JPH0775577A - Gene - Google Patents

Abstract

PURPOSE:To obtain a gene for production of an artificial antibody molecule, having coding regions for a polypeptide having a variable region of an antibody and an ability of expressing a phage surface, and for a polypeptide having an ability of binding to Fc, used for the selection of high affinity antibody and the purification, determination, diagnosis, etc., of an antigen. CONSTITUTION:A novel gene expressed by formula (wherein, X is a gene containing tan integrating position of a gene coding a variable region of an antibody; Y is a gene containing a gene coding a polypeptide having an ability of expressing a phage surface; Z is a gene containing a gene coding a polypeptide having an ability of binding to Fc; (m), (n), (l) each is 1, 0, and at least one of (m) and (n) is 1). This artificial antibody is useful for the purification, determination, diagnosis, etc., of an antigen, since it is possible to select a gene coding an antibody molecule having a high affinity to an objective antigen easily, and to produce a multivalent artificial antibody of mono- or poly clonal type easily from the selected gene.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel gene, and more particularly to a gene useful for selection of high affinity antibody, preparation of artificial antibody molecule and the like.

[0002]

2. Description of the Related Art Antigen specificity of an antibody is V region (V _H ,
_VL ). An antibody library in which a Fab or Fv portion containing the variable region of this antibody is expressed in Escherichia coli has been prepared. That is, _VH and _VL genes derived from mouse and human are
It was amplified by the R (polymerase chain reaction) method and then integrated into an Escherichia coli expression vector for expression [Science, Volume 246, 1275-128].
Page 1 (1989), Nature, 341.
Vol. 544-546 (1989), Journal of Molecular Biology.
ar Biology), Vol. 213, pp. 617-619 (19
90), Nature, Volume 347, Pages 483-485 (1990), Science, Volume 240, 1038-.
1041 (1988), ibid., 240, 1041
-1043 (1988)]. Furthermore, in order to efficiently screen a recombinant expressing an antibody molecule that reacts with an antigen of interest, a so-called phage display antibody library in which the antibody molecule is expressed in the form of a fusion protein with a coat protein of filamentous phage is also available. [Proceedings of the National Academy of Science of the USA (Proceedings of the USA
the National Academy of Sciences of the USA)
Vol. 89, pp. 3576-3580 (1992), ibid.
Vol. 89, pp. 4457-4461 (1992),
Vol. 88, pp. 7978-7982 (1991), ibid.
88, 4363-4366 (1991), Journal of Molecular Biology, 227.
Vol., Pp. 381-388 (1992), ibid., 226.
Vol., 889-896 (1992), ibid., 222
Volume 581-597 (1991), Nature, Volume 352, 624-628 (1991), ibid., 3rd.
48, 522-554 (1990), ibid., 34
9, pp. 293-299 (1991)].

In this phage display antibody library,
Since the antibody molecule-derived polypeptide is present on the surface of the phage, the phage particles can be directly screened with a resin or the like on which the antigen is immobilized, and the phage particles can be propagated by reinfection with E. coli. The phage particles used in the present specification also include phagemid particles. Therefore, not only can a large number of clones be screened in a short time, but also the gene encoding the antibody molecule of interest can be easily recovered. As a method for recovering the antibody molecule from the obtained gene encoding the target antibody molecule, the gene encoding the phage coat protein can be removed by restriction enzyme digestion to express only the antibody molecule. In addition, by inserting an amber mutation between the antibody gene and the phage coat protein gene and changing the type of E. coli, a vector capable of selectively expressing the antibody molecule or the fusion protein of the antibody molecule and the coat protein has been constructed. There is. Further, in order to purify or use the expressed antibody molecule, a short peptide chain is fused to the antibody molecule, and the antibody against the peptide chain can be used to purify or detect the antibody. A protein that has been expressed as a protein and detected by its enzymatic activity has also been developed [Bio / Technolo
gy), Vol. 11, pp. 601-605 (1993),
Vol. 10, pp. 1128-1132 (1992),
Gene, Vol. 122, pp. 361-365 (1
992)].

[0004]

When referring to the affinity between an antibody and an antigen, there are an intrinsic affinity called affinity and a functional affinity called avidity. The former is
The strength of the bond formed at the binding site between the antibody and the antigen, that is, the affinity determined by hydrogen bond, hydrophobic bond, van der Waals force, etc., the latter, in addition to the strength of the affinity, It is the overall affinity when the valency of the binding site of the antigen is multivalent. When an antibody molecule is actually used in an immunological experiment, it is necessary that the affinity be strong and specific. However, a molecule having a higher valence is more practically useful, and an antibody molecule having a lower affinity is However, if the valence is large, it will show a strong affinity as a whole. In a vector used in a phage display antibody library or the like, the antibody molecule is Fa
b fragment or single-chain Fv fragment [expressed in the form of a single-chain polypeptide (hereinafter abbreviated as scFv) by connecting V _H and V _L fragments with a spacer consisting of several amino acid residues] I am letting you. However, it is unknown how many antibody molecules are displayed on one phage particle, and a method for controlling the number is not shown. Further, there is no disclosure of a method of increasing the avidity of the obtained antibody molecule to convert it into a practically useful form. An object of the present invention is to provide a method for selecting a high affinity antibody molecule by controlling the number of displayed antibody function molecules in a phage display antibody expression system. The purpose of the present invention is to provide a gene capable of providing an artificial antibody which has high avidity and can detect an antigen with high sensitivity by providing the above-mentioned binding property.

[0005]

SUMMARY OF THE INVENTION The present invention will be described in brief. The present invention relates to a gene represented by the following general formula (Formula 1).

[0006]

Embedded image X- (Y) _m- (Z) _n

(In the formula, X is a gene containing an integration site of a gene encoding an antibody variable region, Y is a gene containing a gene encoding a polypeptide capable of expressing a phage surface, and Z is Fc (of an antibody molecule). (Constant domain) A gene containing a gene encoding a polypeptide having binding ability, m and n are 1 or 0, and at least one of m and n is 1.)

The present inventors have created a gene represented by the formula (Formula 1), integrated a gene encoding an antibody variable region into the gene, and designed the antibody variable region with a polypeptide capable of expressing a phage surface. If the phage-displayed antibody is expressed in the form of a fusion protein on the surface of the phage, the affinity of the phage-displayed antibody can be easily measured, and the high-affinity antibody variable region can be efficiently selected.
When expressed in the form of a fusion protein with a polypeptide capable of binding, the protein forms a complex with a molecule containing Fc, and the complex exhibits a multivalent antibody titer,
Since it is extremely stable and the like, it was found that it is useful as an artificial antibody with high avidity for immunological use, and the present invention was completed.

The gene of the present invention contains a gene encoding a polypeptide capable of expressing a phage surface on the downstream side of a gene containing an integration site of a gene encoding an antibody variable region (hereinafter referred to as X gene). Gene (hereinafter Y
(Hereinafter referred to as a gene), and a gene containing a gene encoding a polypeptide having Fc-binding ability further downstream (hereinafter, referred to as Z
(Called genes) are arranged side by side. The term “antibody variable region” as used herein refers to any natural or artificial antibody variable region, Fab, scFv, or Fv, but it is preferable to select Fv when the affinity ability is measured by the method described below.

Examples of the polypeptide having the ability to express phage surface include linear phage coat protein III (hereinafter abbreviated as cpIII) and VIII (hereinafter abbreviated as cpVIII), or a part of each of them. There is no particular limitation as long as it is expressed in the form of a fusion polypeptide with the site and the antibody variable region is displayed on the surface of the phage particle. Further, the polypeptide having Fc binding ability includes, for example, protein A and protein G, and the Fc binding domain or Fc binding domain-like structure thereof may be used. Fc binding domain, or Fc
When a binding domain-like structure is used, the number of domains is usually selected in the range of 1-5. Regarding the sequences of these genes, the open reading frame of the polypeptide encoded by the Y gene is linked to the open reading frame of the antibody variable region incorporated in the X gene, and the polypeptide is terminated at the end of the polypeptide encoded by the Y gene. There are codons. Further, a restriction enzyme site exists at the boundary between the X gene and the Y gene, and a restriction enzyme site exists at the boundary between the Y gene and the Z gene. The two restriction enzyme sites may be the same restriction enzyme site or different restriction enzyme sites as long as the ends generated after cleavage, that is, the X gene end and the Z gene end can be connected. Furthermore, the Z gene is connected to the open reading frame of the polypeptide encoded by the Z gene in a form following the open reading frame of the antibody variable region incorporated in the X gene, and a stop codon is present at the end of the polypeptide encoded by the Z gene. Are connected.

The gene of the present invention is used by incorporating it into a vector. Examples of the vector used in the present invention include a phagemid vector as a basic skeleton and an XYZ gene incorporated in the downstream of a promoter, and a phagemid vector as a basic skeleton in which an XY gene is incorporated in the downstream of a promoter. And the phagemid vector as a basic skeleton, X-
There is a vector in which the Z gene is integrated, and these can be used in combination.

As described above, the X gene can incorporate a gene encoding a natural or artificial antibody variable region. The X gene into which the gene encoding the antibody variable region has been incorporated is hereinafter referred to as the X'gene. Similarly, an X-Y-Z gene in which a gene encoding an antibody variable region is incorporated, X
-Y gene and X-Z gene are referred to as X'-Y-Z gene, X'-Y gene and X'-Z gene, respectively.
As used herein, the antibody variable region means a polypeptide containing the antibody variable region in its molecule.

When Escherichia coli carrying a phagemid vector containing an X'-YZ gene in which a gene encoding an antibody variable region, for example, a gene encoding Fv, Fab or scFv is integrated, is infected with helper phages. Phage is formed. At this time, X'- on the vector
From the Y gene, a phage coat protein in which antibody variable regions are linked is expressed and integrated on the phage surface. That is, the polypeptide containing the antibody variable region encoded by the X'-Y gene is displayed on the surface of this filamentous phage. Phage particles displaying an antibody variable region having the desired antigen-binding ability can be easily selected by affinity screening using a carrier on which the desired antigen is immobilized. At this time, if the polypeptide containing the antibody variable region is in the form of an Fv fragment, the phage particles are washed with a solution containing the surfactant N-lauroyl sarcosine sodium (hereinafter abbreviated as sarcosyl) to infect the phage. Fv without compromising sex
The V _H and V _L domains of the fragment can be dissociated. That is, the Fv having the antigen-binding ability displayed on the surface of the phage under these washing conditions
The number of fragments can be adjusted. This allows the valency of the antibody present on the phage surface to be adjusted,
Phage particles presenting antibody molecules with the same valence and high affinity can be selected. The conditions for this washing can be determined, for example, by using a BIA core system (Pharmacia) that utilizes surface plasmon resonance and by introducing a phage solution that has been subjected to various treatments to a sensor chip on which an antigen has been immobilized. You can

By re-infecting Escherichia coli with the obtained phage particles, the plasmid containing the gene encoding the target antibody molecule can be recovered. The recovered plasmid is digested with a restriction enzyme to release the Y gene,
By self-ligation, the fused polypeptide encoded by the X'-Z gene can be converted into an expressed form. By introducing the transformed plasmid vector into E. coli and culturing it, a large amount of the fusion polypeptide encoded by the X'-Z gene can be produced. This polypeptide has Fc binding ability,
For example, it can be easily purified by using a resin to which IgG is bound. Alternatively, a resin having an antigen immobilized thereon may be used.

The antibody variable region thus obtained and Fc
A complex is formed by mixing a polypeptide fused with a polypeptide having binding ability with a molecule containing Fc, for example, human IgG. For example, a polypeptide having an Fc-binding ability comprises a domain structure having an Fc-binding ability consisting of 58 amino acid residues similar to the Fc-binding domain derived from protein A encoded by the gene represented by SEQ ID NO: 2 in the sequence listing. In the case of (SEQ ID NO: 4 in the sequence listing), a molecule containing one Fc can be bound at two positions [European Journal of
Biochemistry (European Journal of Biochemist
ry), Vol. 78, pp. 471-490 (1977), Molecular Immunology,
Volume 17, pp. 1563-1573 (1980), J.
The EMBO Journal, Volume 4, Pages 1075-1080 (1985), Protein Engineering, Volume 1, Volume 2
102-113 (1987)]. Therefore,
The fusion polypeptide containing the Fc binding domain forms, for example, a complex in which two molecules of the fusion polypeptide are added to an IgG1 molecule. That is, a molecule having a divalent antibody titer can be created. The resulting complex is 2
Because of its high value, it has high avidity as an antibody,
Since it contains the c fragment, it can be easily detected by using an anti-Fc fragment antibody, and can be widely applied to ELISA and the like.

Further, when the number of polypeptides having Fc binding ability is doubled, a more stable complex having a high antibody titer is formed. That is, for example, a fusion polypeptide containing a polypeptide containing two Fc binding domain-like structures of protein A encoded by the gene represented by SEQ ID NO: 3 in the sequence listing forms a macromolecule with, for example, human IgG. Not surprisingly, IgG2 molecule and fusion polypeptide 3
Forms a stable complex of molecules. This complex is extremely stable, has a high trivalent antibody titer, and can be detected by an anti-Fc fragment antibody, and the complex is also an artificial antibody that is extremely useful for applications such as ELISA.

The plasmid pM13Fv produced by the present inventors is an example of a vector containing the gene of the present invention having the above-mentioned functions.

A schematic diagram of the gene sequence of the plasmid pM13Fv is shown in FIG. In FIG. 1, the gene of the present invention is
The portion corresponding to the X gene is between HindIII and SalI, and Y
Gene between SalI and SalI, Z gene between SalI and EcoRI
Is equivalent to HindIII-Ec corresponding to this XYZ
The gene sequence between the oRI sites is also shown in SEQ ID NO: 5 in the sequence listing.

The plasmid pM13Fv has the gene X-Y-Z of the present invention inserted between the HindIII and EcoRI sites of the plasmid pTZ19R (Pharmacia), which is a general phagemid vector. The X gene downstream of the vector-derived promoter has an SD sequence (Shine-Dalgarno sequence) followed by a first open reading frame, which is encoded by a fusion polypeptide of a secretory signal peptide and a V _H fragment-like polypeptide. Has been done. Further downstream is the SD sequence, followed by a second open reading frame encoding a secretory signal peptide V _L fragment-like polypeptide. V 3 of _L gene 'terminal portion to the restriction enzyme SalI site exists (nucleotide numbers 891-896 of SEQ ID NO: 5), in the form following the reading frame of the V _L genes, gene (SEQ ID NO: 1 which encode ΔcpIII polypeptide ) Containing Y gene is connected. That is, the second open reading frame is the secretory signal peptide, V _L
It encodes a fragment-like polypeptide, a fusion polypeptide of the ΔcpIII polypeptide. The Y gene is linked to the Z gene at the restriction enzyme SalI site (base numbers 1555-1560 of SEQ ID NO: 5) located further downstream of the ΔcpIII polypeptide gene encoded by the Y gene. Z
The gene is a gene encoding a polypeptide containing two Fc-binding domain-like structures of protein A (SEQ ID NO: 3)
Is a gene containing

PM13Fv having such a structure is a restriction enzyme
When the gene is completely digested with SalI, the Y gene is excised, and when the Y gene is removed and self-ligation is performed, the X gene and the Z gene are linked at the SalI site. At this time, Z
A polypeptide containing two Fc-binding domain-like structures of the protein-encoded gene is linked in the form following the second open reading frame of the X gene. That is, the plasmid p
After removing the Y gene by digesting M13Fv with the restriction enzyme SalI, the second open reading frame encoded by the gene of the present invention is a secretory signal peptide, a _VL fragment-like polypeptide, and an Fc-binding domain-like structure of protein A. Encodes a fusion polypeptide of two containing polypeptides.

In this plasmid pM13Fv, X
The Fv gene derived from a monoclonal antibody against chicken egg white lysozyme (hereinafter abbreviated as HEL) is inserted in the portion corresponding to the gene. Since there is a restriction enzyme site so that the Fv gene can be easily replaced, the inserted Fv gene can be regarded as the X gene by considering it as a mere spacer sequence. That is, the PstI site present at base numbers 115-120 and the base number 4 in the base sequence shown by SEQ ID NO: 5 in the sequence listing shown above.
Plasmid p at the BstEII site present in 38-444
Remove the V _H gene was inserted by cutting the M13Fv, you can insert any V _H gene prepared as reading frame fit. PstI-BstEII that the reading frame fits
The fragment may be DNA synthesized by using a DNA synthesizer, or P may be prepared by a method conventionally used.
DN obtained by digesting a DNA fragment obtained by PCR with PstI and BstEII using a V _H gene amplification primer synthesized so as to contain stI site or BstEII site
A fragment is also acceptable. DNA fragment encoding the V _L gene between SalI site present in the SacI site and the nucleotide numbers 891-896 which is present in the nucleotide sequence of nucleotide numbers 583-588 shown in SEQ ID NO: 5 also Sequence Listing V _L gene in the same manner Can be replaced with However, this plasmid pM1
In the case of 3Fv, since there is a SalI site between the Y gene and the Z gene, SalI digestion causes partial decomposition to prepare a vector.

A method for constructing this plasmid pM13Fv will be briefly described. Monoclonal antibody D against HEL
Plasmid p encoding Fv fragment derived from 1.3
SW1V _H D1.3V _K D1.3 [Journal of Molecular Biology, Volume 213, Volumes 617-6
19 (1990)] is Greg Winter (GregWi
Dr. Nter) (Cambridge, UK).
However, the transferred plasmid was slightly different from the sequence described in the literature. This plasmid pSW1V _H D1.3
V _K D1.3 is the HindIII-EcoR of plasmid pUC19.
A gene encoding an Fv fragment has been inserted between I and I. The sequence of this HindIII-EcoRI insert fragment is shown in SEQ ID NO: 6 in the sequence listing.

This plasmid pSW1V _H D 1.3V _K D
A HindIII-SmaI fragment containing the _VH gene of 1.3 was added to pTZ.
The plasmid T19VH is constructed by inserting it into the HindIII and SmaI sites of 19R (Pharmacia). In addition, the SmaI-EcoRI fragment containing the V _L gene was inserted into the SmaI and EcoRI sites of pTZ18R (Pharmacia) to obtain the plasmid T.
Build 18 VL. Using the plasmid T18VL as a template, the restriction enzyme EcoRI shown in SEQ ID NO: 7 in the sequence listing.
Primer VL3'S having a recognition sequence and a SalI recognition sequence
alI and the primer UPM shown in SEQ ID NO: 8 in the sequence listing
After amplifying DNA by PCR method using CS, HindII
A DNA fragment is obtained by digesting with I and EcoRI. This is inserted into the HindIII and EcoRI sites of the plasmid pTZ18R to obtain the plasmid T18VLS. This plasmid T18
The SmaI-EcoRI fragment containing the V _L gene of VLS is inserted into the SmaI and EcoRI sites of the previously constructed plasmid T19VH to obtain the plasmid T19VHVLS. Next, using plasmid pEZZ18 (Pharmacia) as a template,
Primer ProA5 ′ shown in SEQ ID NO: 9 in the sequence listing
Then, a DNA containing a gene encoding a polypeptide containing two Fc-binding domain-like structures of protein A is amplified by PCR using the primer ProA3 ′ shown in SEQ ID NO: 10. Use this DNA as restriction enzymes SalI and E
After digestion with coRI, Sal of plasmid pTZ18R
I and EcoRI sites were inserted to obtain the plasmid T18PA. Fc of protein A of this plasmid T18PA
A SalI-EcoRI fragment containing a gene encoding a polypeptide containing two binding domain-like structures was ligated to plasmid T
19VHVLS is inserted into SalI and EcoRI sites to obtain plasmid pFv-PP. Then plasmid M13m
By using the p18 as a template and the primer cpIII 5'-1 shown in SEQ ID NO: 11 and the primer cpIII 3 'shown in SEQ ID NO: 12 in the sequence listing, cpIII was obtained by PCR.
Containing a gene encoding the C-terminal polypeptide of Escherichia coli
Amplify A. This DNA is digested with the restriction enzyme SalI and then inserted into the SalI site of the plasmid pFv-PP. At this time, pM13Fv is the plasmid inserted in the direction in which the sequence derived from the primer cpIII 5'-1 was connected following the _VL gene of the plasmid pFv-PP.
Escherichia coli JM109 introduced with the plasmid pM13Fv constructed in this manner is Escherichia coli JM109 / pM13.
Named and displayed as Fv, it has been deposited as FERM P-13698 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

By using the gene of the present invention, the following can be achieved. First, the gene of the present invention is used by incorporating it into a phagemid vector. By inserting a gene encoding an antibody variable region into the X gene of the present invention in the presence of the Y gene, it is possible to construct a so-called phage-display antibody library in which the antibody variable region is displayed on the surface. As a method for obtaining the antibody gene, according to a conventionally used method, RNA is used as a raw material, which is obtained from animals including humans or cultured cells derived from them, and the like, which includes mRNA of the antibody, and then cDNA is synthesized by the PCR method. Genes containing natural antibody variable regions can be amplified. This may be incorporated into the gene of the present invention so that the antibody variable region is expressed. Alternatively, a gene encoding a synthetic antibody variable region prepared so as to encode various kinds of amino acids using synthetic DNA may be used. In this case, particularly in the antibody variable region, there are many amino acid substitutions, and the effect can be expected by introducing many mutations into the hypervariable region that directly acts on the antigen.

When a phage displaying an antibody molecule having the desired antigen-binding ability is selected from the phage-displaying antibody library displaying the antibody variable region constructed as described above, it is confirmed that the X'gene expresses the Fv fragment. It is very useful when prepared. The reason for this is that the present inventors, as a result of diligent efforts, have found a method for controlling the number of Fv molecules capable of binding to an antigen that are expressed on the surface of a phage. The Fv fragment indicating the antigen-binding ability is a case where V _H fragment and the V _L fragment is present as a Fv fragment linked, V _H
The binding ability of the fragment or the _VL fragment alone becomes very low.

By utilizing this property, the Fv fragment expressed on the phage surface is dissociated into a V _H fragment and a V _L fragment to determine the number of Fv molecules having an antigen-binding ability existing on the phage surface. It can be about one molecule. This can be done in a step with a gentle wash treatment with the detergent sarcosyl, and the step has no effect on the infectivity of the phage particles. When an antibody molecule is displayed on the phage particle using a phagemid vector, the number of the antibody molecule cannot be controlled. When two or more antibody molecules are present on the phage surface, the avidity as a whole becomes high even if the affinity of the antibody molecule is low, and it is as if they have high affinity in affinity screening using an antigen. act. At this time, if the number of Fv molecules can be controlled to about 1 molecule without impairing the infectivity of the phage particles, it is expected to obtain phage particles displaying antibody molecules with high affinity. Such control is difficult in scFv in which a V _H fragment and a V _L fragment are covalently linked, or in a phage-displayed antibody library displaying Fab.
In addition, the gene encoding the Fv fragment is recovered from the obtained phage particle displaying the Fv molecule,
It is easy to convert to scFv fragment or Fab fragment. That is, Escherichia coli is infected with the obtained phagemid particles and the plasmid is recovered. The desired V _H gene and V _L gene may be extracted from this plasmid and reinserted into a plasmid constructed so that it can be expressed in the form of scFv fragment or Fab fragment.

After the phage particles displaying the desired antibody variable region were obtained, the gene of the present invention used was X'-Y-Z.
In the case of, it is easy to convert it into a practically useful form. That is, Escherichia coli is infected with the obtained phagemid particles to recover the plasmid. Thereafter, as described above, by removing the Y gene and converting it into the X′-Z form, the plasmid expresses a fusion polypeptide of the polypeptide containing the antibody variable region and the polypeptide having the Fc-binding ability encoded by the Z gene. It is converted to the form.

A polypeptide containing this antibody variable region,
Fusion polypeptides of polypeptides having Fc binding ability are very useful. That is, first of all, the purification is easy. For example, the fusion polypeptide can be recovered from the culture using a resin on which a polypeptide or protein containing Fc is immobilized. Second, the fusion polypeptide is mixed with a Fc-containing polypeptide or protein, such as human IgG, to form a stable multivalent antibody complex. Since it is multivalent, the binding stability to the antigen is higher than when it is monovalent, and it is useful when used for ELISA or Western blotting. In addition, detection is facilitated and useful due to the nature of the mixed Fc-containing polypeptide. When the gene X'-Z of the present invention is used to produce such a fusion polypeptide, the polypeptide containing the antibody variable region encoded by the X'gene is a Fv fragment, scFv fragment, Fab fragment, etc. Anything is valid.

Further, a polyclonal artificial antibody can be prepared by using the gene of the present invention.
That is, a library of genes encoding antibody variable regions is prepared and then incorporated into the X gene to prepare an X'-YZ gene library. Next, the X'-Y gene library is transformed into a phage to select an X'-Y gene group having an antigen-binding property. Next, a polyclonal artificial antibody can be prepared by expressing an X'-Z gene group having the selected X'gene group.
The polyclonal artificial antibody is also useful in the fields of antigen purification, antigen measurement, diagnosis and the like.

As described in detail above, by using the gene of the present invention, an antibody molecule having a particularly high affinity is selected among antibody molecules specific to an antigen,
Furthermore, it has become possible to provide artificial antibodies with high avidity.

[0031]

EXAMPLES Examples will be shown below, which do not limit the present invention.

[0032] Plasmid pSWlV encoding Fv fragments from monoclonal antibodies D1.3 against building HEL Example 1 Expression Plasmid _H D1.3
V _K D1.3 was assigned by Dr. Greg Winter (Cambridge, UK). However, the transferred plasmid was slightly different from the sequence described in the literature. This plasmid pSWlV _H D1.3V _K D1.3 is of pUC19
A gene encoding an Fv fragment is inserted between HindIII and EcoRI. The sequence of this HindIII-EcoRI insert is shown in SEQ ID NO: 6.

(1-1) Plasmid pFv-PP, pFv-
Construction of P Plasmid pSW1V _H D1.3V _K D1.3 was digested with restriction enzyme H
After digestion with indIII and SmaI and separation by agarose gel electrophoresis, a DNA fragment of about 470 bp containing the V _H gene was extracted and purified. This DNA fragment was used as plasmid pTZ19R.
(Pharmacia Co.) HindIII, insert into SmaI site,
The plasmid T19VH was obtained. In addition, the plasmid pSW
1 V _H D1.3 V _K D1.3 was digested with restriction enzymes EcoRI and SmaI, separated by agarose gel electrophoresis, and a DNA fragment of about 450 bp containing the V _L gene was extracted and purified. This DNA fragment was inserted into the EcoRI and SmaI sites of plasmid pTZ18R (Pharmacia) to obtain plasmid T
18 VL was obtained. Using this plasmid T18VL as a template, the primer VL3'SalI having the restriction enzyme EcoRI recognition sequence shown in SEQ ID NO: 7 of the sequence listing and the SalI recognition sequence.
PCR was performed using the primer UPMCS, which is the pTZ18R-derived sequence shown in SEQ ID NO: 8, and the amplified DNA was digested with the restriction enzymes HindIII and EcoRI. After separation by agarose gel electrophoresis, HindIII and EcoRI
The fragment was extracted and purified. This DNA fragment was used as plasmid pT
It was inserted into the HindIII and EcoRI sites of Z18R to obtain plasmid T18VLS. This plasmid T18VLS has a restriction enzyme SalI recognition sequence at the 3 'end side of the V _l gene. This plasmid T18VLS was digested with the restriction enzyme EcoRI,
After digestion with SmaI and separation by agarose gel electrophoresis,
A DNA fragment of about 430 bp containing the _VL gene was extracted and purified. This DNA fragment was inserted into the EcoRI and SmaI sites of the plasmid T19VH having the V _H gene constructed above to obtain the plasmid T19VHVLS. This plasmid T1
9VHVLS has a V _H gene and a V _L gene downstream of the pTZ19Rlac promoter.

Next, using the plasmid pEZZ18 (Pharmacia) as a template, the primer ProA5 'having the restriction enzyme SalI recognition sequence shown in SEQ ID NO: 9 in the sequence listing,
DNA was prepared by PCR using the primer ProA3 ′ having the restriction enzyme EcoRI recognition sequence shown in SEQ ID NO: 10.
Was amplified. The amplified DNA containing a gene (SEQ ID NO: 3) encoding a polypeptide containing two Fc-binding domain-like structures of protein A was digested with restriction enzymes SalI and E.
About 390 after digested with coRI and subjected to agarose gel electrophoresis
The bp DNA fragment was extracted and purified. This DNA fragment is called pT
Z18RSalI and plasmid T introduced into EcoRI site
18 PA was obtained. Next, this plasmid T18PA was digested with restriction enzymes SalI and EcoRI and subjected to agarose gel electrophoresis to extract and purify a DNA fragment of about 390 bp.

This DNA fragment was obtained from the plasmid T1 previously obtained.
9VHVLS was inserted into SalI and EcoRI sites to construct a plasmid pFv-PP. This plasmid pFv
-PP is the C of the _VH fragment and the _VL fragment
It encodes a fusion polypeptide containing two Fc-binding domain-like structures of Protein A (58 amino acid residues of SEQ ID NO: 4) at the end. This plasmid pFv-PP was digested with a restriction enzyme MluI to remove a released DNA fragment of about 170 bp, and then self-ligated to obtain a plasmid pFv-PP.
P was obtained. This plasmid pFv-P has V _H fragment and SEQ ID NO: 2 in the sequence listing at the C terminal of the V _L fragment.
It encodes a fusion polypeptide containing one Fc-binding domain-like structure of protein A encoded by the gene shown in.

(1-2) Construction of plasmid pM13Fv Using plasmid M13mp18 (Takara Shuzo) as a template
The primer cpIII 5'-1 having the restriction enzyme SalI recognition sequence shown in SEQ ID NO: 11 in the sequence listing, and SEQ ID NO: 12
PCR was performed using the primer cpIII 3 ′ having the restriction enzyme SalI recognition sequence and the NheI recognition sequence shown in
DNA encoding the C-terminal polypeptide of III was amplified. This DNA was digested with restriction enzyme SalI and then subjected to agarose gel electrophoresis to extract and purify a DNA fragment of about 650 bp. This DNA fragment was inserted into the SalI site of the plasmid pFv-PP obtained in Example (1-1). Among the obtained plasmids, _VL gene was followed by primer cpII
The plasmid inserted in the direction in which the sequences derived from I 5'-1 were connected was designated as plasmid pM13Fv.

This plasmid pM13Fv is a fusion polypeptide in which a V _H fragment and a polypeptide (ΔcpIII) on the C-terminal side of cpIII, which is encoded by the gene shown in SEQ ID NO: 1 in the sequence listing, are linked to the C-terminal of the V _L fragment. Constructed to express the peptide. Further, a schematic diagram of the DNA fragment inserted into HindIII-EcoRI of this plasmid pM13Fv is shown in FIG. 1, and its nucleotide sequence is shown in SEQ ID NO: 5 in the sequence listing. This plasmid pM13Fv was easily digested with the restriction enzyme SalI and then self-ligated to easily construct the plasmid pFvf constructed in Example (1-1).
It can be in the form of PP. This plasmid pM1
3Fv-introduced E. coli JM109 was transformed into Escherichia coli JM1
It was designated as 09 / pM13Fv and deposited as FERM P-13698 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

Similarly, using the plasmid M13mp18 as a template, the primer cpIII 5'-2 having the restriction enzyme SalI recognition sequence shown in SEQ ID NO: 13 of the sequence listing,
PCR was carried out using the primer cpIII 3'used above, and the amplified DNA was digested with SalI to obtain plasmid pF.
The plasmid pM13 was inserted into the SalI site of v-PP.
ΔFv was constructed. This plasmid pM13ΔFv
It has almost the same gene sequence as pM13Fv, but the termination codon appears in the open reading frame between the _VL gene and the ΔcpIII gene because the termination codon is included in the sequence of the primer cpIII 5′-2. To do. Therefore, p
M13ΔFv is constructed to express only V _H and V _L fragments.

Example 2 Expression of Fv fragment on the surface of phage and analysis of its properties (2-1) Preparation of Fv-displayed phage particle Escherichia coli BMH71-18 was prepared using the plasmid pM13Fv obtained in Example (1-2). Transformed with E. coli BMH71-18 /
pM13Fv was obtained. As a control, Escherichia coli BM transformed with Escherichia coli BMH71-18 using pM13ΔFv
H71-18 / pM13ΔFv was used. 2 x these two types of E. coli
TY medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl) was inoculated and shake-cultured at 30 ° C. overnight. 4 ml of this culture was inoculated into 12 ml of 2 × TY medium and cultured at 30 ° C. for 1 hour. 2 ml of M13K
After adding 07 phage solution (1 × 10 ¹² pfu / ml), the culture was further incubated at 30 ° C. for 1 hour. 500 ml of this culture solution containing 3 ml each of 70 μg / ml of kanamycin.
6 x TY medium was inoculated and cultured at 30 ° C for 24 hours.
Similarly, M13K07 phage was prepared using Escherichia coli BMH71-18 having no plasmid as a host. The cells were removed from each culture by centrifugation to obtain a supernatant.

To 3 liters of culture supernatant, 1 liter of PEG / NaCl (20% polyethylene glycol 6,
000 and 2.5 M NaCl) was added and the resulting mixture was left at 4 ° C. overnight. The phage was centrifuged to produce a pellet. Divide the precipitate into 3 equal parts and add 3 to each pellet.
It was prepared by different methods. That is, firstly, 30
0 ml TE (10 mM Tris-HCl buffer, pH 8.0,
1 mM EDTA) at room temperature for 1 hour, second, 300 ml
TES (TE containing 0.1% sarcosyl) at room temperature for 1 hour, and thirdly, 300 ml TES at room temperature for 18 hours, vortexed. Phage was reprecipitated by adding 100 ml of PEC / NaCl to the phage solution treated with these three kinds. Precipitate was pelleted by NET (10 mM Tris-HCl buffer, pH 8.0, 0.1 M NaCl, and 1
mM EDTA). 170000 phage
It was collected by centrifugation at xg for 3 hours and dissolved in NET. Finally, the phage was purified by centrifugation at 36,000 rpm for 1 hour using the gradient CsCl ultracentrifugation method. The phage bands were collected and dialyzed against NET to obtain a phage solution. The phage obtained from E. coli BMH71-18 / pM13Fv was M13Fv, E. coli BMH71-18 / pM
The phage obtained from 13ΔFv was named M13ΔFv. Three types of phage solutions prepared in this way, M13F
The following analyzes were performed on v, M13ΔF, and helper phage M13K07, which were purified after the above-described three kinds of treatments, respectively.

(2-2) Measurement of infectivity of phage and phagemid The three types of phages (M13Fv, M) obtained in Example (2-1)
13ΔFv, M13K07) with three types of processing (no processing,
The colony forming ability and the plaque forming ability of the phage solution which had been washed with sarcosyl for 1 hour and washed with sarcosyl for 18 hours were measured. That is, these phage solutions were appropriately serially diluted using NET. 100 μl of this phage solution was added to E. coli XLI-Blue in the logarithmic growth phase (OD ₆₀₀ = 0.8).
(Stratagene) culture solution (500 μl) was added and left at room temperature for 45 minutes. Of this, 100 μl is 50 μg /
A 2 × TY medium plate containing ml ampicillin was directly smeared and kept at 37 ° C. overnight to count the number of appeared colonies. Separately, 100 μl was kept warm at 50 ° C. 4
It was mixed with ml of soft agar (agar dissolved in 2 × TY medium so that the concentration was 0.65%), spread on a 2 × TY medium plate and solidified. This was kept at 37 ° C. overnight and the number of plaques that appeared was counted. The results are shown in Table 1. That is, Table 1 is a table comparing the colony forming ability and the plaque forming ability of the obtained phage solution.

[0042]

[Table 1] Table 1 ─────────────────────────────── Clone washing conditions Number of plaques / Number of colonies / 10. D. ₂₆₀ 1O. D. ₂₆₀ ─────────────────────────────── 10 ¹² 10 ¹² M13K07 No treatment 3.3-Washing 1 hour 3.1- Washing 18 hours 3.2-M13Fv no treatment 0.27 0.91 washing 1 hour 0.37 1.2 washing 18 hours 0.32 1.3 M13ΔFv no treatment 0.30 0.96 washing 1 hour 0.27 1 .3 18 hours of cleaning 0.23 1.2 ───────────────────────────────

The molar absorptivity of fdDNA in virions per phosphate group at 260 nm has been determined to be 6750. The length of DNA of M13K07 is 6.
4 kb, and the length of DNA of M13Fv and M13ΔFv is 4.8 kb. D. _{A 280} unit phage (hereinafter abbreviated as DU) should correspond to approximately 1.3 × 10 ¹³ phage particles for M13K07 and approximately 1.8 × 10 ¹³ phage particles for M13Fv and M13ΔFv. is there. From the results of the sample that was not treated with sarcosyl, M13K07 was 0.25PF
U / phage particles, M13Fv and M13ΔF
v was shown to be on the order of 0.06 CFU / phage particles. Plaques formed by bacteria infected with M13Fv as well as M13ΔFv were considered to be derived from helper phage, suggesting that approximately 10% of the phage in the preparation were helper phage. These three
The infectivity of the phage particles of the seeds was also shown by treatment with 0.1% sarcosyl solution for 1 hour (gentle washing)
It was not impaired at all by the treatment of time (sufficient washing).

(2-3) Analysis of Fv Fragment Expressed on Phage Surface Each of M13Fv, M13ΔFv, and M13K07 prepared in Example (2-1) was treated with 3 kinds of phage 11.2DU and treated with 5%. 10 mM Tris-acetate buffer (pH containing SDS, 1% β-mercaptoethanol)
5.4) Dissolved in 1 ml and stirred at room temperature for 24 hours.
After neutralization with 1N NaOH, MgCl ₂ was added to a final concentration of 0.1M. 150,000 × g at 20 ℃
After centrifugation for 12 hours, the supernatant was taken out and boiled for 2 minutes.
Add an equal volume of sample buffer for SDS-PAGE, and add 15 μl (equivalent to 0.075 DU) of 16% SDS-PAGE.
I went to PAGE. After migration, a polyvinylidene fluoride (PVDF) membrane (Millipore) using a semi-dry blotter
Blotted to. This membrane was immersed in 1% bovine serum albumin (BSA) and kept at room temperature for 1 hour for blocking. As a primary antibody, anti-D1.3Fv fragment rabbit antibody (serum obtained by immunizing a rabbit with D1.3Fv was purified by a D1.3Fv-immobilized column, hereinafter abbreviated as anti-D1.3 antibody) was 0.05%. Tween 20
20) containing phosphate buffer (hereinafter abbreviated as PBS / T) and soaked in PBS / T for 4 hours at room temperature
It was washed 3 times. For detection, an ECL Western blotting detection system (Amersham) using an anti-rabbit IgG donkey antibody conjugated with horseradish peroxidase was used. The result is shown in FIG. That is, FIG. 2 shows filamentous phages M13Fv, M13ΔFv, and M1.
It is a figure comparing the amount of Fv fragment-derived polypeptides remaining in the phage coat protein when 3K07 was treated with a solution containing the surfactant sarcosyl.

The Fv fragment in M13ΔFv is M1
Although it is not considered to be adsorbed on the surface of 3 phage,
Western blotting revealed a band corresponding in size to the _VH fragment. But the phage
After washing for 1 hour with a 0.1% sarcosyl solution, this band disappeared completely. On the other hand, in the case of M13Fv,
Two bands were clearly detected, one thick corresponding to the V _H fragment and the other corresponding to the fusion of the _VL fragment with ΔcpIII. Phage 1 with 0.1% sarcosyl solution
Even after washing for a long time, the bands corresponding to V _H became weak, but both bands remained. 1 phage
After thorough washing for 8 hours, the V _H band disappeared completely, but the V _L fragment was removed by ΔcpIII.
The one fused with was retained on the phage without decreasing the amount. As mentioned above, both V _{H and} V _L fragments are expressed on the phage surface in an associated form, and treatment with a sarcosyl solution removes only the V _H fragment from the phage surface.

Next, it was expressed on the surface of phage M13Fv.
To quantify the Fv fragment, 0.075 each
DU phage and D1.3Fv fragment with known concentration
Similarly analyzed by Western blotting
It was The result is shown in FIG. That is, FIG.
Of the Fv fragment expressed on the surface of M13Fv
It is the figure which compared the quantity of the coming polypeptide. Sarkosyl dissolution
Expression on M13Fv phage after washing with liquid for 1 hour
The V _HThe intensity of the band is 1 ng of Fv fragment
It seemed to correspond. Each lane has 0.075 DU
The total protein prepared from the Cage was added. Fv flag
The molecular mass of menthol is 24.7 kDa, so 1 ng of Fv
Fragment is 2.4 × 10^TenCorresponds to individual molecules. this
Therefore, M treated with 0.1% sarcosyl solution for 1 hour
When preparing 13Fv phage, about 2% phage
Were expressed to express the Fv molecule. But V_H
Some of the fragments were washed with Sarkosyl solution.
Therefore, it should have been removed from the phage surface.
In fact, when M13Fv phage was prepared without washing
V_HThe strength of the band is the sump that has been washed for 1 hour.
V in case of Le_HIt was several times higher than the intensity of the band. Washing
The case where M13ΔFv was prepared without performing
Free V_HFragments are not in this fraction
Fv fragment because it may be specifically mixed
It is difficult to estimate the exact amount of. Also, the phage grain
During the process of preparing protein from the offspring, a substantial amount of
There must have been a loss of quality. Therefore, Fv
The true rate of M13Fv expressing the molecule is the above estimate.
Should be much higher. As mentioned above, M
13 Fv phage actually express Fv molecules on their surface
In addition, among the M13Fv phage, the Fv molecule
Is estimated to be 5% or more.
To be done.

(2-4) Analysis of antigen-binding property of Fv fragment expressed on phage surface The antigen-binding property of Fv fragment expressed on the phage surface was analyzed by BIA core system (Pharmacia; Company). HEL or anti-D1.3 antibody was chemically coupled to the biosensor chip support by amine coupling according to the manufacturer's instructions. All experiments are H
BS (10 mM HEPES, pH 7.4, 3.4 mM EDT
A, 150 mM NaCl, and 0.5% Tween 20)
Was performed at 30 ° C. at a flow rate of 5 μl / min.

All of the 9 types of phage solutions obtained in Example (2-1) were dialyzed against HBS and then 5 concentrations (3.
(7, 1.8, 0.9, 0.45, 0.23 DU / ml), and 20 μl was added to the HEL-immobilized sensor chip. The results are shown in FIGS. That is, FIGS. 4 to 12 correspond to FIGS. 4 to 6, FIG. 7 to FIG. 9, and FIG. 10 to FIG. RU), the horizontal axis represents time (seconds). Similarly, after injecting 20 μl of phage having a concentration of 3.7 DU / ml, anti-M13 phage rabbit antibody (serum obtained by immunizing rabbits with M13 phage was purified by M13 phage-immobilized column, The signal was amplified by further adding M13 antibody). The result is shown in FIG. That is, FIG. 13 shows that after the phage was loaded on the HEL-immobilized sensor chip, the anti-M13
It is a figure which showed the result of the surface plasmon resonance sensor when the antibody was further thrown in, and a vertical axis | shaft shows a resonance unit (RU) and a horizontal axis shows time (second). In addition, D1.3Fv on the phage surface
To examine the presence of the fragment-derived polypeptide, 20 μl of a phage solution having a concentration of 3.7 DU / ml was put into a sensor chip on which anti-D1.3Fv antibody was immobilized. The result is shown in FIG. That is, FIG. 14 is a diagram showing the results of the surface plasmamon resonance sensor when phages were put into an anti-D1.3 antibody-immobilized sensor chip, wherein the vertical axis represents resonance units (RU) and the horizontal axis represents time (seconds). ) Is shown.

The results shown in FIGS. 4 to 14 are summarized below. As shown in FIG. 4 and FIG. 5, M13K07 is also M13K07.
ΔFv also did not bind to HEL. The baseline migration is believed to be due to differences in sample conditions, ie solute, pH, and / or ionic strength. The resonance unit (RU) generated by the binding is 3.7
M13Fv clearly bound to HEL even though it was about 200 RU for the DU / ml phage solution, which was much higher (FIG. 6). Judging from the shape of the dissociation curve, part of the phage rapidly dissociated from HEL (K _off
= 3 × 10 ⁻² S ⁻¹ ), and the remaining phages appeared to hardly dissociate (K _off = 1 × 10 ⁻³ S ⁻¹ ). This suggested that these phage contained differently shaped phages. That is,
It was predicted that there might be a phage that expresses only one Fv molecule on its surface and a phage that expresses multiple Fv molecules on the surface of one phage particle. This was supported by the results obtained with the M13Fv preparation that was washed with the sarcosyl solution for 1 hour (FIG. 9). As shown in Figure 9, M13 washed with sarcosyl for 1 hour.
The curve upon dissociation of Fv phage from HEL resulted in the simple pattern expected for a phage antibody with a single antigen binding site ( _Koff = _3x10).
^-2 S ^-1 ). M13Fv thoroughly washed with sarcosyl solution
Completely abolished HEL binding activity (Fig. 12).

The phage antibody bound to HEL on the sensor chip was directly detected by injecting anti-M13 antibody. From FIG. 13, it can be seen that by the injection of the anti-M13 antibody, the RU was increased in the case of M13Fv which was not washed with sarcosyl, the RU was reduced when washed with the sarcosyl solution, and the RU was bound when washed sufficiently. It was suggested that the isolated phage disappeared to the background level. These results directly demonstrated that phage expressing the Fv fragment on the surface bound HEL. It is considered that the effect of washing with the sarcosyl solution on the binding activity to HEL is due to the removal of the V _H fragment from the phage surface. That is, as shown in FIG.
The binding of 3Fv to the anti-D1.3 antibody molecule was not significantly affected by washing with sarcosyl solution. Since the anti-D1.3 antibody binds not only to the V _H fragment but also to the V _L fragment, these results indicate that only V _H fragment was removed from the phage surface by washing with sarcosyl, and ΔcpIII It is suggested that the fused V _L fragment remains adsorbed on the surface of the phage after washing with sarcosyl.
The results of the analysis using the BIAcore system are shown in Example (2-
It is in perfect agreement with the Western blotting result obtained in 3).

As described above, a part of M13Fv phage is composed of two or more Fv phages on the surface of one phage particle.
To express the molecule, and to control the valency of the antibody displayed on the phage surface and the kinetic properties upon dissociation by gently washing with sarcosyl solution. I came to the conclusion that I could do it.

Example 3 Using the Fv fragment fused to a polypeptide containing an Fc binding domain-like structure,
Preparation of artificial antibody with high binding property (3-1) Preparation of Fv fragment fused to polypeptide containing Fc binding domain-like structure Plasmid pFv-P and plasmid pFv-PP constructed in Example (1-1) Plasmid pSW1V _H D1.3V _K
Escherichia coli BMH71-18 was transformed with D1.3 to obtain E. coli BMH71-18 / pSW1V _H D1.3V _K D1.3 and E. coli BM, respectively.
H71-18 / pFv-PP and Escherichia coli BMH71-18 / pFv-P were obtained. These three types of E. coli were treated with 100 μg / ml ampicillin, 0.1
It was inoculated into 2 × TY medium containing% glucose and shake-cultured at 30 ° C. overnight. 1 ml of 1 liter of 10 ml of this culture
00 μg / ml ampicillin, containing 0.1% glucose 2
It was inoculated into a TY medium and shake-cultured at 30 ° C. (each 2 liters). The turbidity of the culture solution was measured by a spectrophotometer. D. ₆₀₀ = 0.
IPTG to reach a final concentration of 1 mM at 8
(Isopropyl-β-D-thiogalactopyranoside) was added, and the mixture was further shake-cultured at 30 ° C. for 36 hours. The cells were removed from each culture by centrifugation to obtain a supernatant.
Two liters of each of the obtained supernatants was filtered with a 0.45 μm filter using a Pellicon cassette (Millipore). After concentrating with a filter having a molecular weight cut-off of 10,000, a binding buffer [10 mM Tris-HCl buffer pH 8.0, 140
mM-NaCl, 1 mM EDTA, 0.1 mM PMSF (phenylmethylsulfonyl fluoride)].

To purify the Fv fragment from this concentrated solution, HEL was used for Reacti-Gel (Pierce).
The affinity gel immobilized on Fv-P and Fv-
IgG Sepharose 6FF (Pharmacia) was used for the purification of PP. That is, 10 to 200 ml of concentrated liquid
ml of each affinity gel was added. The mixture was gently spun at 4 ° C. for 24 hours and washed 5 times with binding buffer. The column was filled with the gel and the Fv fragment bound to the affinity gel was treated with 50 mM glycine / hydrochloric acid buffer (pH 2.5) and 150 mM NaC.
Elute with l. The eluate was rapidly neutralized with 1 M Tris-hydrochloric acid buffer (pH 8.0) and dialyzed against 50 mM sodium phosphate buffer (pH 7.0) containing 200 mM NaCl at 4 ° C. for 2 nights. E. coli BMH71-18 / pSW1V _H D
1.3V _K D1.3, E. coli BMH71-18 / pFv-PP, E. coli BMH71-1
The Fv fragment-derived polypeptides obtained from the 8 / pFv-P culture were designated as Fv, Fv-PP, and Fv-P, and used in the following analysis.

First, the obtained Fv, Fv-P and Fv-PP
A part of the product was separated by SDS-PAGE and then stained with Coomassie Brilliant Blue R250 for detection. The result is shown in FIG. That is, FIG. 15 is a diagram showing the results of SDS-PAGE of Fv, Fv-P, and Fv-PP purified by affinity resin. As shown in FIG. 15, the V _H portion and the V _L portion are associated with each other at a molar ratio of 1: 1 to form an Fv shape. In addition, the results of affinity chromatography showed that the portion derived from protein A had an IgG binding activity.

(3-2) Fv of Fv, Fv-P and Fv-PP
Analysis of physical properties of moieties The association constant (K _A ) and the enthalpy of association (ΔH) when Fv, Fv-P, and Fv-PP bind to HEL,
It was measured by calorimetry using an OMEGA titration calorimeter manufactured by Microcal (this is referred to as DTC analysis). That is, the calorimeter was automatically operated so that 100 μl of a syringe for injection was used to perform 24 injections at intervals of 2 minutes. Within 15 seconds,
5 μl of HEL solution was added to the calorimeter cell containing 1.36 ml of sample solution. Prior to performing the calorimetric measurement, the O. D. HEL by measuring ₂₈₀
Was accurately measured and fixed at 0.02028 mM. The concentrations of Fv, Fv-P, and Fv-PP in the cell of the calorimeter were adjusted to about 0.01 mM and calculated accurately from the data of DTC analysis. Binding experiments using a calorimeter were carried out using 50 mM sodium phosphate buffer (pH 7.0) and 200 mM Na.
Performed in Cl at 30 ° C.

From the data obtained, the values of ΔG and -TΔS were also calculated. The data are summarized in Tables 2 and 3.
That is, Tables 2 and 3 are tables showing thermodynamic and kinetic parameters relating to the binding of Fv fragments to HEL.

[0057]

[Table 2] Table 2 ─────────────────────────────────── K _A ΔH ΔG ───── ────────────────────────────── 10 ⁷ / M kcal / mol kcal / mol Fv 15.9 ± 1.9 −20.3 ± 0.1 −11.37 ± 0.07 Fv-P 9.59 ± 1.20 -19.9 ± 0.1 -11.07 ± 0.08 Fv-PP 8.48 ± 1.00 -19.7 ± 0.1 -10.99 ± 0.07 ──────────────────── ───────────────

[0058]

[Table 3] Table 3 ──────────────────────────────────── −TΔS K _on K _off ── ────────────────────────────────── kcal / mol 10 ⁶ / SM 10 ^-2 / S Fv 8.9 ± 0.1 2.93 ± 0.09 1.88 ± 0.28 Fv-P 8.8 ± 0.2 2.57 ± 0.06 2.73 ± 0.40 Fv-PP 8.7 ± 0.2 2.51 ± 0.07 3.01 ± 0.44 ───────────────────── ────────────────

The values of K _A of Fv-P and Fv-PP are F
Slightly less than the value of K _A for v. These differences are likely due to the slightly reduced enthalpy term when analyzing these fusion proteins. Then F
v, Fv-P, and Fv-PP has a K _on the time of reaction with HEL, stopped-flow apparatus (Model SF.17
It was measured by a fluorescence quenching method using MV, Applied Photo Physics. The HEL concentration was 6 μM
To 16 μM in 2 μM increments, and Fv, Fv−
The concentrations of P and Fv-PP were fixed at 1 μM. next,
50 μl of each solution was mixed at a volume ratio of 1: 1. The wavelength of the excitation light was 280 nm, and fluorescence with a wavelength of 320 nm or more was detected. The experiment was performed 13 times at 30 ° C. and the average value of the data was used for further analysis. Nonlinear regression analysis was performed and the apparent kinetics (K _app ) was measured as described in the software manual provided by Applied Photo Physics.

In this method, a pseudo first-order condition is assumed, and the following equation (Equation 1):

[0061]

[ _Formula 1] K _app = K _on [A] ₀ + K _off

Are assumed to be valid. [A] ₀ in the formula corresponds to the initial concentration of HEL. Experimentally,
The concentration of HEL was changed under the condition that the HEL was far in excess of the antibody, and the value of K _app was measured at each concentration. K _on was calculated from the slope value of the graph of K _app versus [A] ₀ . K _off is the value of K _app when [A] ₀ is equal to _0, but the actual value of K _off was too small to be experimentally measured by this method. So K _off
Was calculated from K _on / K _A. K _A is D as described above
It was measured by TC. Table 3 shows the K _{on and} K _off values for the three molecules. More as molecules shown in Table 3 is large, the value of K _on small, although the value of K _off is large, when it is compared with these molecules, the difference of K _on and K _off The large I couldn't say it. As mentioned above, DTC analysis and stopped
Summarizing the results of the flow analysis, the physical properties of the Fv portion were substantially the same in all of Fv, Fv-P, and Fv-PP.

(3-3) Analysis of IgG and Fv-P, Fv-PP complex The thermodynamic parameters for the association of Fv-P and Fv-PP with human IgG were measured by DTC. First, based on the data obtained in the binding experiment using Fv-P, Fv-PP, and HEL, Fv in the cell of the calorimeter
The concentration of -P was adjusted to 0.0100 mM and the concentration of Fv-PP was adjusted to 0.0050 mM. The concentration of human IgG (Sigma, # I-4506) in a 200 μl syringe was 0.
It was adjusted to about 04 mM. Add 12.5 μl of IgG solution to 1.
Twenty-three times were placed in a calorimeter cell containing 36 ml of Fv-P or Fv-PP solution. IgG concentration is DTC
Calculated from the data obtained in the analysis.

The results of DTC analysis are shown in FIG. 16 for Fv-P and FIG. 17 for Fv-PP. That is, FIG. 16 and FIG. 17 respectively show Fv-P and Fv-P.
It is a figure showing the DTC analysis result about the binding of P and human IgG, and the vertical axis shows the amount of heat (extracted substance kcal / mol and μcal /
Seconds), and the horizontal axis shows the injection number and time (seconds). The thermodynamic parameters calculated therefrom are shown in Tables 4 and 5. That is, Tables 4 and 5 are tables showing thermodynamic parameters relating to the binding of Fv-P and Fv-PP to human IgG.

[0065]

[Table 4] Table 4 ────────────────────────────────── n K _A ──────── ─────────────────────────── 10 ⁷ / M Fv-P 0.608 ± 0.003 0.657 ± 0.054 Fv- PP 0.631 ± 0.004 7.89 ± 2.20 ───────────────────────────────────

[0066]

[Table 5] Table 5 ──────────────────────────────────── ΔH ΔG −TΔS ──── ──────────────────────────────── kcal / mol kcal / mol kcal / mol Fv-P −27.4 ± 0.2 −9.45 ± 0.05 18.0 ± 0.3 Fv-PP −26.9 ± 0.3 −10.93 ± 0.17 16.0 ± 0.5 ─────────────────────────────── ─────

The affinity of Fv-PP for IgG is Fv
Approximately 10 times higher than -P, which seemed to come primarily from the entropy term. However, FIG. 16 and FIG.
Judging from the molar ratio of the concentrations of the reactants at the inflection point in Fig. 7, one IgG molecule has a maximum of two Fv-P molecules, and two IgG molecules have a maximum of two Fv-PPs.
Both Fv-P and Fv-PP protein A domains have Fv-binding activity because they can bind to a molecule. There may also be differences in the stability of the Protein A domain and the physical accessibility of the Fc portion. Fv-
For the combination of PP and IgG, unusual heat adsorption was observed after reaching the saturation point (Fig. 17). In the sequential titration experiment,
The molar ratio is sharp, that is, Fv-PP relative to IgG.
Is changed from the condition in which IgG is excessive to the condition in which IgG is excessive. This heat adsorption suggests that the complex is reorganized when IgG is present in excess relative to Fv-PP.

The molecular shape of the complex formed between Fv-P or Fv-PP and IgG was further investigated by HPLC. Two kinds of HPLC columns, G3000SW
And G2000SW (Tosoh Corporation) were connected in series. Perform all work at room temperature and use 20 as running buffer.
A 50 mM sodium phosphate buffer (pH 7.0) containing 0 mM NaCl was used. 15 μl of 5 μM human IgG solution or 15 μl of 10 μM Fv, Fv-P, or Fv-PP solution was first introduced into the device. When analyzing the shape of the complex, Fv, Fv-P, or Fv-
PP (final concentration of 10 μM in each case) was added to human Ig
G (final concentration, 5 μM) was mixed and incubated at room temperature for 1 hour, then 15 μl of the mixture was loaded on the HPLC instrument. The absorption at 245 nm was monitored. The result is shown in FIG. That is, FIG. 18 shows Fv, Fv-P, Fv-
It is the figure which showed the result by HPLC of PP and these IgG mixtures, and a vertical axis | shaft is O.D. D. ₂₄₅ , the horizontal axis represents time (minutes).

The expected molecular mass was as follows: V _H
Domain, 12,831 Da; V _L domain, 11,877
Da; domain 1 fused to the V _L domain of protein A, 19,341Da; V _L domain was fused to two domains of Protein A, 25,963Da; human IgG,
146,000 Da (average value). Fv, Fv-P, and F
The elution curves of v-PP showed that in each case the _VH and _VL domains were tightly associated with each other. The elution curve of human IgG contained one large peak and one small peak. The smaller peak corresponds to the IgG dimer. As expected, the elution curve for the mixture of Fv and IgG was simply a combination of the separate elution curves for IgG and Fv, indicating that no interaction occurred between these two molecules. . F
When v-P was mixed with IgG, a new substance peak (fraction A in FIG. 18) was eluted slightly before the IgG peak. Judging from the fact that the substances in the fraction of Fv-P and IgG are reduced and the molecular weight, fraction A shows 1 Ig
It corresponds to a complex composed of G molecule and two Fv-P molecules [referred to as (Fv-P) ₂ IgG complex].

When Fv-PP is mixed with IgG,
A new small peak and a large peak (fraction B in FIG. 18) appeared. As with Fv-P and IgG, Fraction B corresponds to a complex of 2 IgG molecules and 3 Fv-PP molecules [referred to as (Fv-PP) ₃ (IgG) ₂ complex]. is doing.

This (Fv-PP) ₃ (IgG) ₂ complex was selectively formed even when the concentration of Fv-PP and IgG in the mixture was reduced 10 times. Also, judging from the elution curves, this (Fv-PP) ₃ (IgG) ₂ complex appeared to be more stable than the (Fv-P) ₂ IgG complex. Most of IgG was found in the complex, and the elution curve of the Fv-PP molecule not forming the complex was extremely sharp, and the fraction B in FIG. It was almost the same as the elution curve of -PP.

In order to confirm that these fractions A and B formed a new complex, they were further analyzed by HPLC under denaturing conditions. That is, the fractions showing the absorption peak (fractions A and B in FIG. 18) were fractionated and freeze-dried.
These were dissolved in 5M guanidine-hydrochloric acid, and then subjected to HPLC analysis under the denaturing conditions on the same column. As the running buffer, 50 mM sodium phosphate buffer (pH 7.0) containing 5 M guanidine-hydrochloric acid was used. As controls, a mixture of IgG and Fv-P, as well as IgG and F
v-PP mixture (molar ratio of each of the two components is 1:
2) was also analyzed.

The results are shown in FIG. That is, FIG.
Is a diagram showing the analysis result by HPLC under denaturing conditions of Fv-P, Fv-PP and IgG complex (fractions A and B in FIG. 18, respectively), and the vertical axis represents O.D. D. ₂₄₅ , the horizontal axis represents time (minutes). As shown in FIG. 19, the complex of Fraction A was split into IgG, one V _L -Protein AF Fc binding domain, and V _H at a molar ratio of 1: 2: 2. In addition, the complex of Fraction B has a molar ratio of 2: 3: 3 IgG, _VL −.
Two protein AFc binding domains and V _H were generated.

As described above, when an IgG molecule is added, the Fv-P molecule and the Fv-PP molecule form a complex having a plurality of antigen binding sites.

(3-4) Analysis of Physical Properties of Fv-P, Fv-PP and IgG Molecule Complex Fv, Fv-P, Fv-PP, or those and IgG
The K _on and K _off of the complex with the HEL that had been bound to the solid support were measured using the BIAcore system. 35 μl of 200 μg / ml HEL solution
It was chemically coupled to the support on the biosensor chip by the amine coupling method according to the manufacturer's instructions. All experiments were performed at 30 ° C. in HBS with a flow rate of 5 μl / min. First, it contains human IgG (molar ratio 2:
1), or 100nM, 50nM, and 25 not containing
20 μl of nM antibody (Fv, Fv-P or Fv-PP) solution was loaded into the biosensor chip, then washed with HBS and finally the chip was regenerated with 100 mM hydrochloric acid. The mixing conditions of human IgG were the same as those shown in Example (3-3) except that HBS was used as a dilution buffer.
It was the same as the case of the analysis by PLC.

The results are shown in FIG. That is, FIG.
HE with and without IgG
FIG. 3 shows the results of a resonance sensor obtained when the association of L with Fv, Fv-P, and Fv-PP and the dissociation of the resulting complex were analyzed using the BIAcore system. The axis represents resonance units (RU) and the horizontal axis represents time (seconds). Values of K _on and K _off were calculated based on these data. The results are shown in Table 6. That is, Table 6 shows B
Fv fragment and H measured by IA core system
9 is a table showing kinetic parameters for EL binding.

[0077]

[Table 6] Table 6 ────────────────────────────────── K _on antibody K _off K _A concentration ── ──────────────────────────────── 10 ⁵ / SM nM 10 ^-3 / S 10 ⁷ / M Fv 1.71 ± 0.14 100 1.98 ± 0.05 8.68 ± 0.93 50 1.81 ± 0.04 9.50 ± 0.97 25 1.43 ± 0.02 12.0 ± 1.2 Fv-P 1.13 ± 0.01 100 2.17 ± 0.04 5.21 ± 0.12 50 2.02 ± 0.04 5.59 ± 0.12 25 1.83 ± 0.02 6.17 ± 0.10 Fv- PP 0.948 ± 0.001 100 2.24 ± 0.04 4.24 ± 0.07 50 2.16 ± 0.03 4.40 ± 0.08 25 2.04 ± 0.02 4.65 ± 0.06 Fv + IgG 1.86 ± 0.21 100 1.60 ± 0.05 11.7 ± 1.7 50 1.42 ± 0.03 13.2 ± 1.7 25 1.20 ± 0.02 15.5 ± 2.0 Fv-P + IgG 0.522 ± 0.021 100 0.647 ± 0.021 8.09 ± 0.59 50 0.790 ± 0.025 6.63 ± 0.47 25 1.10 ± 0.03 4.77 ± 0.32 Fv-PP + IgG 0.511 ± 0.049 100 0.636 ± 0.016 8.06 ± 0.97 50 0.643 ± 0.014 7.96 ± 0.93 25 0.617 ± 0.014 8.31 ± 0.98 ───────────────────────────────────

Fv, Fv-P, and Fv-PP are Ig
K _on measured by BIAcore when G molecule was not added
The values of K _off and K _off are both D shown in Example (3-2).
1 from the value measured by TC and stopped flow equipment
It was about 0 times smaller. These apparent differences are probably due to different complex formation conditions. That is, in the case of the BIA core system, the HEL was chemically bound to the solid support, whereas in the case of DTC and stopped flow analysis the reactants were free in solution. Despite these differences, the value of K _A , which is considered to reflect the affinity as a whole,
They were almost identical to each other. The values of K _off are Fv-P and I
In all cases, lower antibody concentrations were lower, except in the case of gG combinations. This phenomenon is
It seems to be unique to analysis using the IA core system. The stability of the complex formed on the solid support appeared to depend on the physical conditions of the antigen bound to the solid support on the biosensor chip. That is, antibody accessibility was not achieved equally on the chip. The true _Koff should ideally be measured at zero concentration. Therefore, all measurements should be considered empirical predictions.

In the case of Fv, addition of IgG did not cause any change in K _on or K _off . In the case of Fv-P,
When IgG was added, the K _on value was halved and the K _off value was _⅓ . However, if the low concentration of Fv-P, the value of K _off was increased. This phenomenon is (Fv-P)
_{2 It is considered that the} IgG complex is unstable and, as a result, dissociates at low concentrations. Fv-PP
In the case of, the positive effect of the addition of IgG on the reduction of K _off became even clearer. That is, the K _off value was 3.5 times lower at all the antibody concentrations measured. (Fv-P) ₂ observed in BIAcore analysis
This difference in stability between the IgG complex and the (Fv-PP) ₃ (IgG) ₂ complex is consistent with the HPLC and DTC analysis results shown in Example (3-3). is there. Thus, (Fv-PP) ₃ (IgG) ₂
The complex is considered stable and multivalent with respect to antigen binding.

(3-5) Enzyme-Linked Immunosorbent Assay (ELISA) Using Fv-P, Fv-PP and IgG Molecule Complex Dissolved in 20 mM Tris-HCl buffer (pH 8.0) 10
50 μl of 0 μg / ml HEL was put into a 96-well plate (Sumicon Multiplate; Sumitomo Bakelite Co.), and the plate was kept at 40 ° C. overnight. After removing the supernatant, 100 μl of 1% BSA was added and kept at room temperature for 1 hour. The plate was washed 3 times with PBS / T and added to each well.
50 μl of primary antibody solution was added. This primary antibody solution contains an antibody containing or not containing human IgG (Fv, Fv-P, Fv-PP), or human IgG.
Only is serially diluted.

The mixing conditions for human IgG were the same as those for the analysis by HPLC shown in Example (3-3) except that PBS / T was used as the dilution buffer. The plate was incubated at room temperature for 4 hours. PBS / after removing the solution
Wash 3 times with T. Anti-D1.3 antibody as a secondary antibody
50 μl diluted with BS / T was added. After incubating at room temperature for 4 hours, the plate was washed 3 times with PBS / T.
As a tertiary antibody, 50 μl of an anti-rabbit IgG donkey-derived antibody conjugated with horseradish peroxidase diluted with PBS / T was added, and the mixture was incubated at room temperature for 4 hours. PBS /
After washing 4 times with T, 0.04% of o- phenylenediamine (OPDA), 0.02% of H ₂ O _2, citrate 48.5 mm, and Na ^- phosphate (pH 5.0) 50 μl of the following staining solution was added. Staining reaction 2 at room temperature in a dark room
It was allowed to proceed for 0 minutes and stopped by adding 50 μl of 2M sulfuric acid. The absorbance at 492 nm is measured by Titertek [Multiskan, MCC].
Measured by The result is shown in FIG. That is, FIG. 21 shows only human IgG (x), Fv (white circle), Fv-P (white square), Fv-PP (white triangle) and F.
Human IgG added to v (black circle), human IgG added to Fv-P (black square), human Ig added to Fv-PP
It is a figure showing the result of ELISA in which G was added (black triangle) as the primary antibody and detected by anti-D1.3 antibody,
The vertical axis represents OD ₄₉₂ , and the horizontal axis represents the primary antibody concentration (μM).

Similarly, the secondary antibody was changed to a horseradish peroxidase-conjugated anti-human IgG Fc fragment goat antibody (Kappel, hereinafter abbreviated as anti-human Fc antibody), and the secondary antibody was washed to develop color. in this case,
Whether or not human IgG is contained in the primary antibody bound to HEL will be examined. The result is shown in FIG. That is, FIG. 22 is a diagram showing the results of ELISA in which the same antibody as in FIG. 21 was detected with an anti-human Fc antibody as a primary antibody, and the vertical axis represents O.D. D. ₄₉₂ , the horizontal axis represents the primary antibody concentration (μM).

The results of these ELISAs are protein A
Interpretation of the data obtained was somewhat complicated, as it was possible that the portion derived from A.coccus bound to not only the first human IgG molecule, but also the second and third antibodies.
However, in any case, Fv-P and Fv-PP
It was clear that the sensitivity of detection was higher when the mixture of Fv-P and IgG and the mixture of Fv-PP and IgG were used than when used alone. Furthermore,
Equal O. D. Comparing the concentrations of the first antibody showing a value of ₄₉₂ , the sensitivity of detection when the mixture of Fv-PP and IgG was used was one digit or more higher than the sensitivity of detection when Fv was used alone. .

As shown in FIG. 22, anti-F was used as the second antibody.
In the ELISA using the c antibody, it was clearly observed that the sensitivity was increased when IgG was added. Neither Fv-P nor Fv-PP showed a signal when IgG was not added. As mentioned above, F
The mixture of v-P and Fv-PP with IgG is E
It is useful as a reactant used in LISA.

(3-6) Western blotting using Fv-P, Fv-PP and IgG molecule complex 0.5 μg, 0.25 μg, 0.125 μg, 0.0625 μg
After SDS-PAGE, the HEL of Example 1 was blotted on a PVDF membrane using a semi-dry blotter. This film
It was immersed in PBS / T containing 1% BSA at room temperature for 1 hour for blocking. 1 diluted with PBS / T as primary antibody
0 mM Fv, Fv-P or Fv-PP was used.
After incubating at room temperature for 4 hours, the cells were washed 3 times with PBS / T. Anti-D1.
Immersed in PBS / T containing 3 antibodies, kept at room temperature for 4 hours, and washed 3 times with PBS / T. The membrane was further dipped in PBS / T containing an anti-rabbit IgG antibody conjugated with horseradish peroxidase and kept at room temperature for 4 hours. After washing 5 times with PBS / T, ELC Western
It detected using the blotting system (Amersham).

The results are shown in FIG. That is, FIG.
Is anti-D using Fv, Fv-P and Fv-PP as primary antibodies.
HEL detected by 1.3 antibody [0.5 μg (1), 0.25
It is a figure showing the result of Western blotting of μg (2), 0.125 μg (3), 0.0625 μg (4)].

Similarly, Fv-P or Fv-
Western blot with PP and human IgG complex as primary antibody
Blotting was performed. That is, the complex used in the HPLC analysis shown in Example (3-3) was treated with 100% PBS / T.
0-fold diluted (Fv-P, 1 as Fv-PP concentration
0 nM) was used as the primary antibody. An anti-human Fc antibody conjugated with horseradish peroxidase was used as a secondary antibody. After washing with the secondary antibody, detection was performed using the ECL Western blotting system. The result is shown in FIG. That is, FIG. 24 shows HEL detected by anti-human Fc antibody using Fv-P or Fv-PP and human IgG complex as a primary antibody [0.5 μg (1), 0.25 μg (2),
It is a figure showing the result of Western blotting of 0.125 µg (3), 0.0625 µg (4)].

The results of these Western blottings are summarized. When anti-D1.3 antibody was used as the second antibody, Fv-P and Fv-PP showed stronger signals than Fv. When an anti-Fc antibody is used as the second antibody, F
As expected, the complex of v-P or Fv-PP with IgG showed a stronger signal.

[0089]

EFFECTS OF THE INVENTION By using the gene provided by the present invention, it becomes easy to select a gene encoding an antibody molecule having high affinity for a target antigen, and a large number of monoclonal type antibodies containing the selected high affinity antibody can be selected. Also provided is a method for producing a titrated artificial antibody. By using a gene group that encodes an antibody molecule showing affinity for a target antigen, it is easy to produce a polyclonal polyvalent artificial antibody, and these polyvalent artificial antibodies are used for antigen purification, antigen measurement, diagnosis, etc. It is useful in the field.

[0090]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 630 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence: CCATTCGTTT GTGAATATCA AGGCCAATCG TCTGACCTGCCTCAACCTCC TGTCAATGCT 60 GGCGGGGGCT CTGGTGGTGG TTCTGGTGGC GGCTCTGAGGGTGGTGGCTC TGTCGCTGGAGGGTGGTCGCTG GTGGCTCTGG TTCCGGTGAT 180 TTTGATTATG AAAAGATGGC AAACGCTAAT AAGGGGGCTA TGACCGAAAA TGCCGATGAA 240 AACGCGCTAC AGTCTGACGC TAAAGGCAAA CTTGATTCTG TCGCTACTGA TTACGGTGCT 300 GCTATCGATG GTTTCATTGG TGACGTTTCC GGCCTTGCTA ATGGTAATGG TGCTACTGGT 360 GATTTTGCTG GCTCTAATTC CCAAATGGCT CAAGTCGGTG ACGGTGATAA TTCACCTTTA 420 ATGAATAATT TCCGTCAATA TTTACCTTCC CTCCCTCAAT CGGTTGAATG TCGCCCTTTT 480 GTCTTTAGCG CTGGTAAACC ATATGAATTT TCTATTGATT GTGACAAAAT AAACTTATTC 540 CGTGGTGTCT TTGCGTTTCT TTTATATGTT GCCACCTTTA TGTATGTATT TTCTACGTTT 600 GCTAACATAC TGCGTAATAA GGAGTCTTAA 630

SEQ ID NO: 2 Sequence Length: 174 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence: GTAGACAACA AATTCAACAA AGAACAACAA AACGCGTTCT ATGAGATCTT ACATTTACCT 60 AACTTAAACG AAGAACAACG AAACGCTCC ATCCAAAGTT TAAAAGATGA CCCAGCCCTCAG CTCAGGCGCC GAAA 174

SEQ ID NO: 3 Sequence Length: 348 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence: GTAGACAACA AATTCAACAA AGAACAACAA AACGCGTTCT ATGAGATCTT ACATTTACCT 60 AACTTAAACG AAGAACAACG AAACGCCTTC ATCCAAAGTT TAAAAGCTTT CCAAAGATGA CCCAAG CTCAGGCGCC GAAAGTAGAC 180 AACAAATTCA ACAAAGAACA ACAAAACGCG TTCTATGAGA TCTTACATTT ACCTAACTTA 240 AACGAAGAAC AACGAAACGC CTTCATCCAA AGTTTAAAAG ATGACCCAAG CCAAAGCGCT 300 AACCTTTTAG CAGAAGCTAA AAAGCTAAAT GATGCTCAGG

SEQ ID NO: 4 Sequence length: 58 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence: Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu 1 5 10 15 Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 35 40 45 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55

SEQ ID NO: 5 Sequence Length: 1952 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence: AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATAA TGAAATACCT ATTGCCTACG 60 GCAGCCGCTG GATTGTTATT ACTCGCTGCC CAACCAGCGAGTCAGCAGCT GCTGCTGCAGCT GC CCATCACATG CACCGTCTCA 180 GGGTTCTCAT TAACCGGCTA TGGTGTAAAC TGGGTTCGCC AGCCTCCAGG AAAGGGTCTG 240 GAGTGGCTGG GAATGATTTG GGGTGATGGA AACACAGACT ATAATTCAGC TCTCAAATCC 300 AGACTGAGCA TCAGCAAGGA CAACTCCAAG AGCCAAGTTT TCTTAAAAAT GAACAGTCTG 360 CACACTGATG ACACAGCCAG GTACTACTGT GCCAGAGAGA GAGATTATAG GCTTGACTAC 420 TGGGGCCAAG GCACCACGGT CACCGTCTCC TCATAATAAG AGCTATCCCG GGAGCTTGCA 480 TGCAAATTCT ATTTCAAGGA GACAGTCATA ATGAAATACC TATTGCCTAC GGCAGCCGCT 540 GGATTGTTAT TACTCGCTGC CCAACCAGCG ATGGCCGACA TCGAGCTCAC CCAGTCTCCA 600 GCCTCCCTTT CTGCGTCTGT GGGAGAAACT GTCACCATCA CATGTCGAGC AAGTGGGAAT 660 ATTCACAATT ATTTAGCATG GTATCAGCAG AAACAGGGAA AATCTCCTCA GCTCCTGGTC 720 TATTATACAA CAACCTTAGC AGATGGTGTG CCATCAAGGT TCAGTGGCAG TGGATCAGGA 780 ACACAATATT CTCTCAAGAT CAACAGCCTG CAACCTGAAG ATTTTGGGAG TTATTACTGT 840 CAACATTTTT GGAGTACTCC TCGGACGTTC GGTGGAGGCA CCAAGCTGGA GTCGACTCCA 900 TTCGTTTGTG AATATCAAGG CCAATCGTCT GACCTGCCTC AACCTCCTGT CAATGCTGGC 960 GGCGGCTCTG GTGGTGGTTC TGGTGGCGGC TCTGAGGGTG GTGGCTCTGA GGGTGGCGGT 1020 TCTGAGGGTG GCGGCTCTGA GGGAGGCGGT TCCGGTGGTG GCTCTGGTTC CGGTGATTTT 1080 GATTATGAAA AGATGGCAAA CGCTAATAAG GGGGCTATGA CCGAAAATGC CGATGAAAAC 1140 GCGCTACAGT CTGACGCTAA AGGCAAACTT GATTCTGTCG CTACTGATTA CGGTGCTGCT 1200 ATCGATGGTT TCATTGGTGA CGTTTCCGGC CTTGCTAATG GTAATGGTGC TACTGGTGAT 1260 TTTGCTGGCT CTAATTCCCA AATGGCTCAA GTCGGTGACG GTGATAATTC ACCTTTAATG 1320 AATAATTTCC GTCAATATTT ACCTTCCCTC CCTCAATCGG TTGAATGTCG CCCTTTTGTC 1380 TTTAGCGCTG GTAAACCATA TGAATTTTCT ATTGATTGTG ACAAAATAAA CTTATTCCGT 1440 GGTGTCTTTG CGTTTCTTTT ATATGTTGCC ACCTTTATGT ATGTATTTTC TACGTTTGCT 1500 AACATACTGC GTAATAAGGA GTCTTAATCA TGCCAGTTCT TTTGGGTGCT AGCTGTCGAC 1560 TGCGCAACAC GATGAAGCCG TAGACAACA A ATTCAACAAA GAACAACAAA ACGCGTTCTA 1620 TGAGATCTTA CATTTACCTA ACTTAAACGA AGAACAACGA AACGCCTTCA TCCAAAGTTT 1680 AAAAGATGAC CCAAGCCAAA GCGCTAACCT TTTAGCAGAA GCTAAAAAGC TAAATGATGC 1740 TCAGGCGCCG AAAGTAGACA ACAAATTCAA CAAAGAACAA CAAAACGCGT TCTATGAGAT 1800 CTTACATTTA CCTAACTTAA ACGAAGAACA ACGAAACGCC TTCATCCAAA GTTTAAAAGA 1860 TGACCCAAGC CAAAGCGCTA ACCTTTTAGC AGAAGCTAAA AAGCTAAATG ATGCTCAGGC 1920 GCCGAAAGTA GACGCGAATT AGCTGGGAAT TC 1952

SEQ ID NO: 6 Sequence length: 923 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear sequence: AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATAA TGAAATACCT ATTGCCTACG 60 GCAGCCGCTG GATTGTTATT ACTCGCTGCC CAACCAGCGA TGGCCCCTGTAGAGGCAGCT CCATCACATG CACCGTCTCA 180 GGGTTCTCAT TAACCGGCTA TGGTGTAAAC TGGGTTCGCC AGCCTCCAGG AAAGGGTCTG 240 GAGTGGCTGG GAATGATTTG GGGTGATGGA AACACAGACT ATAATTCAGC TCTCAAATCC 300 AGACTGAGCA TCAGCAAGGA CAACTCCAAG AGCCAAGTTT TCTTAAAAAT GAACAGTCTG 360 CACACTGATG ACACAGCCAG GTACTACTGT GCCAGAGAGA GAGATTATAG GCTTGACTAC 420 TGGGGCCAAG GCACCACGGT CACCGTCTCC TCATAATAAG AGCTATCCCG GGAGCTTGCA 480 TGCAAATTCT ATTTCAAGGA GACAGTCATA ATGAAATACC TATTGCCTAC GGCAGCCGCT 540 GGATTGTTAT TACTCGCTGC CCAACCAGCG ATGGCCGACA TCGAGCTCAC CCAGTCTCCA 600 GCCTCCCTTT CTGCGTCTGT GGGAGAAACT GTCACCATCA CATGTCGAGC AAGTGGGAAT 660 ATTCACAATT ATTTAGCATG GTATCAGCAG AAACAGGGAA AATCTCCTCA GCTCCTGGTC 720 TATTATACAA CAACCTTAGC AGATGGTGTG CCATCAAGGT TCAGTGGCAG TGGATCAGGA 780 ACACAATATT CTCTCAAGAT CAACAGCCTG CAACCTGAAG ATTTTGGGAG TTATTACTGT 840 CAACATTTTT GGAGTACTCC TCGGACGTTC GGTGGAGGCA CCAAGCTGGA AATCAAACGG 900 TAATAAGGAT CC923C

SEQ ID NO: 7 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: GCGAATTCTA AGTCGACTCC AGCTTGGTGC CTCC 34

SEQ ID NO: 8 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: CGACGTTGTA AAACGACGGC CAGTG 25

SEQ ID NO: 9 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: GCGTCGACTG CGCAACACGA TGAAGCC 27

SEQ ID NO: 10 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: ACGAATTCCC AGCTAATTCG CGTCTAC 27

SEQ ID NO: 11 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: GAGTCGACTC CATTCGTTTG TGAAT 25

SEQ ID NO: 12 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: CTGTCGACAG CTAGCACCCA AAAGAACTGG 30

SEQ ID NO: 13 Sequence Length: 31 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence: GAGTCGACGT CCATTCGTTT GTGAATATCA A 31

[Brief description of drawings]

FIG. 1 is a schematic diagram of the PM13Fv Hind III-EcoR I insert fragment and its vicinity.

FIG. 2. Filamentous phages M13Fv, M13ΔFv, M1
The figure which compared the polypeptide derived from the Fv fragment which remains in the phage coat protein when 3K07 is treated with sarcosyl.

FIG. 3 shows comparative quantification of Fv fragment-derived polypeptides expressed on the surface of filamentous phage M13Fv.

FIG. 4: HE without phage M13K07 sarcosyl treatment
The figure which showed the surface plasmon resonance sensor analysis result when throwing in the L fixed sensor chip.

FIG. 5: Phage M13ΔFv H without sarcosyl treatment
The figure which showed the surface plasmon resonance sensor analysis result when throwing in the EL fixed sensor chip.

[FIG. 6] HE of phage M13Fv without sarcosyl treatment
The figure which showed the surface plasmon resonance sensor analysis result when throwing in the L fixed sensor chip.

FIG. 7 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13K07 was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 1 hour.

FIG. 8 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13ΔFv was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 1 hour.

FIG. 9 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13Fv was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 1 hour.

FIG. 10 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13K07 was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 18 hours.

FIG. 11 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13ΔFv was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 18 hours.

FIG. 12 is a diagram showing the results of surface plasmon resonance sensor analysis when phage M13Fv was applied to a HEL-immobilized sensor chip by treatment with sarcosyl for 18 hours.

FIG. 13 is a diagram showing the results of surface plasmon resonance sensor analysis when an anti-M13 phage antibody was further added after the phage was added to the HEL-immobilized sensor chip.

FIG. 14 is a view showing the results of surface plasmon resonance sensor analysis when phages were put into an anti-D1.3 antibody-immobilized sensor chip.

FIG. 15: Fv, F purified by affinity resin
The figure which showed the analysis result of vP and Fv-PP by SDS-PAGE.

FIG. 16: DT on binding of Fv-P to human IgG
The figure which showed the C analysis result.

FIG. 17: D regarding binding between Fv-PP and human IgG
The figure which showed the TC analysis result.

FIG. 18: Fv, Fv-P, Fv-PP and their I
The figure which showed the analysis result by HPLC of a gG mixture.

FIG. 19 is a view showing the analysis results by HPLC under denaturing conditions of Fv-P, Fv-PP and IgG complexes.

FIG. 20 is a diagram showing the results of surface plasmon resonance sensor analysis when Fv, Fv-P, Fv-PP, and human IgG added thereto were put into a HEL-immobilized sensor chip.

FIG. 21 is a diagram showing the results of ELISA in which Fv, Fv-P, Fv-PP, and Fv-PP and human IgG added thereto were used as primary antibodies and detected with an anti-D1.3 antibody.

FIG. 22 is a diagram showing the results of ELISA in which Fv, Fv-P, Fv-PP, and Fv-PP and their addition to human IgG were detected by an anti-human Fc antibody as a primary antibody.

FIG. 23 is a diagram showing the results of Western blotting in which Fv, Fv-P, and Fv-PP were used as primary antibodies to detect with anti-D1.3 antibody.

FIG. 24 shows the results of Western blotting in which Fv-P or Fv-PP and human IgG complex were detected as primary antibodies by anti-human Fc antibody.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (3)

[Claims] 1. A gene represented by the following general formula (Formula 1). X- (Y) _m- (Z) _n (where X is a gene containing an integration site of a gene encoding an antibody variable region, Y is a gene encoding a polypeptide capable of expressing a phage surface) , A gene containing a gene encoding a polypeptide having Fc binding ability, m and n are 1 or 0, and m and n
At least one of is 1) 2. In the general formula (Formula 1), Y is a gene containing a gene encoding cpIII or a part thereof, and Z is a gene containing a gene encoding the Fc binding domain of protein A. The gene according to 1. 3. In the general formula (Formula 1), Y is a gene containing the gene represented by SEQ ID NO: 1 in the sequence listing, and Z
The gene according to claim 1, which is a gene containing the gene represented by SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing.