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WO2004031382A1 - Nouveau procede permettant la synthese d'un anticorps monoclonal - Google Patents

Nouveau procede permettant la synthese d'un anticorps monoclonal Download PDF

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Publication number
WO2004031382A1
WO2004031382A1 PCT/JP2003/012658 JP0312658W WO2004031382A1 WO 2004031382 A1 WO2004031382 A1 WO 2004031382A1 JP 0312658 W JP0312658 W JP 0312658W WO 2004031382 A1 WO2004031382 A1 WO 2004031382A1
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Prior art keywords
antigen
mouse
antibody
lymph node
cells
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Japanese (ja)
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Kamon Shirakawa
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Mochida Pharmaceutical Co Ltd
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Mochida Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to a method for producing a mouse monoclonal antibody, a method for producing a hybridoma that produces an antibody having reactivity to a specific antigen, and a method for inducing an immune response to a specific antigen in mice.
  • the first dose is administered into the photonode, and the lymph node-derived cells are used for cell fusion with Mie-mouth, a hybrid to produce the desired antibody.
  • the lymph node-derived cells are used for cell fusion with Mie-mouth, a hybrid to produce the desired antibody.
  • one horse can be obtained with high efficiency.
  • no method has been established in mice to surely enlarge the lymph node by immunization, it is a common practice to use the spleen for cell fusion.
  • the efficiency of monoclonal antibody production is greatly affected by the nature of the antigen.
  • the conventional method for producing monoclonal antibodies in mice is once In some cases, the desired antibody cannot be obtained by cell fusion.In such a case, it is necessary to try cell fusion multiple times by changing conditions such as the immunization period and the amount of antigen.
  • Oligonucleotides containing the unmethylated cytosine-guanidine dinucleotide motif are frequently found in Bacteria-derived DNA, It is known that vertebrates exhibit a leukocyte activating effect (Krieg A, Nature, 374, 546-549, 1995). Unmethylated cytosine-guanine dinucleotide motifs are common in bacterial DNA, but are rare in vertebrate DNA, and the vertebrate immune system is associated with unmethylated cytosine-guanidine dinucleotides. It is thought to have evolved to recognize DNA containing the motif as a sign of infection.
  • CpG oligonucleotides are being studied for use as adjuvants, and optimization of sequences to enhance immunostimulatory effects has been studied (international studies). Published 9 6/0 2 5 No. 55 pan fret, International Publication No. 98/41010 Pan fret, International publication No. 98/18810 pan fret, International Published No. 996-61 No. 6 Pamphlet). CpG oligonucleotide is also commercially available as an adjuvant for producing antibodies in mice. The manual for commercial products states that cell fusion uses the spleen, and that intravenous administration is not recommended because it does not have any beneficial effects. (“ImmunEasy TM Mouse Adjuvant Handbook”, QIAGEN, January 2001, p.8_10).
  • lymph node-derived cells If cell fusion using lymph node-derived cells becomes possible in mice, it would be possible to obtain monoclonal antibodies with high efficiency and in a short period of time, and efficiently even for antigens for which antibody production is difficult. Be expected. However, it is possible to reliably enlarge the lymph node with a normal amount of antigen and irrespective of the type of antigen, and an immunization method that enables cell fusion using lymph node-derived cells has been established. Had not been done.
  • the present invention has been made to solve the above-mentioned problems in the prior art.
  • the mouse is always irrespective of the type of antigen. It has been found that the lymph node can be enlarged and cell fusion using lymph node-derived cells can be performed.
  • the present invention provides a mouse monoclonal antibody, characterized in that CpG oligonucleotide is used as an adjuvant and the first immunization is performed in a mouse footnode. It is intended to provide a production method, a method for producing a hybridoma that produces an antibody reactive with a specific antigen, and a method for inducing an immune response to a specific antigen in mice.
  • the method for producing a mouse monoclonal antibody of the present invention comprises the steps of: using an oligonucleotide containing an unmethylated cytosine-guanidine dinucleotide motif as an adjuvant; By using the cells derived from mouse lymph node immunized against a specific antigen by performing in a pad, a hybridoma is obtained by a cell fusion method, and the desired identification is performed. A mouse monoclonal antibody reactive with an antigen is obtained.
  • the method for producing the mouse monoclonal antibody of the present invention is as follows.
  • the method of the present invention for inducing an immune response against a specific antigen in a mouse is characterized by the following steps (a) and (b): (a) an antigen and a non-methylated cytosine-guanine dinu nu Mixing the oligonucleotide with the oligonucleotide containing the motif; (b) the antigen and the oligonucleotide containing the unmethylated cytosine-guanidine dinucleotide motif; Priming a mouse by administering the mixture with at least the Footnod S. These steps are described in detail below I do.
  • the method for inducing an immune response to a specific antigen in a mouse of the present invention can include, in addition to the above steps, (C) a step of confirming that an immune response to a specific antigen has been induced in the mouse.
  • inducing an immune response to a specific antigen means, specifically, inducing the production of an antibody reactive with a specific antigen in a mouse. Induction of the production of an antibody reactive with a specific antigen in a mouse means that a large amount of B cells that produce an antibody reactive with the specific antigen appear in the mouse body. is there.
  • the method for inducing an immune response against a specific antigen in the mouse of the present invention which is characterized by the above steps (a) and (b), is particularly effective in stimulating lymph node cells.
  • lymph node cells Within which a large number of B cells that produce antibodies reactive with a specific antigen appear. The result is observed as an enlarged lymph node.
  • the mouse lymph node enlargement and / or The increase in the antibody titer in the serum against the original may be confirmed. More preferably, it is confirmed by fusing lymph node-derived cells with myeloma cells and obtaining a hybridoma that produces an antibody reactive with a specific antigen.
  • a preferred embodiment of the method of the present invention for inducing an immune response against a specific antigen in a mouse is as follows: a method for inducing an immune response to a specific antigen in a mouse to enlarge lymph node, comprising: a) a method characterized by comprising the steps of (a) and (b); (a) mixing an antigen with an oligonucleotide containing a non-methylated cytosine-guanine dinucleotide motif. (B) administering a mixture of the antigen and an oligonucleotide containing an unmethylated cytosine-guanidine dinucleotide motif, at least in the foot knot. First immunization of mice.
  • the method of inducing an immune response to a specific antigen in the mouse of the present invention to enlarge the lymph node includes the above steps (a) and (b), and also includes the following steps (c) and / or (d) Niyotsu also characterized by; (c) Ri per one mouse, that can 1 XI 0 7 cells also rather small Ri by any one of the lymph nodes in the acquisition this (D) hybridomas that produce antibodies reactive to a specific antigen can be obtained by fusing the obtained lymph node-derived cells with myeloma cells.
  • the primary immunization is performed by using an oligonucleotide containing an unmethylated cytosine-guanidine dinucleotide motif as an antigen. It is a standard method in the field of monoclonal antibody production, using cells derived from the lymph node of a mouse immunized against a specific antigen by performing it in a tongue. A hybridoma producing a mouse monoclonal antibody reactive to a desired specific antigen is obtained using a cell fusion method.
  • the method for producing a hybridoma according to the present invention is characterized by the following steps (a) to (c): (a) an oligonucleotide containing an antigen and a non-methylated cytosine-guanine dinucleotide motif; Mixing the antigen with an oligonucleotide containing at least the antigen and an unmethylated cytosine-guanidine dinucleotide motif; at least a footprint of the antigen. (C) isolating the lymph node from the mouse, and lysing the lymph node-derived cells with myeloma cells. Stage to obtain high pre-doma.
  • the method for producing a hybridoma of the present invention is not limited to the above steps.
  • Oligonucleotides containing a non-methylated ditocin-guanidine dinucleotide motif used in the present invention have immunostimulatory effects in vertebrates, particularly mice. It is characterized by having. There is no particular limitation on the form of the oligonucleotide as long as it has an immunostimulatory effect in mice and has at least one unmethylated cytosine-guanidine dinucleotide motif in the sequence.
  • CpG oligonucleotides can be double-stranded or single-stranded. In general, double-stranded molecules are more stable in vivo, but single-stranded molecules have stronger immunostimulatory effects.
  • Oligonucleotides are defined as multiple nucleotides (ie, A sugar linked to a phosphate group and a base (either cytosine (C), thymine (T), peracyl (U)), adenine (A), or guanine (G)) (Eg, molecules containing ribose or deoxyribose) form a phosphate ester between the 3′-hydroxyl and 5′-phosphate groups of the sugar residue, resulting in a large number of nucleic acids. It is a nucleotide multimer formed of covalently linked leotides, and is synonymous with nucleic acid.
  • Oligonucleotide includes Oligoliponucleide, and Oligodoxyribonucleotide.
  • the term also includes polynucleosides (ie, polynucleotides excluding phosphoric acid) and any other organic base, including polymers.
  • Nucleic acid molecules can be obtained from existing nucleic acid sources (eg, genomic or cDNA), but are preferably synthesized (eg, produced by oligonucleotide synthesis).
  • An unmethylated cytosine-guanine dinucleotide motif is a nucleotide sequence that is present in an oligonucleotide in the order of (5'—) cytosine-guanine (13 '). This means that at least the carbon or nitrogen constituting the pyrimidine ring of cytosine is not substituted with a methyl group.
  • Nucleotide sequence of CpG oligonucleotide used in the present invention Has an immunostimulatory effect in mice and has at least one unmethylated cytosine-guanidine dinucleotide chief in the sequence, including, but not limited to, the following: Ru C p G O Li Gore j click Les Ochi de der that having a nucleotide sequence shown Ri by the formula: 5, N 1 X 1 X 2 CGX 3 X 4 N 2 3 '; in here, CG is not been methylation; X iX 2 is, GT, GG, GA, AA , AT, AG, CT, CA, CG, TA, TT, and nucleotide sequences der selected Ri by TG Tona Ru group X 3 X 4 is a nucleotide sequence selected from the group consisting of TT, CT, AT, TG, AG, CG, TC, AC, CC, TA, AA, and CA; N 2 is composed of each 1-5 0
  • X! X 2 is X 3 X 4 GA, AA, GG or GT, and its is a TT, ⁇ C or C ⁇ .
  • X i or X 2 or both are bleach and X 3 or X 4 or both are pyrimidines, or X i X 2 is a GA, and X 3 or X 4 or both are pyrimidines.
  • CpG oligonucleotides that can be used in the present invention are known (eg, International Publication No. WO 96/025555, Publication No. 98/41010 Non-Patent, International Publication No. 98/18810 Panplate, International Publication No. Fret), and the sequence can be appropriately selected with reference to known techniques.
  • a preferred example of the CpG oligonucleotide used in the present invention is an oligonucleotide consisting of a nucleotide sequence of TCCATGACGTTCCTGACGTT (SEQ ID NO: 3). The underlined portion indicates an unmethylated cytosine-guanidine dinucleotide motif.
  • the chain length of the CpG oligonucleotide used in the present invention has an immunostimulatory effect in mice and has at least one non-methylated cytosine-guanine dinucleotide in the sequence. It is not limited as long as it has a leotide motif, but preferably has a length of at least 8 residues or more. It is known that sufficient immunostimulatory effect may not be obtained if the length is 7 residues or less (WO 96/025555 Panflet). . Rather than the preferred Ri good is, 8 to; the L 0 0 range of about residues and N 2, respectively;! To about 50 residues), more preferably about 8 to 50 residues ((! And N 2). Are about 1 to 25 residues, respectively).
  • the CpG oligonucleotide used in the present invention includes all kinds of derivatives, including those having a base, a sugar, a phosphoric acid, and a backbone structure, which do not exist in nature. Is included.
  • Examples of the derivatives included in the present invention include phosphodiester bonds and phosphodiester bonds in whole or in part of the skeleton of the oligonucleotide. Hosphorothioate bond, methylphosphonate bond, phosphoroamidate bond, phosphoroamidate bond Derivatives with phosphorodithioate bonds, morpholino groups, etc. (Yoko Tokaibayashi, etc., Cancer and Chemotherapy, Vol. 20, 1989-199, pp. 19, 19) 93 years).
  • DNG Deoxylibonucleodoguanidine
  • examples of derivatives include those substituted with other atoms or substituents, and modified sugar moieties such as ⁇ -ribose (Bertrand JR. Biochem. Biophys). R es. 164, 311, 1989).
  • those in which the sugar moiety is replaced by another substance those in which some bases are replaced by inosine or universal bases (bases that bind to any of A, T, C, and G), Cholesterol, acridine, poly-L-lysine, psoralen, long-chain alkyl at the 5 'or 3' end or inside the oligonucleotide Oligonucleotide derivatives such as those to which the above are bonded are also included in the present invention (G. Degols et al., Nucleic Acid Research. Vol. 17, pp. 9341, 1989). A. Mc Connaghie et al., J. Med. Chem. 38, 348, p. 488, 1993 G. Godard, etc. E ur. J. Biochem. 404 page, 1995).
  • the CpG oligonucleotide used in the present invention preferably has a modified backbone. Modification of the oligonucleotide backbone has effects such as enhancement of nuclease resistance, enhancement of cell uptake, and enhancement of protein binding, and contributes to enhancement of immunostimulatory effects. .
  • the CpG oligonucleotide used in the present invention can be produced by a known method (for example, Stanley T. Cr). ooke and Bernald Lebleu, eds. in Antisense Research and Ap ications, CRC Publishing, Florida, 1993). Typically, the synthesis is performed using a chemical synthesizer (for example, Expedite Model 809, manufactured by Applied Biosystems Japan Co., Ltd.). In this case, the operation is performed according to the manual attached to the chemical synthesizer, and the obtained synthetic product is purified by an HPLC method using reversed phase chromatography or the like. Thus, it is possible to obtain oligonucleotides.
  • a chemical synthesizer for example, Expedite Model 809, manufactured by Applied Biosystems Japan Co., Ltd.
  • the immunostimulatory effect of the CpG oligonucleotide used in the present invention can be evaluated by using a known method (for example, International Publication No. 98/18810 pan). Fret).
  • the immunostimulatory effect is measured by measuring various immune parameters, for example, lymphocyte proliferation, B cell IgM antibody production, and cytodynamic protein production (eg, mouse spleen-derived B cells). Can be evaluated.
  • lymphocyte proliferation for example, lymphocyte proliferation, B cell IgM antibody production, and cytodynamic protein production (eg, mouse spleen-derived B cells).
  • cytodynamic protein production eg, mouse spleen-derived B cells.
  • the growth measurement of the lymphocytes the incorporation of 3 H ⁇ lysine of mouse spleen-derived B cell cultures and indicators, in C p G O Li Gore j click Reochi de presence and absence Measure cell proliferation.
  • Oligonucleotide At least 2 times, preferably 5 times, as compared to oligonucleotides (oligonucleotides without the unmethylated cytosine-guanidine dinucleotide motif) More preferably, it has an immunostimulating effect of 10 times or more.
  • a desired antigen may be used, and the type thereof is not particularly limited.
  • the specific antigen means one specified antigen, and is synonymous with a desired antigen.
  • Antigens broadly include any type of molecule that is recognized as foreign by the host immune system. Examples of antigens include, but are not limited to: cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide mimetics of polysaccharides, lipids, glycolipids , Carbohydrates, viruses, viral extracts and allergens. Preferably, it is a protein, polypeptide or peptide.
  • the protein, polypeptide or peptide can be prepared from cells or tissues that express the protein, and can be prepared using a peptide synthesizer (for example, peptide synthesizer type 133A, It can be prepared by a chemical synthesis method using Dono Systems Japan Co., Ltd.) or by a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes.
  • a peptide synthesizer for example, peptide synthesizer type 133A, It can be prepared by a chemical synthesis method using Dono Systems Japan Co., Ltd.
  • a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes.
  • the step of "mixing the antigen with an unmethylated cytosine-oligonucleotide containing a guanidine dinucleotide motif” comprises the step of adjuvanting the non-methylated cytosine. This is a step of mixing an oligonucleotide containing a cytosine-guanidine dinucleotide motif and a desired antigen prepared in advance for administration to a mouse. Since the adjuvant CpG oligonucleotide can be provided in an aqueous solution, there is no need for the step of forming an emulsion with an antigen unlike FCA.
  • the solution containing CpG oligonucleotide and the solution containing the antigen may be mixed at an appropriate ratio.
  • CpG oligonucleotide is used as an adjuvant, and the first immunization is carried out in a photonode, whereby lymphocytes are efficiently lysed.
  • Large amounts of antigen are not required because the nodes can be enlarged.
  • Protein antigen In this case, about 1 to 5 O ⁇ g per mouse may be used for one administration.
  • immunization the number (mol) of antigens is important.
  • the liquid volume of the mixture of CpG oligonucleotide and antigen is 500 per mouse. It is prepared so as to be not more than L, preferably not more than 20 O ⁇ L.
  • CpG oligonucleotides and antigens include adjuvants other than CpG oligonucleotides and CpG oligonucleotides and antigens. It can also contain substances (resins, porous particles, etc.) that enable stabilization and sustained release in vivo. That is, the “mixture of an antigen and an oligonucleotide containing a non-methylated cytosine-guanidine dinucleotide motif” in the method of the present invention includes (a) an antigen, and (b) ) Means a mixture containing CpG oligonucleotides.
  • Adjuvants other than CpG oligonucleotides include saponin, trehalose dimycocoat, lipopolysaccharide or a derivative thereof, lipid A or a derivative thereof, and FCA. , FIA, Myo Nonon, Hydroxya What is known by those skilled in the art, such as lumidium, can be used. It is preferable to use CpG oligonucleotide in combination with myo-nonon or hydroxylamine, because it can induce a high antibody titer without damaging mouse tissues.
  • a mixture of an antigen and an oligonucleotide containing an unmethylated cytosine monoguanidine nucleotide motif is contained in at least a foot knot. Step of priming a mouse by administration ”will be described.
  • the method of the present invention is characterized by using at least a photonode as a route for administering an antigen to a mouse in the first immunization.
  • administration into the footnode is not effective.
  • the present inventor adjuvanted CpG oligonucleotide.
  • the footnode may be the forelimb or the hindlimb, but is preferably the hindlimb.
  • the administration is preferably performed on both the left and right sides.
  • administration to other sites is also acceptable.
  • Other sites include intramuscular, intraperitoneal, subcutaneous and intradermal.
  • the method of the present invention provides for the mixture of the antigen and an oligonucleotide containing an unmethylated cytosine-guanidine dinucleotide motif with at least a foot.
  • the step of priming a mouse by administering it into a pad '' refers to the step of priming in the step of priming, wherein the mouse is immunized intramuscularly, intradermally, subcutaneously, or intraperitoneally in addition to the footpad.
  • the method is characterized in that a mixture of an antigen and an oligonucleotide containing an unmethylated cytosine guanidine dinucleotide motif is administered.
  • intramuscular particularly preferred is in the thigh muscles of both limbs.
  • intra-footpad and intra-thigh muscle administration in the first immunization, the lumbar and inguinal lymph nodes, in particular, can be efficiently enlarged.
  • mice used in the method of the present invention is not particularly limited, SPF mouse is used for the purpose of producing a monoclonal antibody. It is preferable to use it. Typically, mice of strains such as BALB / c, ddY and C57BL / 6 can be used.
  • the step of administering a mixture of an antigen and an oligonucleotide containing an unmethylated cytosine-guanine dinucleotide motif into a mouse footpad is followed by It may further include the step of performing additional immunization.
  • the booster immunization can be carried out either by administering a mixture of an adjuvant and an antigen or administering only the antigen, but it is preferable to administer the antigen only when administering immediately before cell fusion.
  • the CpG oligonucleotide is used as an imaging and the first immunization is carried out in the peptide, whereby the lymphocyte is efficiently lysed.
  • boosters should be boosted within 2 weeks, preferably within 9 days after the first dose of the mixture of CpG oligonucleotide and antigen. Is possible.
  • a route of administration of the booster it is preferable to use a footnode, but intramuscular, intraperitoneal, intradermal, subcutaneous and intravenous may be used as appropriate.
  • the method of the present invention uses a CpG oligonucleotide as an adjuvant, and the first immunization is performed in the footnode, so that the lymph node is more efficiently used. Can be enlarged.
  • the conventional mouse immunization method that required multiple booster immunizations and an immunization period of at least one month or more (for example, a method using FCA as an adjuvant and spleen-derived cells for cell fusion)
  • the method of the present invention has made it possible to establish immunity in mice within one booster immunization period of two weeks. Whether or not immunity has been established can be confirmed by measuring the titer of an antibody reactive with the administered antigen in mouse blood. Booster immunizations can be performed more than once if necessary.
  • the “step of isolating the lymph node from the mouse and fusing the lymph node-derived cells with myeloma cells to obtain a hybridoma” in the method of the present invention will be described. It is preferred that the mice be killed within a few days after the boost. The sacrificed mice are laparotomized under sterile conditions and the lymph nodes are isolated. There are no particular restrictions on the lymph nodes to be isolated, such as lumbar, inguinal, popliteal, ischial, tail, mesenteric, and Peyer's patches. At least one type of lymph node can be used, such as renal node, thymic node, axillary node, upper arm node, and cervical node. Generally, antigen-reactive lymph nodes are located in the lymph nodes near the antigen administration site. W 200
  • lymph nodes are often enlarged, administration of an antigen to the hind limb footpad may result in inguinal, lumbar, and sciatic lymph nodes. It is preferable to use a node, a tail lymph node, a popliteal lymph node, and the like, and particularly, it is preferable to use a lumbar lymph node and / or an inguinal lymph node. Lymph node-derived cells obtained from a plurality of lymph nodes may be mixed and used for cell fusion.
  • lymph node-derived cell By loosening the isolated lymph node on a cell strainer using a pin set or the like in a medium, it is possible to prepare a lymph node-derived cell. At this point, it is preferable to count the number of obtained lymph node-derived cells. Although the child to get the least for the 1 XI 0 7 or more of the antibody-producing cells also in order to perform the cell fusion efficiency good phrase is important, mouse method of immunization using a conventional FCA adjuvant Roh down doors, etc. Thus, it was difficult to enlarge the lymph node, and a sufficient number of cells could not be obtained from the lymph node.
  • the method of the present invention uses a CpG oligonucleotide as an adjuvant, and the first immunization is carried out in the foot node to efficiently reduce the immunity. Since the child to hypertrophy of the lymph nodes is possible, mouse-out one animal Nitsu, any one of the lymph nodes (if any left and right to match both sides) good Ri, 1 XI 0 7 or more of even rather small Lymph node-derived cells Can be obtained.
  • Myeloma cells used for cell fusion are generally cell lines obtained from mice, for example, 8-azaguanine-resistant mice (derived from BALB / c) Myeloma cell line P3X63Ag8U.l (P3-Ul) [Yelton, DE et al. Current Topics in icroDiology and Immunology, 81, 1-7 (1978)], P3 / NSI / 1 ⁇ Ag4-1 (NS ⁇ 1) [Kohler, G. et al. European J. Immunology, 6, 511 -519 (1976)], Sp2 / 0-Agl4 (SP-2) [Shulman, M. et al.
  • IMDM Iscove's Modified Dulbecco's Medium
  • DMEM Dulbecco's Modified Eagle Medium
  • This method includes, for example, the following procedure. Wash the lymphocyte-derived cells and myeloma well with a serum-free medium (for example, RPMI 164) or phosphate buffered saline (hereinafter referred to as “PBS”). Mix the cells so that the ratio of the number of cells derived from the nodes to the number of cells in the mouth is about 1: 1 to 10: 1, and centrifuge. In this step, it is also possible to mix the lymph node-derived cells and myeloma cells and then wash the cells. I like it because it will be less.
  • RPMI 164 phosphate buffered saline
  • HAT medium a normal medium
  • HAT aminopterin 'thymidine
  • IL_2 mouse interleukin-12
  • the myeoma cell is an 8-azaguanine-resistant strain, that is, a hypoxanthine / guanine / phospholibosyltransferase (HGPRT) -deficient strain
  • HGPRT hypoxanthine / guanine / phospholibosyltransferase
  • the myeloma cell that has not fused
  • fusion cells of myeoma cells cannot survive in HAT-containing medium.
  • fused cells between antibody-producing cells, or hybrids between antibody-producing cells and myeloma cells can survive, but are fused to antibody-producing cells. Has a lifetime. Therefore, by continuing the culture in the HAT-containing medium, only the hybridomas between the antibody-producing cells and the myeloma cells survive, and as a result, the hybridomas can be selected.
  • the step of culturing the obtained hybridoma and recovering the antibody from the culture supernatant in the method of the present invention will be described.
  • This stage The floor further comprises a step of cloning (unifying) the hybridoma, and determining that the obtained hybridoma produces an antibody of interest (an antibody reactive with a desired specific antigen). It may include a confirmation step.
  • the medium is replaced with a medium excluding the aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured by, for example, the ELISA method.
  • HT medium a medium excluding the aminopterin from the HAT medium
  • RIA radioisotope immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • fluorescent antibody fluorescent antibody
  • passive hemagglutination Techniques can be used, but RIA or ELISA is more preferable in terms of detection sensitivity, speed, accuracy, and possibility of automation of operation.
  • the antibody titer can be measured, for example, by the following procedure according to the ELISA method.
  • the antigen is adsorbed on a solid surface such as a 96-well plate for ELISA, and the solid surface on which the antigen is not adsorbed is exposed to a protein that is unrelated to the antigen, for example, peroxyserum albumin ( (BSA), and after cleaning the surface, A sample (eg, a hybridoma culture supernatant, a serum diluent) is brought into contact with the first antibody to bind the antibody in the sample to the above antigen.
  • BSA peroxyserum albumin
  • an antibody against a mouse antibody labeled with an enzyme is added as a second antibody to bind to the mouse antibody.
  • a substrate of the enzyme is added, and a change in absorbance due to color development based on the decomposition of the substrate is measured. Thereby, the antibody titer is calculated.
  • the cloning method is a limiting dilution method in which a plate is diluted so that one hybridoma is contained in one well of the plate, and the colony is cultured in a soft agar medium and cultured.
  • the soft agar method to recover, the method of taking out and culturing individual cells by micro chromatography, and the method of separating one cell by Celso, etc.
  • the limiting dilution method is simple and often used.
  • the cloning by limiting dilution method is repeated 2 to 4 times.
  • the desired antibody is selected as a hybridoma.
  • the hybridoma is cultured by changing the medium from HT medium to normal medium. Large-scale culture is performed by rotary culture using large culture bottles or spinner culture. The antibody can be recovered by purifying the supernatant from this large-scale culture using a method known to those skilled in the art, such as gel filtration.
  • a method known to those skilled in the art such as gel filtration.
  • by growing the hybridoma in the abdominal cavity of a mouse of the same strain (for example, BALB / c) or a NuZNii mouse ascites containing a large amount of the target antibody can be obtained. Can be.
  • Antibody recovery and purification from the culture supernatant of the hybridoma include, for example, treatment with a conventional protein precipitant, ultrafiltration, molecular sieving chromatography (gel filtration), and adsorption chromatography.
  • Various types of chromatographs such as chromatograph, ion exchange chromatograph, affinity chromatograph, high-performance liquid chromatograph (HPLC), etc. Dialysis, dialysis, and combinations thereof.
  • affinity chromatography using a protein A-containing column is particularly useful.
  • the hybridoma obtained by the method for producing a monoclonal antibody and the hybridoma in the present invention is a hybridoma that produces an IgG antibody in a preferred embodiment.
  • the immunization period is two weeks, which is shorter than before, but the CpG oligonucleotide is used as an adjuvant, and the first immunization is performed with the photoimmunoassay.
  • Can produce an antibody with a high affinity in IgG type since it is possible to efficiently induce an immune response to a specific antigen in mice by performing intracellular injection. It became possible to acquire high predoma efficiently. Evening of antibodies produced by hybridomas can be easily performed using a commercially available typing kit or the like.
  • the extracellular domain of the human SJ2368 protein (SEQ ID NO: 1) (in the amino acid residues 1-222 of SEQ ID NO: 1) is used as an administration antigen for antibody production. Equivalent) and histidine tag Or chimera (fusion) protein with the human IgGFc fragment.
  • Sense Primer 1 (5,1 CCGCTCGAGCAGGAAGGCCAT GGCGGGGCCCGAG-3 ') and Antisense Primer 1 (5,1 CGCGGATCCGGCTTCTGGGAC CTCCAGCAAGAA-3,) were synthesized, and the Ministry of Economy, Trade and Industry (METI), an international microbial depository organization, Plasmid sj was commissioned by the Institute of Biotechnology, Industrial Technology, and Technology on September 18, 2003, and transferred to a deposit based on the Busust Convention on September 17, 2002. 0 2 3 6 8 (Accession No.
  • FERMBP — 8 1 8 2. to type I using Pyrobest DNA Polymerase (TAKARA) for 10 seconds at 98 ° C, 30 seconds at 55 ° C, 7 2
  • TAKARA Pyrobest DNA Polymerase
  • the PCR reaction was repeated 30 times at 1 ° C. for 30 minutes.
  • the obtained PCR product of about 0.7 kb was digested with XhoI and BamHI, and the DNA fragment was recovered by agarose gel electrophoresis.
  • pc DNA 3.1 / Myc-His (-) A is replaced with Xhol and Ba After digestion with mHI, the above DNA fragment was ligated.
  • the competent cell JM109 was transformed and human SJ2368-His expression plasmid was prepared according to a standard method. Human SJ 2 3
  • Sense primer 2 (5, 1 C C G G
  • the R product was digested with Ec0RI and BamHI, and the DNA fragment was recovered by agarose gel electrophoresis.
  • the transformant cell JM109 was transformed and human SJ2368-Fc expression plasmid was prepared according to a standard method.
  • the amino acid sequence of the human SJ2368_Fc protein obtained by this construction is as shown in SEQ ID NO: 2.
  • the obtained expression plasmid was introduced into COS cells by the following method. That is: FUGENE 6 (Roche-Diagnostics) 5 and each of the above brass mids DN
  • A12.5 ⁇ g was mixed with the attached protocol, and added to C0S cells grown on a semiconfruent 150 cm 2 flask. 5% C 0 2 presence, the culture supernatant was collected after culturing for 3 days at 3 7 ° C.
  • an anti-histidine tag antibody Penta.His Antibody; QIAGEN
  • peroxidase-labeled anti-mouse immunoglobulin antibody DAK 0
  • anti-Fc antibody peroxidase-labeled anti-human IgA, IgG, 1 gM, Kappa, Lambda antibody; DAKO
  • human SJ2368-His was purified from the culture supernatant using a nickel column, and human SJ2368-Fc was purified using a ProteneA column.
  • An adjuvant-antigen mixture was prepared by the following method. 20 g of the purified SJ2368-Fc protein was dissolved in 50 L of physiological saline, and used as an adjuvant containing CpG oligonucleotide as an adjuvant containing QIAGEN Imm. The unEasy Mouse Adjuvant was added to 50 ⁇ l [] to prepare 100 ⁇ l. Emulsion mixed with 50 ⁇ L of Freund's complete adjuvant (FCA, DIFCO) commonly used as a control and 50 pi L of saline containing 20 g of antigen was prepared.
  • FCA Freund's complete adjuvant
  • ddY mice male, 8 weeks old, SLC
  • mice were each administered 50 L of the adjuvant-antigen mixture into the footpads of both feet via a micromouth syringe.
  • 20 ⁇ g of the antigen diluted with 100 ⁇ L of physiological saline, was administered in a volume of 50 liters each to the footnode.
  • the administration was performed in one CpG group and three FCA groups.
  • mice were squeezed and the lumbar lymph node was aseptically removed.
  • the extracted lymph nodes were replaced with RPMO containing 10% fetal calf serum.
  • the lymphocytes were immersed in I164 medium (GIBCO), and the lymphocytes were separated by squeezing the lymph nodes on a 100-micron cell strainer (FALCON). Measure the cell concentration of the separated lymphocytes, collect the cells by centrifugation, and culture the P3X63-Ag.8.
  • the mixture was mixed at a ratio of 3U1) and 4: 1 (lymphocyte: P3U1) and centrifuged again. After washing twice with serum-free RPM I1640 medium, the medium was removed so that no medium remained. Then warmed to 37 ° C
  • Cell fusion was performed using a 45% polyethylene glycol solution containing 5% DMSO (SIGMA) according to the method of Tamoe Ando et al. (Introduction to single clone antibody experimental procedures, Kodansha).
  • SIGMA polyethylene glycol solution containing 5% DMSO
  • the cells were suspended in 10% fetal bovine serum containing 1 XHAT (GIBCO), 50% Hybridoma-SFM (GIBCO, hereinafter referred to as SFM), and 40% RPMI 1640 medium.
  • the cells were seeded on a 96-well plate at about 0.2 mL / well. Cultured at 37 ° C and 5% C02, and after 1 week, when hybridoma growth was observed, 10% fetal calf serum containing 1XHT, 50% SFM, 40% RPM I
  • the medium was replaced with a 1640 medium.
  • the medium was changed several times. After that, the culture supernatant was sampled to screen for a nodipril dormer producing an antibody against the antigen.
  • DAKO peroxidase-labeled anti-mouse immunoglobulin antibody
  • the lymph node could not be sufficiently enlarged even when administered intravenously, and even when cell fusion was performed, SJ23 was observed. No hybridoma producing 68 antibody was obtained.
  • the group using CpG oligonucleotide as the adjuvant hyperplasia of the lymph node was observed, and a hybridoma producing the SJ2368 antibody was obtained. And came out. (5) Preparation of purified antibody
  • Clones are performed on the wells for which the antibody titer is recognized by the limiting dilution method, and an antibody that reacts with the SJ2368 protein is produced. Hai Puri Doma 10 I got a clone.
  • the selected hybridomas are cultured in 10% fetal calf serum, 50% SFM, and 40% RPMI-164 medium, and then cultured in Hybridoma-SFM medium (GIBC0) to produce antibodies.
  • the antibody was purified using rosep-A column (Millipore). All of the resulting 10 clones could be purified by protein A and were of the IgG type.
  • the time required to establish this antibody was 2 weeks of immunization (from the first immunization to cell fusion, with only one additional dose during this period), 7 days from cell fusion to primary screening, and 7 days To secondary screening It took 10 days, 2 weeks from cell growth to cell freezing, for a total of 1.5 months.
  • S CD14 (1-307) S2886C protein (hereinafter referred to as CD1) prepared by the method described in WO 01/72993 4 (may be written as (S286C)) 15 ⁇ g is dissolved in physiological saline, and QIAGEN is used as an adjuvant containing CpG oligonucleotide. Of ImmunEasy Mouse Adjuvant or 150 L of 50 ⁇ L was added to make 200 L.
  • 15 ⁇ g was diluted with 100 ⁇ L of physiological saline, and each of them was administered in a footpad 5 times.
  • mice were squeezed and the lumbar lymph node was aseptically removed.
  • the excised lymph node was immersed in RPMI medium (0180 ⁇ 0) containing 10% fetal bovine serum, and re-substituted on a 100 micron cell strainer (FALCON).
  • FALCON 100 micron cell strainer
  • the lymph nodes were separated by squeezing the lymph nodes, and cell fusion was performed as in Example 1 (3). After the medium was changed several times, the culture supernatant was sampled to screen the hybridomas producing antibodies against the antigen.
  • ddY mouse (medium) was prepared according to the method described in the attached manual.
  • Antigen 8 weeks old, SLC). That is, 2 / g of CD14 (S286C) protein and 20 L of Immun Easy Mouse Adjuvant were mixed, and the antigen to be administered was prepared to be 1 OOL with physiological saline. . Thigh muscles of both feet 5 O ⁇ L of an adjuvant-antigen mixture was administered into the meat via a micro syringe. Two weeks later, booster immunization was performed with the adjuvant-antigen mixture prepared in the same manner.
  • CRP C-reactive protein, ADVYChemical
  • dd Y mice (8-week-old, SLC) were given 50 ⁇ L of adjuvant-antigen mixture to the microsyringe in the thigh muscles and in the feet of both feet. More administration. 9 days later, antigen protein W
  • a solution obtained by diluting 2 O ⁇ g with 100 L of physiological saline was administered to the footpad in an amount of 50 L each.
  • mice were strangled and the lumbar and inguinal lymph nodes were aseptically removed.
  • the excised lymph node was immersed in RPMI 164 medium (GIBCO) containing 10% fetal bovine serum, and lysed on a 100 micron cell strainer (FALCON). Then, the lymphocytes were separated from each other, and cell fusion was performed in the same manner as in Example 1 (3). After the medium was exchanged several times, the culture supernatant was sampled to screen the hybridomas producing antibodies against the antigen.
  • RPMI 164 medium GEBCO
  • FALCON micron cell strainer
  • CRP was diluted with PBS to l ⁇ g / mL, dispensed into 96-well immunoplates (NUNC, Maxisorp) at 50 L / well, and allowed to stand at 4 C overnight.
  • the antibody titer in the culture supernatant was measured in the same manner as in the method described in Example 1 (4).
  • Table 3 shows the number of lymph node cells measured in (3) and the results of the positive cells measured in (4).
  • the combined use of intra-foot and intra-thigh muscle administration allows the average number of lymph nodes to be increased. Hypertrophy was observed, and the hypertrophy was particularly remarkable in the inguinal and lumbar lymph nodes. There is no difference in the number of cells that can be collected from the inguinal and lumbar lymph nodes and the positive rate, and it is possible to produce antibodies against the target antigen using either of the lymph nodes. Was shown.
  • CpG oligonucleotides are used as adjuvants, and the first immunization is performed in the mouse photonodes, thereby affecting the type of antigen.
  • the lymph node of the mouse could be reliably enlarged, and cell fusion using lymph node-derived cells became possible. This has made it possible to produce mouse monoclonal antibodies with high efficiency and in a short period of time.

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Abstract

L'invention a pour objet l'obtention de cellules d'hybridome à partir de cellules de ganglions lymphatiques de souris et un procédé permettant de produire un anticorps monoclonal. L'invention concerne en particulier un procédé permettant de produire un anticorps monoclonal de souris, caractérisé par les étapes (a) à (d) suivantes consistant à: (a) mélanger l'antigène avec un oligonucléotide contenant un motif de dinucléotide cytosine-guanine non méthylé, (b) préparer une souris en administrant à la souris, au moins dans la ration alimentaire, le mélange d'antigène et d'oligonucléotide à motif dinucléotide cytosine-guanine non méthylé. (c) prélever un ganglion lymphatique de la souris et hybrider les cellules du ganglion avec des cellules de myélome afin de produire des hybridomes, et (d) mettre en culture les hybridomes ainsi produits et récolter un anticorps dans le surnageant de cette culture.
PCT/JP2003/012658 2002-10-02 2003-10-02 Nouveau procede permettant la synthese d'un anticorps monoclonal Ceased WO2004031382A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002555A1 (fr) * 1994-07-15 1996-02-01 The University Of Iowa Research Foundation Oligonucleotides immunomodulateurs
WO1998018810A1 (fr) * 1996-10-30 1998-05-07 The University Of Iowa Research Foundation Molecules d'acide nucleique immunostimulantes
WO1999061056A2 (fr) * 1998-05-22 1999-12-02 Loeb Health Research Institute At The Ottawa Hospital Methodes et produits permettant d'induire une immunite au niveau des muqueuses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002555A1 (fr) * 1994-07-15 1996-02-01 The University Of Iowa Research Foundation Oligonucleotides immunomodulateurs
WO1998018810A1 (fr) * 1996-10-30 1998-05-07 The University Of Iowa Research Foundation Molecules d'acide nucleique immunostimulantes
WO1999061056A2 (fr) * 1998-05-22 1999-12-02 Loeb Health Research Institute At The Ottawa Hospital Methodes et produits permettant d'induire une immunite au niveau des muqueuses

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COYLE P.V. ET AL.: "A simple standardised protocol for the production of monoclonal antibodies reactive with autoantigens", J. IMMUNOL. METHODS, vol. 153, no. 1-2, 1992, pages 81 - 84, XP002976349 *
HOLMDAHL R. ET AL.: "A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens", J. IMMUNOL. METHODS, vol. 83, no. 2, 1985, pages 379 - 384, XP002976350 *

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