JP4304274B2 - Chicken-human chimeric antibody expression vector, method for producing chicken-human chimeric antibody using the same, and use thereof - Google Patents

Description

  The present invention relates to a vector for expression of a chicken-human chimeric antibody, a method for producing a chicken-human chimeric antibody using the same, and the use thereof, and in particular, easily produces a chicken-human chimeric antibody that is highly practical in mammalian cells. Chicken-human chimeric antibody expression vector that can be produced, a production method for efficiently producing a highly practical chicken-human chimeric antibody using the same, and a highly practical chicken obtained by the expression vector or production method -It relates to human chimeric antibodies and the like.

  A monoclonal antibody is a single antibody that recognizes only a specific part of an antigen, and has the advantages of uniform antigen specificity and high antibody titer compared to a polyclonal antibody (antiserum). Therefore, monoclonal antibodies are important as research reagents that are widely used in bioscience research in general, but in recent years, they have been put into practical use, such as diagnostics and therapeutics for various human diseases, from the research and development level. The technology has advanced to the level.

  Monoclonal antibodies are generally produced using mice. A mouse-human chimeric antibody was reported in 1984 as the first technique for utilizing monoclonal antibodies in human clinical practice (see Non-Patent Documents 1 and 2). Since then, various mouse-human chimeric antibody expression vectors using gene recombination techniques have been reported as techniques for easily expressing chimeric antibodies due to technological advances (see Non-Patent Documents 3 to 6).

  Currently, mouse-human chimeric antibodies are put into practical use as therapeutic agents. Specifically, for example, the antibody “Rituximab” used for the treatment of B-cell lymphoma (see Non-patent Documents 7 and 8; the antibody “Infliximab” used for the treatment of refractory chronic rheumatism and Crohn's disease (Non-Patent Document 9). -Refer to 10).

  By the way, when the target antigen is an antigen having high homology among mammals, for example, prion protein, it is very difficult to produce a mouse-human chimeric antibody against the antigen because of immune tolerance. On the other hand, birds have fine immune functions but are phylogenetically far from mammals. Therefore, if birds are selected as immunized animals, it is possible to produce monoclonal antibodies even for antigens that are difficult to produce antibodies in mammals due to immune tolerance.

  When birds are used as immunized animals, specific species include chickens (Gallus gallus). Chickens have been bred for a long time as edible livestock. In particular, chicken eggs can be used as raw materials having high productivity from the viewpoint of producing a material called protein rather than edible. Therefore, chickens can be suitably used as immunized animals because they have reliability as immunized animals and also have antibody production efficiency.

  As a technique for producing a monoclonal antibody using a chicken as an immunized animal, a method using a chicken hybridoma has been reported by the present inventors group (see Non-Patent Document 11). Various techniques using gene recombination have also been proposed. As a production technique of a monoclonal antibody using gene recombination, a method using phage display in which a recombinant antibody is expressed on the surface of a phage has been reported (see Non-Patent Documents 12 and 13). The present inventors have proposed a method using an expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced (see Patent Document 1).

In addition, as a method for expressing a chicken-human chimeric antibody, there is a technique applying the above phage display. For example, chicken V _L gene and human Cκ gene, and a chicken VH gene and human C _H gene was ligated with PCR, Fab-type chicken incorporated into a vector for phage display PCob3 - Methods for expressing human chimeric antibody It is known (see Non-Patent Document 14).
JP 2001-228676 (published September 4, 2001) Boulianne et al., 1984. Nature 312, 643-646 Morrison et al., 1984. Proc Natl Acad Sci USA 81, 6851-6855 Walls et al., 1993. Nucleic Acid Research 21, 2921-2929 Norderhung et al., 1997. J Immunol Methods 204, 77-78 Persic et al., 1997. Gene 187, 9-18 McLean et al., 2000. Mol Immunol 37, 837-845 McLaughlin et al., 1998. J Clin Incol 16, 2825-2833 Tobinai et al., 1998. Ann Oncol 9, 527-534 Elliott et al., 1993. Arthritis Rheum 36, 1681-1690 Targan et al., 1997. N Engl J Med 337, 1029-1035 Nishinaka et al., 1989. Arch. Allergy Appl. Immunol., 89, 416-419 Davis et al., 1995. J. Immunol. Methods, 186, 125-135 Yamanaka et al., 1996. J. Immunol., 157, 1156-1162 Andris-Widhopf et al., 2000. J Immunol Methods 242, 159-181

  However, the production technique of chicken monoclonal antibodies using phage display remains inadequate to effectively produce chicken-human chimeric antibodies with high practicality.

  Specifically, in the method for expressing a Fab-type chicken-human chimeric antibody, a monoclonal antibody fragment can be obtained, but a complete antibody molecule cannot be expressed. A complete antibody molecule has an effector function effective for treatment and has an advantage of a long half-life in vivo. Therefore, in order to efficiently produce a highly practical chicken-human chimeric antibody, establishment of a technique capable of easily producing the antibody molecule itself has been required.

  The present invention has been made in view of the above problems, and an object thereof is to provide a technique for easily producing a highly practical chicken-human chimeric antibody, not an antibody fragment, and a typical method for using the same. There is to do.

  As a result of intensive studies in view of the above problems, the present inventor has completely replaced the constant region, which is the most immunogenic region of the chicken monoclonal antibody, from the chicken type to the human type and expressed in mammalian cells. It has been found that chicken-human chimeric antibody molecules can be easily produced in mammalian cells, and the present invention has been completed.

  That is, the chicken-human chimeric antibody expression vector according to the present invention is a vector capable of expressing a protein using a mammalian cell as a host, and for incorporating at least a promoter and a chicken variable region gene as a DNA segment. It contains an integration site, a chicken immunoglobulin leader sequence, and a human immunoglobulin constant region gene.

  In the chicken-human chimeric antibody expression vector, the human immunoglobulin constant region gene is a gene encoding a heavy chain or light chain constant region. At this time, as the human immunoglobulin heavy chain constant region gene, for example, at least one of the polynucleotides having the base sequence shown in SEQ ID NO: 1 or 2 is used, and as the human immunoglobulin light chain constant region gene, for example, the sequence A polynucleotide having the base sequence shown in No. 3 is used.

  In the above chicken-human chimeric antibody expression vector, for example, a polynucleotide having the base sequence shown in SEQ ID NO: 4 is used as the leader sequence of chicken immunoglobulin.

  In the chicken-human chimeric antibody expression vector, the integration site preferably contains a restriction enzyme site that is recognized by at least one of AscI, BamHI, and HindIII. This integration site may be a multicloning site in which a plurality of restriction enzyme sites are connected.

  In the chicken-human chimeric antibody expression vector, for example, a promoter derived from cytomegalovirus can be used as the promoter.

  The chicken-human chimeric antibody expression vector preferably further has a selection marker for determining whether or not the vector has been introduced into mammalian cells. Examples of the selection marker include a zeocin resistance gene. Or a neomycin resistance gene can be mentioned.

  The chicken-human chimeric antibody expression vector preferably further has a replication origin, and the replication origin is preferably derived from pUC and SV40.

  The chicken-human chimeric antibody expression vector preferably further includes a poly (A) addition signal, and examples of the poly (A) addition signal include those derived from bovine growth hormone.

  The method for producing a chicken-human chimeric antibody according to the present invention is characterized by using the above-mentioned chicken-human chimeric antibody expression vector. Specifically, first, as the above-described chicken-human chimeric antibody expression vector, a human immunoglobulin constant vector is used. Expression vector for heavy chain in which the region gene is a gene encoding the constant region of the heavy chain, and expression vector for light chain in which the human immunoglobulin constant region gene is the gene encoding the constant region of the light chain And a method including a chicken V region introduction step of introducing a chicken heavy chain variable region gene into the heavy chain expression vector and introducing a chicken light chain variable region gene into the light chain expression vector. be able to. Furthermore, the production method preferably includes a transfection step in which the expression vector for heavy chain and the expression vector for light chain into which the chicken variable region gene is introduced are co-expressed in mammalian cells.

  The mammalian cells used at this time are not particularly limited, but for example, CHO cells can be preferably used. Further, the method used in the transfection step is not particularly limited, but the lipofection method can be preferably used.

  Furthermore, the production method preferably includes an antibody recovery step of recovering a chicken-human chimeric antibody from mammalian cells co-expressed with the heavy chain expression vector and the light chain expression vector. It is preferable to include an antibody purification step for purifying the chicken-human chimeric antibody recovered from the cells.

  The present invention also includes a chicken-human chimeric antibody obtained by the above-described method for producing a chicken-human chimeric antibody and having a variable region derived from chicken and a constant region derived from human.

  As a typical utilization method of the present invention, for example, a chicken-human chimeric antibody production kit for carrying out the above-described method for producing a chicken-human chimeric antibody can be mentioned. This chicken-human chimeric antibody production kit comprises at least a heavy chain expression vector in which a human immunoglobulin constant region gene is a gene encoding a heavy chain constant region, and a human immunoglobulin constant region gene having a light chain. A light chain expression vector that is a gene encoding the constant region, and further, a reagent group for introducing the expression vector into mammalian cells, and a chicken variable region gene in the expression vector. It is preferable to include at least one of reagent groups for incorporation. Furthermore, it is more preferable to include at least one of a reagent group for recovering chicken-human chimeric antibodies from mammalian cells and a reagent group for purifying chicken-human chimeric antibodies recovered from mammalian cells.

  Moreover, as a typical application technique of the present invention, a drug using the chicken-human chimeric antibody obtained as described above can be mentioned.

  As described above, the present invention has a leader sequence of chicken immunoglobulin, an integration site for incorporating a human constant region gene and a chicken variable region gene, and the like, and can be expressed in mammalian cells. Furthermore, functional antibody molecules can be generated by co-expression in mammalian cells using light chain and heavy chain as expression vectors. In addition, these expression vectors can be transiently expressed or stably expressed even when introduced into mammalian cells.

  Since chickens are phylogenetically distant from mammals, antibodies can be easily produced against human antigens that have been difficult to produce antibodies in mice or the like due to immune tolerance. Therefore, in the present invention, a monoclonal antibody is prepared using a chicken, which is chimerized (chicken-human chimeric antibody) and expressed in mammalian cells. This makes it possible to easily produce a clinically useful chimeric antibody against a wider range of human antigens. As a result, according to the present invention, there is an effect that the possibility of developing a new antibody drug can be further increased.

  An embodiment of the present invention will be described below with reference to FIGS. 1 to 3, but the present invention is not limited to this. In the present embodiment, the chicken-human chimeric antibody expression vector according to the present invention, the chicken-human chimeric antibody production method according to the present invention, the chicken-human chimeric antibody according to the present invention, and the use of the present invention are used in this order. Will be described in detail.

(I) Vector for expression of chicken-human chimeric antibody according to the present invention A vector for expression of chicken-human chimeric antibody according to the present invention (hereinafter abbreviated as appropriate expression vector) is an expression vector using a mammalian cell as a host, The DNA segment contains at least a promoter, an integration site for incorporating a chicken variable region gene, a chicken immunoglobulin leader sequence, and a human immunoglobulin constant region gene. By introducing this into mammalian cells and expressing it, a chicken-human chimeric antibody (hereinafter abbreviated as a chimeric antibody as appropriate) can be expressed.

<Outline of chicken-human chimeric antibody expression vector>
The specific configuration of the expression vector according to the present invention is not particularly limited. For example, as will be described in the examples described later, the light chain expression vector pcSLCκ shown in FIG. Examples include the heavy chain expression vector pcSLCγ1 or pcLSCγ4 shown in 1 (b).

  The light chain expression vector pcSLCκ contains a cytomegalovirus (CMV) -derived promoter (pCMV in the figure) as a promoter, a HindIII site (H in the figure), an AscI site (A in the figure) as an integration site, and It has a BamHI site (B in the figure). A restriction enzyme site that is recognized and cleaved by a restriction enzyme is simply referred to as a “restriction enzyme name” site. A leader sequence (L in the figure) is located between the HindIII site and the AscI site. Furthermore, pcSLCκ includes a human light chain constant region gene (Cκ in the figure) as a human immunoglobulin constant region gene.

  Furthermore, pcSLCκ includes poly (A) addition signal derived from bovine growth hormone (BGApA in the figure), SV40-derived replication origin and promoter (SV40ori / p in the figure), neomycin resistance gene (Neo in the figure), ampicillin resistance. The gene (Amp in the figure) is included. The neomycin resistance gene and the ampicillin resistance gene are selection markers for determining whether a vector has been introduced into a host cell.

The light chain expression vector pcSLCκ has a size of 6.3 kb, and the sequence of each of the above DNA segments, except for the selectable marker, is a promoter, an integration site / leader sequence, a human immunoglobulin constant region gene, a poly ( A) The order of the additional signal and the replication origin (and promoter) are as follows. Then, a chicken light chain variable region gene (V _{L in the} figure) can be introduced.

The heavy chain expression vectors pcSLCγ1 and pcLSCγ4 have a size of 7.1 kb, but instead of the human light chain constant region gene, the human heavy chain constant region gene (Cγ1 / Cγ4 in the figure) is used. ) Is basically the same as pcSLCκ except that a zeocin resistance gene (Zeo in the figure) is introduced instead of the neomycin resistance gene. Then, a chicken heavy chain variable region gene (V _{H in the} figure) can be introduced.

  As is apparent from the above configuration, pcSLCκ is an expression vector for expressing the light chain of the chimeric antibody, and pcSLCγ1 and pcLSCγ4 are expression vectors for expressing the heavy chain of the chimeric antibody. All the basic configurations are the same. Therefore, the expression vector according to the present invention only needs to contain a promoter, an integration site, a leader sequence, and a human immunoglobulin constant region gene as described above, and the specific configuration thereof is particularly limited. It is not something. Hereinafter, each DNA segment will be specifically described.

<Human immunoglobulin constant region gene>
In the expression vector according to the present invention, the human immunoglobulin constant region gene may be a gene encoding a heavy chain constant region, or a gene encoding a light chain constant region, as described above. It may be. These constant region genes are not particularly limited, and any gene can be used as long as it is a constant region gene derived from a human antibody.

  For example, as the constant region gene used in the examples, the heavy chain constant region gene has a polynucleotide (Cγ1 gene) having the base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2. It is a polynucleotide (Cγ4 gene). The light chain constant region gene is a polynucleotide (Cκ gene) having the base sequence shown in SEQ ID NO: 3.

  Further, the method for obtaining a human antibody-derived constant region gene is not particularly limited, and a conventionally known method can be suitably used. For example, in the examples described later, genomic DNA is prepared from human peripheral blood lymphocytes isolated from human blood, and the target gene is amplified by PCR.

  As described later, in the present invention, a complete chimeric antibody molecule is produced by introducing and co-expressing both a heavy chain expression vector and a light chain expression vector into a host mammalian cell. be able to. Therefore, in the present invention, except for the use for obtaining only the light chain or only the heavy chain of the chimeric antibody, the heavy chain constant region gene and the light chain constant region gene were always obtained as a set, and these were respectively introduced. It is highly preferred to produce a heavy chain expression vector and a light chain expression vector.

<Chicken immunoglobulin leader sequence>
In the expression vector according to the present invention, the leader sequence of chicken immunoglobulin is not particularly limited, and any chicken antibody leader sequence can be used. For example, a polynucleotide having the base sequence shown in SEQ ID NO: 4 is used as the leader sequence used in the examples. Further, the method for obtaining the leader sequence is not particularly limited, and a conventionally known method can be suitably used.

<Integration site for incorporating chicken variable region genes>
In the expression vector according to the present invention, the integration site is not particularly limited. In the present invention, as shown in Examples described later, a specific restriction enzyme is examined by examining whether or not to cleave a chicken variable region gene for a restriction enzyme that does not cleave the vector that is the main skeleton of the expression vector. Determine the site. As a result, it was revealed that AscI, HindIII and BamHI do not cut the chicken variable region genes registered in the database. Therefore, the expression vector according to the present invention has these three restriction enzyme sites as integration sites. Is selected. Therefore, the integration site may contain a restriction enzyme site that is recognized by at least one restriction enzyme of AscI, BamHI, and HindIII.

  In other words, in the present invention, the restriction enzyme site does not cut the main skeleton of the expression vector and the introduced gene (leader sequence, constant region gene) and does not cut the chicken variable region gene. It is not limited to enzymes. In other words, an appropriate restriction enzyme site may be found by the technique described in the examples according to the type of vector (referred to as a base vector for convenience) employed as the main skeleton.

  Here, in the pcSLCκ, pcSLCγ1 and pcLSCγ4, a leader sequence is located between the HindIII site and the AscI site. However, the present invention is not limited to this, and is limited as an integration site. A multiple cloning site in which a plurality of enzyme sites are connected and no other gene or the like is interposed between them may be used. Of course, the embedded site may consist of one restriction enzyme site.

  The design of the integration site can be appropriately changed according to the type of expression vector used, the type of chicken variable region gene to be incorporated, the construction strategy of the expression vector, and the like. In addition, the integration site should just synthesize | combine the oligonucleotide containing a restriction enzyme site by a well-known method, and introduce | transduce this to a vector.

<Promoter>
Since the expression vector according to the present invention is intended to enable expression of a chimeric antibody at least in mammalian cells, a promoter is an essential component. However, the specific promoter is not particularly limited, and a known promoter can be appropriately used according to the type of mammalian cell serving as a host. Specifically, in the above-described pcSLCκ, pcSLCγ1, and pcLSCγ4, a cytomegalovirus-derived CMV promoter is used as the promoter.

<Selection marker>
It is very preferable that the expression vector according to the present invention further contains a selection marker for determining whether or not the expression vector has been introduced into mammalian cells. As this selection marker, an antibiotic resistance gene is usually used. Specifically, in the above-described pcSLCκ, pcSLCγ1 and pcLSCγ4, a zeocin resistance gene or a neomycin resistance gene is used as a selection marker. However, the present invention is not limited to these, depending on the type of mammalian cell serving as a host. A known selection marker can be used as appropriate.

  In general, a plasmid such as an expression vector according to the present invention is allowed to grow in E. coli for gene recombination and for the propagation of the expression vector. Therefore, it is highly preferable that the expression vector according to the present invention contains a selection marker for determining whether or not the vector has been introduced into E. coli. As this selection marker, an antibiotic resistance gene is usually used, and in the above-mentioned pcSLCκ, pcSLCγ1 and pcLSCγ4, an ampicillin resistance gene is used as a selection marker, but the present invention is not limited to this. .

<Replication start point>
It is very preferable that the expression vector according to the present invention has an origin of replication so that transient expression is possible in mammalian cells as a host. The specific type of the replication start point is not particularly limited, and a known one may be appropriately selected according to the type of mammalian cell serving as a host, the type of the main skeleton of the expression vector, and the like. Specifically, in the above-described pcSLCκ, pcSLCγ1 and pcLSCγ4, those starting from SV40 are used as the replication start point, but the present invention is of course not limited thereto.

  In addition, the expression vector according to the present invention preferably also has a replication origin for stable maintenance in Escherichia coli. The specific type of the replication origin for E. coli is not particularly limited, and a known type may be appropriately selected according to the type of E. coli serving as a host, the type of the main skeleton of the expression vector, and the like. Specifically, in the above-described pcSLCκ, pcSLCγ1 and pcLSCγ4, those derived from pUC are used as the replication start point, but the present invention is of course not limited thereto.

<Other DNA segments>
The expression vector according to the present invention may contain known DNA segments other than those described above as long as they contribute to the expression of the chimeric antibody in the host. Specific examples include poly (A) addition signals. The eukaryotic mRNA after transcription has a poly (A) added to the 3 ′ end, which improves the stability of the mRNA and, as a result, the expression of the protein (chimeric antibody in the present invention). Can be improved. Such poly (A) addition signal is not particularly limited, but in the above-mentioned pcSLCκ, pcSLCγ1 and pcLSCγ4, those derived from bovine growth hormone are used as the poly (A) addition signal.

<Methods for producing and producing expression vectors>
The method for producing the expression vector according to the present invention is not particularly limited, and the DNA segments described above may be introduced into a vector (base vector) serving as a main skeleton in a predetermined order. There are no particular limitations on the types of reagents used when introducing these DNA segments, ie, restriction enzymes and ligation enzymes, and commercially available ones may be appropriately selected and used.

  The strategy for producing the expression vector according to the present invention is not particularly limited as long as it is a strategy that allows the DNA segments to be efficiently arranged in a predetermined order. . For example, in the examples described later, as shown in FIG. 2, an expression vector is prepared in three stages by recombining a plasmid vector.

  First, regarding the production strategy of the heavy chain expression vector pcSLCγ1 or pcLSCγ4, as shown in FIG. 2, a human heavy chain constant region gene (Human Cγ1 / Cγ4 in the figure) was cloned from human genomic DNA and cloned into an arbitrary vector. Introduce a constant region gene vector. The vector used at this time is not particularly limited. In Examples described later, a pGEM-T Easy vector (Promega, pGEM in the figure) is used, and pGCγ1 / Cγ4 (size 4.8 kb) is obtained as a constant region gene vector.

  Next, a human heavy chain constant region gene is excised from the constant region gene vector using an appropriate restriction enzyme, and introduced into an arbitrary vector to prepare an intermediate vector. The vector used at this time is not particularly limited, but a vector that is substantially the main skeleton is used in this strategy. In Examples described later, pcDNA4 / myc-HisA (manufactured by Invitrogen) is used, and pcDCγ1 / Cγ4 (size 6.9 kb) is obtained as an intermediate vector.

  Separately, a chicken heavy chain leader sequence (Leader in the figure) is cloned from chicken genomic DNA, and an arbitrary vector is introduced to prepare a leader sequence vector. In Examples described later, pBluescript II SK (−) is used, and pBSL (size 3.2 kb) is obtained as a leader sequence vector.

  A leader sequence is excised from the above leader sequence vector using an appropriate restriction enzyme and introduced into an intermediate vector to obtain a heavy chain expression vector pcSLCγ1 / Cγ4 (size 7.1 kb).

  The production strategy of the light chain expression vector pcSLCκ is basically the same as that for the heavy chain expression vector. As shown in FIG. 2, a human light chain constant region gene is cloned from human genomic DNA and introduced into an arbitrary vector (in this case, pBluescript II SK (−)), and the constant region gene vector (pBCκ, size 3. 6 kb) is produced. From this, the human light chain constant region gene is excised and introduced into an arbitrary vector (in this case pcDNA3 / myc-HisA (manufactured by Invitrogen)) to produce an intermediate vector (pcDCκ, size 6.1 kb).

  Thereafter, the leader sequence is cut out from the leader sequence vector and introduced into an intermediate vector to obtain a light chain expression vector pcSLCκ (size 6.3 kb).

The production method of the expression vector thus constructed, that is, the method of propagating the expression vector produced by the above production method is not particularly limited, and a known method can be used. In general, Escherichia coli may be used as a host and grown in the E. coli cells. At this time, a preferred E. coli type may be selected according to the type of the base vector or the produced expression vector. Further, the method for recovering the expression vector grown from E. coli cells is not particularly limited, and a known method may be used.
(II) Method for Producing Chicken-Human Chimeric Antibody According to the Present Invention The method for producing a chimeric antibody according to the present invention (hereinafter abbreviated as “production method”) is a method using the expression vector described above. Although not particularly limited, preferably, expression vectors for heavy chains and expression vectors for light chains are used, and the variable region regions of chicken antibodies are introduced into these, respectively, and expressed in mammalian cells as hosts. Can be mentioned.

<Mammalian cells as hosts>
The mammalian cells used as a host in the present invention are not particularly limited, and known cultured cells (cell lines) can be suitably used. Specific examples include COS cell line, CHO cell line, SP2 / 0 cell line, NSO cell line and the like. In the examples described later, COS cells and CHO cells are used. In particular, since CHO cells have been put into practical use for the production of erythropoietin, blood coagulation factor VIII and the like by gene recombination technology, they can be used more preferably in the present invention.

  The conditions for culturing the mammalian cells are not particularly limited, and a known medium (culture solution), culture temperature, or the like corresponding to the type of cell line can be appropriately selected and used.

  In addition, the cell which can be used as a host by this invention is not limited to a well-known cell line, The cell acquired from arbitrary mammals can also be cultured and used. In other words, the established culture cell line may be sufficient as the host used by this invention, and the culture cell produced newly may be sufficient as it.

<Chicken V range introduction process>
Since the expression vector used in the production method according to the present invention has an integration site, any chicken variable region (V region) can be introduced according to the target antibody. Therefore, the production method according to the present invention preferably includes a chicken V region introduction step for introducing a chicken variable region gene into the expression vector.

  The specific method for introducing the chicken variable region gene into the expression vector according to the present invention is not particularly limited, and a chicken variable region gene according to the purpose is obtained by a known method or the like, and this is expressed as an expression vector. Should be introduced. At this time, the chicken heavy chain variable region gene is introduced into the heavy chain expression vector, and the chicken light chain variable region gene is introduced into the light chain expression vector. In examples described later, a prion protein is selected as an antigen, and a chicken variable region gene is obtained from a hybridoma or phage display in order to produce a chimeric antibody against the prion protein.

<Transfection process>
In the production method according to the present invention, after the above-described chicken V region introduction step, a transfection step of co-expressing a heavy chain expression vector and a light chain expression vector into which a chicken variable region gene has been introduced in mammalian cells is performed. .

  The method of introducing the expression vector used at this time, that is, the transfection method (transfection method) is not particularly limited, and includes lipofection method, electroporation method, calcium phosphate method, liposome method, DEAE dextran method, microinjection method and the like. Conventionally known methods can be suitably used. In Examples described later, a lipofection method using a Polyfect transfection reagent (Qiagen) method is used.

  In the transfection step, it is preferable to determine whether or not the expression vector has been introduced into the mammalian cell based on the selection marker contained in the expression vector. As described in the description of the expression vector, an antibiotic resistance gene is often used as a selectable marker, so if an antibiotic is added to the mammalian cell culture medium, the grown mammalian cells can be transfected. Can be confirmed.

<Antibody recovery process / antibody purification process>
In the production method according to the present invention, after the transfection step, an antibody recovery step of recovering the chimeric antibody from mammalian cells co-expressed with the heavy chain expression vector and the light chain expression vector is performed. The method for recovering the chimeric antibody performed in the antibody recovery step is not particularly limited, and a known method can be used. If the chimeric antibody is secreted into the culture supernatant as in the examples described later, the culture supernatant may be recovered. However, purification is performed to remove the culture medium components to obtain a high-purity chimeric antibody molecule. May be. Therefore, the production method according to the present invention may include an antibody purification step for purifying the chimeric antibody recovered from the mammalian cells. The purification method of the chimeric antibody performed in the antibody purification step is not particularly limited, and a known method can be used.

In the production method according to the present invention, all of the above steps are not essential, and in the production method according to the present invention, steps other than the above steps may be included.
(III) Chimeric antibody according to the present invention By using the expression vector according to the present invention described above or the production method according to the present invention, a chicken-human chimeric antibody can be easily produced. Specifically, the structure of the chimeric antibody produced in the present invention will be described. As shown in FIG. 3 (a), first, the light chain (L chain) 10 has a light chain variable region derived from a chicken on the N-terminal side ( (Chicken V _L ) 11 and a human-derived light chain constant region (human C _L ) 12 connected to the C-terminal side.

As shown in FIG. 3B, the heavy chain (H chain) 20 is composed of a chicken-derived heavy chain variable region (chicken V _H ) 21 on the N-terminal side and a human-derived heavy chain constant region connected thereto. The structure has (human C _H ) 22 a and human-derived heavy chain constant regions (human C _H ) 22 b and 22 c (C-terminal side) connected via the hinge part 23.

When the light chain 10 and the heavy chain 20 are bonded by an S—S bond, and the heavy chains 20 are further bonded by an S—S bond, as shown in FIG. Fc (crystallizable fragment) 31 is formed from human C _H 22b and 22c, and is separated from Fc via hinge part 23 by light chain 10 and heavy chain 20 chicken V _H 21 and human C _H 22a. An expanding Fab (antigen binding fragment) 32 is formed, and a chimeric antibody molecule 30 is formed. As described above, the chimeric antibody obtained in the present invention has a constitution in which the variable region (V region) is derived from chicken and the constant region (C region) is derived from human, so that it is difficult to produce an antibody for immune tolerance. Antibodies can also be generated effectively against antigens.
(IV) Use of the present invention <Chimeric antibody production kit>
A typical example of the use of the present invention is a chicken-human chimeric antibody production kit (for convenience, abbreviated as a production kit) for carrying out the above production method.

Although the specific structure of a production kit is not specifically limited, For example, the structure containing each following expression vector and reagent group can be mentioned.
(A) Expression vector for heavy chain in which the human immunoglobulin constant region gene is a gene encoding the constant region of the heavy chain (b) Human immunoglobulin constant region gene is a gene encoding the constant region of the light chain Light chain expression vector (c) Reagent group for introducing expression vector into mammalian cells (Reagent group for transfection step)
(D) Reagent group for incorporating the chicken variable region gene into the expression vector (chicken V region introduction step reagent group)
(E) Reagent group for recovering chicken-human chimeric antibody from mammalian cells (reagent group for antibody recovery step)
(F) Reagent group for purifying chicken-human chimeric antibody recovered from mammalian cells (Reagent group for antibody purification step)
The “reagent group” mentioned here may include materials such as instruments and vectors used for a series of operations in addition to general reagents such as enzymes and salts. In addition, the production kit according to the present invention may include a reagent group other than the above (a) to (f).

  Of these reagent groups, (a) and (b) are indispensable components, but other reagent groups may be included as necessary. Preferably, (c) and (d) are included in addition to (a) and (b), and more preferably (e) and (f) are further included.

  Since the reagent groups (a) and (b) are the expression vectors described in the above section (I), their specific description is omitted. The reagent group (c) is a reagent group for transfection, and may contain drugs, buffers, and the like according to the transfection method employed.

  Examples of the reagent group (d) include general reagents for gene recombination. Specific examples include restriction enzymes, ligation enzymes, buffers, oligonucleotides serving as linkers, and the like according to the integration site of the expression vector. Further, the reagent group of (d) may include a reagent group for obtaining a chicken variable region gene. For example, a primer for amplifying a variable region gene, a reagent for PCR, and the like can be mentioned.

  The reagent groups (e) and (f) only need to contain drugs and instruments according to the method for recovering and purifying the chimeric antibody employed. In view of the contents of the antibody recovery step and the antibody purification step, the recovery method and the purification method may overlap in terms of technique (for example, when recovering a chimeric antibody from a culture supernatant using a column or the like, Therefore, the reagent groups (e) and (f) may be combined.

<Application of the present invention>
According to the present invention, chicken-human chimeric antibodies can be easily produced in mammalian cells. Moreover, the resulting chimeric antibody is a complete antibody molecule. A complete antibody molecule has an effector function effective for treatment and has an advantage of a long half-life in vivo. Therefore, the chimeric antibody according to the present invention is expected to be applied in the clinical field.

  More specifically, examples of applicable technologies and products include antibody drugs and diagnostic drugs. Antibodies have the ability to specifically bind to a particular antigen. Therefore, for example, when used as an antibody drug, if an antibody against an antigen generated in a specific cell (cancer cell or the like) in an affected area is prepared, only the specific cell can be killed, and thus a high therapeutic effect can be exhibited. At the same time, side effects can be reduced. In addition, when used as a diagnostic agent, if a protein specific to a specific disease is used as an antigen and an antibody against it is prepared, it can be confirmed whether or not the antigen is present in the sample by an antigen-antibody reaction. .

  Since the present invention can produce antibodies against antigens that have been difficult to produce antibodies by immunological tolerance and has high homology between mammals, it is widely used as the above-mentioned antibody drugs and diagnostic agents. Application is expected.

  Hereinafter, the present invention will be described more specifically based on Examples and FIGS. 1, 2, 4 to 6, but the present invention is not limited thereto.

[Example 1: Construction of expression vector]
(1-1) Cloning of human heavy chain constant region gene Genomic DNA was prepared from human peripheral blood mononuclear cells isolated from heparinized blood of healthy volunteers. The human heavy chain constant region (CH region) gene uses LA Taq DNA polymerase (manufactured by TaKaRa) in GC rich buffer using primer IgGF (SEQ ID NO: 5) containing EcoRI site and IgGR (SEQ ID NO: 6). And was amplified from genomic DNA by PCR. These primers hybridized to the 5 ′ intron of the CH1 exon and the 3 ′ end of the CH3 exon, respectively.

  The purified PCR product was connected to the pGEM-T Easy vector (Promega) and confirmed by sequencing. Sequencing was performed using BigDye Terminator Cycle Sequencing Kit and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). A construct containing the Cγ1 or Cγ4 gene and having no sense mutation was selected and designated as pGCγ1 or pGCγ4, respectively (see FIG. 2).

(1-2) Cloning of human light chain constant region gene The human light chain constant region (Cκ region) gene is expressed as KOD plus DNA using primers IgKF (SEQ ID NO: 7) and IgKR (SEQ ID NO: 8) containing an EcoRI site. Amplification was performed from genomic DNA by PCR using polymerase (TOYOBO). These primers hybridized to the 5 ′ intron and 3 ′ end of the Cκ exon, respectively. The purified PCR product was digested with EcoRI and ligated to the EcoRI / HincII site of pBluescript II SK (−). The correct size insert construct was confirmed by sequencing and designated pBCκ (see FIG. 2).

(1-3) Cloning of chicken heavy chain leader sequence Chicken genomic DNA was prepared from chicken hybridoma HUC2-13 (see Matsuda et al., 1999. FEMS Immunol. Med. Microbiol. 23, 189-194). A chicken heavy chain leader sequence was prepared by using a primer VH3F (SEQ ID NO: 9) containing a HindIII site and a Kozak sequence, and VH-LeR (SEQ ID NO: 10) containing a KpnI and AscI site. ) Was used to amplify from genomic DNA by PCR.

  These primers hybridized to the 5 'end of the heavy chain leader exon and the leader sequence of the leader V intron and 2nd exon, respectively. The purified PCR product was digested with HindIII and KpnI and ligated to the HindIII / KpnI site of pBluescript II SK (−). The correct size insert construct was confirmed by sequencing and designated pBSL (see FIG. 2).

(1-4) Selection of chicken variable region gene integration site Restriction that does not cut any of the expression vector, pcSLCκ, pcSLCγ1, and pcSLCγ4 to determine the restriction enzyme site for incorporating the chicken variable region gene into the expression vector The enzyme was examined to cleave the chicken variable region gene. As a result, it was revealed that AscI, HindIII and BamHI do not cleave the chicken variable region genes registered in the database. Therefore, these three restriction enzyme sites were selected as integration sites.

(1-5) Construction of expression vector 1: pscSLCγ1
The pGCγ1 was digested with EcoRI to excise the Cγ1 gene, and inserted into the EcoRI site of pcDNA4 / myc-HisA. The direction of insertion was confirmed by sequencing, and the correct orientation was obtained as pcDCγ1. The pBSL was digested with HindIII and KpnI to excise the leader sequence and introduced into the HindIII / KpnI site of the pcDCγ1. As a result, pcSLCγ1 as an expression vector was obtained (see FIG. 2).

  The obtained pcSLCγ1 has a size of 7.1 kb, a chicken immunoglobulin leader sequence, a human constant region gene (Cγ1), and a chicken variable region gene integration site, as shown in FIG. Furthermore, it has a zeocin resistance gene (for mammalian cells) and an ampicillin resistance gene (for E. coli) as selection markers. It also has a CMV promoter, a bovine growth hormone poly (A) addition signal, and an SV40 replication origin.

(1-6) Construction of expression vector 2: pscSCLγ4
The pGCγ4 was digested with NotI to excise the Cγ4 gene, and inserted into the NotI site of pcDNA4 / myc-HisA (Invitrogen). The direction of insertion was confirmed by sequencing, and the correct orientation was obtained as pcDCγ4. The pBSL was digested with HindIII and KpnI to excise the leader sequence, and introduced into the HindIII / KpnI site of pcDCγ4. As a result, pcSLCγ4 as an expression vector was obtained (see FIG. 2).

  The obtained pcSLCγ4 has a size of 7.1 kb, a chicken immunoglobulin leader sequence, a human constant region gene (Cγ4), and a chicken variable region gene integration site, as shown in FIG. 1 (b). Furthermore, it has a zeocin resistance gene (for mammalian cells) and an ampicillin resistance gene (for E. coli) as selection markers. It also has a CMV promoter, a bovine growth hormone poly (A) addition signal, and an SV40 replication origin.

(1-7) Construction of expression vector 3: pcSLCK
The pBCκ was digested with EcoRI and XhoI to excise the Cκ gene, and inserted into the EcoRI / XhoI site of pcDNA3 / myc-His (Invitrogen). The direction of insertion was confirmed by sequencing, and the correct orientation was obtained as pcDCκ. The pBSL was digested with HindIII and KpnI to excise the leader sequence and introduced into the HindIII / KpnI site of the pcDCκ. As a result, pcSLCκ as an expression vector was obtained (see FIG. 2).

  The obtained pcSLCκ has a size of 7.1 kb, a chicken immunoglobulin leader sequence, a human constant region gene (Cκ), and a chicken variable region gene integration site, as shown in FIG. 1 (a). Furthermore, it has a neomycin resistance gene (for mammalian cells) and an ampicillin resistance gene (for E. coli) as selection markers. It also has a CMV promoter, a bovine growth hormone poly (A) addition signal, and an SV40 replication origin.

[Example 2: Preparation of chicken-human chimeric antibody using expression vector]
(2-1) Cloning of chicken variable region gene in expression vector The chicken variable region gene was cloned from two different antibodies. One is a gene derived from chicken hybridoma HUNN1, and the other is a gene of scFV, phAb3-15 selected from a phage display library.

Total RNA was extracted from HUN1 by ISOGEN-LS (Nippon Gene). The first cDNA strand was primed with oligo (dT) _12-18 primer (Roche Diagnostics) and synthesized with Superscript II Synthesis cDNA Kit (GIBCO BRL). The variable region gene uses sense primer VH3F (SEQ ID NO: 9) and antisense primer VH4R (SEQ ID NO: 11) for the heavy chain and VL3F (SEQ ID NO: 12) and VL4R (SEQ ID NO: 13) for the light chain. Amplified by PCR using KOD plus DNA polymerase. Both sense primers contain a HindIII restriction site, Kozak sequence, while the antisense primer contains a BamHI restriction site, a splice donor site.

  PhAb3-15 as template, VH5F (SEQ ID NO: 14) and VH4R (SEQ ID NO: 11) containing AscI restriction sites for the heavy chain and VL5F (SEQ ID NO: 15) containing the AscI restriction site for the light chain PCR was performed using VL4R (SEQ ID NO: 13). The amplified variable region gene was first introduced into an intermediate vector, sequenced, and then introduced into an expression vector. HindIII / BamHI or AscI / BamHI sites were used for introduction.

(2-2) Introduction and selection of expression vector into mammalian cells COS-7 and CHO-K1 cell lines were used for transfection. COS-7 cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and kanamycin (60 μg / ml). CHO-K1 cells were maintained in F-12 Ham's medium (Invitrogen) supplemented with 10% FBS.

Antibody production was tested with both transient and stable expression. For transient expression, light chain and heavy chain expression vectors were introduced into COS-7 cells using Polyfect transfection reagent (Qiagen). Specifically, 2.5 μg of plasmid DNA dissolved in 150 μl of serum-free medium was mixed with 15 μl of Polyfect transfection reagent and added to 4 × 10 ⁵ cells. After 72 hours of incubation, culture supernatants were collected and assayed by quantitative ELISA.

For stable expression, both expression vectors were introduced into CHO-K1 cells in the same manner. After 48 hours of incubation, the cells were harvested and resuspended in selective medium containing zeotin (200 μg / ml, Invitrogen) and geneticin (400 μg / ml, Sigma). Cells were cultured for 2 weeks in selective media and the media was changed every 3-4 days. Cells exhibiting resistance were collected, resuspended in a medium in which the antibiotic concentration was halved, and plated in a 96-well culture plate so that there were 1 cell per well. Two weeks later, antibody producing (producing) clones were selected by ELISA. Positive clones that reacted with PrP were selected and further cultured. For evaluation of antibody production, culture supernatants were collected from 10 ⁶ cells cultured for 24 hours and assayed by quantitative ELISA.

(2-3) ELISA
ELISA plates were coated overnight at 4 ° C. using 50 μl of recombinant mouse PrP or BSA (5 μg / ml) as a control antigen. Plates were blocked with 350 μl PBS containing 25% BlockAce at 37 ° C. for 1 hour. The culture supernatant of the transferred cells was added to each well and incubated at 37 ° C. for 1 hour. The binding of the chimeric antibody was detected using a peroxidase-labeled anti-human IgG antibody (Cappel).

After washing the plate, o-phenylenediamine sulfate (manufactured by KATAYMA CHEMICAL) was added, and the absorbance at 490 nm was measured. Antibody production was measured by quantitative ELISA using anti-human Igκ chain antibody for capture and peroxidase-labeled anti-human IgG (Fc) antibody (Zymed) for detection. Purified human IgG ₁ or IgG ₄ (The Binding Site) was used as a standard.

  As a result of the ELISA assay for transient expression, the antibody expression level after 72 hours (3 days) of culture was 1.5 to 2.5 μg / ml.

On the other hand, the result of ELISA assay in stable expression is shown in FIG. The vertical axis of the figure shows the amount of antibody production, and the unit is μg / 10 ⁶ cell / day. The abscissa indicates the selected positive clone name. Among the clone names, “C / H” indicates a chicken-human chimeric antibody, “1” or “4” indicates IgG ₁ or IgG ₄ , and “HU” indicates HUNN1 antibody, and “ph” indicates a phage-derived phAb3-15 antibody. As is clear from the figure, the antibody production level is in the range of 3.5 to 16.4 μg / 10 ⁶ cell / day. In general, the amount of antibody production in CHO-K1 cells is 1 to 10 μg / 10 ⁶ cell / 24 hr. As shown in FIG. 4, in the present example, stable production strains showed antibody production levels similar to or higher than these, so it is considered that high expression levels can be obtained by using this vector. It is done.

(2-4) Western Blotting Chimeric antibody size and IgG subclass were analyzed using standard procedures. About the clone obtained by the stable expression of the above (2-2), the culture supernatant containing 20 ng of the antibody was subjected to SDS-PAGE under reducing or non-reducing conditions. The gel concentration was 10% under reducing conditions and 12.5% under non-reducing conditions. The protein was transferred to an Immun-Blot ^™ PVDF membrane (manufactured by BIO RAD) at 180 mA for 3 hours. Blotting was incubated with various monoclonal antibodies for 1 hour at room temperature and detected by ECL-plus (Amersham Pharmacia Biotech). As monoclonal antibodies, anti-human Igγ chain antibody (manufactured by Sigma), anti-human Igκ chain antibody (manufactured by Sigma), anti-human IgG ₁ antibody (manufactured by Biosciences), anti-human IgG ₄ antibody (manufactured by Biosciences), peroxidase Label donkey anti-chicken IgG antibody (Chemicon International) and peroxidase-labeled goat anti-human IgG antibody (Cappel) were used in the initial reaction. Peroxidase-labeled rabbit anti-goat antibody IgG and goat anti-mouse IgG antibody (Southern Biotech) were used for the next reaction.

  The characteristics of the structure of the chimeric antibody obtained from the culture supernatant of (2-2) above were confirmed by Western blotting. First, it was confirmed whether or not the expressed chimeric antibody had a size estimated by the amino acid sequence. The results of the clones C / H1-ph16 and C / H4-ph24 under non-reducing conditions are shown in FIG. 5A, and the results under reducing conditions are shown in FIG. 5B. Lane 1 in FIG. 5A and lanes 1 and 3 in FIG. 5B show the results for C / H1-ph16, and lane 2 in FIG. 5A and lanes 2 and 4 in FIG. 5B show the results for C / H1-ph24. In FIG. 5B, lanes 1 and 2 show the heavy chain results, and lanes 3 and 4 show the light chain results.

Under non-reducing conditions, a band (H ₂ L ₂ ) having a molecular weight of about 150 kDa or more was detected in lane 2, and a weak band (HL) was found below (between 50 and 100 kDa) for C / H1-ph24. was detected. Thus, it is presumed that two heavy chain molecules and two light chain molecules associate to produce a functional antibody molecule. However, under non-reducing conditions, the mobility in the gel varies depending on the three-dimensional structure, and the band does not move to a position corresponding to the molecular weight.

  On the other hand, under reducing conditions, the heavy chain has a molecular weight of about 50 kDa (lanes 1 and 2), and the light chain has a molecular weight of about 25 kDa (lanes 3 and 4). This is approximately the same size as the molecular weight estimated from the amino acid sequence.

Next, the antigen specificity of the expressed chimeric antibody was confirmed. The results for clones C / H1-ph16 and C / H4-ph24 are shown in FIG. From this result, (i) the chimeric antibody has a chicken part that reacts with the anti-chicken antibody and a human part that reacts with the anti-human antibody, and (ii) for the human part, a strain into which the pcSLCγ1 vector has been introduced. It was revealed that the chimeric antibody produced reacts with the anti-IgG1 antibody, and the chimeric antibody produced by the strain introduced with the pcSLCγ4 vector reacts with the anti-IgG4 antibody.
Therefore, clone C / H1-ph16 has the variable region of phAb3-15 and the constant region of human Cκ · Cγ1, and clone C / H4-ph24, like C / H1-ph16, has Cγ1 Instead, it was found that it has a constant region of Cγ4, and it was revealed that the chimeric antibody produced in the present invention exhibits the expected specificity.

  As described above, according to the present invention, it is possible to express a complete chicken-human chimeric antibody molecule. This opens the way to apply chicken monoclonal antibodies to human clinical practice. Therefore, the present invention can be widely used in industries related to pharmaceuticals, particularly antibody pharmaceuticals and diagnostic agents.

(A) is a schematic diagram showing the configuration of pcSLCκ, which is an example of a light chain expression vector, among the chicken-human chimeric expression vectors according to the present invention, and (b) is an example of a heavy chain expression vector. It is a schematic diagram which shows the structure of a certain pcSLCγ1 or pcSLCγ4. FIG. 2 is a schematic diagram showing a process of producing the expression vector shown in FIGS. 1 (a) and 1 (b). (A) is a schematic diagram showing the structure of the light chain of the chicken-human chimeric antibody according to the present invention, (b) is a schematic diagram showing the structure of the heavy chain, and (c) is a diagram of the chimeric antibody molecule. It is a schematic diagram which shows a structure. It is a graph which shows the result of the ELISA assay when the vector for expression is stably expressed in the host in an example. It is a figure which shows the result of the western blotting of the chimeric antibody expressed in the Example, A is a result when SDS-PAGE is performed under non-reducing conditions, and B is a result when SDS-PAGE is performed under reducing conditions. It is. It is a figure which shows the result of the western blotting which examined the antigen specificity of the chimeric antibody expressed in the Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Light chain of chicken-human chimeric antibody 11 Chicken-derived light chain variable region 12 Human-derived light chain constant region 20 Heavy chain of chicken-human chimeric antibody 21 Chicken-derived heavy chain variable region 22a / 22b / 22c Human-derived heavy chain constant region 30 Chicken -Human chimeric antibody 31 Fc
32 Fab

Claims (23)

A vector capable of expressing a protein using a mammalian cell as a host,
A chicken-human chimeric antibody expression vector comprising, as DNA segments, at least a promoter, an integration site for incorporating a chicken variable region gene, a leader sequence of chicken immunoglobulin, and a human immunoglobulin constant region gene .   2. The chicken-human chimeric antibody expression vector according to claim 1, wherein the human immunoglobulin constant region gene is a gene encoding a heavy chain or light chain constant region.   The chicken-human chimeric antibody expression vector according to claim 2, wherein at least one of the polynucleotides having the nucleotide sequence shown in SEQ ID NO: 1 or 2 is used as the human immunoglobulin heavy chain constant region gene.   The chicken-human chimeric antibody expression vector according to claim 2, wherein a polynucleotide having the base sequence represented by SEQ ID NO: 3 is used as the human immunoglobulin light chain constant region gene.   5. The chicken-human chimeric antibody expression vector according to any one of claims 1 to 4, wherein a polynucleotide having the base sequence represented by SEQ ID NO: 4 is used as a chicken immunoglobulin leader sequence.   The chicken-human chimera according to any one of claims 1 to 5, wherein the integration site contains a restriction enzyme site recognized by at least one restriction enzyme of AscI, BamHI and HindIII. Antibody expression vector.   The chicken-human chimeric antibody expression vector according to claim 6, wherein the integration site is a multicloning site in which a plurality of restriction enzyme sites are connected.   The chicken-human chimeric antibody expression vector according to any one of claims 1 to 7, wherein the promoter is a cytomegalovirus-derived promoter.   The chicken-human chimeric antibody expression vector according to any one of claims 1 to 8, further comprising a selection marker for determining whether or not the vector has been introduced into a mammalian cell. .   10. The chicken-human chimeric antibody expression vector according to claim 9, wherein the selection marker is a zeocin resistance gene or a neomycin resistance gene.   The chicken-human chimeric antibody expression vector according to any one of claims 1 to 10, further comprising a replication origin.   The chicken-human chimeric antibody expression vector according to claim 11, wherein the replication origin is derived from pUC and SV40.   The chicken-human chimeric antibody expression vector according to any one of claims 1 to 12, further comprising a poly (A) addition signal.   The chicken-human chimeric antibody expression vector according to claim 13, wherein the poly (A) addition signal is derived from bovine growth hormone.   A method for producing a chicken-human chimeric antibody, comprising using the chicken-human chimeric antibody expression vector according to any one of claims 1 to 14. The chicken-human chimeric antibody expression vector includes a heavy chain expression vector in which a human immunoglobulin constant region gene is a gene encoding a heavy chain constant region, and a human immunoglobulin constant region gene is a light chain constant region. And a light chain expression vector that is a gene encoding
Introducing a chicken heavy chain variable region gene into the heavy chain expression vector and introducing a chicken light chain variable region gene into the light chain expression vector;
The chicken-human chimeric antibody according to claim 15, further comprising a transfection step in which the expression vector for heavy chain and the expression vector for light chain into which the chicken variable region gene is introduced are coexpressed in a mammalian cell. Production method. The method for producing a chicken-human chimeric antibody according to claim 16, wherein the mammalian cells used are CHO cells. The method for producing a chicken-human chimeric antibody according to claim 16 or 17, wherein in the transfection step, an expression vector is introduced by a lipofection method. 19. The method of claim 15, further comprising an antibody recovery step of recovering a chicken-human chimeric antibody from mammalian cells co-expressed with the heavy chain expression vector and the light chain expression vector. A method for producing the chicken-human chimeric antibody according to item 2. The method for producing a chicken-human chimeric antibody according to claim 19, further comprising an antibody purification step of purifying the chicken-human chimeric antibody recovered from the mammalian cells. A chicken-human chimeric antibody production kit for performing the method of producing a chicken-human chimeric antibody according to any one of claims 15 to 20,
  A heavy chain expression vector in which the human immunoglobulin constant region gene is a gene encoding the heavy chain constant region, and a light chain in which the human immunoglobulin constant region gene is a gene encoding the light chain constant region A chicken-human chimeric antibody production kit comprising an expression vector for use. The method according to claim 21, further comprising at least one of a reagent group for introducing an expression vector into a mammalian cell and a reagent group for incorporating a chicken variable region gene into the expression vector. Chicken-human chimeric antibody production kit. 23. The method according to claim 22, further comprising at least one of a reagent group for recovering a chicken-human chimeric antibody from mammalian cells and a reagent group for purifying a chicken-human chimeric antibody recovered from mammalian cells. The chicken-human chimeric antibody production kit described.

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