JPH04360696A - Recombinant human antibody produced with new human b lymphocyte strain as host - Google Patents

Abstract

PURPOSE:To provide a new antibody-nonproductive cell strain to be used for producing IgM or IgA antibody containing human J-chain protein. CONSTITUTION:The objective antibody-nonproductive cell strain, which does not produce endogenous antibody and H-chain and L-chain as its constituents, obtained by introducing exogenous antibody gene into namaruba cell strain assimilated to a serumless medium. Specifically, antibody-nonproductive mutant cell strain is first obtained, by X-ray irradiation or using a mutating chemical, from the namaruba cells (ATCC CRL 1432) of human B lymphocyte strain, pref. NKG4 strain, and said mutant cell strain is put to cloning using a serumless medium to enable said strain to be cultured in such serumless medium, thus obtaining the objective antibody-nonproductive cell strain sensitive to amidoglycoside-based antibiotic G-418 or mycophenolic acid.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a novel host cell line used for the production of antigen-specific human monoclonal antibodies using genetic engineering techniques, and the characteristics obtained by introducing foreign antibody genes into the cell line. The present invention relates to a transformed strain and a method for producing a recombinant human monoclonal antibody by culturing the transformed strain. More specifically, the present invention relates to a cell line derived from a human B lymphoid cell line that cannot produce or does not secrete its own antibodies and can be cultured in a serum-free medium, and a cell line using the mutant cell line as a host. The present invention relates to a transformed strain obtained by introducing an antibody gene using a DNA introduction method, and a recombinant human monoclonal antibody produced by the transformed strain.

0002

BACKGROUND OF THE INVENTION Monoclonal antibodies are widely used industrially in the medical field for diagnosis and therapeutic applications, and in the industrial field as research reagents and affinity column substrates. Among them, mouse monoclonal antibodies are widely used because they can be easily produced by the mouse/mouse hybridoma method. However, despite the various improvements that have been made to date regarding human monoclonal antibodies, which have high pharmaceutical value for use in human therapy, there is no method for establishing production strains that can be produced efficiently and with high reproducibility over a wide range of areas. However, this has not been established. One reason is that
This is due to the unavailability of suitable cells as fusion partners for hybridoma methods. Several human lymphoid cell lines are known as fusion partners that can be used to create human/human hybridomas. but,
All of these human lymphoid cell lines produce antibodies, H chains, or L chains. If the human lymphoid cell line that is the fusion partner itself produces antibodies, H chains, or L chains derived from that cell, the hybridoma obtained by cell fusion with B lymphocytes that produce the desired antibody will contain both cells. The H chain and L chain derived from the antibody are combined to form a complex hybrid antibody, resulting in a loss of antibody unity and a decrease in antibody binding ability. Furthermore, the cell efficiency of these human lymphoid cell lines and antibody-producing lymphocytes is extremely low, making it extremely difficult to establish specific antibody-producing cell lines. Therefore, in order to obtain human monoclonal antibodies, it is common to establish a human/mouse hybridoma by fusing a mouse lymphoid cell line with a human-derived antibody-producing lymphocyte. However, in human/mouse hybridomas, differences in the sugar chain structure added to the antibody, the J chain between humans and mice, and the presence of mouse-derived contaminants pose problems when producing human antibodies and using them industrially. Become. That is, differences in sugar chain molecules cause changes in the biological activity of antibodies and dynamics within the body, or cause allergies. Furthermore, the presence of mouse-derived proteins makes purification difficult in the production of pharmaceuticals that can be administered to humans. Because of these problems, methods have been developed to obtain fully human monoclonal antibodies by cloning antibody genes from hybridomas and introducing them into human cells.

0003

[Problem to be solved by the invention] However, using this method,
Even when genes are cloned from human/mouse hybridomas and introduced into human cells, the same problems as with hybridomas occur when human lymphoid cells are used as hosts. That is, when human lymphoid cells producing endogenous antibodies are used as hosts, endogenous antibodies, H chains or L
As hybrid antibodies with the chains are formed, antibody unity is lost, resulting in decreased antibody binding ability, similar to human/mouse hybridomas.

0004

[Means for solving the problem] Therefore, the present inventors
For the purpose of obtaining lymphoid cell lines that do not produce endogenous antibodies, H chains, or L chains, human B lymphoid cell lines are treated with mutagenic drugs or through mutagenic treatments such as X-ray irradiation. succeeded in establishing a mutant cell line that does not produce antibodies. Furthermore, by cloning the obtained non-antibody-producing cell line using a serum-free medium, they succeeded in establishing a non-antibody-producing cell line that was adapted to a serum-free medium.

[0005]

Effects of the Invention By using this non-antibody-producing cell line as a host, introducing and expressing a foreign antibody gene, we succeeded in producing a single antibody without interference from endogenous antibodies. Since the antibody is a single specific antibody, the antigen binding ability is not decreased. In addition, by using the non-antibody-producing human B lymphoid cell line of the present invention as a host cell, IgM containing human J chain protein that is non-antigenic to humans,
It has become possible to produce IgA antibodies or antibodies with natural sugar chains that are non-antigenic to humans. Furthermore, by using the non-antibody-producing human cell line of the present invention as a host cell, it has become possible to reduce impurities derived from a foreign animal, which is a problem when using other animal cells. In addition, the non-antibody-producing human B lymphoid cell line of the present invention can be cultured in a serum-free medium and can be cultured in large quantities. It has become possible to simplify the legal process.

The present invention will be described in more detail below. The present inventor established a non-antibody-producing mutant cell line from a human B lymphoid cell line by treatment with a mutant drug or by X-ray irradiation, and then cloned it into a serum-free medium using a serum-free medium. An adapted non-antibody producing cell line was established. The established cell line has advantages for antibody gene expression as described below. That is, i) it does not produce endogenous H chains and L chains, which are the main components of antibodies. ii) Produces J chain protein, which is a component of IgM and IgA antibodies. iii) Sensitive to amide glycoside antibiotic G-418 or mycophenolic acid. iv) Can be cultured in serum-free medium.

[0007] By introducing and expressing foreign antibody genes using such non-antibody-producing cell lines as hosts, it has become possible to obtain antibodies that have high industrial utility value and can be administered to humans. Ta. The terms used in this specification are defined below. The term cell line refers to culture cell lines with unique characteristics or genetic markers that have been isolated by selection or cloning.
line). In addition, cell lines that do not produce antibodies and their components, H chains and L chains, are cases in which the cell line does not produce antibodies and their components, H chains and L chains themselves, and cases in which the cell line does not produce antibodies and its components, H chains and L chains themselves, and cases in which the cell line does produce them but does not use them in the culture medium. This includes both cases where it is not secreted. A fully human antibody means that the H chain, L chain, and J chain (in the case of IgM and IgA), which are the components of the antibody, are all proteins derived from human genes. In addition to the above-mentioned fully human antibodies, human antibodies include the variable region of the H chain and L chain (
There are so-called chimeric antibodies in which the V region (V region) is derived from a mouse and the constant region (C region) is derived from a human, while in the V region, only the CDR region directly involved in antigen binding is derived from a mouse, and the FR region and C region in the V region are derived from a mouse. It refers to antibodies including so-called anthropomorphic (humanized) antibodies derived from humans.

The present inventors first established a non-antibody-producing mutant cell line from a human B lymphoid cell line by treatment with a mutant drug or by X-ray irradiation. The human B lymphoid cell line used is Namalva cell (ATCC CRL 1
432 ) (with IgM (μ, λ) on the cell surface) can be used. As a mutagen, for example, nitrosoguanidine (NTG), ethyl methanesulfonic acid (E
MS), ICR-191 (Wako Pure Chemical Industries), 4-nitroquinoline N oxide (4NQO), etc., but NTG or EMS is preferable. As a treatment for carrying out mutations, a medium for animal cell culture, usually R containing 10% fetal bovine serum,
1-3μg/ml using PMI1640 or MEM medium
of NTG-containing medium for 1 hour or 100-800μ
g/ml EMS-containing medium for 24 hours, 5% CO2.
, and culture at 37°C. Next, it was cultured for 1 to 5 days in a medium containing no mutagen, and the soft agar method (Immunology Experimental Procedures (Japanese Society of Immunology) p.
974-1, 2218) or the limiting dilution method (Immunology Experimental Procedures (Japan Society of Immunology) p. 2226, 2218) to clone a cell line that does not produce antibodies. 8 more
Azaguanine (8AG), 6-thioguanine (6TG),
10-25 in a medium supplemented with drugs such as 5 bromouracil.
HGPRT or TK-deficient strains can be obtained by culturing for days. At this time, it is confirmed by enzyme antibody assay (ELISA), Western blotting, etc. that the obtained non-antibody-producing mutant cell line does not produce human antibodies, H chains, or L chains.

[0009] Thereafter, the obtained antibody-nonproducing mutant cell line is cloned using a serum-free medium to establish a cell line adapted to the serum-free medium. Serum-free media used include Cell Grosser H (Meguro Institute), ASF (Ajinomoto), and S.
FM-101 (Nissui), HB101 (Fujirebio)
are mentioned, but Cell Grosser H is preferred. By the above method, it is possible to obtain human B lymphocytes that do not produce endogenous antibodies, H chains or L chains and can be cultured in a serum-free medium. NK derived from Namalva cells (ATCC CRL1432)
A preferred example is the G4 strain. The NKG4 strain is characterized by having the following traits i) to v). i) Does not produce antibodies. ii) Produces J chain protein, which is a component of antibodies. iii) 8AG resistant. iv) Sensitive to amide glycoside antibiotic G-418 or mycophenolic acid. v) Can be cultured in serum-free medium Cell Grosser H.

[0010] Using the above-mentioned non-antibody-producing human B lymphoid cell line as a host, a fully human antibody-producing cell line can be obtained by expressing a human-specific antibody gene by a conventional DNA introduction method. First, antibody genes are cloned from human/mouse hybridomas using standard genetic engineering methods (Molecular Cl
oning, Cold Spring Harbo
r Laboratory Press1989)
. In other words, genomic DNA is extracted from monoclonal antibody-producing cells.
Prepare A, human genomic DNA is digested with various restriction enzymes, transferred to a filter, and Southern Hybridization is performed using the constant region or J region of the antibody gene as a probe.
The size of each restriction enzyme fragment of the specific antibody gene of the hybridoma is determined by the cation method. After collecting fragments of a specific size and concentrating the antibody genes by low melting point agarose gel electrophoresis, the genomic DNA is extracted from EMBL3, EM
Combine with phage vectors such as BL4, λL47, and λOngC to form phage particles (in vitro
o Packaging (A. Becker and
M. Gold. Proc. Natl. Aca
d. Sci. U. S. A. Volume 72, 581 (1975
)) Create a gene library by infecting E. coli. Plaque hybridization method (WD) using human antibody J gene or C gene (JCRB) as a probe.
Benton and R. W. Daris.
Science. 196, Vol. 180 (1977)), the target gene is obtained and its sequence is determined.

[0011] Antibody genes can be obtained from known hybridomas, but are preferably obtained from hybridomas directed against various antigens of Pseudomonas aeruginosa. In addition, hybridoma HI223 (Feikokenjoyori 3rd
No. 213), hybridoma IN2A8 (Feikoken Article No. 2741) and MH4H7 (Feikoken Article No. 2402)
Preferably, the gene encoding the following amino acid sequence obtained from No.

[0012] H chain Sequence number 1 and

[0013]L chain Sequence number 2 or

[0014]H chain Sequence number 3 and

[0015]L chain Sequence number 4 or

[0016] H chain Sequence number 5 and

[0017] L chain Sequence number 6

[0018] The thus obtained foreign antibody gene is ligated to an expression vector in animal cells to construct an expression plasmid. At that time, pSV
2dhfr (S. Subramani et al.,
Mol. Cell. Biol. Volume 1, 854 (198
1)), pSV2neo (P.J. Southern
&P. Berg. J. Mol. Appl. Genet
．． , vol. 1, 327 (1982)), pSV2gp
t(R.C.Mulligan & P.Berg.S
science. 209, 1422 (1980) is possible. A method for introducing the constructed expression plasmid into the non-antibody producing cell line of the present invention is the red blood cell ghost method (Proc. Natl. Acid. Sci.,
77, 2163 (1980)), DEAE-dextran method (Nature, 293, 79 (1981))
, Calcium phosphate method (Virology, 52, 4
56 (1973)), protoplast fusion method (Mol
ecular and cellular biolo
gy, 743 (1981)), electroporation method (EMBO J., 1, 841 (1982)),
Lipofection method (Proc. Natl. Acid.
Sci. , 84, 7413 (1987)). Enzyme-linked immunosorbent assay (ELISA) (Immunology Experimental Procedures (Japan Society of Immunology) p. 1137, 3165) is used as an effective means for analyzing antibodies produced by transformed strains. The antibody amount can be determined by ELISA using a plate adsorbed with an antibody against the constant region, the antigen binding activity can be determined by ELISA using a plate adsorbed with antigen, and the specific activity of the antibody can be calculated. In the conventional method of producing antibodies using human B lymphocytes as hosts,
When a hybrid antibody was produced between an antibody derived from a host cell and a specific antibody derived from a foreign gene, the specific activity of the antibody decreased. Therefore, in the present invention, human /
As a result of calculating the specific activity of the antibody produced by the recombinant by setting the specific activity of the antibody produced by the mouse hybridoma to 1, they succeeded in obtaining an antibody whose biological activity does not decrease.

[0019]

[Examples] The present invention will be explained in more detail with reference to Examples below, but it goes without saying that the present invention is not limited to these specific examples. Example 1 Establishment of mutant cell line derived from human B lymphoid cell line The present invention was carried out to detect IgM (μ
The amount of chain) was measured. Anti-human IgM (μ chain) antibody (Kappel Catalog No. 0201-020
2) in phosphate buffer (pH 7.2, composition NaCl (8g
/l), KCl (0.2g/l), NaHPO4 12
H2O (2.99g/l) and KH2PO4 (
0.2g/l)) (hereinafter abbreviated as PBS) to 10μg/m
The solution was dissolved at a concentration of 100 μl per well in a 96-well microplate (Falcon #3912) (hereinafter abbreviated as microplate), and kept at 37° C. for 2 hours. 1% bovine serum albumin (BSA)
The unadsorbed portion of the microplate was blocked by dispensing 120 μl of the PBS solution containing the microplate (abbreviated as ) per well and incubating it at 37° C. for 2 hours. The sample for which you want to measure the amount of antibody is mixed with PBS containing 0.05% Tween20 (
Appropriately diluted with PBST (hereinafter abbreviated as PBST) at 50% per well.
μl was added and kept at 37°C for 2 hours. The sample was then removed and washed three times with PBST. Next, add 100 μl of the second antibody solution per well, and
It was kept warm at ℃ for 2 hours. 500 in PBST as the second antibody.
~1,000-fold diluted phosphatase-labeled affinity-purified anti-human immunoglobulin μ chain antibody (Kirkega
ard&PerryLab. Inc. ) was used. Second
After removing the antibody and washing 3 times with PBST, add the chromogenic substrate solution (3
mg of P-nitrophenyl phosphate-2-sodium salt to 1 ml of NaN3 (0.2 mg/ml) MgCl2
・Aqueous solution dissolved in 10% diethanolamine buffer pH 9.1 containing 6H2O (0.1 mg/ml)
100 μl was added per well and reacted at 37°C. Multi-scan (Titer) OD405 after reaction
tek).

[0020] Furthermore, the amount of λ chain was measured in the same manner as the amount of IgM (μ chain) described above. Anti-human λ chain antibody (Kappel Catalog No. 0201-028
1) was dissolved in PBS to a concentration of 10 μg/ml, and dispensed into a microplate at 100 μl per well.
It was kept warm at 37°C for 2 hours. The unadsorbed portion of the microplate was blocked by dispensing 120 μl of 1% BSA-PBS solution per well and incubating at 37° C. for 2 hours. A sample whose antibody amount was to be measured was appropriately diluted with PBST, 50 μl was added per well, and the mixture was incubated at 37° C. for 2 hours. The sample was then removed and washed three times with PBST. Subsequently, 100 μl of the second antibody solution was added to each well, and the mixture was incubated at 37° C. for 2 hours. PB as second antibody
Phosphatase-labeled affinity-purified anti-human immunoglobulin λ chain antibody (K
irkegaard & PerryLab. Inc.) was used. After removing the second antibody and washing three times with PBST, I
The chromogenic substrate solution used for gM quantification was added at 10 gM per well.
0 μl each was added and reacted at 37°C. OD4 after reaction
05 was measured using multiscan.

(1) Establishment of Namalva cell-derived non-antibody-producing strain Namalva cells (ATCC CRL 1432) were
Using RPMI1640 (Nissui Pharmaceutical) medium containing 0% fetal bovine serum (Hyclone) (hereinafter abbreviated as FCS),
% CO2 at 37°C, then 240 μg/
ml of ethyl methanesulfonic acid (Hani Chemicals) (EM
The cells were cultured for 3 days in the above-mentioned medium containing (abbreviated as S). Then E
The cells were cultured in a medium containing no MS for 3 days, and antibody-producing cloning was performed by the agarose method or limiting dilution method. Six clones (N2D1, N2G5, NPA10, NPD
2, NPE3, NCG7), and then 8AG (8-
Azaguanine, Wako Pure Chemical) treatment was performed as follows. 1000 cells/well in one 96-well plate
Each non-antibody producing strain was inoculated at a ratio of 8A every 3 days.
By replacing half the volume with G-added medium, the 8AG concentration was increased from 0 to 5.
The concentration was gradually increased to →7.5 → 14 → 16 μg/ml and cultured for 14 days, and 5 strains were obtained from NPA10. The results of measuring μ chain and lambda chain for each culture supernatant of the 5 clones are summarized in the following table.

(2) Establishment of cell line NKG4 adapted to serum-free medium The NPA10C5 strain established in Example 1-(1) was cloned in order to acclimatize it to serum-free medium. For cloning, cells were suspended in serum-free medium Cell Grosser H (Meguro Institute) and seeded in a 96-well plate, and cell lines that proliferated in serum-free medium were selectively selected and NPA10C
5G4 strain (hereinafter abbreviated as NKG4 strain) was obtained.

Example 2 Cloning of antibody gene and construction of expression plasmid (1) Cloning of HI223 antibody gene (1-1)
Isolation of HI223 genomic DNA Crush human anti-Pseudomonas aeruginosa antibody-producing cells HI223 (108 cells) with a glass rod,
mM Tris-HCl (pH 8.0)/10mM EDTA
The cells were suspended in a buffer containing /10mM NaCl. Proteinase K (Boehringer Mannheim) 2
00 μg/ml, SDS 0.5%, 9 at 37°C.
After incubating for 0 minutes, an equal amount of water-saturated phenol was added, and after stirring, the phenol phase and the aqueous phase were separated by centrifugation. The aqueous phase was removed and 2 volumes of cold ethanol were added. The interface was slowly stirred with a glass rod, and the precipitated DNA was wound up around the glass rod. After drying the DNA under reduced pressure, TE buffer (10mM
Tris-HCl pH 8.0/1mM EDTA) 3m
1, ribonuclease A (Sigma) was added to give a concentration of 50 μg/ml, and the mixture was incubated at 37° C. for 30 minutes. An equal volume of a 1:1 mixture of phenol and chloroform was added, and after stirring, the phases were separated by centrifugation to obtain an aqueous phase. 2 to this
of cold ethanol, and use the glass rod as before to DN.
I wound up A. After drying under reduced pressure and suspending in 2 ml of TE buffer, approximately 600 μg of human genomic DNA was obtained.

(1-2) Preparation of HI223 gene library Example 2 - Human genomic DNA 10 obtained in (1-1)
μg was incubated with 150 units of restriction enzyme SacI at 37°C for 2 hours, and then further treated at 65°C for 5 minutes to stop the reaction and obtain a complete decomposition product.
-7kb fragment was recovered. In addition, human genome DN
TA buffer (33mM Tris acetate (pH 7.9)/66mM potassium acetate/10mM
Magnesium/0.5mM DTT) medium restriction enzyme Hind
50 units of III were added, kept at 37°C for 2 hours, and further treated at 65°C for 5 minutes to obtain a complete decomposition product. After recovering a 4 to 5 kb fragment by low melting point agarose gel electrophoresis, the 5' overhang was filled in by 2 bases by polymerase I (Takara Shuzo) reaction in the presence of dATP and dGTP. These were mixed with 10mM Tris-HCl pH 8.0/0.1mM
After treatment with 2 units of bovine small intestine alkaline phosphatase in EDTA at 37°C for 30 minutes, phenol extraction and HI
223 genomic DNA was recovered by ethanol precipitation. The λOngC vector for H chain gene cloning was decomposed in advance by incubating 10 μg of it in TA buffer with 100 units of restriction enzyme SacI at 37°C for 2 hours, and then recovering it by phenol extraction and ethanol precipitation. Genomic DNA (5-7kbSac
I fragment). For L chain gene cloning, 10 μg of the λOngC vector was digested in TA buffer with 100 units of restriction enzyme
The 5' overhang was filled in by 2 bases with polymerase I in the presence of TTP, and the 4-5 kb HI-223 genomic DNA
Connected with. For the ligation reaction, 2 μg of vector DNA,
Genomic DNA degradation product 1 μg, T4 DNA ligase 5
66mM Tris-HCl (pH 7.6) containing 00 units/
6.6mM MgCl2/10mM DTT/1mMAT
The reaction was carried out by reacting 100 μl of a solution of P at 16°C for 16 hours. Then, the obtained DNA was in
Vitro Packaging (A. Becker
and M. Gold. Proc. Natl
．． Acad. Sci. U. S. A. Volume 72, 581 (
(1975)) to obtain a human gene library. (1-3) Screening of HI-223μ chain gene Using the SacIλOngC library obtained in Example 2-(1-2), 530,000 plaques were formed using Escherichia coli LE392 (JCRB) as an indicator bacteria. . The phage on this plaque were transferred to a nitrocellulose filter by replica, and then the plaque hybridization method (W.D. Benton and R.W.
Daris. Science. 196 Volume 180 (19
77) Human antibody genes were screened by the following method. Human antibody JH gene and Cμ gene (JCR
B) was labeled with 32P using a random priming kit (Amersham) and then subjected to plaque hybridization. SacIλOngC library 53
Twenty-five phage clones hybridizing with the human antibody JH gene probe were obtained from the ten million phages.

(1-4) Screening of HI-223λ chain gene Example 2 - HindIII-λ obtained in (1-2)
Escherichia coli LE392 (JC
RB) was used as an indicator bacterium to form 1.5 million plaques. Screening was performed using the human Cλ2 probe by the plaque hybridization method shown in Example 2-(1-3), and 80 phage clones that hybridized with the probe were obtained.

(1-5) Analysis of phage clones containing antibody genes A phage lysate was prepared from the phage clones obtained in Example 2-(1-3, 4), and phage DNA
(T. Maniatis .EF Fri
tsh and J. Sambrook. “
"Molecular Cloning" (1982)
Cold Spring Harbor Lab
oratory). Phage DNA 0.5 μg
Add 5 units of restriction enzyme per tube, add 3 units of restriction enzyme in TA buffer
After being decomposed by keeping it warm at 7℃ for 2 hours, it becomes 0.8 to 2.4.
The DNA degradation product was separated by electrophoresis using % agarose. Thereafter, the DNA was stained by immersion in 1 μg/ml ethidium bromide and then irradiated with ultraviolet rays to observe the fluorescence of the DNA-ethidium bromide complex. By comparing the mobility with the λDNAHindIII decomposition product (Takara Shuzo) that was electrophoresed at the same time, we determined the length of the decomposition product (in kilobase units: k
b) was determined. Furthermore, the cleavage sites of various restriction enzymes were similarly determined using various restriction enzymes, and a so-called restriction enzyme map was created. In addition, the electrophoresed various restriction enzyme cleavage fragments were transferred to a nitrocellulose filter and subjected to Southern hybridization (E. Southern. J.M.
ol. Biol, Vol. 98, 503 (1975))
I did it. Using the human JH gene, human Cμ, or human Cλ gene as a probe labeled with 32P,
The V region and C region in the obtained phage clone were identified. (1-6) Determination of the base sequence of the HI-223 antibody gene The base sequences of the λ chain and μ chain V region genes were determined as follows. First, EcoRI-H containing the λ chain V region
Cloning the indIII (both vector-derived restriction enzyme sites) fragment (Figure 1 (B)) into the vector pUC
19 (C.Yanisch-Perron.J.Vi
eira. and J. Messing. Gen
e, vol. 33, 103 (1985); Takara Shuzo) H
The SacI fragment containing the μ chain V region (Fig. 1(A)) was inserted into the SacI and EcoRI sites of pUC19.
subcloned to the site. That is, 100 units of restriction enzymes HindIII and EcoRI were added to 2 μg of DNA of a phage clone containing the λ chain gene, and 100 units of restriction enzyme SacI were added to 2 μg of DNA of a phage clone containing the μ chain gene, and 20 μl of TA was added.
It was incubated in a buffer at 37°C for 2 hours for decomposition. 6
After inactivating the enzyme by treating at 5° C. for 5 minutes, DNA was collected by ethanol precipitation and suspended in 10 μl TE buffer. 0.5 μg of pUC19 DNA was added to E
coRI, HindIII 5 units each, or Sa
It was decomposed with 10 units of cI under the same conditions as above and recovered by ethanol precipitation. This pUC19 DNA was mixed with 0.2 unit of bovine small intestine alkaline phosphotase in Example 2-(1-2
), followed by phenol extraction, ethanol precipitation, and suspension in 5 μl TE buffer. 1 μg of the thus obtained λ chain gene digest and 0.2 μg of the vector digest were added to 20 μl of a reaction solution containing 100 units of T4 DNA ligase (see Example 2-(1-2)) at 16°C for 16 min. They were linked by reacting for a period of time. This ligation was carried out using Escherichia coli JM83 (C.Yanisch-
Perron. J. Vieira and J.
．． Messing. Gene. Volume 33, 103 (1
985); JCRB). 2mM isopropylthiogalacide, 40μg/mlX-ga
1, white colonies resistant to 50 μg/ml ampicillin were selected, and after culturing, plasmid DNA was extracted by the alkaline solution method and treated with restriction enzymes HindIII and E.
After being digested with coRI or SacI, it was subjected to agarose gel electrophoresis (see Example 2-(1-5)). The desired plasmid was selected based on the DNA pattern after staining with ethidium bromide, and plasmid pUCHIL containing a fragment of the λ chain V region and plasmid 153SacIpUC19 containing a fragment of the μ chain V region were obtained. The cleavage sites of various restriction enzymes in these plasmids were identified and a restriction enzyme map was created. Furthermore, various deletion derivatives of the V region gene were created using various restriction enzyme sites on the V region gene. That is, cut points are inserted into the V region gene and the multi-cloning site on the vector pUC19 by restriction enzyme treatment, and then religated with T4 DNA ligase to introduce various deletions into the V region gene starting from the multi-cloning site. did. If the restriction enzyme that cut the V region gene and the restriction enzyme that cut the multi-cloning site are different, after cutting, T4D
Repair synthesis was performed using NA polymerase to make the cut ends blunt, and then religation was performed. After transforming the recombinant thus obtained into Escherichia coli DH1, 50 μg/m
An ampicillin-resistant strain was selected, plasmid DNA was extracted, and the desired deletion derivative was selected by observing cleavage patterns with various restriction enzymes. Next, the various deletion derivatives obtained were denatured with 0.2N sodium hydroxide, recovered by ethanol precipitation, and the base sequence of each part of the V region gene was decoded by the chain terminator method using didexyoxynucleotides ( This was done using Takara Shuzo's M13 Sequencing Kit according to the attached protocol). By joining together the partial base sequences obtained in this way, the base sequence of the V region gene and its surroundings was determined (Figures 2 and 3) (Reference: J.
Messing in“Methods in
Enzymoligy” 101 (1983) 20-7
8). Furthermore, the amino acid sequence predicted from the determined base sequence and the known amino acid sequence of the V region (EA Ka
bat et al., “Sequences of Prot.
eins of immunological
Interest” (1983) NIH) and determined the exact position of the V region of HI-223.C
A restriction enzyme map of the μ chain gene lacking the region and the entire λ chain gene encoding the V region and C region is shown in FIG.

(2) Cloning of IN2A8 antibody gene Cloning of the antibody gene of HI223 was carried out in the same manner. (2-1) Isolation of IN2A8 genomic DNA Human anti-Pseudomonas aeruginosa antibody-producing cells IN2A8 (108 cells) were crushed with a glass rod, and after proteinase K treatment, the DNA was rolled up with a glass rod in ethanol in the same manner as HI223. . After drying under reduced pressure and suspending in 2 ml of TE buffer, approximately 600 μ
g human genomic DNA was obtained.

(2-2) Preparation of IN2A8 gene library Example 2 - Human genomic DNA 10 obtained in (2-1)
μg of restriction enzyme BamHI in 150 units at 37°C, 2
Incubate for an hour and further treat at 65°C for 5 minutes to stop the reaction.
A complete decomposition product was obtained. Separately, 10 μg of human genomic DNA
and restriction enzyme HindIII50 in TA buffer.
Unit was added, incubated at 37°C for 2 hours, and further treated at 65°C for 5 minutes to obtain a complete decomposition product, and a fragment of 8 to 8.5 kb was collected by low melting point agarose gel electrophoresis. These were mixed with 10mM Tris-HCl pH 8.0/0.1m
Bovine small intestine alkaline phosphatase 2 in M EDTA
After processing at 37℃ for 30 minutes, phenol extraction was carried out.
IN2A8 genomic DNA was recovered by ethanol precipitation, and the BamHI site of phage vector EMBL3DNA (Promega Biotech) or phage vector λL was collected.
HindIII of 47DNA (Promega Biotech)
Connected to the site. The EMBL3 vector was prepared by incubating 50 μg of the vector in TA buffer with 100 units of restriction enzymes BamHI and 100 units of EcoRI at 37°C for 2 hours to decompose it, and then inactivating the enzyme at 65°C for 5 minutes. For the λL47 vector, 50 μg of it was injected with 100 units of restriction enzymes BamHI and 100 units of HindIII.
It was prepared by incubating the enzyme together with the unit in TA buffer for 2 hours at 37°C to decompose it, and then inactivating the enzyme at 65°C for 5 minutes. For the ligation reaction, 24 μg of vector DNA, genomic DNA
66mM Tris-HCl (pH 7.6)/6.6mM containing 3 μg of A-digested product and 500 units of T4 DNA ligase
MgCl2/10mM DTT/1mM ATP solution 1
The reaction was carried out by reacting 00 μl at 16°C for 16 hours. Then, the obtained DNA was in vitro
Packaging (A. Becker and
M. Gold. Proc. Natl. Acad
．． Sci. U. S. A. Volume 72, 581 (1975)
), and a human gene library was obtained.

(2-3) IN2A 8μ chain gene screening Example 2 - BamHI-EMBL obtained in (2-2)
E. coli LE392 (JCRB
) was used as an indicator bacterium to form 800,000 plaques. After the phage on this plaque was transferred to a nitrocellulose filter by replica, human antibody genes were screened by plaque hybridization using 32P-labeled human antibody JH gene and human Cμ gene as probes. Five phage clones hybridizing with the human antibody JH gene probe were obtained from the 800,000 phage BamHI-EMBL3 library.

(2-4) Screening of IN2A8λ chain gene Example 2 - HindIII-λ obtained in (2-2)
Escherichia coli LE392 (JCR
B) was used as an indicator bacterium to form 200,000 plaques, and screening was performed by plaque hybridization using a human Cλ2 probe to obtain three phage clones that hybridized with the probe. (2-5) Analysis of phage clones containing antibody genes Example 2 - A phage lysate was prepared from the phage clones obtained in (2-3, 4), phage DNA was extracted from it, and a restriction enzyme map was created. (Example 2-(1-5
)reference). In addition, the electrophoresed various restriction enzyme fragments were transferred to a nitrocellulose filter, and Southern hybridization was performed using the human JH gene, human Cμ, or human Cλ gene as a probe labeled with 32P, and the obtained phage clone The middle V area,
The C region was identified. The phage clone containing the above-mentioned λ chain gene was named λλIN1, and the phage clone containing the μ chain gene was named λμIN1.

(2-6) Determination of the base sequence of the IN2A8 antibody gene The base sequences of the λ chain and μ chain V region genes were determined as follows. First, HindII containing the λ chain V region
I, BglI fragment (arrow fragment in Figure 4(B)) was cloned into the cloning vector pUC18 (C.Yanisch-
Perron. J. Vieira. and J
．． Messing. Gene, vol. 33, 103 (19
85) Reference; Takara Shuzo) HindIII, BamHI
BamHI-HindII site containing μ chain V region
I 4.0kb fragment, BamHI- containing μ chain C region
The HindIII 9.5kb fragment (arrow fragment in Figure 4(A)) was inserted into BamHI-HindIII of pUC18.
The plasmid pμIN1 containing a λ chain V region fragment, the plasmid pμIN1V containing a μ chain V region fragment, and the plasmid pμIN1C containing a μ chain C region fragment were obtained. The cleavage sites of various restriction enzymes in these plasmids were identified and a restriction enzyme map was created. Furthermore, various deletion derivatives of the V region gene were created using the sites of various restriction enzymes on the V region gene. Next, the various deletion derivatives obtained were denatured with 0.2N sodium hydroxide, recovered by ethanol precipitation, and the base sequence of each part of the V region gene was decoded by the chain terminator method using didexyoxynucleotides ( This was done using Takara Shuzo's M13 Sequencing Kit according to the attached protocol). By joining together the partial base sequences obtained in this way, the base sequence of the V region gene and its surroundings was determined (Figures 5 and 6
) (Reference: J. Messing in “Method
s in Enzymoligy”101(19
83) 20-78). Furthermore, the amino acid sequence predicted from the determined base sequence and the known amino acid sequence of the V region (EA Kabat et al., “Sequences
f Proteins of Immunol
logical Interest” (1983) NI
H) and determined the exact position of the V region of IN-2A8. A restriction enzyme map of the entire μ chain gene and lambda chain gene including the V region and C region is shown in FIG.

(3) Cloning of MH4H7 antibody gene Cloning of the HI223 antibody gene was carried out in the same manner. (3-1) Isolation of MH4H7 genomic DNA Human anti-Pseudomonas aeruginosa antibody-producing cells MH4H7 (108 cells) were crushed with a glass rod, and after proteinase K treatment, the DNA was rolled up with a glass rod in ethanol in the same manner as in HI223. . After drying under reduced pressure and suspending in 2 ml of TE buffer, approximately 600 μ
g human genomic DNA was obtained.

(3-2) Preparation of MH4H7 gene library Example 2 - Human genomic DNA 10 obtained in (3-1)
μg of restriction enzyme BamHI in 150 units at 37°C, 2
Incubate for an hour and further treat at 65°C for 5 minutes to stop the reaction.
A complete decomposition product was obtained. Separately, 10 μg of human genomic DNA
Add 50 units of restriction enzyme EcoRI in TA buffer, incubate at 37°C for 2 hours, and then incubate at 65°C for 5 hours.
After treatment for 1 minute, a complete decomposition product was obtained. These were treated with 2 units of bovine small intestine alkaline phosphatase in 10mM Tris-HCl pH 8.0/0.1mM EDTA at 37°C for 30 minutes, followed by phenol extraction and MH4H7 genomic DNA.
was recovered by ethanol precipitation, and the phage vector EMBL
It was ligated to the BamHI site of 3DNA (Promega Biotech) or the EcoRI site of phage vector EMBL4DNA (Promega Biotech). EMB
The L3 and 4 vectors were prepared by incubating 50 μg of the restriction enzymes in TA buffer with 100 units of BamHI and 100 units of EcoRI at 37°C for 2 hours to decompose the enzymes, and then inactivating the enzymes at 65°C for 5 minutes. The ligation reaction involved 24 μg of vector DNA and genomic DNA.
66mM Tris-HCl (pH 7.6)/6.6mM containing 3 μg of digested product and 500 units of T4 DNA ligase
MgCl2/10mM DTT/1mM ATP solution 10
The reaction was carried out by reacting 0 μl at 16°C for 16 hours. Thereafter, the obtained DNA was in vitro
Packaging (A. Becker and
M. Gold. Proc. Natl. Acad
．． Sci. U. S. A. Volume 72, 581 (1975)
), and a human gene library was obtained.

(3-3) MH4H7μ chain gene screening Example 2 - BamHI-EMBL obtained in (3-2)
E. coli LE392 (JCRB
) was used as an indicator bacterium to form 1.6 million plaques. The phage on this plaque was transferred to a nitrocellulose filter by replica, and human antibody genes were screened by plaque hybridization using 32P-labeled human antibody JH gene and Cμ gene as probes. Three phage clones that hybridized with the human antibody JH gene probe were obtained from the 1.6 million phage BamHI-EMBL3 library.

(3-4) MH4H7λ chain gene screening Example 2 - EcoRI-EMBL obtained in (3-2)
E. coli LE392 (JCRB) using 4 libraries
was used as an indicator bacterium to form 600,000 plaques, and screening was performed by plaque hybridization using a Cλ2 probe to obtain 21 phage clones that hybridized with the probe.

(3-5) Analysis of phage clones containing antibody genes Example 2 - A phage lysate was prepared from the phage clones obtained in (3-3, 4), and phage DNA was
was extracted and a so-called restriction enzyme map was created (Example 2
- (1-5)). Further, the electrophoresed various restriction enzyme-cleaved fragments were transferred to a nitrocellulose filter, and Southern hybridization was performed using the human JH gene, human Cμ, or human Cλ gene labeled with 32P as a probe. The V region and C region in the obtained phage clone were identified. The phage clone containing the above-mentioned λ chain gene was named λλMH1, and the phage clone containing the μ chain gene was named λμMH1.

(3-6) Determination of the nucleotide sequence of the MH4H7 antibody gene The nucleotide sequences of the λ chain and μ chain V region genes were determined as follows. First, HindII containing the λ chain V region
I-EcoRI fragment (arrow fragment in Figure 7(B)) was cloned into the cloning vector pUC18 (C.Yanisch
-Perron. J. Vieira. andJ. M
essing. Gene, vol. 33, 103 (1985
), the BamHI fragment containing the μ chain VC region (see Figure 7 (A
)) was cloned into the BamHI site of pUC19 to create a plasmid pλMH1 containing a fragment of the λ chain VC region,
Plasmid pμMH1 containing a fragment of the μ chain VC region was obtained. The cleavage sites of various restriction enzymes in these plasmids were identified and a restriction enzyme map was created. Furthermore, various deletion derivatives of the V region gene were created using the sites of various restriction enzymes on the V region gene. Next, the various deletion derivatives obtained were denatured with 0.2N sodium hydroxide, recovered by ethanol precipitation, and the base sequence of each part of the V region gene was decoded by the chain terminator method using didexyoxynucleotides ( This was done using Takara Shuzo's M13 Sequencing Kit according to the attached protocol). By joining the partial base sequences obtained in this way, the base sequence of the V region gene and its surroundings was determined (Figures 8 and 9) (Reference: J.Mess
ing in“Methods in Enzy
Furthermore, the amino acid sequence predicted from the determined base sequence and the known amino acid sequence of the V region (EA Kabat et al., "Sequences of Proteins
of Immunological Int.
erest” (1983) NIH), and MH
The exact location of the V region of 4H7 was determined. A restriction enzyme map of the entire μ chain gene and lambda chain gene including the V region and C region is shown in FIG.

(4) Construction of expression plasmid Vector pSV2dhfr (P.J. Sout
Hern &P. Berg. J. Mml. Appl
．． Genet. 1, 327 (1982): Bethesda Research) 5 μg of DNA was incubated at 37° C. for 2 hours in 20 μl TA buffer with 20 units of restriction enzyme EcoRI or BamHI, and then recovered by ethanol precipitation to obtain an EcoRI and BamHI digest. After treatment with alkaline phosphatase under the reaction conditions of Example 2-(1-2), vector DNA was recovered by phenol extraction and ethanol precipitation, and then treated with T4 DNA ligase under the reaction conditions of Example 2-(1-2) with the following antibody gene. were combined to construct an antibody gene expression plasmid. (4-1) Construction of HI223 antibody expression plasmid IN2
Plasmid DNA with A8-derived μ chain (pμIN1C)
was digested with HindIII and treated with alkaline phosphatase to obtain 153SacIpUC containing the HI223VH region.
It was ligated with 1 μg of HindIII digested product of No. 19. A BamHI fragment containing the VH Cμ region was recovered from the obtained plasmid and transformed into the vector pSV2dhf.
Mix 0.1 μg of rBamHI decomposition product and
In 20 μl reaction solution with unit T4 DNA ligase,
The reaction was carried out at 16° C. for 16 hours (see Example 2-(1-2)) to obtain these conjugates. This reaction solution was added to E. coli DH1
Plasmid DNA was extracted from the resulting ampicillin-resistant strain, analyzed by restriction enzyme digestion, and a plasmid containing the μ chain gene and pSV2dhfr was selected and named pHIH1. 2 μg of plasmid DNA (pUCHIL) containing a fragment of the VC region of the HI223 λ chain was digested with the restriction enzyme HindIII and then treated with 1 unit of T4 DNA polymerase, 67 mM Tris-HCl, and 67 mM MgC.
l2, 10mM 2-mercaptoethanol, 6.7μ
MEDTA, 16.6mM (NH4)2SO4,
330μM each dCTP, dATP, dGTP, dTTP
The DNA was reacted for 30 minutes at 37°C in 30 µl of a reaction solution containing 30 µl of DNA to make sticky ends into blunt ends, and then the DNA was recovered by phenol extraction and ethanol precipitation. 1μg of this DNA and E
0.1 μg of coRI linker was added to T4 DNA ligase 10
0 unit and 20 μl reaction solution (Example 2-(1-2)
After incubating at 16°C for 16 hours in
transformed into. The plasmid DNA of the obtained ampicillin-resistant strain was extracted and selected by observing the degradation products with the restriction enzyme EcoRI.
I got LE. EcoRI containing HI223λ chain VC region
The fragment was collected, ligated with the aforementioned pSV2dhfr vector, and transformed into E. coli DH1 to obtain pHIL1.

(4-2) Construction of IN2A8 antibody expression plasmid BamHI-digested product of phage clone λμIN1 1μ
g and 0.1 μg of the vector pSV2dhfrBamHI digested product were mixed and reacted together with 100 units of T4 DNA ligase in a 20 μl reaction solution at 16° C. for 16 hours (see Example 2-(1-2)) to form a ligated product. Obtained. This reaction solution was transformed into Escherichia coli DH1, and plasmid DNA was extracted from the resulting ampicillin-resistant strain and analyzed by restriction enzyme digestion to identify the μ chain gene and pSV2d.
A plasmid containing hfr was selected and named pINH1. Next, λλIN1 HindIII decomposition product 1 μ
g and puc18 HindIII decomposition product 0.1 μ
g was ligated under the conditions of Example 2 to obtain pλIN1. pλ
After IN1 was digested with EcoRI, it was ligated with T4 ligase and pλ
IN1△ was obtained. pλIN1△ is restriction enzyme HindII
After I degradation, repair synthesis is performed using T4 DNA polymerase to make the cut ends blunt, and the plasmid DNA (pλIN) is ligated with EcoRI linker DNA.
1E) was obtained (see Example 2-(4-1)). λ chain VC
A fragment containing EcoRI at both ends of the antibody gene containing the region was recovered, ligated with the aforementioned pSV2dhfr vector, and transformed into E. coli DH1 to obtain pINL1.

(4-3) Construction of MH4H7 antibody expression plasmid 1μ of BamHI digested product of phage clone λμMH1
g and 0.1 μg of the vector pSV2dhfrBamHI digested product were mixed and reacted together with 100 units of T4 DNA ligase in a 20 μl reaction solution at 16° C. for 16 hours (see Example 2-(1-2)) to form a ligated product. Obtained. This reaction solution was transformed into Escherichia coli DH1, and plasmid DNA was extracted from the resulting ampicillin-resistant strain and analyzed by restriction enzyme digestion to identify the μ chain gene and pSV2d.
A plasmid containing hfr was selected and named pMHH1. Next, EcoRI-HindIII of λλMH1
1 μg of degraded product and puc18 EcoRI-Hin
0.1 μg of the dIII decomposition product was ligated under the conditions of Example 2 to obtain pλMH1. pλMH1 is the restriction enzyme Hind
III After degradation, repair synthesis is performed using T4 DNA polymerase to make the cut ends blunt, and then ligated with EcoRI linker DNA to create plasmid DNA (pλ
MH1E) was obtained (see Example 2-(4-1)). A fragment containing EcoRI at both ends of the antibody gene containing the λ chain VC region was collected, ligated with the aforementioned pSV2dhfr vector, and transformed into E. coli DH1 to obtain pMHL1. Example 3 Obtaining antibody gene expression recombinant (1) EL
Measurement of anti-Pseudomonas aeruginosa antibody titer by ISA The anti-Pseudomonas aeruginosa surface antigen antibody titer was measured by the following method. Pseudomonas aeruginosa was suspended in PBS so that the absorbance at a wavelength of 600 nm was 0.2, and 50 μl per well was dispensed into a microplate and centrifuged at 2,000 rpm for 15 minutes. 2% glutaraldehyde at 50% per well
Microbial cells were immobilized on a microplate by adding μl each. After removing the bacterial cell solution from the microplate, add 3% BSA-
Dispense 120 μl of PBS solution per well.
The unadsorbed portions of bacterial cells were blocked by incubating at ℃ for 30 minutes. The microplate was used as an antigen adsorption plate for subsequent operations. If necessary, it was stored at -20°C at this stage. Antigen adsorption plates were washed three times with PBST before assay. P the sample whose antibody titer you want to measure.
Appropriately dilute with BST and add 50 μl per well.
It was kept warm at ℃ for 2 hours. The sample was then removed and washed three times with PBST. Next, add the second antibody solution at 10% per well.
0 μl each was added and kept at 37° C. for 2 hours. As a second antibody, a phosphatase-labeled affinity-purified anti-human immunoglobulin μ chain antibody diluted 500-1000 times with PBST (Kirkegaard & PerryLab.In
c. ) was used. After removing the second antibody and washing three times with PBST, 100 μl of a chromogenic substrate solution was added per well according to Example 1-(1), and the mixture was reacted at 37°C. Multiscan OD405 after reaction (Titertek
).

(2) Establishment of IN2A8 antibody-producing cell line by electroporation method 40 μg of the above expression plasmid pINH1, pINL
1 20 μg, pSV2neo (S. Subrama
ni et al. Mol. Cell. Biol.
Volume 1. 854 (1981): Bethesda Research Inc.) 10
37 μg of the restriction enzyme PvuII in 300 μl of TA buffer/150 mM KCl reaction solution with 500 units of restriction enzyme PvuII.
After reacting at 65°C for 3 hours, the mixture was treated at 65°C for 5 minutes, and DNA was recovered by ethanol precipitation. This DNA was diluted with modified phosphate buffered saline (hereinafter abbreviated as mPBS) (NaCl
0.58g/l, KCl 11.6g/l, Na
2 HPO4 1.15g/l, KH2PO4
The suspension was suspended in 100 μl of 0.2 g/l, 5 mM MgSO4, and incubated at 65° C. for 20 minutes. Namalva cells (ATCC
CRL1432) (NK) or the Namalva cell mutant strain (NKG4) described in Example 1 was cultured to the logarithmic growth phase in a serum-free medium Cell Grosser H, and then grown to 107 cells.
ls was centrifugally washed with the above mPBS and further suspended in 400 μl of the same buffer. After adding and mixing 100 μl of the above DNA solution to this cell suspension, the cell suspension was transferred to a plastic cell equipped with aluminum foil electrodes on both sides. The distance between the electrodes was approximately 7 mm. After leaving it in ice for 10 minutes, 2.
After applying a high voltage pulse of 9 kVcm-1, it was left on ice for an additional 10 minutes. Add 10 m to this cell-DNA suspension.
1 of Cell Grosser H medium was added, and after incubation at 37°C for 2 hours in the presence of 5% CO2, the mixture was centrifuged, suspended in 20 ml Cell Grosser H-10% FCS, and dispensed into 10 96-well plates. This was cultured for 2 days at 37° C. and 5% CO 2 , and antibiotic G418 (Gibco) was added to give a concentration of 400 μg/ml. Thereafter, half of the medium was replaced every 3 to 4 days. G-418-resistant colonies appeared from around day 20, and the expression of human antibodies in the culture supernatant was also examined using colonies obtained after that. Part of the results of ELISA using a type B Pseudomonas aeruginosa cell adsorption plate are shown below.

(3) Establishment of MH4H7 antibody-producing cell line by electroporation method According to Example 3-(2), expression plasmid pMHH1
, pMHL1 was transformed into NK strain or NKG4 strain. Specifically, 40 μg of pMHH1 DNA, pMHL
20 μg of pSV2neo DNA and 10 μg of pSV2neo DNA were digested with restriction enzyme PvuII and suspended in 100 μl of mPBS. For human lymphoblastoid cells NK strain or NKG4 strain cultured to the logarithmic growth phase, 107 cells were centrifugally washed with the above-mentioned improved PBS, and then diluted with 400μ of the same buffer.
Suspended in l. Add 100μ of the above DNA solution to this cell suspension.
After mixing, the mixture was transferred to a plastic cell equipped with aluminum foil electrodes on both sides. The distance between the electrodes was approximately 7 mm. After being left in ice for 10 minutes, the power supply (ISCO, Model 494) was applied to 2.9 kVc.
After applying a high voltage pulse of m-1, the sample was left on ice for an additional 10 minutes. Add 10 ml of Cell Grosser H medium to this cell-DNA suspension and incubate for 37 hours in the presence of 5% CO2.
After incubating at ℃ for 2 hours, centrifuge, suspend in 20 ml Cell Grosser H-10% FCS, and transfer to 96-well plate 10.
Dispense into sheets. This was cultured for 2 days at 37℃ with 5% CO2, and antibiotic G-418 (Gibco) was added to
The medium was adjusted to μg/ml, and half of the medium was replaced every 3 to 4 days thereafter. G418-resistant colonies appeared from around day 20, and the expression of human antibodies in the culture supernatant was also examined using colonies obtained after that. Some of the results of ELISA using a Pseudomonas aeruginosa type G cell adsorption plate are shown below.

(4) HI223 by lipofectin method
Establishment of antibody-producing cell line Namalva cells (NK) or NK-derived antibody non-producing cells NKG4 were cultured in serum-free medium Cell Grosser H until the logarithmic growth phase. Collect 5 x 106 cells by centrifugation.
．． The suspension was suspended in 5 ml of Cell Grosser H and placed in a 6-well plate. Expression plasmid pHIL described in Example 2
1 6μg, pHIH1 12μg and pSV2ne
o 3 μg was digested with restriction enzyme PvuI to obtain linear DNA.
A was suspended in 250 μl of Cell Grosser H. 50 μl of Lipofectin (purchased from BRL) was diluted with 200 μl of Cell Grosser H, mixed with DNA solution, and DNA/
A lipofectin complex was created. The DNA/lipofectin complex was added dropwise to the cells in a 6-well plate, and the mixture was incubated at 37°C.
The cells were cultured under 5% CO2 conditions. After 10, 12, or 24 hours, cells were harvested and suspended in Cell Grosser H containing 10% FCS at a cell density of 5 x 104 cells/ml.
100 μl each was added to a well plate. 1-2 days later,
800 μg/ml G-418-containing Cell Grosser H
00 μl was added. Thereafter, 400 μg every 2-3 days
/mlG-418-containing Cell Grosser H to change the solution.
-418 resistant colonies were obtained. G- from around the 20th day
418-resistant colonies appeared, and the expression of human antibodies in the culture supernatant was also examined using the colonies obtained thereafter. Part of the results of ELISA, in which the amount of antibody produced was calculated using HI223 of known concentration as a standard, is shown below.

(5) IN2A8 by lipofectin method
Establishment of antibody-producing cell line According to Example 3-(4), the expression plasmid pINH1
, pINL1 and pSV2neo were transformed into NK strain or NKG4 strain. Namalva cells (NK) or NK-derived non-antibody producing cells NKG4 were cultured in a serum-free medium Cell Grosser H until the logarithmic growth phase. 5×10 6 cells were collected by centrifugation, suspended in 0.5 ml of Cell Grosser H, and placed in a 6-well plate. The antibody expression plasmid and the drug resistance marker gene pSV2neo were digested with the restriction enzyme PvuI to form linear DNA, suspended in 250 μl of Cell Grosser H, and then mixed with lipofectin to form a DNA/lipofectin complex. The DNA/lipofectin complex was dropped onto cells in a 6-well plate and cultured at 37°C and 5% CO2, and then the cells were collected by centrifugation and cultured in a 96-well plate. After 1-2 days, G-418-containing cell grosser H
Gene-introduced cells were selected. G-4 from around the 20th day
18 resistant colonies appeared, and the expression of human antibodies in the culture supernatant was also examined using the colonies obtained thereafter. Part of the results of ELISA, in which the amount of antibody produced was calculated using IN2A8 of known concentration as a standard, is shown below.

(6) MH4H7 by lipofectin method
Establishment of antibody-producing cell line According to Example 3-(4), expression plasmid pMHH1
, pMHL1 and pSV2neo were transformed into NK strain or NKG4 strain. Namalva cells (NK) or NK-derived antibody non-producing cells NKG4 were cultured in a serum-free medium Cell Grosser H until the logarithmic growth phase. 5×10 6 cells were collected by centrifugation, suspended in 0.5 ml of Cell Grosser H, and placed in a 6-well plate. The antibody expression plasmid and the drug resistance marker gene pSV2neo were digested with the restriction enzyme PvuI to form linear DNA, suspended in 250 μl of Cell Grosser H, and then mixed with lipofectin to form a DNA/lipofectin complex. The DNA/lipofectin complex was dropped onto cells in a 6-well plate and cultured at 37°C and 5% CO2, and then the cells were collected by centrifugation and cultured in a 96-well plate. After 1-2 days, G-418-containing cell grosser H
Gene-introduced cells were selected. G-4 from around the 20th day
18 resistant colonies appeared, and the expression of human antibodies in the culture supernatant was also examined using the colonies obtained thereafter. Part of the results of ELISA in which the amount of antibody produced was calculated using MH4H7 of known concentration as a standard is shown below.

(7) Amplification of antibody gene and increase in antibody production amount by MTX Example 3 - The antibody producing cells shown in (2, 3, 4, 5, 6) were suspended in Cellgrosser H medium containing 50 nMMTX. -105 cells/200 μl was cultured in a 96-well plate. When the culture conditions are such that colonies appear in 30-60 out of 96 wells 10-25 days after the start of culture, the amount of antibody in the culture supernatant of each well is screened by ELISA according to Example 3-(1). did. Expand the culture scale of cells in wells with high antibody production and further increase
A cell line resistant to MTX-containing medium was established. For MTX-resistant strains, cells were suspended in Cell Grosser H medium containing MTX at the same concentration and cultured at 96 cells at 0.5-1 cells/well.
It was cultured and cloned using a well plate. When the culture conditions are such that colonies appear in 30-60 out of 96 wells 10-25 days after the start of culture, the amount of antibody in the culture supernatant of each well was determined by EL according to Example 3-(1).
After screening by ISA, the culture scale was expanded and the amount of antibody produced was determined by ELISA. The antibody production amounts of the MTX-resistant strain and the cell line after cloning are summarized in the table below.

Example 4 Mass culture of antibody-producing cell line Mass culture of Namalva cell-transformed cells H4-10-800-3A4 was carried out using a jar fermenter (capacity 6L, model number MCT-3S, Marubishi Bioengineering). went. The commercially available serum-free medium Cell Grosser H was used as the medium. 4.4
Cell density 2.6 x 105 cells/m in the above medium of L
After the cells were precipitated by temporarily stopping stirring every 2 to 3 days, the cell-free culture supernatant at the top was replaced with fresh medium to perform continuous culture. After 5 days of culture, the cell density is 7.9 x 105 cells.
s/ml was reached.

Example 5 Purification of antibody 65 ml of H62-RE5-200 culture supernatant was filtered using an ultrafiltration device (Amicon) equipped with an ultrafiltration membrane YM30.
Gel filtration column SU concentrated to ml and equilibrated with PBS
It was applied to PEROSE6, and IgM was eluted with PBS at a flow rate of 0.5 ml/min and collected in 1 ml portions. Each fraction was subjected to SDS-PAGE (Daiichi Kagaku PAG Plate 1
0/20) Served with CBB (Kumaji Brilliant Blue)
The IgM fraction was detected by staining. H4-10-200
60 ml of the culture supernatant was also concentrated to 1 ml by ultrafiltration as described above, and then applied to a gel filtration column SUPEROSE6 to obtain an IgM fraction.

Example 6 Biochemical analysis of antibodies (1) Examination of antibodies produced by transformed cells by Western blotting Namalva cells are known to produce IgM antibodies on the cell surface (Int. J. Cancer 12 39
6-408 (1973)). Culture supernatants of NK strain and NKG4 strain were analyzed by Western blotting using anti-human μ chain antibody and anti-human λ chain antibody.
It was confirmed that the chain was secreted. The presence or absence of the Namalva-derived λ chain in the antibody produced by the DNA-transformed cell line was confirmed by Western blotting. Specifically, the culture supernatant was electrophoresed under reducing conditions (Daiichi Kagaku PAG plate 10/20), and filtered with a Durapore filter (Millipore).
After blotting with alkaline phosphatase-labeled anti-human λ chain antibody (Tago 2498), BCIP (5-B
romo 4-chloro 3-indolyl p
(BioRad), NBT (Nit
ro Blue Tetrazolium) (BioR
λ chain was detected in ad). The results are shown in FIG. N
H62-RE5-, a cell line in which the antibody gene was introduced into the K strain
200, 3B2-H11-100, etc., λ chains derived from Namalva cells were detected. On the other hand, H4-10-800-3A4 is a cell line in which an antibody gene has been introduced into the NKG4 strain.
, 4E2-B5-400, etc., the λ chain derived from Namalva cells could not be detected.

(2) Examination of the J chain of the antibody produced by the transformed cell line The antibody produced by the transformed cell line was filtered onto a gel filtration column (SU
When analyzed using PEROSE6), it was confirmed that IgM was eluted at the molecular weight position of 1 million, forming a multimer. It was confirmed by Western blotting that the multimeric IgM contained human J chain. That is, purified antibodies were electrophoresed under non-reducing conditions (Pharmacia 2-16
gradient gel), anti-human J chain antibody (rabbit Nordic) after blotting onto a Durapore filter,
Detection was performed using an alkaline phosphatase-labeled anti-rabbit antibody. As shown in Figure 17, human J
It was confirmed that the chain was included.

Example 7 Measurement of antigen binding ability of antibodies (1)
) Comparison of specific activities of antibodies produced by transformed cells and hybridomas Purified antibodies derived from hybridomas and purified antibodies derived from transformed cells were appropriately diluted with PBST, and ELISA was performed to determine the OD of the transformed cell-derived antibodies relative to the hybridoma-derived antibodies.
The ratio of the 405 values was determined. Furthermore, regarding the ratio of OD405 values, the ratio of the value of the Pseudomonas aeruginosa adsorption plate to the value of the anti-μ chain antibody plate was determined and used as the ratio of specific activity. The results are shown in the table below. Antibody H62- with λ chain derived from host Namalva cell NK strain
RE5-200 (see Example 6-(1)) has lower specific activity compared to hybridoma-derived antibodies, and DNA transformation was performed using the NKG4 strain (see Example 1), which does not produce host-derived antibodies, as a host. cell line H4-10-20
At 0, no decrease in specific activity was observed.

[0052]

[Sequence list] Sequence number: 1 Sequence length: 119 Sequence type: Amino acid Sequence type: Peptide sequence Glu Glu Gln Leu Val Glu S
er Gly Gly Gly Leu Val Ly
s Pro Gly Gly 1
5
10 15 S
er Leu Arg Leu Ser Cys Al
a Ala Ser Gly Phe Thr Phe
Ser Asn Ala
20 25
30 Trp Me
t Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu
Trp Val 35
40
45 Gly Arg Ile Leu
Ser Lys Thr His His Gly
Ala Thr Asp Tyr Ala Ala
50 5
5 60 Pro
Val Lys Gly Arg Phe Thr
Ile Ala Arg Asp Asp Ser L
ys Ser Thr 65
70
75 80
Leu Tyr Leu Gln Met Asn S
er Leu Lys Thr Glu Asp Th
r Ala Val Tyr
85
90 95 T
yr Cys Thr Thr Asp Ala Gl
u Val Pro Arg Ile Trp Gly
Gln Gly Thr
100 105
110 MetVa
l Thr Val Ser Ser Gly
115 Sequence number: 2 Sequence length: 110 Sequence type: Amino acid sequence type: Peptide sequence Gln Ala Val Val Thr Gln G
lu Pro Ser Leu Thr Val Se
r Pro Gly Gly 1
5
10 15 T
hr Val Thr Leu Thr Cys Gl
y Ser Ser Thr Gly Ala Val
Thr Ser Gly
20 25
30 His Ty
r Ala Tyr Trp Phe Gln Gln
Lys Pro Gly Gln Ala Pro
Arg Thr 35
40
45 Leu Ile Tyr Asp
Thr Ser Asn Lys His Ser
Trp Thr Pro Ala Arg Phe
50 5
5 60 Ser
Gly Ser Leu Leu Gly Gly
Lys Ala Ala Leu Thr Leu S
er Gly Ala 65
70
75 80
Gln Pro Glu Asp Glu Ala G
lu Tyr Tyr Cys Leu Leu Se
r Tyr Ser Ala
85
90 95 T
hr Arg Val Phe Gly Gly Gl
y Thr Lys Leu Thr Val Leu
Gly 100
105
110 Sequence number: 3 Sequence length: 122 Sequence type: Amino acid sequence type: Peptide sequence Glu Val Gln Leu Val Glu S
er Gly Gly Asp Leu Val Gl
n Pro Gly Gly 1
5
10 15 S
er Leu Arg Leu Ser Cys Al
a Ala Ser Gly Phe Thr Phe
Ser Ser Tyr
20 25
30 Glu Me
t Asp Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu
Trp Ile 35
40
45 Ser Tyr Ile Ser
Asp Ser Gly Ser Ser Ile
Phe Tyr Ala Asp Ser Val
50 5
5 60 Lys
Gly Arg Phe Thr Ile Ser
Arg Asp Lys Ala Lys Lys S
er Leu Tyr 65
70
75 80
Leu Gln Met Asn Asn Leu A
rg Ala Glu Asp Thr Ala Va
l Tyr Tyr Cys
85
90 95 A
la Arg Glu Pro Ala Val Al
a Gly Asn Pro His Phe Asp
Tyr Trp Arg
100 105
110 Gln Gl
y Thr Leu Val Thr Val Ser
Ser Gly 115
120 Sequence number: 4 Sequence length: 110 Sequence type: Amino acid sequence type: Peptide sequence Gln Ser Ala Leu Thr Gln G
lu Ala Ser Val Ser Gly Th
r Val Gly Gln 1
5
10 15 L
ys Val Thr Leu Ser Cys Th
r Gly Asn Ser Asn Asn Ile
Gly Thr Tyr
20 25
30 Ala Va
l Gly Trp Tyr Gln Gln Ile
Ser His Gly Ala Pro Lys
Pro Val 35
40
45 Met Phe Gly Asn.
Ser Pro Pro Ser Gly Ile
Pro Asp Arg Phe Ser Gly
50 5
5 60 Ser
Lys Ser Gly Thr Thr Ala
Ser Leu Thr Ile Ser Gly L
eu Gln Pro 65
70
75 80
Glu Asp Glu Ala Asp Tyr T
yr Cys Ser Thr Trp Asp Se
r Ser Leu Ser
85
90 95 V
al Lys Val Phe Gly Gly Gl
y Thr Lys Leu Thr Val Leu
Gly 100
105 Sequence number: 5 Sequence length: 112 Sequence type: Amino acid Sequence type: Peptide sequence Gln Val Pro Leu Val Glu S
er Gly Gly Gly Val Val Gl
n Pro Gly Arg 1
5
10 15 S
er Leu Arg Leu Ser Cys Gl
u Ala Ser Gly Phe Thr Phe
Ser Thr Tyr
20 25
30 Ala Me
t Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu
Trp Val 35
40
45 Ala Val Ile Ser
Tyr Asp Gly Ser Thr Ile
Tyr Tyr Gly Asp Ser Val
50 5
5 60 Lys
Gly Arg Phe Thr Ile Ser
Arg Asp Asn Pro Gln Asn T
hr Leu Tyr 65
70
75 80
Leu Gln Leu Asn Ser Leu L
ys Ser Glu Asp Thr Ala Le
u Tyr Tyr Cys
85
90 95V
al Arg Glu Glu Tyr Ala Ty
r Gly Leu Gly Ser Phe Asp
Ile Trp Gly
100 105
110 Gln Gl
y Thr Met Val Thr Val Ser
Ser Gly 115
120 Sequence number: 6 Sequence length: 111 Sequence type: Amino acid sequence type: Peptide sequence Gln Ser Ala Leu Thr Gln P
ro Ala Ser Val Ser Gly Se
r Pro Gly Gln 1
5
10 15 S
er Ile Thr Ile Ser Cys Th
r Gly Thr Ser Ser Ser Asp Ile
Gly Gly Tyr
20 25
30 Asn Ty
r Val Ser Trp Tyr Gln Gln
His Pro Gly Lys Ala Pro
Lys Leu 35
40
45 Val Ile Tyr Ala
Val Ser Asn Arg Pro Ser
Gly Val Ser His Arg Phe
50 5
5 60 Ser
Gly Ser Lys Ser Gly Asn
Thr Ala Ser Leu Thr Ile S
er Gly Leu 65
70
75 80
Gln Ala Glu Asp Glu Ala A
sp Tyr Tyr Cys Asn Ser As
p Ala Ser Thr
85
90 95 S
er Lys Trp Val Phe Gly Gl
y Gly Thr Lys Leu Thr Val
Leu Gly 100
105
110

[Brief explanation of the drawing]

FIG. 1 (A) shows the μ chain of HI223, and FIG. 1 (B) shows the μ chain of HI223.
A restriction enzyme map of the λ chain of HI223 is shown. S is SacI
, Ps is PstI, Bs is BstEII, Sm is SmaI, Bg is BglII, H is HindIII
, Ba represents BamHI.

FIG. 2 shows the nucleotide sequence and amino acid sequence of the μ chain V region of HI223.

FIG. 3 shows the base sequence and amino acid sequence of the λ chain V region of HI223.

FIG. 4: (A) μ chain of IN2A8, (B) HI
223 represents a restriction enzyme map of the λ chain of 223. S stands for SacI,
Ps is PstI, Bs is BstEII, Sm is Sm
aI, Bg represents BglII, H represents HindIII, Ba represents BamHI, and E represents EcoRI.

FIG. 5 shows the nucleotide sequence and amino acid sequence of the μ chain V region of IN2A8.

FIG. 6 shows the base sequence and amino acid sequence of the λ chain V region of IN2A8.

FIG. 7: (A) MH4H7 μ chain, (B) HI
Represents the restriction enzyme map of the λ chain of 223. Bs is Bst
EII, Bg is BglII, H is HindIII
, Ba represents BamHI, and E represents EcoRI.

FIG. 8 shows the base sequence and amino acid sequence of the μ chain V region of MH4H7.

FIG. 9 shows the base sequence and amino acid sequence of the λ chain V region of MH4H7.

FIG. 10 depicts the construction of an expression plasmid for the μ chain of HI223.

FIG. 11 depicts the construction of an expression plasmid for the HI223 λ chain.

FIG. 12 depicts the construction of an expression plasmid for the μ chain of IN2A8.

FIG. 13 depicts the construction of an expression plasmid for the λ chain of IN2A8.

FIG. 14 depicts the construction of an expression plasmid for the μ chain of MH4H7.

FIG. 15 depicts the construction of an expression plasmid for the λ chain of MH4H7.

FIG. 16 shows Western blotting using anti-human λ chain antibody. The numbers above represent the lane numbers. In (A), lane 1 is hybridoma HI223
, Lane 2 shows H4-10-800-3A4 (NGK4 strain), Lane 3 shows H62-RE5-200 (NK strain), and Lane 4 shows Namalva NK. In (B), lane 1 is hybridoma IN2A8 and lane 2 is IN5F8.
(NGK4 strain), lane 3 shows IN5-H2 (NGK4 strain), and lane 4 shows Namalva NK. In (C),
Lane 1 is hybridoma MH4H7, lane 2 is 4E
2-B5-400 (NGK4 strain), lane 3 is 3B2-
H11-100 (NK strain), lane 4 shows Namalva NK.

FIG. 17 shows Western blotting using anti-human J chain antibody. The numbers above represent the lane numbers. Lane 1 is IN1-4-200 (IN-2A8), Lane 2 is H4-10-800 (HI223), Lane 3 is
indicates H6-5F6-100 (HI223), lane 4 indicates hybridoma HI223.

Claims (1)

[Scope of Claim] [Claim 1] Endogenous antibody and its components H chain, L
It is characterized by culturing a transformed strain that does not produce any of the recombinant human monoclonal antibodies and that produces a recombinant human monoclonal antibody obtained by introducing a foreign antibody gene into a Namalva cell line that has been adapted to a serum-free medium. [Claim 2] The method for producing a human monoclonal antibody according to Claim 1, wherein the foreign antibody gene is an antibody gene against Pseudomonas aeruginosa [Claim 3] The foreign antibody gene 4. The method for producing a human monoclonal antibody according to claim 1, wherein the foreign antibody gene is an antibody gene directed against the O antigen of Pseudomonas aeruginosa.
23 (Feikoken Joyori No. 3213) The method for producing a human monoclonal antibody according to claim 1, characterized in that the antibody gene is derived from the following antibody gene: [Claim 5] The V region of the H chain and L chain of the foreign antibody gene is The method for producing a human monoclonal antibody according to claim 1, characterized in that the foreign antibody gene encodes the amino acid sequence of H chain SEQ ID NO: 1 and L chain SEQ ID NO: 2. [Claim 7] The method for producing a human monoclonal antibody according to claim 1, characterized in that the foreign antibody gene is derived from hybridoma IN2.
A method for producing a human monoclonal antibody according to claim 1, characterized in that the antibody gene is derived from A8 (Feikoken Joyori No. 2741).Claim 8: The V regions of the H chain and L chain of the foreign antibody gene are as follows: The method for producing a human monoclonal antibody according to claim 1, characterized in that the foreign antibody gene encodes the amino acid sequence H chain SEQ ID NO: 3 and SEQ ID NO: 4 L chain. [Claim 9] The foreign antibody gene is an antibody gene against Pseudomonas aeruginosa LPS outer core. The method for producing a human monoclonal antibody according to claim 1, characterized in that the foreign antibody gene is derived from hybridoma MH
A method for producing a human monoclonal antibody according to claim 1, characterized in that the antibody gene is derived from 4H7 (Feikoken Joyori No. 2402).Claim 11: The V regions of the H chain and L chain of the foreign antibody gene are as follows: A method for producing a human monoclonal antibody according to claim 1, which is characterized in that it encodes an amino acid sequence H chain SEQ ID NO: 5 and L chain SEQ ID NO: 6.Claim 12: An endogenous antibody and its component H chain;
[Claim 13] A transformed strain that does not produce any L chain and produces a recombinant human monoclonal antibody obtained by introducing a foreign antibody gene into a Namalva cell line that has been adapted to a serum-free medium. 13. The transformed strain according to claim 12, wherein the antibody gene is an antibody gene against Pseudomonas aeruginosa. [Claim 14] The transformed strain according to claim 12, wherein the foreign antibody gene is an antibody gene against O antigen of Pseudomonas aeruginosa. 12. The transformed strain according to claim 12, wherein the foreign antibody gene is derived from hybridoma HI.
12. The transformed strain according to claim 12, characterized in that the antibody gene is derived from No. 223 (Feikoken Joyori No. 3213). [Claim 16] The V regions of the H chain and L chain of the foreign antibody gene have the following amino acid sequences: [Claim 17] The transformed strain H chain SEQ ID NO: 1 and L chain SEQ ID NO: 2 according to claim 12, wherein the foreign antibody gene is an antibody gene against Pseudomonas aeruginosa flagella. [Claim 18] The transformed strain according to claim 12, wherein the foreign antibody gene is derived from hybridoma IN
12. The transformed strain according to claim 12, characterized in that the antibody gene is derived from 2A8 (Feikoken Joyori No. 2741). [Claim 19] The V regions of the H chain and L chain of the foreign antibody gene have the following amino acid sequences: H chain SEQ ID NO: 3 and SEQ ID NO: 4 L chain of the transformed strain according to claim 12, characterized in that the foreign antibody gene encodes Claim 12
[Claim 21] The foreign antibody gene is derived from hybridoma MH.
12. The transformed strain according to claim 12, which is an antibody gene derived from 4H7 (Feikoken Joyori No. 2402). [Claim 22] The V regions of the H chain and L chain of the foreign antibody gene have the following amino acid sequences: [Claim 23] The transformed strain according to claim 12, characterized in that it encodes H chain SEQ ID NO: 5 and L chain SEQ ID NO: 6. [Claim 23] The transformed strain according to claim 12 is cultivated and produced. Recombinant human monoclonal antibody.Claim 24: A recombinant human monoclonal antibody characterized by culturing and producing the transformed strain according to claim 13.Claim 25: Cultivating and producing the transformed strain according to claim 14. [Claim 26] A recombinant human monoclonal antibody characterized in that it is produced by culturing the transformed strain according to claim 15. [Claim 27] A recombinant human monoclonal antibody characterized in that it is produced by culturing the transformed strain according to claim 16. [Claim 28] A recombinant human monoclonal antibody produced by culturing the transformed strain of Claim 17. [Claim 29] The transformation of Claim 18. [Claim 30] A recombinant human monoclonal antibody characterized in that it is produced by culturing the transformed strain of Claim 19.[Claim 31] A recombinant human monoclonal antibody characterized in that it is produced by culturing the transformed strain of Claim 19. [Claim 32] A recombinant human monoclonal antibody characterized by culturing and producing the transformed strain of Claim 21. [Claim 32] A recombinant human monoclonal antibody characterized by culturing and producing the transformed strain of Claim 21. 33. A recombinant human monoclonal antibody produced by culturing the transformed strain according to claim 22. Claim 34: An endogenous antibody and its component H chain,
A Namalva cell line that does not produce any L chain and is adapted to a serum-free medium.Claim 35:Claims 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, or 33. A prophylactic/therapeutic agent for microbial infection comprising the human monoclonal antibody of 28, 29, 30, 31, 32, or 33.Claim 36.Claim 24, 25, 26, 27, 28, 29,
Pseudomonas aeruginosa infection preventive/therapeutic agent containing human monoclonal antibody 30, 31, 32 or 33