Method for producing immortalized cells

Abstract

The present invention addresses the problem of providing: a method for immortalizing an antibody-producing cell; and an immortalized transformed cell produced by the method. The present invention relates to: a method for producing an immortalized cell, the method comprising introducing at least one gene selected from the group consisting of a SV40T antigen gene, a Bcl-2 family gene, a Myc family gene and a cyclin D family gene into an antibody-producing cell that has not been immortalized yet to produce the immortalized cell; and a method for producing an immortalized antibody-producing cell, the method comprising collecting an antibody-producing cell from a mammal, performing primary culture of the cell to produce a primary antibody-producing cell, and introducing at least one gene selected from the group consisting of a SV40T antigen gene, a Bcl-2 family gene, a Myc family gene and a cyclin D family gene into the primary antibody-producing cell to produce an immortalized antibody-producing cell.

Description

[Technical Field]

[0001] The present invention relates to a method for immortalizing antibody-producing cells and immortalized transformed cells obtained by said method. This application claims priority based on Japanese Patent Application No. 2022-195145, filed on December 6, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] Some cells have the ability to proliferate, while others do not. Even cells with the ability to proliferate are usually genetically limited in the number of times they can divide, and have a lifespan. For this reason, for example, primary culture cells, which are cells extracted from animal tissue and cultured, can only divide a limited number of times, even if grown under appropriate conditions, and then lose their ability to proliferate. For this reason, attempts have been made to remove the limit on the number of times cells with useful traits can divide and give them the ability to divide indefinitely, a process known as immortalization. Generally, cells acquire immortalization potential through transformation.

[0003] Monoclonal antibodies are widely used as reagents for detecting target substances using immune reactions. In recent years, human monoclonal antibodies have also been used as active ingredients in antibody drugs, making monoclonal antibodies valuable for industrial use. Monoclonal antibodies are often produced by antibody-producing cells, and to ensure a stable supply of monoclonal antibodies, it is necessary to immortalize antibody-producing cells without impairing their antibody-producing ability, so that they can be cultured in vitro at an industrial level.

[0004] The hybridoma method has already been established for the production of rodent monoclonal antibodies. Hybridomas are established by immunizing an animal, isolating a single B cell that produces the desired monoclonal antibody, culturing it in primary culture, and fusing it with myeloma cancer cells (myeloma). The resulting hybridomas are immortalized while retaining their antibody-producing ability. However, since there is no suitable hybridoma method for animals other than rodents, the immortalization of antibody-producing cells requires transformation using complex genetic engineering techniques.

[0005] Known methods for immortalizing primary cultured cells include, for example, a method in which three cancer-related genes, the Bcl-2 (B-cell / CLL lymphoma 2) gene, the Myc gene, and the Ccnd1 gene, are introduced into pro B cells for transformation (Non-Patent Document 1).Another known method is a method in which two cancer-related genes, the c-Myc gene and the SV40 (simian virus 40) large T antigen gene, are introduced into human fetal fibroblasts for transformation (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Nakagawa, et al., Haematologica, 2011, vol.96(9), p.1318-1326. [Non-patent document 2] Kim, et al., Experimental and Molecular Medicine, 2001, vol.33(4), p.293-298. Summary of the Invention [Problem to be solved by the invention]

[0007] Unlike proB cells, antibody-producing cells, which are terminally differentiated cells, cannot acquire immortalization ability even when transformed by introducing the Bcl-2 gene, Myc gene, and Ccnd1 gene. Furthermore, when antibody-producing cells are transformed by introducing the c-Myc gene and the SV40 large T antigen gene, they acquire the ability to proliferate and become immortalized, but the phenotype cannot be maintained, and immortalized cells that maintain antibody production ability cannot be obtained.

[0008] An object of the present invention is to provide a method for immortalizing antibody-producing cells and immortalized transformed cells obtained by said method. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors discovered that antibody-producing cells collected from a mammal immunized with an antigen can be immortalized while maintaining their antibody-producing ability by transforming them with an SV40 T antigen gene, a Myc family gene, a Bcl-2 family gene, and a cyclin D family gene, and thus completed the present invention.

[0010] That is, the present invention provides the following drugs and the like: [1] A method for producing immortalized cells, which comprises introducing one or more genes selected from the group consisting of the SV40 T antigen gene, the Bcl-2 family gene, the Myc family gene, and the cyclin D family gene into non-immortalized antibody-producing cells to produce immortalized cells. [2] The method for producing immortalized cells according to [1] above, wherein the Myc family gene is a c-Myc gene or an L-Myc gene. [3] The method for producing an immortalized cell according to [1] or [2] above, wherein the Bcl-2 family gene is a Bcl-2 gene or a Bcl-XL gene. [4] The method for producing an immortalized cell according to any one of [1] to [3] above, wherein the cyclin D family gene is a cyclin D1 gene or a cyclin D3 gene. [5] A method for producing an immortalized cell according to any one of [1] to [3] above, comprising introducing an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene into the non-immortalized antibody-producing cell. [6] A method for producing immortalized antibody-producing cells, comprising collecting antibody-producing cells from a mammal and performing primary culture, and then introducing one or more genes selected from the group consisting of an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene into the obtained primary antibody-producing cells to produce immortalized antibody-producing cells. [7] The method for producing immortalized antibody-producing cells according to [6], wherein the mammal is an animal immunized with an antigen, and the immortalized antibody-producing cells are screened for immortalized antibody-producing cells that produce an antibody that recognizes the antigen. [8] A method for producing an antibody, comprising producing immortalized antibody-producing cells by the method for producing immortalized antibody-producing cells according to [6] or [7] above, culturing the immortalized antibody-producing cells, and recovering the produced antibody. [9] A transformed cell in which a non-immortalized antibody-producing cell has been immortalized by introducing one or more genes selected from the group consisting of an exogenous SV40 T antigen gene, an exogenous Bcl-2 family gene, an exogenous Myc family gene, and an exogenous cyclin D family gene.

[10] The transformed cell according to [9] above, which has the ability to produce an antibody. [Effects of the Invention]

[0011] The method for producing immortalized cells according to the present invention allows antibody-producing cells obtained by primary subculture or the like to be immortalized relatively easily, thereby obtaining transformed cells that have acquired immortalization ability while maintaining antibody production ability. The transformed cells thus obtained are useful, for example, for producing monoclonal antibodies. [Brief explanation of the drawings]

[0012] [Figure 1]1 shows a cumulative growth curve of transformed cells immortalized from plasma cells producing anti-rCRP rabbit monoclonal antibody collected from rabbits in Example 1. [Figure 2] FIG. 1 shows the time course of the amount of anti-rCRP rabbit monoclonal antibody in transformed cells immortalized from anti-rCRP rabbit monoclonal antibody-producing plasma cells collected from rabbits in Example 1. [Figure 3] 1 shows an HPLC chromatogram of the culture supernatant of immortalized transformed cells producing anti-rCRP rabbit monoclonal antibody in Example 1. [Figure 4] FIG. 1 shows the results of SDS-PAGE of purified anti-rCRP rabbit monoclonal antibody under reducing conditions (R in the figure) and non-reducing conditions (NR in the figure) in Example 1. [Figure 5] FIG. 1 shows the results of Western blotting using purified anti-rCRP rabbit monoclonal antibody in Example 1. [Figure 6] FIG. 1 shows cumulative growth curves of transformed cells into which each expression cassette was introduced in Example 2. [Figure 7] FIG. 1 shows the results of flow cytometry analysis of cell groups stained with anti-rabbit IgG antibody, of transformed cells into which each expression cassette was introduced in Example 2. [Figure 8] FIG. 1 shows cumulative growth curves of transformed cells into which each expression cassette was introduced in Example 3. [Figure 9] FIG. 1 shows cumulative growth curves of transformed cells into which each expression cassette was introduced in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0013] The method for producing immortalized cells according to the present invention involves introducing one or more genes selected from the group consisting of an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene into non-immortalized antibody-producing cells to produce immortalized cells. By introducing and expressing a combination of these cancer-related genes into non-immortalized antibody-producing cells, the limit on the number of cell divisions is lifted, and the cells are given the ability to divide, resulting in immortalized cells. While differentiated plasma cells are generally unable to divide in vitro, the method for producing immortalized cells according to the present invention makes it possible to artificially produce immortalized cells with a doubling capacity comparable to that of cancer cells such as CHO cells.

[0014] In the method for producing immortalized cells according to the present invention, three types of genes, namely, an SV40 T antigen gene, a Bcl-2 family gene, and a Myc family gene, may be introduced into non-immortalized antibody-producing cells; three types of genes, namely, an SV40 T antigen gene, a Bcl-2 family gene, and a cyclin D family gene, may be introduced; or four types of genes, namely, an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene, may be introduced. Any combination of genes can confer immortalization ability to antibody-producing cells. In the method for producing immortalized cells according to the present invention, introduction of four types of genes, namely, an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene, is particularly preferred, since this allows for the production of a larger number of transformed cells that have acquired immortalization ability while retaining antibody production ability.

[0015] The SV40 T antigen gene is a viral gene known to contribute to the immortalization of cells of many species by inactivating tumor suppressor genes that induce replicative senescence in cells. In the present invention and this specification, both the SV40 large T antigen and the SV40 small T antigen are collectively referred to as "SV40 T antigen."

[0016] The term "SV40 large T antigen" includes all proteins that, like native SV40 large T antigen, function to inactivate tumor suppressor genes that induce cellular replicative senescence within cells. The term "native" refers to proteins encoded in the genomic DNA of any organism. Specifically, the term includes not only proteins consisting of the same amino acid sequence as native SV40 large T antigen, but also mutants of native SV40 large T antigen, i.e., proteins consisting of the same amino acid sequence as native SV40 large T antigen but with one or more amino acids deleted, substituted, or added, and that retain their tumor suppressor gene inactivation ability. Furthermore, the SV40 large T antigen encoded by the SV40 T antigen gene introduced into non-immortalized cells in the present invention may be a fusion protein in which various tags or other structural proteins are linked to native SV40 large T antigen or its mutants, either directly or indirectly via a suitable linker sequence. The amino acid sequence of the native SV40 large T antigen is registered in the International Nucleotide Sequence Database (INSD) (INSD accession number: AAB59924).

[0017] The term "SV40 small T antigen" includes all proteins that, like native SV40 small T antigen, bind to protein phosphatase 2 (PP2A) and inactivate PP2A. Specifically, it includes not only proteins consisting of the same amino acid sequence as native SV40 small T antigen (INSD Accession No. AAB59925), but also mutants of native SV40 small T antigen, i.e., proteins consisting of the same amino acid sequence as native SV40 small T antigen but with one or more amino acids deleted, substituted, or added, and that retain their PP2A inactivation ability. Furthermore, the SV40 small T antigen encoded by the SV40 T antigen gene introduced into non-immortalized cells in the present invention may be a fusion protein in which various tags or other structural proteins are linked to native SV40 small T antigen or its mutants, either directly or indirectly via an appropriate linker sequence.

[0018] In the present invention and this specification, the term "SV40 T antigen gene" refers to a gene encoding a protein that, like natural SV40 large T antigen, functions to inactivate tumor suppressor genes that induce cellular replicative senescence in cells, and refers to a nucleic acid containing a nucleotide sequence encoding the SV40 T antigen. The "SV40 T antigen gene" used in the present invention may be a nucleic acid containing both a nucleotide sequence encoding the SV40 large T antigen and a nucleotide sequence encoding the SV40 small T antigen, or a nucleic acid containing only a nucleotide sequence encoding the SV40 large T antigen. The SV40 T antigen gene to be introduced into non-immortalized cells in the present invention is not particularly limited as long as it is a gene that can express SV40 T antigen when introduced into mammalian cells. It may contain introns and express both SV40 large T antigen and SV40 small T antigen, or it may consist of only exons. Furthermore, the nucleotide sequence encoding the SV40 large T antigen or the SV40 small T antigen in the SV40 T antigen gene may be modified in various ways, such as by changing degenerate codons to those frequently used in finitely proliferative antibody-producing cells (hereinafter sometimes simply referred to as "finitely proliferative cells") into which the SV40 T antigen gene or the like is introduced for immortalization. Codon modification can be carried out by known gene sequence mutation techniques or artificial gene synthesis.

[0019] In the present invention and specification, the term "Myc family" refers to a family of transcription factors containing a basic helix-loop-helix (bHLH) and leucine zipper (LZ) motif. The human Myc family includes three transcription factors: c-Myc (INSD accession number: AAA36340), L-Myc (INSD accession number: BAG58834), and N-Myc (INSD accession number: AAP36048). In the present invention and this specification, the term "Myc family" includes not only naturally occurring Myc family members, but also variants obtained by modifying naturally occurring Myc family proteins while retaining the transcription factor function similar to human c-Myc. Specifically, the Myc family includes naturally occurring Myc family members such as human c-Myc, human L-Myc, and human N-Myc, as well as their homologs. An example of a homolog of human c-Myc is rabbit c-Myc (INSD accession number: AJC97784). A homolog of human L-Myc is An example of a Myc family member is rabbit L-Myc (INSD accession number: XP_002715238). In addition to proteins consisting of the same amino acid sequence as these native Mycs, the present invention also includes mutants thereof, i.e., proteins consisting of the same amino acid sequence as native Myc but with one or more amino acids deleted, substituted, or added, and retaining their transcriptional activity. An example of a Myc family member is the T58N mutant of human c-Myc. In the present invention, the Myc family member encoded by the Myc family gene introduced into non-immortalized cells may be a modified form, such as a fusion protein, in which various tags or other structural proteins are linked to the native Myc family member or a mutant thereof, directly or indirectly via an appropriate linker sequence.In the present invention, the Myc family gene to be introduced into non-immortalized cells is preferably a gene for c-Myc (human c-Myc and its orthologues, and mutants thereof), L-Myc (human c-Myc and its orthologues, and mutants thereof), or a gene for a modified version thereof, and more preferably a gene for human c-Myc, human L-Myc, or a mutant version thereof, or a gene for a modified version thereof.

[0020] In the present invention and the present specification, the term "Myc family gene" refers to a nucleic acid containing a nucleotide sequence encoding a Myc family protein. The Myc family gene to be introduced into non-immortalized cells in the present invention is not particularly limited as long as it is a gene that can express a Myc family protein when introduced into mammalian cells, and may contain introns or may consist of only exons. Furthermore, the nucleotide sequence encoding a Myc family protein in a Myc family gene may be modified in various ways, such as by changing degenerate codons to codons frequently used in finitely proliferating cells. Codon modification can be achieved by known gene sequence mutation techniques or artificial gene synthesis.

[0021] In the present invention and this specification, the term "Bcl-2 family" refers to a family of mitochondrial outer membrane proteins that have one or more BH (Bcl-2 homology) domains and a highly hydrophobic transmembrane region at the C-terminus and have anti-apoptotic activity. Examples of human Bcl-2 family proteins with anti-apoptotic activity include Bcl-2 (INSD accession number: AAH27258), Bcl-XL (INSD accession number: AAP35872), and MCL-1 (Myeloid cell leukemia sequence 1) (INSD accession number: AAD13299).

[0022] In the present invention, the Bcl-2 family gene to be introduced into non-immortalized cells is not particularly limited as long as it has one or more BH domains and a transmembrane region and is a protein with anti-apoptotic activity. For example, in the present invention and the present specification, the term "Bcl-2 family" includes not only native Bcl-2 family members but also modified versions of native Bcl-2 family proteins that retain the anti-apoptotic activity of human Bcl-2. Specifically, the Bcl-2 family with anti-apoptotic activity includes native Bcl-2 family members such as human Bcl-2, human Bcl-XL, and human MCL-1, as well as their homologs. Examples of human Bcl-2 homologs include rabbit Bcl-2 (INSD accession number: XP_008259661). Examples of human Bcl-XL homologs include rabbit Bcl-XL (INSD accession number: XP_008254359). In addition to proteins consisting of the same amino acid sequence as these native Bcl-2 family members, the present invention also includes mutants thereof, i.e., proteins consisting of the same amino acid sequence as the native Bcl-2 family members but with one or more amino acids deleted, substituted, or added, and which retain their anti-apoptotic activity. Furthermore, the Bcl-2 family members encoded by the Bcl-2 family genes introduced into non-immortalized cells in the present invention may be modified proteins such as fusion proteins in which various tags or other structural proteins are linked to the native Bcl-2 family members or their mutants, either directly or indirectly via an appropriate linker sequence. The Bcl-2 family genes introduced into non-immortalized cells in the present invention are preferably genes for Bcl-2 (human Bcl-2 and its orthologues, and mutants thereof), Bcl-XL (human Bcl-XL and its orthologues, and mutants thereof), or mutants thereof, and more preferably genes for human Bcl-2, human Bcl-XL, mutants thereof, or mutants thereof.

[0023] In the present invention and the present specification, the term "Bcl-2 family gene" refers to a nucleic acid containing a nucleotide sequence encoding a Bcl-2 family protein. The Bcl-2 family gene to be introduced into non-immortalized cells in the present invention is not particularly limited, as long as it is a gene that can express a Bcl-2 family protein when introduced into mammalian cells. It may contain introns or may consist of only exons. Furthermore, the nucleotide sequence encoding a Bcl-2 family protein in a Bcl-2 family gene may be modified in various ways, such as by changing degenerate codons to those frequently used in finitely proliferating cells. Codon modification can be performed by known gene sequence mutation techniques or artificial gene synthesis.

[0024] In the present invention and the present specification, the term "cyclin D family" refers to a family of proteins that contain a cyclin box domain that binds to cyclin-dependent kinases (CDKs) and an N-terminal RB (Retinoblastoma tumor suppressor protein)-binding domain, whose synthesis begins in the G1 phase of the cell cycle and controls the transition from G1 to S phase. Examples of the human cyclin D family include cyclin D1 (INSD accession number: AAA58392), cyclin D2 (NSD accession number: AAA51926), and cyclin D3 (NSD accession number: AAA52137).

[0025] In the present invention, the cyclin D family gene to be introduced into non-immortalized cells is not particularly limited as long as it is a protein that has the same S-phase transition regulatory activity as human cyclin D1. For example, in the present invention and the present specification, the term "cyclin D family" includes not only naturally occurring cyclin D family members but also mutants of naturally occurring cyclin D family proteins that retain their S-phase transition regulatory activity. Specifically, naturally occurring cyclin D family members include human cyclin D1, human cyclin D2, and human cyclin D3, as well as their homologs. Furthermore, in addition to proteins consisting of the same amino acid sequence as these naturally occurring cyclin Ds, the term also includes mutants thereof, i.e., proteins consisting of the same amino acid sequence as naturally occurring cyclin D but with one or more amino acids deleted, substituted, or added, and that retain their S-phase transition regulatory activity. Furthermore, the cyclin D family encoded by the cyclin D family gene introduced into non-immortalized cells in the present invention may be a modified form such as a fusion protein in which various tags or other structural proteins are linked to a naturally occurring cyclin D family member or a mutant thereof, either directly or indirectly via an appropriate linker sequence. The cyclin D family gene introduced into non-immortalized cells in the present invention is preferably a gene for cyclin D1 (human cyclin D1 and its orthologues, and mutants thereof), cyclin D3 (human cyclin D3 and its orthologues, and mutants thereof), or a modified form thereof, and more preferably a gene for human cyclin D1, human cyclin D3, or a mutant or modified form thereof.

[0026] In the method for producing immortalized cells according to the present invention, the Myc family genes, Bcl-2 family genes, and cyclin D family genes introduced into non-immortalized cells may all be derived from the same or different species as the cells into which they are introduced. Furthermore, these three genes may be derived from the same or different species. For example, the SV40 T antigen gene, human c-Myc gene, human Bcl-2 gene, and human cyclin D1 gene may be introduced into lymph node cells collected from a rabbit; the SV40 T antigen gene, mouse c-Myc gene, human Bcl-2 gene, and human cyclin D1 gene may be introduced into spleen cells collected from a mouse; or the SV40 T antigen gene, human c-Myc gene, human Bcl-2 gene, and human cyclin D1 gene may be introduced into epithelial cells collected from a human.

[0027] In the method for producing immortalized cells according to the present invention, the cells to be transformed into immortalized cells are not particularly limited as long as they are non-immortalized finitely proliferative cells, and the species or tissue from which they are derived is not particularly limited. The finitely proliferative cells used in the present invention are preferably cells collected from animals or primary cultures thereof, more preferably cells collected from mammals or primary cultures thereof. The mammals in question are not particularly limited, but include humans, laboratory animals such as mice, rats, and monkeys, and livestock or pet animals such as rabbits, dogs, cats, cows, horses, and sheep, with humans being particularly preferred. Collection and primary culture of cells from animals can be carried out using conventional methods.

[0028] In the method for producing immortalized cells of the present invention, non-immortalized plasma cells are preferably used as the cells to be transformed into immortalized cells. Here, plasma cells are mononuclear cells similar to lymphocytes that are distributed in the spleen, lymph nodes, bone marrow, connective tissue, etc., and acquire the ability to produce antibodies upon stimulation with an antigen. The plasma cells to be transformed in the method for producing immortalized cells of the present invention may be plasma cells before acquiring the ability to produce antibodies, plasma cells that retain the ability to produce antibodies after acquisition, or plasma cells that have lost the ability to produce antibodies after acquisition. Those skilled in the art can select plasma cells using an appropriate method, such as flow cytometry. Selected plasma cells may be confirmed by hematoxylin-eosin staining and optical microscopic images of the nuclear shape and cytoplasm.

[0029] In the method for producing immortalized cells according to the present invention, immortalized transformed cells can be produced by introducing an SV40 T antigen gene, a Myc family gene, a Bcl-2 family gene, and a cyclin D family gene into non-immortalized cells by genetic engineering techniques. All four of these genes may be introduced into non-immortalized cells simultaneously or sequentially (in any order).

[0030] In the method for producing immortalized cells according to the present invention, the SV40 T antigen gene or the like may be introduced into the chromosome of the cells to be immortalized, or may be introduced as an extrachromosomal gene. By introducing the SV40 T antigen gene or the like into the chromosome, transformed cells with excellent maintenance stability over successive generations can be obtained.

[0031] The genetic engineering method used to introduce an SV40 T antigen gene or the like into antibody-producing cells to be immortalized may be any known method for introducing a foreign gene into finitely proliferating cells, or an appropriate modification of a known method may be used. For example, such a method includes introducing a vector incorporating an expression cassette for the foreign gene into cells. The SV40 T antigen gene or the like may be expressed in a polycistronic or monocistronic form.

[0032] An expression cassette is a combination of DNA necessary to express a protein of interest, and includes a structural gene encoding the protein of interest and a promoter that functions in finitely proliferating cells. An expression cassette may contain only one type of structural gene, or two or more types.

[0033] When expressing in a monocistronic form, specific examples of expression cassettes used in transformation for immortalization include an SV40 T antigen expression cassette containing an SV40 T antigen gene and a promoter, a Myc family expression cassette containing a Myc family gene and a promoter, a Bcl-2 family expression cassette containing a Bcl-2 family gene and a promoter, and a cyclin D family expression cassette containing a cyclin D family gene and a promoter.

[0034] Two or more of the SV40 T antigen gene, Myc family gene, Bcl-2 family gene, and cyclin D family gene may be incorporated into an expression cassette (1) for polycistronic expression. For example, immortalized transformed cells can be obtained by introducing into finitely proliferating cells an expression cassette (1) in which the SV40 T antigen gene, Myc family gene, Bcl-2 family gene, and cyclin D family gene are sequentially linked downstream of a promoter (1) via a linker sequence containing a nucleotide sequence encoding a self-cleaving peptide. The self-cleaving peptide can be appropriately selected from known self-cleaving peptides such as P2A peptide, E2A peptide, and T2A peptide. The linker sequence linking each gene may contain an internal ribosome entry site (IRES) sequence instead of a self-cleaving peptide. The IRES sequence may be derived from a viral genome or an animal cell genome, and may be appropriately selected from known IRES sequences.

[0035] The promoter in the expression cassette may be any promoter that functions in finitely proliferative cells, and may be a promoter native to the finitely proliferative cells or a promoter not native to the finitely proliferative cells. When the finitely proliferative cells are antibody-producing plasma cells, an example of a promoter native to the finitely proliferative cells is the promoter of the Blimp1 gene (Blimp1 promoter), which is specifically expressed in plasma cells. Examples of promoters not native to the finitely proliferative cells include promoters derived from animal cell viruses and artificial promoters modified from such promoters. Preferred promoters for use in the present invention include the Piggyback promoter, CAG promoter, CBh promoter, SV40 promoter, SRα promoter, and hCMV promoter, due to their widespread use and extensive track record. The promoters for the SV40 T antigen gene and the like may all be of the same type or may be heterologous.

[0036] The expression cassette may further contain one or more of a terminator that functions in finitely proliferative cells, an enhancer that functions in finitely proliferative cells, a 5'-untranslated region, and a 3'-untranslated region. The terminator may be a terminator that is naturally present in finitely proliferative cells or a terminator that is not naturally present in finitely proliferative cells. Examples of such enhancers include the Eu enhancer.

[0037] A non-viral vector such as a plasmid vector can be used as a vector to incorporate an expression cassette containing an SV40 T antigen gene or the like. A plasmid vector can be produced by incorporating the expression cassette into a vector having a circular DNA structure. Instead of a plasmid vector, a vector having a linear DNA structure into which an expression cassette has been incorporated can also be used.

[0038] When preparing a transformant in which the expression cassette is maintained as an extrachromosomal gene in a finitely proliferative cell, the vector is preferably a plasmid containing an autonomously replicating sequence (ARS), i.e., a sequence for replication within the finitely proliferative cell. On the other hand, when preparing a transformant in which the expression cassette is integrated into the chromosome of a finitely proliferative cell, the vector is preferably a vector with a linear DNA structure that does not contain an ARS. Alternatively, the vector may be a plasmid vector without an ARS that contains a restriction enzyme recognition sequence for cutting into linear DNA when introduced into a finitely proliferative cell.

[0039] When the vector is introduced into the chromosome of a finitely proliferative cell, the vector has homologous recombination sites upstream and downstream of the expression cassette, each consisting of a nucleotide sequence that allows homologous recombination with a target site of homologous recombination in the chromosome of the finitely proliferative cell. The homologous recombination site can be appropriately designed based on the nucleotide sequence information of the genomic DNA of the finitely proliferative cell. The target site for homologous recombination in the chromosome of the finitely proliferative cell may be only one site in the chromosome of the finitely proliferative cell, or multiple sites in the chromosome may be used as target sites. When multiple sites in the chromosome are used as target sites, it is preferable to use a transposon as the target site. For example, by introducing into a finitely proliferative cell a vector incorporating an expression cassette for polycistronic expression containing an SV40 T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family gene and the cyclin D family gene, between the 5' transposon-specific repeat sequence and the 3' transposon-specific repeat sequence, the expression cassette is incorporated into multiple transposons present in the chromosome of the finitely proliferative cell, resulting in the production of immortalized transformed cells (antibody-producing cells).

[0040] When the vector into which an expression cassette containing an SV40 T antigen gene or the like is incorporated is a non-viral vector, introduction into finitely proliferating cells can be carried out by appropriately selecting from known transfection methods such as electroporation, microinjection, lipofection, calcium phosphate method, etc.

[0041] The vector into which the expression cassette containing the SV40 T antigen gene or the like is incorporated may be a viral vector. Various known viral vectors used for gene transfer into primary culture cells, such as lentiviral vectors, adeno-associated viral vectors, and retroviral vectors, can be used as the viral vector. Synthesis of each viral vector and subsequent production of a virus into which an expression cassette containing the SV40 T antigen gene or the like has been incorporated can be carried out by standard methods using a commercially available viral expression kit.

[0042] The cultivation of finitely proliferative cells before and during the introduction of a vector incorporating an expression cassette containing an SV40 T antigen gene, etc., and the cultivation of transformed cells obtained after the introduction of the vector can be carried out using the same culture medium and under the same culture conditions as those used to cultivate cells of the same type as the finitely proliferative cells subjected to immortalization.

[0043] The method for producing immortalized cells according to the present invention enables the production of immortalized transformed cells capable of producing antibodies. The biological species of antibody-producing cells is preferably mammalian, and more preferably animal species used to produce monoclonal antibodies that have traditionally been used as reagents, such as humans, mice, rats, rabbits, donkeys, horses, sheep, and goats.

[0044] For example, antibody-producing cells are collected from a mammal and subjected to primary culture, and the resulting primary antibody-producing cells are then introduced with an SV40 T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family gene and cyclin D family gene to produce transformed cells that produce immortalized antibodies. Furthermore, antibodies against the causative microorganism of an infectious disease are produced in the body of a patient suffering from an infectious disease. Therefore, by introducing an SV40 T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family gene and cyclin D family gene into plasma cells capable of producing antibodies collected from a patient suffering from an infectious disease, transformed cells that have acquired the ability to be immortalized while retaining the ability to produce antibodies against the causative microorganism of the infectious disease can be obtained.

[0045] For example, a mammal is immunized with an antigen, and antibody-producing cells are formed in the animal's body. Then, cells are collected from tissues containing antibody-producing cells, such as lymph nodes. The collected lymph node cell population is transfected with an SV40 T antigen gene, a Bcl-2 family gene, and at least one of a Myc family gene and a cyclin D family gene to obtain immortalized transformed cells. From the resulting population of immortalized transformed cells, transformed cells (immortalized antibody-producing cells) that produce antibodies that recognize the antigen used in immunization are screened. This allows for the stable production of antibodies against the target antigen. The resulting immortalized antibody-producing cells are then cultured and the produced antibodies are collected, allowing for the stable production of antibodies against the target antigen. Antibodies produced by immortalized antibody-producing cells, like other antibodies, can be used directly as research reagents and as raw materials for pharmaceutical compositions, hygiene products (e.g., masks), decontamination-related products, and the like. [Example]

[0046] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0047] [Example 1] Anti-rCRP antibody-producing B cells (antibody-producing cells) were isolated from rabbits immunized with C-reactive protein (rCRP), and then immortalized to obtain transformed cells.

[0048] (1) Preparation of immunizing antigens and screening antigens rCRP (manufactured by Oriental Yeast Co., Ltd.) was used as an immunizing antigen, and the same antigen was also used as a screening antigen in the ELISA used later.

[0049] (2) Animals and antigen immunization methods Female rabbits (Japanese white, 2.5-3.0 kg) were used as immunization animals. Immunizations were performed at 2-week intervals using an emulsion prepared by mixing equal amounts of antigen and adjuvant. The first immunization was performed using Freund's complete adjuvant, with the amount of immunizing antigen being 1 mg per rabbit. Subsequent immunizations were performed using Freund's incomplete adjuvant, with the amount of immunizing antigen being 0.5 mg per rabbit.

[0050] [2] Gene transfer and screening (1) Production of expression vectors Expression cassette A (SEQ ID NO: 1: CAG promoter (residues 1-1679), human cyclin D1 gene (residues 1734-2618), human cyclin D1 gene (residues 2619-2684), P2A peptide-encoding sequence (residues 2685-3362), rabbit Bcl-2 gene (residues 3363-3428), P2A peptide-encoding sequence (residues 3429-4745), rabbit c-Myc gene (including a stop codon) (residues 3429-4745), and bovine growth hormone poly(A) signal (residues 4789-5013) was inserted downstream of the CAG promoter in a mammalian gene expression vector (VectorBuilder). Linkages were performed using seamless cloning. Furthermore, as an expression cassette for the SV40 T antigen gene, expression cassette B (sequence number 2: positions 1 to 330 are the SV40 promoter, positions 344 to 2816 are the large T antigen exon, positions 344 to 868 are the small T antigen exon, positions 590 to 935 are the large T antigen intron, and positions 2839 to 2973 are the SV40 poly A signal) was prepared by excising a region containing the SV40 promoter, SV40 T antigen gene, and SV40 poly A signal from SV40 viral DNA, and this was incorporated upstream of the CAG promoter of the expression vector.

[0051] (2) Collection of antibody-producing cells Blood was collected from the immunized animals over time, and the antibody titer of anti-rCRP antibody in the serum was measured by ELISA. After a sufficient increase in antibody titer was confirmed, the popliteal lymph nodes were removed and a suspension of popliteal lymph node cells was prepared according to standard methods.

[0052] (3) Gene transfer A plasmid vector incorporating expression cassette A was introduced into popliteal lymph node cells by electroporation. After gene introduction, the popliteal lymph node cells were suspended in RPMI1640 medium (Sigma) containing 1% by mass methylcellulose (Nacalai Tesque), 30% by volume FBS (fetal bovine serum), and 1% by mass penicillin / streptomycin, and then cultured in a 100 mm dish.

[0053] (4) Cell proliferation The 100 mm dish was cultured stationary for 14 days in a humidified incubator at 37°C and 5% CO2. The cells that proliferated and formed colonies in the medium were transformed cells into which the gene had been introduced and were monoclonal cells. Individual colonies were picked from the 100 mm dish using a pipette under a stereomicroscope, transferred to RPMI 1640 medium (Sigma) containing 10% FBS and 1% P / S, and cultured in a microtiter plate until confluent.

[0054] (5) ELISA The anti-rCRP antibody in the culture supernatant obtained by culturing the transformed cells was detected by ELISA. First, rCRP (0.5 μg / mL) was immobilized on an ELISA plate, which was then blocked with 1% by mass of BSA. The culture supernatant from a 100-mm dish, the medium of which had been replaced the day before, was dispensed onto the ELISA plate and incubated for a predetermined period of time to allow the reaction. This allowed the anti-rCRP rabbit monoclonal antibody in the culture supernatant to bind to the rCRP immobilized in each well of the ELISA plate. Next, horseradish peroxidase-labeled anti-rabbit IgG antibody was dispensed into each well of the ELISA plate and incubated for a specified period of time. The enzyme substrate, peroxidase, was then dispensed, and the fluorescence intensity was measured using a plate reader to detect transformed cells producing anti-rCRP rabbit monoclonal antibody.

[0055] The resulting transformed cells were passaged three times a week in 6-well plates to confirm their proliferation ability and antibody production ability. The number of transformed cells and the amount of anti-rCRP rabbit monoclonal antibody were measured over time. The number of transformed cells was counted under a microscope. The amount of anti-rCRP rabbit monoclonal antibody was measured by ELISA as described above. The cumulative growth curve of the number of transformed cells is shown in Figure 1, and the measurement results of the amount of anti-rCRP rabbit monoclonal antibody (μg / mL) are shown in Figure 2.

[0056] As shown in Figure 1, the resulting transformed cells proliferated for nearly 650 days. Furthermore, the doubling time was approximately 16 hours, and no decline in proliferation rate was observed. In other words, in this example, we artificially produced immortalized suspension cells with a doubling capacity comparable to that of cancer cells such as CHO cells. Furthermore, as shown in Figure 2, the resulting transformed cells produced anti-rCRP rabbit monoclonal antibodies for nearly 60 days. This demonstrates that immortalized transformed cells that maintain antibody production can be obtained simply by introducing the SV40 T antigen gene, Myc family genes, Bcl-2 family genes, and cyclin D family genes into primary cultured antibody-producing cells.

[0057] [3] Purification and electrophoresis of monoclonal antibodies (1) Refining Transformed cells capable of producing antibodies were cultured for expansion. The culture conditions were the same as those described above. Anti-rCRP antibodies were then purified from the culture supernatant using a Protein A column according to standard methods. The purified antibodies were subjected to HPLC at a flow rate of 0.7 mL / min using a size exclusion chromatography (SEC) column (TSKgel G3000SWXL, manufactured by Toshiba). The HPLC chromatogram is shown in Figure 3. The chromatogram showed only one peak, confirming that there was no problem with purity.

[0058] (2) SDS-PAGE SDS-PAGE was performed using the purified antibody according to standard methods. The gel used was "Perfect Nt Gel" (DRC), 1 μg of purified antibody, and "Optibopt Blue" (Abcam) as the staining solution. SDS-PAGE was performed on the reduced antibody (reducing conditions) and the non-reduced antibody (non-reducing conditions). The results of SDS-PAGE are shown in Figure 4. In Figure 4, "R" indicates the lane in which the reduced antibody was run, and "NR" indicates the lane in which the non-reduced antibody was run. The band around 160 kDa corresponds to the complete antibody (an antibody consisting of two antibody heavy chains (H chains) and two antibody light chains (L chains)), the band around 50 kDa corresponds to one H chain, and the band around 25 kDa corresponds to one L chain. These results confirmed that bands at the target molecular weights were obtained under both reducing and non-reducing conditions.

[0059] (3) Western blot The purified antibody was used to transfer the immunizing antigen, rCRP, to a PVDF membrane by SDS-PAGE, and then Western blot analysis was performed on the resulting PVDF membrane according to standard procedures. The PVDF membrane was prepared using "iblot Gel Transfer Stacks PVDF, MINI" (Invitrogen), the blocking solution was "Blocking One" (Nacalai), the secondary antibody was "Anti-IgG, Rabbit, Goat-Poly, HRP" (GeneTex), and the coloring solution was "KPL TMB Membrane Peroxidase Substrate (1-c)" (Sera Care). The results of the Western blot are shown in Figure 5. In Figure 5, lane 1 is the lane containing the positive control "Anti-C Reactive Protein Antibody [Y284] (ab32412)" (Abcam), lane 2 is the lane containing the purified antibody sample, and lane M is the lane containing the molecular weight marker. As shown in FIG. 5, the purified antibody bound to the rCRP band in the PVDF membrane and developed a color.

[0060] (4) Antibody gene sequencing To confirm that the antibodies produced by the immortalized transformed cells were monoclonal, the antibody gene sequences were confirmed. First, RNA was extracted from the cells, and cDNA was synthesized by reverse transcription. RNA was extracted from the immortalized transformed cells using NucleoSpin® RNA, and reverse transcription was performed using PrimeScript IV 1st strand cDNA Synthesis Mix (manufactured by TaKaRa). Next, PCR was performed using the cDNA as a template and primer sets (Table 1) specific to the H chain and L chain of the rabbit antibody gene, respectively, and polymerase (PrimeSTAR Max DNA Polymerase, manufactured by TaKaRa) to amplify the antibody genes.

[0061] [Table 1]

[0062] Each PCR fragment obtained was purified using "NucleoSpin" (manufactured by TaKaRa) and then its nucleotide sequence was confirmed by direct sequencing using the primers listed in Table 1. As a result, it was found that the H-chain cDNA of the anti-rCRP antibody produced by the immortalized transformed cells consisted of the nucleotide sequence shown in SEQ ID NO: 7, and the L-chain cDNA consisted of the nucleotide sequence shown in SEQ ID NO: 9. From these nucleotide sequence results, it was found that the anti-rCRP antibody produced by the immortalized transformed cells has the same H-chain amino acid sequence (SEQ ID NO: 8: 1-19 is a signal sequence, 20-43 is frame 1, 44-51 is CDR1, 52-68 is frame 2, 69-75 is CDR2, 76-111 is frame 3, 112-130 is CDR3, 131-141 is frame 4, and 142-464 is γ-chain cDNA). The antibody was confirmed at the genetic level to be a monoclonal antibody based on the amino acid sequence of the L chain (sequence number 10: 1-22 is the signal sequence, 23-48 is frame 1, 49-55 is CDR1, 56-72 is frame 2, 73-75 is CDR2, 76-111 is frame 3, 112-123 is CDR3, 124-133 is frame 4, and 134-237 is the κ chain constant region).

[0063] [Example 2] The SV40 T antigen gene, Bcl-2 family gene, Myc family gene, and cyclin D family gene were transfected into primary cultures of rabbit spleen cells, and the effects on immortalization and antibody production were examined.

[0064] [1] Gene transfer and screening (1) Production of expression vectors Expression vectors were prepared by incorporating the expression cassettes of each gene into one plasmid vector in the combinations shown in Table 2. In Table 2, a "○" in the column for each expression cassette indicates that the expression cassette was incorporated into the expression vector. Expression vector NC is a vector (control vector) in which no expression cassette was incorporated.

[0065] [Table 2]

[0066] Expression vector T was produced by incorporating the expression cassette B used in Example 1 into a control vector as the expression cassette for the SV40 T antigen gene. The expression vector TB was produced by incorporating an expression cassette for the rabbit Bcl-2 gene (sequence number 11: positions 1 to 1679 are the CAG promoter, positions 1734 to 2414 are the rabbit Bcl-2 gene (including the stop codon), and positions 2458 to 2682 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TC was constructed by incorporating an expression cassette for the human cyclin D1 gene (sequence number 12: positions 1 to 1679 are the CAG promoter, positions 1734 to 2621 are the human cyclin D1 gene (including the stop codon), and positions 2665 to 2889 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TM was produced by incorporating an expression cassette for the rabbit c-Myc gene (sequence number 13: positions 1 to 1679 are the CAG promoter, positions 1734 to 3050 are the rabbit c-Myc gene (including the stop codon), and positions 3094 to 3318 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TBC was constructed by incorporating an expression cassette for the human cyclin D1 gene and rabbit Bcl-2 gene (SEQ ID NO: 14: nucleotides 1-1679 are the CAG promoter, nucleotides 1734-2618 are the human cyclin D1 gene, nucleotides 2619-2684 are the nucleotide sequence encoding the P2A peptide, nucleotides 2685-3365 are the rabbit Bcl-2 gene (including the stop codon), and nucleotides 3409-3633 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TBM was produced by incorporating an expression cassette for the rabbit Bcl-2 gene and rabbit c-Myc gene (sequence number 15: positions 1 to 1679 are the CAG promoter, positions 1734 to 2411 are the rabbit Bcl-2 gene, positions 2412 to 2477 are the nucleotide sequence encoding the P2A peptide, positions 2478 to 3794 are the rabbit c-Myc gene (including the stop codon), and positions 3838 to 4062 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TCM was constructed by incorporating an expression cassette for the human cyclin D1 gene and rabbit c-Myc gene (sequence number 16: nucleotides 1-1679 are the CAG promoter, nucleotides 1734-2618 are the human cyclin D1 gene (including the stop codon), nucleotides 2619-2684 are the nucleotide sequence encoding the P2A peptide, nucleotides 2685-4001 are the rabbit c-Myc gene (including the stop codon), and nucleotides 4045-4269 are the bovine growth hormone poly(A) signal) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TBCM was produced by incorporating expression cassettes for the human cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene (expression cassette A used in Example 1) and an expression cassette for the SV40 T antigen gene (expression cassette B) into a control vector. The expression vector BCM was constructed by incorporating expression cassettes (expression cassette A) for the human cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene into a control vector.

[0067] (2) Gene transfer Spleens were removed from rabbits, and suspensions of spleen cells were prepared according to standard procedures. Each expression vector was introduced into the spleen cells by electroporation. The spleen cells were suspended and subsequently cultured in the same manner as in Example 1 for the popliteal lymph node cells after electroporation, and the transfected cells were grown to form colonies.

[0068] (3) Evaluation of cell proliferation Using a stereomicroscope, individual colonies were picked from the 100 mm culture dish with a pipette and transferred to RPMI 1640 medium (Sigma) containing 10% FBS and 1% P / S. The cell mass in the medium was measured by counting under a microscope. Figure 6 shows the cumulative growth curve based on the results of measuring the cumulative cell number over time after gene transfer by electroporation.

[0069] As shown in Figure 6, in transformed cells transfected with expression vector NC (control vector), no proliferation was observed until day 27 of culture after gene transfection. In contrast, in transformed cells transfected with expression cassettes for at least four genes, cell proliferation was observed by day 32 of culture, confirming that plasma cells (antibody-producing cells), which do not normally proliferate in vitro, had acquired immortalization ability. Figure 6 also confirms that the cell proliferation rate differs depending on the type and combination of transfected genes. Expression vector T and expression vector TB did not proliferate until day 19 of culture, while expression vector TC did not proliferate until day 12 of culture, showing significantly slower proliferation rates. Transformed cells transfected with expression vectors containing the Bcl-2 gene expression cassette (TBC, TBCM, TBM, BCM) showed significantly faster cell proliferation rates than transformed cells transfected with expression vectors not containing the Bcl-2 gene expression cassette (TM, TCM). Transformed cells transfected with expression vector TBC, expression vector TBCM, or expression vector TBM showed particularly high proliferation potential.

[0070] (4) Identification of antibody-producing cells The cumulative cell proliferation curve shown in Figure 6 measured the proliferation of all cells obtained by transfection of the recovered spleen cells, including cells other than plasma cells (antibody-producing cells). Therefore, the proportion of antibody-producing cells in the cell population after 19, 25, or 32 days of culture following gene transfer was examined by flow cytometry. Specifically, transformed cells into which each expression cassette had been introduced were fixed and permeabilized using the membrane permeabilization reagent "PerFix nc" (Beckman Coulter), and then stained with a fluorescently labeled goat anti-rabbit IgG antibody ("Goat Anti Rabbit IgG H&L" (AlexaFluor® 647, Abcam). Antibody-producing cells possess endogenous IgG in the cytoplasm and are therefore fluorescently stained with the fluorescently labeled goat anti-rabbit IgG antibody. The stained cells were analyzed by flow cytometry to determine the percentage (%) of fluorescently stained cells (antibody-producing cells) relative to the total cell count. Flow cytometry was performed using a flow cytometer "BD Accuri® C6 Plus" (Becton Dickinson).

[0071] Figure 7 shows the results of flow cytometry of cells cultured for 32 days after gene transfer. Figures 7(A) to 7(I) show the results of flow cytometry of transformed cell groups transfected with expression vector T, expression vector TB, expression vector TC, expression vector TM, expression vector TBC, expression vector TBM, expression vector TCM, expression vector TBCM, and expression vector BCM, respectively. In the figure, "M1" indicates the cell group fluorescently stained with fluorescently labeled goat anti-rabbit IgG antibody, i.e., the antibody-producing cell group.

[0072] The percentage of antibody-producing cells (IgG ratio) (%) was calculated from the flow cytometry results and is shown in Table 3. As a result, proliferation of antibody-producing cells was confirmed only in transformed cells transfected with the expression vector TBC, expression vector TBCM, or expression vector TBM. This demonstrated that antibody-producing cells can be immortalized while maintaining their antibody-producing ability by transfecting them with one or more genes selected from the group consisting of the SV40 T antigen gene, Bcl-2 family genes, Myc family genes, and cyclin D family genes. In particular, cells transfected with the expression vector TBCM, i.e., cells transfected with all four genes (SV40 T antigen gene, Bcl-2 family genes, Myc family genes, and cyclin D family genes), showed a high percentage of antibody-producing cells, demonstrating that transfection of these four genes enables the effective production of immortalized antibody-producing cells.

[0073] [Table 3]

[0074] [Example 3] In Example 2, one gene selected from the group consisting of Bcl-2 family genes, Myc family genes, and cyclin D family genes was replaced with another gene in the same gene family, and the effects on immortalization ability and antibody production ability were examined.

[0075] [1] Gene transfer and screening (1) Production of expression vectors Expression vectors were prepared by incorporating the expression cassettes of each gene into one plasmid vector in the combinations shown in Table 4. In Table 4, a "○" in the column for each expression cassette indicates that the expression cassette was incorporated into the expression vector. Expression vector NC is a vector (control vector) in which no expression cassette was incorporated.

[0076] [Table 4]

[0077] The expression vector TBCM was produced in the same manner as in Example 2. The expression vector TB'CM was constructed by incorporating expression cassettes for the human cyclin D1 gene, human Bcl-XL gene, and rabbit c-Myc gene (SEQ ID NO: 17: nucleotides 1-1679 are the CAG promoter, nucleotides 1734-2618 are the human cyclin D1 gene, nucleotides 2619-2684 are the sequence encoding the P2A peptide, nucleotides 2685-3383 are the human Bcl-XL gene, nucleotides 3384-3449 are the sequence encoding the P2A peptide, nucleotides 3450-4766 are the rabbit c-Myc gene (including the stop codon), and nucleotides 4810-5034 are the bovine growth hormone poly(A) signal) and an SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TBC'M was constructed by incorporating expression cassettes for the human cyclin D3 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene (SEQ ID NO: 18: nucleotides 1-1679 are the CAG promoter, nucleotides 1734-2609 are the human cyclin D3 gene, nucleotides 2610-2675 are the sequence encoding the P2A peptide, nucleotides 2676-3353 are the rabbit Bcl-2 gene, nucleotides 3354-3419 are the sequence encoding the P2A peptide, nucleotides 3420-4736 are the rabbit c-Myc gene (including the stop codon), and nucleotides 4780-5004 are the bovine growth hormone poly(A) signal) and an SV40 T antigen gene (expression cassette B) into a control vector. The expression vector TBCM' was constructed by incorporating expression cassettes for the human cyclin D1 gene, rabbit Bcl-2 gene, and rabbit L-Myc gene (SEQ ID NO: 19: nucleotides 1-1679 are the CAG promoter, nucleotides 1734-2618 are the human cyclin D1 gene, nucleotides 2619-2684 are the sequence encoding the P2A peptide, nucleotides 2685-3362 are the rabbit Bcl-2 gene, nucleotides 3363-3428 are the sequence encoding the P2A peptide, nucleotides 3429-4613 are the rabbit L-Myc gene (including a stop codon), and nucleotides 4657-4881 are the bovine growth hormone poly(A) signal) and an SV40 T antigen gene (expression cassette B) into a control vector.

[0078] (2) Gene transfer Each gene was introduced into rabbit spleen cells in the same manner as in Example 2 to obtain transformed cells.

[0079] (3) Evaluation of cell proliferation The cell mass of each transformed cell was measured over time after gene transfer to evaluate the cell proliferation ability in the same manner as described in Example 2. The cumulative proliferation curve based on the measurement results is shown in Figure 8.

[0080] As shown in Figure 8, in the transformed cells into which the expression vector NC was introduced, no proliferation was observed up to 29 days after gene introduction. In contrast, in the transformed cells into which the expression vectors (TB'CM, TBC'M, TBCM') were introduced, cell proliferation was observed, similar to the transformed cells into which the expression vector (TBCM) was introduced, confirming that they had acquired immortalization ability. Furthermore, Figure 8 confirmed that the cell proliferation rate differed depending on the type and combination of genes introduced, as in Example 2. The transformed cells introduced with the expression vectors (TB'CM, TBC'M) showed a cell proliferation rate similar to that of the transformed cells introduced with the expression vector (TBCM). Although the cell proliferation rate of the transformed cells introduced with the expression vector (TBCM') was slightly slower than that of the transformed cells introduced with the expression vectors (TBCM, TB'CM, TBC'M), this rate was not problematic for commercial use.

[0081] (4) Identification of antibody-producing cells The cumulative cell proliferation curve shown in Figure 8 measured the proliferation of all cells obtained by transfecting the recovered spleen cells, including cells other than plasma cells (antibody-producing cells). Therefore, the proportion of antibody-producing cells in the cell population after 14, 21, or 28 days of culture following gene transfer was examined by flow cytometry. Specifically, the procedure was similar to that described in Example 2.

[0082] The percentage of antibody-producing cells (IgG ratio) (%) was calculated from the flow cytometry results and is shown in Table 5. As a result, proliferation of antibody-producing cells was confirmed in all combinations. These results demonstrated that immortalization was possible while maintaining antibody production ability, regardless of whether the Bcl-2 family gene used was replaced from the Bcl-2 gene to the Bcl-XL gene, the Myc family gene used was replaced from the c-Myc gene to the L-Myc gene, or the cyclin D family gene used was replaced from the cyclin D1 gene to the cyclin D3 gene. In other words, it was revealed that the Bcl-2 family genes, Myc family genes, and cyclin D family genes used in the present invention were capable of immortalizing plasma cells while maintaining antibody production ability, regardless of whether any gene from each gene family was used.

[0083] [Table 5]

[0084] [Example 4] In Example 2, the SV40 T antigen gene of the expression vector TBCM was examined for its effect on immortalization ability when it contained only the SV40 large T antigen gene, only the SV40 small T antigen gene, or both the SV40 large T antigen gene and the SV40 small T antigen gene.

[0085] [1] Gene transfer and screening (1) Production of expression vectors Expression vectors were prepared by incorporating the expression cassettes of each gene into one plasmid vector in the combinations shown in Table 6. In Table 6, a "○" in the column for each expression cassette indicates that the expression cassette was incorporated into the expression vector. Expression vector NC is a vector (control vector) in which no expression cassette was incorporated.

[0086] [Table 6]

[0087] The expression vector (largeT+smallT) was produced in the same manner as in Example 2 for the expression vector TBCM. The expression vector (largeT) was produced by incorporating into a control vector an expression cassette for the human cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene (expression cassette A used in Example 1), and an expression cassette for the SV40 large T antigen gene (sequence number 20: SV40 promoter from positions 1 to 330, gene encoding large T antigen (including stop codon) from positions 344 to 2470, SV40 poly(A) signal from positions 2493 to 2627). The expression vector (smallT) was produced by incorporating into a control vector an expression cassette for the human cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene (expression cassette A used in Example 1), and an expression cassette for the SV40 T antigen gene (sequence number 21: SV40 promoter at positions 1 to 330, gene encoding small T antigen (including stop codon) at positions 344 to 868, SV40 poly(A) signal at positions 891 to 1025).

[0088] (2) Gene transfer Each gene was introduced into rabbit spleen cells in the same manner as in Example 2 to obtain transformed cells.

[0089] (3) Evaluation of cell proliferation The cell mass of each transformed cell was measured over time after gene transfer to evaluate the cell proliferation ability in the same manner as described in Example 2. The cumulative proliferation curve based on the measurement results is shown in Figure 9.

[0090] As shown in Figure 9, in the transformed cells into which the expression vector NC was introduced, no proliferation was observed up to 28 days after gene introduction. In contrast, in all transformed cells into which the expression vectors (largeT+smallT, largeT, smallT) were introduced, cell proliferation was confirmed, confirming that they had acquired immortalization ability. Figure 9 confirms that the cell proliferation rate differs depending on the type and combination of genes introduced. Specifically, transformed cells introduced with the expression vector (largeT + smallT) showed the fastest proliferation rate, with the cell proliferation rate varying in the following order: expression vector (largeT + smallT) > expression vector (largeT) > expression vector (smallT). For transformed cells introduced with the expression vector (smallT), proliferation was confirmed only around day 10 of culture. When only the SV40 small T antigen gene was introduced, proliferation was promoted but very slow. These results clearly demonstrate that the SV40 T antigen gene introduced into plasma cells in the present invention preferably contains the SV40 large T antigen gene. While only the SV40 large T antigen gene may be used, it is particularly preferable to contain both the SV40 large T antigen gene and the SV40 small T antigen gene.

Claims

1. A method for producing immortalized cells, comprising introducing one or more genes selected from the group consisting of an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene into non-immortalized antibody-producing cells ex vivo to produce immortalized cells.

2. The method for producing immortalized cells according to claim 1, wherein the Myc family gene is a c-Myc gene or an L-Myc gene.

3. The method for producing immortalized cells according to claim 1, wherein the Bcl-2 family gene is a Bcl-2 gene or a Bcl-XL gene.

4. The method for producing immortalized cells according to claim 1, wherein the cyclin D family gene is a cyclin D1 gene or a cyclin D3 gene.

5. 2. The method for producing immortalized cells according to claim 1, wherein an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene are introduced into the non-immortalized antibody-producing cells.

6. A method for producing immortalized antibody-producing cells, comprising introducing one or more genes selected from the group consisting of an SV40 T antigen gene, a Bcl-2 family gene, a Myc family gene, and a cyclin D family gene into antibody-producing cells collected from a mammal, or into primary antibody-producing cells obtained by primary culturing the antibody-producing cells, to produce immortalized antibody-producing cells.

7. the mammal is an animal immunized with an antigen, The method for producing immortalized antibody-producing cells according to claim 6, wherein the immortalized antibody-producing cells are screened for immortalized antibody-producing cells that produce an antibody that recognizes the antigen.

8. A method for producing an antibody, comprising producing immortalized antibody-producing cells by the method for producing immortalized antibody-producing cells according to claim 6 or 7, culturing the immortalized antibody-producing cells, and recovering the produced antibody.

9. A transformed cell in which one or more genes selected from the group consisting of an exogenous SV40 T antigen gene, an exogenous Bcl-2 family gene, an exogenous Myc family gene, and an exogenous cyclin D family gene have been introduced into a non-immortalized antibody-producing cell, thereby immortalizing the cell.

10. The transformed cell according to claim 9, which has the ability to produce an antibody.

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