビーズとFGFR1－Fc固相化ビーズとの領域の同定を行った。最適な分取条件にてソーティングを行い、それぞれ50万粒子をプロテインローバインディングチューブに回収した。その後、13,000xgで10分間、25℃にて遠心分離を行った。遠心後、プロテインローバインディングチューブを磁気プレート上に室温にて10分間静置し、その後、上清を除去して20μLの水に溶解した。この溶解液をcnvK NewYtag for poly A（配列番号7）とT7 omega new（配列番号8）をプライマーに使用して98℃で2分間の後、98℃で10秒、68℃で5秒、72℃で35秒を25サイクル行い、７２℃で１分の条件でPCRによって増幅させ、それぞれR5－1－1－FACSライブラリ、R5－1－2－FACSライブラリ、R5－2－1－FACSライブラリ及びR5－2－2－FACSライブラリとした。 The regions of the magnetic beads were identified based on the particle size fractionated by FACS, and the regions of the hFcF beads and the FGFR1-Fc immobilized beads were identified from the fluorescence intensity due to 488 nm laser irradiation. Sorting was performed under the optimum preparative conditions, and 500,000 particles were collected in protein low binding tubes for each. After that, centrifugation was performed at 13,000 xg for 10 minutes at 25°C. After centrifugation, the protein low binding tube was allowed to stand on a magnetic plate at room temperature for 10 minutes, after which the supernatant was removed and dissolved in 20 µL of water. This lysate was treated with cnvK NewYtag for poly A (SEQ ID NO: 7) and T7 omega new (SEQ ID NO: 8) as primers at 98°C for 2 minutes, followed by 98°C for 10 seconds, 68°C for 5 seconds, 72 25 cycles of 35 seconds at ° C., amplified by PCR under the conditions of 72 ° C. for 1 minute, respectively, R5-1-1-FACS library, R5-1-2-FACS library, R5-2-1-FACS library and This was used as the R5-2-2-FACS library.
（２）ＮＧＳ解析
　試験管内淘汰サイクルによるDNAライブラリの収束度を詳細に確認するために、次世代シーケンサー（以下、「NGS」ということがある。）を利用したシーケンス解析を行った。シーケンスサンプルの調製は、illumina社が提供している2－step PCR Amplicon Library Preparationの方法を参考に行った。 (2) NGS Analysis In order to confirm in detail the degree of convergence of the DNA library by the in vitro selection cycle, sequence analysis was performed using a next-generation sequencer (hereinafter sometimes referred to as "NGS"). Sequencing samples were prepared by referring to the 2-step PCR Amplicon Library Preparation method provided by Illumina.
　最初に、上記の各セレクション後のPCR産物を鋳型とし、下記表６に示すプライマー（配列番号9及び10）をプライマーとして用いて、Amplicon PCRを行い、増幅産物1を得た。Amplicon PCR条件は、(s1)98℃で1分間の後、(s2)98℃で10秒間、62℃で5秒間、72℃で35秒を15サイクル、そして(s3)72℃で1分間とした。得られたPCR産物をAgencourt AMPure XPの説明書に従って精製した後、付属の説明書に従ってIndex PCR（イルミナ社製）を行い、増幅産物2を得た。得られた増幅産物2をAgencourt AMPure XPの説明書に従って精製した後、NanoPad DS-11FX(DeNovix)により定量した。引き続き、MiSeq（イルミナ社製）及びMiSeq Reagent Nano kit v2 (500 cycles)の説明書に従って配列解析を行い、得られたDNA配列をVHHアミノ酸配列に翻訳し、出現頻度で順位付けをした。 First, using the PCR products after each of the above selections as templates and the primers (SEQ ID NOs: 9 and 10) shown in Table 6 below, Amplicon PCR was performed to obtain amplification product 1. Amplicon PCR conditions were (s1) 98°C for 1 minute, (s2) 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, 72°C for 35 seconds, and (s3) 72°C for 1 minute. did. After purifying the resulting PCR product according to the instructions of Agencourt AMPure XP, Index PCR (manufactured by Illumina) was performed according to the attached instructions to obtain amplification product 2. The resulting amplification product 2 was purified according to the instructions of Agencourt AMPure XP and then quantified by NanoPad DS-11FX (DeNovix). Subsequently, sequence analysis was performed according to the instructions of MiSeq (manufactured by Illumina) and MiSeq Reagent Nano kit v2 (500 cycles), the obtained DNA sequences were translated into VHH amino acid sequences, and ranked by appearance frequency.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（３）ファージディスプレイ法を用いたヒット化合物のスクリーニング
　上記の実施例3の(1)及び(2)に記載したスクリーニングを行ってR5で得られた各ライブラリは、Initialと比較すると目的分子の含有量が増加しており、cDNAディスプレイ法は目的化合物の濃縮ができることが示された。次いで、これらの濃縮DNAライブラリをファージディスプレイ法に供してさらに濃縮をしつつ、目的分子（以下、「ヒット化合物」ということがある。）の取得を行った。ファージディスプレイの方法を図3に模式的に示す。 (3) Screening of hit compounds using phage display method Each library obtained in R5 by performing the screening described in (1) and (2) of Example 3 above contains the target molecule when compared with Initial. The amount was increased, indicating that the cDNA display method is capable of enriching target compounds. Then, these enriched DNA libraries were subjected to a phage display method to obtain target molecules (hereinafter sometimes referred to as "hit compounds") while further enriching the libraries. The method of phage display is shown schematically in FIG.
（３－１）ファージミド及び各DNAライブラリの制限酵素処理
　上記実施例3の（１）及び（２）で得られた各ライブラリに、ファージミドベクターとのライゲーション用の制限酵素認識配列を以下のようにして付加した。まず、上記各ライブラリをテンプレートとし、上記表７に示した制限酵素認識用配列を付加するために、上記表７に示すプライマー、Alp-to-pPK4-Nco-Sfi-VHH-F（フォラードプライマー、配列番号11）及びAlp-GGGS-HisTag-to-pPK4-R（リバースプライマー）（配列番号12）を用いて、アニーリング温度55℃、伸長反応60秒の条件でPCRを行って増幅産物を得た。この増幅産物を、AMPure XP(Beckman社製)で精製した。 (3-1) Restriction enzyme treatment of phagemid and each DNA library Each library obtained in (1) and (2) of Example 3 above was given a restriction enzyme recognition sequence for ligation with a phagemid vector as follows. added. First, using each of the above libraries as a template, the primers shown in Table 7, Alp-to-pPK4-Nco-Sfi-VHH-F (Forard primer , SEQ ID NO: 11) and Alp-GGGS-HisTag-to-pPK4-R (reverse primer) (SEQ ID NO: 12), PCR was performed under the conditions of an annealing temperature of 55 ° C and an extension reaction of 60 seconds to obtain an amplified product. rice field. This amplified product was purified with AMPure XP (manufactured by Beckman).
　引き続き、ファージミド及び各DNAライブラリにFastDigest BamHIを加え、37℃にて1時間反応させ、その後FastDigest SfiI（いずれの制限酵素もThermo Fisher Scientific社製）を加え、50℃にて1時間反応させ、制限酵素処理ファージミド（以下、「ファージミドベクター1」ということがある。）を得た。その後、AMPure XPを使用して、上記ファージミドベクターを添付された説明書に従って粗精製した。 Subsequently, FastDigest BamHI was added to the phagemid and each DNA library, reacted at 37°C for 1 hour, then FastDigest SfiI (both restriction enzymes are manufactured by Thermo Fisher Scientific) was added, reacted at 50°C for 1 hour, and restricted. An enzyme-treated phagemid (hereinafter sometimes referred to as "phagemid vector 1") was obtained. Then, using AMPure XP, the phagemid vector was crudely purified according to the attached instructions.
　1xGel green（富士フイルム和光純薬(株)）を含む1％アガロースゲルに上記ファージミドベクター1をロードし、100Vで30分電気泳動した。その後、FastGene Gel/PCR Extraction Kit（日本ジェネティクス(株)）を使用し、添付の説明書に従って抽出し、さらに精製した。この精製したファージミドベクター1に、FastAP Thermosensitive Alkaline Phosphatase（Thermo Fisher Scientific社製）を加えて、37℃にて60分間反応させ脱リン酸した後に、75℃にて5分反応させて酵素を失活させた。 A 1% agarose gel containing 1xGel green (Fujifilm Wako Pure Chemical Industries, Ltd.) was loaded with the above phagemid vector 1 and subjected to electrophoresis at 100V for 30 minutes. Then, using FastGene Gel/PCR Extraction Kit (Nippon Genetics Co., Ltd.), it was extracted and further purified according to the attached instructions. FastAP Thermosensitive Alkaline Phosphatase (manufactured by Thermo Fisher Scientific) was added to this purified phagemid vector 1, reacted at 37°C for 60 minutes to dephosphorylate, and then reacted at 75°C for 5 minutes to deactivate the enzyme. let me
　上記ファージミドベクター1と上記のように制限酵素処理したライブラリ（VHH抗体遺伝子断片）を、１：5～10（モル比）で混合し、Ligation high Ver. 2 (TOYOBO社製)を加え、16℃にて一晩反応させ、上記VHH遺伝子断片が組み込まれたファージミドベクター2を得た。得られたファージミドベクター2をエタノール沈殿させて濃縮し、その後、このファージミドベクター2をエレクトロポレーションして、大腸菌TG－1（ファージディスプレイ用コンピテントセル、Lucigen社製）を形質転換した。形質転換したTG－1を2YTAG寒天培地プレートに播種して30℃にて一晩、インキュベータ中で培養した。 The above phagemid vector 1 and the library (VHH antibody gene fragment) treated with restriction enzymes as above were mixed at a ratio of 1:5 to 10 (molar ratio), Ligation high Ver. to obtain phagemid vector 2 into which the above VHH gene fragment was integrated. The resulting phagemid vector 2 was concentrated by ethanol precipitation, and then electroporated to transform Escherichia coli TG-1 (phage display competent cells, manufactured by Lucigen). The transformed TG-1 was seeded on 2YTAG agar plates and cultured overnight at 30°C in an incubator.
　上記寒天培地上に出現したコロニーをすべてピッキングして、500mLの三角フラスコ中の2YTAG 培地に回収し、O.D.600が0.5～1になるまで30℃にて培養し、大腸菌数を計数した。その後、ヘルパーファージを大腸菌の20倍量加えてヘルパーファージを大腸菌に感染させ、30℃にて一晩培養した。この培養液を、4,000 x gで、30分間、4℃にて遠心分離し、上清を回収した。回収した上清を含むチューブに2.5 MのNaClを含有する20％PEG溶液を添加し、蓋をして転倒混和した。その後、このチューブを1時間、氷上にて冷却し、その後、4,000 x gで、30分間、4℃にて遠心分離して上清を除去した。得られた沈殿物に、10％グリセロールを含むPBS(Phosphate Buffered Saline)を加えてこれを溶解し、VHHを提示したファージを含むファージディスプレイ溶液を得た。 All the colonies that appeared on the above agar medium were picked, collected in 2YTAG medium in a 500 mL Erlenmeyer flask, cultured at 30°C until O.D.600 reached 0.5 to 1, and the number of E. coli was counted. After that, the helper phage was added in an amount 20 times that of E. coli to infect E. coli with the helper phage, and cultured overnight at 30°C. This culture was centrifuged at 4,000 xg for 30 minutes at 4°C, and the supernatant was collected. A 20% PEG solution containing 2.5 M NaCl was added to the tube containing the collected supernatant, and the tube was capped and mixed by inversion. The tube was then chilled on ice for 1 hour and then centrifuged at 4,000 xg for 30 minutes at 4°C to remove the supernatant. PBS (Phosphate Buffered Saline) containing 10% glycerol was added to the obtained precipitates to dissolve them to obtain a phage display solution containing VHH-displaying phages.
（３－２）ELISAプレートを用いたバイオパニング
　FGFR1－Fc及びFGFR1－HisをPBSで10μｇ/mLに希釈し、FGFR1固定化溶液を調製した。100μLの上記FGFR1固定化溶液を96ウェルELISAプレート(Immuno Clear Standard Modules_C8_MaxiSorp: Cat # 445101、Thermo Scientific社製)の各ウェルに入れ、4℃にて一晩インキュベートして固定化した。ネガティブコントロールにはPBSを使用し、同様の操作を行った。 (3-2) Biopanning using ELISA plate FGFR1-Fc and FGFR1-His were diluted with PBS to 10 μg/mL to prepare an FGFR1-immobilized solution. 100 μL of the FGFR1 immobilization solution was placed in each well of a 96-well ELISA plate (Immuno Clear Standard Modules_C8_MaxiSorp: Cat # 445101, manufactured by Thermo Scientific) and incubated overnight at 4° C. for immobilization. PBS was used as a negative control, and the same procedure was performed.
　インキュベート後、各ウェルから溶液を除き、PBSを加えて3回洗浄し、その後、200μLのブロッキング溶液（3％スキムミルクを含有するPBS）を加え、室温にて1時間静置し、ブロッキングを行った。その後、ブロッキング溶液を捨て、各ウェルにPBSを加える操作を3回繰り返して洗浄した。3％スキムミルク及び5％BSAを含む1mLのPBSに上記（３－１）で作製した50μLのファージディスプレイ溶液を加えて混合液1とし、この混合液1を上記ELISAプレートの各ウェルに100μLずつ添加し、室温にて1時間反応させた。 After incubation, the solution was removed from each well, PBS was added and washed 3 times, then 200 μL of blocking solution (PBS containing 3% skimmed milk) was added and allowed to stand at room temperature for 1 hour for blocking. . After that, the blocking solution was discarded, and the operation of adding PBS to each well was repeated three times for washing. Add 50 μL of the phage display solution prepared in (3-1) above to 1 mL of PBS containing 3% skim milk and 5% BSA to make Mixture 1, and add 100 μL of this Mixture 1 to each well of the ELISA plate. and reacted at room temperature for 1 hour.
　この反応の終了後に、各ウェルから上記混合液を除き、200μLのPBS－Tを各ウェルに加える操作を4回繰り返して洗浄した。その後、200μLのPBS－Tをさらに各ウェルに加えて5分間振とうして洗浄した。以上の洗浄操作をもう一度繰り返した。その後、100μLの100mMトリメチルアミン溶液を各ウェルに加えて回収し、さらに100μLの100mMトリメチルアミン溶液を各ウェルに加え、室温で10分間静置して、結合しているファージを溶出させた。得られた溶出液を直ちに100μLの0.5 M Tris-HCl(pH6.8)で中和した。 After the reaction was completed, the mixture was removed from each well, and 200 μL of PBS-T was added to each well four times for washing. After that, 200 μL of PBS-T was added to each well and shaken for 5 minutes to wash. The above washing operation was repeated once more. After that, 100 μL of 100 mM trimethylamine solution was added to each well for recovery, and 100 μL of 100 mM trimethylamine solution was added to each well and allowed to stand at room temperature for 10 minutes to elute bound phages. The obtained eluate was immediately neutralized with 100 μL of 0.5 M Tris-HCl (pH 6.8).
　このように中和した溶出液を1,200μLの大腸菌TG－1と混合し、30℃にて1時間静置して混合液2とした。10μLのこの混合液2を、2YTAG寒天培地（1.5％、10cmディッシュ）に播種した。上記混合液2の残りを4000 x gで20分間、25℃にて遠心分離し、上清を除去した。得られた沈殿物（大腸菌TG－1）を、上記と同じ2YTAG寒天培地（15cmディッシュ）に播種した。 The eluate neutralized in this way was mixed with 1,200 μL of E. coli TG-1 and allowed to stand at 30°C for 1 hour to obtain mixed solution 2. 10 μL of this mixture 2 was plated on 2YTAG agar medium (1.5%, 10 cm dish). The rest of the mixture 2 was centrifuged at 4000 xg for 20 minutes at 25°C and the supernatant was removed. The resulting precipitate (Escherichia coli TG-1) was seeded on the same 2YTAG agar medium (15 cm dish) as above.
　上記のように大腸菌を播種した各ディッシュを、30℃にて一晩培養した。上記（３－１）と同様の操作を行い、ヘルパーファージを大腸菌の20倍量加えてこれを大腸菌に感染させ、30℃で一晩培養した。増殖した大腸菌を含む培養液を上記（３－１）と同様に遠心して上清をチューブに回収し、2.5MのNaCl含有20％PEG溶液をここに添加し、蓋をして転倒混和した。その後、1時間氷上にて冷却し、上記（３－１）と同様に遠心分離して上清を除去し、沈殿物を得た。この沈殿物に10％グリセロールを含むPBSを加えて溶解させ、ファージディスプレイを作成し、もう一度バイオパニングを行った。 Each dish seeded with E. coli as described above was cultured overnight at 30°C. E. coli was infected with helper phage by adding 20 times the amount of E. coli and cultured overnight at 30°C. The culture solution containing the grown E. coli was centrifuged in the same manner as in (3-1) above, and the supernatant was collected in a tube. Thereafter, the mixture was cooled on ice for 1 hour, centrifuged in the same manner as in (3-1) above, and the supernatant was removed to obtain a precipitate. PBS containing 10% glycerol was added to this precipitate to dissolve it, phage display was prepared, and biopanning was performed again.
（３－３）イムノチューブを用いたバイオパニング
　FGFR1－FcをPBSで10μｇ/mLに希釈してFGFR1固定化溶液（以下、単に「固定化溶液」ということがある。）を調製し、1mLをイムノチューブ（MAXISORP NUNC-IMMUNO TUBE 5.0 mL:Cat. #444202,Thermo Scientific社製）に取って、4℃にて一晩CO2インキュベータ中にて固定化した。上記固定化用得液を捨て、ここにPBSを加えて洗浄するという操作を3回繰り返し、その後、5mLのブロッキング溶液（3％スキムミルク含有PBS）をこのチューブに加え、室温にて1時間静置してブロッキングを行った。
　その後、PBSを加えて除去するという洗浄操作を5回繰り返した。引き続き、3mLのアルブミン溶液（3％スキムミルク、5％BSA及び5％ヒト血清アルブミン(HSA)を含む)に上記（３－１）で作製した100μLのファージディスプレイ溶液を混合して混合液3を調製し、その後、この混合液3の全量をイムノチューブに添加し、室温にて1時間反応させた。 (3-3) Biopanning using an immunotube Dilute FGFR1-Fc with PBS to 10 μg/mL to prepare an FGFR1 immobilization solution (hereinafter sometimes simply referred to as “immobilization solution”), and dispense 1 mL. It was taken in an immunotube (MAXISORP NUNC-IMMUNO TUBE 5.0 mL: Cat. #444202, manufactured by Thermo Scientific) and immobilized overnight at 4°C in a CO 2 incubator. Discard the immobilization solution, add PBS to wash, and repeat the procedure three times. After that, add 5 mL of blocking solution (PBS containing 3% skimmed milk) to the tube and let stand at room temperature for 1 hour. blocking.
After that, a washing operation of adding and removing PBS was repeated five times. Subsequently, mix 100 μL of the phage display solution prepared in (3-1) above with 3 mL of albumin solution (containing 3% skim milk, 5% BSA and 5% human serum albumin (HSA)) to prepare mixture 3. After that, the whole amount of this mixed solution 3 was added to the immunotube and reacted at room temperature for 1 hour.
　上記反応終了後、上記イムノチューブ内の溶液を捨て、5mLのPBS－Tを加えて洗浄するという操作を10回繰り返し、さらに5mLのPBS－Tを加え、蓋をして30分間振とうして洗浄した。引き続き、5mLのPBS－Tを加えて洗浄するという操作を10回繰り返した。その後、1mLの100 mMトリメチルアミン溶液をこの洗浄後のイムノチューブに加えて新しいチューブに回収し、さらに再度同量の100 mM トリメチルアミン溶液をこのイムノチューブに加え、室温にて10分間静置して、結合しているファージを溶出させた。 After completion of the above reaction, discard the solution in the immunotube, add 5 mL of PBS-T for washing, repeat this procedure 10 times, add 5 mL of PBS-T, cover and shake for 30 minutes. washed. Subsequently, the operation of adding 5 mL of PBS-T and washing was repeated 10 times. After that, add 1 mL of 100 mM trimethylamine solution to this immunotube after washing and collect it in a new tube. Bound phage were eluted.
　得られた溶出液は、直ちに1mLの0.5 M Tris-HCl (pH6.8)で中和した。中和後の溶出液を、10mLの大腸菌TG－1と混合し、30℃にて1時間静置して混合液4とした。この混合液4を上記と同様に遠心分離し、得られた沈殿物（大腸菌TG－1）を、上述した2YTAG寒天培地（15cmディッシュ）に播種して、30℃にて一晩、CO2インキュベータ中にて培養した。 The resulting eluate was immediately neutralized with 1 mL of 0.5 M Tris-HCl (pH 6.8). The eluate after neutralization was mixed with 10 mL of Escherichia coli TG-1 and allowed to stand at 30° C. for 1 hour to obtain mixed solution 4. This mixture 4 was centrifuged in the same manner as above, and the resulting precipitate (Escherichia coli TG-1) was seeded on the above-described 2YTAG agar medium (15 cm dish) and placed in a CO 2 incubator at 30°C overnight. cultivated in the
　このディッシュ中の寒天培地上に出現したすべてのコロニーを状時期と同様に液体培地をいれたフラスコ中に回収し、O. D. 600が0.5～1になるまで30℃にてCO2インキュベータ中で培養し、大腸菌数を計数した。その後、ヘルパーファージを大腸菌の20倍量加えてヘルパーファージを大腸菌に感染させ、30℃にてCO2インキュベータ中で一晩培養した。 All the colonies that appeared on the agar medium in this dish were collected in a flask containing a liquid medium in the same manner as during the first stage, and cultured in a CO 2 incubator at 30°C until the O.D.600 reached 0.5-1. , the number of E. coli was counted. After that, helper phage was added in an amount 20 times that of E. coli to infect E. coli with the helper phage, and cultured overnight at 30°C in a CO 2 incubator.
　この培養液を、上記（３－２）と同様に処理して上清を新たなチューブ中に回収し、25M NaCl含有20％PEG溶液をここに加え、蓋をして転倒混和した。その後、1時間氷上にて冷却し、上記と同様に遠心分離して上清を除去し、沈殿物を得た。得られた沈殿物に10％グリセロール含有PBSを加えて溶解し、ファージディスプレイを作成し、第2回バイオパニングを行った。第2回バイオパニングでは、FGFR1－Fcに代えてFGFR1－Hisをイムノチューブに固定化した以外は、上記と同様の操作を行い、第2ライブラリを得た。 This culture medium was treated in the same manner as in (3-2) above, the supernatant was collected in a new tube, a 20% PEG solution containing 25M NaCl was added, and the tube was covered and mixed by inversion. Thereafter, the mixture was cooled on ice for 1 hour, centrifuged in the same manner as above, and the supernatant was removed to obtain a precipitate. The resulting precipitate was dissolved in PBS containing 10% glycerol, phage display was prepared, and a second round of biopanning was performed. In the second biopanning, the same procedure as above was performed except that FGFR1-His was immobilized on the immunotube instead of FGFR1-Fc to obtain a second library.
（３－４）ファージELISA
　上記のようにして得られた第2ライブラリ中に、FGFR1に結合するVHHが濃縮されているか否かを確認するために、ポリクローナルの状態でファージELISAを行った。
　まず、FGFR1－Fc及びFGFR1－HisをPBSで10μｇ/mLに希釈しFGFR1固定化溶液を調製した。100μLの上記FGFR1固定化溶液を96ウェルELISAプレートの各ウェルに添加し、4℃にて一晩インキュベートし、これらを固定化した。各ウェルから上記固定化溶液を除去し、PBSを加えて洗浄する操作を3回繰り返し、その後、上記（３－３）と同様にブロッキングを行った。ブロッキングの終了後、各ウェルからブロッキング溶液を除去し、PBSを加えて洗浄する操作を3回繰り返した。 (3-4) Phage ELISA
In order to confirm whether VHHs that bind to FGFR1 are enriched in the second library obtained as described above, phage ELISA was performed in a polyclonal state.
First, FGFR1-Fc and FGFR1-His were diluted with PBS to 10 μg/mL to prepare an FGFR1-immobilized solution. 100 μL of the above FGFR1 immobilization solution was added to each well of a 96-well ELISA plate and incubated overnight at 4° C. to immobilize them. An operation of removing the immobilization solution from each well, adding PBS and washing was repeated three times, and then blocking was performed in the same manner as in (3-3) above. After blocking, the blocking solution was removed from each well, and PBS was added for washing, which was repeated three times.
　各ラウンドで得られたファージディスプレイから160μLを取り、これを160μLの10％BSA含有PBSと混合して混合液5とした。その後、50 μLの上記混合液5を各ウェルに添加し、室温で1時間反応させた。各ウェルから上記混合液5を除去し、その後、各ウェルに200μLのPBS－Tを加えて洗浄する操作を5回繰り返した。抗M13－mAb－HRP (Sinobiological社製)を、PBS－Tで3,000倍に希釈した抗体溶液を各ウェルに50μL加え、室温で1時間反応させた。 　160 µL was taken from the phage display obtained in each round and mixed with 160 µL of 10% BSA-containing PBS to obtain Mixture 5. After that, 50 μL of the mixed solution 5 was added to each well and reacted at room temperature for 1 hour. The operation of removing the above mixture 5 from each well and then adding 200 μL of PBS-T to each well for washing was repeated five times. Anti-M13-mAb-HRP (manufactured by Sinobiological) was diluted 3,000-fold with PBS-T and 50 μL of the antibody solution was added to each well and reacted at room temperature for 1 hour.
　その後、上記抗体溶液を各ウェルから除去し、200μLのPBS－Tを加えて洗浄する操作を5回繰り返した。引き続き、OPDタブレット(cat# 155-02161、富士フィルム和光純薬(株)製)を10 mLの0.1 M NaH２PO４で溶解し、各ウェルに100μLずつ分注し、遮光して15分間、室温にてインキュベートした。100μLの1 M 硫酸を添加して反応を停止させ、直ちにマイクロプレートリーダー Infinite 200Pro M PLEX(TECAN)を用いて、各ウェルの吸光度を490nmで測定した。 Thereafter, the above antibody solution was removed from each well, and 200 μL of PBS-T was added to wash the wells, which was repeated 5 times. Subsequently, OPD tablet (cat# 155-02161, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of 0.1 M NaH 2 PO 4 , 100 μL was dispensed into each well, shielded from light for 15 minutes, Incubated at room temperature. 100 μL of 1 M sulfuric acid was added to stop the reaction, and the absorbance of each well was immediately measured at 490 nm using a microplate reader Infinite 200Pro M PLEX (TECAN).
　上記（３－２）でバイオパニングしたVHH提示ファージをエッペンドルフチューブに入れ、ここに大腸菌TG－1を加えて、30℃にて1時間静置しF-E混合液とした。このF-E混合液を4000 x gで20分間25℃にて遠心分離し、得られた沈殿物（大腸菌TG－1）を2YTAGに溶解させて溶解液とした。その後、この溶解液を、上記と同様に2YTAG寒天培地に播種し、寒天培地上に出現したコロニーをシングルピッキングし、クローン化してファージディスプレイを回収した。 The VHH-displaying phages biopanned in (3-2) above were placed in an Eppendorf tube, E. coli TG-1 was added, and the mixture was allowed to stand at 30°C for 1 hour to form an F-E mixture. This F-E mixture was centrifuged at 4000×g for 20 minutes at 25° C., and the resulting precipitate (Escherichia coli TG-1) was dissolved in 2YTAG to obtain a lysate. Thereafter, this lysate was seeded on a 2YTAG agar medium in the same manner as described above, and colonies that appeared on the agar medium were single-picked and cloned to recover phage display.
　ELISAプレートにFGFR1－HisをPBSで2μg/mLに希釈した溶液を使用した点を除き、ファージELISAを同様に行った。その後、レスポンスが得られたクローンについてはファージミドを大腸菌から抽出し、サンガー法で、提示されたVHHに対応するDNAの配列解析を行った。この配列解析は、ユーロフィンジェノミクスに委託した。以上の解析により、2つのVHHクローン、＃1及び＃2が同定された。 Phage ELISA was performed in the same manner, except that FGFR1-His diluted with PBS to 2 μg/mL was used in the ELISA plate. After that, the phagemids of the responding clones were extracted from E. coli, and DNA sequences corresponding to the displayed VHHs were analyzed by the Sanger method. This sequence analysis was contracted to Eurofins Genomics. The above analysis identified two VHH clones, #1 and #2.
（実施例４）VHH（単量体）の産生
　上記実施例３（３－４）で得られたVHHクローン＃1及び＃2について、コリネバクテリウム・グルタミカム（C. glutamicum）を用いて、以下のようにして単量体VHH（以下、「sVHH」ということがある。）を発現させ、これを精製した。
（１）sVHH発現用プラスミドの構築
　上記VHHクローン＃1及び＃2のファージミドをテンプレート、下記表７に示すようにpSLiCE_PK4_SMDPhage_F （配列番号13）、pSMDPhage_#1_R （配列番号14）又はpSMDPhage_#2_R （配列番号15）をプライマーとし、アニーリング温度を55℃、伸長反応5秒の条件でPCRを行って増幅産物としてsVHH発現用プラスミドを得た。得られた増幅産物は、Gel/PCR Extraction Kit（日本ジェネティクス社製）を使用し、添付されたマニュアルに従って精製し、精製DNAを得た。 (Example 4) Production of VHH (monomer) VHH clones #1 and #2 obtained in Example 3 (3-4) above were treated with Corynebacterium glutamicum (C. glutamicum) as follows. A monomeric VHH (hereinafter sometimes referred to as "sVHH") was expressed and purified as described above.
(1) Construction of sVHH Expression Plasmid Using the phagemids of the above VHH clones #1 and #2 as templates, pSLiCE_PK4_SMDPhage_F (SEQ ID NO: 13), pSMDPhage_#1_R (SEQ ID NO: 14) or pSMDPhage_#2_R (sequence No. 15) as primers, PCR was performed under the conditions of an annealing temperature of 55°C and an extension reaction of 5 seconds to obtain an sVHH expression plasmid as an amplified product. The resulting amplified product was purified using a Gel/PCR Extraction Kit (manufactured by Nippon Genetics) according to the attached manual to obtain purified DNA.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　50mM Tris-HCl(pH7.5)、10mM MgCl２、1mM ATP、及び1mM DTTを含むSLiCEバッファーを入れた8連PCRチューブ中に、上記の精製DNA、C. glutamicum発現用プラスミド、及び大腸菌抽出用SLiCE反応液を加え、37℃にて15分間のIn vitro相同組換えを行った。得られた相同組換え体でコンピテントセルJM109（タカラバイオ社製）を形質転換し、この形質転換体からプラスミドを抽出した。抽出したプラスミドのDNA配列解析の結果、VHHクローン＃1又は＃2の遺伝子が、それぞれこのプラスミド中に組み込まれていることが確認された。 The above purified DNA, C. glutamicum expression plasmid, and E. coli extraction were added to an 8-strip PCR tube containing SLiCE buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM ATP, and 1 mM DTT. The SLiCE reaction solution was added, and in vitro homologous recombination was performed at 37°C for 15 minutes. A competent cell JM109 (manufactured by Takara Bio Inc.) was transformed with the obtained homologous recombinant, and a plasmid was extracted from this transformant. DNA sequence analysis of the extracted plasmid confirmed that the gene of VHH clone #1 or #2, respectively, was integrated into this plasmid.
（２）C. glutamicumを用いたsVHHの発現
　エレクトロポレーションにより各プラスミドをC. glutamicumへ導入し、形質転換を行った。得られた形質転換体をCM2G培地に植菌し、30℃にて一晩前培養を行った。その後、この前培養液をPM1S培地（96ディープウェルプレート;Thermo Fisher社製）に1/20量継代し、25℃にて72時間で培養し、上記形質転換体に培養上清へVHH（単量体）を分泌させた。培養終了後、96ウェルディープウェルプレートを4,000ｘgで30分間、20℃にて遠心し、培養上清を回収した。回収した培養上清を0.22μｍのフィルターで処理し、上清からの菌体を除去した。菌体を除去した培養上清は－80℃にて凍結保存し、適宜、精製に使用した。 (2) Expression of sVHH using C. glutamicum Each plasmid was introduced into C. glutamicum by electroporation to effect transformation. The resulting transformant was inoculated into CM2G medium and precultured overnight at 30°C. Then, 1/20 volume of this preculture was subcultured in PM1S medium (96 deep well plate; manufactured by Thermo Fisher) and cultured at 25°C for 72 hours. (monomer) was secreted. After completion of the culture, the 96-well deep well plate was centrifuged at 4,000 xg for 30 minutes at 20°C to collect the culture supernatant. The collected culture supernatant was treated with a 0.22 μm filter to remove the cells from the supernatant. The culture supernatant from which the cells were removed was stored frozen at -80°C and used for purification as appropriate.
（３）sVHHの精製
　上記（２）のように凍結保存した培養上清を解凍し、終濃度10mMとなるようにイミダゾールを添加した。その後、100μLのNi Sepharose 6 Fast Flow (Cytiva社製、以下、単に「担体」ということがある。）をここに添加して混合し、蓋をして4℃にて1時間転倒混和した。500ｘgで1分間、4℃にて遠心して上記担体を沈殿させ、最終液量が700μL程度になるように上清を除去した。 (3) Purification of sVHH The culture supernatant that had been cryopreserved as in (2) above was thawed, and imidazole was added to a final concentration of 10 mM. After that, 100 μL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva, hereinafter sometimes simply referred to as “carrier”) was added thereto, mixed, covered, and mixed by inversion at 4° C. for 1 hour. The carrier was precipitated by centrifugation at 500 xg for 1 minute at 4°C, and the supernatant was removed so that the final liquid volume was about 700 µL.
　懸濁液をスピンカラム（EconoSpin;Ajinomoto Bio-Pharma社製）に移し、100ｘgで30秒間、4℃にて遠心し、担体を回収した。引き続き、600μLの洗浄バッファー（300 mM NaCl及び30 mMイミダゾールを含む50 mM Tris-HCl (pH7.5)）を加えて、上記と同様の条件で遠心する操作を2回繰り返し、その後、700μLの溶出バッファー（300 mMのNaCl及び500 mMのイミダゾールを含む50 mM Tris-HCl(pH7.5)）を加えて、結合されたVHHの溶出を行った。 The suspension was transferred to a spin column (EconoSpin; manufactured by Ajinomoto Bio-Pharma) and centrifuged at 100 xg for 30 seconds at 4°C to recover the carrier. Subsequently, add 600 μL of washing buffer (50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 30 mM imidazole), repeat the centrifugation operation twice under the same conditions as above, and then elute with 700 μL. Bound VHHs were eluted by adding buffer (50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 500 mM imidazole).
　溶出されたsVHHの純度はSDS－PAGEにて確認した（図5参照）。SDS-PAGEは4%濃縮ゲル、15%分離ゲルにて行った。各ウェルに4μL分のサンプルをアプライした後、150Vで1時間の条件で電気泳動した。分子量マーカーにはPrecision Plus ProteinTM Standards（BIO-RAD）を用いた。電気泳動後、クマシーブリリアントブルー（CBB）染色した。 The purity of the eluted sVHH was confirmed by SDS-PAGE (see Figure 5). SDS-PAGE was performed using a 4% stacking gel and a 15% separating gel. After applying 4 μL of sample to each well, electrophoresis was performed at 150 V for 1 hour. Precision Plus Protein ™ Standards (BIO-RAD) were used as molecular weight markers. After electrophoresis, it was stained with Coomassie brilliant blue (CBB).
　上記のようにして得た溶出液含まれる高濃度のイミダゾールを除去するために、脱塩カラム(Zeba Spin Desalting Columns；Thermo Fisher社製）を通して溶出液のバッファーをPBSに置換した。ウシ血清アルブミン（富士フイルム和光純薬(株)製）を標準タンパク質とし、Pierce BCA Protein Assay Kit（Thermo Fisher社製）を用いたBCA法により、sVHHの濃度を定量した。 In order to remove the high-concentration imidazole contained in the eluate obtained as described above, the eluate buffer was replaced with PBS through a desalting column (Zeba Spin Desalting Columns; Thermo Fisher). The concentration of sVHH was quantified by the BCA method using bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard protein and Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher).
（実施例５）sVHHの親和性測定
　sVHHの親和性測定は、表面プラズモン共鳴(SPR)法共鳴法を用いて試験した。
　Series S Sensor Chip CAP(Cytiva社製)に固相化したVHHクローン＃1又は＃2のFGFR1－Fcに対する結合活性をｍ、Biacore（商標）T200(Cytiva社製)を用いて測定し、CAP Single-cycle Kinetics法で解析した。温度は25℃に設定した。ランニング緩衝液として、HBS－EP+（150 mM NaCl、0.5mM EDTA及び0.05% surfactant P20を含む10 mM HEPES (pH 7.4)) (Cytiva社製)を用いた。ラン毎の測定順は以下の通りとし、試薬はすべてCytiva社製を使用した。 (Example 5) Affinity measurement of sVHH Affinity measurement of sVHH was tested using surface plasmon resonance (SPR) resonance method.
The binding activity to FGFR1-Fc of VHH clone #1 or #2 immobilized on Series S Sensor Chip CAP (manufactured by Cytiva) was measured using Biacore (trademark) T200 (manufactured by Cytiva), and CAP Single -Analyzed by cycle kinetics method. The temperature was set at 25°C. As a running buffer, HBS-EP+ (10 mM HEPES (pH 7.4) containing 150 mM NaCl, 0.5 mM EDTA and 0.05% surfactant P20) (manufactured by Cytiva) was used. The measurement order for each run was as follows, and all reagents were manufactured by Cytiva.
　1)ビオチン化FGFR1－Fcの固相化：流速を2μL/mLに設定し、Biotin CAPture Reagent を300秒間添加し、その後流速を10μL/mLに上げ、ランニング緩衝液で希釈したFGFR1－Fc溶液を120秒間添加することで140RU固定化した。
　2)結合活性の測定：流速を30μL/mLに設定し、ランニング緩衝液で1.85, 5.56, 16.67, 及び50nMに希釈したVHHクローン＃1又は＃2をAssociation Time 120秒間で結合させ、Dissociation Timeを600秒間に設定して相互作用させた。 1) Immobilization of biotinylated FGFR1-Fc: Set the flow rate to 2 μL/mL, add Biotin CAPture Reagent for 300 seconds, then increase the flow rate to 10 μL/mL, and add the FGFR1-Fc solution diluted with running buffer. 140 RU was immobilized by adding for 120 seconds.
2) Measurement of binding activity: Set the flow rate to 30 μL/mL, bind VHH clone #1 or #2 diluted to 1.85, 5.56, 16.67, and 50 nM with running buffer for an association time of 120 seconds, and set the dissociation time to The interaction was set to 600 seconds.
　3)センサーチップ表面の再生：流速を10μL/mLに設定し、Reaneration stock 1とRegeneration stock 2とを3：1で混合した溶液を添加して120秒間置き、固相化されたHisタグ付きVHHを溶離させた。Biacore（商標）T200 Evaluation（ソフトウェアバージョン2.0）を用いて1：1結合モデルによる解析を行い、結合活性を算出した（図3及び図4参照）。その結果、VHHクローン＃1のFGFR1－Fcに対する平衡乖離定数(KD)は1.74x10－９M（ka＝8.19x10６（1/Ms）、kd＝1.42x10－３（1/s））であった。VHHクローン＃2のFGFR1に対する平衡乖離定数(KD)は1.02x10－９M（ka＝1.11x10６（1/Ms）、kd=1.14x10－３（1/s））であった。 3) Regeneration of the sensor chip surface: Set the flow rate to 10 μL/mL, add a 3:1 mixture of Reaneration stock 1 and Regeneration stock 2, leave for 120 seconds, and immobilize His-tagged VHH. was eluted. Analysis was performed with a 1:1 binding model using Biacore™ T200 Evaluation (software version 2.0) and binding activity was calculated (see Figures 3 and 4). As a result, the equilibrium dissociation constant (KD) of VHH clone #1 for FGFR1-Fc was 1.74×10 −9 M (ka=8.19×10 6 (1/Ms), kd=1.42×10 −3 (1/s)). rice field. The equilibrium dissociation constant (KD) of VHH clone #2 for FGFR1 was 1.02×10 −9 M (ka=1.11×10 6 (1/Ms), kd=1.14×10 −3 (1/s)).
　本発明は、医学、診断薬等の技術分野において有用である。 The present invention is useful in technical fields such as medicine and diagnostic agents.
配列番号１：目的分子用プライマーの塩基配列
配列番号２：目的分子用プライマーの塩基配列
配列番号３：目的分子用プライマーの塩基配列
配列番号４：オーバーラップPCR用プライマーの塩基配列
配列番号５：オーバーラップPCR用プライマーの塩基配列
配列番号６：ｃｎｖＫリンカーの主鎖の塩基配列
配列番号７：プライマー（cnvK NewYtag）の塩基配列
配列番号８：プライマー（T7ΩNew）の塩基配列
配列番号９：NGS Fw 1st PCR primerの塩基配列
配列番号１０：NGS Rv 1st PCR primerの塩基配列
配列番号１１：制限酵素認識配列付加用プライマーの塩基配列
配列番号１２：制限酵素認識配列付加用プライマーの塩基配列
配列番号１３：プライマー（pSLiCE_PK4_SMDPhage_F）の塩基配列
配列番号１４：プライマー（pSMDPhage_#1_R）の塩基配列
配列番号１５：プライマー（pSMDPhage_#2_R）の塩基配列 SEQ ID NO: 1: Base sequence of target molecule primer SEQ ID NO: 2: Target molecule primer base sequence SEQ ID NO: 3: Target molecule primer base sequence SEQ ID NO: 4: Overlap PCR primer base sequence SEQ ID NO: 5: Overlap Nucleotide sequence of primer for wrap PCR SEQ ID NO: 6: base sequence of main chain of cnvK linker SEQ ID NO: 7: base sequence of primer (cnvK NewYtag) SEQ ID NO: 8: base sequence of primer (T7ΩNew) SEQ ID NO: 9: NGS Fw 1st PCR Base sequence of primer SEQ ID NO: 10: Base sequence of NGS Rv 1st PCR primer SEQ ID NO: 11: Base sequence of primer for addition of restriction enzyme recognition sequence SEQ ID NO: 12: Base sequence of primer for addition of restriction enzyme recognition sequence SEQ ID NO: 13: Primer ( pSLiCE_PK4_SMDPhage_F) base sequence SEQ ID NO: 14: base sequence of primer (pSMDPhage_#1_R) SEQ ID NO: 15: base sequence of primer (pSMDPhage_#2_R)

Claims (10)

  1. 　(a)無細胞翻訳系を使用して目的分子のDNAを選択してライブラリを構成し、前記ライブラリに含まれるDNA断片の中から、前記目的分子のDNAを選択し、濃縮して淘汰することによって一次ライブラリを作製する、選択・淘汰工程と；
    　(b)前記選択・淘汰工程で得られた一次ライブラリに含まれる前記目的分子から作られるタンパクのうち、生物において作製可能なタンパクをコードするDNA断片を、細胞を用いて発現させて選択する発現・選択工程と；
    　を備える、目的分子のスクリーニング方法。     (a) Using a cell-free translation system to select target molecule DNAs to construct a library, and then selecting, concentrating and eliminating the target molecule DNAs from DNA fragments contained in the library. A selection and selection step to create a primary library by;
    (b) Expression using cells to express and select a DNA fragment encoding a protein that can be produced in an organism, among the proteins produced from the target molecule contained in the primary library obtained in the selection/selection step - a selection step;
    A method of screening for a molecule of interest, comprising:
  2. 　前記選択・淘汰工程では、主鎖と側鎖とを有するcDNAディスプレイ用リンカーを使用する請求項１に記載の目的分子のスクリーニング方法：ここで、
    　前記リンカーの主鎖は、
    　　(m1)前記リンカーを固相へ固定するための固相結合部位と；
    　　(m2)固相から前記リンカーを切り離すための切断部位と；
    　　(m3)前記主鎖にmRNAを結合させるための光架橋塩基と；
    　　(m4)側鎖結合部位と；
    　　(m5)逆転写開始部位と；を含み、
    　前記側鎖は、
    　　(s1)前記主鎖に結合されたmRNAに対応して合成されたペプチドを結合するためのペプチド結合部位と；
    　　(s2)標識結合部位と；
    　　(s3)前記標識結合部位に結合する検出用蛍光分子と；
    　　(s4)前記主鎖の前記側鎖結合部位に結合する主鎖結合部位と；
    を備える。     The method for screening a target molecule according to claim 1, wherein the selection/selection step uses a cDNA display linker having a main chain and a side chain;
    The main chain of the linker is
    (m1) a solid phase binding site for anchoring the linker to a solid phase;
    (m2) a cleavage site for cleaving the linker from the solid phase;
    (m3) a photocrosslinking base for binding mRNA to the backbone;
    (m4) a side chain binding site;
    (m5) a reverse transcription initiation site;
    The side chain is
    (s1) a peptide binding site for binding a peptide synthesized corresponding to the mRNA bound to the backbone;
    (s2) a labeled binding site;
    (s3) a detectable fluorescent molecule that binds to said label binding site;
    (s4) a main chain binding site that binds to the side chain binding site of the main chain;
    Prepare.
  3. 　前記リンカーにおいて、
    　前記固相結合部位は、ビオチン、ストレプトアビジン、及びそれらの類縁体からなる群から選ばれる分子で構成され；
    　前記切断部位は、リボＧ又はイノシンで構成され；
    　前記光架橋塩基は、シアノビニルカルバゾール又はその類縁体で構成され；
    　前記ペプチド結合部位は、ピューロマイシン又はその類縁体で構成され；
    　前記検出用蛍光分子は、フルオレセイン、フルオレセインイソチオシアネート、テトラクロロフルオレセイン、５'－ヘキサクロロ－フルオレセイン－ＣＥホスホロアミダイト、Cy3、Cy5、Alexa568、及びAlexa647からなる群から選ばれる；
    請求項２に記載の目的分子のスクリーニング方法。     In the linker,
    said solid phase binding site is composed of a molecule selected from the group consisting of biotin, streptavidin, and analogues thereof;
    the cleavage site is composed of ribo-G or inosine;
    The photocrosslinking base is composed of cyanovinylcarbazole or an analogue thereof;
    said peptide binding site is composed of puromycin or an analog thereof;
    The fluorescent molecule for detection is selected from the group consisting of fluorescein, fluorescein isothiocyanate, tetrachlorofluorescein, 5′-hexachloro-fluorescein-CE phosphoramidites, Cy3, Cy5, Alexa568, and Alexa647;
    The method of screening for a target molecule according to claim 2.
  4. 　前記一次ライブラリは、1010～1014のライブラリサイズのcDNAライブラリから、前記リンカーを用いて構成される請求項１又は２に記載の目的分子のスクリーニング方法。 3. The method for screening a target molecule according to claim 1, wherein the primary library is constructed from a cDNA library with a library size of 10 10 to 10 14 using the linkers.
  5. 　前記選択・淘汰工程は、
    　(a1)目的分子のmRNAを調製するmRNA調製工程と；
    　(a2)300～365nmの波長を15秒～60秒間照射して、前記(a1)工程で得られたmRNAと前記リンカーとを前記光架橋塩基で連結させて、リンカー－mRNA連結体を作製する連結工程と；
    　(a3)前記リンカー－mRNA連結体を固相に結合させ、その後無細胞系で翻訳し、リンカー－mRNA－ペプチド連結体を形成する翻訳工程と；
    　(a4)前記リンカー－mRNA－ペプチド連結体をPCRで増幅させ、逆転写してmRNA/cDNA－タンパク質連結体を得る逆転写工程と；
    　(a5)前記増幅されたcDNAディスプレイ分子を前記固相から固相切断部位で切断し、一次ライブラリを得る、一次ライブラリ作製工程と；
    　(a6)前記一次ライブラリに含まれる目的分子のDNAをセレクションして濃縮する濃縮工程と；
    を備える請求項１～４のいずれかに記載の目的分子のスクリーニング方法。     The selection/selection step includes:
    (a1) an mRNA preparation step of preparing mRNA of the target molecule;
    (a2) irradiating for 15 to 60 seconds with a wavelength of 300 to 365 nm to ligate the mRNA obtained in step (a1) and the linker with the photocrosslinking base to prepare a linker-mRNA conjugate. a concatenating step;
    (a3) a translation step of binding the linker-mRNA conjugate to a solid phase and then translating in a cell-free system to form a linker-mRNA-peptide conjugate;
    (a4) a reverse transcription step of amplifying the linker-mRNA-peptide conjugate by PCR and reverse transcription to obtain an mRNA/cDNA-protein conjugate;
    (a5) a primary library preparation step of cleaving the amplified cDNA display molecule from the solid phase at a solid phase cleavage site to obtain a primary library;
    (a6) an enrichment step of selecting and enriching target molecule DNAs contained in the primary library;
    The method for screening a target molecule according to any one of claims 1 to 4, comprising
  6. 　前記固相は、セファロースビーズ、シリカビーズ及びラテックスビーズからなる群から選ばれる磁性粒子である請求項５に記載の目的分子のスクリーニング方法。 The method for screening a target molecule according to claim 5, wherein the solid phase is magnetic particles selected from the group consisting of sepharose beads, silica beads and latex beads.
  7. 　前記前記選択・淘汰工程は、
    　(b1)上記一次ライブラリ中のDNAをファージミドと混合してファージミドベクターを形成させるファージミドベクター形成工程と；
    　(b2)前記ファージミドベクター中に目的分子の遺伝子断片を組み込み、前記細胞を形質転換させる形質転換工程と；
    　(b3)前記形質転換工程で得られた細胞を培養し、ヘルパーファージを加えて上記細胞に感染させ、培養して上記目的分子を提示したファージを含むファージライブラリ作製工程と、
    　(b4)前記ファージライブラリに含まれるファージをバイオパニングして上記目的分子と結合するファージを選択するバイオパニング工程と；
    　を備える請求項１～６のいずれかに記載の目的分子のスクリーニング方法。     The selection/selection step includes:
    (b1) a phagemid vector forming step of mixing the DNA in the primary library with a phagemid to form a phagemid vector;
    (b2) a transformation step of integrating a gene fragment of the target molecule into the phagemid vector to transform the cell;
    (b3) culturing the cells obtained in the transformation step, adding a helper phage to infect the cells, culturing and preparing a phage library containing phages displaying the target molecule;
    (b4) a biopanning step of biopanning phages contained in the phage library to select phages that bind to the target molecule;
    The method for screening a target molecule according to any one of claims 1 to 6, comprising
  8. 　前記細胞は、コンピテントセルである請求項１～７のいずれかに記載の目的分子のスクリーニング方法。 The method for screening a target molecule according to any one of claims 1 to 7, wherein the cells are competent cells.
  9. 　前記コンピテントセルは、大腸菌である請求項８のいずれかに記載の目的分子のスクリーニング方法。 The method for screening a target molecule according to any one of claims 8, wherein the competent cells are Escherichia coli.
  10. 　前記（a1）～（a6）工程を繰り返す回数が５回以内である請求項５又は６のいずれかに記載の目的分子のスクリーニング方法。
          7. The method for screening a target molecule according to claim 5, wherein steps (a1) to (a6) are repeated 5 times or less.
PCT/JP2022/024080 2021-06-21 2022-06-16 Double-screening method WO2022270396A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021102744 2021-06-21
JP2021-102744 2021-06-21

Publications (1)

Publication Number Publication Date
WO2022270396A1 true WO2022270396A1 (en) 2022-12-29

Family

ID=84544320

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/024080 WO2022270396A1 (en) 2021-06-21 2022-06-16 Double-screening method

Country Status (1)

Country Link
WO (1) WO2022270396A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007306866A (en) * 2006-05-19 2007-11-29 National Institute Of Advanced Industrial & Technology Three-finger-like protein library
WO2017170776A1 (en) * 2016-03-30 2017-10-05 株式会社Ｅｐｓｉｌｏｎ Ｍｏｌｅｃｕｌａｒ Ｅｎｇｉｎｅｅｒｉｎｇ High-speed in vitro screening method
JP2018513670A (en) * 2015-01-29 2018-05-31 テクノファージ， インベスティガサン エ デセンボルビメント エム ビオテクノロジア，エスエー Antibody molecule and peptide delivery system for use in Alzheimer's disease and related disorders
JP2019509023A (en) * 2016-01-20 2019-04-04 ザ・スクリップス・リサーチ・インスティテュート ROR2 antibody composition and related methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007306866A (en) * 2006-05-19 2007-11-29 National Institute Of Advanced Industrial & Technology Three-finger-like protein library
JP2018513670A (en) * 2015-01-29 2018-05-31 テクノファージ， インベスティガサン エ デセンボルビメント エム ビオテクノロジア，エスエー Antibody molecule and peptide delivery system for use in Alzheimer's disease and related disorders
JP2019509023A (en) * 2016-01-20 2019-04-04 ザ・スクリップス・リサーチ・インスティテュート ROR2 antibody composition and related methods
WO2017170776A1 (en) * 2016-03-30 2017-10-05 株式会社Ｅｐｓｉｌｏｎ Ｍｏｌｅｃｕｌａｒ Ｅｎｇｉｎｅｅｒｉｎｇ High-speed in vitro screening method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHABROL ERIC; STOJKO JOHANN; NICOLAS ALEXANDRE; BOTZANOWSKI THOMAS; FOULD BENJAMIN; ANTOINE MATHIAS; CIANFéRANI SARAH; FERRY : "VHH characterization.Recombinant VHHs: Production, characterization and affinity", ANALYTICAL BIOCHEMISTRY, vol. 589, 30 October 2019 (2019-10-30), Amsterdam, NL , XP085934170, ISSN: 0003-2697, DOI: 10.1016/j.ab.2019.113491 *
ZIMMERMANN IWAN : "Synthetic single domain antibodies for the conformational trapping of membrane proteins", ELIFE, vol. 7, no. e34317, 1 January 2018 (2018-01-01), pages 1 - 32, XP093016090 *

Similar Documents

Publication Publication Date Title
CA2583009C (en) Ubiquitin or gamma-crystalline conjugates for use in therapy, diagnosis and chromatography
JP6073038B2 (en) Stabilized immunoglobulin variable domain selection method and application of selected domains
US20180017573A1 (en) Polynucleotide construct capable of displaying fab in a cell-free translation system, and method for manufacturing and screening fab using same
CN109970858B (en) CD22 single domain antibody, nucleotide sequence and kit
CN108753792B (en) Encoding gene of green fluorescent protein nano antibody and preparation method and application thereof
MX2011003381A (en) Improved method of rna display.
JP2015531597A5 (en)
JP2011502507A (en) Directed evolution using proteins containing unnatural amino acids
US20200332280A1 (en) Use of lambda-gam protein in ribosomal display technology
JP2010515463A (en) Combinatorial library of conformationally constrained polypeptide sequences
EP3077508A2 (en) Methods of utilizing recombination for the identification of binding moieties
US20160069897A1 (en) Selection of fab fragments using ribosomal display technology
Schaefer et al. Construction of scFv fragments from hybridoma or spleen cells by PCR assembly
US20220228138A1 (en) Method for preparing phage library
JP2008271903A (en) Ribosome display
WO2022270396A1 (en) Double-screening method
Batonick et al. Platform for high-throughput antibody selection using synthetically-designed antibody libraries
JP2009112286A (en) Method for selecting polypeptide binding to target polypeptide
JP6478392B2 (en) Nucleic acid linker
US20240027436A1 (en) Cell-free expression of antibodies, antigen-binding fragments thereof, and antibody derivatives
Leonard et al. Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli
CN109942704B (en) HSA single domain antibodies, nucleic acids and kits
EP3702495A1 (en) Antibody like protein
CN110003334B (en) Polypeptide, CD19 single domain antibody, preparation method thereof, nucleotide sequence and kit
US10870926B2 (en) Antibody like protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22828313

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE