WO2018043996A1 - Nouvelle cellule de myélome pour le développement d'un hybridome - Google Patents

Nouvelle cellule de myélome pour le développement d'un hybridome Download PDF

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WO2018043996A1
WO2018043996A1 PCT/KR2017/009311 KR2017009311W WO2018043996A1 WO 2018043996 A1 WO2018043996 A1 WO 2018043996A1 KR 2017009311 W KR2017009311 W KR 2017009311W WO 2018043996 A1 WO2018043996 A1 WO 2018043996A1
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xiap
protein
myeloma cells
cells
present
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김지혜
이영하
고봉국
김규태
이종서
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앱클론(주)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte

Definitions

  • the present invention relates to novel myeloma cells for hybridoma development. Specifically, it relates to a myeloma cell overexpressing XIAP protein and a method for producing the same. More specifically, the present invention relates to a myeloma cell overexpressing a XIAP protein capable of producing an antibody-producing hybridoma with higher efficiency and a method for producing the same.
  • Monoclonal antibodies are prepared from hybridomas prepared by fusion of splenocytes producing antibodies with myeloma cells.
  • Myeloma cells used for hybridoma production are hypoxanthine-Aminopterin-Thymine (HAT) because they lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) genes. DNA synthesis cannot continue in the medium and dies.
  • HAT hypoxanthine-Aminopterin-Thymine
  • HGPRT hypoxanthine-guanine phosphoribosyl transferase
  • TK thymidine kinase
  • hybridoma cells are fused with splenocytes carrying the HGPRT gene, so they can continue to synthesize DNA using the salvage pathway of normal cells (Arthur and Massey, 1981), and have a HAT-resistant HAT medium. Proliferation is also possible. Thus, hybridoma cells can be screened with unfused myeloma cells.
  • Hybridoma production technology is the most widely used technique for producing antibodies for diagnostic and therapeutic purposes.
  • B lymphocytes and myeloma cells isolated from living bodies are treated with polyethylene glycol (PEG) or electrofusion.
  • PEG polyethylene glycol
  • the PEG treatment or the electrofusion process causes damage to the cells, so the survival rate of the fused hybridoma is low. Therefore, the production of hybridomas is very inefficient, and the antibody production capacity of hybridomas is not constant. Accordingly, the necessity of developing new myeloma cells for hybridoma production with excellent fusion efficiency and excellent antibody production ability of fused hybridomas has been raised.
  • the present inventors introduced various genes related to cell survival and proliferation into myeloma cells in order to produce myeloma cells having excellent fusion efficiency and hybridoma antibody production ability.
  • XIAP X-linked inhibitor of apoptosis gene was introduced into myeloma cells, and as compared with other myeloma cells which did not introduce the gene, it was confirmed that it showed high survival rate and antibody (IgG) expression rate and completed the present invention.
  • US 4720459 A discloses myeloma cells for the production of human / human hybridomas.
  • the myeloma cells do not secrete EBNA-1 protein derived from Epstein Barr virus and myeloma immunoglobulin to detectable levels, but can fusion with human lymphocytes to form human / human hybridomas to continuously secrete antibodies.
  • Human myeloma cells are disclosed.
  • US 20070141064 A1 also discloses human lymphocyte cell lines, human myeloma cell lines, human fusion partner cell lines and methods for producing hybridomas using them.
  • An object of the present invention is to provide a myeloma cell overexpressing XIAP (X-linked inhibitor of apoptosis) protein.
  • Another object of the present invention is to provide a method of producing myeloma cells overexpressing XIAP protein, comprising transfecting myeloma cells with a recombinant vector comprising a nucleotide sequence encoding the XIAP protein.
  • Still another object of the present invention is to provide a hybridoma prepared by fusion of myeloma cells expressing XIAP protein with antibody-producing B lymphocytes.
  • Another object of the present invention to provide a recombinant vector kit for the production of XIAP overexpressing myeloma cells comprising a recombinant vector comprising a nucleotide sequence encoding a XIAP protein as an active ingredient.
  • the present invention provides a myeloma cell overexpressing XIAP (X-linked inhibitor of apoptosis) protein.
  • XIAP X-linked inhibitor of apoptosis
  • the present invention provides a method for producing myeloma cells overexpressing XIAP protein comprising transfecting myeloma cells with a recombinant vector comprising a nucleotide sequence encoding the XIAP protein.
  • the inventors of the present invention intensively tried to solve the problem that the myeloma cells are fused with antibody-producing B lymphocytes to produce hybridomas, causing damage to the cells, thereby lowering the survival rate of the fused hybridomas.
  • the XIAP gene which is a gene related to apoptosis, was introduced into myeloma cells and overexpressed, the survival rate during the fusion of the myeloma cells was increased, and the antibody production capacity was increased.
  • X-linked inhibitor of apoptosis refers to a gene that is structurally characterized by three baculovirus IAP repeat domains and a RING domain, and is a direct method of cell-death protease. It is known as an important modulator of inhibitors and caspases.
  • the caspase is a family of cysteine protease (cysteine protease) also called ICE (interleukin-1 ⁇ converting enzyme) and proteolytic enzymes, it is essential for apoptosis (apoptosis), that is, programmed cell death Enzymes known as proteins that carry out the death penalty of cells.
  • cysteine proteins that are involved in many physiological phenomena, including post-processing of cytokines (IL-1 ⁇ or IFN- ⁇ -inducing factor) that are important for apoptosis and inflammatory responses, cell proliferation, migration and differentiation Means enzymes.
  • Caspase has been a potential therapeutic target since its discovery in the mid-1990s, and by November 2009, 12 caspases were found in humans.
  • Caspases that cause apoptosis are divided into two types: initiator caspases, which cut inactivated effector caspases, and effector caspases that induce apoptosis killing by breaking down other proteins as substrates. Inhibition of caspase cascade is known to be regulated by caspase inhibitors.
  • myeloma cell refers to tumorized tumor cells, which are morphologically diverse from immature to mature, and are rich in various cell biological properties. It is closely related to the clinical picture, course, and prognosis, and the immunoglobulin produced is characterized by monoclonal.
  • hybridoma is a hybrid cell formed by cell fusion between myeloma cells and antibody producing cells, and is a cell capable of continuously producing a monoclonal antibody.
  • Wheat stain (Milstein) and Kohler (Kohler) fabricated the first time in 1975, this is 10 6 -10 (monoclonal antibody) monoclonal antibody by proliferation to separate the eight antigen-specific antibody producing cells present in one of the frequency by It is a breakthrough way to produce.
  • polyethylene glycol is used as a cell fusion agent, hybridoma recovery efficiency is increased and the method is relatively simple.
  • Myeloma cells used for hybridoma production are hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK), and are hypothaxine-Aminopterin-Thymine (HAT).
  • HGPRT hypoxanthine-guanine phosphoribosyl transferase
  • TK thymidine kinase
  • HAT hypothaxine-Aminopterin-Thymine
  • the term "over-expression" means that a particular gene is expressed at an increased level relative to the average expression level of the wild-type protein of the gene. In the case of the present invention, it refers to the case of expressing the XIAP protein at an increased level than the average expression amount of the XIAP protein in the wild type.
  • the myeloma cells of the present invention are myeloma cells derived from mammalian animals, specifically, mice, rats, hamsters, rabbits, dogs, cats, goats, pigs, cattle, sheep, primates, humans And myeloma cells derived from these are P3X63Ag8, p3-U1, NS-1, MPC-11, SP2 / 0, F0, P3x63Ag8. V653, R-210 and S194.
  • mouse myeloma cells were fused with NS0, P3-X63-Ag8.653, and SP2 / 0-Ag14, which were derived from MOPC21 cancer cells expressing IgG1 and removed the H and L chains of immunoglobulins. It is widely used as a cell line (fusion partner cell).
  • Mouse myeloma cells used in specific examples of the invention are SP2 / 0-Ag14 (ATCC, CRL-1581).
  • the XIAP protein of the present invention may be a XIAP protein derived from the various mammalian animals, specifically, mouse, rat, hamster, rabbit, dog, cat, goat, pig, cow, sheep , Primates, human-derived XIAP protein, most specifically mouse-derived XIAP protein, but is not limited thereto.
  • the XIAP protein of the present invention is a protein having an amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto.
  • myeloma cells overexpressing the XIAP protein of the present invention is characterized in that the resistance to apoptosis (apoptosis) is increased.
  • the increase in apoptosis resistance means a state in which apoptosis does not occur relatively in comparison with the average apoptosis level of wild-type myeloma cells that do not overexpress XIAP protein, and thus the cell survival rate is high, and overexpression of the XIAP gene of the present invention By inhibiting Caspase 3 activation in myeloma cells.
  • the XIAP gene of the present invention is synthesized on the basis of ExPASy database and then amplified by PCR (polymerase chain reaction) method (sense primer: 5'-ggccTCTAGAgccaccatgactttcaacagtttgag-3 '/ antisense primer: 5'-ggccTCTAGAtcaagacatgaaaatttt-3 ') and PCR purification kit (iNtRON, Cat No. 17286) were used to purify XIAP DNA.
  • PCR polymerase chain reaction
  • Mouse XIAP gene was cut using XbaI (NEB, R0145L) restriction enzyme and then ligated to pLenti6 / V5-DEST (Invitrogen, V49610) and transformed into DH5 ⁇ host cells to transform large amounts of DNA. Secured.
  • the XIAP gene of the obtained mouse may specifically have a nucleotide sequence disclosed in SEQ ID NO: 2, but is not limited thereto.
  • Techniques related to cloning of the XIAP gene, transformation, colony picking, recovery of plasmid DNA comprising the XIAP gene, and the like may be performed by various methods well known in the art.
  • the present invention is characterized by producing a myeloma cell overexpressing the XIAP protein by transfecting the nucleotide sequence encoding the obtained XIAP protein into myeloma cells.
  • the nucleotide sequence may be in the form contained in naked DNA or vector.
  • vector may be used interchangeably with the term “gene carrier.”
  • vector refers to DNA fragments, nucleic acid molecules, and the like delivered into a cell, and said vector can replicate DNA, can be independently remanufactured in a host cell
  • expression vector “Expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism, generally used in a vector
  • base sequences such as a selection marker and a fluorescent protein expression gene, may be further included.
  • the vector of the present invention may include a nucleotide sequence encoding a XIAP protein, the vector is specifically a vector expressed in animal cells.
  • the animal cell expression vector includes a non-viral (plasmid or liposome) or viral vector capable of expressing the gene by passing the gene in the animal cell.
  • it may be a viral vector such as a retrovirus, lentivirus, adenovirus, adenovirus, and more specifically, it is a lentiviral vector.
  • 1 of the present invention discloses the lentiviral vector pLenti6 / V5-DEST expressing XIAP, which is designed to use blasticidin and ampicillin as selection markers. .
  • transfection refers to a method of directly introducing DNA into an animal cell to change the genetic trait of the cell, and to introduce a nucleotide sequence encoding the XIAP protein of the present invention into the cell. There is no limit as long as you can.
  • the XIAP protein in the method may be a protein having an amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto, the nucleotide sequence encoding the XIAP protein is due to the degeneracy of the codon Combinations of nucleotide sequences.
  • the nucleotide sequence encoding the XIAP protein may specifically have a nucleotide sequence disclosed in SEQ ID NO: 2, but is not limited thereto. Accordingly, the nucleotide sequence used in the present invention is interpreted to include nucleotide sequences showing substantial identity to the nucleotide sequence in addition to the sequences disclosed in SEQ ID NO: 2.
  • This substantial identity is at least 80 when the nucleotide sequence of the present invention is aligned with the maximal correspondence of any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art.
  • nucleotide sequence exhibiting% homology more specifically at least 90% homology, most specifically at least 95% homology.
  • Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol.
  • BLAST can be accessed through the BLAST page of the ncbi website. Sequence homology comparisons using this program can be found on the BLAST help page on the ncbi website.
  • the XIAP gene of the present invention can be introduced into myeloma cells by the various vectors described above.
  • the XIAP gene was ligated into pLenti6 / V5-DEST, which is one of the lentiviral vectors described above, and introduced into SP2 / 0 myeloma cells, which are expressed in packaging cells (293T cells) and myeloma cells by Western blot.
  • XIAP protein was detected (FIG. 2).
  • myeloma cells overexpressing the XIAP gene of the present invention express XIAP protein up to about 13-fold as compared to normal myeloma cells (FIGS. 3A and 3B).
  • myeloma cells overexpressing the XIAP gene of the present invention are equal in cell growth rate and slightly superior in cell viability compared to normal myeloma cells.
  • myeloma cells overexpressing the XIAP gene of the present invention exhibits HAT sensitivity like normal myeloma cells, and thus can differentiate myeloma cells that are not fused with myeloma cells fused to B lymphocytes. It can be usefully used for the production of hybridomas for monoclonal antibodies for antibody production.
  • myeloma cells overexpressing the XIAP gene of the present invention exhibited a high survival rate compared to normal myeloma cells in resistance to apoptosis by sodium azide (FIGS. 6A and 6B), Annexin V staining As a result of analysis through the early and late phase apoptosis is reduced by more than 40% showed a result that can be expected higher survival rate when used in hybridoma production (Fig. 7a and 7b).
  • the present invention provides a hybridoma prepared by fusion of myeloma cells overexpressing XIAP protein with antibody-producing B lymphocytes.
  • hybridomas prepared using myeloma cells overexpressing the XIAP protein of the present invention showed an average survival rate of 1.36 times when fused with splenocytes of normal mice compared to normal myeloma cells. , The production rate of colonies was also confirmed to be excellent 1.35 times (Table 4).
  • the invention provides a kit for the production of XIAP overexpressing myeloma cells comprising a nucleotide sequence encoding a XIAP protein as an active ingredient.
  • the nucleotide sequence may be naked DNA or a form included in a vector.
  • the vector includes a non-viral (plasmid or liposome) or viral vector capable of expressing the gene in an animal cell.
  • a viral vector such as a retrovirus, lentivirus, adenovirus, adenovirus, and more specifically, it is a lentiviral vector.
  • Kit for the production of XIAP overexpressing myeloma cells of the present invention may include a composition comprising one or more other components suitable for introduction into the host cell of the XIAP gene, the structure or trans of naked DNA or recombinant vector comprising the same Additional buffers or reactions may be included that maintain the stability of the specification activity. In addition, in order to maintain stability, it may be provided in a state where a low temperature of -80 °C to 4 °C is maintained.
  • the nucleotide sequence encoding the XIAP protein in the kit for production of XIAP overexpressing myeloma cells of the present invention may include, but is not limited to, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.
  • the nucleotide sequence encoding the amino acid sequence of the first sequence may include a nucleotide sequence substantially the same by degeneracy of the codon, specifically may include a nucleotide sequence of the second sequence of the sequence list It is not limited.
  • the recombinant vector of the present invention may include a PCR primer for amplifying the nucleotides encoding the XIAP protein.
  • the sequence of the primer may be the same as the nucleotide sequence disclosed in the Examples to be described later, but is not necessarily limited thereto.
  • the recombinant vector of the present invention may be provided in a state in which the nucleotides encoding the XIAP protein are ligated separately from the recombinant vector or with each other.
  • the component for the binding of the nucleotide or XIAP protein of the present invention in vitro or in vivo, or a component for searching for the position thereof may further comprise.
  • Such components include restriction enzymes, ligases, and the like, for inserting the XIAP encoding nucleotides of the present invention into a vector.
  • the component is a known compound for labeling the XIAP protein, or an antibody binding to the peptide of the present invention or the XIAP protein of the present invention or a secondary antibody thereof for the detection through antigen-antibody reaction, and for the detection thereof. It may be a reagent.
  • Kit for production of XIAP overexpressing myeloma cells of the present invention may include a packaging cell line for packaging the recombinant vector into which the XIAP gene is introduced as infectious virus particles.
  • the packaging cell line may be any packaging cell, including 293T cells and HT1080 cells, but is specifically 293T cells.
  • the recombinant vector into which the XIAP gene is introduced may be introduced into and cultured in a suitable packaging cell to produce virus particles into which the XIAP gene is introduced, and the produced XIAP gene-introducing virus particles are infected with a myeloma cell line intended to induce overexpression of the XIAP gene.
  • XIAP overexpressing myeloma cells which are one embodiment of the present invention, can be prepared.
  • the recombinant vector comprising the nucleotide sequence encoding the XIAP protein may be a lentiviral vector.
  • the lentiviral vector ligates a nucleotide sequence encoding a XIAP protein into the vector and then transfects it into packaging cells to produce a packaged lentiviral particle.
  • another embodiment of the present invention is a method for producing myeloma cells, the production of a myeloma cells for production of a vector comprising the recombinant vector is overlapping because the present invention is the same as the content of one embodiment Do not explain.
  • the present invention relates to a myeloma cell overexpressing XIAP protein and a method for producing the same.
  • myeloma cells overexpressing the XIAP protein acquire resistance to apoptosis due to the inhibition of caspase 3 activation, so that the fusion efficiency is increased due to the increased survival rate when the hybridoma is produced, and thus the antibody.
  • the production capacity is improved.
  • FIG. 1 is a diagram showing a vector map of the lentiviral vectors pLenti6 / V5-DEST used for the introduction of the XIAP gene of the present invention.
  • Figure 2 is a diagram confirming the amount of XIAP expression in the packaging cells (293T) of the lentiviral vector containing the XIAP gene of the present invention and myeloma cells (SP2 / 0) transfected with the XIAP gene by Western blot.
  • 3A and 3B are diagrams comparing the expression levels of XIAP of 12 subclones obtained by overexpressing the XIAP gene.
  • GAPDH is a house keeping gene in which the expression level in a cell is maintained substantially constant. By comparing the expression level of the GAPDH gene, GAPDH can objectively quantify the expression level of the XIAP gene.
  • Figure 4 is a graph showing the cell growth rate and survival rate of myeloma cells (SP2 / XIAP) overexpressed the XIAP gene compared to normal myeloma cells (SP2 / 0).
  • Figures 5a and 5b shows the survival rate and microscopy of cells over time incubated in HAT medium to compare the sensitivity to HAT of myeloma cells (SP2 / XIAP) and normal myeloma cells (SP2 / 0) overexpressed XIAP gene The photograph shows.
  • Figures 6a and 6b shows the survival rate and microscope of cells over time to compare the resistance to apoptosis by sodium azide of myeloma cells (SP2 / XIAP) overexpressing the XIAP gene and normal myeloma cells (SP2 / 0)
  • Figure shows the microscopic picture.
  • 7A and 7B are graphs showing the resistance of apoptosis by sodium azide to FACS through Annexin V staining.
  • FIG. 8 is a diagram showing the survival rate and antibody production capacity of hybridomas prepared by fusion of B lymphocytes of mice immunized with BSA antigen and myeloma cells (SP2 / XIAP) overexpressed XIAP gene of the present invention. .
  • FIG. 9 is a diagram showing the survival rate and antibody production ability of hybridomas prepared by fusion of B lymphocytes of mice immunized with GST antigen and myeloma cells (SP2 / XIAP) overexpressed XIAP gene of the present invention. .
  • FIG. 10 is a diagram showing a comparison of the survival rate and antibody production capacity of hybridomas prepared by fusion of B lymphocytes of mice immunized with hIgG as antigen and myeloma cells (SP2 / XIAP) overexpressed XIAP gene of the present invention. .
  • X-linked inhibitor of apoptosis (XIAP) genes in mice are synthesized based on the ExPASy database and amplified by PCR (polymerase chain reaction) (sense primer: 5'-ggccTCTAGAgccaccatgactttcaacagtttgag-3 '/ antisense primer: 5'- ggccTCTAGAtcaagacatgaaaatttt-3 ') and PCR purification kit (iNtRON, Cat No. 17286) were used to purify XIAP DNA.
  • PCR polymerase chain reaction
  • Mouse XIAP gene was cut using XbaI (NEB, R0145L) restriction enzyme and then ligated to pLenti6 / V5-DEST (Invitrogen, V49610) and transformed into DH5 ⁇ host cells to transform large amounts of DNA. Secured.
  • XIAP gene containing a viral vector production and activity measurement (Virus production and titration)
  • 293T cells were prepared by incubating in 10% FBS / DMEM medium at 4 ⁇ 10 6 cells / 10 ml in a 100 mm culture vessel (Corning, 430147). Prepare two tubes containing 500 ⁇ l of Opti-MEM medium (Gibco, 31985-070), and add DNA into one tube.
  • the two tubes were reacted separately at room temperature for 5 minutes, and then the lipofectamine mixture was transferred to the DNA tube, mixed carefully, and further reacted at room temperature for 20 minutes.
  • the prepared 293T cells were replaced with 5 ml of Opti-MEM medium for transfection after 24 hours of incubation, and the DNA and lipofectamine mixtures were slowly dropped onto the cells for transfection. After 8 hours, 10% FBS / DMEM medium was replaced and cultured for 48 hours to obtain the lentiviral of the culture supernatant, and then the lentiviral was concentrated 10 times using Lenti-X concentrator (Takara, 631231).
  • A549 cells were prepared as 2 ⁇ 10 5 cells in 6-well plates (Corning, 3516) the day before. Remove the medium for viral infection and dilute the virus supernatant containing 6 ⁇ g / ml polybrene (Sigma, H9268-5G) (10: 1, 10: 2, 10: 3, 10: 4, 10 : 5) put it. After 24 hours it was replaced with fresh medium and incubated for an additional 24 hours. The cells were incubated for about 20 days while being replaced with a medium containing blasticidin (Gibco / R210-01, 10ug / ml), which is a selection marker of pLenti6 / V5-DEST vector.
  • a medium containing blasticidin Gibco / R210-01, 10ug / ml
  • the mouse myeloma cells were cultured with SP2 / 0 cells, the medium was removed on the day of transfection, and the lenti containing the XIAP gene prepared in 1-2 above together with the medium.
  • Virus was added to SP2 / 0 cells with MOI (1, 2, 5, 10), and then polybrene was added at a final concentration of 6 ⁇ g / ml, followed by incubation at 37 ° C. in a CO 2 incubator.
  • NC membrane was blocked for 1 hour at room temperature with blocking buffer (1X PBS / 5% Scheme Milk) in order to reduce non-specific reactions, washed three times with 1X TBS / T for 5 minutes each, and then the rabbit as a primary antibody in the blocking buffer.
  • (Rabbit) anti-XIAP (Santa Cruz, sc-55551) is 1: 1000
  • the loading control mouse anti-GAPDH (Abcam, ab9484) is diluted 1: 2000 and then overnight at 4 ° C. -night) reacted.
  • the expression of XIAP was the highest in # 3E4 and # 5E11 clones among 12 subclones obtained from XIAP, and the expression amount of XIAP relative to SP2 / 0 GAPDH was 1.
  • # 3E4 clone was about 13 times and # 5E11 clone was about 2.5 times higher expression rate was confirmed. From the results, the # 3E4 clone with the highest XIAP expression was selected and the following experiment was conducted, which was named SP2 / XIAP.
  • SP2 / 0 and SP2 / XIAP cells were aliquoted into 2.5 ⁇ 10 5 / ml, 100 ⁇ l in 96-well plates and cultured in DMEM medium containing 10% DMEM and HAT (Sigma, H0262).
  • CCK-8 (Dojindo, CK-04-13) was added to 10% of the culture solution at 24, 48, and 72 hours after incubation, followed by reaction at 37 ° C for 3 hours, followed by spectrophotometer (PerkinElmer, Cat. 2030-0030) was used to measure absorbance at 450 nm. It was also examined under a microscope for visual observation.
  • SP2 / XIAP myeloma cell line
  • SP2 / XIAP myeloma cell line
  • SP2 / XIAP myeloma cell line
  • SP2 / 0 and SP2 / XIAP were diluted to 2.5 ⁇ 10 5 / ml with 10% FBS / DMEM medium and 100 ⁇ l were dispensed into 96 well plates (Corning, 3596).
  • Sodium azide (Sigma, S2002) was added to 10% FBS / DMEM medium at 4 and 8 mM concentrations, respectively, and filtered through a 0.2 ⁇ m syringe filter (Sartorius, 16534-K), followed by 10% FBS / DMEM medium. 100 ⁇ l was dispensed at a final concentration of 2, 4 mM. The control group not treated with sodium azide was further dispensed in 100ul of 10% FBS / DMEM medium. 24 and 48 hours after treatment with sodium azide, CCK-8 (Dojindo, CK-04-13) was added as 10% of the culture. After 3 hours of reaction at 37 ° C., the absorbance was measured at 450 nm using a spectrophotometer (Perkin Elmer, Cat. No. 2030-0030). It was also examined under a microscope for observation by the naked eye.
  • apoptosis resistance by sodium azide of the two cell lines was observed by Fluorescence activated cell sorter (FACS). After treating the same two cell lines (SP2 / 0, SP2 / XIAP) with 0.5% sodium azide for 6 hours, apoptosis was observed through staining with Annexin V (Southern Biothed, 10010-02).
  • FACS Fluorescence activated cell sorter
  • Cells were recovered at 1, 2, 4, and 8 hours after dexamethasone treatment, washed twice with 1X PBS, and then cytolyzed using 1X RIPA buffer (Biosolution, BR002) containing a protease inhibitor (Sigma, P8340). Concentrations were measured using the BCA Protein Assay Kit (Pierce, 23227). 30 ⁇ g of each lysate was separated by 12% SDS-PAGE and transferred to NC membrane. Membranes were blocked for 1 hour at room temperature with blocking buffer (1X PBS / 5% Scheme Milk) and washed three times with 1X TBS / T for 5 minutes each to protect the non-specific reactions.
  • SP2 / 0 the amount of cleaved caspase 3, the active form, was increased from 4 hours after dexamethasone treatment, but the cleaved caspase 3 increased from 8 hours after dexamethasone treatment in SP2 / XIAP. Therefore, SP2 / XIAP cell line was found to exhibit resistance to apoptosis by inhibiting caspase 3 activation.
  • the proteins used for mouse immunization are BSA (Millipore, 82-100-6), GST, hIgG, and 100 ⁇ g of each protein per BALB / c mouse is mixed with adjuvant (Sigma, P5506) intraperitoneally. , IP) injection. Two weeks later, 100 ⁇ g of the same antigen was mixed in 1 ⁇ PBS and injected intraperitoneally to boost the immune response. Three days later, the spleens of the mice were removed to separate B lymphocytes.
  • the isolated B lymphocytes were mixed 5: 1 with SP2 / 0-Ag14 (ATCC, CRL-1581) or SP2 / XIAP cell lines overexpressing the XIAP of the present invention, and mixed with PEG-1500 (Roche, 783641) solution. Fusion.
  • the fused cells were selected by culturing in HAT medium (HAT supplement, Sigma, H0262) to which hypoxanthin, aminopterine, and thymidine were added. On the 5th day of culturing myeloma and B lymphocytes in HAT medium, each well was observed under a microscope, and the number of hybridoma colonies growing in a cluster was counted to compare the production rate of hybridomas.
  • HAT medium HAT supplement, Sigma, H0262
  • hybridomas selected in 3-3 above were further cultured for 7 days and then identified as hybridoma cells producing antibodies using an indirect ELISA method.
  • each antigen was dispensed into a half area 96 well ELISA plate (Corning, 3690) at a concentration of 1 ⁇ g / mL in a half area 96 well ELISA plate (Corning, 3690) and coated overnight at 4 ° C. After washing three times with TBS-T (0.05% Triton X-100) and blocking with 200 ⁇ l of TBS-T / SM (2% Scheme Milk) for 30 minutes at room temperature, the blocked plate was washed three times and then hybridized.
  • TBS-T 0.05% Triton X-100
  • TBS-T / SM 2% Scheme Milk
  • a cutting board culture sample was added and the antigen-antibody reaction was induced for 2 hours at 37 ° C.
  • the anti-mouse IgG-HRP (AbClon, Abc-5001) was diluted 1: 10,000 in TBS-T / SM with secondary antibody, and the antibody was bound for 1 hour at 37 ° C.
  • TMB solution (Biofx, TMBC-1000) was added and developed for 20 minutes at room temperature. After the coloring was stopped by adding 1N sulfuric acid (DukSan, 254), the spectrophotometer (Perkin Elmer, 2030- Absorbance at 450 nm was used to select whether to produce an antibody that specifically binds to the antigen.
  • hybridomas that produce antibodies specific for each antigen prepared in Example 3 XIAP expression and antibody production rate were compared by Western blot. XIAP expression was confirmed in the same manner as described above in Example 2-1, and for the expression of the antibody, each hybridoma cell was cultured in 10% FBS / DMEM medium at 1 ⁇ 10 6 / ml 1 Culture supernatants on days 2, 3 and 4 were separated by 12% SDS-PAGE and transferred to NC membranes. After blocking the NC membrane at room temperature for 1 hour, anti-mouse IgG-HRP (AbClon, Abc-5001) was treated at 1: 5000 and bound at room temperature for 2 hours.
  • Anti-mouse IgG-HRP AbClon, Abc-5001
  • the AbSignal substrate (Abclon, Abc-3001) was treated to determine the degree of luminescence of the cell line using the ChemiDoc TM Touch Imaging system. It was confirmed that the hybridoma cell line in which XIAP expression was maintained was higher in IgG expression level than the hybridoma cell line in which SP2 / 0 or XIAP expression was lost. Therefore, it was confirmed that overexpression of the XIAP protein increases the viability of the hybridoma and thus can maintain the antibody expression for a long time.

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Abstract

La présente invention concerne une cellule de myélome surexprimant une protéine XIAP, et un procédé de production correspondant. Selon la présente invention, la cellule de myélome surexprimant une protéine XIAP acquiert une résistance à l'apoptose résultant de l'inhibition de l'activation de la caspase 3, et ainsi, lorsqu'elle est produite en tant qu'hybridome, a pour effet d'acroître l'efficacité de fusion de celui-ci en conséquence de l'augmentation du taux de survie ; par conséquent, la capacité de production d'anticorps de l'hybridome augmente.
PCT/KR2017/009311 2016-08-29 2017-08-25 Nouvelle cellule de myélome pour le développement d'un hybridome WO2018043996A1 (fr)

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US20090181426A1 (en) * 2007-01-08 2009-07-16 Millipore Corporation Cell culture methods for producing recombinant proteins in the presence of reduced levels of one or more contaminants
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115947861A (zh) * 2022-07-25 2023-04-11 南京佰抗生物科技有限公司 一种高效杂交瘤融合方法
CN115947861B (zh) * 2022-07-25 2023-11-17 南京佰抗生物科技有限公司 一种高效杂交瘤融合方法

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