JPS63245690A - Production of human type antibody - Google Patents

Abstract

PURPOSE:To safely and stably obtain a human type antibody useful for measuring antigenic substances, etc., by cultivating an animal cell transformed by a specific recombinant. CONSTITUTION:A vector pG31VK-neo containing a human type antibody L-chain (two light chains) gene is introduced into an animal cell P3U1 to provide a transformant 26-103. A vector pG3101 (gamma1) containing H-chain (two heavy chains) gene is then introduced into the transformant 26-103 to afford a transformant G3101-F. Cells of the resultant transformant are subsequently inoculated into a complete culture medium prepared by adding bovine fetal blood serum to an RPMI-1640 culture medium, etc., and cultivated. The resultant culture is separated to afford a supernatant, which is then treated with (NH4)2SO4, etc., to provide protein precipitates. The obtained protein is purified by an ion exchanger, gel filtration, etc., to isolate a human type antibody, such as IgG1 or IgG3, corresponding to Fischer's serotype classification type 4 (F4) of Pseudomonas aeruginosa.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing human antibodies using genetic engineering techniques.

[Conventional technology]

Antibodies are used as reagents for measuring various antigenic substances, as well as methods that directly attack tumors by administering monoclonal antibodies, and methods that utilize the specificity of monoclonal antibodies by binding anticancer drugs to monoclonal antibodies. Methods of directly administering antibodies to the human body, such as methods of concentrating anticancer drugs in tumors, are being considered. When antibodies from animals other than humans are administered in such applications, the production of antibodies against the administered antibodies becomes a problem. Therefore, there is a strong desire to develop a method for producing human antibodies. As a method for this purpose, a method using human-human hybridomas is being considered. However, it is difficult to subculture human-human hybridomas for a long period of time while maintaining their antibody-producing ability. As a method for eliminating such drawbacks of the human-human hybridoma method, a method is known in which human cells capable of producing a specific antibody are transformed with Epstein-Parr virus to be immortalized. However, it is not easy to obtain antibodies of any class and subclass with the desired specificity by this method.

For this reason, methods for producing antibodies using genetic engineering techniques are being considered.

JP-A-61-134324 describes the expression of an incomplete antibody containing only the human antibody Hfi.

JP-A-61-47500 and JP-A-61-13432
No. 5 describes the expression of a human-mouse chimeric antibody consisting of HM and LH. However, these specifications do not describe the expression and secretion of human antibodies.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method capable of producing a human antibody of any class or subclass having a specific specificity of interest.

[Means for solving problems]

The above purpose can be achieved by using a recombinant containing the gene encoding the human antibody Ljff and a recombinant containing the gene encoding the human antibody Hffi, or by combining the gene encoding the L chain of the human antibody and the human antibody. The problem is solved by a method for producing human antibodies, which comprises secreting human antibodies by culturing animal cells transformed with a recombinant containing a gene encoding an antibody H chain, and recovering the antibodies. be done.

[Specific explanation]

a) The basic units of the 11 Hango Δ Koto antibody are two heavy @ (H1) and two light g (L
Each chain consists of a variable region (V region) and a constant region (C region).If the base sequence or amino acid sequence of the human antibody gene is known, chemical can be synthesized into In addition, for those that are only partially elucidated or that are unsuitable for chemical synthesis, chromosomal DNA can be obtained from cells derived from various human organs according to known conventional methods.
There are methods of obtaining Tll RNA directly, methods of first obtaining Tll RNA, and then preparing cDNA. Here, the source of the human antibody gene may be any cell derived from a human organ, but preferably one derived from lymphoid cells.

In order to prepare a gene encoding a variable region having specificity for a specific antigen, a hybridoma producing a specific antibody having a gene in which the rearrangement of the antibody gene has been completed, a cell established by a transformation method, Myeloma cells, etc. whose antigens are known are preferred. Generally, undifferentiated antibody genes include the vo gene, D gene, and J gene, which constitute the gene encoding the H chain variable region, and the C8 gene, which encodes the constant region. They are arranged apart, and these form a D-J bond, then a V-D-J bond, and then V.

-DH-JH-CH, and the expressed gene is established. Subsequently, V-J coupling occurs between the VL gene encoding the L chain variable region and the JL gene with respect to the gene encoding Lfi, and finally the expression type gene of type or λ type LH is established. Therefore, the preparation of genes encoding the variable regions described above is obtained from cells after such expressed antibody genes have been established.

On the other hand, when obtaining a gene encoding a constant region (referred to as CH gene for Hfi and cL gene for Lfs), the source may be any human-derived cells or an established lymphoid cultured cell line. In this case, it is not necessarily necessary to use a cell line that produces a specific antibody.

The CL gene has a C gene and a Cλ gene that code for the ni-type and λ-type chains, respectively, and the co gene encodes the μ chain, δ chain, γ chain, ε chain, and α chain that determine the antibody class. cli gene, Cε gene, Cγ gene, C
There is an ε gene and a Cα gene. Therefore, in the present invention, any type of L chain gene can be constructed by linking either the Cγ gene or the C gene with a variable region gene of interest, and on the other hand, any type of L chain gene can be constructed from the Cμ gene to Any type of H chain gene can be obtained by linking any of the Cα genes with the desired variable region gene.

The IgG class antibody, or γ immune gene, is γl in humans,
γ2. γ5. There are four known types of γ, IgG+ and Tg, respectively, by linking the variable region genes.
There are antibodies with different constant regions: Gz, IgG*, and IgG. Because these constant sites are different, IgG
It is known that the properties of the
Cos+prehensive Immunology
, 5, PP187, PLENUM MEDICAL
BOOK COMPANY, N, Y, 8ND LON
DON (1987), la++unology in
Medicine, PP50. (1983) Grune
and 5 Tratton, London), for example, the complement fixation ability is strong in IgG and IgG2, but weak in IgG2, and it is not observed in IgG, and the affinity for monocytes and neutrophils is not found in IgG and IgG. It is seen in some cases, and hardly seen in others. Therefore, by producing a specific subclass antibody, it is possible to supply the specific subclass antibody for therapeutic purposes.

Furthermore, combinations of these various L chain genes and H chain genes can be selected and expressed within the same cell to secrete the antibody.

b) Cloning It is usually convenient to clone the above-mentioned L chain gene and Hg gene by inserting them into separate cloning vectors.
A is extracted, cut using conventional means such as restriction enzymes and shearing force, and inserted into cloning vectors such as plasmids pBR322 and ptlc13 according to conventional methods. Create a gene library by transforming
This flycatcher library is then screened by conventional means, such as colony hybridization.

In other methods, R
PolyA'' RNA is concentrated by extracting NA and treating this extract with an oligo dT column. Next, using this polyA'' RNA as a template, a first cDNA complementary to this is extracted using a conventional method. Then, after decomposing and removing the template RNA, a second cDNA is synthesized using the first cDNA as a template.
Synthesize NA and insert it into the vector to create double strand c
Obtain recombinant DNA containing DNA. A gene library is created by transforming E. coli with this plasmid, for example. Next, by screening this library as described above, the desired cD
Plasmids containing NA inserts can be selected.

C) ■Appearance reward? ≦Sword” H chain gene and L cloned as described above
The H gene may be inserted into separate expression vectors and the host cells may be transformed with the two expression vectors, or both the H chain gene and the Lth\ gene may be inserted into the same expression vector and this one A host may be transformed with a different type of recombinant.

In either case, any vector that satisfies the following points can be used.

i) It is possible to produce the desired antibody gene DNA and recombinant specimens.

ii) Ability to proliferate within the target host cell or integrate into the host chromosome.

iii) It can be introduced into target host cells, and iv) It can specifically detect or select cells containing recombinant DNA.

There are a wide variety of vectors that satisfy these points, examples of which include the SV40 series, psV2
neo, psV2gpt, psV3neo,
pSV3gpt, pSV5neoP.

SV5gpt, pCD-XP, 5L-TK;
As a BKV system, pBKTK-1; as a BPV system,
pBPVssT(52-1), pBPUssT-S
VzgDt.

pMLIIPU-11TK; as a 7 deno system, d-
SVR1-26.

ΔEl/d1309; pHEB as an EB virus system
o; pP2-103, DGX as 11sV system
58; '7 Cuccinia '7 As an virus system, p
HBs4; As a retrovirus system, pZIP-Ne
oSV(0), pTK-SNV; pMusV-
Examples include vectors using gpt and retroviruses.

Methods for producing recombinant DNA are known, such as (1) a method using restriction enzymes; (2) a method in which sticky ends or blunt ends produced by cutting double-stranded DNA are joined together with DNA ligase; and (2) a method using terminal transferase. In this method, single-stranded nucleotide tails that are complementary to each other are added to the ends of the vector DNA and foreign DNA, respectively (for example, polyAM to the vector, polyT strand to the foreign DNA), and the single strands of both are linked to each other. In one example of the present invention, a recombinant DNA containing an H chain gene and a recombinant containing an L chain gene were constructed. More specifically, it will be described in Examples.

d) Admiration (Yoshiki e-1) There are several known methods for introducing a target gene into animal cells into a vector that can be expressed in animal cells, including the following: However, in reality, the conditions on the host and vector sides must match.
It is necessary to select an appropriate introduction method depending on each combination.

i) Calcium phosphate-DNA complex method (calcium phosphate method) (M, 141g1in et al, Ce
1l.

25 (1978)), ii) Protoplast fusion method (1'1.5 chatfr
er, Proc.

Natl, ^cad, sci, Us^, 77.1980
), 1ii) DEAE-dextran method CG, Mi
1ean, M, lIerz-berg, So+ma
Lic Ce1l Geneti, 7161 (198
1)), iv) Microinjection method (Microinjection method) [^, Graessman et al., Methods i
n Enzymol,.

Competition, 816 (1980)), V) Red blood cell ghost fusion method (T, l1no et al., Exp.

Ce1l Res, 148, 475 (1985))
, vi) Pricking method CF, Yamamoto et al., Exp, Ce11Res, 142.79 (1982)
), vii) Electric pulse method [E, Newmann et al., The EMBOJ.

841 (1982)], Ribosome fusion method [R, Fraley et al., J, Bi
ol.

Chew+, 255, 10431 (1980
) ), etc. are possible methods.

All methods for selecting cells into which a target gene has been introduced (transformed cells) from all treated cells generally involve adding markers to vector DNA that are easy to detect and select. All of the vectors mentioned are also DNA containing various markers. Vectors that can be used in animal cells include those with drug resistance markers, those using HAT medium, and those using EcogpL. As a drug resistance marker, neomycin (G418
), and the resistance gene includes aminoglycoside 3'-phosphotransferase of transboson Tn5. Regarding methotrexate resistance,
The dihydrofolate reductase gene is involved in this resistance. In either case, cells into which a vector containing this resistance gene has been introduced exhibit resistance to these drugs, thereby being able to grow in these drugs and becoming selectable. With HAT medium, HGPRT- or T
When K-cells are used as hosts, they cannot survive and proliferate in HAT medium; by introducing a vector containing the HGPRT or TK gene as a marker into these cells, transformed cells can proliferate in HAT medium and can be selected. Become. For those that use Ecogpt, mycophenolic acid inhibits IMP→XMP in nucleic acid precursor synthesis, so cells cannot proliferate unless guanine or xanthine is supplied.
uses guanine and hypoxanthin as substrates, whereas E
, Ecogpt of E. coli uses xanthine. Utilizing this difference, it is possible to select only animal cells into which a vector containing Ecogpt derived from E. coli has been introduced in a medium containing xanthine.

Select one of the host-vector systems described above, and use the appropriate introduction method described above to introduce the expression vector containing the antibody gene of interest into animal cells to obtain the desired transformant using a conventional method. be able to.

When introducing expression vectors containing such genes into animal cells, vectors containing either the H chain gene or the L chain gene may be introduced into the cells and expressed, but vectors containing both genes may be prepared in advance. This may also be introduced into cells.

In one example of the present invention, first, the vector pSV3-neo-ε containing the H chain gene was introduced into animal cells P and Ul to obtain transformant A-3. Although expression was confirmed in these cells, the presence of HM could not be confirmed in the culture supernatant (Hjl was not secreted).

Next, this A-3, 14! ! Vector pG containing the gene
31Vk-gpt was introduced to obtain transformant 8B1. When the cells were cultured according to a conventional method and the supernatant was analyzed, it was confirmed that human IgE antibodies were secreted.

In another example of the present invention, first, the vector pG31VK-neo containing the LSI gene was injected into animal cells P3U1.
transformant 28-103 was obtained. The gene was expressed in these cells, and the presence of the L chain was confirmed in the culture supernatant. Next, vectors pG3101(γ+) and pG containing the H chain gene were added to 26-103.
3102 (γ2) or G3103 (γ,) was introduced to generate transformants G3101-F and G3102, respectively.
-8 or G3103-8 was obtained. The cells were cultured according to a conventional method, and the supernatant was analyzed, and it was found that IgG human antibodies against Pseudomonas aeruginosa Fisher's serotype 4 (F) were detected.

It was confirmed that they secreted IgG and IgG.

In yet another embodiment of the present invention, the vector pH7232L-neo containing the L chain gene is first introduced into animal cell P3U1 to obtain transformant H7L35.

Note that expression was confirmed in these cells, and the presence of LM was confirmed in the culture supernatant. Next, to this H7L35, H
A transformant ++7LBH was obtained by introducing the vector pH720511-gpt containing the M gene. When these cells were cultured according to a conventional method and the supernatant was analyzed, it was confirmed that anti-Pseudomonas aeruginosa antibodies were secreted.

Therefore, in one aspect of the present invention, one of the vectors containing each gene prepared in advance is used to
After the host is transformed, further transformation can be performed using the other vector to obtain a transformant that produces the desired antibody.

e) If a Tora Ikusaiki α Futsuyu transformant is obtained, this cell can be cultured under normal cell culture conditions that allow the host itself to be cultured (conditions that further include a selection agent if necessary). . Therefore, it is possible to culture the transformed cells according to the culture conditions of the host cells, and to obtain the desired expressed and secreted antibodies or fragments thereof from the culture supernatant. For example, R.P.
A complete MI-1640 medium containing 10% fetal bovine serum can be used. Furthermore, if necessary, the culture conditions can be changed to serum-free culture, which facilitates the subsequent recovery and purification of the products (human antibodies and fragments thereof). This serum-free medium is manufactured by KC Co., Ltd.
C20008/Fujirebio HBIOI■, HB102@
, HB103L9, HB104 [F], Sanko Pure Chemical Industries 1o, Funakoshi HL-1@, LKB ULTR
OSER-G”, Boehringer Mannheim Yamanouchi Company,
Nutridomo-HU', Nutridoma
-5P@, etc. can be used. Radioimmunoassay (R1^) and enzyme-linked immunosorbent assay (ELIΔ) are used to confirm the detection of secreted antibodies.
can be used.

Furthermore, the antibody or its fragment can be recovered from the medium in the same manner as in the usual antibody purification process.

That is, the appropriate ammonium sulfate saturation level of the culture supernatant is 4.
Precipitation recovery of the antibody protein by 0 to 60% (preferably 50%), purification using an ion exchanger, and purification using gel filtration are possible.

In addition, it is possible to purify using DEAE-^ff1Gel Blue■, high performance liquid chromatography, and the like.

〔Effect of the invention〕

According to the method of the present invention, safe human antibodies can be stably produced.

According to the method of the present invention, the gene encoding the V chain having the desired specificity and the gene encoding the constant region determining any class or subclass can be linked, so that the desired specificity can be achieved. Humanized antibodies of any class and subclass can be produced.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

As an example of a gene encoding the human H chain C region, the ε1 gene derived from human sperm was cloned, and its expression was confirmed.

L Itsuji N @ Tool 1 2% 2-mercaptoethanol (2-M
E), 0.01M Tris-HCl buffer (hereinafter referred to as Tris-
11C1) pH8,0,0,1M NaCl1,0,0
Solution 14 consisting of Company M EDT^ and 0.5% SDS
m1 was added and kept at 50°C for 30 minutes. Next, 10-tease K (Boehringer Mannheim) was added to this solution to give a final concentration of 0.2 mg/μl, and after reaction for 2 hours, water-saturated ethanol was added, and then phenol was added. After recovering the aqueous phase by centrifugation, 2.5 times the volume of ethanol was added to obtain a precipitate (gene). And this precipitate is 0.01MTris
-IC1' (plI8.0), 1mMEDT^ solution 1
ml (330 μg/ml!).

Next, 50 μg of the above gene was digested with restriction enzyme Ram II (Takara) [hereinafter, the restriction enzyme is underlined and written as Baa III. ] Digested with 80 units.

Then, 10-40% sucrose density gradient centrifugation (Hitachi RPS2
7-2, 2) krp transfection, 24 hours) to perform size fractionation of gene fragments. Parts of both obtained fractions were subjected to agarose gel electrophoresis, and both fractions containing a fragment of approximately 3 kbp (
1,76 Mg/lal) (hereinafter referred to as "rBa+sHI fragment") was recovered. Note that both portions contain the human ε1 gene fragment (The EMBO Journal
al., 1.655 (1982)).

2. -Ibra1- Plasmid pBR322 (Feikoken Article No. 235; however, deposited in E. coli as 12C600 (pB8322))
After 3 μg was digested with 13 Mm Hl, it was treated with alkaline phosphatase (Takara) at 65° C. for 30 minutes. Next, perform phenol extraction twice and ether extraction five times to prepare a cloning vector according to a conventional method.

This cloning vector and the above Ram HIUr piece (molar ratio 1:1) were treated with T4 DNA ligase (Takara).
was used to obtain recombinant DNA. Next, using this recombinant DNA, we injected E. coli with 12C600
No. 5) was transformed using the Kushner method (Genetic E
ngi-neering, 1978.17 (1978)
). and 2.5X10' CFU/μ
gDNA Live-7! I got J-.

A@41 Based on the above The ENBOJourna L chain, the 21mar oligonucleotide shown below was synthesized using the solid phase synthesis method (Nucleic Ac1ds Research,
8.5491 (1980)). In addition,
This fragment corresponds to the 3'-untranslated region of the human ε1 gene.

5'-TCCCAGG to CTCCATCCAGCT
Screening for the ε gene was carried out using the above nucleotide as a probe according to the method described below.

, 20 pmol of the above probe was added to γ-32P, ^TP(N
EN) 90 μCi and T4 polynucleotide kinase (
Boehringer Mannheim) was used for 32p labeling to obtain 3 x 10'cps/20 picomole of the labeled compound.

Then, colony hybridization was performed using this to obtain a desired transformant. Next, after preparing a plasmid from this transformant, the restriction enzyme cleavage pattern was confirmed and the Maxam-Gilbert method (Methods i
n Enzymology, 65, 499-560 (1
The nucleotide sequence was determined by 980)) and found to be consistent with the EMBOJournal, confirming that the plasmid contained the human ε1 gene. The plasmid obtained here is pOsDE234 (
It is called FIG. 1 a). In the figure, the shaded area is ε, the gene, and C111 to CH4 indicate the domains of the constant region.

B, VDJ -;,,' 5KO-007 (U-268^R1) [^TCCNo,
CRL8033) 10” to T, Maniati
The method of S et al.
Approximately 1 g of cellular DNA was obtained according to BJ-^, Laboratory Manual 1).

2. DNA Library 101 20 μg of the above cell DNA was digested with 24 units of 5alI (Takara) and 18 units of EcoRI (Takara), and then subjected to 0.4% agarose gel electrophoresis (40V).

1.5 hours). After collecting the gel near Skb, 3.4μg DNA of one company was extracted by electroelution.
was recovered.

On the other hand, plasmid pUc13 (Pharmacia) 10μ
After digesting 1 and 5a11 of DNAUrg, the longer DNAUr piece (3
, 4 μg/ml") was collected. Then, in the same manner as above, this fragment was ligated with the above cell DNA to obtain recombinant DNA, and this was used to transform 12C600 into Escherichia coli.
Transformants (8×10′ CFU/μg DNA) were obtained.

Shizuko: Ta1mimachi [Bam the plasmid pO5DE234 of 20ttg]
After digestion with HI (Takara) and Nippon Gene), 6% polyacrylamide gel electrophoresis (100V,
DNA fragments (approximately 1 μg) were collected according to a conventional method. Next, Nick Translation Killa I-(
A probe was prepared by labeling this fragment with co2P (3 x 10'cpm/ugD N
A).

4. Screening for VDJ E Hybridization was performed using the above probe from about 2,500 transformant colonies to obtain positive clones.

After preparing 1 rasmid in the same manner as above, the restriction enzyme cleavage pattern was confirmed and the base sequence was determined (these results are published in The EMBO Journal, Pr.
oc, Natl, ^cad, sci.

USA, 79.3834 (1982), 1bid, 79
．． 6661 (1982). ) It was confirmed that the target VDJ gene was present. The plasmid containing this VDJ gene is hereinafter referred to as pF208 (Figure 2).

In the figure, C and LV mean the following.

C: Part of the constant region (Cε,) LV, leader (signal) sequence region and variable region plasmid pOsDE2341 μg and plasmid p
Bam1[I
After digestion with T4, these were mixed at a molar ratio of 1:1 and T4
Treated with DNA ligase. The plasmid pOsDE12341J has the ε1 gene in the same direction as the Nac Z gene of pHc13 (Fig. 1b).
) was obtained.

Next G: pOSDE1234U 10 μg, pF2
After digesting 0810 μg each with EcoRI and 5alt, 0.6% agarose gel electrophoresis (60V,
2 hours), one piece of DNA was recovered from each plasmid.

pOsDE1234U Karaha e + 3fl gene (3μ
g>, pF208 is VD J3ft gene (5 μg
) were collected, and then these gene fragments were mixed in a molar ratio of 1:
Fragment A) to be ligated according to the conventional method in step 1.

2. Preparation of 5V3neo-ε1 pSV3
-neo (J, Mol, appl, Genet
, supra, 327 (1982); available from Ball Barb] 3 μg was digested with EcoRl and then treated with alkaline phosphatase (65° C., 60 minutes). Then, phenol extraction and ether extraction were performed to obtain DN.
Fragment A (fragment B) was recovered. Then, Fragment A was treated with EcoRl and Fragment B was ligated to obtain recombinant DNA. Then, using this, host bacteria (E
, after transforming coli 12C600),
Twelve positive transformants were obtained by colony hybridization using the probe.

After preparing a plasmid from this transformant,
Cutting patterns according to sales ■, oto I, 肚口■, and Pstl were investigated. and plasmid vector psV3-
Four plasmids were obtained in which antibody genes were cloned in the opposite direction to the SV40 T antigen gene in neo. This was then used as a recombinant for gene expression. Below, this is the plasmid psV3-neo-ε1 (Figure 3)
It is called. Escherichia coli K12C6 containing this plasmid
00/ pSV3-neo-e, was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Microtechnology Research Institute No. 8904 (FEBN
P8904).

In the figure, SV40-Tag indicates the 5V40T antigen gene, 5V40-or i indicates the SV40 replication origin, and Neo-Tag indicates neomycin resistance (an aminoglycoside antibiotic with a structure similar to gentamicin, neomycin, and nakamycin). resistant) gene, L
■, C are the leader, variable region, and constant region (C
1) is shown.

3. 20 μg of the above plasmid psV3-neo-ε was subjected to ethanol precipitation, and after washing with 70% ethanol, 500 μg of the above plasmid was subjected to ethanol precipitation.
Dissolved in 1 liter of water.

Next, after adding 250mM CaClz 500μf to this, 2805M NaCl, 50eM I
IEPES (N-2-11ydroxyethyl
p 1peraz 1ne-N'-2-ethane
sulf on 1cacid, Sigma) 1.5
Mixed solution 1 of mM NaJP04 (pff7.05)
．． To obtain microprecipitates of phosphate-calcium-DNA by continuously adding 100 μl of 0w+1 within 5 minutes (hereinafter referred to as 1 "microprecipitation"), host P, υ1# cells (mouse tumor cells; Flow Inc.) 1.5 x 10 After centrifugal washing, pe
It was set as llet. After adding the fine precipitate, the mixture was stirred for 5 minutes and then allowed to stand for 30 minutes. Next, add complete medium to this (
RPNI-1640 medium (contains 10% tallow serum) 10
After adding n+N, the mixture was dispensed into a 96-well plate (Nunc) at a rate of 0.12 ml/well, and then left overnight at room temperature. And after leaving Cenetisin'e
G418 (Gibco) 2000. ug/la L chain was added at a ratio of 0.12 ml/well and left in a carbon dioxide incubator. After 15 days, 1 well of transformant A-3 showing good growth was obtained from 96 wells.

4. Confirmation of 8-chain residue Goat anti-human IgE antibody (Cabell) 50 μl diluted 500 times in a 96-well immunoplate (Nunc)
Immobilization was carried out in proportion to the wells. Then this is PSB (-)
- After washing with Tween 20 (hereinafter used for washing), a reaction was performed for 2 hours with a sample containing IgE (50 μl/well).

After the reaction, horseradish peroxidase (II
PO)-conjugated goat anti-human IgE antibody (Cabell)
50 μm/well of the 0-fold diluted solution was added and the reaction was carried out for 2 hours. After the reaction is completed, wash with 0.1M citric acid aorr solution (pH 4,2), ^BTS (2,5mM), H
100 μl/well of a substrate solution consisting of 25 mM of 2O was added, and the reaction was carried out for 5 to 15 minutes at room temperature. 2mM Na
OD after stopping the reaction by adding N3 at a rate of 100 μl/well. ,. , was measured using Titertic Multiscan 0 (Flow Co., Ltd.), and I g E ' + H
The ε chain was detected (hereinafter referred to as "ELISA method").

The samples used here were A-3 culture supernatant stock solution, 10 times concentrated solution, and A-3 # cell lysate '(Cel l
Iysate (cell lysate) was used. In addition,
A-3# cell lysate was prepared as follows, fS, A-
38 cells were cultured in complete medium containing 800 μg/ml of G418 and 2.6×10” cells were collected.

This cell was transformed into 2X10' cells/- using PflS(-).
The concentration was adjusted to 2% (V/
V)/nidet (Np-40) to 1/1/2 of the previous cell suspension.
After adding 3 amounts (final concentration Np-400, 5%) and stirring,
Insoluble matter was removed by centrifugation. And the soluble part is PBS (-'
The dialysate was then lyophilized and water was added to give a 4 ml solution. When IgE was measured in this cell lysate and culture supernatant, it was found that IgE was found in the culture supernatant.
gE was not observed, and A-3 did not secrete Hε chain to the outside of the cells. On the other hand, regarding the A-3 cell lysate, a considerable amount (6,4n
The presence of gI gE/10@cells) was observed. This confirmed that pSV3-neo-C1 was expressed and synthesized the ε chain in A-3 cells, but was not secreted outside the cells.

The Lfi gene of an anti-Pseudomonas aeruginosa human monoclonal antibody was prepared and secreted and expressed using genetic engineering techniques.

A. Monoclonal antibody-producing cells G3 against Pseudomonas aeruginosa F4 according to the method disclosed in JP-A No. 61-91134.
1 (for IgG2/).

1X10'' G3-1 cells were cultured in 10 M street solution (0.5
After treatment with MEDTΔ (pl+8.0 > ioo μg/ml protease K (Boehringer Mannheim) and 0.5% sarcosyl at 50°C for 3 hours, an equal volume of water-saturated phenol was added. Then centrifugation (8000 x g, 1
0 min) to separate the aqueous phase, which was mixed with 50 mM) Tris-1ic1 (pH 8,0),
Dialyzed against 10mM EDT^, 10mM NaCN. This was heated or treated with bonuclease A (Boehringer Mannheim) to a final concentration of 100 μg/l111
After processing at 37°C for 3 hours, phenol extraction and dialysis were performed again as above to obtain 712 μg.
/ml of chromosomal DNA (G3-1-DNA) was obtained.

(2) 10 μg of the above G3-1-DNA was added to the restriction enzyme EcoRI.
Digestion was performed at 37° C. for 2 hours using either BamHI or BamHI (Tsubofuji-2). After digestion, 0.8% agarose gel electrophoresis was performed and the DNA [! The 7r piece was separated. Next, the gel obtained here was treated with 0.25N HCI.
for 15 minutes at 0.5 N Na0)l 1.5 M
0.5 M Tris-HCl (p
H17, 4) 1.5M NaCj! for 30 minutes. The DNA was then transferred to a nitrocellulose filter (S&S) using 20xSSC (0.3M sodium citrate, 3M NaCff1).

On the other hand, regarding the coding region encoding the human chain constant region (C) on chromosomal DNA, when digested with the nucleotide sequence and restriction enzyme EcoRI, a fragment of approximately 2.5 kb contains the coding region in C. It is known that there are [:
Ce1l, 22, l97-207 (1980)
).

Therefore, in order to obtain a Wangan I-digested DNA fragment containing the coding region in C, and use a nick translation kit (BRL) to use 0.5 μg of the 2.5 kb fragment as a probe
It was labeled with 32p using the following method (Company).

Then, Southern hybridization was performed between this and the above filter. The results at this time are shown in FIG.

In the same figure, the numbers indicate the length of base pairs, and EcoRI and h-U mean that they were digested using this restriction enzyme. From this result, bands were confirmed at approximately 2.5 kbρ and 8.5 kbp for the DNAI gene digested with Ilamll I and Ilamll I, respectively. It is known that chromosomal DNA without V and J rearrangement gives a band at approximately 10 kbp when digested with BamtlI (The Journal of Biologics
al Chemistry, 25, 1518-15
22 (1982), l.

Therefore, 8,5 obtained by Bam1l I treatment
The kbp DNA AlJi fragment was confirmed to be a DNA fragment containing the L chain coding region including the rearranged VJ-coding region.

After digesting 20 μg of G3-1 DNA from the ItoJ library with 10-
40% sucrose density gradient centrifugation (Hitachi RPS 40T35k
size fractionation was carried out by (rpm, 14 hours). The desired DNA fraction (rich mH) was then isolated by agarose gel electrophoresis and DNA dot hybridization.
The digested 7-9 kb fragment) was recovered (4 μg/ml>
.

On the other hand, plasmid ρυC13 (commercially available from Pharmacia) was digested with Bam11 and treated with alkaline phosphatase to prepare a linear plasmid.

Next, a reaction between this and the above DNA fraction was carried out at 4°C overnight using T4 DNA ligase (Takara Shuzo), and the obtained recombinant DNA was used to transform E. coli with 12C600 to transform G3-1. Gene library (2x 10'CF
υ/μg DNA) was created.

4) Screening of LDNA 5 x 10' pieces of the above-transformed E. coli were subjected to colony hybridization method (Molecular C1on
-ing-＾Laboratory Mannua1)
Accordingly, using the 32p-labeled human C gene as a probe, a plasmid into which a DNA fragment encoding the L chain was inserted was selected, and this plasmid was transformed into pG31V.
Named K.

5,,l:
3 of the above plasmid pG31VK with EcoRI,
Ba+*HI Oyohi 11indllll was digested with Tubon Gene Co., Ltd.) and the cleavage pattern was analyzed. In addition, the anal cavity fragment (approximately 2.9 kb) containing the variable region (VJ)
) to Hin of plasmid pUc13 (Pharmacia)
dll [cloned into the site (hereinafter the resulting plasmid will be referred to as pG31VK-H2), pG
31VK-H2 as Sst I, Pst I or Nc
ol (Pon Gene) to create a restriction enzyme cleavage map. The results are shown in Figure 5. In this figure, VJ indicates the coding region encoding the variable region, C indicates the coding region encoding the constant region, and the numbers on the top row indicate the length of the DNA fragment. Show that.

As is clear from this figure, this DNA fragment is a complete VJ
A code region and a code region are included in C.

The fragment obtained by digesting pG31VK-H2 with Pstf was helicloned into the Pst1 site of plasmid pUC13. The Ba butterfly 1 and 11indI [[ sites of the plasmid obtained by this cloning, and pG31V
K-112) After 32p labeling of Nco1 site, Maxam-Gilbert method (Methods in Enzy
The base sequence was determined by N+ology, 85499-560 (1980)).

The results of base sequencing are shown in Figures 6-1 and 6-2. These figures show the base sequence of the VJ-coding region and its vicinity. This sequence includes the 5' non-coding region, the leader sequence ), a ■-coding region (indicated by V), a J-coding region (indicated by J), and a following 3' non-coding region.

Each letter of the alphabet at the bottom of the base sequence is an abbreviation for an amino acid and has the following meanings.

N: Asn S: 5er D: Asp E: Glu C: Cys P: Pr.

L:Leu H:His G:GIy Y:Tyr V: Val M: MetI:Ile
A: AIa K: Lys Q: Gln R: Arg W: TrpF: Phe The double underlined portion in the amino acid sequence is a region required for antigen binding.

The Vg range of human immunoglobulin chains is broadly classified into four subgroups [for example, Nippon Seikagaku Shokuma, Seikagaku Data Book II p1022].

The area determined according to the present invention is suggested to belong to a subgroup. In addition, in the base number shown above in the base sequence Figure 6, the Set at position 821 is as follows:
The original subgroup ■ is the same as the subgroup ■ except that it is Phe.

The amino acids that are enclosed in the middle part of the alphabet below the base sequence are amino acids that are characteristic of subgroup ■.

psV2-gpt, or 5V2-neo (J, Noi
, ^pp1. cenet,,l.

327 (1982); obtained from Mr. Ballbarb] with Oji 1 and the fragment obtained by digesting pG31v with b-blade U- in a conventional manner to create plasmid pG31VK-gpt containing the Lfin gene, or pG31VK
-neo was obtained. It was confirmed in the same manner as in Example 1 that this plasmid contained the L chain gene. Escherichia coli (E. coli) containing this plasmid
scherichia cologne) K12C800/p
G31V-gpt has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology as FERNP-8905.

2. of;.

The above plasmid pG31VK-gpt DNA 2
After ethanol precipitation of 0 μg, 1 mM Tris,
Dissolved in 100ul of 0.1+sMEDT^. G,: 500mM CaCl2100)te was added,
80mM NaCl', 50mM HEPES, 1.5
Solution 200 containing mM Naz11PO+ (pH), 05)
μN was added dropwise over 2 minutes while stirring. Then 37
C. for 5 minutes to form a fine precipitate of DNA-Ca-phosphoric acid.

On the other hand, as a host, P, RP
After washing with Mf-1640 medium, it was made into a pellet, and the above-mentioned fine precipitate was added thereto and slowly stirred. 37℃, 1
40 complete medium containing f&10% tallow serum that had been allowed to rise for 0 minutes was added. This is 96 at 0.1 m1/well.
The cells were seeded in each well of 4 wool plates and cultured for 2 days at 37°C and 5% CO7 in a carbon dioxide gas incubator. 2
After 1 day, mycophenolic acid 10 μl/sha, xanthine 25
Complete medium containing 0 μg/la L chain was added at 0 or 1 ml/well for selection. Transformed cells that proliferated well were obtained after about two weeks (Table 1).

Further, DNA-Ca-phosphate microprecipitate 3 was prepared for pG31VK-neo DNA2C)tcg in the same manner as described above, and introduced into P, U1 cells (2×10 cells).

Thereafter, the cells were suspended in 20mj+ complete medium, plated on two 96-well plates, and incubated for 2 days. Then, Gencticin@41B was added to each well at a concentration of 800 μg/ml for selection. After about 3 weeks, transformed cells that proliferated well were obtained (Table 2).

3. Confirmation of Lori child oi super engineering LM (nimata) using ELIS method. That is, a 500-fold diluted goat anti-human (kappa) chain antibody was immobilized on each well of a polystyrene NUNC immunoblate [F], and after washing with PBS-Tween, 50 μL of a sample containing the chain was immobilized. were added and reacted. After 2 hours, after washing with PBS-Tween, HP O-conjugated anti-human chain antibody (TAGO) x 5
000 times diluted solution. 2 hours later, PBS-Tw
After washing with een, ^BTS substrate solution (0.1M citrate buffer (pH 4.2), 8BTS (2.5mM), H2O
2 (5mM)) was added to each well, 5-
The reaction was allowed to proceed for 15 minutes at room temperature. After stopping the reaction with 2mM Na and Nt, 0D4o, n was obtained using Titertic Multiscan @ (Flow Co., Ltd.).

This was taken as the chain measurement. Among the transformed cells obtained, 26 strains with good proliferation were selected, and the amount of L chain secretion in the culture supernatant was calculated using the above method. that's the first
Shown in the table. The production amount (μg/mZ) in the table is Bence-
Jones's protein was calculated as a standard substance.

Below are the margins: pG31VK-transformed cells pG31VK-gpt
- Neo transformed cells 19-31 and 2
For 6-37, cell concentration 5 x 105cells
/mf, as a result of re-measurement under 24-hour culture conditions, 19-
151μg/company for 31, huff 4ttg for 2B-37
/vat, so L te 26-103 te 35 tt g
Secretion of strands was observed in /ml.

Preparation of transformants of plasmid pG31V-gpt DNA40AL
After ethanol precipitation of g, wash with 70% ethanol,
Dissolve in 100μm' of ris-EDTA solution, add 5
00mMCaCl2100μm”!?m added, 280m
M Macl, 50mM HEPES, 1.5mM N
200 μN of a solution containing aJPO< (pH 7,05),
Add gradually with stirring. The required time was 3 minutes. Thereafter, the mixture was allowed to stand at 37°C for 10 minutes to form a DNA-Ca-phosphate precipitate. On the other hand, A-3 (P2O
pSV3-neo-εI transformed cells of 3) IXIO'
After washing the cells with RPMI-1640 medium, they were made into pellets, to which a DNA-Ca-phosphate solution was added and slowly stirred. The cells were left at 37°C for 30 minutes with occasional gentle shaking to incorporate the DNA into the cells. RPMI-1640 medium containing 10% tallow serum (complete medium >10a+i
' and further neomycin G418 500μg7
Suspend in complete medium containing the mL chain and place in 96-well plate 8.
The plate was seeded at 0.1 tan/well. After 2 days, mycophenolic acid 20μg/ml, xanthine 250ttg/
Add 0.1 ml/well of complete medium containing mL chain,
Selected. Approximately 2 weeks later, transformed cells proliferating in one well were obtained (8B 1 >, this 8B1 was treated with neomycin (: 4181000 μg/company, mycophenolic acid 2).
Good proliferation (average doubling time 15-25 hours) was shown in the presence of 0 μg/vsl xanthine 250 μg/comp.

2, 8 [Confirmation of L chain gene expression Examples 1 and 2
Regarding the 8B1 culture supernatant described in , IgE-EL
When subjected to IS^ and -ELIS^, it reacted to both and was found to be positive. The amount of antibody produced in the epidermis is 0.5 μg/ml as a chain and 0.3 μg/ml as IgE.
It was ml. In A3, the ε chain was not secreted to the outside of the cell, but in 8B1, the ε chain was also secreted to the outside of the cell together with the 2 chain, and a method for secretory production of human IgE antibodies was established.

In addition, the culture supernatant of 8μ1m cells obtained here was subjected to a conventional method (D
After purification according to EAE cellulose column chromatography, affinity column chromatography, gel filtration, etc.), the human antibodies of the present invention were purified by SDS polyacrylamide electrophoresis, and the results showed that the human antibodies of the present invention are II, L 2 type human monoclonal antibodies. was suggested.

Monoclonal antibody-producing cells G3 against Pseudomonas aeruginosa F according to the method disclosed in JP-A-61-91134.
1 (IgGz/to) was obtained.

G3-1# cells 2.5 x 10” in 10mf jllll
Medium (0,5M EDT^ (pH 8,0), 100μs/
1lZ70f7-zeK (Boehringer Mannheim),
After treatment with 0.5% Sarkosyl for 3 hours at 50°C, an equal volume of water-saturated phenol was added. Then centrifuge (8000
x g for 10 min), and this was heated to 505
M Tris-HCl buffer (Tris-tlci') (pH
8,0), 10mM ED T^, 10mM NaCl. This can be heated or treated with bonuclease A.
(Boehringer Mannheim) at a final concentration of 100 μg/
After treatment at 37°C for 3 hours, phenol extraction and dialysis were performed again in the same manner as above.
．． 1 mg of chromosomal DNA (G3-I DNA) was obtained.

2. The above G3-IDNA10. czg, restriction enzyme EcoR
I (Takara Sake Brewery) or BamHI Nippon Gene Co.)
Or at 37°C using Hindlll and Tupongene).
Digestion was performed for 5 hours. After digestion, 0.6% agarose gel electrophoresis was performed to separate DNA fragments. The gel obtained here was then soaked in 0.25N 11CN for 15 minutes at 0.25N.
5M Na011-1.5'M NaCl for 30 minutes;
0.5NTris-11CI (pH17,4) -1,
Treated with 5M Nace for 30 minutes. 20XSS in Rye
DN to a nitrocellulose filter (S&S Co., Ltd.) using C (0.3M sodium citrate, 3M NaC1).
Transferred A.

In order to prepare the probe,
A plasmid was prepared from Escherichia coli deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as ERN P-8906''), and the P of the DNA encoding the C region in this plasmid was
The vuII-Smal fragment was 32p-labeled using a nick translation kit (BRL).

Southern hybridization was performed between this probe and the above filter. The results at this time are shown in FIG.

In the figure, the numbers indicate the length of base pairs, EcoRI, 1
lindl [I is G3-1 using these restriction enzymes.
This means that the DNA of From this result, E
For the DNA genes digested with coRI and old ndII, bands were confirmed at positions of 18.5 kbp and 6.2 kbp, respectively.

After digesting 100 μg of G3-1 DNA from the L'ni library with EcoRI, size fractionation was performed by 0.6% agarose gel electrophoresis. Cut out the gel around 20 kbp and collect the DNA (12 μg). On the other hand, EMBL4 (Funakoshi, E
MBL 4 kits) [lamll I, EcoR
The mixture was digested with I and a portion of the stuffer was removed according to a conventional method.

Next, a reaction between this and the above DNA fraction was carried out at 4°C overnight using T4 DNA ligase (Takara Shuzo), and the obtained recombinant DNA was packaged into phage coat protein (PromeHa 13iotec (using a manufacturer's kit).

The result is 2.6X10 pfu/t-t gEMBL
A library of 4 DNA was obtained.

4 Screening of HDDNA Using the packaged recombinant phage, Braak hybridization method [book Molecular Cl
oning, ^Laboratory Mannua1
), using the above-mentioned co2ptM-recognized G3-1 cDNA fragment as a probe, the DNA encoding the I chain was detected.
Select the phage into which the fragment has been inserted and insert this phage into #
It was named 383.

The above phage #383 was digested with the restriction enzyme EcoRI, B
amHI, and l1indllll)
and analyzed the cleavage pattern. In addition, the EcoRT-1lindl fragment containing the variable region (VDJ) (
Approximately 4.8 kb) was cloned into the EcoRI and l1ind■ sites of plasmid pUc13 (Pharmacia) (hereinafter the resulting plasmid will be referred to as pGVl). pGVL chain with restriction enzymes Dral, Pstl
(Boehringer Mannheim), XI+ol (Takara Shuzo), and Smal (Ppon Gene) to create a restriction enzyme breakpoint map. The results are shown in FIG.

In this figure, VDJ indicates a code region encoding a variable region, and C10 indicates a code region encoding a constant region. The number at the top indicates the length of the DNA fragment.

As is clear from this figure, this DNA fragment is a complete VD
It comprises a J code region and a Cγ2 code region.

psV2-gpt or pSV2-neo with EcoRI
The digested fragment and #383 phage DNA were
The fragments digested with I are ligated according to a conventional method to create a Boo7 smid pSV2g 72E or psV2 containing the H chain gene.
nγ2E, also psvz-gpt or pSV2-
A fragment obtained by digesting neo with EcoRI and BamHI and a fragment obtained by digesting #383 with EcoRI and BamHI were ligated according to a conventional method to create a plasmid p containing the H chain gene.
sV2g 72EB or psV2n r 2EB was obtained.

Then, this plasmid was prepared in the same manner as in Example 4.
It was confirmed that H chain was included.

Plasmid pSV2nγ2EB obtained by the method (C) above
DNA gene g was introduced into L chain gene transformed cells 19-31 (3×10' cells) by the same method as in (A) above. Genetic in@G418 in 2 days
(Gibco) was added at a concentration of 500 μg/le. Transformed cells were obtained about 3 weeks later (3rd
table).

Similarly, psV2n72E201-1g was added to 26-6
6 cells' (3 x 10'' cells) to obtain transformed cells (Table 2). Next, 20 tt g (7) pSV2g
r2E was introduced into 26-103 (1.5 x 10 cells). Two days later, mycophenolic acid was added at 10 μg/ml and xanthine was added at 250 μg/ml for selection. Transformed cells were obtained after about 2 weeks (Table 3).

20 μg of plasmid FpG31VK-gpt DNA obtained by the method of Example 3 (B) and Example 4 (C) and 20 μg of psV2g72E (1) DNA were mixed,
After ethanol precipitation, 1+*M Tris, 0.1 mM
Dissolved in EDT^iooμx. It was introduced into p3UI cells (1.5 x 10' cells) in the same manner as in the method (A) above. After 2 days, mycophenolic acid 10μg/11L
Selection was made by adding xanthine at a concentration of 250 μg/ml. Transformed cells were obtained after about 2 weeks (Table 3).

The reactivity of the antibody against type 1 Pseudomonas aeruginosa Fisher serotype 1 (F4) was confirmed using the ELIS method.

Regarding the detection method of anti-Pseudomonas aeruginosa antibodies, please refer to JP-A-61-911.
The method described in Publication No. 34 was used.

Each well of a polystyrene INUNc immunoplate 0 was coated with voryley lysine (10 μg/va1),
After washing, use formaldehyde-treated F4 type bacterial solution (5 x 10
50 μL chains (1/ml) were added and centrifuged at 3500 rpm for 15 minutes to collect bacterial cells on the well surface. A 50 μL chain of glutaraldehyde (0.5%) was further added thereto, reacted at room temperature for 15 minutes, and then washed. This well is 1
% BSA and 100 mM glycine in PBS (-) to prepare an anti-Pseudomonas aeruginosa antibody detection plate.

Pour 50μ of the sample containing anti-Pseudomonas aeruginosa antibody into each well of this plate! and reacted for 2 hours.

Then, after washing the plate three times with PBS-Tween, HPO-conjugated anti-IgG antibody (TAO Company) 500
50 μL of the 0-fold diluted solution was added and reacted. 2 hours later,
After washing 5 times with PBS-Tween, IITS substrate solution 1
00 μL chain was added to each well and allowed to react for 5-15 minutes at room temperature. After stopping the reaction with 2mM NaN, the OD was determined by Titertic Multiscan e (Flow Co., Ltd.).
°snm was measured.

Anti-Pseudomonas aeruginosa human IgG was detected in the culture supernatant of each transformed cell using the above ELIS^.

As shown in Table 2, the OD A of the transfected cells measured as a control. ,. , was 0.15-0.21, whereas in the culture supernatant of transformed cells, it was 0.38-1.
Humanized antibodies against P. aeruginosa types 36 and F4 were detected, and a method for secretory production of anti-P. aeruginosa human antibodies was established.

Note that the plasmid pSV2. Escherichia coli E containing γ2E, coli K12C600 (pSV2-gy 2
E) is FERN P-90
36) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Margin below Plan-”=”1 ★As a control, measure the culture medium of the following cells.

P3 old culture supernatant 0.16 19-31 Horn 0.1526-86
Not O, 172B-103 Noah
0.21 Takahashi et al.'s method (Cell, 2
9, 671-679 (1982)), the human antibody γ2. γ2 and γ genes were obtained. 1 μg of these genetic reserves was further treated with restriction enzymes 11indll and B.
am1l I, and this was transformed into vector pUc1
3 into the same restriction enzyme cleavage point to create Cγ1-containing plasmid pG101 and Cγ2-containing plasmid pG10, respectively.
2 and Cγ3-containing plasmid pG103 was constructed. These plasmids were prepared using restriction enzymes Sma1 and Bi as usual.
The correctness of the structure was confirmed using Gl ■, Hindll, etc.

Next, the plasmid pGVI (G3-11[
plasmid containing g gene) with restriction enzyme 11indll.
3 μg of DNA fragment containing the VDJ region was cut with l and EcoRI and subjected to 0.8% agarose gel electrophoresis.
was recovered. On the other hand, the plasmid pG101 mentioned above
.

Restriction enzyme old nd from pG102 and pG103 respectively
DN containing C10, C10 and Cγ by cutting with l[I and Bam1lI and electrophoresing on 0.8% agarose gel.
Next, the expression vector pSV2-gpt (donated by Mr. Ballbarb) was cut with restriction enzymes EcoRI and 13 amlll, and the above pGV1-derived VDJ DNA and pG1
01-derived Cr + Alui contains pG102-derived Cγ2 or pG103 Yamato Cγs DNA at a weight ratio of 2
:1:2, and reacted with T4 ligase in the usual manner to obtain H chain expression plasmid pG310.
1-gpt, pG]02-gpt and pG3103
-gpL was obtained. pG3101-gpt obtained here
contains VDJ region DNA and Cγ1 gene, pG31
02-FIpt contains VDJ region DNA and the Cγ2 gene, and pG3103-gpL contains VDJ region DNA
and Cγ, the restriction enzyme Sma1 containing the gene
, HindI[I, EcoRI, and BamH
It was confirmed using I.

X1 Takumiori humanoid ゛2 20Bg pG3101-gpt, pG3t02-
2B- gpt or pG3103-gpt, respectively.
103 (1,5×10' pieces) were introduced. After 2 days, mycophenolic acid 10 μg 7 ml and xanthine 2
It was selected by adding 50 Bg/company. Transformed cells were obtained after about 2 weeks. (Table 4) Escherichia coli containing plasmid pG3101-gpt
(pG3101-pgt) is the microtechnical research institute serial number 9289 (
FERM P-9289) and plasmid p
Escherichia coli containing G3103-gpt (pG310
3-pgt) is FERN P
-9288) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Dan-5MJljJJ human type 7 ■ 11 present! The reactivity of antibodies against P. aeruginosa Fisher serotype 4 (F4) was confirmed using the ELTS method.

Regarding the detection method of anti-Pseudomonas aeruginosa antibodies, please refer to JP-A-61-911.
The method described in Publication No. 34 was used.

Each well of a polystyrene NUNC immunoplate 0 was coated with voryleyridin (10 μg/ml), washed, and treated with formaldehyde to form an F4 type bacterial solution (5 x 10').
Cells were collected on the surface of the wells by adding 50 μL chains (cells/ml) and centrifuging at 3500 rpm for 15 minutes. To this, 50 μr of glutaraldehyde (0.5%) was further added and reacted at room temperature for 15 minutes.
It was blocked with PBS (-) containing SA and 100geM glycine to prepare an anti-Pseudomonas aeruginosa antibody detection plate.

Pour 50μ of the sample containing anti-Pseudomonas aeruginosa antibody into each well of this plate! and reacted for 2 hours.

Next, after washing the plate three times with PBS-Tween, HPO-conjugated anti-human IgG antibody (TAGO) 500
0x diluted solution 50μ! Alternatively, anti-human IgG
Antibody, anti-human IgG, antibody, anti-human IgG 3 antibody (Bio-Yeda) and 50 μL chain of HPO-conjugated anti-mouse antibody (KPL) were added and reacted. After 2 hours, the reaction was carried out with P[lS-Tween. After washing 5 times, 100 μL of BTS substrate solution was added to each well and reacted for 5-15 minutes at room temperature. After stopping the reaction with 2mM NaN+, the OD4 was determined by Titertic Multiscan @ (Flow Co., Ltd.).
°snm was measured.

Anti-Pseudomonas aeruginosa human IgG was detected in the culture supernatant of each transformed cell using the above ELIS^.

Some of them are shown in Table 4.The measured values in Table 0 are 40
This is the OD value at 5 nm. In this measurement, all control values ranged from 0.14 to 0.17. Therefore, in the culture supernatant of transformed cells, F4 is 0.24 to 0.85.
Human antibodies of different subclasses against P. aeruginosa were detected, and a method for secretory production of anti-P. aeruginosa human antibodies was established.

The following is a margin plan: Transformed cells C, G3101-F, C3102-8, and 3103-8 with pG3101-gpL, pG3102-gpt, and pG3103-gpt.
The 1g G (IgG+, tgc,
, IgGs) was measured using the ELIS^ method. That is, goat anti-human IgG diluted 500 times was added to each well of a polystyrene NUNC immunoplate.
After immobilizing the antibody (CTAGO) and washing with PBS-Tween, the antibody was immobilized (3101-F, 3102-8).
50 μL of the culture supernatant of G3103-11 was added and reacted. On the other hand, to create a standard curve, standard human IgCI, 1gG2 or IgG was added to the same plate instead of the culture supernatant.
s (each from 5EROTEC) was added to react by adding 50 μL chains of 1 gg and 7 ml of serially diluted samples. 2 hours later, P.
After washing with BS-TWeen, HPO-conjugated anti-human Ig
It was reacted with a 2500-fold dilution of G antibody (TAGO). After 2 hours, wash with PI) S-TMeen and transfer to BTS.
Substrate solution (0.1M citrate MiI solution (pH 4, 2),
0TS (2,5giM), H,0□(5-M))100
μL chains were added to each well and allowed to react for 5-15 minutes at room temperature. After stopping the reaction with 2mM Naps, 5LT2
10 (SLT-LABINSTRUNENTS) G:
.

0Dnosna was measured from [gG+, IgGz
and IgGs were calculated. It is shown in Table 5.

The anti-Pseudomonas aeruginosa human monoclonal antibody L gene was prepared and secreted and expressed using genetic engineering techniques.

'' Monoclonal antibody-producing cells H7-2 (I g Gz/ni) against Pseudomonas aeruginosa Honma serotype type II (H1) were obtained according to the method disclosed in JP-A No. 61-91134.

One H7-2al cell (1 x 10') was placed in 10 ml of M street solution (
0.5M EDT^ (pH 8.0), 100μg/ml
Protease K (Boehringer Mannheim), 0.5
After treatment with % Sarkosyl for 3 hours at 50°C, an equal volume of water-saturated phenol was added. Then centrifuge (8000xg,
10 minutes) to separate the aqueous phase, which was then mixed with 50 mM Tris-HCl buffer (Tris-HC1) (pH 8,0), 1
Dialyzed against 0mM ED T^, 10mM NaCl. This was heated and bonuclease A (Boehringer Mannheim) was added to the final concentration of 100 μg/infusion, and after treatment at 37°C for 3 hours, phenol extraction and dialysis were performed again in the same manner as above to yield 710 μg. /i chromosomal DNA (H7-2-DNA) was obtained.

2 10 μg of the above H7-2-DNA of the L chain DNA was treated with restriction enzyme E.
coRI or Ba…! Digestion was carried out at 37° C. for 2 hours using one of the following methods: After digestion, 0
．． DNA fragments were separated by 8% agarose gel electrophoresis. Then, the gel obtained here was treated with 0.25N HCl for 15 minutes, 0.5N NaOH-1, 5M NaCl
0.5 M Tris-11cj for 30 minutes! (pH
17,4)-treated with 1,5M NaCl for 30 minutes. Then 20XSSC (0.3M sodium citrate, 3M
using a nitrocellulose filter (S
& S Company).

On the other hand, regarding the coding region encoding the human chain constant region (C) on chromosomal DNA, when digested with the nucleotide sequence and restriction enzyme EcoRI, a fragment of approximately 2.5 kb contains the coding region in C. It is known that there are (C
ell, 22.197-207 (1980)).

Therefore, EcoR (EfH D) containing the coding region in this C
To obtain the NA@ fragment and use the nick translation kit (
32p labeling using BRL).

Then, Southern hybridization was performed between this and the above filter. The results at this time are shown in FIG. In the figure, the numbers indicate the length of base pairs, and EcoRI and Bamll I mean that the restriction enzymes were used for digestion. From this result, oRI and Bam1l
For each DNA gene digested with I, approximately 2.5
Bands were confirmed at kbp and 8.5 kbp. In addition, the chromosomal DNA in the case without VJ rearrangement is Bam1
Ili digestion is known to give a band at approximately 10 kbp (The Journal of Ili.
Logical Cbemistry, 257.15
16-1522 (1982)).

Therefore, 8,5 obtained by 13amll I treatment
The kbp DNA fragment was confirmed to be a DNA fragment containing the L chain coding region including the rearranged VJ-coding region.

(Incorporated Foundation)) Preparation of 4-gene library - After digesting 10 μg of H7-2-DNA with Genus I, 0.
Size fractionation was performed by electrophoresis using 8% agarose gel. Then agarose gel electrophoresis and DN
Target D by A dot hybridization
The NA fraction (1) amHI-digested 7-9 kb fragment) was collected (1.1 μg/ml').

On the other hand, plasmid pυC13 (commercially available from Pharmacia) was digested with Bam1l I and treated with alkaline phosphatase to prepare a linear plasmid.

Next, the reaction between this and the above DN'A fraction was performed using T4DN.
A ligase (Takara Shuzo) was used at 4°C overnight, and the resulting recombinant DNA was used to transform Escherichia coli with 12C600 to transform the H7-2 gene library (2.5X 1
0'CFU/μgDNA) All preparations.

Screening of 4 L DNA 5 x 10' pieces of the above-transformed E. coli were subjected to colony hybridization method (Molecular CIoni
ng-^Laboratory Manual), the 32P-labeled human C gene was used as a probe to select a plasmid into which a DNA fragment encoding LM had been inserted, and this plasmid was incubated at pH 7232.
Named it L.

5th grade 6th grade 9. In Figure h, the above plasmid pH7232L is Bam1, Bam11
I and 1lindllll were digested with PPONGENE Inc.) and the cleavage pattern was analyzed. In addition, the variable region (
VJ) was cloned into the HindI [I site of plasmid pUc13 (Pharmacia). !, Digested with Ei or Ncor Nippon Gene Co., Ltd.),
A restriction enzyme cleavage map was created. The result at that time is the 19th
As shown in the figure.

In this figure, VJ indicates the coding region encoding the variable region, C indicates the coding region encoding the constant region, and the number at the top indicates the length of the DNA fragment.

As is clear from this figure, this DNAffr piece comprises a complete VJ coding region and a C coding region.

A fragment obtained by digesting Deaf L1'1υmu Maruyake pH7232L-6 with Psti was helicloned at the Pst1 site of plasmid pUc13. The mHi and Hindln sites of the plasmid obtained by this recloning, as well as the pH 232L
After 32p labeling of the Nco1 site of -6, Maxam
Methods in Enzymol
ogy, 65499-560 (1980)).

The results of base sequencing are shown in Figure 20. These figures are V
The base sequence of the J-coding region and its vicinity is shown. This sequence includes a 5' non-coding region, a leader sequence, a -coding region, a J-coding region, and the following 3' non-coding region.

The single letter alphabet at the bottom of the base sequence is the abbreviation for the amino acid, as described above.

Also, from the amino acid sequence, this variable region can be found in the Japan Biochemical Diet, Biochemical Data Book I, pp1022, E, ^.

5equence orPro edited by Kabat et al.
teins of immunological In
terest (1983) (National In
5fi-tute of Ifealth, Beth
esda) belongs to group ■.

pSV2-neo [J, No I, ^pp1. G
encit, 1.327 (1982) obtained from Mr. H. Bohlberg] was digested with Ba+*III I, and a fragment obtained by digesting pH7232L with Bam1l I was ligated according to a conventional method to obtain a fragment containing the L chain gene. Plasmid pH
7232L-neo was obtained. It was confirmed that this plasmid contained the L chain gene using the same method as in Example 8-A.

20. (5, - of - 20μ of DNA of the above plasmid pH7232L-neo
After ethanol precipitation of g, 155M Tris, 0.1m
Dissolved in MEDT^100μN. 500m to this
Add 2100 u 1 M CaC4', 80 m
M NaCl, 50mM HEPES, 1.5mM Na
200μm of liquid containing 211r'0<(p117.05)
was added dropwise over 2 minutes while stirring. Then 37℃5
The mixture was allowed to stand for a minute to form a fine precipitate of DNA-Ca-phosphoric acid. On the other hand, as a host, P, U14 cells (5 x 10' cells)
was washed with RPMI-1640 medium and pelleted,
The above-mentioned fine precipitate was added to this and slowly stirred.

After incubating at 37° C. for 10 minutes, 40 ml of complete medium containing 10% tallow serum was added. This was inoculated at 0.1 ml/well into each well of four 96-well plates, and cultured in a carbon dioxide gas incubator at 37° C. and 5% CO□ for 2 days. Two days later, Geneticin'C418 was added to each well at 800 Mg/l and selected. After about 3 weeks, transformed cells that proliferated well were obtained (Table 6).

3. L-no! Lfi was confirmed using the ELIS method. That is, goat anti-human diluted 500 times (
Kappa) chain antibody was immobilized, and after washing with PBS-Tween, 50 μL of a sample containing the Kappa chain was added thereto for reaction. After 2 hours, after washing with PBS-Tween,
HP O-linked anti-human chain antibody (TAGO) x 50
It was reacted with a 1:00 diluted solution. After 2 hours, PBS-Twe
After washing with en, BTS substrate solution (0.1M citrate buffer (pH 4°2), 8BTS (2.5mM), HzOz
(5mM)) was added to each well and 5
- 15 minutes at room temperature. After stopping the reaction with 2m, M NaNi, titertic multiscan 0 (
The chain was measured by measuring the OD4°snm using Flow Co., Ltd.). Among the transformed cells obtained, 10 strains with good proliferative properties were selected, and the L[ni type in the culture supernatant was measured by the method described above.

This is shown in Table 6. The measured value in Table 6 is the OD at 405 na+.

Transformation cells (1-1
Monoclonal antibody-producing cells H7 against Pseudomonas aeruginosa H1 were prepared according to the method disclosed in Japanese Patent Application Laid-Open No. 61-91134.
-2 (IgG2/) was obtained.

I x io” H7-2 cells were cultured in 10 different buffers (
0.5M EDT^ (pH 8.0), 100μg/r*
& Protease K (Boehringer Mannheim), 0.
After treatment with 5% Sarkosyl for 3 hours at 50°C, an equal volume of water-saturated phenol was added. Then centrifuge (8000Xg
, 10 min) to separate the aqueous phase, which was then diluted with 50 mM Tris-HCl MEI solution (Tris-11C1) (pH 8, O
), 10mM ED T^, 10mM Mace. This was heated or treated with bonuclease A (Boehringer Mannheim) to a final concentration of 100 μg/+ai.
After processing at 37°C for 3 hours, phenol extraction and dialysis were performed again in the same manner as above to obtain 710μ
g/ml of chromosomal DNA (H7-2 DNA) was obtained.

2. 10 μg of the above H7-2 DNA was treated with the restriction enzyme EcoRT (
Takara Sake Brewery) or [lamHI Nippon Gene Co., Ltd.]
Alternatively, digestion was performed at 37° C. for 5 hours using old ndl [Nippon Gene Co., Ltd.]. After digestion, 0.6% agarose gel electrophoresis was performed to separate DNA fragments. The gel obtained here was then diluted with 0.25N HCl for 15 minutes.
NaOH-1,5M NaCl for 30 minutes, 0,5M
Tris-HCl (pif7.4) 1.5M Na
Cj! for 30 minutes. Then 20XSSC (0,
Using 3M sodium citrate, 3M NaC1'),
DNA was transferred to a nitrocellulose filter (S&S).

On the other hand, it is known that the human H chain enhancer region of chromosomal DNA exists between the variable region and the constant region, and plays an important role in gene expression (Natural
Plasmid pF208 was obtained according to the method described in Japanese Patent Application No. 61-270953 (Re, 306.809-812 (1983)). This plasmid DNA was cut with the restriction enzymes tlindll [and BstE II] to approximately 8
0.5 μg of DNA containing a 70 bp enhancer region was prepared and labeled with 32p using a nick translation kit (BRL) for use as a probe.

Then, Southern hybridization was performed between this and the above filter. The results at this time are shown in Figures 1 and 2.
In the figure, the numbers indicate the length of base pairs, Ec
oRI and llindll mean that this restriction enzyme was used for digestion. From this result, for the DNA genes digested with EcoRI and old ndI, respectively,
Bands were confirmed at approximately 20 kbp and above and at approximately 8 kbp.

Fire 1 Eel stop] -Ibra l-'s H2 2 DNA 10. After digesting ug with old ndl[[, 0.8% agarose gel electrophoresis was performed to obtain the desired DNA fraction (7 to 9 kb
fragment) was collected (1.1 μs/sj).

On the other hand, plasmid pUc13 (commercially available from Pharmacia) was digested with llindll and treated with alkaline phosphase to prepare a linear plasmid.

Next, a reaction between this and the above DNA fraction was carried out at 4°C overnight using T4 DNA ligase (Takara Shuzo), and the obtained recombinant DNA was used to infect Escherichia coli with the 12C600 (FERN P-115) trait. Transformation was performed to create an H7-2 gene library (3×10′CFυ/μg DNA).

(Screening of 2HD DNA) 1.5 x 10' of the transformed Escherichia coli were subjected to colony hybridization method [Book Molecular C
1oning A Laboratory Mannu
According to a1), the above 32Pi! DNA encoding HM was detected using the ml enhancer DNA as a probe.
A plasmid into which the A fragment was inserted was selected, and this plasmid was named pH720511.

Real JllJ-B-11720511-t・psV
2-gpt (Proc, Nat l, ^cad, s
ci, USA, 78.2072 (1981), obtained from Mr. Ballpurg] was digested with EcoR [and BamHI, and pH7205H was digested with EcoRI and 1f.
ind[[ fragment digested with
Plasmid pSV obtained according to the method shown in No. 953
l1indll and Bam1l I from 2g r 2E
The C10-containing fragment obtained by digestion with
1-gpt was obtained.

It was confirmed by the same method as in Example 9 that this plasmid contained the Hi gene.

The above plasmid pH7205H was digested with the restriction enzyme EcoRI.

ll1ndll[, Pst I, Neo I,
After digesting with PvuI and BstEII (Boehringer) to create a restriction enzyme breakpoint map and identifying the same restriction enzyme cleavage site as 32p, the Maxam-Gilbert method (Me
thods in Enzy++++ology.

499-560 (1980)), the base sequence was determined. This restriction enzyme breakpoint map is
As shown in the figure.

Figure 23 shows the results of the base sequencing of Kagarigoji Nikuji @ Akihiro. Figure 23 is VD
The base sequence of the J-coding region and its vicinity is shown. This sequence includes a 5'-noncoding region, a leader sequence, a -coding region, a J-coding region, and the following 3'-noncoding region.

The single letter alphabet at the bottom of the base sequence is the abbreviation for the amino acid, as described above. Based on this amino acid sequence, this variable region has been identified in the Japanese Biochemical Diet, Biochemical Data Book I.
5 edited by I pp1022, E, ^, Kobat et al.
sequence of proteins or im
munological Ink (1983) (Nati
onal In5fiftcs of 1lealt
h.

Bethesdi) belongs to group ■.

Transfer λ 20 μg of DNA of plasmid pH7205H-gpt obtained in the above method was introduced into Ll gene transformed cells (9×10′ cells) by the same method as above. Two days later, 10 μg/infusion of mycophenolic acid! , xanthine 250
It was selected by adding 7'al in μg. Transformed cells were obtained after about 2 weeks (Table 7).

The reactivity of the antibody against serotype I (H1) of human Pseudomonas aeruginosa weight was confirmed using the ELIS^ method. The method described in Japanese Patent No. 61-91134 was used.

Each well of a polystyrene NUNC immunoplate 0 was coated with poly-L-lysine (10 μg/ml), washed, and treated with formaldehyde to form an H1 type bacterial solution (5 x 10').
50 µL chain was added and centrifuged at 3500 rpm for 15 minutes to collect bacterial cells on the well surface. A 50 μL chain of glutaraldehyde (0.5%) was further added thereto, reacted at room temperature for 15 minutes, and then washed. ' The wells were treated with P containing 1% BSA, 100mM glycine.
The plate was blocked with BS (-') to prepare an anti-Pseudomonas aeruginosa antibody detection plate.

50μ2 of a sample containing an anti-Pseudomonas aeruginosa antibody was added to each well of this plate and allowed to react for 2 hours.

Next, after washing the plate three times with PBS-Tween, HPO-conjugated anti-human IgG antibody (TAGO) 500
50 μL of the 0-fold diluted solution was added and reacted. 2 hours later,
After washing 5 times with PBS-Tween, add BTS substrate solution 1 to
00 μL chain was added to each well and allowed to react for 5-15 minutes at room temperature.

After stopping the reaction with 2iaM NaN and 1, the OD was 4° using Titertic Multiscan @ (Flow Corporation).

1 was measured.

Anti-Pseudomonas aeruginosa human IgG was detected in the culture supernatant of each transformed cell using the above ELIS^.

As shown in Table 7, the transfection cells measured as a control had an OD4゜sn++ of 0.14, whereas the culture supernatant of the transformed cells had an OD4゜sn++ of 1.20 to >2.00.
Humanized antibodies against P. aeruginosa type 1 have been detected, and a method for secreting and producing anti-P. aeruginosa type I antibodies has been established.

The following margin is 1.'' Knee regression - 51 The transformed cells shown in B and the original H7-2@cells were each incubated 5x in RPMI-1640 (containing to% FC8) medium.
10' cells were cultured for 24 hours, and the amount of antibody in the culture supernatant was measured. Polystyrene immunoplate O (N
Goat anti-human r diluted 500 times in each well of UNC)
ga antibody (TAGO) was immobilized and PBS-Twee
After washing with n, H7-2, 117L116, l
l7LI112.

1 (7L1115, H7LI116, +17L
50 μL of culture supernatant of H17 and H7LH21 was added for reaction. On the other hand, to create a calibration curve, standard human IgGf! : 5 μL chains sequentially diluted from 1 μg/ml were added and reacted. 2 hours later, PB
After washing with S-Tween, HP O-conjugated anti-human Ig
It was reacted with a 2500-fold dilution of G antibody (TAGO). After 2 hours, after washing with PBS-Tween, ^13TS
Base ffi solution [: 0.1M citrate buffer (pH 4,2)
, 813'rS (2,5mM>, +1202 (5mM
) ) 100. Add cz light chain to each well;
The reaction was allowed to proceed for 5-15 minutes at room temperature.

After stopping the reaction with 2+nM NaN, OD was determined using Titertic Multiscan [F] (Flow Corporation). ,
. , and the amount of antibody was calculated from the standard curve. Shown in Table 8.

On the other hand, using the same Pseudomonas aeruginosa-fixed plate as in F, EIJS^ of the same culture supernatant shown above was performed in the same manner as shown in B. ,. The dilution factor of the culture supernatant showing 1.0 was determined. The reciprocal of this dilution ratio is shown in Table 8. The specific activity of the antibody against Pseudomonas aeruginosa H1 was calculated by dividing the reciprocal of the dilution ratio by the antibody weight. While H7-2 had a value of 14.5, the calculated value showed a value of 10.7 to 15.6, making it clear that it was an anti-Pseudomonas aeruginosa human antibody with the same activity. Shown in Table 8.

Immune activity of human antibodies obtained from transformed cells

[Brief explanation of drawings]

Figures 1(a) and (b) show plasmid pOsD, respectively.
Structures of E234t-5 and pOsDE12341J are shown. Figure 2 shows the structure of plasmid pF208. FIG. 3 shows the structure of plasmid psV3-neo-ε. FIG. 4 shows the autoradiogram results of Southern hybridization in screening for the gene of anti-Aeruginosa IIa F4 antibody. Figure 5 shows plasmid p (:31V and L chain DNA).
A restriction enzyme cleavage map of the gene is shown. Figures 6-1 and 6-2 show the base sequence of the L chain variable region of the anti-Pseudomonas aeruginosa F4 antibody. Figures 7 and 8 show pG31V-gpL and pG3
1V shows the structure of -neo. FIG. 9 shows the autoradiogram results of Southern hybridization for detecting the H chain gene of anti-Pseudomonas aeruginosa F antibody. Figure 10 shows the DN encoding the H chain of anti-Pseudomonas aeruginosa F antibody.
A restriction enzyme cleavage map of a DNA fragment containing A is shown. FIGS. 11, 12, 13, and 14 show tsure ('tLpsV2gy2E, psV2nγ2E,
The structures of psV2rr2EI3 and psV2nγ2EB are shown. Figure 15 shows the DN encoding the H chain of anti-Pseudomonas aeruginosa F antibody.
Showing the structure and partial base sequence of a DNA fragment containing A,
Numbers indicate length in base pairs from EcoRI. FIG. 16 shows the autoradiogram results of Southern hybridization for detecting the H chain gene of anti-Pseudomonas aeruginosa F antibody. Figure 17 shows the I (D encoding chain) of anti-Pseudomonas aeruginosa F4 antibody.
A restriction enzyme cleavage map of a DNA fragment containing NA is shown. FIG. 18 shows the autoradiogram results of Southern hybridization in screening for the gene of anti-Pseudomonas aeruginosa H1 antibody. Figure 19 shows 1) which encodes the L chain of anti-Pseudomonas aeruginosa H1 antibody.
A restriction enzyme cleavage map of a DNA fragment containing NA is shown. FIG. 20 shows the base sequence of the Lg variable region and its vicinity of the anti-Pseudomonas aeruginosa H1 antibody. FIG. 21 shows the autoradiogram results of Southern hybridization in screening for the HM gene of anti-Pseudomonas aeruginosa H1 antibody. Figure 22 shows the DN encoding the H chain of anti-Pseudomonas aeruginosa H1 antibody.
A restriction enzyme cleavage map of a DNA fragment containing A is shown. FIG. 23 shows the base sequence of the H@variable region and its vicinity of the anti-Pseudomonas aeruginosa H1 antibody.

Claims (1)

[Scope of Claims] 1. By a recombinant containing a gene encoding the L chain of a human antibody and a recombinant containing a gene encoding the H chain of a human antibody, or encoding the L chain of a human antibody A human antibody is secreted by culturing an animal cell transformed with a recombinant containing a gene encoding a human antibody and a gene encoding an H chain of a human antibody, and the antibody is recovered. Method for producing human antibodies. 2. The human antibody is derived from Pseudomonas aeruginosa.
The method according to claim 1, which is a humanized antibody against S. aeruginosa. 3. The human antibody is derived from Pseudomonas aeruginosa.
3. The method according to claim 2, which is a humanized antibody against Fisher's serotype 4 (F_4) of S. aeruginosa. 4. The constant regions of the human antibody are γ_1, γ_2 and γ
_3 The method according to claim 2, which is a human antibody corresponding to the immunoglobulin gene. 5. The human antibody is derived from Pseudomonas aeruginosa.
aeruginosa) Honma serotype type I (H1
2. The method according to claim 2, which is a humanized antibody directed against ).