Method for manufacturing immortalized cells

Abstract

The present invention aims to provide a method for immortalizing antibody-producing cells and the immortalized transformed cells obtained by this method. The present invention relates to a method for manufacturing immortalized cells, which involves introducing one or more genes selected from the group consisting of the SV40T antigen gene, the Bcl-2 family gene, and the Myc family gene and the Cyclin D family gene into non-immortalized antibody-producing cells. The present invention also relates to a method for manufacturing immortalized antibody-producing cells, which involves collecting antibody-producing cells from mammals, performing primary culture, and introducing one or more genes selected from the group consisting of the SV40T antigen gene, the Bcl-2 family gene, and the Myc family gene and the Cyclin D family gene into the obtained primary antibody-producing cells to create immortalized antibody-producing cells.

Description

Methods for manufacturing immortalized cells Technical Field

[0001] This 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 Japan Patent Application No. 2022-195145 filed on December 6, 2022, the contents of which are incorporated herein by reference. Background Technology

[0003] Cells exist in both proliferative and non-proliferative forms. Even proliferative cells typically have a genetically limited number of divisions and a finite lifespan. Therefore, for example, primary cultured cells, obtained by culturing cells from animal tissues, can only divide a limited number of times even under suitable conditions, and subsequently lose their proliferative capacity. Thus, for cells possessing useful traits, attempts have been made to remove the limitation on the number of divisions, achieving the so-called immortalization ability—the capacity to divide indefinitely. Generally, cells acquire immortalization capabilities through transformation.

[0004] Monoclonal antibodies are widely used as reagents for detecting target substances using immune reactions. Furthermore, in recent years, human monoclonal antibodies have shown industrial value as an effective component in antibody pharmaceuticals. In most cases, monoclonal antibodies are produced through antibody-producing cells. To ensure a stable supply of monoclonal antibodies, these cells need to be immortalized without impairing their antibody-producing capacity, enabling industrial-scale in vitro culture.

[0005] Hybridoma technology has been established for the production of monoclonal antibodies in rodents. Hybridoma technology involves immunizing animals, isolating single B cells that produce the desired monoclonal antibodies, culturing them in primary culture, and fusing these B cells with myeloma cells. The resulting hybridoma is immortalized while retaining its antibody-producing ability. However, suitable hybridoma technology is lacking for rodents; therefore, the immortalization of antibody-producing cells requires complex genetic engineering techniques for transformation.

[0006] As a method for immortalizing primary cultured cells, one known method is to introduce three cancer-associated genes—Bcl-2 (B-cell / CLL lymphoma 2), Myc, and Ccnd1—into proB cells for transformation and immortalization (Non-Patent Literature 1). Another known method is to introduce two cancer-associated genes—c-Myc and the SV40 (simian virus 40) large T antigen gene—into human fetal fibroblasts for transformation and immortalization (Non-Patent Literature 2).

[0007] Existing technical documents

[0008] Non-patent literature

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

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

[0011] The problem the invention aims to solve

[0012] Unlike proB cells, antibody-producing cells, which are terminally differentiated cells, cannot acquire immortalization ability even after transformation with the introduction of the Bcl-2, Myc, and Ccnd1 genes. Furthermore, while transformation with the c-Myc gene and the SV40 large T antigen gene can achieve proliferative capacity and immortalization, it cannot maintain its characteristics, and immortalized cells that retain antibody-producing ability cannot be obtained.

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

[0014] means for solving problems

[0015] To solve the above problems, the inventors discovered through in-depth research that by introducing the SV40T antigen gene, Myc family gene, Bcl-2 family gene, and Cyclin D (cyclin D) family gene into antibody-producing cells collected from mammals immunized with antigens, 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 pharmaceutical agents.

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

[0018] [2] The method for manufacturing immortalized cells as described in [1], wherein the Myc family gene is the c-Myc gene or the L-Myc gene.

[0019] [3] The method for manufacturing immortalized cells as described in [1] or [2], wherein the Bcl-2 family gene is the Bcl-2 gene or the Bcl-XL gene.

[0020] [4] The method for manufacturing immortalized cells as described in 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 manufacturing immortalized cells as described in any one of [1] to [3], 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.

[0022] [6] A method for manufacturing immortalized antibody-producing cells, wherein the method involves collecting antibody-producing cells from mammals, culturing them in primary culture, and introducing one or more genes from the group consisting of SV40T antigen gene, Bcl-2 family gene, and genes selected from Myc family gene and Cyclin D family gene into the obtained primary antibody-producing cells to manufacture immortalized antibody-producing cells.

[0023] [7] The method for manufacturing immortalized antibody-producing cells as described in [6], wherein the mammal is an animal immunized with an antigen, and immortalized antibody-producing cells that generate antibodies that recognize the antigen are selected from the immortalized antibody-producing cells.

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

[0025] [9] A transformed cell that achieves immortalization 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 a non-immortalized antibody-producing cell.

[0026]

[10] Transformed cells as described in [9] have the ability to produce antibodies.

[0027] Invention Effects

[0028] The method for manufacturing immortalized cells based on the present invention allows for a relatively simple method of immortalizing antibody-producing cells obtained through primary passage culture, thereby obtaining transformed cells that retain their antibody-producing ability while achieving immortalization. The resulting transformed cells are useful, for example, for the manufacture of monoclonal antibodies. Attached Figure Description

[0029] Figure 1 is a graph showing the cumulative growth curve of transformed cells that were immortalized from plasma cells collected from rabbits and produced anti-rCRP rabbit monoclonal antibodies in Example 1.

[0030] Figure 2 is a graph showing the change over time in the amount of anti-rCRP rabbit monoclonal antibody in the transformed cells that were immortalized from plasma cells collected from rabbits in Example 1 and produced anti-rCRP rabbit monoclonal antibodies.

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

[0032] Figure 4 shows the results of SDS-PAGE of the 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 shows the results of Western blotting using purified anti-rCRP rabbit monoclonal antibody in Example 1.

[0034] Figure 6 is a graph showing the cumulative growth curves of transformed cells in which each expression cassette was introduced in Example 2.

[0035] Figure 7 is a graph showing the flow cytometry analysis results of the cell population stained with anti-rabbit IgG antibody in Example 2, in which the transformed cells introduced with each expression cassette were stained.

[0036] Figure 8 is a graph showing the cumulative growth curves of transformed cells in Example 3 in which each expression cassette was introduced.

[0037] Figure 9 is a graph showing the cumulative growth curves of transformed cells in Example 4 in which each expression cassette was introduced. Detailed Implementation

[0038] The method for manufacturing immortalized cells of the present invention involves introducing one or more genes selected from the group consisting of SV40T antigen gene, Bcl-2 family genes, and genes selected from the Myc family and Cyclin D family into non-immortalized antibody-producing cells. By introducing and expressing these combinations of cancer-related genes into non-immortalized antibody-producing cells, the limitation on the number of cell divisions is removed, endowing cells with the ability to divide and thus obtaining immortalized cells. Normally, differentiated plasma cells cannot divide in vitro, but the method for manufacturing immortalized cells of the present invention can artificially create immortalized cells with the same degree of multiplication ability as cancer cells such as CHO cells (Chinese hamster ovary cells).

[0039] In the method for manufacturing immortalized cells of the present invention, three genes—SV40T antigen gene, Bcl-2 family gene, and Myc family gene—can be introduced into non-immortalized antibody-producing cells; alternatively, three genes—SV40T antigen gene, Bcl-2 family gene, and Cyclin D family gene—can be introduced into non-immortalized antibody-producing cells; or all four genes—SV40T antigen gene, Bcl-2 family gene, Myc family gene, and Cyclin D family gene—can be introduced into non-immortalized antibody-producing cells. Regardless of the combination of genes introduced, immortalization ability can be conferred upon antibody-producing cells. From the viewpoint of being able to produce more transformed cells that have acquired immortalization ability while maintaining their antibody-producing ability, the introduction of four genes—SV40T antigen gene, Bcl-2 family gene, Myc family gene, and Cyclin D family gene—is particularly preferred in the method for manufacturing immortalized cells of the present invention.

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

[0041] The term "SV40 large T antigen" refers to a protein, like the natural SV40 large T antigen, that contains all the proteins that inactivate tumor suppressor genes that induce cellular replication and senescence. Furthermore, "natural" means a protein encoded by the genomic DNA of any biological species. Specifically, in addition to proteins containing the same amino acid sequence as the natural SV40 large T antigen, it also includes mutants of the natural SV40 large T antigen, i.e., proteins whose amino acid sequences are obtained by deleting, substituting, or adding one or more amino acids to the same amino acid sequence as the natural SV40 large T antigen, and which retain the ability to inactivate the tumor suppressor gene. Furthermore, the SV40 large T antigen encoded by the SV40T antigen gene introduced into non-immortalized cells in this invention can also be a fusion protein obtained by directly or indirectly linking various tags or other structural proteins to the natural SV40 large T antigen or its mutants via appropriate adapter sequences (linking sequences). Additionally, the amino acid sequence of the natural SV40 large T antigen has been registered in the International Base Sequence Database (INSD) (INSD accession number: AAB59924).

[0042] The so-called "SV40 small T antigen" contains, like the natural SV40 small T antigen, all proteins that have the function of binding to and inactivating protein phosphatase 2 (PP2A). Specifically, in addition to containing proteins with the same amino acid sequence as the natural SV40 small T antigen (INSD accession number: AAB59925), it also contains mutants of the natural SV40 small T antigen, i.e., proteins whose amino acid sequence is 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 which retain their PP2A inactivation ability. Furthermore, the SV40 small T antigen encoded by the SV40T antigen gene introduced into non-immortalized cells in this invention can also be a fusion protein obtained by directly or indirectly linking various tags or other structural proteins to the natural SV40 small T antigen or its mutants through appropriate linker sequences.

[0043] In this invention and the specification of this application, the term "SV40T antigen gene" refers to a nucleic acid containing the base sequence encoding the SV40 large T antigen, similar to the natural SV40 large T antigen. This gene encodes a protein that inactivates tumor suppressor genes that induce cellular replication and senescence within cells. The "SV40T antigen gene" used in this invention can 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 can be a nucleic acid containing only the base sequence encoding the SV40 large T antigen. When introducing the SV40T antigen gene into non-immortalized cells in this invention, there are no particular limitations as long as the gene can express the SV40T antigen, especially in the case of mammalian cells. It can be a gene containing introns and expressing both the SV40 large T antigen and the SV40 small T antigen, or it can be a gene consisting only of exons. Furthermore, various alterations (modifications) can be made to the base sequences encoding the SV40 large T antigen and the SV40 small T antigen in the SV40T antigen gene. For example, degenerate codons can be changed to codons frequently used in limited-proliferation antibody-producing cells (hereinafter sometimes referred to as "limited-proliferation cells") for the purpose of immortalization, such as introducing the SV40T antigen gene into these cells. Codon alterations (codon modifications) can be performed using well-known gene sequence variation techniques or artificial gene synthesis.

[0044] In this invention and this application, 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 this invention and this application, the "Myc family" includes not only the natural Myc family but also variants (modified forms) obtained by altering natural Myc family proteins while maintaining the same transcription factor function as human c-Myc. Specifically, in the Myc family, in addition to human c-Myc, human L-Myc, and human N-Myc, their homologs can also be cited as examples of the natural Myc family. For example, rabbit c-Myc (INSD accession number: AJC97784) can be cited as a homolog of human c-Myc. Examples of homologs of human L-Myc include rabbit L-Myc (INSD accession number: XP_002715238). In addition to proteins comprising the same amino acid sequence as these natural Mycs, mutants of these Mycs are also included; that is, proteins whose amino acid sequences are obtained by deleting, substituting, or adding one or more amino acids to the same amino acid sequence as natural Myc and which retain their transcriptional activity. Examples of mutant Myc families include, for instance, the T58N mutant of human c-Myc. Furthermore, the Myc family genes encoded by Myc family genes introduced into non-immortalized cells in this invention can also be variants such as fusion proteins obtained by directly or indirectly linking various tags or other structural proteins to the natural Myc family or its mutants via appropriate linker sequences. The Myc family gene introduced into non-immortalized cells in this invention is preferably a gene of c-Myc (human c-Myc and its orthologs and their mutants), L-Myc (human c-Myc and its orthologs and their mutants), or a variant of all of these, and more preferably a gene of human c-Myc, human L-Myc, or a mutant of these, or a variant of all of these.

[0045] In this invention and the specification of this application, the term "Myc family gene" refers to a nucleic acid containing a base sequence encoding the Myc family. As for the Myc family gene introduced into non-immortalized cells in this invention, in the case of mammalian cells, there are no particular limitations as long as the gene can express the Myc family; it can be a gene containing introns or a gene consisting only of exons. Furthermore, various alterations (modifications) can be applied to the base sequence encoding Myc family proteins in the Myc family gene, such as changing degenerate codons to codons with higher frequency of use in determinate proliferating cells. Codon alterations (modifications) can be performed using known gene sequence variation techniques or artificial gene synthesis.

[0046] In this invention and the present application specification, the term "Bcl-2 family" refers to a family of mitochondrial outer membrane proteins that possess one or more BH (Bcl-2 homology) domains and a highly hydrophobic membrane permeation region on the C-terminal side, and that exhibit anti-apoptotic activity. Examples of anti-apoptotic proteins in the human Bcl-2 family include Bcl-2 (INSD accession number: AAH27258), Bcl-XL (INSD accession number: AAP35872), and MCL-1 (Myeloid cellleukemia sequence 1) (INSD accession number: AAD13299).

[0047] The Bcl-2 family genes introduced into non-immortalized cells in this invention are not particularly limited to proteins possessing one or more BH domains and membrane-transfer regions and exhibiting anti-apoptotic activity. For example, in this invention and the specification of this application, "Bcl-2 family" includes not only the natural Bcl-2 family but also variants obtained by modifying natural Bcl-2 family proteins to maintain the same anti-apoptotic activity as human Bcl-2. Specifically, among the Bcl-2 family with anti-apoptotic activity, examples of the natural Bcl-2 family include human Bcl-2, human Bcl-XL, and human MCL-1, as well as their homologs. Examples of homologs of human Bcl-2 include rabbit Bcl-2 (INSD accession number: XP_008259661). Examples of homologs of human Bcl-XL include rabbit Bcl-XL (INSD accession number: XP_008254359). In addition to proteins comprising the same amino acid sequences as these natural Bcl-2 families, the invention also includes mutants of these proteins, i.e., proteins whose amino acid sequences are obtained by deleting, substituting, or adding one or more amino acids in the same amino acid sequences as the natural Bcl-2 family, and which retain their anti-apoptotic activity. Furthermore, the Bcl-2 family encoded by the Bcl-2 family gene introduced into non-immortalized cells in this invention can also be a variant such as a fusion protein obtained by directly linking various tags or other structural proteins to the natural Bcl-2 family or its mutants, or indirectly through suitable linker sequences. As the Bcl-2 family gene introduced into non-immortalized cells in this 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, more preferably a gene of human Bcl-2, human Bcl-XL, or a mutant of these, or a variant of all of these.

[0048] In this invention and the specification of this application, the term "Bcl-2 family gene" refers to a nucleic acid containing a base sequence encoding the Bcl-2 family. As for the Bcl-2 family gene introduced into non-immortalized cells in this invention, in the case of mammalian cells, there is no particular limitation as long as it is a gene capable of expressing the Bcl-2 family; it can be a gene containing introns or a gene containing only exons. Furthermore, the base sequence encoding Bcl-2 family proteins in the Bcl-2 family gene can be altered in various ways, such as changing degenerate codons to codons with higher frequency of use in determinate proliferating cells. Codon alterations can be performed using known gene sequence variation techniques or artificial gene synthesis.

[0049] In this invention and the specification of this application, the term "Cyclin D family" refers to a family of proteins that possess a cyclin box domain that binds to a cyclin-dependent kinase (CDK) and an RB (Retinoblastoma tumor suppressor protein) binding domain at the N-terminus, and that are synthesized at the start of the G1 phase of the cell cycle and control 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).

[0050] As for Cyclin D family genes introduced into non-immortalized cells in this invention, there are no particular limitations as long as they are proteins that have the same S-phase transfer control function as human Cyclin D1. For example, in this invention and the specification of this application, "Cyclin D family" includes not only the natural Cyclin D family, but also variants obtained by modifying natural Cyclin D family proteins while maintaining their S-phase transfer control function. Specifically, in addition to human Cyclin D1, human Cyclin D2, and human Cyclin D3, their homologs can also be cited as examples of the natural Cyclin D family. Furthermore, in addition to proteins composed of the same amino acid sequence as these natural Cyclin Ds, mutants of these proteins are also included, i.e., proteins whose amino acid sequence is obtained by deleting, substituting, or adding one or more amino acids in the same amino acid sequence as natural Cyclin D, and which retain their S-phase transfer control function. Furthermore, the Cyclin D family gene encoded by the Cyclin D family gene introduced into non-immortalized cells in this invention can also be a variant of a fusion protein obtained by directly or indirectly linking various tags, other structural proteins, to the natural Cyclin D family or its mutants through appropriate adapter sequences. Preferably, the Cyclin D family gene introduced into non-immortalized cells in this invention is a 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 a variant of all of these, and more preferably a gene of human Cyclin D1, human Cyclin D3, or their mutants or a variant of all of these.

[0051] In the method for manufacturing immortalized cells of the present invention, the genes introduced into non-immortalized cells, including Myc family genes, Bcl-2 family genes, and Cyclin D family genes, can be genes from the same biological species as the introduced cells, or genes from different biological species. Furthermore, these three genes can be genes from the same biological species or genes 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 manufacturing immortalized cells of the present invention, the cells used for immortalization are not particularly limited as long as they are non-immortalized limited proliferative cells, and there are no particular limitations on the biological species or tissues from which they originate. As limited proliferative cells used in the present invention, cells collected from animals or primary cultured cells obtained from such cells are preferred, and cells collected from mammals or primary cultured cells obtained from such cells are more preferred. The mammal is not particularly limited, but experimental animals such as humans, mice or rats, and monkeys are more preferred, as are livestock or pets such as rabbits, pigs, cats, cattle, horses, and sheep, with humans being particularly preferred. The collection and primary culture of cells derived from animals can be carried out using conventional methods.

[0053] In the method for manufacturing immortalized cells of the present invention, non-immortalized plasma cells are preferably used as cells for immortalization transformation. Here, plasma cells are mononuclear cells similar to lymphocytes distributed in the spleen, lymph nodes, bone marrow, connective tissue, etc., which acquire the ability to produce antibodies upon stimulation by antigens. The plasma cells used for transformation in the method for manufacturing immortalized cells of the present invention can be any of the following: plasma cells before acquiring antibody-producing ability, plasma cells that acquire antibody-producing ability after acquisition, and plasma cells that lose antibody-producing ability after acquisition. Those skilled in the art can appropriately select methods such as flow cytometry to screen plasma cells. For confirmation of the selected plasma cells, hematoxylin-eosin staining, nucleus shape, and cytoplasm can be confirmed using optical microscopy images.

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

[0055] In the method for manufacturing immortalized cells of the present invention, the SV40T antigen gene or the like can be introduced into the chromosome of the immortalized cells, or it can be introduced as an extrachromosomal gene. By introducing the SV40T antigen gene or the like into the chromosome, transformed cells with excellent stability can be obtained through passage.

[0056] The genetic engineering method used to introduce the SV40T antigen gene, etc., into immortalized antibody-producing cells can be any known method of introducing a foreign gene into cells with limited proliferation, or a method that appropriately modifies a known method can be used. For example, one method is to introduce a vector integrating an expression cassette containing a foreign gene into cells. The SV40T antigen gene, etc., can be expressed in a polycistronic or monocistronic form.

[0057] An expression cassette is a combination of DNA needed to express a target protein, containing the structural gene encoding the target protein and a promoter that functions in cells with limited proliferation. An expression cassette may contain only one structural gene or two or more structural genes.

[0058] In the case of monocistronic expression, the expression cassettes used in transformations for immortalization can specifically include: SV40T antigen expression cassettes containing SV40T antigen genes and promoters, Myc family expression cassettes containing Myc family genes and promoters, Bcl-2 family expression cassettes containing Bcl-2 family genes and promoters, and Cyclin D family expression cassettes containing Cyclin D family genes and promoters.

[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 an expression cassette and expressed in a polycistronic form. For example, immortalized transformed cells can be obtained even by introducing an expression cassette containing the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D family genes sequentially through a linker sequence encoding a self-cleaving peptide downstream of a promoter into determinate proliferating cells. The self-cleaving peptide can be appropriately selected and used from known self-cleaving peptides such as P2A peptide, E2A peptide, and T2A peptide. An IRES (internal ribosome entrysite) sequence can also be included in the linker sequence connecting the genes to replace the self-cleaving peptide. The IRES sequence can be an IRES sequence derived from a viral genome or an IRES sequence derived from an animal cell genome; an appropriate selection from known IRES sequences can be used.

[0060] As a promoter in the expression cassette, any promoter that functions within delimited proliferating cells is acceptable. It can be a promoter inherent in delimited proliferating cells or one not inherently present in them. In the case where the delimited proliferating cells are antibody-producing plasma cells, an example of a promoter inherent in delimited proliferating cells is the promoter of the Blimp1 gene, specifically expressed by plasma cells (Blimp1 promoter). Examples of promoters not inherent in delimited proliferating cells include promoters derived from animal cell viruses and artificial promoters modified from those promoters. From the viewpoint of widespread use and proven effectiveness, the Piggyback promoter, CAG promoter, CBh promoter, SV40 promoter, SRα promoter, and hCMV promoter are preferred as promoters used in this invention. Promoters for genes such as the SV40T antigen gene can be all of the same species or can be of different species.

[0061] The expression cassette may also include a terminator that functions in delimited-proliferation cells, an enhancer that functions in delimited-proliferation cells, and any one or more of the 5'-untranslated region and the 3'-untranslated region. As the terminator, terminators that are inherent to delimited-proliferation cells or terminators that are not inherent to delimited-proliferation cells can be used. Examples of enhancers include the Eu enhancer.

[0062] As a vector integrating an expression cassette containing the SV40T antigen gene, non-viral vectors such as plasmid vectors can be used. Plasmid vectors can be created by integrating this expression cassette into a vector with a circular DNA structure. Alternatively, vectors with a linear DNA structure that integrate the expression cassette can be used instead of plasmid vectors.

[0063] When creating transformants that maintain the expression cassette as an extrachromosomal gene within delimited proliferating cells, it is preferable that the vector contains a plasmid with a sequence for replication within the delimited proliferating cells, i.e., an autonomously replicating sequence (ARS). On the other hand, when creating transformants that integrate the expression cassette into the chromosome of delimited proliferating cells, a vector with a linear DNA structure lacking an ARS is preferred. Alternatively, a plasmid vector without an ARS may be used, containing a restriction endonuclease recognition sequence for cutting the linear DNA upon introduction into delimited proliferating cells.

[0064] When this vector is introduced into the chromosome of a degenerate cell, the expression cassette in the vector has homologous recombination sites upstream and downstream. These homologous recombination sites are constructed from base sequences that are capable of homologous recombination at target sites on the chromosome of the degenerate cell. The homologous recombination sites can be appropriately set based on the base sequence information of the genomic DNA of the degenerate cell. There can be only one target site for homologous recombination in the chromosome of the degenerate cell, or multiple sites can be used as target sites. When multiple sites are used as target sites, transposons are preferred. For example, by introducing a vector containing an expression cassette for polycistronic expression, which integrates at least one of the genes from the SV40T antigen gene, the Bcl-2 family gene, the Myc family gene, and the Cyclin D family gene, between the 5' transposon-specific repeat sequence and the 3' transposon-specific repeat sequence, into determinate proliferating cells, the expression cassette can be integrated into multiple transposons present in the chromosome of the determinate proliferating cells, thereby obtaining immortalized transformed cells (antibody-producing cells).

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

[0066] The vector integrating an expression cassette containing the SV40T antigen gene, etc., can be a viral vector. Various known viral vectors used for introducing genes into primary cultured cells, such as lentiviral vectors, adeno-associated virus vectors, and retroviral vectors, can be used. The synthesis of each viral vector and the subsequent generation of viruses integrating expression cassettes containing the SV40T antigen gene, etc., can be performed using commercially available viral expression kits and conventional methods.

[0067] For the culture of delimited proliferating cells before and during the introduction of a vector that integrates an expression cassette (including the SV40T antigen gene, etc.) and for the culture of transformed cells obtained after the introduction of the vector, it is possible to use the same culture medium as the culture medium for the delimited proliferating cells supplied for immortalization and to carry out the culture under the same culture conditions.

[0068] The immortalized cell manufacturing method of the present invention enables the production of immortalized transformed cells capable of producing antibodies. As the biological species for antibody-producing cells, mammals are preferred, and more preferably, animal species such as humans, mice, rats, rabbits, donkeys, horses, sheep, and goats, which have traditionally been used in the manufacture of monoclonal antibodies as reagents.

[0069] For example, antibody-producing cells can be collected from mammals and cultured in their primary form. By introducing the SV40T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family and Cyclin D family into the resulting primary antibody-producing cells, immortalized antibody-producing transformed cells can be created. Additionally, antibodies against the pathogen of an infectious disease can be produced in the body of a patient with such an infection. Therefore, by introducing the SV40T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family and Cyclin D family into plasma cells capable of producing antibodies collected from an infected patient, a transformed cell can be obtained that achieves immortalization while maintaining its ability to produce antibodies against the pathogen of the infectious disease.

[0070] For example, after immunizing a mammal with an antigen and forming antibody-producing cells in the animal, cells are collected from tissues containing these antibody-producing cells, such as lymph nodes. Immortalized transformed cells can be obtained by introducing the SV40T antigen gene, a Bcl-2 family gene, and at least one gene selected from the Myc family and Cyclin D family into the collected lymph node cell clusters. Transformed cells (immortalized antibody-producing cells) that produce antibodies recognizing the antigen used for immunization are screened from the resulting immortalized transformed cell clusters. Thus, immortalized antibody-producing cells that produce antibodies recognizing the target antigen can be obtained. These immortalized antibody-producing cells are cultured, and the produced antibodies are recovered, thereby enabling the stable production of antibodies against the target antigen. Antibodies produced using immortalized antibody-producing cells, like other antibodies, can be used directly as research reagents and can also be used as raw materials in pharmaceutical compositions, hygiene products (masks, etc.), and pollution removal-related products.

[0071] Example

[0072] The present invention will then be described in further 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 rabbits immunized with C-reactive protein (rCRP) to obtain transformed cells that were immortalized.

[0075] (1) Preparation of immunoantigens and screening antigens

[0076] rCRP (manufactured by Orient Yeast Industry Co., Ltd., Japan) was used as the immunogenic antigen. Furthermore, the same antigen was used subsequently for screening using ELISA.

[0077] (2) Animals and antigen immunization methods

[0078] Female rabbits (Japanese White, 2.5–3.0 kg) were used as the immunization animals. For immunization, rabbits were immunized at two-week intervals using an emulsion prepared by mixing equal volumes of antigen and adjuvant. Freund's complete adjuvant was used as the adjuvant to achieve an immunogenicity of 1 mg per rabbit for the primary immunization. Freund's incomplete adjuvant was used as the adjuvant to achieve an immunogenicity of 0.5 mg per rabbit for subsequent immunizations.

[0079] [2] Gene introduction and screening

[0080] (1) Manufacturing of carriers for expression

[0081] Expression cassette A, obtained by sequentially linking the human Cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene with a linker sequence containing the P2A peptide as a self-cleaving peptide, was integrated downstream of the CAG promoter in a mammalian gene expression vector (VectorBuilder). (Sequence number 1: sequences 1-1679 are the CAG promoter, sequences 1734-2618 are the human Cyclin D1 gene, sequences 2619-2684 are the P2A peptide encoding sequences, sequences 2685-3362 are the rabbit Bcl-2 gene, sequences 3363-3428 are the P2A peptide encoding sequences, sequences 3429-4745 are the rabbit c-Myc gene (containing a stop codon), and sequences 4789-5013 are the bovine growth hormone tailing signal [polyA signal]). Seamless cloning was used for ligation. In addition, as an expression cassette for the SV40T antigen gene, an expression cassette B (sequence number 2: 1-330 are the SV40 promoter, 344-2816 are large T antigen exons, 344-868 are small T antigen exons, 590-935 are large T antigen introns, and 2839-2973 are SV40 tailing signals) was prepared from the SV40 viral DNA and integrated upstream of the CAG promoter of the expression vector.

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

[0083] Blood was collected from immunized animals over time, and the antibody titer of anti-rCRP antibodies in the serum was measured using ELISA. After confirming a sufficient increase in antibody titer, the popliteal lymph nodes were removed, and a suspension of popliteal lymph node cells was prepared using standard methods.

[0084] (3) Gene introduction

[0085] The plasmid vector integrating expression cassette A was introduced into popliteal lymph node cells using electroporation. The resulting cell suspension and subsequent culture were performed in 100 mm culture dishes containing 1% methylcellulose (NACALAITESQUE, Inc.), 30% FBS (fetal bovine serum), and 1% penicillin / streptomycin.

[0086] (4) Cell proliferation

[0087] The 100mm culture dishes were cultured for 14 days in a humidified culture vessel at 37°C and 5% CO2. The cells that proliferated and colonized (colonized) in this medium were transformed cells with introduced genes and were monoclonal cells. Colonies were collected from the 100mm culture dishes using a stereomicroscope and pipettes, transferred to RPMI 1640 medium (Sigma-Aldrich) containing 10% FBS and 1% P / S, and cultured using microtiter plates until confluent.

[0088] (5) ELISA

[0089] Anti-rCRP antibodies in the culture supernatant obtained from cultured and transformed cells were detected using the ELISA method.

[0090] First, rCRP (0.5 μg / mL) was immobilized on an ELISA plate, which was then blocked with 1% (w / w) BSA. Subsequently, the culture supernatant from 100 mm culture dishes that had undergone culture medium exchange the previous day was aliquoted into the ELISA plate, and the reaction was carried out after a specified incubation period. 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.

[0091] Next, horseradish peroxidase-labeled anti-rabbit IgG antibodies were injected into each well of the ELISA plate, and the cells were cultured for a specified time. The fluorescence intensity was measured using a plate reader to detect the transformed cells that produced anti-rCRP rabbit monoclonal antibodies.

[0092] To confirm the proliferative capacity and antibody production ability of the transformed cells, 6-well plates were passaged three times per week. The cell count and the amount of anti-rCRP rabbit monoclonal antibody were measured over time. Cell count was performed under a microscope. The amount of anti-rCRP rabbit monoclonal antibody was measured using the same ELISA method as described above. Figure 1 shows the cumulative growth curve of the transformed cell count, and Figure 2 shows the measurement results of the anti-rCRP rabbit monoclonal antibody amount (μg / mL).

[0093] As shown in Figure 1, the resulting transformed cells proliferated for approximately 650 days. Furthermore, the doubling time was about 16 hours, and no decline in the proliferation rate was observed. That is, in this embodiment, immortalized cells with a doubling capacity similar to that of cancer cells such as CHO cells can be artificially created. Additionally, as shown in Figure 2, the obtained transformed cells also produced anti-rCRP rabbit monoclonal antibodies for approximately 60 days. This demonstrates that simply introducing the SV40T antigen gene, Myc family genes, Bcl-2 family genes, and Cyclin D family genes into primary cultured antibody-producing cells can obtain immortalized transformed cells while maintaining their antibody-producing capacity.

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

[0095] (1) Purification

[0096] Transformed cells capable of producing antibodies were used for expanded culture. Culture conditions were set as described above. Subsequently, the anti-rCRP antibody was purified from the resulting culture supernatant using a Protein A column according to standard methods. The purified antibody was analyzed by high-performance liquid chromatography (HPLC) using a size exclusion chromatography (SEC) column (TSKgel G3000SWXL, Tosoh Corporation) at a flow rate of 0.7 mL / min. The HPLC chromatogram is shown in Figure 3. The chromatogram showed only one peak, confirming that purity was not an issue.

[0097] (2) SDS-PAGE

[0098] Purified antibodies were subjected to SDS-PAGE using standard methods. The gel used was "Perfect Nt Gel" (manufactured by DRC Corporation), 1 μg of purified antibody was used, and "Optibopt Blue" (manufactured by Abcam) was used as the staining solution. SDS-PAGE was performed separately on the reduced antibody (reduced conditions) and the unreduced antibody (non-reduced conditions). The results of SDS-PAGE are shown in Figure 4. In Figure 4, "R" represents the lane through which the reduced antibody flowed, and "NR" represents the lane through which the unreduced antibody flowed. The band near 160 kDa represents the band of the intact antibody (composed of two antibody heavy chains (H chains) and two antibody light chains (L chains)), the band near 50 kDa represents the band of one H chain, and the band near 25 kDa represents the band of one L chain. The results confirm that bands were obtained at the target molecular weight under both reducing and non-reducing conditions.

[0099] (3) Western blotting

[0100] Purified antibodies were used to perform SDS-PAGE on rCRP, which served as an immunogenic antigen, and the resulting PVDF membrane was transferred to a PVDF membrane. The PVDF membrane was then subjected to Western blotting according to standard procedures. The PVDF membrane used was "iblot Gel Transfer Stacks PVDF, MINI" (Invitrogen), the blocking buffer was "Blocking One" (NACALAITESQUE), the secondary antibody was "Anti-IgG, rabbit, goat-Poly, HRP" (Gene Tex), and the chromogenic buffer was "KPL TMB Membrane Peroxidase Substrate (1-c)" (SeraCare). Figure 5 shows the results of the Western blotting. In Figure 5, lane 1 is the lane through which "Anti-CReactive Protein Antibody [Y284] (ab32412)" (Abcam) flowed as a positive control, lane 2 is the lane through which the purified antibody sample flowed, and lane M is the lane through which the molecular weight marker flowed. As shown in Figure 5, the purified antibody binds to the rCRP band in the PVDF membrane and develops color.

[0101] (4) Antibody gene sequence

[0102] To confirm that the antibodies produced by the immortalized transformed cells were monoclonal antibodies, the sequence of the antibody gene was determined. First, RNA was extracted from the cells, and cDNA was synthesized via reverse transcription. RNA extraction from the immortalized transformed cells was performed using NucleoSpin RNA (registered trademark), and the reverse transcription reaction was performed using PrimeScript IV 1st strand cDNA Synthesis Mix (TaKaRa). Next, using the cDNA as a template, PCR was performed using a primer set (Table 1) specific to the H and L strands of the rabbit antibody gene, respectively, and a polymerase (Prime STAR Max DNA Polymerase, TaKaRa) to amplify the antibody gene.

[0103] [Table 1]

[0104]

[0105] After purifying the obtained PCR fragments using NucleoSpin (manufactured by TaKaRa), the base sequences were confirmed by direct sequencing using the primers listed in Table 1. The results showed that the H chain cDNA of the anti-rCRP antibody produced by immortalized transformed cells consisted of the base sequence shown in SEQ ID NO: 7, and the L chain cDNA consisted of the base sequence shown in SEQ ID NO: 9. Based on these base sequence results, it was confirmed at the gene level that the anti-rCRP antibody produced by immortalized transformed cells consisted of the amino acid sequence of the H chain (SEQ ID NO: 8: 1-19 are signal sequences, 20-43 are frame 1, 44-51 are CDR1, 52-68 are frame 2, 69-75 are CDR2, 76-111 are frame 3, 112-130 are CDR3, 131-141 are frame 4, 14...). Monoclonal antibodies consisting of amino acid sequences of the γ chain (2-464 being the γ chain constant region) and the L chain (Sequence No. 10: sequences 1-22 are the signal sequence, sequences 23-48 are frame 1, sequences 49-55 are CDR1, sequences 56-72 are frame 2, sequences 73-75 are CDR2, sequences 76-111 are frame 3, sequences 112-123 are CDR3, sequences 124-133 are frame 4, and sequences 134-237 are the κ chain constant region).

[0106] [Example 2]

[0107] The SV40T antigen gene, Bcl-2 family genes, Myc family genes, and Cyclin D family genome were combined and introduced into primary cultured rabbit spleen cells to study their effects on immortalization ability and antibody production capacity.

[0108] [1] Gene introduction and screening

[0109] (1) Manufacturing of carriers for expression

[0110] Expression cassettes of each gene were integrated into a plasmid vector according to the combinations shown in Table 2 to prepare expression vectors. In Table 2, "○" in the column for 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 cassettes (control vector).

[0111] [Table 2]

[0112]

[0113] Using expression cassette B, which was used in Example 1, as the expression cassette for the SV40T antigen gene, the expression cassette was integrated into the control vector to prepare expression vector T.

[0114] The expression cassette of the rabbit Bcl-2 gene (sequence number 11: sequences 1-1679 are the CAG promoter, sequences 1734-2414 are the rabbit Bcl-2 gene (containing the stop codon), and sequences 2458-2682 are the bovine growth hormone tailing signal) and the expression cassette of the SV40T antigen gene (expression cassette B) were integrated into the control vector to prepare expression vector TB.

[0115] The expression cassette of the human Cyclin D1 gene (sequence number 12: sequences 1-1679 are CAG promoters, sequences 1734-2621 are the human Cyclin D1 gene (containing stop codons), and sequences 2665-2889 are bovine growth hormone tailing signals) and the expression cassette of the SV40T antigen gene (expression cassette B) were integrated into the control vector to prepare the expression vector TC.

[0116] The expression cassette of the rabbit c-Myc gene (sequence number 13: sequences 1-1679 are the CAG promoter, sequences 1734-3050 are the rabbit c-Myc gene (containing the stop codon), and sequences 3094-3318 are the bovine growth hormone tailing signal) and the expression cassette of the SV40T antigen gene (expression cassette B) were integrated into the control vector to prepare expression vector TM.

[0117] Expression cassettes of the human Cyclin D1 gene and the rabbit Bcl-2 gene (sequence number 14: sequences 1-1679 are the CAG promoter, sequences 1734-2618 are the human Cyclin D1 gene, sequences 2619-2684 are the base sequence encoding the P2A peptide, sequences 2685-3365 are the rabbit Bcl-2 gene (containing the stop codon), and sequences 3409-3633 are the bovine growth hormone tailing signal) and the SV40T antigen gene (expression cassette B) were integrated into the control vector to prepare expression vector TBC.

[0118] Expression cassettes of the rabbit Bcl-2 gene and the rabbit c-Myc gene (sequence number 15: sequences 1-1679 are the CAG promoter, sequences 1734-2411 are the rabbit Bcl-2 gene, sequences 2412-2477 are the base sequence encoding the P2A peptide, sequences 2478-3794 are the rabbit c-Myc gene (containing the stop codon), and sequences 3838-4062 are the bovine growth hormone tailing signal) and the expression cassette of the SV40T antigen gene (expression cassette B) were integrated into the control vector to prepare the expression vector TBM.

[0119] Expression cassettes of the human Cyclin D1 gene and the rabbit c-Myc gene (Sequence No. 16: sequences 1-1679 are the CAG promoter, sequences 1734-2618 are the human Cyclin D1 gene (containing a stop codon), sequences 2619-2684 are the base sequence encoding the P2A peptide, sequences 2685-4001 are the rabbit c-Myc gene (containing a stop codon), and sequences 4045-4269 are the bovine growth hormone tailing signal) and the SV40T antigen gene (expression cassette B) were integrated into a control vector to prepare the expression vector TCM.

[0120] The expression vector TBCM was prepared by integrating the expression cassettes of the human Cyclin D1 gene, the rabbit Bcl-2 gene, and the rabbit c-Myc gene (expression cassette A used in Example 1) and the expression cassette of the SV40T antigen gene (expression cassette B) into the control vector.

[0121] Expression vector BCM was prepared by integrating expression cassettes (expression cassette A) of human Cyclin D1 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene into a control vector.

[0122] (2) Gene introduction

[0123] Spleens were harvested from rabbits, and a suspension of spleen cells was prepared using standard methods. Each expression vector was then introduced into the spleen cells via electroporation. The same procedures were followed for the electroporated popliteal lymph node cells in Example 1 to prepare the gene-transformed spleen cell suspension and subsequent culture, allowing the transformed cells to proliferate and colonize.

[0124] (3) Evaluation of cell proliferation capacity

[0125] Colonies were collected from 100 mm culture dishes using a pipette under a stereomicroscope and transferred to RPMI 1640 medium (Sigma) containing 10% FBS and 1% P / S. Cell count was measured under a microscope. Figure 6 shows the cumulative growth curve based on the cumulative cell count measured over time since gene introduction via electroporation.

[0126] As shown in Figure 6, for transformed cells introduced with expression vector NC (control vector), no cell proliferation was observed until day 27 of culture after gene introduction. In contrast, for transformed cells introduced with expression cassettes containing at least four genes, cell proliferation was observed at day 32 of culture, confirming that plasma cells (antibody-producing cells) that did not proliferate in vitro had acquired immortalization capabilities. Furthermore, Figure 6 shows that the cell proliferation rate varied depending on the type and combination of introduced genes. No proliferation was observed with expression vectors T and TB until day 19 of culture, and no proliferation was observed with expression vector TC until day 12 of culture, indicating a very slow proliferation rate. Transformed cells introduced with expression vectors containing the Bcl-2 gene (TBC, TBCM, TBM, BCM) showed significantly faster cell proliferation rates compared to transformed cells introduced with expression vectors not containing the Bcl-2 gene (TM, TCM), especially those introduced with expression vectors TBC, TBCM, or TBM.

[0127] (4) Confirmation of antibody-producing cells

[0128] The cumulative cell growth curve shown in Figure 6 is a measurement of the growth of the entire cell population obtained after transforming the recovered spleen cells, including cells other than plasma cells (antibody-producing cells). Therefore, flow cytometry was used to investigate the proportion of antibody-producing cells in the cell populations after 19, 25, or 32 days of culture following gene introduction. Specifically, transformed cells containing the various expression cassettes were fixed and subjected to cell membrane permeation using the membrane permeation treatment reagent "PerFix nc" (manufactured by BECKMAN COULTER). The cells were then stained with fluorescently labeled goat anti-rabbit IgG antibody ("Goat Anti Rabbit IgG H&L" (Alexa Fluor 647, manufactured by Abcam). Since antibody-producing cells retain endogenous IgG in their cytoplasm, they were stained with fluorescently labeled goat anti-rabbit IgG antibody. Flow cytometry was used to analyze the stained cells and determine the percentage (%) of fluorescently stained cells (antibody-producing cells) in the total cell count. Flow cytometry was performed using a "BD Accuri C6 Plus" flow cytometer (manufactured by Becton Dickinson).

[0129] Figure 7 shows the flow cytometry results of cells cultured for 32 days after gene introduction. Figures 7(A)–(I) show the flow cytometry results of transformed cell populations introduced with expression vectors T, TB, TC, TM, TBC, TBM, TCM, TBCM, and BCM, respectively. In the figure, "M1" represents the cell population stained with fluorescently labeled goat anti-rabbit IgG antibody, i.e., the antibody-producing cell population.

[0130] Table 3 shows the percentage of antibody-producing cells (IgG ratio) calculated based on flow cytometry results. According to these results, only transformed cells infused with expression vectors TBC, TBCM, or TBM showed confirmed antibody-producing cell proliferation. This indicates that by introducing one or more genes from the group consisting of the SV40T antigen gene, Bcl-2 family genes, and genes selected from the Myc and Cyclin D family, antibody-producing cells can be immortalized while maintaining their antibody-producing ability. In particular, cells infused with expression vector TBCM—that is, cells infused with all four genes (SV40T antigen gene, Bcl-2 family genes, Myc family genes, and Cyclin D family genes)—had a higher proportion of antibody-producing cells, indicating that introducing these four genes can effectively create immortalized antibody-producing cells.

[0131] [Table 3]

[0132]

[0133] [Example 3]

[0134] In Example 2, the effects on immortalization ability and antibody production ability were investigated by replacing one gene selected from the group consisting of Bcl-2 family genes, Myc family genes and Cyclin D family genes with other genes within the same gene family.

[0135] [1] Gene introduction and screening

[0136] (1) Manufacturing of carriers for expression

[0137] Expression vectors were prepared by integrating the expression cassettes of each gene into a plasmid vector according to the combinations shown in Table 4. In Table 4, "○" in the column for each expression cassette indicates that the expression cassette has been integrated into the expression vector. Expression vector NC is a vector that has not integrated any expression cassette (control vector).

[0138] [Table 4]

[0139]

[0140] The expression vector TBCM was prepared using the same method as described in Example 2.

[0141] Expression cassettes of the human Cyclin D1 gene, human Bcl-XL gene, and rabbit c-Myc gene (Sequence No. 17: sequences 1-1679 are CAG promoters, sequences 1734-2618 are human Cyclin D1 gene sequences, sequences 2619-2684 are P2A peptide sequences, sequences 2685-3383 are human Bcl-XL gene sequences, sequences 3384-3449 are P2A peptide sequences, sequences 3450-4766 are rabbit c-Myc gene sequences (containing stop codons), and sequences 4810-5034 are bovine growth hormone tailing signals) and the SV40T antigen gene (expression cassette B) were integrated into a control vector to prepare the expression vector TB'CM.

[0142] Expression cassettes of the human Cyclin D3 gene, rabbit Bcl-2 gene, and rabbit c-Myc gene (Sequence No. 18: sequences 1-1679 are the CAG promoter, sequences 1734-2609 are the human Cyclin D3 gene, sequences 2610-2675 are the P2A peptide, sequences 2676-3353 are the rabbit Bcl-2 gene, sequences 3354-3419 are the P2A peptide, sequences 3420-4736 are the rabbit c-Myc gene (containing a stop codon), and sequences 4780-5004 are the bovine growth hormone tailing signal) and the SV40T antigen gene (expression cassette B) were integrated into a control vector to prepare the expression vector TBC'M.

[0143] Expression cassettes of the human Cyclin D1 gene, rabbit Bcl-2 gene, and rabbit L-Myc gene (Sequence No. 19: sequences 1-1679 are the CAG promoter, sequences 1734-2618 are the human Cyclin D1 gene, sequences 2619-2684 are the P2A peptide encoding sequences, sequences 2685-3362 are the rabbit Bcl-2 gene, sequences 3363-3428 are the P2A peptide encoding sequences, sequences 3429-4613 are the rabbit L-Myc gene (containing a stop codon), and sequences 4657-4881 are the bovine growth hormone tailing signal) and the SV40T antigen gene (expression cassette B) were integrated into a control vector to prepare the expression vector TBCM'.

[0144] (2) Gene introduction

[0145] Using the same procedure as described in Example 2, each gene was introduced into rabbit spleen cells to obtain transformed cells.

[0146] (3) Evaluation of cell proliferation capacity

[0147] Using the same procedure as described in Example 2, the cell mass of each transformed cell was measured over time after gene introduction to evaluate cell proliferation capacity. Figure 8 shows the cumulative growth curve based on the measurement results.

[0148] As shown in Figure 8, for the transformed cells introduced with the expression vector NC, no proliferation was observed until day 29 of culture after gene introduction. In contrast, for the transformed cells introduced with the expression vectors (TB'CM, TBC'M, TBCM'), cell proliferation (growth) was observed, confirming the acquisition of immortalization ability, just like for the transformed cells introduced with the expression vector (TBCM).

[0149] Furthermore, Figure 8 confirms that the cell proliferation rate, similar to that in Example 2, varies depending on the type and combination of the introduced genes. Transformed cells incorporating expression vectors (TB'CM, TBC'M) showed the same cell proliferation rate as those incorporating expression vector (TBCM). Transformed cells incorporating expression vector (TBCM') exhibited a slightly slower proliferation rate compared to those incorporating expression vectors (TBCM, TB'CM, TBC'M), but this rate is acceptable for commercial use.

[0150] (4) Confirmation of antibody-producing cells

[0151] The cell cumulative growth curve shown in Figure 8 is a curve measuring the growth of the entire cell population obtained by transforming the recovered spleen cells, including cells other than plasma cells (antibody-producing cells). Therefore, the proportion of antibody-producing cells in the cell populations after 14, 21, or 28 days of culture following gene introduction was studied using flow cytometry. Specifically, the study was performed using the same procedures as described in Example 2.

[0152] Table 5 shows the percentage of antibody-producing cells (IgG ratio) calculated based on flow cytometry results. According to these results, antibody-producing cell proliferation was confirmed in all combinations. These results demonstrate that immortalization was possible while maintaining antibody-producing capacity, even when any of the following cases were performed: replacing the Bcl-2 family gene with the Bcl-XL gene, replacing the Myc family gene with the L-Myc gene, or replacing the Cyclin D family gene with the Cyclin D1 gene. That is, it is thus determined that for the Bcl-2 family genes, Myc family genes, and Cyclin D family genes used in this invention, plasma cells can be immortalized while maintaining their antibody-producing capacity, even when using any gene from each gene family.

[0153] [Table 5]

[0154]

[0155] [Example 4]

[0156] The effects on immortalization ability were investigated under the following conditions: the expression vector TBCM in Example 2 was set to only the SV40 large T antigen gene, only the SV40 small T antigen gene, and both the SV40 large T antigen gene and the SV40 small T antigen gene.

[0157] [1] Gene introduction and screening

[0158] (1) Manufacturing of carriers for expression

[0159] 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 for each expression cassette indicates that the expression cassette has been integrated into the expression vector. Expression vector NC is a vector that has not integrated any expression cassette (control vector).

[0160] [Table 6]

[0161]

[0162] The expression vector (large T + small T) was prepared using the same method as described in Example 2 for the expression vector TBCM.

[0163] The expression cassettes of the human Cyclin D1 gene, the rabbit Bcl-2 gene, and the rabbit c-Myc gene (expression cassette A used in Example 1) and the expression cassette of the SV40 large T antigen gene (sequence number 20: 1-330 are the SV40 promoter, 344-2470 are the gene encoding the large T antigen (containing the stop codon), and 2493-2627 are the SV40 tailing signal) were integrated into the control vector to prepare the expression vector (large T).

[0164] The expression cassettes of the human Cyclin D1 gene, the rabbit Bcl-2 gene, and the rabbit c-Myc gene (expression cassette A used in Example 1) and the expression cassette of the SV40T antigen gene (sequence number 21: sequences 1-330 are the SV40 promoter, sequences 344-868 are the gene encoding the small T antigen (containing the stop codon), and sequences 891-1025 are the SV40 tailing signal) were integrated into the control vector to prepare the expression vector (small T).

[0165] (2) Gene introduction

[0166] Using the same procedure as described in Example 2, each gene was introduced into rabbit spleen cells to obtain transformed cells.

[0167] (3) Evaluation of cell proliferation capacity

[0168] Using the same procedure as described in Example 2, the cell mass of each transformed cell was measured over time after gene introduction to evaluate cell proliferation capacity. Figure 9 shows the cumulative growth curve based on the measurement results.

[0169] As shown in Figure 9, for the transformed cells introduced with the expression vector NC, no proliferation was confirmed until day 28 of culture after gene introduction. In contrast, the transformed cells introduced with expression vectors (large T + small T, large T, small T) all showed cell proliferation and were confirmed to have acquired immortalization capabilities.

[0170] Figure 9 confirms that cell proliferation rates vary depending on the type and combination of introduced genes. Specifically, it was found that transformed cells incorporating the expression vector (large T + small T) proliferated fastest, with the proliferation rate following the order of expression vector (large T + small T) > expression vector (large T) > expression vector (small T). Transformed cells incorporating the expression vector (small T) only showed proliferation around day 10 of culture. While proliferation was promoted with only the SV40 small T antigen gene introduced, it was very slow. Based on these results, it is determined that the SV40 T antigen gene introduced into plasma cells in this invention preferably includes the SV40 large T antigen gene. Although it can be only the SV40 large T antigen gene, it is particularly preferred to include both the SV40 large T antigen gene and the SV40 small T antigen gene.

Claims

1. A method for manufacturing immortalized cells, comprising 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 in vitro to manufacture immortalized cells, wherein the antibody-producing cells are B cells or plasma cells, the Bcl-2 family gene is the Bcl-2 gene or the Bcl-XL gene, the Myc family gene is the c-Myc gene or the L-Myc gene, and the Cyclin D family gene is the Cyclin D1 gene or the Cyclin D3 gene.

2. The method for manufacturing immortalized cells as described in claim 1, wherein, The SV40T antigen gene, Bcl-2 family gene, Myc family gene, and Cyclin D family gene were introduced into the non-immortalized antibody-producing cells.

3. A method for manufacturing immortalized antibody-producing cells, comprising 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 antibody-producing cells collected from mammals or primary antibody-producing cells obtained by primary culture of said antibody-producing cells, wherein the antibody-producing cells are B cells or plasma cells, the Bcl-2 family gene is the Bcl-2 gene or the Bcl-XL gene, the Myc family gene is the c-Myc gene or the L-Myc gene, and the Cyclin D family gene is the Cyclin D1 gene or the Cyclin D3 gene.

4. The method for manufacturing immortalized antibody-producing cells as described in claim 3, wherein, The mammal is an animal that has been immunized with an antigen, and immortalized antibody-producing cells that produce antibodies that recognize the antigen are selected from the immortalized antibody-producing cells.

5. A method for manufacturing an antibody, wherein, Immortalized antibody-producing cells are manufactured using the method for manufacturing immortalized antibody-producing cells according to claim 3 or 4, the immortalized antibody-producing cells are cultured, and the generated antibodies are recovered.

6. A transformed cell, which achieves immortalization 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 a non-immortalized antibody-producing cell, wherein the antibody-producing cell is a B cell or a plasma cell, the Bcl-2 family gene is the Bcl-2 gene or the Bcl-XL gene, the Myc family gene is the c-Myc gene or the L-Myc gene, and the Cyclin D family gene is the Cyclin D1 gene or the Cyclin D3 gene.

7. The transformed cell as described in claim 6, wherein, The transformed cells have the ability to produce antibodies.

Images