Method for producing immortalized cells

Abstract

The purpose of the present invention is to provide a method for causing cell immortalization of an antibody, and an immortalized transformed cell obtained by the method. The present invention relates to: a method for producing an immortalized cell by introducing, into a non-immortalized antibody-producing cell, an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of a Myc family gene and a Cyclin D family gene; the invention also relates to a manufacturing method of the immortalized antibody producing cell. An immortalized antibody-producing cell is produced by collecting an antibody-producing cell from a mammal, performing primary culture, and introducing, into the obtained primary antibody-producing cell, an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of a Myc family gene and a Cyclin D family gene.

Description

Technical Field

[0001] The present invention relates to a method for immortalizing antibody-producing cells and immortalized transformed cells (immortalized transformed cells) obtained by the method.

[0002] This application claims priority based on Japanese Patent Application No. 2022-195145 filed in Japan on December 6, 2022, and the contents of that application are incorporated herein by reference. Background Art

[0003] There are cells with proliferation ability and cells that do not proliferate in cells. Even for cells with proliferation ability, the number of times that they can usually divide is genetically limited and has a limited lifespan. Therefore, for example, primary culture cells collected from animal tissues and cultured cells can only divide a limited number of times even if they proliferate under appropriate conditions, and then lose their proliferation ability. Therefore, for cells with useful traits, attempts have been made to remove the restrictions on the number of divisions to obtain the so-called immortalization of the ability to divide without limit. Generally speaking, cells obtain immortalization ability through transformation.

[0004] Monoclonal antibodies are widely used as a reagent for detecting target substances using immune reactions. In addition, in recent years, human monoclonal antibodies have been used as active ingredients in antibody medicines, and monoclonal antibodies have industrial utilization value. In most cases, monoclonal antibodies are produced by antibody-producing cells. In order to stably supply monoclonal antibodies, antibody-producing cells are required to be immortalized without damaging their antibody-producing ability so that industrial-scale in vitro culture can be achieved.

[0005] Hybridoma technology has been established for the production of monoclonal antibodies in rodents. Hybridoma is a method of immunizing animals, isolating single B cells that produce the desired monoclonal antibodies, culturing them in primary culture, and fusing the B cells with myeloma cells (myeloma). The resulting hybridoma is immortalized while maintaining its ability to produce antibodies. However, there is no suitable hybridoma technology for animals other than rodents, so the immortalization of antibody-producing cells requires the use of complex genetic engineering methods for transformation.

[0006] As a method for immortalizing primary cultured cells, for example, there is a method known in which three cancer-related genes, namely, the Bcl-2 (B-cell / CLL lymphoma 2) gene, the Myc gene, and the Ccnd1 gene, are introduced into proB cells for transformation to achieve immortalization (Non-Patent Document 1). In addition, there is a method known in which two cancer-related genes, namely, the c-Myc gene and the SV40 (simian virus 40) large T antigen gene, are introduced into human fetal fibroblasts for transformation to achieve immortalization (Non-Patent Document 2).

[0007] Prior Art Documents

[0008] Non-Patent Documents

[0009] Non-Patent Document 1: Nakagawa, et al , Haematologica, 2011, vol.96(9), p.1318-1326.

[0010] Non-Patent Document 2: Kim, et al , Experimental and Molecular Medicine, 2001,vol.33(4), p.293-298. Summary of the Invention

[0011] Problems to be Solved by the Invention

[0012] Antibody-producing cells, which are terminally differentiated cells, unlike proB cells, cannot acquire immortalization ability even when transformed by introducing the Bcl-2 gene, the Myc gene, and the Ccnd1 gene. In addition, when the c-Myc gene and the SV40 large T antigen gene are introduced into antibody-producing cells for transformation, although they can acquire proliferation ability and become immortal, they cannot maintain their characteristics and cannot obtain immortalized cells that retain antibody production ability.

[0013] An object of the present invention is to provide a method for immortalizing antibody-producing cells and immortalized transformed cells obtained by this method.

[0014] Means for Solving the Problems

[0015] To solve the above problems, the present inventors conducted in-depth research and found that by introducing the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D (cyclin D) family genes into antibody-producing cells collected from mammals immunized with an antigen for transformation, immortalization can be achieved while maintaining their antibody-producing ability, thus completing the present invention.

[0016] That is, the present invention provides technical solutions such as the following medicaments.

[0017] [1] A method for producing immortalized cells, which produces immortalized cells by introducing the SV40T antigen gene, Bcl-2 family genes, and one or more genes selected from the group consisting of Myc family genes and Cyclin D family genes into non-immortalized antibody-producing cells.

[0018] [2] The method for producing immortalized cells according to [1], wherein the Myc family gene is the c-Myc gene or the L-Myc gene.

[0019] [3] The method for producing immortalized cells according to [1] or [2], wherein the Bcl-2 family gene is the Bcl-2 gene or the Bcl-XL gene.

[0020] [4] The method for producing immortalized cells according to any one of [1] to [3], wherein the Cyclin D family gene is the Cyclin D1 gene or the Cyclin D3 gene.

[0021] [5] The method for producing immortalized cells according to any one of [1] to [3], wherein the SV40T antigen gene, Bcl-2 family genes, Myc family genes, and Cyclin D family genes are introduced into the non-immortalized antibody-producing cells.

[0022] [6] A method for producing immortalized antibody-producing cells, which produces immortalized antibody-producing cells by collecting antibody-producing cells from a mammal for primary culture and introducing the SV40T antigen gene, Bcl-2 family genes, and one or more genes selected from the group consisting of Myc family genes and Cyclin D family genes into the obtained primary antibody-producing cells.

[0023] [7] The method for producing immortalized antibody-producing cells according to [6], wherein the mammal is an animal immunized with an antigen, and immortalized antibody-producing cells that produce antibodies recognizing the antigen are screened from the immortalized antibody-producing cells.

[0024] [8] A method for producing an antibody, wherein immortalized antibody-producing cells are produced by the method for producing immortalized antibody-producing cells described in [6] or [7], the immortalized antibody-producing cells are cultured, and the produced antibody is recovered.

[0025] [9] A transformed cell, which is an immortalized transformed cell obtained by introducing an exogenous SV40 T antigen gene, an exogenous Bcl-2 family gene, and one or more genes selected from the group consisting of an exogenous Myc family gene and an exogenous Cyclin D family gene into non-immortalized antibody-producing cells.

[0026]

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

[0027] Advantages of the Invention

[0028] Based on the method for producing immortalized cells of the present invention, it is possible to relatively simply immortalize antibody-producing cells obtained by primary passage culture, etc., and to obtain transformed cells that have acquired immortalization ability while maintaining their antibody-producing ability. The transformed cells thus obtained are useful, for example, for the production of monoclonal antibodies. Brief Description of the Drawings

[0029] Figure 1 It is a graph showing the cumulative growth curve of transformed cells immortalized from rabbit-derived plasma cells producing anti-rCRP rabbit monoclonal antibodies in Example 1.

[0030] Figure 2 It is a graph showing the time-course change in the amount of anti-rCRP rabbit monoclonal antibody of transformed cells immortalized from rabbit-derived plasma cells producing anti-rCRP rabbit monoclonal antibodies in Example 1.

[0031] Figure 3 It is a chromatogram of high performance liquid chromatography of the culture supernatant of immortalized transformed cells producing anti-rCRP rabbit monoclonal antibodies in Example 1.

[0032] Figure 4 It is a graph showing 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.

[0033] Figure 5 It is a graph showing the results of Western blotting using purified anti-rCRP rabbit monoclonal antibody in Example 1.

[0034] Figure 6 It is a graph showing the cumulative growth curve of transformed cells into which each expression cassette has been introduced in Example 2.

[0035] Figure 7 This figure shows the results of flow cytometry analysis of a cell population in which transformed cells into which each expression cassette was introduced were stained with anti-rabbit IgG antibody in Example 2.

[0036] Figure 8 This figure shows the cumulative growth curves of transformed cells into which each expression cassette was introduced in Example 3.

[0037] Figure 9 This figure shows the cumulative growth curves of transformed cells into which each expression cassette was introduced in Example 4. Detailed implementation mode

[0038] The method for producing immortalized cells of the present invention is a method for producing immortalized cells by introducing the SV40T antigen gene, the Bcl-2 family gene, and one or more genes selected from the group consisting of the Myc family gene and the Cyclin D family gene into non-immortalized antibody-producing cells. By introducing and expressing these combinations of cancer-related genes into non-immortalized antibody-producing cells, the limit on the number of cell divisions is lifted, and the ability to divide is imparted, enabling the acquisition of immortalized cells. Generally, differentiated plasma cells cannot divide in vitro, but by the method for producing immortalized cells of the present invention, immortalized cells with a doubling ability comparable to that of cancer cells such as CHO cells (Chinese hamster ovary cells) can be artificially produced.

[0039] In the method for producing immortalized cells of the present invention, the three genes of the SV40T antigen gene, the Bcl-2 family gene, and the Myc family gene can be introduced into non-immortalized antibody-producing cells, or the three genes of the SV40T antigen gene, the Bcl-2 family gene, and the Cyclin D family gene can be introduced into non-immortalized antibody-producing cells, or the four genes of the SV40T antigen gene, the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene can be introduced into non-immortalized antibody-producing cells. No matter which combination is introduced, the ability to immortalize antibody-producing cells can be imparted. For the method for producing immortalized cells of the present invention, from the perspective of being able to produce more transformed cells that have acquired immortalization ability while maintaining their antibody-producing ability, it is particularly preferred to introduce the four genes of the SV40T antigen gene, the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene.

[0040] The SV40T antigen gene is a viral gene, which is known to promote the immortalization of cells of a large number of biological species by inactivating tumor suppressor genes that induce replicative senescence of cells. In the present invention and the specification of this application, both the SV40 large T antigen and the SV40 small T antigen are collectively referred to as the "SV40T antigen".

[0041] The so-called "SV40 large T antigen" includes all proteins having the function of inactivating tumor suppressor genes that induce replicative senescence in cells, just like the natural SV40 large T antigen. In addition, the meaning of "natural" refers to proteins encoded by genomic DNA of any biological species. Specifically, in addition to proteins composed of the same amino acid sequence as the natural SV40 large T antigen, it also includes mutants of the natural SV40 large T antigen, that is, proteins composed of amino acid sequences obtained by deleting, substituting or adding one or more amino acids in the same amino acid sequence as the natural SV40 large T antigen and retaining the ability to inactivate the tumor suppressor gene. In addition, the SV40 large T antigen encoded by the SV40T antigen gene introduced into non-immortalized cells in the present invention can also be a fusion protein obtained by directly or indirectly connecting various tags and other structural proteins to the natural SV40 large T antigen or its mutants through an appropriate linker sequence (linking sequence). In addition, the amino acid sequence of the natural SV40 large T antigen has been registered in the international nucleotide sequence database (INSD) (INSD accession number: AAB59924).

[0042] The so-called "SV40 small T antigen" includes all proteins having the function of binding to protein phosphatase 2 (PP2A) and inactivating PP2A, just like the natural SV small T antigen. Specifically, in addition to proteins composed of the same amino acid sequence as the natural SV40 small T antigen (INSD accession number: AAB59925), it also includes mutants of the natural SV40 small T antigen, that is, proteins composed of amino acid sequences obtained by deleting, substituting or adding one or more amino acids in the same amino acid sequence as the natural SV40 small T antigen and retaining its ability to inactivate PP2A. In addition, the SV40 small T antigen encoded by the SV40T antigen gene introduced into non-immortalized cells in the present invention can also be a fusion protein obtained by directly or indirectly connecting various tags and other structural proteins to the natural SV40 small T antigen or its mutants.

[0043] In the present invention and the specification of the present application, the so-called "SV40 T antigen gene" is, like the natural SV40 large T antigen, a gene encoding a protein having the function of inactivating a tumor suppressor gene that induces cellular replicative senescence in cells, and means a nucleic acid containing a base sequence encoding the SV40 T antigen. As the "SV40 T antigen gene" used in the present invention, it may be a nucleic acid containing both the base sequence encoding the SV40 large T antigen and the base sequence encoding the SV40 small T antigen, or it may be a nucleic acid containing only the base sequence encoding the SV40 large T antigen. As the SV40 T antigen gene introduced into non-immortalized cells in the present invention, when introduced into mammalian cells, there is no particular limitation as long as it is a gene capable of expressing the SV40 T antigen. It may be a gene containing introns and expressing both the SV40 large T antigen and the SV40 small T antigen, or it may be a gene composed only of exons. In addition, various alterations (modifications) may be applied to the base sequence encoding the SV40 large T antigen and the base sequence encoding the SV40 small T antigen in the SV40 T antigen gene, such as changing degenerate codons to codons with a high codon usage frequency in limited proliferative antibody-producing cells (hereinafter sometimes simply referred to as "limited proliferative cells") into which the SV40 T antigen gene is introduced for the purpose of immortalization, and the like. The codon alteration (codon modification) can be carried out by known gene sequence variation techniques or artificial gene synthesis.

[0044] In the present invention and the specification of the present application, the so-called "Myc family" refers to a family of transcription factors containing basic helix-loop-helix (bHLH) and leucine zipper (LZ) motifs. The human Myc family has three transcription factors, namely c-Myc (INSD accession number: AAA36340), L-Myc (INSD accession number: BAG58834), and N-Myc (INSD accession number: AAP36048). In the present invention and the specification of the present application, the "Myc family" includes not only the natural Myc family but also variants (modified forms) obtained by modifying the natural Myc family proteins while maintaining the same transcription factor function as human c-Myc. Specifically, in the Myc family, examples of the natural Myc family include human c-Myc, human L-Myc, and human N-Myc, and their homologs can also be cited. As a homolog of human c-Myc, for example, rabbit c-Myc (INSD accession number: AJC97784) can be cited. As a homolog of human L-Myc, for example, rabbit L-Myc (INSD accession number: XP_002715238) can be cited. In addition, it includes not only proteins composed of the same amino acid sequences as these natural Mycs but also mutants thereof, that is, proteins composed of amino acid sequences obtained by deleting, substituting, or adding one or more amino acids in the same amino acid sequence as the natural Myc and maintaining their transcriptional activity ability. As a mutant of the Myc family, for example, the T58N mutant of human c-Myc can be cited. In addition, in the present invention, the Myc family encoded by the Myc family gene introduced into non-immortalized cells can also be a variant such as a fusion protein obtained by directly or indirectly connecting various tags (Tags) or other structural proteins to the natural Myc family or its mutants via an appropriate linker sequence. As the Myc family gene introduced into non-immortalized cells in the present invention, genes of c-Myc (human c-Myc and its orthologs and their mutants), L-Myc (human c-Myc and its orthologs and their mutants), or variants of all of these are preferred, and genes of human c-Myc, human L-Myc, or their mutants or variants of all of these are more preferred.

[0045] In the present invention and the specification of the present application, the so-called "Myc family gene" means a nucleic acid containing a base sequence encoding the Myc family. As the Myc family gene introduced into non-immortalized cells in the present invention, when introduced into mammalian cells, as long as it can express the Myc family gene, there is no particular limitation. It can be a gene containing introns or a gene composed only of exons. In addition, various changes (modifications) can be made to the base sequence encoding the Myc family protein in the Myc family gene, such as changing degenerate codons to codons with a high frequency of use in limited proliferative cells. The codon change (modification) can be carried out by known gene sequence variation techniques or artificial gene synthesis.

[0046] In the present invention and the specification of the present application, the so-called "Bcl-2 family" means a family of mitochondrial outer membrane proteins having one or more BH (Bcl-2 homology) domains and a highly hydrophobic transmembrane region on the C-terminal side and having an anti-apoptotic effect. In the human Bcl-2 family, as family proteins having an anti-apoptotic effect, Bcl-2 (INSD accession number: AAH27258), Bcl-XL (INSD accession number: AAP35872), MCL-1 (Myeloid cell leukemia sequence 1) (INSD accession number: AAD13299), etc. can be cited.

[0047] The Bcl-2 family gene introduced into non-immortalized cells in the present invention is not particularly limited as long as it is a protein having one or more BH domains, a transmembrane region, and an anti-apoptotic effect. For example, in the present invention and the specification of the present application, the "Bcl-2 family" includes not only the natural Bcl-2 family but also variants obtained by modifying natural Bcl-2 family proteins while maintaining the anti-apoptotic effect in the same manner as human Bcl-2. Specifically, among the Bcl-2 family having an anti-apoptotic effect, as the natural Bcl-2 family, in addition to human Bcl-2, human Bcl-XL, and human MCL-1, their homologs can also be cited. As a homolog of human Bcl-2, for example, rabbit Bcl-2 (INSD accession number: XP_008259661) etc. can be cited. As a homolog of human Bcl-XL, for example, rabbit Bcl-XL (INSD accession number: XP_008254359) etc. can be cited. In addition, in addition to proteins composed of the same amino acid sequence as these natural Bcl-2 families, it also includes mutants thereof, that is, proteins composed of amino acid sequences obtained by deleting, substituting, or adding one or more amino acids in the same amino acid sequence as the natural Bcl-2 family and maintaining their anti-apoptotic effect. In addition, the Bcl-2 family encoded by the Bcl-2 family gene introduced into non-immortalized cells in the present invention can also be a variant such as a fusion protein obtained by directly or indirectly connecting various tags or other structural proteins to the natural Bcl-2 family or its mutants through an appropriate linker sequence. As the Bcl-2 family gene introduced into non-immortalized cells in the present invention, it is preferably a gene of Bcl-2 (human Bcl-2 and its orthologs and their mutants), Bcl-XL (human Bcl-XL and its orthologs and their mutants), or a variant of all of these, and more preferably a gene of human Bcl-2, human Bcl-XL, or their mutants, or a variant of all of these.

[0048] In the present invention and the specification of the present application, the term "Bcl-2 family gene" means a nucleic acid containing a base sequence encoding the Bcl-2 family. As the Bcl-2 family gene introduced into non-immortalized cells in the present invention, when introduced into mammalian cells, as long as it is a gene capable of expressing the Bcl-2 family, it is not particularly limited and can be a gene containing introns or a gene containing only exons. In addition, various modifications such as changing degenerate codons to codons with a high codon usage frequency in limited proliferative cells can also be applied to the base sequence encoding the Bcl-2 family protein in the Bcl-2 family gene. The codon change can be carried out by known gene sequence variation techniques or artificial gene synthesis.

[0049] In the present invention and the specification of the present application, the so-called "Cyclin D family" refers to a family of proteins that have a cyclin box domain that binds to cyclin-dependent kinases (CDKs: cyclin-dependent kinases) and have an RB (Retinoblastoma tumor suppressor protein) binding domain on the N-terminal side, and are synthesized at the start of the G1 phase of the cell cycle and control the transition from the G1 phase to the S phase. In the human Cyclin D family, Cyclin D1 (INSD accession number: AAA58392), Cyclin D2 (NSD accession number: AAA51926), Cyclin D3 (NSD accession number: AAA52137), etc. can be cited.

[0050] As the Cyclin D family gene introduced into non-immortalized cells in the present invention, there is no particular limitation as long as it is a protein having the same S-phase transition control function as human Cyclin D1. For example, in the present invention and the specification of the present application, as the "Cyclin D family", in addition to including the natural Cyclin D family, it also includes variants obtained by modifying the natural Cyclin D family protein while maintaining the S-phase transition control function. Specifically, as the natural Cyclin D family, in addition to human Cyclin D1, human Cyclin D2, and human Cyclin D3, their homologs can also be cited. In addition, in addition to including proteins composed of the same amino acid sequence as these natural Cyclin Ds, it also includes mutants thereof, that is, proteins composed of an amino acid sequence obtained by deleting, substituting, or adding one or more amino acids in the same amino acid sequence as the natural Cyclin D and maintaining its S-phase transition control function. In addition, the Cyclin D family encoded by the Cyclin D family gene introduced into non-immortalized cells in the present invention can also be a variant such as a fusion protein obtained by directly or indirectly connecting various tags and other structural proteins to the natural Cyclin D family or its mutants through an appropriate linker sequence. As the Cyclin D family gene introduced into non-immortalized cells in the present invention, it is preferably the gene of Cyclin D1 (human Cyclin D1 and its orthologs and their mutants), Cyclin D3 (human Cyclin D3 and its orthologs and their mutants), or variants of all of these, and more preferably the gene of human Cyclin D1, human Cyclin D3, or their mutants, or variants of all of these.

[0051] In the method for producing immortalized cells of the present invention, among the genes introduced into non-immortalized cells, as Myc family genes, Bcl-2 family genes, and Cyclin D family genes, they can all be genes derived from the same biological species as the introduced cells, or they can be genes derived from different biological species. Additionally, these three genes can be genes derived from the same biological species or genes derived from different biological species. For example, the SV40T antigen gene, human c-Myc gene, human Bcl-2 gene, and human Cyclin D1 gene can be introduced together into lymph node cells collected from rabbits, the SV40T antigen gene, mouse c-Myc gene, human Bcl-2 gene, and human Cyclin D1 gene can be introduced together into spleen cells collected from mice, and the SV40T antigen gene, human c-Myc gene, human Bcl-2 gene, and human Cyclin D1 gene can be introduced into epithelial cells collected from humans.

[0052] In the method for producing immortalized cells of the present invention, as the cells to be transformed into immortalized cells, as long as they are non-immortalized finite-proliferative cells, there are no particular limitations, and there are no particular limitations on the biological species, tissues, etc. from which they are derived. As the finite-proliferative cells for use in the present invention, cells collected from animals or primary cultured cells obtained by culturing such cells are preferred, and cells collected from mammals or primary cultured cells obtained by culturing such cells are more preferred. As the mammal, there are no particular limitations, but experimental animals such as humans, mice, rats, and monkeys, livestock or pets such as rabbits, pigs, cats, cows, horses, and sheep are more preferred, and humans are particularly preferred. For the collection and primary culture of cells derived from animals, it can be carried out by conventional methods.

[0053] 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, a plasma cell is a mononuclear cell similar to a lymphocyte distributed in the spleen, lymph nodes, bone marrow, connective tissue, etc., and acquires the ability to produce antibodies through antigen stimulation. As the plasma cells for transformation in the method for producing immortalized cells of the present invention, they can be any of the plasma cells before acquiring the antibody-producing ability, the plasma cells having the ability to produce antibodies after acquisition, and the plasma cells that have lost the ability to produce antibodies after acquisition. Those skilled in the art can appropriately select methods such as flow cytometry to screen plasma cells. For the confirmation of the selected plasma cells, hematoxylin-eosin staining, the shape of the nucleus, and the cytoplasm can be confirmed using optical microscope images.

[0054] In the method for producing immortalized cells of the present invention, immortalized transformed cells can be produced by introducing the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D family genes into non-immortalized cells by using genetic engineering methods. These four genes can be all introduced simultaneously or sequentially (in different orders) into non-immortalized cells.

[0055] In the method for producing immortalized cells of the present invention, the SV40T antigen gene, etc. can be introduced into the chromosomes of the cells to be immortalized, or can be introduced as extrachromosomal genes. By introducing the SV40T antigen gene, etc. into the chromosomes, transformed cells with excellent stability in maintaining passage can be obtained.

[0056] As the genetic engineering method used for introducing the SV40T antigen gene, etc. into antibody-producing cells to be immortalized, it can be any known method for introducing foreign genes into limited-proliferative cells, or a method obtained by appropriately modifying a known method. As such a method, for example, a method of introducing a vector containing an expression cassette integrated with a foreign gene into cells can be cited. The SV40T antigen gene, etc. can be expressed in a polycistronic form or a monocistronic form.

[0057] An expression cassette is a combination of DNAs required for expressing a target protein, and includes a structural gene encoding the target protein and a promoter that functions in limited-proliferative cells. In one expression cassette, only one structural gene can be included, or two or more structural genes can be included.

[0058] In the case of monocistronic expression, as the expression cassette used for immortalizing transformation, specifically, the following can be cited: an SV40T antigen-expressing cassette containing the SV40T antigen gene and a promoter, a Myc family-expressing cassette containing a Myc family gene and a promoter, a Bcl-2 family-expressing cassette containing a Bcl-2 family gene and a promoter, and a Cyclin D family-expressing cassette containing a Cyclin D family gene and a promoter.

[0059] Two or more of the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D family genes can be integrated into one expression cassette and expressed in a polycistronic form. For example, even by introducing an expression cassette in which the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D family genes are successively linked via a linker sequence containing a base sequence encoding a self-cleaving peptide downstream of a single promoter into a limited-proliferative cell, immortalized transformed cells can be obtained. As the self-cleaving peptide, it is possible to appropriately select and use from known self-cleaving peptides such as P2A peptide, E2A peptide, and T2A peptide. In the linker sequence connecting the respective genes, an IRES (internal ribosome entry site) sequence can also be included instead of the self-cleaving peptide. As the IRES sequence, it can be an IRES sequence derived from the genome of a virus or an IRES sequence derived from the genome of an animal cell, and can be appropriately selected and used from known IRES sequences.

[0060] As the promoter in the expression cassette, any promoter that functions in a limited-proliferative cell can be used. It can be a promoter that the limited-proliferative cell originally has, or a promoter that the limited-proliferative cell does not originally have. When the limited-proliferative cell is a plasma cell that produces an antibody, as a promoter that the limited-proliferative cell originally has, the promoter of the Blimp1 gene (Blimp1 promoter) specifically expressed in plasma cells can be cited. As a promoter that the limited-proliferative cell does not originally have, for example, a promoter derived from an animal cell virus or an artificial promoter obtained by modifying the promoter can be cited. From the viewpoints of wide use and many usage records, the Piggyback promoter, CAG promoter, CBh promoter, SV40 promoter, SRα promoter, hCMV promoter, etc. are preferred as the promoters used in the present invention. The promoters of the SV40T antigen gene, etc. can all be of the same type or of different types from each other.

[0061] The expression cassette can also further contain a terminator that functions in a limited-proliferative cell, an enhancer that functions in a limited-proliferative cell, and any one or more of a 5'-untranslated region and a 3'-untranslated region. As the terminator, a terminator that the limited-proliferative cell originally has or a terminator that the limited-proliferative cell does not originally have can be used. As the enhancer, the Eu enhancer, etc. can be cited.

[0062] As a vector integrating an expression cassette containing a gene such as the SV40T antigen gene, non-viral vectors such as plasmid vectors can be used. By integrating the expression cassette into a vector having a circular DNA structure, a plasmid vector can be produced. A vector having a linear DNA structure integrated with the expression cassette can also be used instead of the plasmid vector.

[0063] When producing a transformant that maintains the expression cassette as an extrachromosomal gene in a limited-proliferative cell, it is preferable that the vector is a plasmid containing a sequence for replication in a limited-proliferative cell, that is, an autonomous replication sequence (ARS). On the other hand, when producing a transformant that integrates the expression cassette into the chromosome of a limited-proliferative cell, a vector having a linear DNA structure without ARS is preferable. It can also be a plasmid vector without ARS having a restriction endonuclease recognition sequence for cleaving the linear DNA when introduced into a limited-proliferative cell.

[0064] When the vector is introduced into the chromosome of a limited-proliferative cell, homologous recombination sites are present upstream and downstream of the expression cassette in the vector, and the homologous recombination sites are composed of base sequences capable of homologous recombination with the target sites of homologous recombination in the chromosome of the limited-proliferative cell. For the homologous recombination sites, they can be appropriately set according to the base sequence information of the genomic DNA of the limited-proliferative cell. As the target sites of homologous recombination in the chromosome of the limited-proliferative cell, there can be only one site in the chromosome of the limited-proliferative cell, or multiple sites in the chromosome can be used as target sites. When multiple sites in the chromosome are used as target sites, it is preferable to use transposons as target sites. For example, by introducing a vector obtained by integrating an expression cassette for polycistronic expression containing at least one of the SV40T antigen gene, Bcl-2 family gene, Myc family gene, and Cyclin D family gene between the 5'-side transposon-specific repeat sequence and the 3'-side transposon-specific repeat sequence into a limited-proliferative cell, the expression cassette can be integrated into multiple transposons present in the chromosome of the limited-proliferative cell, thereby obtaining an immortalized transformed cell (antibody-producing cell).

[0065] When the vector integrating the expression cassette containing a gene such as the SV40T antigen gene is a non-viral vector, for introduction into a limited-proliferative cell, an appropriate method can be selected from known transfection methods such as electroporation, microinjection, liposome transfection, and calcium phosphate method.

[0066] As a vector integrating an expression cassette containing a gene such as the SV40T antigen gene, it can be a viral vector. As a viral vector, various well-known viral vectors for gene introduction into primary cultured cells such as lentiviral vectors, adeno-associated viral vectors, and retroviral vectors can be used. For the synthesis of each viral vector and subsequent integration to generate a virus containing an expression cassette such as the SV40T antigen gene, a commercially available virus expression kit can be used and carried out by conventional methods.

[0067] For the culture of finite-proliferative cells before and at the time of introduction of a vector integrating an expression cassette (including a gene such as the SV40T antigen gene) and the culture of transformed cells obtained after the introduction of the vector, a medium for culturing cells of the same type as those for culturing and supplying immortalized finite-proliferative cells can be used and carried out under the same culture conditions.

[0068] By the method for producing immortalized cells of the present invention, immortalized transformed cells having the ability to produce antibodies can be produced. As the biological species of antibody-producing cells, mammals are preferred, and more preferably animal species such as humans, mice, rats, rabbits, donkeys, horses, sheep, and goats that have been used in the production of monoclonal antibodies for use as reagents and the like.

[0069] For example, antibody-producing cells are collected from a mammal and subjected to primary culture. Genes such as the SV40T antigen gene, Bcl-2 family gene, and at least one gene selected from the Myc family gene and the Cyclin D family gene are introduced into the obtained primary antibody-producing cells, and immortalized antibody-producing transformed cells can be produced. In addition, antibodies against pathogenic microorganisms of the infectious disease are produced in the body of a patient suffering from an infectious disease. Therefore, by introducing the SV40T antigen gene, Bcl-2 family gene, and at least one gene selected from the Myc family gene and the Cyclin D family gene into plasma cells having the ability to produce antibodies collected from an infectious disease patient, a transformed cell can be obtained that has acquired immortalization ability while maintaining the ability to produce antibodies against the pathogenic microorganisms of the infectious disease.

[0070] For example, after immunizing a mammal with an antigen and forming antibody-producing cells in the body of the animal, cells are collected from tissues containing antibody-producing cells, such as lymph nodes. By introducing genes encoding SV40 T antigen, Bcl-2 family genes, and at least one gene selected from Myc family genes and Cyclin D family genes into the collected group of lymph node cells, immortalized transformed cells can be obtained. Transformed cells (immortalized antibody-producing cells) that produce antibodies recognizing the antigen used for immunization are screened from the obtained group of immortalized transformed cells. Thus, immortalized antibody-producing cells that produce antibodies recognizing the target antigen can be obtained. The obtained immortalized antibody-producing cells are cultured, and the produced antibodies are recovered, enabling the stable production of antibodies against the target antigen. Similar to other antibodies, the antibodies produced using immortalized antibody-producing cells can be directly used as research reagents, and can also be used as raw materials for pharmaceutical compositions, hygiene products (such as masks), and pollution removal-related products.

[0071] Example

[0072] Next, the present invention will be further described in detail by way of examples, but the present invention is not limited to the following examples.

[0073] [Example 1]

[0074] B cells (antibody-producing cells) that produce anti-rCRP antibodies were collected from a rabbit immunized with C-reactive protein (rCRP), and immortalized transformed cells were obtained.

[0075] (1) Preparation of Immunizing Antigen and Screening Antigen

[0076] rCRP (manufactured by Oriental Yeast Co., Ltd., Japan) was used as the immunizing antigen. In addition, the same antigen was used as the screening antigen for ELISA used thereafter.

[0077] (2) Animals and Antigen Immunization Method

[0078] Female rabbits (Japanese white breed, 2.5 - 3.0 kg) were used as the immunized animals. For immunization, emulsions prepared by mixing equal amounts of the antigen and adjuvant were used for immunization at two-week intervals. Freund Complete Adjuvant was used as the adjuvant for the primary immunization so that the amount of immunizing antigen per rabbit reached 1 mg. Freund Incomplete Adjuvant was used as the adjuvant for the second and subsequent immunizations so that the amount of immunizing antigen per rabbit reached 0.5 mg.

[0079] [2] Gene Introduction and Screening

[0080] (1) Preparation of expression vector

[0081] An expression cassette A (SEQ ID NO: 1: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2618 are the human Cyclin D1 gene, nucleotides 2619 to 2684 are the nucleotide sequence encoding the P2A peptide, nucleotides 2685 to 3362 are the rabbit Bcl-2 gene, nucleotides 3363 to 3428 are the nucleotide sequence encoding the P2A peptide, nucleotides 3429 to 4745 are the rabbit c-Myc gene (including the stop codon), nucleotides 4789 to 5013 are the bovine growth hormone polyadenylation signal [polyA signal]) obtained by sequentially linking the human Cyclin D1 gene, the rabbit Bcl-2 gene, and the rabbit c-Myc gene through a linker sequence containing the P2A peptide as a self-cleaving peptide was integrated downstream of the CAG promoter of a mammalian gene expression vector (manufactured by VectorBuilder). Ligation was performed using the seamless cloning method. In addition, as an expression cassette for the SV40 T antigen gene, an expression cassette B (SEQ ID NO: 2: nucleotides 1 to 330 are the SV40 promoter, nucleotides 344 to 2816 are the large T antigen exon, nucleotides 344 to 868 are the small T antigen exon, nucleotides 590 to 935 are the large T antigen intron, nucleotides 2839 to 2973 are the SV40 polyadenylation signal) obtained by cutting out the region containing the SV40 promoter, the SV40 T antigen gene, and the SV40 polyadenylation signal from the SV40 virus DNA was prepared and integrated upstream of the CAG promoter of the expression vector.

[0082] (2) Collection of antibody-producing cells

[0083] Blood was collected from the immunized animal over time, and the antibody titer of the anti-rCRP antibody in the serum was measured by ELISA. After confirming a sufficient increase in the antibody titer, the popliteal lymph nodes were excised, and a suspension of popliteal lymph node cells was prepared by a conventional method.

[0084] (3) Gene transfer

[0085] The plasmid vector integrated with the expression cassette A was introduced into the popliteal lymph node cells by electroporation. The cell suspension of the popliteal lymph node cells after gene transfer and subsequent culture were suspended in RPMI1640 medium (manufactured by Sigma) containing 1% by mass of methyl cellulose (manufactured by NACALAITESQUE), 30% by volume of FBS (fetal bovine serum), and 1% by mass of penicillin / streptomycin, and cultured in a 100 mm culture dish.

[0086] (4) Cell proliferation

[0087] The 100 mm Petri dish was statically cultured for 14 days in an incubator at 37 °C, 5% CO₂ by volume, and in a humid state. The cells that proliferated and colonized (formed colonies) in this culture medium were transformed cells into which genes had been introduced and were monoclonal cells. From the 100 mm Petri dish during culture, each colony was collected using a pipette under a stereomicroscope and transferred to RPMI 1640 medium (manufactured by Sigma Corporation) containing 10% FBS and 1% P / S, and cultured in a microtiter plate until confluent.

[0088] (5) ELISA

[0089] The anti-rCRP antibody in the culture supernatant obtained by culturing the transformed cells was detected by the ELISA method.

[0090] First, rCRP (0.5 μg / mL) was immobilized on an ELISA plate, and then the ELISA plate was blocked with 1% BSA by mass. Thereafter, the culture supernatant of the 100 mm Petri dish in which the culture medium had been exchanged the previous day was dispensed into this ELISA plate and reacted by culturing for a specified time. Thereby, the anti-rCRP rabbit monoclonal antibody in the culture supernatant was bound to the rCRP in each well immobilized on the ELISA plate.

[0091] Next, by dispensing a horseradish peroxidase-labeled anti-rabbit IgG antibody into each well of the ELISA plate, culturing for a specified time, dispensing peroxidase as an enzyme substrate, and measuring the fluorescence intensity using a microplate reader, the transformed cells that produced the anti-rCRP rabbit monoclonal antibody were detected.

[0092] For the obtained transformed cells, in order to confirm the proliferation ability and antibody production ability, subculture was performed three times a week using a 6-well plate. The cell number of the transformed cells and the amount of the anti-rCRP rabbit monoclonal antibody were measured over time. The cell number of the transformed cells was counted under a microscope. The amount of the anti-rCRP rabbit monoclonal antibody was measured by the same ELISA as above. Figure 1 The cumulative growth curve of the cell number of the transformed cells is shown, Figure 2 The measurement results of the amount (μg / mL) of the anti-rCRP rabbit monoclonal antibody are shown.

[0093] As Figure 1 shown, as a result, the obtained transformed cells also proliferated even at around 650 days. In addition, the doubling time was about 16 hours, and no attenuation of the proliferation rate was observed. That is, in this example, immortalized cells of a suspension cell line having a doubling ability comparable to that of cancer cells such as CHO cells could be artificially produced. In addition, as Figure 2As shown, the obtained transformed cells produced anti-rCRP rabbit monoclonal antibodies for nearly 60 days, indicating that immortalized transformed cells can be obtained while maintaining their antibody-producing ability simply by introducing the SV40T antigen gene, Myc family gene, Bcl-2 family gene, and Cyclin D family gene into primary cultured antibody-producing cells.

[0094] [3] Purification and electrophoresis of monoclonal antibodies

[0095] (1) Purification

[0096] Transformed cells with the ability to produce antibodies were used for expansion culture. The culture conditions were set to be cultured in the same manner as described above. Thereafter, the anti-rCRP antibody was purified from the obtained culture supernatant by a Protein A column according to a conventional method. For the purified antibody, a high performance liquid chromatography (HPLC) analysis (HPLC) was performed using a size exclusion chromatography (SEC) column (TSKgel G3000SWXL, manufactured by Tosoh Corporation) at a flow rate of 0.7 mL / min. Figure 3 The chromatogram of high performance liquid chromatography is shown in . In this chromatogram, there is only one peak, and it is confirmed that there is no problem with the purity.

[0097] (2) SDS-PAGE

[0098] Using purified antibodies, SDS-PAGE was performed according to conventional methods. The gel used was "Perfect Nt Gel" (manufactured by DRC Co., Ltd.), 1 μg of purified antibodies was used, and the staining solution was "Optibopt Blue" (manufactured by Abcam). SDS-PAGE was performed on antibodies that had been reduced (reducing conditions) and antibodies that had not been reduced (non-reducing conditions). Figure 4 The results of SDS-PAGE are shown in FIG. Figure 4 In the above, "R" is a lane where the antibody subjected to reduction treatment flows, and "NR" is a lane where the antibody subjected to non-reduction treatment flows. The band near 160 kDa is a band of a complete antibody (an antibody composed of two antibody heavy chains (H chains) and two antibody light chains (L chains)), the band near 50 kDa is a band of one H chain, and the band near 25 kDa is a band of one L chain. As a result, it was confirmed that a band of the target molecular weight was obtained under both reducing and non-reducing conditions.

[0099] (3) Western blotting

[0100] After subjecting rCRP as an immunizing antigen to SDS-PAGE using a purified antibody and transferring it to a PVDF membrane, Western blotting was performed on the obtained PVDF membrane by a conventional method. The PVDF membrane used was "iblot Gel TransferStacks PVDF, MINI" (manufactured by Invitrogen), the blocking solution used was "Blocking One" (manufactured by NACALAITESQUE), the secondary antibody used was "Anti-IgG, rabbit, Goat-Poly, HRP" (manufactured by Gene Tex), and the color developing solution used was "KPL TMB Membrane Peroxidase Substrate(1-c)" (manufactured by SeraCare). Figure 5 The results of Western blotting are shown in Figure 5 . In Figure 5 , lane 1 is the lane through which "Anti-CReactive Protein antibody [Y284] (ab32412)" (manufactured by Abcam) was run as a positive control, lane 2 is the lane through which the purified antibody sample was run, and lane M is the lane through which the molecular weight marker was run. As

[0101] (4) Sequence of the antibody gene

[0102] To confirm that the antibody produced by the immortalized transformed cells is a monoclonal antibody, the sequence of the antibody gene was confirmed. First, RNA was extracted from the cells, and cDNA was synthesized by reverse transcription reaction. Extraction of RNA from the immortalized transformed cells was performed using "NucleoSpin (registered trademark) RNA", and the reverse transcription reaction used "PrimeScript IV 1st strand cDNA Synthesis Mix" (manufactured by TaKaRa). Then, using the cDNA as a template, PCR was performed using primer sets specific for the H chain and L chain of the rabbit antibody gene (Table 1) and a polymerase ("Prime STAR Max DNA Polymerase", manufactured by TaKaRa) to amplify the antibody gene.

[0103] [Table 1]

[0104] After purifying each of the obtained PCR fragments using "NucleoSpin" (manufactured by TaKaRa), the base sequences were confirmed by direct sequencing using the primers shown in Table 1. As a result, it was found that the cDNA of the H chain of the anti-rCRP antibody produced by the immortalized transformed cells consisted of the base sequence shown in SEQ ID NO: 7, and the cDNA of the L chain consisted of the base sequence shown in SEQ ID NO: 9. Based on the results of these base sequences, it was confirmed at the gene level that the anti-rCRP antibody produced by the immortalized transformed cells was a monoclonal antibody composed of the amino acid sequence of the H chain (SEQ ID NO: 8: positions 1 to 19 are the signal sequence, positions 20 to 43 are framework 1, positions 44 to 51 are CDR1, positions 52 to 68 are framework 2, positions 69 to 75 are CDR2, positions 76 to 111 are framework 3, positions 112 to 130 are CDR3, positions 131 to 141 are framework 4, and positions 142 to 464 are the γ-chain constant region) and the amino acid sequence of the L chain (SEQ ID NO: 10: positions 1 to 22 are the signal sequence, positions 23 to 48 are framework 1, positions 49 to 55 are CDR1, positions 56 to 72 are framework 2, positions 73 to 75 are CDR2, positions 76 to 111 are framework 3, positions 112 to 123 are CDR3, positions 124 to 133 are framework 4, and positions 134 to 237 are the κ-chain constant region).

[0105] [Example 2]

[0106] The SV40T antigen gene, Bcl-2 family genes, Myc family genes, and Cyclin D family genes were combined and introduced into primary cultured cells of rabbit spleen cells, and the effects on the immortalization ability and antibody production ability were studied.

[0107] [1] Gene introduction and screening

[0108] (1) Production of expression vectors

[0109] The expression cassettes of each gene were integrated into a single plasmid vector according to the combinations shown in Table 2 to prepare expression vectors. In Table 2, "○" in the column of each expression cassette indicates that the expression cassette has been integrated into the expression vector. Expression vector NC is a vector without any integrated expression cassette (control vector).

[0110] [Table 2]

[0111] Using expression cassette B used in Example 1 as the expression cassette for the SV40T antigen gene, this expression cassette was integrated into the control vector to prepare expression vector T.

[0112] The expression cassette of rabbit Bcl-2 gene (SEQ ID NO: 11: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2414 are the rabbit Bcl-2 gene (including the stop codon), nucleotides 2458 to 2682 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TB.

[0113] The expression cassette of human Cyclin D1 gene (SEQ ID NO: 12: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2621 are the human Cyclin D1 gene (including the stop codon), nucleotides 2665 to 2889 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TC.

[0114] The expression cassette of rabbit c-Myc gene (SEQ ID NO: 13: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 3050 are the rabbit c-Myc gene (including the stop codon), nucleotides 3094 to 3318 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TM.

[0115] The expression cassette of human Cyclin D1 gene and rabbit Bcl-2 gene (SEQ ID NO: 14: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2618 are the human Cyclin D1 gene, nucleotides 2619 to 2684 are the nucleotide sequence encoding P2A peptide, nucleotides 2685 to 3365 are the rabbit Bcl-2 gene (including the stop codon), nucleotides 3409 to 3633 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TBC.

[0116] The expression cassette of rabbit Bcl-2 gene and rabbit c-Myc gene (SEQ ID NO: 15: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2411 are the rabbit Bcl-2 gene, nucleotides 2412 to 2477 are the nucleotide sequence encoding P2A peptide, nucleotides 2478 to 3794 are the rabbit c-Myc gene (including the stop codon), nucleotides 3838 to 4062 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TBM.

[0117] The expression cassette of human Cyclin D1 gene and rabbit c-Myc gene (SEQ ID NO: 16: the 1st to 1679th is the CAG promoter, the 1734th to 2618th is the human Cyclin D1 gene (including the stop codon), the 2619th to 2684th is the base sequence encoding the P2A peptide, the 2685th to 4001st is the rabbit c-Myc gene (including the stop codon), and the 4045th to 4269th is the bovine growth hormone polyadenylation signal) and the expression cassette of the SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TCM.

[0118] The expression cassette of human Cyclin D1 gene, rabbit Bcl-2 gene and rabbit c-Myc gene (Expression cassette A used in Example 1) and the expression cassette of the SV40T antigen gene (Expression cassette B) were integrated into the control vector to prepare the expression vector TBCM.

[0119] The expression cassette of human Cyclin D1 gene, rabbit Bcl-2 gene and rabbit c-Myc gene (Expression cassette A) was integrated into the control vector to prepare the expression vector BCM.

[0120] (2) Gene introduction

[0121] The spleen was removed from the rabbit, and a suspension of spleen cells was prepared by a conventional method. Using the electroporation method, each expression vector was introduced into the spleen cells. The cell suspension of the spleen cells after gene introduction and subsequent culture were carried out in the same manner as the popliteal lymph node cells after electroporation in Example 1, so that the transformed cells into which the gene was introduced proliferated and colonized.

[0122] (3) Evaluation of cell proliferation ability

[0123] From the 100 mm culture dish during culture, each colony was collected using a pipette through a stereomicroscope and transferred to RPMI1640 medium (manufactured by Sigma) containing 10% FBS and 1% P / S. The cell amount in the medium was measured by counting under a microscope. Figure 6 The cumulative growth curve showing the measurement results based on the cumulative cell number measured over time starting from after gene introduction by electroporation is shown.

[0124] As Figure 6 shown, for the transformed cells into which the expression vector NC (control vector) was introduced, no cell proliferation was confirmed until the 27th day of culture after gene introduction. In contrast, for the transformed cells into which the expression cassette of any one of at least four genes was introduced, cell proliferation was confirmed at the time point of the 32nd day of culture, and it was confirmed that plasma cells (antibody-producing cells) that do not normally proliferate in vitro acquired the immortalization ability. In addition, from Figure 6It was confirmed that the cell proliferation rate varies depending on the type and combination of the introduced genes. No proliferation was confirmed for expression vector T and expression vector TB until the 19th day of culture, and no proliferation was confirmed for expression vector TC until the 12th day of culture, and the proliferation rate was very slow. As for the transformed cells of the expression vectors (TBC, TBCM, TBM, BCM) into which the expression cassette containing the Bcl-2 gene was introduced, the cell proliferation rate was significantly faster than that of the transformed cells of the expression vectors (TM, TCM) into which the expression cassette not containing the Bcl-2 gene was introduced. In particular, the proliferation ability of the transformed cells into which expression vector TBC, expression vector TBCM, or expression vector TBM was introduced was high.

[0125] (4) Confirmation of antibody-producing cells

[0126] Figure 6 The cell cumulative growth curve shown in the figure is the growth curve of all the cells obtained by transforming the recovered spleen cells, and also includes cells other than plasma cells (antibody-producing cells). Therefore, the proportion of antibody-producing cells in the cell population after culturing for 19 days, 25 days, or 32 days after gene introduction was investigated by flow cytometry. Specifically, the transformed cells into which each expression cassette was introduced were fixed using the membrane permeabilization treatment reagent "PerFix nc" (manufactured by BECKMAN COULTER) and subjected to membrane permeabilization treatment, and stained with a fluorescently labeled goat anti-rabbit IgG antibody ("Goat Anti Rabbit IgG H&L (AlexaFluor (registered trademark) 647, manufactured by Abcam). Since antibody-producing cells have endogenous IgG in the cell quality, they were fluorescently stained with the fluorescently labeled goat anti-rabbit IgG antibody. The stained cells were analyzed by flow cytometry, and the proportion (%) of the cells stained with fluorescence (antibody-producing cells) in the total cell number was determined. Flow cytometry was performed using the flow cytometer "BD Accuri (registered trademark) C6 Plus" (manufactured by Becton Dickinson).

[0127] Figure 7 The results of flow cytometry of the cells after culturing for 32 days after gene introduction are shown. Figure 7 (A) to (I) in the figure are the results of flow cytometry of the transformed cell populations into which 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 were introduced. In the figure, "M1" indicates the cell population stained by fluorescence staining using the fluorescently labeled goat anti-rabbit IgG antibody, that is, the antibody-producing cell population.

[0128] Table 3 shows the results of calculating the proportion of antibody-producing cells (IgG ratio) (%) based on the results of flow cytometry. According to these results, only the transformed cells into which the expression vector TBC, the expression vector TBCM, or the expression vector TBM was introduced showed proliferation of antibody-producing cells. From this, it can be seen that by introducing the SV40T antigen gene, the Bcl-2 family gene, and one or more genes selected from the group consisting of the Myc family gene and the Cyclin D family gene into antibody-producing cells, it is possible to immortalize them while maintaining their antibody-producing ability. In particular, in the cells into which the expression vector TBCM was introduced, that is, the cells into which all four genes, namely the SV40T antigen gene, the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene, were introduced, the proportion of antibody-producing cells was high, indicating that immortalized antibody-producing cells can be effectively produced by introducing these four genes.

[0129] [Table 3]

[0130] [Example 3]

[0131] In Example 2, by replacing one gene selected from the group consisting of the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene with other genes within the gene family, the effects on the immortalization ability and antibody-producing ability were investigated.

[0132] [1] Gene introduction and screening

[0133] (1) Preparation of expression vectors

[0134] Expression cassettes of each gene were integrated into a single plasmid vector according to the combinations shown in Table 4 to prepare expression vectors. In Table 4, "○" in the column of each expression cassette indicates that the expression cassette has been integrated into the expression vector. The expression vector NC is a vector (control vector) into which no arbitrary expression cassette has been integrated.

[0135] [Table 4]

[0136] An expression vector TBCM was prepared by the same procedure as that for the expression vector TBCM described in Example 2.

[0137] The expression cassettes of human Cyclin D1 gene, human Bcl-XL gene and rabbit c-Myc gene (SEQ ID NO: 17: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2618 are the human Cyclin D1 gene, nucleotides 2619 to 2684 are the nucleotide sequence encoding P2A peptide, nucleotides 2685 to 3383 are the human Bcl-XL gene, nucleotides 3384 to 3449 are the nucleotide sequence encoding P2A peptide, nucleotides 3450 to 4766 are the rabbit c-Myc gene (including the stop codon), nucleotides 4810 to 5034 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression Cassette B) were integrated into the control vector to prepare the expression vector TB’CM.

[0138] The expression cassettes of human Cyclin D3 gene, rabbit Bcl-2 gene and rabbit c-Myc gene (SEQ ID NO: 18: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2609 are the human Cyclin D3 gene, nucleotides 2610 to 2675 are the nucleotide sequence encoding P2A peptide, nucleotides 2676 to 3353 are the rabbit Bcl-2 gene, nucleotides 3354 to 3419 are the nucleotide sequence encoding P2A peptide, nucleotides 3420 to 4736 are the rabbit c-Myc gene (including the stop codon), nucleotides 4780 to 5004 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression Cassette B) were integrated into the control vector to prepare the expression vector TBC’M.

[0139] The expression cassettes of human Cyclin D1 gene, rabbit Bcl-2 gene and rabbit L-Myc gene (SEQ ID NO: 19: nucleotides 1 to 1679 are the CAG promoter, nucleotides 1734 to 2618 are the human Cyclin D1 gene, nucleotides 2619 to 2684 are the nucleotide sequence encoding P2A peptide, nucleotides 2685 to 3362 are the rabbit Bcl-2 gene, nucleotides 3363 to 3428 are the nucleotide sequence encoding P2A peptide, nucleotides 3429 to 4613 are the rabbit L-Myc gene (including the stop codon), nucleotides 4657 to 4881 are the bovine growth hormone polyadenylation signal) and the expression cassette of SV40T antigen gene (Expression Cassette B) were integrated into the control vector to prepare the expression vector TBCM’.

[0140] (2) Gene transfer

[0141] Using the same operation as described in Example 2, each gene was transferred into rabbit spleen cells to obtain transformed cells.

[0142] (3) Evaluation of cell proliferation ability

[0143] The same operation as the method described in Example 2 was adopted, and the cell amount of each transformed cell was measured over time starting from after gene introduction, and the cell proliferation ability was evaluated. Figure 8 The cumulative growth curve based on the measurement results is shown.

[0144] As Figure 8 shown, for the transformed cells into which the expression vector NC was introduced, no proliferation was confirmed until the 29th day of culture after gene introduction. In contrast, for the transformed cells into which the expression vectors (TB’CM, TBC’M, TBCM’) were introduced, cell proliferation (growth) was confirmed in the same manner as the transformed cells into which the expression vector (TBCM) was introduced, and it was confirmed that the immortalization ability was obtained.

[0145] In addition, it was confirmed that Figure 8 the cell proliferation rate differed depending on the type and combination of the introduced genes in the same manner as in Example 2. As the transformed cells into which the expression vectors (TB’CM, TBC’M) were introduced, the same cell proliferation rate as the transformed cells into which the expression vector (TBCM) was introduced was shown. As the transformed cells into which the expression vector (TBCM’) was introduced, the cell proliferation rate was slightly slower than that of the transformed cells into which the expression vectors (TBCM, TB’CM, TBC’M) were introduced, but this rate is not a problem for commercial use.

[0146] (4) Confirmation of antibody-producing cells

[0147] Figure 8 The cell cumulative growth curve shown was obtained by measuring the growth of all the cells obtained by transforming the recovered spleen cells, and also includes cells other than plasma cells (antibody-producing cells). Therefore, the proportion of antibody-producing cells in the cell population after culturing for 14, 21, or 28 days after gene introduction was investigated by flow cytometry. Specifically, the investigation was carried out using the same operation as the method described in Example 2.

[0148] The results of the proportion of antibody-producing cells (IgG ratio) (%) calculated based on the results of flow cytometry are shown in Table 5. According to these results, the proliferation of antibody-producing cells was confirmed in all combinations. From these results, it was found that even when the Bcl-2 family gene used was replaced from the Bcl-2 gene with the Bcl-XL gene, the Myc family gene used was replaced from the c-Myc gene with the L-Myc gene, or the Cyclin D family gene used was replaced from the Cyclin D1 gene with the Cyclin D3 gene, immortalization could be achieved while maintaining the antibody-producing ability. That is, it was thus found that for the Bcl-2 family gene, Myc family gene, and Cyclin D family gene used in the present invention, even when any gene within each gene family was used, plasma cells could be immortalized while maintaining their antibody-producing ability.

[0149] [Table 5]

[0150] [Example 4]

[0151] For the cases where the SV40 T antigen gene of the expression vector TBCM in Example 2 was set only to the SV40 large T antigen gene, only to the SV40 small T antigen gene, and to both the SV40 large T antigen gene and the SV40 small T antigen gene, the effects on the immortalization ability were studied for each case.

[0152] [1] Gene introduction and screening

[0153] (1) Preparation of expression vectors

[0154] The expression cassettes of each gene were integrated into a plasmid vector according to the combinations shown in Table 6 to prepare expression vectors. In Table 6, "○" in the column of each expression cassette indicates that the expression cassette has been integrated into the expression vector. Expression vector NC is a vector without any integrated expression cassette (control vector).

[0155] [Table 6]

[0156] An expression vector (large T + small T) was prepared using the same procedure as that for the expression vector TBCM described in Example 2.

[0157] The expression cassettes of human Cyclin D1 gene, rabbit Bcl-2 gene and rabbit c-Myc gene (expression cassette A used in Example 1) and the expression cassette of SV40 large T antigen gene (SEQ ID NO: 20: nucleotides 1 to 330 are SV40 promoter, nucleotides 344 to 2470 are the gene encoding large T antigen (including stop codon), nucleotides 2493 to 2627 are SV40 polyadenylation signal) were integrated into a control vector to prepare an expression vector (large T).

[0158] The expression cassettes of human Cyclin D1 gene, rabbit Bcl-2 gene and rabbit c-Myc gene (expression cassette A used in Example 1) and the expression cassette of SV40 T antigen gene (SEQ ID NO: 21: nucleotides 1 to 330 are SV40 promoter, nucleotides 344 to 868 are the gene encoding small T antigen (including stop codon), nucleotides 891 to 1025 are SV40 polyadenylation signal) were integrated into a control vector to prepare an expression vector (small T).

[0159] (2) Gene introduction

[0160] Using the same operation as described in Example 2, each gene was introduced into rabbit spleen cells to obtain transformed cells.

[0161] (3) Evaluation of cell proliferation ability

[0162] Using the same operation as described in Example 2, the cell amount of each transformed cell was measured over time starting from after gene introduction, and the cell proliferation ability was evaluated. Figure 9 The cumulative growth curves based on the measurement results are shown.

[0163] As Figure 9 shown, for the transformed cells transfected with expression vector NC, no proliferation was confirmed even until the 28th day of culture after gene introduction. In contrast, as the transformed cells transfected with expression vectors (large T + small T, large T, small T), cell proliferation was confirmed, and the acquisition of immortalization ability was confirmed.

[0164] According to Figure 9It was confirmed that the cell proliferation rate differed depending on the type and combination of the introduced genes. Specifically, it was confirmed that the proliferation rate of the transformed cells into which the expression vector (large T + small T) was introduced was the fastest, and the cell proliferation rate differed in the order of expression vector (large T + small T) > expression vector (large T) > expression vector (small T). As for the transformed cells into which the expression vector (small T) was introduced, proliferation was finally confirmed only around the 10th day of culture. In the case where only the SV40 small T antigen gene was introduced, although proliferation was promoted, it was very slow. From these results, it was found that as the SV40 T antigen gene introduced into plasma cells in the present invention, it preferably contains the SV40 large T antigen gene. Although it may be only the SV40 large T antigen gene, it is particularly preferred to contain both the SV40 large T antigen gene and the SV40 small T antigen gene.

Claims

1. A method for producing immortalized cells, which comprises introducing an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of Myc family genes and Cyclin D family genes into non-immortalized antibody-producing cells to produce immortalized cells.

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

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

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

5. The method for producing immortalized cells according to claim 1, wherein the SV40T antigen gene, the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene are introduced into the non-immortalized antibody-producing cells.

6. A method for producing immortalized antibody-producing cells, which comprises collecting antibody-producing cells from a mammal, performing primary culture, and introducing an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of Myc family genes and Cyclin D family genes into the obtained primary antibody-producing cells to produce immortalized antibody-producing cells.

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

8. A method for manufacturing an antibody, wherein, Immortalized antibody-producing cells are produced by the method for producing immortalized antibody-producing cells according to claim 6 or 7, the immortalized antibody-producing cells are cultured, and the produced antibodies are recovered.

9. A transformed cell, which is immortalized by introducing an exogenous SV40T antigen gene, an exogenous Bcl-2 family gene, and one or more genes selected from the group consisting of exogenous Myc family genes and exogenous Cyclin D family genes into non-immortalized antibody-producing cells.

10. The transformed cell according to claim 9, wherein the transformed cell has the ability to produce antibodies.

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