WO2010061892A1 - Novel variable lymphocyte receptor, method for screening for phage capable of binding to target antigen, and method for producing variable lymphocyte receptor for target antigen - Google Patents

Abstract

Disclosed is a novel variable lymphocyte receptor (VLR) which can recognize an antigen that cannot be recognized by known molecules VLRA and VLRB.  Also disclosed is a method for screening for a phage capable of binding to a target antigen, which utilizes the VLR. The VLR comprises one LRRNT, one LRRCT, and at least one LRR arranged between the LRRNT and the LRRCT, wherein the LRRCT has a motif comprising either the amino acid sequence (a) and/or the amino acid sequence (b) shown below. (a) Val-Lys-X1-Val-Asn-(X8)-Cys (b) Val-Lys-X1-Val-X1-Thr-(X7)-Cys  (In the formulae (a) and (b), X represents an amino acid residue; and Xn represents an amino acid sequence composed of n amino acid residues Xs which are the same as or different from each other, wherein n represents a natural number.)

Description

Novel variable antigen-binding molecule, method for screening target antigen-binding phage, and method for producing variable antigen-binding molecule for any target antigen

The present invention relates to one or more leucine-rich repeats between one leucine-rich repeat N-terminal adjacent region (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and the LRRNT and the LRRCT. The present invention relates to a novel variable antigen-binding molecule (VLR) having a sequence (LRR), a method for screening a target antigen-binding phage using the molecule, and a method for producing a VLR against an arbitrary target antigen.

In the immune system of higher animals, various antigen-binding regions are formed by reconstituting the V (D) J region of the B cell genome, which is a kind of lymphocyte, in order to recognize and eliminate various pathogens. The On the other hand, the round vertebrate, the lowest vertebrate, is known to have acquired immunity by gene rearrangement, although rearrangement of the V (D) J region is not observed. Recently, a variable antigen-binding molecule (variable lymphocyte receptor) (VLR) has been discovered in a round mouth (Patent Document 1, Non-Patent Document 1), and two isotypes of VLRA and VLRB have been identified so far ( Non-patent document 2).

It is known that VLR specifically binds to or recognizes a target antigen, which is a specific protein or other molecule, due to the diversity of its amino acid sequence (Non-Patent Document 3 or 4). For example, for VLRB, a monoclonal antibody VLRB specific for anthrax spore shell BclA protein has been prepared (Non-patent Document 5). In addition, these VLRs are known to be excellent in repetitive epitope recognition, and it has been suggested that they recognize lipids and sugar chain antigens that are difficult to recognize by immunoglobulins that are mammalian antibodies ( Non-patent document 6).

On the other hand, a phage display method has been proposed as one of methods for providing various proteins having an arbitrary amino acid sequence. In the phage display method, a foreign DNA consisting of an arbitrary sequence is inserted into a gene encoding a phage coat protein, and the protein encoded by the foreign DNA is displayed on the surface of the phage particle as a fusion protein with the coat protein (display). ). By using a random synthetic DNA as a foreign gene, a polypeptide library comprising a group of phages on which various polypeptides are displayed on the surface of the phage particle can be obtained. By applying this technique and screening a polypeptide library using a target antigen, phage particles displaying a polypeptide that binds to the target antigen can be recovered (Non-patent Document 7).

International Publication WO2006 / 083275 Pamphlet

However, the sequence of the recombination unit of the LRR that defines the antigen binding specificity of the VLR is considered to vary depending on the VLR isotype, and there may be an epitope that is not recognized by the known VLRA or VLRB, and a new VLR isotype is required. It has been. In addition, by creating a phage display library that expresses the LRR region of a new VLR isotype and selecting phage particles that display LRR that specifically bind to the target antigen, epitopes that are not recognized by known VLRA or VLRB are recognized. The production of a new VLR is expected.

The present invention has been made to solve the above-mentioned problems, and includes a novel VLR that recognizes an antigen that is not recognized by a known VLRA or VLRB, a method for screening a target antigen-binding phage, and a VLR against an arbitrary target antigen. The object is to provide a manufacturing method.

The variable antigen-binding molecule (VLR) according to the present invention includes one leucine rich repeat N-terminal adjacent region (LRRRNT), one leucine rich repeat C-terminal adjacent region (LRRRCT), the LRRNT and the LRRCT, A VLR having one or a plurality of LRRs in between, wherein the LRRCT has a motif consisting of at least one of the following amino acid sequences (a) and (b):
(A) Val-Lys-X1-Val-Asn- (X8) -Cys (SEQ ID NO: 1),
(B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys (SEQ ID NO: 2),
In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.

In the present invention, LRRNT is Cys-X1-Cys- (X12) -Cys (SEQ ID NO: 3).
{In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
It may have a motif consisting of the amino acid sequence of

The variable antigen binding molecule (VLR) according to the present invention includes one leucine-rich repeat N-terminal adjacent region (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), the LRRNT and the A variable antigen binding molecule (VLR) having one or more leucine-rich repeat sequences (LRR) with LRRCT, wherein the LRRCT has the amino acid sequence of SEQ ID NO: 4 or one or more conservative amino acid substitutions The amino acid sequence of SEQ ID NO: 4 is included.

In the present invention, LRRNT may include the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.

The method for screening a target antigen-binding phage according to the present invention comprises 1 leucine-rich repeat N-terminal adjacent region (LRRRT), 1 leucine-rich repeat C-terminal adjacent region (LRRRCT), the LRRNT and the A variable antigen-binding molecule (VLR) having one or a plurality of LRRs with LRRCT, wherein the LRRCT has a motif consisting of at least one of the following amino acid sequences (a) and (b): Is displayed on the surface of the phage by a phage display method to produce an antigen-binding phage library, and the antigen-binding phage library is reacted with the target antigen to obtain the target antigen and the antigen-binding phage library. Second step to remove unbound antigen-binding phage , And a third step of growing an antigen-binding phage bound to the target antigen of the antigen-binding phage library was infected with E. coli;
(A) Val-Lys-X1-Val-Asn- (X8) -Cys (SEQ ID NO: 1),
(B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys (SEQ ID NO: 2),
In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.

In the present invention, LRRNT is Cys-X1-Cys- (X12) -Cys (SEQ ID NO: 3).
{In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
It may have a motif consisting of the amino acid sequence of

The method for screening a target antigen-binding phage according to the present invention comprises 1 leucine-rich repeat N-terminal adjacent region (LRRRT), 1 leucine-rich repeat C-terminal adjacent region (LRRRCT), the LRRNT and the A variable antigen binding molecule (VLR) having one or more leucine-rich repeat sequences (LRR) with LRRCT, wherein the LRRCT has the amino acid sequence of SEQ ID NO: 4 or one or more conservative amino acid substitutions A first step of producing an antigen-binding phage library by displaying a VLR containing the amino acid sequence of SEQ ID NO: 4 on the surface of a phage by a phage display method, and reacting the antigen-binding phage library with a target antigen, Among the binding phage libraries, the target antibody Has a second step of removing the antigen-binding phage that do not bind, and a third step of growing said antigen binding phage bound to the target antigen of the antigen-binding phage library was infected with Escherichia coli and.

In the present invention, LRRNT may include the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.

In addition, the in vitro method for producing a variable antigen-binding molecule (VLR) against an arbitrary target antigen according to the present invention comprises 1 leucine-rich repeat N-terminal adjacent region (LRRNT) and 1 leucine-rich repeat C-terminal adjacent region. (LRRRCT) and one or more leucine rich repeat sequences (LRR) between the LRRNT and the LRRCT, wherein the LRRCT is at least one of the following (a) and (b) A first step of producing an antigen-binding phage library by displaying a VLR having a motif consisting of an amino acid sequence on a phage surface by a phage display method, reacting the antigen-binding phage library with a target antigen, and Antigens that do not bind to the target antigen in the library A second step of removing the combined phage, a third step of infecting Escherichia coli with the antigen-binding phage bound to the target antigen in the antigen-binding phage library, and an antigen-binding phage binding to the target antigen A fourth step of recombinantly producing a VLR by recombining a gene encoding the VLR contained in
(A) Val-Lys-X1-Val-Asn- (X8) -Cys,
(B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys,
In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.

In the present invention, LRRNT is Cys-X1-Cys- (X12) -Cys (SEQ ID NO: 3).
{In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
It may have a motif consisting of the amino acid sequence of

Further, the in vitro method for producing a variable antigen-binding molecule (VLR) against an arbitrary target antigen according to the present invention comprises 1 leucine rich repeat N-terminal adjacent region (LRRNT) and 1 leucine rich repeat C-terminal adjacent region. (LRRCT) and a variable antigen binding molecule (VLR) having one or more leucine rich repeat sequences (LRR) between the LRRNT and the LRRCT, wherein the LRRCT is the amino acid sequence of SEQ ID NO: 4 or A first step of producing an antigen-binding phage library by displaying a VLR comprising the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions on the phage surface by a phage display method, and the antigen-binding phage library and the target Reacting with an antigen to produce the antigen-binding fur A second step of removing an antigen-binding phage that does not bind to the target antigen in the library; and a third step of infecting E. coli with an antigen-binding phage that binds to the target antigen in the antigen-binding phage library to proliferate. And a fourth step of recombinantly producing a VLR by recombining a gene encoding a VLR contained in an antigen-binding phage bound to the target antigen.

In the present invention, LRRNT may include the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.

According to the present invention, a novel VLR that recognizes an antigen that is not recognized by a known VLRA or VLRB can be provided, whereby a new clinical test reagent, an experimental reagent, an inhibitor that specifically inhibits a biological reaction, and A kit and a device for screening a target antigen-binding phage using the VLR or the like for an arbitrary target antigen or the novel VLR can be produced.

It is a figure which shows embodiment at the time of integrating and using VLR which concerns on this invention in pCANTAB5E vector. 1 is a diagram showing an alignment of EST amino acid sequences obtained in Example 1. FIG. It is a figure which shows the alignment of the amino acid sequence of the full length sequence determined using RACE method in Example 2 (3). In addition, about LRRV of the following description, only what exists in the C terminal side is shown. It is a figure which shows the alignment of the deduced amino acid sequence obtained in Example 2 (4). It is a figure which shows the adjacent joint phylogenetic tree of the deduced amino acid sequence obtained in Example 2 (4). It is a figure which shows the VLRA, VLRB, and VLRC neighboring joint phylogenetic tree constructed | assembled using the amino acid sequence of LRRNT, LRR1, LRRVe, and LRRCT.

Hereinafter, embodiments of a variable antigen-binding molecule, a method for screening a target antigen-binding phage according to the present invention, and a method for producing a variable antigen-binding molecule against an arbitrary target antigen will be described in detail.

A variable antigen-binding molecule (VLR) according to the present invention is a rearrangeable peptide having one or a plurality of leucine rich repeat sequences (LRR) expressing the diversity of amino acid sequences. A polypeptide capable of functioning in immunity. The length of the polypeptide ranges from about 190 to about 250. One or more LRRs are located between the N-terminal cap unit leucine-rich repeat N-terminal adjacent region (LRRRNT) and the C-terminal cap unit leucine-rich repeat C-terminal adjacent region (LRRRCT) In the invention, it is used separately from LRRNT and LRRCT. That is, the VLR according to the present invention has LRRNT, one or a plurality of LRRs, and LRRCT in that order. Although LRRNT and LRRCT may be included in LRR, LRRNT and LRRCT are used separately from LRR in the present invention.

Furthermore, LRR is LRR1 (LRR first unit) flanking or close to LRRNT, LRRV1 (LRR second unit), LRRV2 (LRR second unit) located on the C-terminal side of LRR1 and continuously in the LRRCT direction. 3 units), LRRV3 (LRR 4th unit), LRRV4 (LRR 5th unit), LRRV5 (LRR 6th unit), LRRV6 (LRR 7th unit), LRRV7 (LRR 8th unit) ..., LRRVn (1st) n + 1 units; n is a natural number), LRRV-end (LRRVe; LRR final unit), and connecting peptide (CP) units.

LRR (leucine-rich repeat) is a leucine-rich sequence and is a motif involved in protein-protein interaction (Kobe et al., Trends. Biochem. Sci. Vol. 19, pages 415-421, 1994), in the present invention, an amino acid sequence containing a leucine rich repeat, or a domain or a partial structure of a protein comprising the amino acid sequence may be included in this. In general, LRR has a leucine residue as a skeleton and a β-strand structure. These are repeated many times to form a protein tertiary structure called “horse-shoe structure” as a whole. In VLR, it is considered that various target antigens are specifically bound or recognized due to the diversity of amino acid sequences present on the concave surface of the horseshoe structure.

In the present invention, “antigen” refers to a molecule such as a protein, lipid, or sugar chain antigen that binds to the VLR according to the present invention, and “binding” includes “recognition”, “specific interaction”, and “selection”. The concept of “interactive interaction” is included. That is, it does not matter whether the antigen is immunogenic or not, and the antigen contains a hapten.

In the present invention, “antigen-binding molecule” is used interchangeably with “antigen-recognizing molecule”, “antibody”, “antigen receptor”, or “lymphocyte receptor”. Here, “antibody” generally refers to a glycoprotein molecule produced by lymphocyte B cells that recognizes and binds to an antigen. In the present invention, it is produced by lymphocyte B cells. Does not mean a glycoprotein molecule, but simply a molecule that binds to an antigen.

LRRCT refers to a C-terminal cap unit rich in cysteine residues that are flanking or close to LRR on the C-terminal side and are invariant during LRR shuffling. A disulfide bond is formed in these cysteine residues, and the structure on the C-terminal side of the VLR becomes stable. The LRRCT of the VLR according to the present invention includes the amino acid sequence VKXVNXXXXXXXXXC (V is valine, K is lysine, N is asparagine, C is cysteine, X is any amino acid that is the same or different), and VKXVXXXXXXXXXXC (V is valine, K Lysine, T is threonine, C is cysteine, and X is the same or different amino acid) as a motif, or has the amino acid sequence of SEQ ID NO: 4 or one or more conservative amino acid substitutions It contains the amino acid sequence of SEQ ID NO: 4.

LRRNT refers to an N-terminal cap unit rich in cysteine residues that are flanking or close to the LRR on the N-terminal side and is an invariant moiety during shuffling of the LRR. Like the LRRCT, these cysteine residues A disulfide bond is formed, and the structure on the N-terminal side of the VLR becomes stable. Examples of the LLRNT of the VLR according to the present invention include, for example, those having a motif of the amino acid sequence CXCXXXXXXXXXC (C is cysteine, X is any same or different arbitrary amino acid) that is one of the LRRAs of VLRA, One having an amino acid sequence CXXXCXC (C is cysteine, X is any same or different arbitrary amino acid) which is one of LRRNT as a motif, amino acid sequence CXCXXXXXXXXXXXXXXX (C is cysteine, X is any amino acid which is the same or different) The amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions. Those containing the amino acid sequence of SEQ ID NO: 5 having an amino acid sequence or one or more conservative amino acid substitutions of those or SEQ ID NO: 5 having a motif XXXXXC are preferred.

In the present invention, a conservative amino acid substitution is a range that can generally be made without changing the physiological activity of the resulting molecule, that is, a conservative substitution (such as Watson et al., Molecular biology of Gene, etc.). For example, acidic amino acids of aspartic acid and glutamic acid; basic amino acids of lysine, arginine and histidine; nonpolar amino acids of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; glycine, asparagine, glutamine, Uncharged polar amino acids of cysteine, serine, threonine and tyrosine; amino acids with similar side chains such as phenylalanine, tryptophan and tyrosine aromatic amino acids Substitutions that occur in the family (within the amino acid family). Similarly, acidic amino acids of aspartic acid and glutamic acid; basic amino acids of lysine, arginine and histidine, aliphatic amino acids of glycine, alanine, valine, leucine, isoleucine, serine and threonine (classified as aliphatic-hydroxyamino acids of serine and threonine Can be classified); aromatic amino acids of phenylalanine, tyrosine and tryptophan; amides of asparagine and glutamine); sulfur-containing amino acids of cysteine and methionine.

The VLR according to the present invention can be expressed in a form having various regions. For example, as shown in FIG. 3, it has signal peptide (SP), LRRNT, LRR1, LRRV1 to LRRVn (n is a natural number), LRRVe, CP, LRRCT, 3′-terminus (3′-terminal) in that order. It can be expressed in an aspect. 3'-terminus (3'-terminal) has a region rich in threonine, serine, and proline on the N-terminal side, and a region rich in hydrophobic amino acids on the C-terminal side. The VLR according to the present invention may have a glycosyl-phosphatidyl-inositol anchor (GPI anchor) for binding to the membrane surface.

In the present specification, the LRRCT has at least one of the motifs, or the LRRCT includes the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions. And a novel VLR comprising the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions in the LRRNT, Called.

Hereinafter, a method for producing a VLR according to the present invention will be described in more detail. The VLR fabrication method according to the present invention mainly includes:
(1) Process of performing homology search for identifying the VLR gene according to the present invention (2) Process of cloning the VLR gene according to the present invention (3) Process of performing amino acid analysis of the VLR gene according to the present invention It has three strokes.

(1) Process of performing homology search for identifying the VLR gene according to the present invention Mainly, a plurality of known EST (expressed sequence tag) base sequences are extracted from a public database and arbitrarily selected from known VLRs This is a process of performing a homology search with a base sequence and determining an EST base sequence of a new VLR. The EST base sequence is a short sequence corresponding to a part of a gene transcript, and serves as a mark for the transcript. Since VLR has diversity in amino acid sequence, it is preferable to identify a novel VLR gene by obtaining an EST base sequence.

Examples of known databases include websites such as National Center Biotechnology Information (NCBI), DNA Data Bank of Japan (DDBJ), Ensembl, and FANTOM3. For the construction of the EST database, known database construction software can be used. For example, Variant Reporter (ABI), DNASIS Pro (Hitachi Software), Lasergene (DNASTAR), GENETYX-PDB (Genetics) Etc. Further, the homology search can be performed by a well-known algorithm or program incorporated in the software, for example, BLAST, BLAST-2, ALIGN, or JALVIEW software. One skilled in the art can determine appropriate parameters in order to measure sequences containing any necessary algorithms.

(2) Process of cloning the VLR gene according to the present invention After synthesizing the target cDNA mainly by a conventional method, a gene-specific primer is prepared by a conventional method based on the EST base sequence obtained by the process of (1). This is the process of designing, amplifying the target gene fragment, and cloning. Hereinafter, in the present invention, molecular biological experimental methods such as extraction and purification of genomic DNA and mRNA, preparation of cDNA by RT-PCR, PCR, ligation into a vector, determination of DNA base sequence, primer design and synthesis, etc. Basically, the biochemical experimental method can be carried out according to the description in a normal experiment document. As such an experimental document, for example, Sambrook et al. , Molecular Cloning, A laboratory manual, 2001, Eds. Sambrook, J .; & Russell, DW. And Cold Spring Harbor Laboratory Press.

The target is not particularly limited as long as there is a possibility of having a novel VLR gene. However, RNA extracted from leukocytes of the crustacea which are lower animals having acquired immunity is preferable. For RNA extraction, for example, QIAamp RNA kit (Qiagen), Concert Plant RNA Regent (Invitrogen), QuickPrep Total RNA Extraction Kit (GE Healthcare), RNeasy Mini Kit (RNeasy Mini Kit) Can do. In addition, RT-PCR (Reverse Transcription Polymer Chain Chain Reaction) includes, for example, Expand High Fidelity ^PLUS PCR System (Roche), Pyrobest (Takara Bio), ThermoScript-MrPCRP. Also, Gibco), ABI Prism 7000, 7900 (both are ABI), Superscript III First Strand Synthesis System (Invitrogen), and the like can be used.

For amplification of the target gene fragment, the PCR method (PolymeraseｍｅChain 主 Reaction) is mainly used, but the LAMP method (Loop-mediated Isomatic Amplification) may be used instead. As the cloning vector, those capable of autonomous replication in Escherichia coli are suitable. For example, ZAP Express (Stratagene), pBluescript II SK (+) (Nucleic Acids Research), Lambda ZAP II (Stratagene), λgt10, λgt11 (both DNA Cloning, A Practical Approach), λTriplEx (Clontech), λExCell, pT7T318U, pTrc99A (both Pharmacia), pcD2 (H.OkaP) PUC118, pUC119, pUC18, pUC19, pSTV28, pSTV29 Misalignment is Takara Bio Inc.), pEG400 (J. Bac.), PQE-30, pQE-80 (all are QIAGEN), pTA2 (TOYOBO), pCR2.1-TOPO (Invitrogen), pGEM-T, pGEM -T Easy (both from Promega). Furthermore, various shuttle vectors can be used in addition to vectors capable of autonomous growth in two or more host microorganisms such as Escherichia coli and Pseudomonas. In this case, the fragment can be obtained by cleaving with the restriction enzyme.

Further, for gene cloning, for example, according to Current 記載 Protocols in Molecular Biology unit 2.4 or DNA Cloning 1 (Core Techniques, A Practical Approach, Second Edition, Oxford Univsity, described in 19), etc. For example, DNeasy Blood & TissueｕKit (QIAGEN), The ilstra bacteria genomicPrep Mini Spin Kit (GE Healthcare), PrepMan Ultra Reagent (ABI) can be used. Further, full-length cDNA can also be obtained by cloning the deletion portion cDNA using the RapidＲAmplification of cDNA ends (RACE) method. Here, the RACE method refers to a method of repairing and amplifying cDNA of a deletion portion using a PCR method recently developed by Citas, Inc. of the United States. For example, 5′-FullＦRACE Core Set 3′-Full RACE Core Set (Takara Bio), Cells-to-cDNA Kit FirstChoice RLM-RACE Kit (Ambion), GeneRacer RACE-Ready cDNA Kit (Invitrogen), etc.

(3) Process of performing amino acid analysis of the VLR gene according to the present invention Process of aligning the deduced amino acid sequence of the obtained full-length cDNA with a known VLR amino acid sequence and preparing a phylogenetic tree using a phylogenetic analysis method It is. For alignment, for example, Clustal W, Clustal X ver. 2.0 (both Larkin et al.), T-COFFEE (EMBnet), DIALIGN (Brudno et al., Nuc. Acids. Res. 32: 41-44 (2004)), Match-Box (Depiereux, E.). et al., Prot. Enng. 4 (6): 603-613 (1991)), MAP (Huang, X. On Global Sequence Alignment. Computer Applications in the Biosciences (10), 227-235 (1994). Can be used. Moreover, the phylogenetic analysis method used for the preparation of the phylogenetic tree is not particularly limited. For example, the maximum saving method, the maximum likelihood method, the Bayes method, the neighborhood joining method (NJ method; Saito, N. et al., Molecular Biology and Evolution ( 4) 406-425 (1987)). For example, MEGA package (Kumar, S. et al., Molecular evolutionary analysis analysis version 4.1. The Pennsylvania State University, 19). ), MicroSeq Microbial Identification System (ABI), or the like can be used.

Next, the method for screening a target antigen-binding phage according to the present invention will be described in more detail. The method for screening a target antigen-binding phage according to the present invention mainly comprises:
(1) Process of producing an antigen-binding phage library by presenting the VLR according to the present invention on the phage surface (2) The antigen-binding phage library and the target antigen are reacted and do not bind to the target antigen in the antigen-binding phage library Step of removing antigen-binding phage (3) The above-mentioned three steps of the step of infecting Escherichia coli with an antigen-binding phage bound to the target antigen in the antigen-binding phage library and propagating it.

(1) Process of producing an antigen-binding phage library by presenting the VLR of the present invention on the phage surface This process uses the DNA encoding the VLR of the present invention and the VLR of the present invention or the poly having the same In this process, peptides are displayed on the surface of phage particles by the phage display method (Smith GP, Science, 228, 1315-1317 (1985), etc.) to produce an antigen-binding phage library. That is, a DNA encoding a VLR according to the present invention or a polynucleotide encoding a protein that hybridizes with a base sequence complementary to the VLR under stringent conditions and has an ability to bind to an antigen is coated with a phage. Insert into one of the protein genes and incorporate it into the phage. Then, an antigen-binding phage library can be prepared by expressing a fusion protein of the VLR according to the present invention or a polypeptide having the VLR and a phage coat protein and displaying it on the surface of the phage particle. In addition, by infecting a host such as E. coli with this phage, the genomic DNA of the phage can be efficiently replicated.

Here, the “stringent conditions” refers to the conditions of 6M urea, 0.4% SDS, 0.5 × SSC, or hybridization conditions equivalent thereto, except for special cases. Accordingly, conditions with higher stringency may be applied, and examples include 6M urea, 0.4% SDS, 0.1 × SSC, or equivalent hybridization conditions. Under each condition, the temperature can be about 40 ° C. or higher, and may be about 50 ° C. to 65 ° C., for example, if conditions with higher stringency are required. Hybridization was performed in Sambrook et al. , Molecular Cloning (2nd edition) and the like.

The phage used in the present invention is not particularly limited, and examples thereof include T4 phage, T7 phage, λ phage, and filamentous phage, and filamentous phage is preferable. In addition, examples of filamentous phages include f1, fd, M13, etc., and have circular single-stranded genomic DNA, and gene 3 protein (g3p, the same applies hereinafter), g6p, g7p , G8p, and g9p are more preferably a rod-shaped M13 phage in which five coat proteins are assembled.

When M13 phage is used, the VLR according to the present invention or the polypeptide having the same is displayed on the surface of the phage particle by hybridizing with the DNA encoding it or a complementary base sequence under stringent conditions. This can be achieved by inserting a polynucleotide encoding a protein that has the ability to bind to an antigen and to an antigen into any of the coat protein genes of g3p, g6p, g7p, g8p, and g9p. From the viewpoint that the fusion protein can be displayed without destroying the physical packaging, it is preferably fused and displayed with g3p or g8p, more preferably fused with g3p and displayed on the surface of the phage particle as a fusion protein. .

Further, the vector used in the present invention may be used as a vector by improving the single-stranded genome of phage, or a foreign plasmid vector may be used. Examples of the foreign plasmid vector include a phagemid vector containing a packaging signal for phage. When using a phagemid vector, it is necessary to infect a helper phage in order to package it as a single-stranded DNA in a phage particle, whereby a single-stranded DNA can be obtained from the culture supernatant. In addition, pCANTAB5E can be mentioned as a preferable phagemid vector, When using a pCANTAB5E vector, as shown in FIG. 1, restriction enzyme sites SfiI and NotI can be utilized.

In addition, the transformed host can be cultured using a medium that can be appropriately selected by those skilled in the art, whereby a high concentration antigen-binding phage solution can be obtained.

Here, for the display of the VLR according to the present invention or the polypeptide having the same on the surface of the phage particle, a polynucleotide encoding LRR may be arbitrarily selected or used. T.A. The degenerate polynucleotide was synthesized from the obtained full-length cDNA according to the above-mentioned method (Stumppp MT et al., J. Mol. Biol. 332 (2) 471-487 (2003)). Also good.

The LRR polynucleotide synthesized or degenerately synthesized can be used as a VLR by ligating with LRRNT, LRRCT, and other LRR polynucleotides. In this case, for example, the usefulness of the prepared VLR can also be examined using immunostaining, flow cytometry, immunoprecipitation, or the like.

(2) Process of reacting antigen-binding phage library and target antigen to remove antigen-binding phage that does not bind to target antigen in antigen-binding phage library This process includes the antigen-binding phage library prepared in process (1) After reacting with the target antigen, an antigen-binding phage that does not bind to the target antigen is removed to obtain an antigen-binding phage that binds to the target antigen.

For example, when the target antigen is immobilized on a solid phase carrier such as a solid phase substrate, film, tape, or bead and this step is performed, after incubating the solution containing the antigen-binding phage library on the surface of the solid phase carrier, The solid phase carrier surface was washed, and phage particles presenting a polypeptide having no affinity for the target antigen were removed, whereby the solid phase carrier was captured by the interaction between the target antigen and the polypeptide. The process of recovering phage particles can be mentioned. Furthermore, for example, by biotinylating the target antigen and immobilizing it via the immobilized streptavidin, the target antigen can be immobilized irrespective of the nature of the target antigen and without affecting its structure. Specific antigen-binding phages can be concentrated.

In addition, for example, when performing this step for the target antigen contained in the liquid phase, after adding a solution containing the antigen-binding phage library to the liquid phase and incubating, a polypeptide having no affinity for the target antigen is displayed by centrifugation. For example, a step of removing the phage particles to be collected, adding phage particles that display a polypeptide having an affinity for the target antigen, and then collecting the precipitate by centrifugation is exemplified. Furthermore, antigen-binding phages specific to the target antigen can be concentrated by binding a carrier such as Sepharose beads to phage particles that display a polypeptide having affinity for the target antigen.

(3) Process of infecting E. coli with antigen-binding phage that has bound to the target antigen in the antigen-binding phage library and proliferating In this process, the antigen-binding phage bound to the target antigen concentrated in the process (2) is transformed into host E. coli. It is the process of infecting and amplifying. For example, if an antigen-binding phage recovered by acid treatment or the like is amplified by infecting host Escherichia coli, a target having an affinity for the target antigen is displayed by a series of operations (2) to (3). Antigen-binding phages bound to the antigen are further concentrated, and antigen-binding phage particle groups having high affinity for the target antigen can be obtained. A more concentrated group of antigen-binding phage particles bound to the target antigen can be obtained by a method of performing steps (2) to (3) a plurality of times (biopanning method; Affinity selection).

Next, the in vitro production method of VLR for any target antigen according to the present invention will be described in more detail. The method for screening a target antigen-binding phage according to the present invention mainly comprises:
(1) Process of producing an antigen-binding phage library by presenting the VLR according to the present invention on the phage surface (2) The antigen-binding phage library and the target antigen are reacted and do not bind to the target antigen in the antigen-binding phage library Step of removing antigen-binding phage (3) Step of infecting Escherichia coli with antigen-binding phage bound to target antigen in the antigen-binding phage library and propagating (4) Encoding VLR contained in antigen-binding phage bound to target antigen The above four steps of the process of recombinantly producing a VLR by recombining the gene to be processed are included. In the configuration of the present invention, the description of the same or corresponding configuration as the method for screening a target antigen-binding phage described above is omitted.

(4) A step of recombining a VLR-encoding gene contained in an antigen-binding phage bound to a target antigen to produce a VLR by recombination This step consists of antigen-binding phage particles bound to the target antigen concentrated in step (3). This is a process for producing a VLR by recombination from a gene encoding the VLR shown in FIG. This recombinant production of VLR is described, for example, in Sambrook et al. , Molecular Cloning, A laboratory manual, 2001, Eds. Sambrook, J .; & Russell, DW. Methods described in various experimental manuals widely known to those skilled in the art, such as Cold Spring Harbor Laboratory Press, can be used.

More specifically, for example, host cells transformed with a VLR-encoding gene or a vector containing this gene are cultured under appropriate conditions, and the culture mixture is recovered. Can be purified accordingly. This purification method can be appropriately selected by those skilled in the art. For example, salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography And various affinity chromatography such as antibody chromatography, chromatofocusing method, adsorption chromatography, reverse phase chromatography and the like. Moreover, HPLC etc. can also be used as needed.

Hereinafter, a method for producing a VLR according to the present invention will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Homology search to identify novel VLR genes>
In order to identify a new VLR gene, an expressed sequence tag (EST) database derived from a petrel (petromyzon marinus) was constructed, and a homology search was performed using a known VLR gene as a query.

From the public database NCBI (National Center for Biotechnology) Information trace archives homepage (http://www.ncbi.nlm.nih.gov/Trace/trace.cgi) Database construction software GENETYX-PDB Ver. 5 (Genetics Co., Ltd.) was used to build a Umiyamatsu EST database. A homology search was performed using a sequence arbitrarily selected from the amino acid sequences of the VLRA gene and the VLRB gene of lampreys (Hampfish) registered in NCBI as a query. This homology search is performed using GENETYX-PDB Ver. 5 was carried out using the TBLASTN program built in 5.

As a result, as shown in FIG. 2, an EST base sequence having a sequence different from known VLRA and VLRB was obtained. The base sequence is shown in SEQ ID NO: 6. This confirmed the possibility of the presence of a novel VLR gene, which was named VLRC.

<Cloning of VLRC gene>
Based on the EST base sequence obtained in Example 1, cloning of the VLRC gene was attempted by the following method.

(1) Synthesis of cDNA Anemonefish (Letheteron japonicum) was anesthetized with 100 mg / L of tricaine methanesulfonate (TMS, MS222; Sigma), and blood was collected by tail cutting. This blood was layered on Histopack 1077 (Sigma), and leukocytes were collected by centrifugation at 4 ° C. and 1500 rpm for 30 minutes, and then leukocyte-derived RNA was extracted using RNeasy Mini Kit (Qiagen). . Subsequently, RT-PCR was performed using Superscript III First Strand Synthesis System (Invitrogen) to synthesize cDNA from 1 μg of total RNA.

(2) Acquisition of full-length cDNA containing VLRC gene Two oligonucleotides were synthesized based on the EST sequence obtained in Example 1. The array is
5′-caactgcaga gtgttcctga cgg-3 ′ (SEQ ID NO: 7)
5′-catccgtgtg ctcatgggtgt gc-3 ′ (SEQ ID NO: 8)
It is represented by

PCR was performed under the following conditions using the oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers.
-Reaction reagent-
A reaction solution was prepared according to the protocol described in Bio Experiment Illustrated Volume 3 (Shyujunsha). For the reaction, AmpliTaq Gold (ABI) was used as a DNA polymerase, and a solution attached to this enzyme was used. The cDNA equivalent to 5 ng obtained in this Example (1) was used as a template.
-Reaction conditions-
After the reaction at 95 ° C. for 10 minutes, each reaction of 95 ° C. for 15 seconds, 37 ° C. for 1 minute and 72 ° C. for 30 seconds was performed for 3 cycles, and then 95 ° C. for 15 seconds and 57 ° C. for 1 Min., Each reaction for 30 seconds at 72 ° C. was performed for 40 cycles, and a reaction was further performed at 72 ° C. for 7 minutes. As a result, amplification of a DNA fragment of about 300 bp was confirmed.

Subsequently, this amplified DNA fragment was TA cloned into the plasmid vector pGEM-T Easy using pGEMＧT-Easy Vector System I (Promega). The operation was performed according to the attached manual. The transformant was applied to an LB agar medium containing ampicillin (final concentration 100 μg / mL), and statically cultured at 37 ° C. overnight. The resulting colonies were inoculated into 2 mL of LB medium and cultured overnight at 37 ° C., and then the pGEM-T Easy-LjVLRC_c2 plasmid was prepared. The base sequence is shown in SEQ ID NO: 9.

Next, in order to obtain sequence information of the full-length VLRC cDNA, a Rapid Amplification A cDNA A Ends (RACE) method was performed. Specifically, using GeneRacer® RACE-Ready® cDNA® kit (Invitrogen) and operating according to the attached manual, the cDNA of the deletion portion was cloned.

First, in order to amplify the 5 ′ end of the gene, two kinds of oligonucleotides used for the first reaction and the second reaction were synthesized. The array is
5'-gagacaaaag gcatgtttaca caca-3 '(SEQ ID NO: 10)
5′-atgttagcagg ggtggggagac gatagc-3 ′ (SEQ ID NO: 11)
It is represented by

Next, in order to amplify the 3 ′ end of the gene, two kinds of oligonucleotides used for the first reaction and the second reaction were synthesized. The array is
5′-gcatcgtctc cacccctgc tacat-3 ′ (SEQ ID NO: 12)
5'-tgtgtgtacac atgccttttg tctc-3 '(SEQ ID NO: 13)
It is represented by

PCR was performed under the same conditions as described above using the oligonucleotides of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 as primers, and amplification of a DNA fragment of about 700 bp was performed at the 5 'end. 'Amplification of a DNA fragment of about 500 bp was confirmed for each end. The amplified DNA fragments at the 5 'end and 3' end were TA cloned by the same method as described above. The transformant was applied to an LB agar medium containing ampicillin (final concentration 100 μg / mL), and statically cultured at 37 ° C. overnight. The resulting colonies were inoculated into 2 mL of LB medium and cultured at 37 ° C. overnight to prepare pGEM-T Easy-LjVLRC_5RACE_A5T9002 plasmid and pGEM-T Easy-LjVLRC_3RACE_A3c4 plasmid. Their base sequences are shown in SEQ ID NO: 14 and SEQ ID NO: 15.

Subsequently, the base sequence of each DNA fragment was determined using the pGEM-T Easy-LjVLRC_c2 plasmid, pGEM-T Easy-LjVLRC_5RACE_A5T9002 plasmid, and pGEM-T Easy-LjVLRC_3RACE_A3c4 plasmid as templates. This base sequence was determined by the dideoxy method according to a conventional method using various synthetic DNAs as primers and using CEQ2000XL®DNA®Analysis®System (Beckman®Coulter).

(3) Determination of full-length VLRC cDNA sequence From the result of sequence analysis of the above pGEM-T Easy-LjVLRC_c2 plasmid, pGEM-T Easy-LjVLRC_5RACE_A5T9002 plasmid, and pGEM-T Easy-LjVLRC_3RACE_A3c4 Confirmed to be composed. The base sequence is shown in SEQ ID NO: 16. In addition, as shown in FIG. 3, alignment with the amino acid sequences of VLRA and VLRB was performed using Clustal X ver. 2.0 (http://www.clustal.org/download/current/, Larkin et al., Bioinformatics 23: 2947-2948 (2007)). The base sequence of SEQ ID NO: 16 contains three LRRVs, but FIG. 3 shows only the LRRV present on the C-terminal side.

(4) Isolation of Diversity Region of VLRC Two types of oligonucleotides were synthesized based on the determined full-length cDNA sequence obtained in this Example (2). The array is
5′-aggtgtgtgtc ccgtgcgagc-3 ′ (SEQ ID NO: 17)
5′-ggtgggagac gatagctgtaa-3 ′ (SEQ ID NO: 18)
It is represented by

PCR was performed under the following conditions using the oligonucleotides of SEQ ID NO: 17 and SEQ ID NO: 18 as primers.
-Reaction reagent-
A reaction solution was prepared according to the manual attached to the Expanded High Fidelity PCR System (Roche Applied Science). For the reaction, 15 ng of cDNA prepared in this Example (1) was used as a template.
-Reaction conditions-
One cycle of reaction at 94 ° C for 2 minutes, then 35 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute each cycle, and 72 minutes at 72 ° C for 7 minutes Went.

The amplified DNA fragment was ligated to a plasmid vector pCR2.1-TOPO (Invitrogen) or a plasmid vector pGEM-T using TOPO-TA-Cloning-kit (Invitrogen) or pGEM-T-Easy-Vector-Systems-I (Promega). TA cloning was performed in Easy (Promega). The operation was performed according to the attached manual. The transformant was applied to an LB agar medium containing ampicillin (final concentration 100 μg / mL) and cultured at 37 ° C. overnight. The resulting colony was inoculated into 2 mL of LB medium and cultured overnight at 37 ° C., and then a plurality of plasmid DNAs were prepared.

Subsequently, the base sequence of the DNA fragment was determined using plasmid DNA as a template. The base sequence was determined according to a conventional method using CEQ2000XL®DNA®Analysis®System (Beckman®Coulter) using various synthetic DNAs as primers. The base sequence of a plurality of plasmid DNAs was determined by the dideoxy method using various synthetic DNAs as primers for the TA-cloned insert DNA. Their base sequences are shown in SEQ ID NOs: 19 to 37. As a result, 19 VLRC genes were successfully cloned.

<Amino acid sequence analysis of VLRC>
The deduced amino acid sequence was determined from SEQ ID NOs: 19 to 37 obtained in Example 2 (4). Their amino acid sequences are shown in SEQ ID NOs: 38-56. The amino acid sequences of SEQ ID NOs: 38 to 56 are all in the LRRCT with the VKXVNXXXXXXXXXC (V is valine, K is lysine, N is asparagine, C is cysteine, X is any amino acid that is the same or different) (SEQ ID NO: 1 ) Or VKXVXTXXXXXXXC (V is valine, K is lysine, T is threonine, C is cysteine, X is the same or different amino acid) as a motif, and LRRNT has CXCXXXXXXXXXXXXC (C is The amino acid sequence (SEQ ID NO: 3) of cysteine and X are the same or different arbitrary amino acids) as a motif. Furthermore, the amino acid sequences of SEQ ID NOs: 38 to 56 all include the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions in LRRCT, and the amino acid sequence of SEQ ID NO: 5 in LRRNT Or comprises the amino acid sequence of SEQ ID NO: 5 with one or more conservative amino acid substitutions.

The amino acid sequences of SEQ ID NOs: 38 to 56 were aligned using the Clustal® X. The result is shown in FIG. In addition, we constructed a neighborhood phylogenetic tree using the amino acid sequence. The phylogenetic tree was constructed using MEGA ver 4.1 Beta program (http://www.megasoftware.net/). This is shown in FIG. The VLRB antibody already reported has a variable region rich in diversity as shown in FIG. 1, and it is known that this variable region binds to an antigen. Therefore, it was strongly suggested that VLRC has the ability to bind to an antigen via its variable region, similar to VLRB.

In addition, using the amino acid sequences of LRRNT, LRR1, LRRVe, and LRRCT, a neighboring phylogenetic tree of VLRA, VLRB, and VLRC was similarly constructed. This is shown in FIG. FIG. 6 shows that VLRC forms a different cluster from VLRA and VLRB.

According to the present embodiment as described above, a new VLR that recognizes an antigen that is not recognized by a known VLRA or VLRB can be provided. Inhibitors and the like, or kits and devices for screening for target antigen-binding phages using this novel VLR can be produced.

In addition, the VLR according to the present invention, the method for screening a target antigen-binding phage, and the method for producing a VLR for any target antigen are not limited to the above-described examples, and can be appropriately changed. For example, the VLR according to the present invention does not function as an antibody, but may function as an epitope peptide, inhibitory peptide, or antagonist (agonist) peptide. Further, instead of the phage display method, for example, the bacteria two-hybrid method, the yeast two-hybrid method, the in vitro virus method and the like may be used.

Claims (12)

1. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT A variable antigen-binding molecule (VLR) having the motif, wherein the LRRCT has a motif consisting of at least one of the following amino acid sequences (a) and (b):
   (A) Val-Lys-X1-Val-Asn- (X8) -Cys,
   (B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys,
   In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.
2. LRRNT is Cys-X1-Cys- (X12) -Cys
   {In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
   The VLR according to claim 1, which has a motif consisting of the amino acid sequence of
3. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT Wherein the LRRCT comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions.
4. 4. The VLR of claim 3, wherein the LRRNT comprises the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.
5. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT A variable antigen-binding molecule (VLR) having a motif in which the LRRCT has a motif consisting of at least one of the following amino acid sequences (a) and (b): A first step of preparing an antigen-binding phage library;
   A second step of reacting the antigen-binding phage library with a target antigen to remove an antigen-binding phage that does not bind to the target antigen from the antigen-binding phage library;
   A method for screening a target antigen-binding phage having a third step of infecting Escherichia coli with an antigen-binding phage bound to the target antigen in the antigen-binding phage library;
   (A) Val-Lys-X1-Val-Asn- (X8) -Cys,
   (B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys,
   In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.
6. LRRNT is Cys-X1-Cys- (X12) -Cys
   {In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
   A method for screening a target antigen-binding phage according to claim 5, which has a motif consisting of the amino acid sequence of
7. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT A VLR comprising a variable antigen binding molecule (VLR) having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions. A first step of generating an antigen-binding phage library displayed on the surface;
   A second step of reacting the antigen-binding phage library with a target antigen to remove antigen-binding phage that does not bind to the target antigen in the antigen-binding phage library;
   A method of screening a target antigen-binding phage having the third step of infecting Escherichia coli with an antigen-binding phage that binds to the target antigen in the antigen-binding phage library.
8. The method for screening a target antigen-binding phage according to claim 7, wherein LRRNT comprises the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.
9. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT A variable antigen-binding molecule (VLR) having a motif in which the LRRCT has a motif consisting of at least one of the following amino acid sequences (a) and (b): A first step of preparing an antigen-binding phage library;
   A second step of reacting the antigen-binding phage library with a target antigen to remove an antigen-binding phage that does not bind to the target antigen from the antigen-binding phage library;
   A third step of infecting Escherichia coli with an antigen-binding phage that has bound to the target antigen in the antigen-binding phage library; and
   A fourth step of recombining a VLR-encoding gene contained in an antigen-binding phage bound to the target antigen to produce a VLR by recombination;
   (A) Val-Lys-X1-Val-Asn- (X8) -Cys,
   (B) Val-Lys-X1-Val-X1-Thr- (X7) -Cys,
   In the formulas (a) and (b), X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of the same or different arbitrary amino acids X of n residues.
10. LRRNT is Cys-X1-Cys- (X12) -Cys
    {In the formula, X represents an arbitrary amino acid, and Xn (n is a natural number) represents an amino acid sequence composed of arbitrary amino acids X of the same or different n residues. }
    An in vitro method for producing a VLR against any target antigen according to claim 9, which has a motif consisting of the amino acid sequence of:
11. One or more leucine-rich repeat N-terminal adjacent regions (LRRRNT), one leucine-rich repeat C-terminal adjacent region (LRRRCT), and one or more leucine-rich repeat sequences (LRR) between the LRRNT and the LRRCT A VLR comprising a variable antigen binding molecule (VLR) having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one or more conservative amino acid substitutions. A first step of generating an antigen-binding phage library displayed on the surface;
    A second step of reacting the antigen-binding phage library with a target antigen to remove antigen-binding phage that does not bind to the target antigen in the antigen-binding phage library;
    A third step of infecting Escherichia coli with an antigen-binding phage that has bound to the target antigen in the antigen-binding phage library;
    A fourth step of recombining a gene encoding a VLR contained in an antigen-binding phage bound to the target antigen to produce the VLR by recombination, and producing an VLR in vitro for any target antigen.
12. The method for producing a VLR against an arbitrary target antigen according to claim 11, wherein the LRRNT comprises the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.

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