Protein synthesis method

Abstract

The present invention addresses the problem of providing a novel technique that makes it possible to stably and efficiently produce a protein that has activity in vivo. The present invention is a protein production method that involves introducing an adenovirus vector that codes for a protein into a cultured cell line from a malignant tumor.

Description

Method for protein synthesis

[0001] The present invention relates to a method for the biosynthesis of proteins.

[0002] In vivo, nerve growth factors and angiogenesis factors in the body are functional active molecules, and their expression in the body is strictly controlled both temporally and spatially. The breakdown of this control has caused various diseases, but it is predicted that some diseases can be alleviated by supplementing these active molecules. However, since the protein structure of active molecules, including their post-translational modifications, is species-specific, it is difficult to extract them from other species and administer them to humans due to lack of evidence and imagination of being beneficial. Although mammalian cells can express human recombinant proteins to obtain molecules equivalent to those in the human body, it is often difficult to obtain them in the microgram order.

[0003] To eliminate the deficiency of biomolecules in the human body, it is difficult to synthesize equivalent molecules in large quantities in vitro. This problem affects basic research, the preparation of research antibodies, and clinical research. The main factors are the three-dimensional structure and folding of proteins (Non-Patent Document 1), the lack of intracellular auxiliary proteins and folding factors (Non-Patent Document 2), the difficulty of post-translational modification (Non-Patent Document 3), and the complexity and diversity of proteins (Non-Patent Document 4). [[ID=十一]]

[0004] Successful examples include recombinant protein production (Non-Patent Literature 5), cell-free translation systems (Non-Patent Literature 6), and protein production by synthetic biology (Non-Patent Literature 7). However, these technologies have challenges in terms of quality and quantity. For example, in recombinant protein production, post-translational modification and folding may not occur properly (Non-Patent Literature 8). In cell-free translation systems, post-translational modification and proper folding may not be guaranteed, making mass production difficult (Non-Patent Literature 9). In synthetic biology, the optimization of gene circuits and metabolic pathways affects the amount of protein produced, and if the gene circuits and metabolic pathways are inefficient, mass production of proteins may be difficult (Non-Patent Literature 10). These are synthesis methods that consider the balance between quality and quantity, but to synthesize protein molecules that are more equivalent to human biomolecules, it is still considered most effective to use mammalian cells. However, there are also several challenges in the synthesis of human bioactive proteins using mammalian cells. First, production efficiency tends to be low and costs high, which is due to the slow proliferation rate and difficulty in maintenance of mammalian cells (Non-Patent Literature 11). Second, although the selection and stabilization of appropriate cell lines are important, this can sometimes be difficult (Non-Patent Literature 12). Furthermore, controlling post-translational modifications can be difficult, as the pattern of glycosylation may differ depending on the cell line and culture conditions (Non-Patent Literature 13). There are also challenges with protein secretion and recovery, as protein recovery and purification inside and outside the cell can be difficult (Non-Patent Literature 14). In addition, there is a risk of viral contamination, requiring rigorous viral testing and removal processes to ensure the safety of the production process (Non-Patent Literature 15). Thus, even considering just one expression system, the technology for synthesizing physiologically active proteins in vitro from the human body presents many challenges.

[0005] Anfinsen, C. B. (1973). Principles that govern the folding of protein chains. Science, 181(4096), 223-230.Hartl, F. U., Bracher, A., & Hayer-Hartl, M. (2011). Molecular chaperones in protein folding and proteostasis. Nature, 475(7356), 324-332.Walsh, C. T., Garneau-Tsodikova, S., & Gatto, G. J. Jr. (2005). Protein posttranslational modifications: the chemistry of proteome diversifications. Angewandte Chemie International Edition, 44(45), 7342-7372.Dill, K. A., & MacCallum, J. L. (2012). The protein-folding problem, 50 years on. Science, 338(6110), 1042-1046.Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., ... & Riggs, A. D. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proceedings of the National Academy of Sciences, 76(1), 106-110.Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda,T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751-755.Cameron, D. E., Bashor, C. J., & Collins, J. J. (2014). A brief history of synthetic biology. Nature Reviews Microbiology, 12(5), 381-390.Gupta, S. K., & Shukla, P. (2018). Microbial platform technology for recombinant antibody fragment production: A review. Critical Reviews in Microbiology, 44(1), 1-13.Zemella, A., Thoring, L., Hoffmeister, C., & Kubick, S. (2015). Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. Chembiochem, 16(17), 2420-2431.Nielsen, J., & Keasling, J. D. (2016). Engineering cellular metabolism. Cell, 164(6), 1185-1197.Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature biotechnology, 22(11), 1393-1398.Birch, J. R., & Racher, A. J. (2006). Antibody production. Advanced drug delivery reviews, 58(5-6), 671-685.Stanley, P., & Schachter,H. (2010). Mammalian N-glycan branching and disease. In Essentials of Glycobiology (pp. 215-224). Cold Spring Harbor Laboratory Press. Shukla, A. A. , & Thommes, J. (2010). Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends in biotechnology, 28(5), 253-261. Merten, O. W. (2002). Virus contaminations of cell cultures-A biotechnological view. Cytotechnology, 39(2), 91-116. ,

[0006] When using normal cells to produce active proteins in vivo, the low proliferative capacity necessitates prolonged and expensive culture nutrients for maintenance, resulting in high costs and very low production efficiency. Furthermore, methods involving the introduction of plasmids encoding biomolecules into cultured cells make it difficult to produce biomolecules stably and efficiently. Therefore, the present invention aims to provide a novel technology that enables the stable and efficient production of proteins that exhibit activity in vivo.

[0007] The present invention, which solves the above problems, is as follows: [Item 1] A method for producing a protein, comprising introducing an adenovirus vector encoding a protein into a cultured cell line derived from a malignant tumor.

[0008] [Item 2] The method according to Item 1, wherein the protein is a secreted protein.

[0009] [Item 3] The method according to Item 2, wherein the secreted protein is a growth factor having heparin-binding activity and / or lectin-binding activity, and the secreted protein is purified from the culture supernatant of a malignant tumor-derived cell line into which the adenovirus vector has been introduced by affinity chromatography using a carrier supporting heparin and / or lectin.

[0010] [Item 4] The method according to Item 2, wherein the secreted protein is netrin and the malignant tumor-derived cultured cell line is a glioma cultured cell line.

[0011] [Item 5] The method according to Item 1, wherein the protein is a non-secretory protein.

[0012] [Item 6] The method according to any one of Items 1 to 5, wherein the animal species from which the protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species.

[0013] [Item 7] The method according to any one of Items 1 to 6, wherein the protein is of human origin and the malignant tumor-derived cultured cell line is of human origin.

[0014] [Item 8] The method according to any one of items 1 to 7, comprising culturing the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced in serum-free medium.

[0015] [Item 9] The method according to any one of Items 1 to 8, wherein the protein is endogenously produced by a specific tissue, and the malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue.

[0016] [Item 10] The method according to any one of items 1 to 9, comprising: seeding the malignant tumor-derived cultured cell line in a serum-containing medium; introducing an adenovirus vector encoding the protein into the malignant tumor-derived cultured cell line; and, after the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced has reached confluence, replacing the medium with serum-free medium and culturing the cells.

[0017] [Item 11] A method for producing a composition comprising the protein produced by the method described in any one of Items 1 to 10, comprising: introducing an adenovirus vector encoding the protein into a malignant tumor-derived cultured cell line; purifying the protein from the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced or from the culture supernatant thereof; and incorporating the purified protein into the composition, wherein the animal species from which the protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species; the protein is endogenously produced by a specific tissue; the malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue; and the composition is selected from pharmaceutical compositions, food compositions, cosmetic compositions, aquaculture feed compositions, and experimental reagent compositions.

[0018] [Item 12] A method for producing an antibody against the protein, comprising producing the protein by the method described in any one of Items 1 to 10, comprising: introducing an adenovirus vector encoding the protein into a malignant tumor-derived cultured cell line; purifying the protein from the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced or from the culture supernatant thereof; and producing the antibody using the purified protein as an antigen, wherein the animal species from which the protein originates and the animal species from which the malignant tumor-derived cultured cell line originates are the same animal species, the protein is endogenously produced by a specific tissue, and the malignant tumor-derived cultured cell line originates from a malignant tumor of the specific tissue.

[0019] [Item 13] A composition comprising the protein produced by the method described in any one of Items 1 to 10.

[0020] [Item 14] The composition according to Item 13, wherein the composition is any of a pharmaceutical composition, a food composition, a cosmetic composition, aquaculture feed composition, or a laboratory reagent composition.

[0021] [Item 15] The composition according to Item 14, which is a pharmaceutical composition for the treatment of a disease in which a decrease in the expression level of the protein in vivo is observed, comprising the protein as an active ingredient.

[0022] [Item 16] The composition according to Item 15, wherein the protein is netrin and the disease is ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease.

[0023] [Item 17] A method for treating a disease, comprising administering a pharmaceutical composition containing the protein produced by any one of Items 1 to 10 to a patient suffering from a disease in which a decrease in the in vivo expression level of the protein is observed.

[0024] [Item 18] The treatment method according to Item 17, wherein the protein is netrin and the disease is ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease.

[0025] [Item 19] The protein produced by any one of Items 1 to 10, for use in the treatment of a disease in which a decrease in the expression level of the protein in vivo is observed.

[0026] [Item 20] The protein according to Item 19, wherein the protein is netrin and the disease is ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease.

[0027] [Item 21] Use of the protein produced by any one of Items 1 to 10 in the manufacture of a therapeutic agent for a disease in which a decrease in the in vivo expression level of the protein is observed.

[0028] [Item 22] The use described in Item 21, wherein the protein is netrin and the disease is ischemic stroke, Alzheimer's disease, or Parkinson's disease.

[0029] According to the present invention, proteins can be produced in large quantities and stably.

[0030] Micrograph (×100) showing the confluent state of human glioma (astrocytoma) U-373MG. (Left) A figure showing the purification of the target glycoprotein, human netrin-1, by dividing the elution fraction of human netrin-1 produced by heparin column chromatography into six parts and performing SDS-PAGE (CBB staining) and Western blotting (anti-human netrin-1 antibody) on a portion of the fraction. (Right) A figure showing the confirmation of 1 microgram of netrin-1 purified according to the protocol of the invention, scaled up, by SDS-PAGE (CBB staining). It was confirmed that secretion expression into the culture supernatant and purification were possible with good reproducibility. A figure showing the angiogenic vitality of human netrin-1 in human umbilical vein endothelial cells (HUVECs) by Migration Assay. Figure 4 shows the results of purifying the culture supernatant from a stable expression cell line prepared by introducing the pCDNA-3.1(+)-human netrin-1 plasmid expression vector into the human non-small cell lung cancer cell line H1299, and confirming its expression using SDS-PAGE (CBB staining) and Western blotting (anti-human netrin-1 antibody), similar to Figure 2. The purified protein (CBB staining) shows more impurities than those observed in the adenovirus expression system using U373-MG cells. Figure 4 shows the results of transfecting various malignant tumor cells with the pcDNA3.1(+) plasmid-mediated human netrin-1 expression vector, confirming its expression in the same manner as the test in Figure 4. In actual human tissues, netrin-1 is frequently expressed in the colon in addition to the brain, but is hardly expressed in colorectal cancer cell lines such as HCT116 and LS174T. For some cell lines, the gene transfer rate by transfection is significantly lower compared to adenovirus. This figure shows the results of confirmation tests of the kinase activity of Src, Akt, and MAPK, demonstrating that the generated human netrin-1 has survival signaling activity.

[0031] The present invention relates to a method for producing a protein. Specifically, an adenovirus vector encoding a protein is introduced into a cultured cell line derived from a malignant tumor, and the cell line is allowed to produce the protein. The protein can be obtained by purifying the cell line or its culture supernatant.

[0032] When using normal cells to synthesize proteins present in the body, the low proliferative capacity necessitates prolonged and expensive culture nutrients for maintenance, resulting in high costs and very low production efficiency. This invention solves these problems by using cultured cell lines derived from malignant tumors. Unlike normal cells, the established cultured cell lines derived from malignant tumors have a remarkably fast proliferation rate and proliferate sterically without the occurrence of contact inhibition, which is proliferation control through cell contact. Therefore, according to this invention, large-scale protein synthesis can be achieved stably and at low cost.

[0033] Furthermore, in order to synthesize proteins in an active form within a living organism, it is necessary to reproduce post-translational modifications such as glycosylation and disulfide bonding that occur in vivo, but protein synthesis systems using E. coli cannot reproduce these modifications. This invention uses a cell line instead of E. coli for protein synthesis, and can produce active proteins within a living organism.

[0034] Furthermore, when culturing normal human cells, the risk of viral infection increases with prolonged cultivation. Normal cells are highly susceptible to viral infection, and infection significantly affects their proliferative capacity. To overcome this, the present invention uses adenovirus vectors with strong expression promoters in highly proliferative malignant tumor cell lines for biomolecule synthesis, thereby reducing the effects of viral infection.

[0035] As described above, the present invention makes it possible to improve production efficiency, reduce costs, stabilize culture, control specific post-translational modifications, and lower the risk of infection by using an adenovirus vector and a malignant tumor cell line for protein synthesis.

[0036] Post-translational modifications such as glycosylation are known to be species-specific. Therefore, it is preferable that the animal species from which the protein introduced by the adenovirus vector originates and the animal species from which the malignant tumor-derived cultured cell line originates are the same. This makes it possible to produce a protein derived from the same animal species that is active in vivo.

[0037] The type of malignant tumor cell line used in this invention is not particularly limited. For example, the following cell lines can be used as human malignant tumor cell lines. Cell lines for brain and nervous system tumors such as glioma cell lines like U-373MG, U-343MG, NCH421K, and NCH644; astrocytoma cell lines like CCF-STTG1; and neuroblastoma cell lines like SK-N-LO; breast cancer cell lines such as BT-20, COLO-824, and ZR-75-1; intestinal and colon cancer cell lines such as colon adenocarcinoma cell lines like Caco-2 and COLO-205; duodenal adenocarcinoma cell lines like HuTu-80; bladder cancer cell lines such as 5637, ECV-304, and HB-CLS-1; skin cancer cell lines such as cutaneous melanoma cell lines like A-375 and A-431; cutaneous sarcoma cell lines like HS1-CLS; and cutaneous fibroblast-like cell lines like WS1; Hepatocellular carcinoma cell lines such as Hep-G2 and PLC-PRF-5, and endothelial carcinoma cell lines such as SK-HEP-1; Burkitt lymphoma cell lines such as EB1, acute lymphoblastic leukemia (ALL) cell lines such as CCRF-CEM, erythroblastic leukemia cell lines such as HEL-92.1.7, acute myeloid leukemia (AML) cell lines such as HL-60, T-cell lymphoma cell lines such as HSB, B-lymphocyte cell lines such as RPMI-1788, monocytic leukemia cell lines such as THP-1, and lymphoblast cell lines such as TK6; Head and neck cancer cell lines (larynx, pharynx, nasal cavity, thyroid) such as A64-CLS (submandibular gland adenoma cell line), C-643 (thyroid cancer cell line), CLS-117 (Struma sarcoma cell line), CLS-354 (oral cancer cell line), COLO-680N (esophageal cancer cell line), Detroit-562 (pharyngeal cancer cell line), HNSCC (oropharynx, tonsil) cell line, HNO41 (oropharynx, tonsil) cell line, HNO97 (oral cavity; tongue) cell line, HNSCC (larynx) cell line, HNO210 (larynx) cell line, HNSCC (tongue) cell line, HNO223 (htonia) cell line, RPMI-2650 (nasal septum cancer cell line), Y-79 (retinoblastoma cell line), and other such cells; Lung cancer cell lines such as A-427, A-549, Cal-1, CLS-54, small cell lung cancer cell lines such as DMS-79, histiocytoma lung cell lines such as GCT, fibroblast lung cell lines such as HEL-299, mesothelioma lung cell lines such as H-Put-1, and large cell lung cancer cell lines such as LCLC-97TM1;Osteosarcoma cell lines such as CADO-ES1 and MHH-ES1, osteosarcoma cell lines such as HOS (TE-85) and KHOS-240S, and osteochondrosarcoma cell lines such as SW-1353; gastric cancer cell lines such as AGS and CLS-145, and gastric cancer cell lines such as HGC-27 and KATO-III; renal cancer cell lines such as 769-P, 786-0, and A-498, renal clear cell carcinoma cell lines such as RCC (KTCTL-13), and renal (Wilms tumor) cell lines such as SK-NEP-1; adrenal cancer cell lines such as NCI-H295R and Huamun adrenal adenocarcinoma cell lines such as SW-13; Pancreatic cancer cell lines such as AsPC-1, Capan-1, DAN-G, and Panc-1; Rhabdomyosarcoma cell lines such as A-204, A-673, and HS-729; Fibrosarcoma cell lines such as HT-1080, Fibrosarcoma cell lines such as SW-684, Male liposarcoma cell lines such as SW-872, Synovial sarcoma cell lines such as SW-982; AN3 Reproductive organ-related tumor cell lines (uterus, placenta, ovary) such as endometrial adenocarcinoma cell lines like CA, placental choriocarcinoma cell lines like BEWO, cervical cancer cell lines like CaSki, DEER-186, CERV-215, ovarian adenocarcinoma cell lines like NIH:OVCAR-3, OAW-42, ovarian teratoma cell lines like PA-1, vulvar leiomyosarcoma cell lines like SK-LMS-1, and uterine leiomyosarcoma cell lines like SK-UT-1, SK-UT-1B; prostate cancer cell lines like DU-145, LNCaP, PC-3.

[0038] While human malignant tumor-derived cell lines have been exemplified above, many types of malignant tumor-derived cell lines from other animal species are also commercially available and can be used without limitation for the purposes of this invention.

[0039] Adenoviruses are introduced into host cells via the coxsackievirus-adenovirus receptor (CAR). Therefore, it is preferable that the malignant tumor-derived cell lines used express the CAR protein.

[0040] Moreover, even in a cell line that does not express the CAR protein, transduction with an adenovirus vector can be performed by introducing a vector encoding the CAR protein to express the CAR protein. Since kits for introducing the CAR protein to improve the transduction efficiency with an adenovirus vector are commercially available, these may also be used. Examples of such commercial products include CAR Receptor Booster (Clontech) and ViraDuctin Adenovirus Gene Transfer Kit (Cell biolabs).

[0041] In the present invention, the type of protein to be produced is not particularly limited, and it may be a secreted protein secreted extracellularly or a non-secreted protein localized intracellularly or in the cell membrane.

[0042] When producing a secreted protein in the present invention, the type thereof is not limited, and it may be any protein released extracellularly, such as an extracellular matrix protein, a blood protein, a digestive enzyme, a hormone, an antibody, etc. The present invention is preferably applied for the production of growth factors or cytokines.

[0043] Examples of secreted proteins include insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), angiopoietin, angiostatin, granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), and acid fibroblast growth factor. One of the transforming growth factor a superfamily including follicular growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and TGF-II), and TGFa, activin, inhibin, or any of bone morphogenetic proteins (BMPs) BMP1-15, one of the neuregulin family including neuregulins 1-4, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin. Examples include hormones and growth and differentiation factors such as NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), one of the families of Neurturin, Agrin, Semaphorin / Collapsin, Netrin including Netrin-1 and Netrin-2, hepatocyte growth factor (HGF), Ephrin, Noggin, and Sonic hedgehog (SHH).

[0044] In one embodiment, an adenovirus vector encoding a human antibody is introduced into a cultured cell line derived from a human malignant tumor to produce a human antibody. In such an embodiment, it is superior to the conventional method in the following aspects 1) to 6). 1) Since it is synthesized from human cells (such as human plasmacytoma-derived strains), the humanization step is completely eliminated. Since the antibody synthesized according to the present invention is based on human-derived proteins, it is possible to reduce the immunogenicity risk associated with the use of non-human-derived proteins. (2) It is possible to produce neutralizing antibodies during a pandemic, and it is possible to obtain an expression level several tens of times higher and much faster than that produced by CHO. As a result, neutralizing antibodies that exert effects prior to the vaccine are distributed to patients. 3) A humanized antibody can be produced in a very short period (within 5 months compared to the existing technology of CHO cells) and with high expression, so that prototype antibody production can be readily prepared, and the clinical trial period can be significantly shortened. 4) Since it does not require as much time, space, and culture scale as CHO cells, all resources including personnel can be significantly reduced. 5) It is not forced to perform long-term culture like CHO cells. Therefore, many maintenance steps, checks for mutations and infections, etc. at the GMP level are eliminated, and safety is also highly ensured. 6) Since the expression level can be controlled by MOI, the amount of antibody synthesized in cells can be strictly controlled according to the properties of the antibody. In the present invention, by adjusting MOI (Multiplicity of Infection), it is possible to scale up or down according to the required amount of antibody, and the transition from clinical trials to commercial production is smooth.

[0045] When producing non-secretory proteins in the present invention, the types are not limited. The non-secretory proteins produced by the present invention may exhibit any localization, such as proteins localized in the cytoplasm, nucleus, other intracellular organelles, or membrane proteins localized in the cell membrane. In addition, the non-secretory proteins produced by the present invention may exhibit any functionality, such as enzymes, structural proteins, transport proteins, transcription factors, receptors, etc.

[0046] Examples of non-secretory proteins include glucocerebrosidase, α-galactosidase A, α-L-iduronidase, acid α-glucosidase, alglucosidase, iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, and lysosomal acid lipase. Examples include lipase and tripeptidyl peptidase I.

[0047] In the present invention, a preferred embodiment is one in which an adenovirus vector encoding a protein endogenously produced by a specific tissue is introduced into a cultured cell line derived from a malignant tumor of the specific tissue. Here, "protein endogenously produced by a specific tissue" refers to a protein that is naturally produced by any of the cells constituting the specific tissue when the specific tissue is functioning normally in vivo. By adopting this embodiment, it is possible to mimic cell-type-specific post-translational modifications that occur in vivo and to produce active proteins.

[0048] In specific embodiments of the present invention, an adenovirus vector encoding netrin, more specifically netrin-1, is introduced into a cultured cell line derived from glioma. When producing netrin as an active ingredient in a pharmaceutical product for administration to humans, an adenovirus vector encoding human netrin, more specifically human netrin-1, is introduced into a cultured cell line derived from human glioma.

[0049] Furthermore, examples have confirmed that netrin produced and purified by the method of the present invention can be stably stored at 4°C for more than one year while maintaining its activity.

[0050] The adenovirus vector encoding the protein used in the present invention can be prepared by conventional methods. For example, the adenovirus vector can be prepared by the COS-TPC method or the full-length DNA introduction method. The COS-TPC method is a method for producing recombinant adenovirus by simultaneously transfecting 293 cells with a recombinant cosmid incorporating the target cDNA or expression cassette and the parental viral DNA-terminal protein complex (DNA-TPC), and utilizing homologous recombination that occurs within the 293 cells (Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA introduction method is a method for producing recombinant adenovirus by restricting digestion of recombinant cosmid containing the target gene, and then transfecting it into 293 cells (Miho Terashima, Takaki Kondo, Hiromi Kanegae, and Izumi Saito (2003) Experimental Medicine 21(7) 931). The COS-TPC method can be performed using the Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.) or the Adenovirus gene DNA-TPC (Takara Bio Inc.). The full-length DNA introduction method can also be performed using the Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.).

[0051] Furthermore, the proteins incorporated into the adenovirus vector may optionally be tagged with a FLAG tag or 3×FLAG. This facilitates purification using affinity columns or other methods. Additionally, fluorescent proteins such as GFP may be added. This allows for easy confirmation, using a fluorescence microscope, that the protein is expressed in cells infected with the adenovirus vector.

[0052] In this invention, the above-mentioned adenovirus vector is introduced into a cultured cell line derived from a malignant tumor. There are no particular restrictions on the introduction method, and it can be carried out by conventional methods.

[0053] Cultured cells into which an adenovirus vector has been introduced can be cultured using known methods. That is, using a medium suitable for the malignant tumor-derived cell line being used, and changing the medium as appropriate, CO ２ It can be cultured in an incubator.

[0054] For seeding cultured cells, any known medium can be used depending on the type of cultured cell. For example, D-MEM / F12 medium, glutamine-supplemented RPMI 1640 medium, etc., can be used. Reagents commonly used in this field, such as HEPES, EDTA, antibiotics (e.g., penicillin, gentamicin, streptomycin / penicillin, etc.), and bovine serum, may be added to the medium.

[0055] In a preferred embodiment of the present invention, serum-free medium is used as the culture medium. By culturing in serum-free medium, the synthesized protein can be easily recovered. Furthermore, when the protein produced by the present invention is provided as a pharmaceutical product, culturing in serum-free medium prevents contamination by biomolecules such as other proteins derived from serum, thereby ensuring the quality of the protein preparation as a pharmaceutical product.

[0056] Furthermore, the growth rate of the malignant tumor-derived cell lines used in this invention is not affected even when cultured in serum-free medium.

[0057] When growth factors are used as proteins introduced into malignant tumor-derived cultured cell lines via adenovirus vectors, autocrine activity is exerted by the growth factors secreted into the culture supernatant, which is advantageous when culturing in serum-free medium. Specifically, using serum-free medium allows for protein synthesis without the inclusion of foreign serum components, and growth factor proteins, in particular, can continuously nourish the cells synthesizing them through autocrine action. Therefore, cells infected with growth factor-expressing adenovirus vectors can survive in serum-free medium without undergoing cell death due to nutrient deficiency under serum-free conditions, continuing to biosynthesize the target protein. Regarding the activity of the biosynthesized growth factors, it has been confirmed that growth signals are transmitted in experiments where the generated growth factors are added to cultured cells under serum-free conditions (see Figure 6).

[0058] In one embodiment, a malignant tumor-derived cell line into which an adenovirus vector has been introduced is cultured in a serum-containing medium after it reaches confluence, and then the medium is changed to a serum-free medium. More specifically, a malignant tumor-derived cell line into which an adenovirus vector has been introduced is cultured in a serum-containing medium, and after it reaches confluence, the medium is changed to a serum-free medium and the cells are cultured. When cells are seeded, they consume a lot of energy for proliferation. In addition, when an adenovirus vector is introduced, a large amount of secreted proteins are produced within the cells, which also consumes a lot of energy. If culture is started in a serum-free medium from the cell seeding stage, the nutrients in the medium may be rapidly depleted due to cell proliferation and the large amount of secreted proteins produced. In this embodiment, energy is supplied by using a nutrient-rich serum-containing medium during the cell proliferation stage, when there is a large energy demand for cells, and then the medium is changed to a serum-free medium once the cells reach confluence and the energy demand for cell proliferation is gone. This prevents cell death due to energy depletion and prevents contamination of biomolecules such as foreign proteins derived from serum, thereby ensuring the quality of the protein preparation as a pharmaceutical product.

[0059] When secreted proteins are produced according to the present invention, the secreted proteins synthesized in the above-mentioned malignant tumor-derived cultured cell line are secreted into the culture supernatant. The secreted proteins present in the culture supernatant obtained after culturing can be purified by known methods. For example, they can be purified by affinity chromatography using a column supported with a substance (specific ligand) that has affinity for the target protein, ion exchange chromatography or chromatofocusing that isolates using surface electrolysis, hydrophobic interaction chromatography that isolates using the hydrophobicity of the protein, or gel filtration chromatography that isolates based on the size of the protein. Furthermore, purification may be performed only once or multiple times.

[0060] Furthermore, when producing non-secretory proteins according to the present invention, non-secretory proteins can be extracted and purified from the above-mentioned malignant tumor-derived cultured cell lines by known methods. Protein extraction can be performed by physical disruption methods, such as sonication, homogenization, freeze-thaw cycles, or bead milling, which physically disrupt cells, or by chemical disruption methods, which dissolve cell membranes using surfactants such as Triton X-100 or SDS. As the buffer used during extraction, it is preferable to use a pH-adjusted solution such as Tris-HCl or PBS. A protease inhibitor may be added to the buffer to prevent protein degradation during extraction. Examples of purification of the extracted target protein include the affinity chromatography, ion exchange chromatography, chromatofocusing, hydrophobic interaction chromatography, and gel filtration chromatography described above.

[0061] In a preferred embodiment of the present invention, the target protein is purified by affinity chromatography.

[0062] The column used can be appropriately selected depending on the protein being synthesized. For example, if the protein has heparin-binding activity, a heparin column can be used.

[0063] Furthermore, with the exception of erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF), only a very small number of growth factors lack heparin-binding activity. Therefore, the present invention, in a form that includes a purification step by affinity chromatography using a heparin-supported carrier, can be applied to the production of almost all growth factors (insulin, VEGF group, HGF group, FGF group, NGF, etc.), including high molecular weight dimers.

[0064] Furthermore, a lectin column supported with lectins, which are sugar-recognizing proteins, may be used as the column. Since most proteins secreted extracellularly are glycoproteins, a lectin column can be suitably used to purify the secreted proteins produced by the present invention.

[0065] Furthermore, lectin columns may be used in combination with heparin columns. Using them together allows for higher affinity chromatography, resulting in highly purified proteins.

[0066] The synthesis method according to the present invention may include other known steps in addition to the steps described above.

[0067] The protein produced by the method according to the present invention undergoes post-translational modifications, such as glycosylation, equivalent to those of the same protein active in vivo. Therefore, it is preferable to incorporate the protein produced by the method according to the present invention into various compositions used for administration or ingestion to living organisms. In other words, the present invention also relates to compositions containing the protein produced by the method described above.

[0068] Specific forms of the composition of the present invention include, for example, pharmaceutical compositions, food compositions, cosmetic compositions, and aquaculture feed compositions.

[0069] The present invention also relates to a method for producing a composition, which includes purifying the protein produced by the method described above and incorporating it into a pharmaceutical composition, a food composition, a cosmetic composition, or aquaculture feed composition.

[0070] In one embodiment, the present invention includes the step of purifying the protein produced by the method described above and adding it to the raw materials of a pharmaceutical composition, a food composition, a cosmetic composition, or an aquaculture feed composition.

[0071] The pharmaceutical composition of the present invention contains a protein produced by the method described above as an active ingredient. The pharmaceutical composition of the present invention is preferably used for the treatment of diseases in which a decrease in the expression level of the protein in vivo is observed.

[0072] Examples of raw materials for the pharmaceutical composition to be combined with the protein include surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Water or aqueous solutions for dispersing these can also be used as raw materials. The pharmaceutical composition may be provided as a freeze-dried preparation or in liquid form.

[0073] For example, when netrin is produced by the method according to the present invention, it can be used as an active ingredient in a pharmaceutical composition for the treatment of diseases in which a decrease in netrin secretion is observed, such as ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease.

[0074] The pharmaceutical composition of the present invention is preferably administered via parenteral routes. For example, it can be in the form of an injection, eye drops, nasal, pulmonary, or transdermal dosage form. The specific formulation for each dosage form can be appropriately designed according to the target disease and site of the disease.

[0075] Examples of raw materials for the food composition to be combined with the aforementioned protein include other proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. When the food composition of the present invention is manufactured as a beverage, examples of raw materials include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts.

[0076] A specific form of the food composition of the present invention is, for example, cultured meat. That is, it can be provided in the form of a cultured meat food product containing the protein produced by the method described above.

[0077] Examples of raw materials for the cosmetic composition to be combined with the aforementioned protein include surfactants, chelating agents, plant-derived extracts, animal-derived extracts, microbial-derived extracts, zinc compounds, thickeners, oils, dyes, pigments, antibacterial agents, humectants, pH adjusters, antioxidants, UV absorbers, moisturizing ingredients, enzymes, and fragrances. The cosmetic composition may be provided in dosage forms such as lotions, emulsions, creams, ointments, hard ointments, poultices, and aerosols.

[0078] When the present invention is provided in the form of a cosmetic composition, it is preferable to select a growth factor having the effect of regenerating skin tissue and preventing aging as the protein to be incorporated as an active ingredient. Furthermore, it is preferable that the cosmetic composition be in the form of a topical skin preparation.

[0079] Examples of raw materials for the aquaculture feed composition to be combined with the aforementioned protein include plant-derived raw materials such as seeds, grains, leaves, roots, tubers, flowers, pods, shells, oils, soybean meal, soybean protein isolates, potato starch powder, wheat, barley, corn, soybean oil, and corn gluten powder, as well as animal-derived raw materials such as fish meal, fish oil, milk powder, skim milk powder, bone extract, meat extract, and blood extract. The specific raw materials and their forms of provision can be appropriately determined depending on the animals being farmed.

[0080] There are no particular restrictions on the animals to which the aquaculture feed composition of the present invention is administered, but examples include fish and mammals, and it can be applied to aquaculture and livestock farming. In this case, it is preferable that the animal species from which the protein produced by the method of the present invention originates, the animal species from which the malignant tumor-derived cultured cell line originates, and the animal species to which the aquaculture feed composition of the present invention is administered are the same or closely related species. Furthermore, it is preferable that the protein to be incorporated into the aquaculture feed composition of the present invention is a growth factor.

[0081] Furthermore, when applying the present invention as a composition for experimental reagents, examples include adding it to the culture medium during the culture of various stem cells such as iPS cells, primary cells, passaged cells, and established cell lines, as well as cultured tissues.

[0082] Furthermore, since the secreted protein produced by the present invention has an active structure in vivo, it can be used as an antigen to prepare antibodies with high titers. As long as the secreted protein produced by the present invention has an active structure in vivo, the embodiments are not particularly limited as long as it is used as an antigen.

[0083] In one embodiment, a secreted protein produced by the method of the present invention is injected as an antigen into an immunized animal (such as a mouse, rat, rabbit, goat, sheep, or chicken). After an immunization period, B cells are collected from the spleen or lymph nodes of the immunized animal and fused with an immortalized malignant tumor-derived cultured cell line, such as myeloma, to produce a hybridoma. From the obtained hybridomas, hybridomas that produce antibodies specifically binding to the secreted protein produced by the method of the present invention are screened. By culturing and growing the hybridomas that produce the desired antibody, a monoclonal antibody that specifically recognizes the secreted protein produced by the method of the present invention can be prepared.

[0084] In one embodiment, antibodies that specifically bind to an antigen (a secreted protein produced by the method of the present invention) are screened from a phage display antibody library expressing Fv or Fab antibodies. This makes it possible to prepare monoclonal antibodies that specifically recognize secreted proteins produced by the method of the present invention without using immunized animals.

[0085] In one embodiment, an antibody that specifically binds to an antigen (a secreted protein produced by the method of the present invention) is screened from a phage display antibody library expressing Fv or Fab antibodies. The antibody can be produced by the method of the present invention using an adenovirus vector encoding the gene for this antibody.

[0086] In one embodiment, antibodies that specifically bind to human-derived antigen proteins (secreted proteins produced by the method of the present invention) are screened from a phage display antibody library expressing human Fv or Fab antibodies using a human-derived malignant tumor-derived cultured cell line. Antibodies can be produced by the method of the present invention using an adenovirus vector encoding the gene for this antibody and a human-derived malignant tumor-derived cultured cell line. When human antibodies are produced by the method according to this embodiment, there are advantages to conventional methods in the points 1) to 6) described above.

[0087] The following examples show the production of netrin-1, a human nerve axon-guiding factor and angiogenic factor, as a secreted protein.

[0088] (1) Cultured human malignant tumor cell line U-373MG cells (ATCC), which are derived from glial cells that express Netrin-1 in the human body, are processed in 3.0 × 10⁻⁶ units. ６ Cells were seeded in eight 15cm diameter plastic culture dishes. The culture medium used was DMEM containing culture antibiotics and 10% FBS (20 mL / dish). The seeded cells were incubated at 37°C in 5% CO2. ２ The cells were cultured for 24 hours to confirm colonization, and then the process of infecting them with an adenovirus vector was carried out. (At this time, the number of cells was 1.0 × 10 per culture dish.) ７ (I confirmed beforehand that it was at the level of "cells".)

[0089] (2) Adenovirus vector infection: Adenovirus vector (Adenovyrus Dual Expression Kit, Takara Baio) encoding the cDNA of human netrin-1 (NCBI Gene ID: 9423) was used to infect the cells at 5 MOI and then cultured for 24 hours.

[0090] (3) Serum-free culture and culture supernatant recovery. After 24 hours of infection, the cells were confirmed to be confluent under a microscope (Figure 1), and the culture medium was aspirated and discarded. Subsequently, the cultured cells were washed three times with 10 mL PBS(-), and the culture medium was replaced with 30 mL serum-free DMEM (containing antibiotics), and the culture was kept at 37°C and 5% CO2. ２The culture was continued below. After 72 hours of serum-free culture, all serum-free culture medium was collected and stored at 4°C. Another 30 mL of serum-free culture medium was added and the culture was continued. Once the cells began to float, the culture medium was collected again and stored at 4°C.

[0091] (4) Preparation of culture supernatant containing target secreted protein The culture supernatant stored at 4°C was processed entirely at 4°C thereafter. The harvested culture medium was centrifuged at 1000 × g for 15 minutes, and the supernatant was centrifuged at 10,000 × g for 20 minutes. The supernatant after centrifugation was passed through a 0.22 μm filter using a peristaltic pump. The supernatant after passing through the filter was subjected to affinity column chromatography.

[0092] (5) The entire supernatant of the affinity column-purified culture was flowed onto heparin column #1 using a peristaltic pump and adsorbed at a flow rate of 500 μL / min. After adsorption of all the culture supernatant, 30 mL of washing buffer #2 was flowed to wash the column. After washing, 10 mL of elution buffer #3 was flowed to obtain the eluted fraction, which was concentrated by centrifugation using an ultrafiltration membrane (50 kDa or higher) and stored at 4°C. #1 Heparin column: HiTrap™ Heparin HP (Amersham Biosciences 17-0406-01) #2 Washing buffer: 10 mM phosphorate buffer (pH 7.0) #3 Elution buffer: 1 M NaCl in 10 mM phosphorate buffer (pH 7.0)

[0093] (6) Quantification, storage, and confirmation of purified protein. Since the eluted fraction contained high concentrations of salt, it was diluted with DDW and protein quantification was performed using the Bradford method. After quantification, the eluted fraction was adjusted to 1 mg / mL using an elution buffer and stored at 4°C (it was possible to store it at 4°C for one year while maintaining its activity).

[0094] 20 μg (5 μg for silver staining) was dissolved in a 5× SDS-PAGE sample buffer, boiled for 2 mins, then SDS-PAGE was performed on an 8% acrylamide gel, followed by CBB staining (or silver staining). Human netrin-1 protein was identified as a single band with a molecular weight of approximately 80 kDa (Figure 2). Using this method, 1.0 × 10⁻⁶ ７ Approximately 100–120 μg of human netrin-1 could be produced from cells U373-MG cells (one 15 cm diameter culture dish).

[0095] (7) Confirmation of biomolecular activity: Netrin-1 not only acts as an axon-guiding factor but also possesses angiogenic activity. Therefore, a Migration Assay (Park, K. W., 2004) was performed using human umbilical vein endothelial cells (HUVECs). Human VEGF was used as a positive control to confirm its activity for angiogenesis (Figure 3).

[0096] (8) Comparison with expression systems using plasmid-introduced stable expression cells In order to prove that the expression system of the present invention is the best method compared to other existing plasmid expression biosynthesis methods using mammalian cells, a human non-small cell lung cancer cell line H1299, which is easy to create stable expression cells from, was introduced with a pCDNA-3.1(+) plasmid expression vector encoding human netrin-1, and a cell line that stably expresses human netrin-1 at high levels was established by antibiotic selection.

[0097] Similar to the U373-MG described above, the established human netrin-1 stable-expressing H1299 cell clone was cultured in serum-free conditions, and the human netrin-1 secreted into the culture supernatant was purified (Figure 4).

[0098] It was found that about one-quarter the amount of U-373MG could be recovered with a similar culture volume, but because cell selection leads to a decrease in proliferation capacity, plasmid-based expression cannot be definitively called a stable biosynthesis system.

[0099] Furthermore, stable expression strains were created using plasmids from malignant tumor cell lines derived from multiple tissues, and their expression levels were confirmed. However, stable expression could not be confirmed in most cells, suggesting the efficiency of forced expression using adenovirus vectors (Figure 5).

[0100] (9) Confirmation of Growth Factor Activity The presence of Netrin-1 as a growth factor in signal transduction was confirmed by examining the kinase activity of Src, Akt, and MAPK. Human non-small cell lung cancer cell line H1299 was cultured in a culture medium containing serum, then the culture medium was changed to one without serum. After 24 hours of starvation, Netrin-1 was added to the culture medium at a concentration of 500 ng / mL. Cells were frozen and recovered over time, and cell lysates were performed by SDS-PAGE followed by Western blotting. Confirmation of the kinase activity of each kinase using phosphorylation antibodies showed that Src and Akt were activated over time after the addition of Netrin-1. This demonstrated that Netrin-1 synthesized and purified by adenovirus possesses survival signal activity (Figure 6).

[0101] This invention can be applied to technologies for producing proteins as active ingredients in pharmaceuticals, foods, cosmetics, aquaculture feed, and experimental reagents.

Claims

1. A method for producing a protein, comprising introducing an adenovirus vector encoding the protein into a cultured cell line derived from a malignant tumor.

2. The method according to claim 1, wherein the protein is a secreted protein.

3. The method according to claim 2, wherein the secreted protein is a growth factor having heparin-binding activity and / or lectin-binding activity, and comprises purifying the secreted protein from the culture supernatant of a malignant tumor-derived cell line into which the adenovirus vector has been introduced by affinity chromatography using a carrier supporting heparin and / or lectin.

4. The method according to claim 2, wherein the secreted protein is netrin and the malignant tumor-derived cultured cell line is a glioma cultured cell line.

5. The method according to claim 1, wherein the protein is a non-secretory protein.

6. The method according to any one of claims 1 to 5, wherein the animal species from which the protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species.

7. The method according to any one of claims 1 to 6, wherein the protein is of human origin, and the malignant tumor-derived cultured cell line is of human origin.

8. The method according to any one of claims 1 to 7, comprising culturing the malignant tumor-derived cell line into which the adenovirus vector has been introduced in serum-free medium.

9. The method according to any one of claims 1 to 8, wherein the protein is endogenously produced by a specific tissue, and the malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue.

10. The method according to any one of claims 1 to 9, comprising: seeding the malignant tumor-derived cell line in a serum-containing medium; introducing an adenovirus vector encoding the protein into the malignant tumor-derived cell line; and, after the malignant tumor-derived cell line into which the adenovirus vector has been introduced has reached confluence, replacing the medium with serum-free medium and culturing the cell line.

11. A method for producing a composition comprising the protein produced by the method of any one of claims 1 to 10, comprising: introducing an adenovirus vector encoding the protein into a malignant tumor-derived cultured cell line; purifying the protein from the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced or from the culture supernatant thereof; and incorporating the purified protein into the composition, wherein the animal species from which the protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species; the protein is endogenously produced by a specific tissue; the malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue; and the composition is selected from pharmaceutical compositions, food compositions, cosmetic compositions, aquaculture feed compositions, and experimental reagent compositions.

12. A method for producing an antibody against the protein, comprising producing the protein by the method according to any one of claims 1 to 10, comprising: introducing an adenovirus vector encoding the protein into a malignant tumor-derived cultured cell line; purifying the protein from the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced or from the culture supernatant thereof; and producing the antibody using the purified protein as an antigen, wherein the animal species from which the protein originates and the animal species from which the malignant tumor-derived cultured cell line originates are the same animal species, the protein is endogenously produced by a specific tissue, and the malignant tumor-derived cultured cell line originates from a malignant tumor of the specific tissue.

13. A composition comprising the protein produced by the method according to any one of claims 1 to 10.

14. The composition according to claim 13, wherein the composition is any one of a pharmaceutical composition, a food composition, a cosmetic composition, aquaculture feed composition, or a laboratory reagent composition.

15. The composition according to claim 14, which is a pharmaceutical composition for the treatment of a disease in which a decrease in the in vivo expression level of the protein is observed, comprising the protein as an active ingredient.

16. The composition according to claim 15, wherein the protein is netrin, and the disease is ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease.

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