JP2000342279A - Production of monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the secretory capacity and produce the subject antibody by transducing an extraneous DNA capable of encoding the same amino acid sequence as that of a rearranged endogenous immunoglobulin heavy chain gene into a cell having the rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene and culturing the resultant transformed cell. SOLUTION: This method is to produce a monoclonal antibody and comprises transducing an extraneous DNA containing a gene capable of encoding an amino acid sequence of an immunoglobulin heavy chain polypeptide derived from a rearranged endogenous immunoglobulin heavy chain gene into a cell having the rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene and capable of secreting a monoclonal antibody comprising the immunoglobulin heavy chain polypeptide derived from the rearranged endogenous immunoglobulin heavy chain gene and an immunoglobulin light chain polypeptide derived from the rearranged endogenous immunoglobulin light chain gene, providing a transformed cell transformed with the extraneous DNA, culturing the resultant cell, obtaining the monoclonal antibody secreted into the cell culture fluid, thereby increasing the secretory capacity and affording the monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a monoclonal antibody, particularly to a method for increasing the amount of secreted monoclonal antibody from cells, and to cells produced by the method.

[0002]

2. Description of the Related Art Mammalian living organisms contain various antigens (for example, foreign antigens (viruses, bacterial toxins, and Humoral, a defense system that specifically captures and eliminates autoantigens (eg, autoreactive lymphocytes; tumor cells; excess in vivo factors (cytokines, hormones, growth factors, etc.)) In humoral immunity, so-called antibodies (also called immunoglobulins) play a leading role.

[0003] Antibodies (immunoglobulins) are composed of four long polypeptide chains (immunoglobulin heavy chains; IgH chains) and two short polypeptide chains (immunoglobulin light chains; IgL chains). It has a Y-type basic structure composed of peptide chains. This Y-type structure is formed by connecting one IgL chain to each of two IgH chains cross-linked by disulfide bonds by disulfide bonds.

[0004] Because of the function of capturing and eliminating antigens (pathogens) harmful to living organisms, these antibodies have been used as antibody pharmaceuticals from an early age. The earliest antibody drugs are so-called antisera, which are sera themselves (ie, polyclonal antibodies) in which various types of antibodies against specific antigens (eg, bacterial toxins and snake venoms) are mixed. However, since the method of obtaining the antiserum is limited to a method based on recovery from serum, the supply of the antiserum was necessarily limited. In addition, it has been extremely difficult to isolate a single type of antibody molecule having a specific antigen specificity, that is, a monoclonal antibody, from this antiserum in which various types of antibodies are mixed.

[0005] These problems are posed by the successful production of monoclonal antibodies by so-called hybridomas by Koehler and Milstein in 1975 (Nature, Vol. 256, p. 4).
95-497, 1975). This method
That is, immortalized B cells are obtained by fusing cells that produce a specific type of monoclonal antibody (B cells such as spleen cells) collected from a non-human mammal immunized with an antigen with myeloma cells and immortalizing the cells. (Hybridoma) was obtained, and the monoclonal antibody was purified and obtained from a cell culture by culturing the hybridoma. Although this method alone does not always achieve the desired mass production of the desired monoclonal antibody, it is an epoch-making technique in that the desired monoclonal antibody can be obtained as desired. This technology has opened the way for the use of monoclonal antibodies as antibody drugs.

[0006] Monoclonal antibodies are particularly superior in the antigen specificity and stability as compared with the above-mentioned antisera (polyclonal antibodies), and antigens involved in the onset or exacerbation of diseases (eg, viruses and bacteria). Exogenous antigens such as toxins; various in vivo factors such as cytokines, hormones or growth factors; and cell surface molecules such as receptors, cell adhesion molecules and signaling molecules)
By specifically binding to, the biological activity of the antigen can be regulated (inhibition of activity, enhancement of activity, inhibition of signal transduction, signal transduction instead of ligand, or inhibition of cell-cell adhesion, etc.). It is used as an extremely useful drug for the prevention and treatment of various diseases.

On the other hand, the production amount of the monoclonal antibody by the above-mentioned hybridoma is not always high, and only the method of culturing the hybridoma and purifying and isolating the monoclonal antibody from the cell culture solution, It was difficult to mass-produce the monoclonal antibody. Therefore, a method for producing a larger amount of a monoclonal antibody from a hybridoma has been studied in order to sufficiently and inexpensively supply a monoclonal antibody that is extremely useful as a pharmaceutical.

For example, it has been reported that culturing human antibody-producing hybridomas in a medium supplemented with interleukin-2 increases the amount of antibody produced (Cell).
ularimmunology, Vol. 115, p. 325-333, 1988)). Also, as an attempt using genetic engineering technology, Ochi
Reported the following (Proc. Natl. Ac
ad. Sci. USA vol.80, p.6351, 1983). Hapten (TN
P; 2,4,6-trinitrophenyl) IgH chain and IgL of the anti-TNP monoclonal antibody cloned from hybridoma Sp6 producing mouse IgM monoclonal antibody
(Κ) chain IgH chain gene (μ) and IgL chain gene (κ) encoding each chain were introduced into a plasmacytoma X63Ag8 producing an IgG monoclonal antibody against an unknown antigen different from the anti-TNP monoclonal antibody. The recombinant cells
The anti-TNP antibody is produced together with the IgG antibody. Further, a cell strain that is a mutant derived from the hybridoma Sp6, secretes only the κ chain, which is the light chain of the TNP antibody, and does not express the heavy chain and thus does not secrete the anti-TNP antibody, the anti-TNP antibody The recombinant cells obtained by introducing the IgH gene encoding the heavy chain of the above produce the anti-TNP antibody. However, according to the results of the experiments by Ochi et al., The amount of the anti-TNP antibody produced by each of the above recombinant cells was 10 to 25% of the amount of the anti-TNP antibody produced by the hybridoma Sp603 subcloned from the hybridoma Sp6. However, it has failed to increase the secretion amount of the target monoclonal antibody.

In general, methods for increasing the number of cells per culture and methods for improving the production of substances per cell have been studied as means for producing large amounts of monoclonal antibodies by host cells into which hybridomas or antibody genes have been incorporated. I have. Increasing the number of cells per culture is a preferred method, but increasing the number of cells does not always lead to high antibody production, but rather selects and isolates a single antibody-producing cell line. It is important to culture the single cell line. Although a great deal of effort is required for the selection and isolation of cell lines with high antibody productivity (such as hybridomas and recombinant cells), they are extremely important factors for increasing the productivity of the desired monoclonal antibody. .

On the other hand, in the production of a desired protein using a transgenic cell, in order to increase the expression efficiency of a gene encoding the desired protein introduced into the recombinant cell,
By introducing a dihydrofolate reductase (DHFR) gene or a glutamate synthase (GS) gene into the recombinant cell together with the gene encoding the desired protein, and amplifying the copy number of the gene for the desired protein, A method for increasing the amount of the protein produced is used (International Patent Application Publication Nos. WO81 / 02426 and WO87 / 04462). DHFR
Taking a gene as an example, these methods include the steps of: constructing an expression vector having a DHFR gene inserted in the vicinity of a gene encoding a desired protein; transforming a host cell with the expression vector; Is cultured in the presence of a drug (eg, methotrexate (MTX), phosphinothricin, methionine sulfoximine, etc.) to select a drug-resistant strain. In the obtained drug-resistant strain, the copy number of the introduced dhfr gene is increased (gene amplification), and at the same time, the gene adjacent to the dhfr gene is amplified. As a result of amplification of the copy number of the gene encoding the desired protein, an increase in the production of the desired protein is expected.

[0011] Immortalized B cells obtained by immortalizing monoclonal antibody-producing B cells isolated from a mammal immunized with an antigen (for example, a hybridoma obtained by fusion of the B cells and myeloma cells described above) In, the rearranged immunoglobulin gene (IgH gene) and rearranged immunoglobulin light chain gene (IgL
Genes) are both integrated into the genome. In order to amplify the IgH gene and the IgL gene using the gene amplification genes described above, it is necessary to identify the location of the respective genes on the genome and to insert the gene amplification genes in the vicinity of each gene. . However, although this method is theoretically feasible, it requires a great deal of time and effort, and it is not practical to accurately target the gene amplification gene to a desired position on the genome of the immortalized B cell. Is impossible.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to increase the efficiency of monoclonal antibody production by antibody-producing cells, particularly immortalized B cells (hybridomas), with simpler operations. That is, an object of the present invention is to provide a novel method that can increase the efficiency of monoclonal antibody expression by the immortalized B cells (hybridomas), which has been considered difficult so far.

[0013]

Means for Solving the Problems The present inventors have compared the expression of immunoglobulin light chain gene (IgL chain gene) in the production of monoclonal antibodies by hybridomas.
Focusing on the fact that the expression of the immunoglobulin heavy chain gene (IgH chain gene) is often unstable, and in some cases the secretion of the antibody molecule stops, and the amount of antibody molecule secretion depends on the expression level of the IgH chain gene. I thought.

Based on this idea, the present inventors have conducted intensive studies on a method for increasing the amount of secreted antibody molecules by improving (enhancing) the expression of the IgH chain gene. The resulting immortalized B cells (hybridomas) are obtained by introducing an IgH-encoding cDNA having the same nucleotide sequence as the rearranged endogenous IgH gene encoding the heavy chain polypeptide of the monoclonal antibody cloned from the hybridomas. In the recombinant hybridoma, the secretion amount of the monoclonal antibody is significantly increased, and the production amount of the monoclonal antibody is also increased by introducing a gene amplification gene such as a DHFR gene together with the IgH-encoding cDNA into the hybridoma. And found that the present invention was completed.

By using the method of the present invention, the amount of the monoclonal antibody produced by the monoclonal antibody-producing cells can be significantly increased. That is, the method for producing the monoclonal antibody of the present invention and cells produced by the method are:
It is extremely useful in the production of monoclonal antibodies useful as pharmaceuticals. That is, the present invention is a method and a cell as described below.

(1) A method for producing a monoclonal antibody, comprising the steps of: (a) having a rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene; The immunoglobulin heavy chain polypeptide is derived from cells that secrete a monoclonal antibody comprising an immunoglobulin heavy chain polypeptide derived from the immunoglobulin heavy chain gene and the immunoglobulin light chain polypeptide derived from the endogenous immunoglobulin light chain gene. Exogenous DNA containing a gene encoding the same amino acid sequence as the peptide
And obtaining a transformed cell transformed with the exogenous DNA; and (b) culturing the transformed cell to obtain the monoclonal antibody secreted in a cell culture solution. A method for producing a monoclonal antibody. (2) The gene according to (1), wherein the gene encoding the same amino acid sequence as the amino acid sequence of the immunoglobulin heavy chain polypeptide is a gene having the same base sequence as the endogenous immunoglobulin heavy chain gene. )). (3) The production method according to (1) or (2), wherein the cells are immortalized B cells derived from mammalian B cells. (4) The production method according to (3), wherein the immortalized B cells are fusion cells obtained by fusing the B cells with myeloma cells or recombinant myeloma cells. (5) The method according to (3) or (4), wherein the mammal is a non-human mammal. (6) The method according to (3) or (4), wherein the mammal is a human. (7) The production method according to (3) or (4), wherein the mammal is a transgenic non-human mammal capable of producing a human antibody. (8) the endogenous immunoglobulin heavy chain gene is a human immunoglobulin heavy chain gene;
(1) The method according to any one of (4), (6) and (7). (9) The endogenous immunoglobulin light chain gene is a human immunoglobulin light chain gene.
(1) The method according to any one of (4), (6) and (7). (10) The method according to any one of (1) to (5), wherein the monoclonal antibody is a monoclonal antibody of a non-human mammal. (11) The above (1) to (4), wherein the monoclonal antibody is a human monoclonal antibody.
The production method according to any one of (6) and (7). (12) The method according to any one of (1) to (11), wherein the exogenous DNA further contains a gene amplification gene. (13) The production method according to (12), wherein the gene amplification gene is a dihydrofolate reductase (DHFR) gene. (14) A transformed cell produced by any one of the above (1) to (13).

Hereinafter, the present invention will be described in more detail by clarifying the meanings of terms used in the present invention and specific embodiments of the present invention.

In the present invention, the term "mammal" refers to a human,
Refers to mammals such as cows, sheep, pigs, goats, rabbits, rats, hamsters, guinea pigs, and mice,
Further, a "transgenic non-human mammal capable of producing a human antibody" described later is also included. In the present invention, a "non-human mammal" means a mammal other than a human, and specifically, a mammal such as a cow, sheep, pig, goat, rabbit, rat, hamster, guinea pig, and mouse. And a transgenic non-human mammal capable of producing a human antibody as described below.
Is also included.

In the present invention, the term "antigen" means any substance that the immunocompetent cells in a mammalian organism recognize as non-self, and a foreign antigen that is foreign to the organism and the organism Includes any endogenous substance of the organism that can produce antibodies. Examples of the foreign antigen include various viruses, bacteria, bacterial toxins and chemical substances. When the living body is a specific mammal, the foreign body may be a different individual or a different animal species from the living body. Any substance derived therefrom (eg, tissues, cells, proteins, fragments thereof, body fluids, etc.) is included. As endogenous substances,
In some cases, various cytokines, growth factors, hormones, cell surface molecules (for example, receptors, channel molecules, signaling molecules, etc.), autoreactive lymphocytes, and the like, which are produced in excess in the living body.

In the present invention, the "monoclonal antibody" is any monoclonal antibody reactive with the above-mentioned antigen. The "monoclonal antibody" is a natural antibody obtained by immunizing a mammal such as a mouse, rat, hamster, guinea pig or rabbit with the above-mentioned antigen, a chimeric monoclonal antibody which can be produced using a genetic recombination technique described below. (Chimeric antibodies) and human monoclonal antibodies (human antibodies; CDR-grafted antibodies), and human monoclonal antibodies (human antibodies) that can be produced using transgenic animals that produce human antibodies. In addition, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgG
Monoclonal antibodies having any isotype such as A, IgD or IgE are also included. Preferably,
IgG (IgG1, IgG2, IgG3, IgG4) or IgM.

The term "cells secreting a monoclonal antibody" used in the present invention has a rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene, Any cell that secretes a monoclonal antibody consisting of an immunoglobulin heavy chain polypeptide derived from the endogenous immunoglobulin heavy chain gene and an immunoglobulin light chain polypeptide derived from the endogenous immunoglobulin light chain gene. Preferably, it is any "monoclonal-producing immortalized B cell" described in detail below, and more preferably a hybridoma obtained by cell fusion of a monoclonal antibody-producing B cell with a myeloma cell or the like.

[0022] The term "monoclonal antibody-producing immortalized B cells" in the present invention refers to B cells that produce monoclonal antibodies against the antigen, which are produced in the above-mentioned mammalian organism when the organism is immunized with the antigen. And immortalized B cells produced by immortalizing the B cells by a desired method. The "monoclonal antibody-producing B cells" and "monoclonal antibody-producing immortalized B cells" can be produced by an existing general production method. That is, for example, a mammal, preferably a mouse, a rat, a hamster, a guinea pig, a rabbit, a cat, a dog, a pig, a goat, a horse, a cow, or a human described below, together with an antigen, if necessary, with Freund's Adjuvant. A transgenic non-human mammal created to produce antibodies from another animal, such as an antibody-producing transgenic non-human mammal, is immunized. Immunization is performed by subcutaneous, intramuscular, intravenous, footpad or intraperitoneal injection or transplantation of the mammal one or several times or by immunization. Usually, immunization is performed 1 to 4 times about every 1 to 14 days from the initial immunization, and further, if necessary, on the day before or two days before the collection of the monoclonal antibody-producing cells.

B cells, which are antibody-producing cells, are recovered from the immunized mammal as described above from the spleen, lymph node, bone marrow, tonsil, or the like, preferably from the spleen, according to a conventional method. The antibody-producing B cells are preferably used in accordance with the method of Kohler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and a modification method analogous thereto, preferably mouse, rat, guinea pig, hamster, rabbit or human. And immortalized to obtain hybridomas by cell fusion with myeloma cells having no autoantibody-producing ability derived from mammals, more preferably mice, rats or humans, to obtain antibody-immortalized B cells. Examples of myeloma cells used for cell fusion include, for example, mouse-derived myeloma P3 / X63.
-AG8.653 (653; ATCC No.CRL1580), P3 / NSI / 1-Ag4-1
(NS-1), NSO, P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14
(Sp2 / 0, Sp2), PAI, F0, BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, G
M1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM
-T15 can be used.

Screening of the hybridomas producing monoclonal antibodies (monoclonal antibody-producing immortalized B cells) produced as described above is performed by culturing the hybridomas in, for example, a microtiter plate and culturing wells in which proliferation has been observed. The reactivity of the supernatant to the immunizing antigen used in the mouse immunization described above, for example, RIA or ELISA
And the like.

In the present invention, the term "human antibody" or "human immunoglobulin" refers to the variable region (V _H ) of the H chain and the constant region (C _H ) of the H chain constituting the above-mentioned immunoglobulin.
And the variable region of the light chain (V _L ) and the constant region of the light chain (C _L )
Are all immunoglobulins derived from genes encoding human immunoglobulins. In other words, it means an antibody in which the H chain is derived from the human immunoglobulin heavy chain gene and the light chain is derived from the human immunoglobulin light chain gene. The human antibody can be prepared by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into a genetic locus of a non-human mammal such as a mouse with an antigen according to a conventional method. The monoclonal antibody can be produced in the same manner as described above. For example, transgenic mice producing human antibodies have already been reported (Nature
Genetics, Vol.15, p.146-156, 1997; Nature Genetic
s, Vol. 7, p. 13-21, 1994; Table 4-504365; International Application Publication WO94 / 25585; Nikkei Science, June,
40 to 50, 1995; Nature, Vol. 368, p. 8
56-859, 1994; and JP-A-6-500233).

The method for producing the monoclonal antibody of the present invention comprises the following steps (a) and (b). (A) an immunoglobulin heavy chain polypeptide comprising a rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene, and an immunoglobulin heavy chain polypeptide derived from the endogenous immunoglobulin heavy chain gene; A cell that secretes a monoclonal antibody comprising an immunoglobulin light chain polypeptide derived from an immunoglobulin light chain gene, and a foreign DNA containing a gene encoding an amino acid sequence identical to the amino acid sequence of the immunoglobulin heavy chain polypeptide
And obtaining a transformed cell transformed with the exogenous DNA. (B) culturing the transformed cells to obtain the monoclonal antibody secreted in a cell culture solution.

As used herein, the term "gene encoding the same amino acid sequence as that of the immunoglobulin heavy chain polypeptide" refers to a cell producing the above-described monoclonal antibody (preferably, an antibody-producing B cell or an antibody-immortalizing B cell). A gene (preferably cDNA) encoding an amino acid sequence identical to the amino acid sequence of an immunoglobulin heavy chain polypeptide encoded by an endogenous immunoglobulin heavy chain gene possessed by a cell (such as a hybridoma). It can be prepared as follows using a cell engineering technique and a gene recombination technique according to a conventional method.

(1) From a hybridoma producing a desired monoclonal antibody, a commercially available reagent (eg, FastTrac
k2.0kit (manufactured by INVITROGEN)) according to the standard method.
Extract and purify lyA ⁺ RNA. Specifically, for example, it is as follows. The hybridoma that has been frozen and stored is dissolved in a cell lysis buffer (Lysis Buffer), and the cells are disrupted with a commercially available cell lysis reagent (for example, POLYTRON or the like) to be solubilized. After incubating the lysate at a suitable temperature (eg, about 45 ° C.), Oligo (dT)
Add cellulose and shake gently for an appropriate amount of time (eg, about 1 hour). Next, after washing Oligo (dT) cellulose, P
Elute olyA ⁺ RNA with Ellution Buffer. Po eluted
lyA ⁺ RNA is ethanol precipitated and dissolved in an appropriate amount of Tris-EDTA buffer. The concentration of the resulting PolyA ⁺ RNA is determined by measuring the absorbance at the appropriate wavelength (eg, 260 nm).

(2) Using the obtained PolyA ⁺ RNA as a template, a commercially available reagent (for example, Marathon cDNA Amplification Kit)
(CLONTECH) by using RACE-PCR method to synthesize cDNA by a conventional method ("Gene amplification PCR method, basic and new development", 1992, 2nd printing, published by Kyoritsu Shuppan Co., p.13
-15). Specifically, as described below, an appropriate amount of purified P
Using olyA ⁺ RNA (for example, about 1 to 5 μg) as a template, 1st str
and cDNA and 2nd strand cDNA are sequentially synthesized. The cDNA
Is subjected to extraction using phenol / chloroform / isoamino alcohol and chloroform. Then cD
NA is ethanol precipitated and ligated to the adapter DNA.
Dilute the resulting DNA reaction to an appropriate concentration (eg, 1/25
0) as a template, using a primer designed based on the base sequence encoding the amino acid sequence of the constant region of the heavy chain of the monoclonal antibody produced by the hybridoma and a primer designed based on the base sequence of the adapter DNA And perform PCR by a conventional method. The obtained PCR product is fractionated by agarose gel electrophoresis, and cDNA is recovered.

(3) cDNA encoding the full-length or partial amino acid sequence of the heavy chain of the obtained monoclonal antibody
Base sequence of a commercially available reagent (eg, Dye Terminator Cyc
le Sequence Kit (PE-Applied Biosystems) and PR
ISM377 DNA Sequencer (PE-Applied Biosystems)
Is determined using (4) cDNA encoding the full-length amino acid sequence of the heavy chain polypeptide by PCR based on the base sequence of the cDNA encoding a part of the amino acid sequence of the heavy chain of the monoclonal antibody
Synthesize a pair of primer DNAs as possible, using the pair of primers, performing PCR in the same manner as described above using the purified PolyA ⁺ RNA as a template, and cDNA encoding the full length of the heavy chain polypeptide. Get.

The method for producing the monoclonal antibody of the present invention comprises the steps of: "gene encoding an amino acid sequence identical to the amino acid sequence of the immunoglobulin heavy chain polypeptide" described in detail above; The cells secreting the antibody (preferably, the monoclonal antibody-producing hybridoma itself used as the source in the preparation of the gene, that is, synonymous with the above-described monoclonal antibody-producing B cells or immortalized B cells) are subjected to genetic engineering techniques according to a conventional method. And transforming the transformed cells with the gene, selecting and isolating the transformed cells, and culturing the transformed cells to purify and obtain the monoclonal antibody from a cell culture solution.

To introduce the gene (eg, cDNA) into the cell, a plasmid vector-integrated expression vector is prepared so that the gene can be expressed in a host cell by a conventional method, and the host is then transfected with the expression vector. This is done by transforming the cells. As a method of incorporating cDNA into a plasmid, for example, the method of Maniatis et al. (Molecular Cloning,
A Laboratory Manual, second edition, Cold Spring
Harbor Laboratory, p. 1.53, 1989). Methods for introducing a plasmid into a host are described in (Molecular Cloning, A Laboratory Manual, seco
nd edition, Cold Spring Harbor Laboratory, Vol.1.7
4, 1989), the calcium chloride method, the calcium chloride / rubidium chloride method, and the electroporation method.

The vector used in the present invention is not particularly limited as long as it can maintain replication or self-replicate in a host cell (preferably eukaryotic cell), and includes a plasmid vector and a phage vector. Specifically, as the plasmid, for example, pLS407, pBR322, pBR3
25, pUC12, pUC13, pUC19, pSH19, pSH15, pUB110, pTP
5, pC194, pcD2, pBSV, CMD, pSV2, and further pMAL C2, pE
F-BOS (Nucleic Acid Research)
Research), vol. 18, p. 5322, 1990 etc.)
Alternatively, pME18S (Experimental Medicine Separate Volume “Genetic Engineering Handbook”, 1992, etc.) and the like are exemplified. Bacteriophages such as λ phage and animal and insect viruses such as retrovirus, vaccinia virus and nucleopolyhedrovirus (pVL1393,
Manufactured by Invitrogen).

When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally contains at least a promoter-
It is composed of an operator region, an initiation codon, a gene encoding the desired immunoglobulin heavy chain (IgH chain), a termination codon, a terminator region, and a replicable unit. When using yeast, animal cells or insect cells as a host, the expression vector is at least a promoter,
Start codon, the desired immunoglobulin heavy chain (IgH
Chain), and preferably includes a stop codon. DN encoding a signal peptide
A, enhancer sequence, 5 'and 3' untranslated regions of the gene encoding the above-mentioned gene encoding the desired immunoglobulin heavy chain (IgH chain), splicing junction, polyadenylation site, selection marker region Alternatively, a copyable unit may be included. Further, it may contain a gene amplification gene which is usually used depending on the purpose.

Examples of the promoter for expressing the gene encoding the desired immunoglobulin heavy chain (IgH chain) in eukaryotic cells such as mammalian cells include, for example, a chicken β-actin promoter and an SV40-derived promoter. , A retrovirus promoter, a heat shock promoter and the like. Preferably, a chicken β-actin promoter, an SV40-derived promoter, SV40, and a retrovirus promoter. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression, and a preferred enhancer is a CMV enhancer. The promoter-operator-region for expressing the gene encoding the above-described desired immunoglobulin heavy chain (IgH chain) in bacteria includes a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). It is a thing. For example, when the host is Escherichia, preferably a Trp promoter, a lac promoter,
Examples include those containing the recA promoter, λPL promoter, lpp promoter, tac promoter, and the like. Examples of a promoter for expressing a gene encoding the above-described desired immunoglobulin heavy chain (IgH chain) in yeast include a PH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter. Means SL01 promoter, SP02 promoter, pe
nP promoter and the like.

A preferred initiation codon is, for example, methionine codon (ATG). Examples of the stop codon include commonly used stop codons (eg, TAG, TGA, TAA). As the terminator region, a commonly used natural or synthetic terminator can be used.

A replicable unit is defined as the total D in the host cell.
A DNA capable of replicating an NA sequence, which includes a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from the natural plasmid), a synthetic plasmid, and the like. Suitable plasmids include plasmid pBR322 in E. coli,
Alternatively, an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) is a yeast 2μ plasmid or a yeast chromosomal DNA in yeast.
However, in mammalian cells, plasmid pRSVneo ATCC 371
98, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV
-MMTneo ATCC 37224, plasmid pSV2neo ATCC 37149 and the like.

As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, for example, those derived from SV40 can be used. The above-described expression vector is prepared by the above-described promoter, start codon, and the above-described expression vector.
It can be prepared by linking a gene encoding an IgH chain and / or a terminator region to an appropriate replicable unit in a continuous and circular manner. At this time,
If desired, an appropriate DNA fragment (for example, a linker or another restriction enzyme cleavage site) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

In selecting a host cell transformed with the above-mentioned expression vector, that is, a transformed cell, a desired selection marker (for example, a drug resistance gene) is inserted into the expression vector, and the transformed cell is selected. It is possible by culturing in the presence of the drug. As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include antibiotic resistance genes such as tetracycline, ampicillin, and kanamycin.

In the above-described method for producing the monoclonal antibody of the present invention, a gene amplification gene is inserted together with the gene encoding the desired heavy chain polypeptide of the monoclonal antibody inserted into the expression vector. An aspect of the present invention also includes an embodiment in which the copy number of the gene encoding the heavy chain polypeptide is amplified to further increase the efficiency of producing the desired monoclonal antibody by the transformed cell.

The gene amplification genes that can be used in the present invention include dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase (GS) gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin- Examples include the B-phosphotransferase gene and the aspartate transcarbamylase gene. Preferably, it is a DHFR gene or a GS gene.

In the method for producing a monoclonal antibody of the present invention, cells (host cells) transformed by the “gene encoding the same amino acid sequence as that of the immunoglobulin heavy chain polypeptide” described above are That is, any cell may be used as long as it is "a cell that secretes a monoclonal antibody comprising the immunoglobulin heavy chain polypeptide", and particularly preferably, the monoclonal antibody-producing hybridoma itself used as a source in the preparation of the gene (that is, the above-described hybridoma). Monoclonal antibody producing B cells or immortalized B cells).

Examples of host cells other than the hybridoma include various cells such as natural cells or artificially established recombinant cells (for example, bacteria (Escherichia, Bacillus) as long as the desired monoclonal antibody is produced. Genus), yeast (Saccharomyces, Pichia, etc.), animal cells, insect cells, etc. If the cells produce the desired monoclonal antibody, the origin of the cells is, for example, Escherichia coli ( DH5α, TB
1, HB101 etc.), mouse-derived cells (COP, L, C127, Sp2 /
0, NS-1 or NIH3T3), rat-derived cells (PC12, PC12
h), hamster-derived cells (such as BHK and CHO), monkey-derived cells (such as COS1, COS3, COS7, CV1 and Velo) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells and HepG2) Etc.).

The introduction (transformation (transfection)) of the expression vector into which the gene encoding the desired immunoglobulin heavy chain described above has been inserted into host cells can be carried out by a conventionally known method. For example, in the case of animal cells,
For example, Graham's method (Virology, Vol. 52, p. 456, 1973)
In the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad.
USA., Vol. 69, p. 2110, 1972), protoplast method (Mo
l. Gen. Genet., Vol. 168, p. 111, 1979) or by the competent method (J. Mol. Biol., Vol. 56, p. 209, 1971), in the case of Saccharomyces cerevisiae, for example, Hinnen
Natl. Acad. Sci. USA., Vol. 75, p. 1
927, 1978) and the lithium method (J. Bacteriol., Vol. 153, p.
163, 1983), in the case of insect cells, for example, Summe
rs et al. (Mol. Cell. Biol., Vol. 3, p. 2156-2165,
1983).

The "transformed cells" in the above-described method for producing the monoclonal antibody of the present invention, which are transformed with the gene encoding the desired immunoglobulin heavy chain, can be cultured by a conventional method. This can be done as follows. By such culture, a desired monoclonal antibody can be obtained from the cell culture solution.

The cells transformed with the gene encoding the heavy chain of the desired immunoglobulin are hybridized with the monoclonal antibody-producing hybridoma itself (the above-mentioned monoclonal antibody-producing B cell or immortalized B cell). Synonymous), that is, when the obtained transformed cells are recombinant hybridomas, it can be carried out in the same manner as a general method for culturing hybridomas.

Specifically, the recombinant hybridoma is
In vitro or in vivo, such as in a mouse, rat, guinea pig, hamster or rabbit, preferably in a mouse or rat, more preferably in a mouse ascites, and isolated from the resulting culture supernatant or mammalian ascites Can be performed. When culturing in vitro, the hybridoma is grown, maintained, and stored in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culturing method, and the monoclonal antibody is produced in the culture supernatant. It can be carried out using a known nutrient medium or any nutrient medium derived and prepared from a known basal medium.

As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium can be used. The basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

For isolation and purification of the monoclonal antibody from the recombinant hybridoma, the above-mentioned culture supernatant or ascites fluid was subjected to saturated ammonium sulfate, euglobulin precipitation, caproic acid, caprylic acid, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as an anti-immunoglobulin column or a protein A column.

On the other hand, cells transformed with the gene encoding the heavy chain of the desired immunoglobulin can be used, for example, in the production of the above-mentioned recombinant protein such as CH
In the case of a host cell such as an O cell, it can be carried out in the same manner as in a general culture method for producing a recombinant protein.

That is, it can be produced by culturing the transformed cells in a nutrient medium. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose.Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and the like.
Examples include meat extract, soybean meal, potato extract and the like. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. . The culturing is performed by a method known in the art. Culture conditions, such as temperature, pH of the medium, and culture time, are appropriately selected so that the desired monoclonal antibody of the present invention is produced in large quantities from the transformed cells.

Specific media and culture conditions used according to the host cell are shown below, but the present invention is not limited thereto. When the host is a bacterium, actinomycete, yeast, or filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8. When the host is E. coli, a preferable medium is L.
B medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spr.
ing Harbor Laboratory, p.431, 1972). In such a case, the cultivation can be carried out usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary. When the host is a bacterium belonging to the genus Bacillus, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours, if necessary, with aeration and stirring.

When the host is yeast, the medium may be, for example, Burkholder minimum culture (Bostian, Proc. Natl. Acad.
i. USA, Vol. 77, p. 4505, 1980), and the pH is 5-
8 is desirable. Culture is usually about 14 at about 20-35 ° C.
144144 hours, and if necessary, aeration and stirring can be performed. When the host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum (Science, Vol.
2, p.501, 1952), DMEM medium (Virology, Vol.8, p.39)
6, 1959), RPMI1640 medium (J. Am. Med. Assoc., Vol. 1
99, p.519, 1967), 199 medium (proc. Soc. Exp. Biol.
Med., Vol. 73, p. 1, 1950) can be used.
The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about pH 8.
The reaction is carried out at 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed. When the host is an insect cell, for example, Grace's medium (Proc. Natl. Acad.
i. USA, Vol. 82, p. 8404, 1985).
Is preferably about 5-8. Culture is usually about 20-40 ° C
For 15 to 100 hours, and if necessary, aeration and stirring can be performed. The desired monoclonal antibody in the present invention can be obtained from the culture supernatant of the transformed cells thus cultured by the above-mentioned general method for purifying a monoclonal antibody.

[0054]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to the embodiments described in the Examples.

Example 1 Preparation of Hybridoma Producing Monoclonal Antibody The hybridoma producing a human monoclonal antibody against human IL-8 described in the previous report was used in the following tests (Nature genetics Vol. 15, p. 146-156, 1997). And International Patent Application Publication No. WO 96/33735). The hybridoma was produced as follows. DNA that inactivates the endogenous loci of each of the mouse heavy and light chains and that contains the human immunoglobulin heavy (Cμ and Cγ ₂ ) and light chain (κ) loci, respectively. the previously reported transgenic mice producing human IgG ₂ / kappa monoclonal antibody prepared by incorporating into the genome were used as animals to be immunized (Nature Genetics, Vol.15,
p.146-156, 1997; Nature Genetics, Vol.7, p.13-21,
1994; Table 4-504365; International Application Publication WO94 / 25585; Nikkei Science, June, pages 40-50, 1
995; Nature, Vol. 368, p. 856-859, 1994; and JP-T-Hei 6-500233).

Recombinant human IL-8 (25 μg) was added to the human IgG ₂ / κ antibody-producing transgenic mouse (8 to 10 weeks old).
Was administered intraperitoneally together with complete Freund's adjuvant for the first immunization. Every 2 weeks after the first immunization, booster the IL-8 with incomplete Freund's adjuvant (3 times)
The cells were finally immunized 4 days before the following cell fusion. After the final immunization, the spleen and lymph nodes of the immunized mouse were collected to collect lymphocytes (including antibody-producing B lymphocytes). The antibody-producing lymphocytes were converted to mouse myeloma cells (NSO-bsl2 cell line) that did not produce any autoantibodies according to a conventional method.
And cell fusion. Hybridomas obtained by cell fusion were selected by the HAT selection method according to a conventional method. The presence or absence of the reactivity of the human monoclonal antibody produced by the obtained hybridoma to human IL-8 was measured by ELISA according to a conventional method, and a plurality of hybridomas producing an anti-human IL-8 human monoclonal antibody were obtained. Each hybridoma was cryopreserved.

Example 2 Isolation of Endogenous IgH Gene from Monoclonal Antibody Producing Hybridoma An anti-human IL-8 human IgG ₂ / κ monoclonal antibody producing hybridoma prepared as described above (clone: K2.2.
The frozen cells of 1) were lysed in a cell lysis buffer (Lysis Buffer), and the cells were disrupted with POLYTRON and solubilized.
Next, a commercially available RNA extraction kit (F
PolyA ⁺ RNA was extracted and purified using astTrack 2.0 kit (manufactured by INVITROGEN). After incubating the cell lysate at 45 ° C., Oligo (dT) cellulose was added, and the mixture was gently shaken for about 1 hour. Next, after washing Oligo (dT) cellulose, PolyA ⁺ RNA was eluted with Elution Buffer. The eluted PolyA ⁺ RNA was precipitated with ethanol and dissolved in Tris-EDTA buffer. The concentration of the resulting PolyA ⁺ RNA was determined by measuring the absorbance at a wavelength of 260 nm.

Using the obtained PolyA ⁺ RNA as a template, commercially available Mar
Using athon cDNA Amplification Kit (CLONTECH)
CDNA was synthesized by the RACE-PCR method according to a conventional method ("Gene amplification PCR method, basics and new development", second edition, 1992, published by Kyoritsu Shuppan Co., Ltd., p.13-15). That is, using PolyA ⁺ RNA (1 to 5 μg) purified from the hybridoma as a template,
1st strand cDNA and 2nd strand cDNA were sequentially synthesized.
The cDNA was subjected to extraction once each using phenol / chloroform / isoamino alcohol and chloroform. Next, the cDNA was precipitated with ethanol and ligated to the adapter DNA provided with the kit. Using the resulting dilution of the DNA reaction product as a template, 5'RACE-PCR was performed by a conventional method using synthetic primers to prepare cDNA encoding a part of the endogenous immunoglobulin heavy chain polypeptide. For the PCR, a primer HG2-3-437 (SEQ ID NO: 3) designed based on the nucleotide sequence encoding the amino acid sequence of the immunoglobulin heavy chain constant region and a primer designed based on the nucleotide sequence of the adapter DNA were used. .

The PCR product was fractionated by agarose gel electrophoresis, and the DNA was recovered. Determination of the nucleotide sequence of the obtained cDNA was performed using a commercially available DyeTerminator Cycle Sequencing FS Kit (P
E-Applied Biosystems) and PRISM377 DNA Sequencer
(Manufactured by PE-Applied Biosystems). In addition,
As a sequencing primer for this sequencing, the primers used in the aforementioned PCR were used. CDNA encoding a part of the determined heavy chain polypeptide of the immunoglobulin
And a pair of primers VH4-21 (SEQ ID NO: 4) and CG2-1 based on the base sequence (including the base sequence near the translation start point)
(SEQ ID NO: 5) was synthesized. Using this pair of primers, PCR was further performed in the same manner as described above. P obtained
The nucleotide sequence of the cDNA obtained from the CR product in the same manner as described above was determined in the same manner as described above, and the full length of the heavy chain polypeptide (IgH) of the anti-human IL-8 human monoclonal antibody produced by the hybridoma K2.2.1 was determined. An encoding cDNA was obtained (base sequence:
SEQ ID NO: 1, and amino acid sequence SEQ ID NO: 2).

Example 3 IgH to hybridoma K2.2.1
Introduction of cDNA and Production of Monoclonal Antibody by Recombinant Hybridoma Anti-human IL-8 Secreted by Hybridoma K2.2.1 Obtained Above
The cDNA (SEQ ID NO: 1) encoding the full length of the heavy chain polypeptide (IgH) of the human monoclonal antibody was obtained by a conventional method.
Using DNA ligase, the plasmid was inserted into the EcoRI restriction enzyme site of plasmid pLS407 carrying the CMV enhancer / chicken B actin promoter and the DHFR gene and ligated to prepare an expression vector pDH502 (FIG. 1). The DHFR gene is a marker gene as well as a marker gene,
The presence of the gene enables selection of transformed cells by the expression vector by culturing the cells in the presence of methotrexate (MTX). The IgH gene expression vector pDH502 was introduced into hybridoma K2.2.1 by electroporation. Then, the hybridomas were transformed with a selective medium (IMDM (JRHBIOSCUENCE); 10% FBS and 300mM).
M MTX), and about 100 transformants (recombinant hybridomas) that grew and were selected and obtained. Each of the selected recombinant hybridomas was cultured in a 96-well microplate.

The amount of human monoclonal antibody (IgG ₂ ) produced in the culture supernatant of each transformant (recombinant hybridoma) cultured in each well was measured by a sandwich ELISA according to a conventional method. In addition, anti-human IgG (Fc) (Organon Teknika) was used as a solid phase antibody (primary antibody).
Anti-human Igκ antibody (PROTOS) labeled with horseradish peroxidase (HRP) as a detection antibody (secondary antibody)
IMMUNORESEARCH). In addition, human IgG / κ (The Binding Site) was used as a control standard. Figure 2 shows the results.
Shown in As a result, the production amount of monoclonal antibodies of most clones of the parent hybridoma K2.2.1 was extremely low, whereas the production amount of monoclonal antibodies was significantly increased in most clones of recombinant hybridomas. Was done. Table 1 shows the production amounts of monoclonal antibodies of several clones of the recombinant hybridoma.

[0062]

[Table 1]

Next, among the recombinant hybridoma clones, clones which showed production of a monoclonal antibody at a particularly high concentration (clone Nos. 12, 15, 41 and 89) were subcloned by limiting dilution and subcloned. (Sub-clon
ed) Recombinant hybridoma was obtained (clone No. 12-6, 1
5-4, 15-12, 41-2, and 89-5). The production amount of the monoclonal antibody of each sub-cloned recombinant hybridoma was measured by the same ELISA as described above. Table 2 (top)
Shown in

[0064]

[Table 2]

As a result, the production amount of the monoclonal antibody of each sub-cloned recombinant hybridoma was significantly higher than that of the parent recombinant hybridoma. For example, the antibody production of the parent recombinant hybridoma (No. 15) was about 37.3 μg / ml, whereas the antibody production of the recombinant hybridoma (No. 15-4) subcloned from the parent strain was: It increased to about 95.3 μg / ml.

Example 4 Production of Monoclonal Antibody from Gene-Amplified Recombinant Hybridoma The presence or absence of the effect of the amplification of the IgH gene by the DHFR gene on increasing the production efficiency of the monoclonal antibody was examined. The sub-cloned recombinant hybridoma (clone
No.12-6, 15-4, 15-12, 41-2, and 89-5)
MTX-resistant cell lines were selected and obtained by further culturing in a nutrient medium containing 1, 2, or 5 μM of methotrexate (MTX). The following MTX-resistant cell lines were obtained from each sub-cloned recombinant hybridoma. <Clone No.12-6> Clone No.12-5μ-96-8 <Clone No.15-4> Clone No.15-1μ-82-1 <Clone No.15-12> Clone No.15-1μ -87-4 <Clone No.41-2> Clone No.41-2μ-75-4 <Clone No.89-5> Clone No.89-2μ-2-5, and
89-2μ-33-12 The monoclonal antibody production of each MTX-resistant recombinant hybridoma clone was determined using the same sandwich EL method as described above.
Measured by ISA. The results are shown in Table 2 (lower). As a result, the production efficiency of monoclonal antibodies per cell number of each recombinant hybridoma selected by MTX was as follows:
Considering that the production amount of the monoclonal antibody of the wild-type parent hybridoma K2.2.1 before transformation with the IgH gene was extremely low (FIG. 2), although lower than that before MTX selection (before gene amplification by DHFR). The monoclonal antibody production efficiency of the MTX-selected recombinant hybridoma was significantly increased as compared to that of the wild-type parent hybridoma.

[0067]

As described above, the use of the method of the present invention makes it possible to easily and significantly increase the amount of monoclonal antibodies produced by monoclonal antibody-producing cells in the production of monoclonal antibodies useful as pharmaceuticals. Become. In particular, when monoclonal antibody production efficiency of monoclonal antibody producing immortalized B cells (hybridoma) generally used in the production of monoclonal antibodies is low depending on the instability or low expression of endogenous IgH gene In addition, by using the method of the present invention, it is possible to significantly increase the secretion amount of the monoclonal antibody of the parent hybridoma. Therefore, the method for producing the monoclonal antibody of the present invention and the monoclonal antibody-producing transformed cells (recombinant cells) obtained by the method are extremely useful means for producing an antibody drug.

[0068]

[Sequence List] SEQUENCE LISTING <110> Japan Tobacco Inc <120> A Process To Produce Monoclonal Antibody <130> J1-A0001Y1 <140><141><150> JP11-087929 <151> 1999-03-30 <160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 1507 <212> DNA <213> Homo sapiens <220><221> CDS <222> (12) .. (1400) <400> 1 gaattcggct t atg aaa cac ctg tgg ttc ttc ctc ctc ctg gtg gca gct 50 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala 1 5 10 ccc aga tgg gtc ctg tcc cag gtt cag cta cag cag tgg ggc gca gga 98 Pro Arg Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly 15 20 25 ctg ttg aag cct tcg gag acc ctg tcc ctc acc tgc gct gtc tat ggt 146 Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly 30 35 40 45 ggg tcc ttc agt ggt tac tac tgg acc tgg atc cgc cag ccc cca ggg 194 Gly Ser Phe Ser Gly Tyr Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly 50 55 60 aag ggg ctg gag tgg att ggg gaa atc att cat cat gga aac acc aac 242 Lys Gly Leu Glu Trp Ile Gly Glu Ile Ile His His Gly Asn Thr Asn 65 70 75 tac aac ccg tcc ctc aag agt cga gtc tcc ata tca gtt gac acg tcc 290 Tyr Asn Pro Ser Leu Lys Ser Arg Val Ser Ile Ser Val Asp Thr Ser 80 85 90 aag aac cag ttc tcc ctg aca ctg agc tct gtg acc gcc gcg gac acg 338 Lys Asn Gln Phe Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr 95 100 105 gct gtg tat tac tgt gcg aga ggg gga gca gtg gct gcg ttt gac tac 386 Ala Val Tyr Tyr Cys Ala Arg Gly Gly Ala Val Ala Ala Phe Asp Tyr 110 115 120 125 tgg ggc cag gga acc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 434 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg gtc ttc ccc ctg gcg ccc tgc tcc agg agc acc tcc gag agc 482 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg 530 Thr Ala Ala Leu Gly Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 160 165 170acg gtg tcg tgg aac t
ca ggc gct ctg acc agc ggc gtg cac acc ttc 578 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 175 180 185 cca gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 626 Pro Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 190 195 200 205 acc gtg ccc tcc agc aac ttc ggc acc cag acc tac acc tgc aac gta 674 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val 210 215 220 gat cac aag ccc agc aac acc aag gtg gac aag aca gtt gag cgc aaa 722 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 225 230 235 tgt tgt gtc gag tgc cca ccg tgc cca gca cca cct gtg gca gga ccg 770 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 240 245 250 tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 818 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 255 260 265 cgg acc cct gag gtc acg tgc gtg gtg gtg gac gtg agc cac gaa gac 866 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 270 275 280 280 285 ccc g ag gtc cag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 914 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 gcc aag aca aag cca cgg gag gag cag ttc aac agc acg ttc gtg 962 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 305 310 315 gtc agc gtc ctc acc gtt gtg cac cag gac tgg ctg aac ggc aag gag 1010 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu 320 325 330 tac aag tgc aag gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa 1058 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys 335 340 345 acc atc tcc aaa acc aaa ggg cag ccc cga gaa cca cag gtg tac acc 1106 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 350 355 360 365 ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc 1154 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 tgc ctg gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag 1202 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 agc aat ggg cag ccg gag aac aac tac aag acc aca cct ccc atg ctg 1250 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 400 405 410 gac tcc gac ggc tcc ttc ttc actc agc ctc acc gtg gac aag 1298 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 415 420 425 agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 1346 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 430 435 440 445 gct ctg cac aac cac tac acg cag aag ag agc ctc tcc ctg tct ccg ggt 1394 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 460 aaa tga gtgccacggc cggcaagccc ccgctcccca ggctctcggg gtcgcgtgag 1450 Lys gatgcttggc acgtaccccg tgtacatact tcccaggcac ccagcaaagc cgaattc 1507 <210> 2 <211> 462 <212> PRT <213> Lea Ue Au Hap Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Ph e 35 40 45 Ser Gly Tyr Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Ile His His Gly Asn Thr Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Val Ser Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Gly Gly Ala Val Ala Ala Phe Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 225 230 235 240 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 350 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <460> 3 <21 1> 23 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Artificially synthesized primer sequence, HG2-3-437 <400> 3 gtgtaggtct gggtgccgaa gtt 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Artificially synthesized primer sequence, VH4-21 <400> 4 atgaaacacc tgtggttctt cct 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Artificially synthesized primer sequence, CG2-1 <400> 5 gctgggtgcc tgggaagtat gta 23

"Sequence List Free Text" SEQ ID NO: 3 Other information: Description of artificial sequence: Artificially synthesized primer sequence HG2-3-437. SEQ ID NO: 4 Other information: Description of artificial sequence: Artificially synthesized primer sequence VH4-21. SEQ ID NO: 5 Other information: Description of artificial sequence: Artificially synthesized primer sequence CG2-1.

[0070]

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a structure and a restriction enzyme map of an expression vector pDH502.

FIG. 2 is a graph showing the amount of anti-human IL-8 monoclonal antibody produced by a recombinant hybridoma. The vertical axis indicates the amount of monoclonal antibody produced, and the horizontal axis indicates the type of each recombinant hybridoma clone in each well in the microplate.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/02 C07K 16/18 C12P 21/08 C12N 5/00 B // C07K 16/18 15/00 C (72) Inventor Atsushi Fukushima 1-13-2 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Japan F-term in the Pharmaceutical Research Laboratory, Tobacco Inc. 4B024 AA01 BA41 BA56 CA04 DA02 DA06 DA07 DA12 EA04 FA02 FA06 FA10 FA13 GA03 GA14 GA18 4B064 AG27 CA10 CA19 CA20 CC24 DA01 4B065 AA92X AA99X AB01 AB05 AC10 AC14 AC20 BA03 BA08 CA25 CA44 4H045 AA11 AA20 AA30 BA10 CA40 DA76 EA20 FA72 FA74

Claims (14)

[Claims] 1. A method for producing a monoclonal antibody, comprising the steps of: (a) having a rearranged endogenous immunoglobulin heavy chain gene and a rearranged endogenous immunoglobulin light chain gene; The immunoglobulin heavy chain polypeptide is secreted into cells that secrete a monoclonal antibody consisting of an immunoglobulin heavy chain polypeptide derived from the immunoglobulin heavy chain gene and an immunoglobulin light chain polypeptide derived from the endogenous immunoglobulin light chain gene. Exogenous DNA containing a gene encoding the same amino acid sequence as
And obtaining a transformed cell transformed with the exogenous DNA; and (b) culturing the transformed cell to obtain the monoclonal antibody secreted in a cell culture solution. A method for producing a monoclonal antibody. 2. The gene encoding the same amino acid sequence as the immunoglobulin heavy chain polypeptide is a gene having the same base sequence as the endogenous immunoglobulin heavy chain gene. Item 1. The production method according to Item 1. 3. The method according to claim 1, wherein the cells are immortalized B cells derived from mammalian B cells. 4. The method according to claim 3, wherein the immortalized B cells are fusion cells obtained by fusing the B cells with myeloma cells or recombinant myeloma cells. 5. The method according to claim 3, wherein the mammal is a non-human mammal. 6. The method according to claim 3, wherein the mammal is a human. 7. The method according to claim 3, wherein the mammal is a transgenic non-human mammal capable of producing a human antibody. 8. The endogenous immunoglobulin heavy chain gene,
The method according to any one of claims 1 to 4, 6, or 7, wherein the method is a human immunoglobulin heavy chain gene. 9. The endogenous immunoglobulin light chain gene comprises:
8. The method according to claim 1, wherein the gene is a human immunoglobulin light chain gene. 10. The method according to claim 1, wherein the monoclonal antibody is a monoclonal antibody of a non-human mammal. 11. The method according to claim 1, wherein the monoclonal antibody is a human monoclonal antibody. 12. The exogenous DNA according to claim 1, further comprising a gene amplification gene.
The production method according to any one of the above. 13. The method according to claim 12, wherein the gene amplification gene is a dihydrofolate reductase (DHFR) gene. 14. A transformed cell produced by the method according to any one of claims 1 to 13.