JP2832358B2 - Increasing monoclonal antibody sales - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for increasing the yield of a monoclonal antibody (hereinafter sometimes abbreviated as MoAb).

Conventional technology Among MoAbs, human MoAb is a useful animal cell culture product used for diagnosis and treatment of various human diseases in the body, but a method for establishing a human MoAb-producing strain is not always well established. In addition, it is difficult to say that the antibody production ability of the obtained cell line is lower than that of a mouse hybridoma line, etc.
The industrial production process is not always well established.

Recently, it has been reported that, when antibodies are produced by human-human hybridomas, the amount of antibody production increases when the cells can be cultured in a medium supplemented with interleukin-2 [cellular immunolog (cellular immunolog).
y), 115 , 325-33 (1988)]. However, even with this method, it is difficult to say that the amount of antibody produced is sufficient.

Problems to be Solved by the Invention In culturing animal cells, as a means for improving the material productivity, a method of increasing the number of cells per culture solution and a method of improving the material productivity per cell are considered. It is generally preferable to increase the number of cells per culture, but in the production of MoAb, the increase in the number of cells does not always reflect antibody production, and it is necessary to increase the number of cells in a state with high antibody productivity. It is. That is, it is extremely important to improve the productivity per cell.

Means for Solving the Problems In view of the circumstances described above, the present inventors have conducted various studies, and as a result, have found that human interleukin-2 (IL-2) receptor polypeptide can be added to human-human hybridomas in the cells. Introduce the encoding gene into an expressible state,
By culturing in a medium supplemented with IL-2, human MoAb
The present inventors have found that the productivity of the present invention is remarkably improved, and have conducted further studies based on this finding to complete the present invention.

That is, the present invention provides a method for producing a monoclonal antibody by culturing a hybridoma producing a monoclonal antibody in a medium supplemented with interleukin-2, wherein the polypeptide of the interleukin-2 receptor is contained in the cell as the hybridoma. A method for increasing the yield of monoclonal antibodies, characterized in that a gene encoding a gene is introduced into an expressible state.

As the hybridoma producing MoAb used in the method of the present invention, cancer cells or cell lines derived from permanent animals or other animals, and non-permanently capable of antibody production, lymphocytes, polyethylene, A fused cell which has an antibody-producing ability and is capable of permanent growth by being fused by glycol treatment or electroporation is used.

The antibody in the hybridoma producing the monoclonal antibody is not particularly specified.

The human MoAb-producing hybridoma strain used in the method of the present invention includes, for example, a human B lymphoblastoid cell line WIL-
2, WIL-2NS (ATCC CRL8155), TAW-925 (Biochemical B
iophysical Research Communication 142, 805 1987
Year), UC729-6 (Proc. Natl. Acad. Sci. USA 80, 6327, 1)
983), LICR-LON-HMy2 (Eur. J. Immunol. 12, 641)
982), mouse emiroma P3 / NS1 / 1-Ag4-1 (NS-
1) (ATCC TIB 18). Mouse myeloma P3 x 63AgU.1
(P3U1) (ATCC CRL 1597), mouse myeloma SP2 / O
−Ag14 (SP2) (ATCC CRL 1581), rat myeloma
Y3-Ag1.2.3 (ATCC CLR 1631), rat myeloma YB
It is produced by fusing 2 / O (ATCC CRL 1662) or the like with peripheral blood lymphocytes (PBL) or EB virus transformants of PBL. Cell fusion is performed by polyethylene glycol (PEG) treatment or electrofusion according to a conventional method. Examples of the thus obtained human-human hybridoma include an anti-hepatitis B virus surface antigen (HBsA
g) producing human MoAb against W471-7.24 (Biochem. B
iophys.Res.Commun. 142, 805, 1987), HBW-4.16
(Proceedings of the 6th Symposium on Next-Generation Industrial Technology: 67 pages, 1
988), HBW-6.20 (Proceedings of the 6th Next-Generation Industrial Technology Symposium, p. 67, 1988), anti-tetanus toxoid (T.
T.) Human MoAb-producing strains TTW-1.10 and TTW-2.1 (Preprints of the 4th Symposium on Next-Generation Industrial Technology, p. 51, 1986)
And the like.

Interleukin-2 used in the method of the present invention
Examples of the gene encoding the receptor polypeptide include a gene that is expressed on the cell surface and encodes a protein capable of binding to IL-2 and transmitting the signal. IL-2
The receptor is thought to be composed of two types of proteins, p55 and p75 (Metabolism, Volume 25 Special Issue, Immunity '88, No. 41
P.) Include genes encoding either or both proteins.

Examples of the gene encoding the polypeptide of the interleukin-2 receptor used in the present invention include, for example, from No. 1 to 252 in FIG. 2 of JP-A-61-52293.
And a gene that encodes a polypeptide consisting of the amino acid sequence. This gene is obtained as a complementary DNA (cDNA) of messenger RNA (m-RNA) obtained from T cells expressing the IL-2 receptor polypeptide, but can also be obtained by chemical synthesis. Even if they differ slightly at the base sequence and amino acid sequence levels, they can be used in the present invention as long as the resulting polypeptide essentially acts as an IL-2 receptor.

A gene encoding the IL-2 receptor polypeptide is introduced into an expressible state.

It is preferred to introduce the gene into a constitutively expressible state.

To introduce the gene into a constitutively expressible state, the genes encoding the constitutively operable promoter, translation initiation codon (ATG) and the IL-2 receptor polypeptide were arranged in this order. An expression vector is introduced into the hybridoma and transformed.

Examples of the expression vector include pSVL [Mol. Cell. Biol. 4 , 817 (1984)] and pCH110.
[J. Mol. App. Gen. 101 (1983)], [Cell. 39 , 653 (198)
5)], pKSV-10 [Cell. 23 , 175 (1981)], pSV2 [Proc. N
atl.Acad.Sci, USA 78, 2072,1982], pBTV 69T [Methods i
n Enzymol. 101 , 387 (1983)], pHEBo [Mol. Cell. Biol.
5 , 410 (1985)], pZIP-NeoSV [Cell. 37 , 1053 (198
4)], but are not limited to these.

The promoter upstream of the translation initiation codon may be any promoter as long as it is appropriate for the host used for gene expression. For example, SV
(Simian virus) 40 promoter regions [Okayama et al.
Molecular and Cellular Biology, 3
280-289 (1983)] and various retrovirus LTRs.
(Long terminal repeat) region.

Examples of the promoter derived from the retrovirus LTR region include the following.

Eberson mouse leukemia virus (A-MuLV)
[Goff, SP et al., Cell, Vol. 22, pp. 777-785 (1980)], Moloney murine leukemia virus (M-MuLV) [Tamba et al., Cell, Vol. 32, pp. 1105-1113 (1983)], Adult T cell leukemia virus (ATLV) [Yoshida et al., Processing of National Academy of Science USA, Vol. 79, pp. 6899-6902 (1982)], Avian sarcoma virus (ASV) [Kitamura et al., Nature, II.
97, 205-208 (1982)].

In the present invention, one or more promoters described above may be used.

Further, in the expression vector, the -21 in FIG. 2 of JP-A-61-52293 is located upstream of the 5 'end of the above gene.
It may have a base sequence that encodes a signal peptide consisting of the first to -1st codons.

The 3 'end of the gene has a translation termination codon.
It may have TAA, TGA or TAG, with TAG being particularly preferred.

The expression vector used for hybridoma transformation in the present invention may further have an enhancer. Examples of the enhancer include enhancers derived from viruses, for example, an enhancer present in the SV40 promoter region [Okayama et al., Supra] and an enhancer present in the retrovirus LTR region. Enhancers are preferred.

Examples of retrovirus-derived enhancers include the following.

 A-MuLV [Cobb, S.P. et al., Supra], M-MuLV [Tamba et al., Supra], ATLV [Yoshida et al., Supra], ASV [Kitamura et al., Supra].

In the present invention, one or more enhancers described above may be used.

Transformation can be performed, for example, by co-transformation [Cell, 16 : 7
77 (1979)] and the like, and a desired animal cell transformant can be advantageously selected and separated (cloned).
That is, the gene (A) that serves as a marker for selecting a transformant
(Eg, a gene that inactivates an antibiotic such as an ampicillin-resistant or neomycin-resistant gene) and a plasmid that carries the gene (B) to be introduced, using a larger amount of (B) than (A). Cells are simultaneously introduced by electroporation or the like. When a clone that grows by culturing in the presence of a resistance drug of the gene (A) and is selected is selected, the gene (A)
And a strain having the gene (B) at the same time.

The method for producing a human MoAb of the present invention can be carried out by culturing the above-mentioned transformed hybridoma in a medium supplemented with IL-2, allowing the antibody protein to secrete and accumulate in the medium, and collecting the antibody protein.

IL-2 used in the method of the present invention includes IL-
A substance having an activity similar to that of 2, that is, a substance having an action capable of maintaining T cells for a passage while maintaining their functions. Specifically, for example, a polypeptide (I) (human IL-2) having the amino acid sequence shown in FIG. 1 of JP-A-60-115528, and a polypeptide required for its biological or immunological activity It may be a polypeptide consisting of a partial amino acid sequence. As the polypeptide, for example, one amino acid (EP
C Publication No. 91539), those lacking four amino acids (Japanese Patent Application Laid-Open No. 60-126088), those lacking several amino acids at the carboxyl terminal, and the like. Furthermore, the first
Polypeptide (I) having the amino acid sequence shown in the figure
May be partially deleted or replaced with another amino acid, for example, a cysteine residue at position 125 replaced with a serine residue [Japanese Patent Laid-Open No. 59-93093].

In particular, in the present invention, it is preferable to use human IL-2 having the same amino acid sequence as the polypeptide (I). In this case, it is preferable to use a human IL-2 having or not having a methionine residue (Met) at its amino terminus. A mixture [Japanese Unexamined Patent Publication No. Sho 60-115528, Japanese Unexamined Patent Publication No. Sho 61-78799] may be used.
Those starting with a) [JP-A-61-78799] may be used.

 Further, those having a sugar chain may be used.

For culturing the hybridoma strain, a medium for animal cells known per se, for example, a medium (IsF medium) obtained by adding 1 to 20% fetal calf serum to an equal mixture of Iscove-modified Dulbecco medium and Ham F12 medium, Iscove-modified Dulbecco medium 0.1 to 100 μg / ml insulin, 0.1 to 200 μg / ml transferrin, 0.1 to 20 mM in a 1: 1: 2 mixture of ham F12 medium and L-15 medium.
ITL-2 medium supplemented with ethanolamine and 1-10 × 10 ^-8 M selenous acid, and a medium (GITL- medium) supplemented with 0.1-5 mg / ml GFS (55-70% ammonium sulfate precipitation fraction of bovine serum).
2 medium). About 5-500 ng /
ml, more preferably about 50-2500 ng / ml IL-2,
After seeding the cells, they are cultured at about 30 to 40 ° C, more preferably at about 34 to 38 ° C, for about 2 to 10 days, more preferably for about 4 to 7 days.

As the culture vessel, a culture flask, a spinner flask, a culture tank, or the like is appropriately selected and used, but immobilized culture using hollow fibers, ceramic cores, or the like is also applied as necessary. In the case of perfusion culture, it is possible to continuously culture for several months while renewing the medium, and to collect the product.

The human MoAb produced and accumulated in the medium can be purified and isolated from the culture obtained after removing the cells by appropriately combining known separation and purification methods.

These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly for differences in molecular weight. , Methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as reversed-phase high-performance liquid chromatography, A method utilizing the difference between isoelectric points, such as isoelectric point electrophoresis, may be used.

The human MoAb produced according to the present invention has the same physiological activity as the MoAb produced by a known method, and can be used as a pharmaceutical or the like.

The anti-HBsAg antibody obtained in the examples described below has the same biological activity as a known anti-HBsAg antibody produced using the blood of an HBV infected person as a raw material, and prevents the HB virus infection in the same manner as the anti-HBsAg antibody. It can be used as a therapeutic agent and as a material of a diagnostic kit (for example, an antibody for detecting HBsAg small particles).

When a base or the like is indicated by an abbreviation in the present specification,
IUPAC-IUB Commission on Biochemical Nomenclature
Or abbreviations used in the art, examples of which are given below.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid The transformed human hybridoma HBW-4.16-459-6 strain obtained in Example 1 of the latter method is Deposited with the Fermentation Research Institute (IFO) on March 24, 2001 under the accession number IFO 50186. In addition, the hybridoma strain was sent to the Research Institute of Microbial Industry and Technology (FRI) of the Ministry of International Trade and Industry of Japan in March 2001.
It has been deposited as a deposit based on the Budapest Treaty as accession number FERM BP-2366 from March 30.

Examples The present invention will be described more specifically with reference to the following reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 Production of IL-2 Receptor Gene Expression Plasmid Plasmid pTB271 obtained in Example 1 (iii) of JP-A-61-63282 was digested with restriction enzymes BamH I and Cla I to obtain a 2.56 kb DNA. Fragment () was isolated. The same plasmid was digested with restriction enzymes Pst I and Cla I, and a 1.6 kb D
The NA fragment () was isolated.

On the other hand, the plasmid Tac-2 having the IL-2 receptor gene obtained in Example 1 (vii) of JP-A-61-52293 was digested with restriction enzymes HindIII and BamHI (at the BamHI cleavage site). Is described in Plasmid Tac-2 in JP-A-61-52293.
It is located downstream near the restriction map shown in FIG. ), A 1.8 kb DNA fragment was isolated and further partially digested with PstI to obtain a 1.3 kb DNA fragment (). these,
, And the three DNA fragments were connected by T4 DNA ligase to
An expression plasmid pTB459 containing the two receptor gene was obtained (see FIG. 1).

Example 1 i) Introduction of IL-2 receptor gene into human-human hybridoma Anti-HBsAg human MoAb producing human-human hybridoma strain HBW
-Suspended in a 0.3 M sucrose solution (sterile) of -4.16 at a rate of 1 x 10 ⁷ cells / ml, 100 µ of the suspension was treated with 25 µg of the plasmid pTB459 obtained in Reference Example 1 treated with the restriction enzyme Cla I,
Plasmid pRSVneo [Science, 221 551
P. 1983] treated with the restriction enzyme BamHI and 5 μg of the mixture. This mixed solution was put into a centrifugal chamber of an electroporation device manufactured by DEP, centrifuged at 2,000 rpm for 2 minutes, and then once applied with a voltage of 1000 V / 3 mm for 30 seconds and left in ice water for 10 minutes. Antibiotic G418
(Sigma, USA) at 1 × 10 ⁵ cells / ml in IsF medium supplemented with 1 mg / ml, and electroporated cells were suspended in a 96-well multiplate.
Each of the cells was inoculated, and cultured for 2 weeks in a 37 ° C. carbon dioxide incubator to select clones that proliferate.

ii) Detection of transgene Cells (1 × 10 ⁶ ) were prepared by adding 10 mM Tris-HCl buffer (pH 7.
4) Suspend in 200μ, add an equal volume of 0.4M Tris-HCl buffer (pH 8.0), 100mM EDTA, 1% SDS, 200μg / ml protease K (BRL, USA) and treat at 60 ° C for 1 hour did. An equal amount of phenol / chloroform is added, and the supernatant is collected.
Further, an equal volume of chloroform was added, and the supernatant was again collected. 0.1 volume of 3M NaOH is added to the obtained DNA solution, and
After treating for an hour, the mixture was cooled to room temperature and neutralized by adding an equal amount of 2M-ammonium acetate. Adsorbed to a nitrocellulose filter pre-moistened with 1M ammonium acetate using a Bio-Rad dot blotting device,
Dry at 80 ° C. for 2 hours.

On the other hand, the plasmid pTB459 was treated with the restriction enzyme BamHI, and then subjected to agarose gel electrophoresis to cut out a band containing the Tac gene. Ultrafree C3HV (Millipore, USA) and purified using oligo labeling kit (manufactured by Pharmacia, Sweden) using a ³² P-dCTP
And by random priming.

Nitrocellulose filter and prehybridization solution [50% formamide, 5
× SSC, 5 × Denhardt's solution, 50mM NaHPO ₄ (pH6.5), 250ng / m
l single-stranded DNA] and incubated at 42 ° C for 8 hours. Next, the solution was subjected to a hybridization solution [50% formamide, 5 × SSC, 1 × Denhardt's solution, 20 mM NaHPO ₄ (pH 6.
5), 10% dextran sulfate, 100 μg / ml single-stranded DNA, heat-treated probe] and incubated at 42 ° C. for 15 hours. The filter was washed twice with 2 × SSC, 0.1% SDS for 10 minutes and then twice with the same buffer heated to 68 ° C. for another 20 minutes. After washing the filter twice with 2 × SSC, the water was removed with paper and subjected to autoradiography (−70 ° C., 2 days).

Note: As a result of autoradiography, human hybridoma HBW-4.16-459-6 giving a clearly stronger autoradiogram as compared to control cells into which plasmid was not introduced, as a result of SSC 0.15 M salt, 15 mM sodium citrate (pH 7.0)
(IFO 50186, FERM BP-2366) strain, HBW-4.16-459-25
And the HBW-4.16-459-66 strain were selected.

iii) Production of MoAb The three types of clones obtained in ii) were seeded at a rate of 105 cells / ml in a medium without addition of recombinant human IL-2 and in a medium in which 2 μg / ml was added to IsF medium, respectively. The cells were cultured in a 24-well multiwell plate in a 5% carbon dioxide incubator at 37 ° C. for 4 days. The number of cells in the obtained culture solution and the amount of antibody production were compared, and the results shown in Table 1 were obtained.

Example 2 (Effect of addition of IL-2) Cloned human hybridoma HBW- obtained in Example 1
4.16-459-6 (IFO 501 86, FERM BP-2366)
2 medium and various concentrations of IL-2 were added to the medium at a rate of 1 × 10 ⁴ cells / ml, and incubated at 35 ° C. in a 5% -carbon dioxide incubator.
For 7 days, the results shown in Table 2 were obtained.

Example 3 (Effect of addition of IL-2) Cloned human hybridoma HBW- obtained in Example 1
4.16-459-6 (IFO 501 86, FERM BP-1366)
ITL-2 medium (A) and 1 μg / ml human IL-
2 were added to the medium (B) at a rate of 1 × 10 ⁵ cells / ml and cultured at 35 ° C. for 5 days. After completion of the culture, the cells are removed by centrifugation, and the resulting supernatant is purified using
It was concentrated to 300 ml. Ammonium sulfate (ammonium sulfate) was added to the mixture so as to be 37% saturated, and the mixture was allowed to stand for 1 hour, followed by removing the precipitate by centrifugation. Add more ammonium sulfate to the supernatant and add 50
The precipitate formed after% saturation was collected by centrifugation. After dissolving in water, dialyzed against 10 mM Na ₂ HPO ₄ (pH 8.0)
The solution was passed through a DE-52 column equilibrated with the same buffer, and the flow-through fraction was collected. Next, the protein A (Seikagaku Corporation, Japan) column equilibrated with 50 mM Tris-HCl buffer (pH 8.6) containing 0.15 M salt was adsorbed, and 50 mM containing 0.15 M salt was added.
After washing with M sodium acetate buffer (pH 6.0), elution was carried out with the same buffer at pH 30, and the active fraction was collected. 0.15
It was dialyzed against a phosphate buffer (pH 7.2) containing M salt to obtain a purified sample. The amount of anti-HBsAg antibody in the purified sample was 11.8 mg in the medium (A), whereas IL-2 was added in the medium (B).
76.2 mg of the same antibody was obtained from the medium. No difference was observed between the obtained antibodies in physicochemical properties and biological activity.

Effect of the Invention Since the method of the present invention can improve the antibody-producing ability of a monoclonal antibody-producing strain, the method of the present invention can be advantageously used when industrially mass-producing a monoclonal antibody.

[Brief description of the drawings]

FIG. 1 shows a construction diagram of plasmid pTB459 obtained in Reference Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (1)

(57) [Claims] 1. A method for producing a monoclonal antibody by culturing a hybridoma producing a monoclonal antibody in a medium supplemented with interleukin-2, wherein the polypeptide of the interleukin-2 receptor is introduced into the cell as the hybridoma. A method for increasing the yield of a monoclonal antibody, comprising using a gene in which an encoded gene has been introduced so that it can be expressed.