JPH08280386A - Antibody and antibody cdna - Google Patents

Abstract

PURPOSE: To obtain a new antibody, specifically reactive with human lung cancer without reacting with a human normal cell strain and utilizable for application in the medical field such as clinical diagnosis or immunotherapy of cancer and purification, etc., of a cancer-specific antigen and a cDNA for production, etc., of the antibody. CONSTITUTION: This new monoclonal antibody is specifically reactive with human lung cancer without reacting with a human normal cell, reactive with a human lung cancer tissue and utilizable for application in the medical field such as clinical diagnosis or immunotherapy of cancer or as a purifying means for a cancer-specific antigen. Furthermore, the new cDNA is respectively represented by formulas I and II and useful for genetic engineering production, etc., of the human monoclonal antibody. The antibody is obtained by fusing a lymphocyte of a patient suffering from the lung cancer to a parent cell strain for preparing a human hybridoma, screening the resultant hybridoma, cloning a strain capable of producing the antibody and then culturing the cloned strain. The cDNA is prepared by collecting its MRNA and synthesizing the cDNA and cloning the synthesized cDNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human monoclonal antibody, cDNA for the antibody, and DNA encoding the CDR of the antibody. The human monoclonal antibody can be used in clinical diagnosis of cancer, application in the medical field such as immunotherapy, and as a means for purifying a cancer-specific antigen. Further, the cDNA can be used for industrial-scale mass production of the human monoclonal antibody using a genetic engineering technique.

[0002]

[Prior Art and Problems to be Solved by the Invention] Kohl
er, G and Milstein, K monoclonal antibody production technology (Nature, Vol.256, p495-497, (1975)) has been developed, and many cancers including leukemia cells are said to be lung cancer and blood cancer. Mouse monoclonal antibodies against cells have been produced. Among the fields of application of such monoclonal antibodies that are said to be cancer-specific, the most promising ones are the so-called missile therapeutic agents, which bind anticancer agents and the like and administer them to the body to treat cancer, or This is a medical field in which a monoclonal antibody labeled with a radioisotope that emits a gamma ray is accumulated in a cancer lesion in the body to perform in-vivo diagnosis of cancer.

When the monoclonal antibody used is a heterologous protein for the human body such as mouse, a human antibody (Human anti-mouse antibody: H) against this mouse antibody is used.
It is known that AMA) is frequently produced. This HAMA not only inactivates the administered mouse monoclonal antibody, but also has a risk of causing an allergic reaction. In order to avoid this problem, for example, attempts have been made to remove the antigenicity that causes the generation of HAMA while retaining the antigen-binding ability of mouse antibodies, but the fundamental solution has not been reached. On the other hand, when a human monoclonal antibody is used, such an antigenicity problem is not considered from the beginning, and thus it is superior to the mouse monoclonal antibody.

In order to obtain a hybridoma producing a mouse monoclonal antibody, in many cases, a substance or cell serving as an antigen is immunized in the body of a mouse to facilitate B lymphocytes sensitized with the antigen. As it can be obtained, it can be used to achieve its purpose. On the other hand, for ethical and health reasons, the human body cannot be positively immunized, and therefore a human monoclonal antibody having a desired specificity cannot be easily obtained.

As a result of energetic research so far, the present inventors have succeeded in obtaining a human monoclonal antibody which is difficult to obtain as described above (Japanese Patent Publication No.
29357). This human monoclonal antibody has specific reactivity to lung cancer cells and does not react to normal cells, and has ideal properties for the purpose of application in the medical field mentioned above. There is.

Further, since the human-human hybridoma described in Japanese Patent Publication No. 29357/1992 can stably produce an antibody in a serum-free medium, an inexpensive and highly pure human monoclonal antibody can be obtained. In mass production on an industrial scale, a more stable and highly productive antibody-producing cell is advantageous.

On the other hand, genetic engineering techniques have provided techniques for producing a large amount of various useful proteins including insulin up to now on an industrial scale. The application of this genetic engineering technique to the production of monoclonal antibodies begins with obtaining chromosomal DNA or complementary DNA (cDNA) of the monoclonal antibody.

Among the antibodies (immunoglobulin),
The basic structure and gene of human IgM relevant to the present invention will be briefly described.

[0009] An IgM molecule is a polypeptide in which a monomer composed of two heavy chains each having a molecular weight of about 65,000 and two short chains each having a molecular weight of about 25,000 is called a J chain. To form a pentamer with five linked via. The heavy chain-light chain and the heavy chain-heavy chain are linked by SS bonds. Approximately 100 amino acid sequences from the amino terminus of the heavy and light chains are antigen-specific and this region binds antigen. This region is called a "variable region". The regions that follow it have a constant structure called the "constant region" and consist of sequences of about 440 and about 150 amino acids, respectively. The constant region of the heavy chain of the IgM molecule is composed of Cμ1, Cμ2, Cμ3, Cμ4, and the light chain is Cκ, Cλ as in the IgG molecule.
, The heavy chains are μ chains, the light chains are κ chains, and λ chains.
Called a chain.

A particular monoclonal antibody is a homogeneous population of antibody molecules in which the heavy and light chain variable regions each have a particular amino acid sequence, and the amino acid sequences of the variable regions differ greatly from monoclonal antibody to monoclonal antibody. The variable region is a group of complementarity determining regions (CDR: Complementar).
Hyper variable region (Hype)
r variable region) and a relatively common group of framework residues (FR). The heavy chain and the light chain each have three CDRs, and the antigen-binding site is formed by the CDRs assembled by the folding of the heavy chain and the light chain. Therefore, the antigen binding specificity and binding strength of the monoclonal antibody are mainly defined by the amino acid sequence of CDR.

On the other hand, the variable region is composed of V gene and D in the heavy chain.
It is known that genes, J genes, and light chains are encoded by V genes and J genes. In human heavy chain, 100 or less V genes, 6 D genes, 12 J genes
In human κ chain, there are 100 or less V genes and 5 J genes. With the differentiation of B cells, V in heavy chain,
Any one gene from each of the D and J gene groups is flanked by rearrangements to form a gene corresponding to the variable region. Similarly in the light chain, rearrangement of the V and J genes forms a variable region gene.

The gene encoding the constant region is located downstream of the variable region gene group. When the recombination of the variable region gene is completed, this gene is expressed continuously with the variable region gene, and heavy and light chain polypeptides are produced.

Based on these findings, a method for rapidly cloning a complementary DNA (cDNA) encoding a variable region of an antibody from a hybridoma producing a human monoclonal antibody has been developed (James W. Larrick et a.
l., Biochem. Biophys. Res. Commun., Vol. 160, p. 1250
-1256 (1989); James W. Larrick et al. , Bio / Technolo
gy, Vol.7, p.934-938 (1989)).

On the other hand, the present inventors have invented an in vitro immunization method for human lymphocytes using a cancer cell line (Japanese Patent Application Laid-Open No. 4-281).
799: Kawahara, K. et al. , Human Antibodies and
Hybridomas, Vol.3, p.8-13 (1992)), by fusing human peripheral blood lymphocytes subjected to this method with parent cells, they react with human cancer cells and become human normal fibroblasts. Succeeded in obtaining a human-human hybridoma producing a human monoclonal antibody which does not react (Japanese Patent Laid-Open No. Hei 6)
-153984). In addition, from complementary DNA (cDNA) prepared based on messenger RNA (mRNA) collected from the human-human hybridoma, cDNA encoding the variable regions of the heavy and light chains constituting the human monoclonal antibody was selected. Each was successfully cloned.
Furthermore, they have succeeded in elucidating the nucleotide sequences of these cDNAs and determining the amino acid sequences.

[Means for Solving the Problems]

The present inventors are now further characterized by a heavy or light chain having a specific amino acid sequence,
The present invention was completed by finding a human monoclonal antibody that reacts specifically with human lung cancer, does not react with a normal human cell line, and reacts with human lung cancer tissue, and the cDNAs of the variable regions of the heavy and light chains of the antibody. It was done. Furthermore, the present invention provides a DNA encoding the CDR of an antibody, which can be subjected to a genetic engineering technique.

That is, according to the present invention, a cDNA encoding a human monoclonal antibody and a variable region of an antibody light chain are characterized in that the cDNA encoding the variable region of the antibody heavy chain contains the nucleotide sequence shown in FIG. Coded cDN
A is a cDNA encoding a human monoclonal antibody, characterized in that it comprises the nucleotide sequence shown in FIG.
The variable region of the antibody heavy chain is represented by the amino acid sequence shown in FIG. 3, and the variable region of the antibody light chain is shown in FIG.
The antibody characterized by being represented by the amino acid sequence described in 1., the DNA characterized by encoding the variable region of the antibody heavy chain consisting of the amino acid sequence described in FIG. 3, and the amino acid sequence described in FIG. DNA encoding the variable region of an antibody light chain, CDR-1 and C of an antibody heavy chain
DR-2 and CDR-3 are encoded by the nucleotide sequences shown in FIG. 5, FIG. 6 and FIG. 7, respectively.
1, CDR-2 and CDR-3 are shown in FIG. 8 and FIG. 9, respectively.
And the cDNA according to claim 2, which is encoded by the nucleotide sequence shown in FIG. 10, and CDR-1, CDR-2 and CDR-3 of the antibody heavy chain in FIGS. 11, 12 and 13, respectively. An antibody having the described amino acid sequence, CDR-1 of the antibody light chain,
Antibodies characterized by having the amino acid sequences set forth in Figure 14, Figure 15 and Figure 16 as CDR-2 and CDR-3, respectively, Figure 11, Figure 12 and Figure 13, respectively.
CDR-1 of the antibody heavy chain consisting of the amino acid sequence of
DNA characterized by encoding CDR-2 and CDR-3, and FIGS. 14, 15 and 16, respectively.
CDR-1 of the antibody light chain consisting of the amino acid sequence described in
DNA characterized in that it encodes CDR-2 and CDR-3.

The human-human hybridoma producing the antibody of the present invention is a parent cell derived from human peripheral blood lymphocytes obtained by in vitro immunization of human lymphocytes using a cancer cell line or lymphocytes obtained from lung cancer patients. It can be prepared by cell fusion with a strain. As one specific example of such a hybridoma, human-human hybridoma HB4C5 (Microtechnological Research Institute Contribution No. No .: FERM BP-1879).

The present invention will be specifically described below with reference to examples.

[0019]

【Example】

Example 1 Hybridoma Production by Cell Fusion Mixing lymphocytes (3 × 10 ⁶ cells) obtained from a lung cancer patient and parental cell line NAT-30 (Hokoku 5-51277) for human hybridoma production (3 × 10 ⁷ cells) The mixed cells were then collected by centrifugation at 400 xg for 5 minutes. After removing the centrifugation supernatant, add 1 ml to the cell pellet.
50% polyethylene glycol 4000 (Boehringer
(Made by the company) was added over 1 minute, and it incubated at 37 degreeC after that for 1 minute. Further, 9 ml of DME medium (Dainippon Pharmaceutical Co., Ltd.) was added at a rate of 1 ml per minute. 400 x g, 5
Centrifuge for 1 minute, discard the supernatant, and suspend the cells in 20 ml of DME medium (containing 15% FCS).
Well plates were seeded. The culture was carried out at 37 ° C., and 15% FCS-DME medium containing 100 mM hypoxanthine, 0.4 mM aminopterin, and 16 mM thymidine so-called HAT was added for the purpose of selecting only the fused cells. Colonies formed by hybridomas began to appear 10 days after the day of cell fusion.

Example 2 C. Producing human monoclonal antibody reactive with cancer cells
Screening and cloning of ibridomas The reactivity of antibodies produced by hybridomas was examined using the indirect fluorescent antibody method described below. The lung adenocarcinoma cell line PC-8 was cultured in a 96-well plate or a tissue culture chamber (Nunc) for 1 to 5 days in a 5% carbon dioxide gas incubator at 37 ° C. and attached thereto. After culturing, the cells were fixed with 0.05% glutaraldehyde or 4% formaldehyde.
After fixation, PBS washed with phosphate buffered saline (hereinafter referred to as PBS) three times and containing 3% bovine serum albumin
Was added and left at room temperature for 30 minutes. After standing, the plate was washed 5 times with PBS, 100 μl of the medium in the well in which the hybridoma was growing was added, and the plate was left standing at 37 ° C. in a 5% carbon dioxide gas incubator for 30 minutes. After that, the cells were washed 5 times with PBS, and a fluorescently labeled (FITC) goat human antibody (Tago) was diluted 50 times with PBS and added.
The mixture was left to stand in a 5% carbon dioxide gas incubator at 30 ° C for 30 minutes. After standing, it was washed 5 times with PBS and immediately observed with a fluorescence microscope. As a result of examining more than 4000 hybridomas, one hybridoma producing an antibody whose specific fluorescence was observed in 50% or more of the cells in the well of PC-8 cells was obtained. This hybridoma was cloned by the limiting dilution method to obtain a clone that stably produces an IgM class monoclonal antibody. The human-human hybridoma producing this monoclonal antibody was designated as HB4C5. The short chain subclass of this monoclonal antibody was κ when examined by ELISA.

Example 3 Reactivity of Monoclonal Antibody to Lung Cancer Tissue The hybridoma obtained above was used at a cell density of 3 × 10 ⁵ cells / ml, 5 μg / ml bovine insulin, 35 μg / ml human transferrin, 20 μM ethanolamine, 2 ．
A culture supernatant containing a monoclonal antibody was obtained by seeding in an ERDF medium containing 5 nM selenite and 2 mg / ml human serum albumin and culturing at 37 ° C. for 7 days.
When the concentration of the monoclonal antibody in the culture supernatant was measured by ELISA for measuring human IgM, it was 2 μm.
It was g / ml. This culture supernatant was spread on the cancer tissue and incubated at room temperature for 2 hours. After the incubation, the cancer tissue was washed with PBS three times for 5 minutes. A 1,000-fold diluted peroxidase-labeled goat anti-human IgM antibody (Tago
(Manufactured by the company) was placed on the cancer tissue and incubated at room temperature for 1 hour. The cancer tissue was washed in the same manner as described above, and 0.5 mg / ml DAB (manufactured by Dojindo Co., Ltd., Cat.
No. 347-00904) and containing 0.01% H ₂ O ₂ 5
The reactivity of the antibody to the cancer tissue was examined using 0 mM Tris-HCL (pH 7.4).

[0022]

[Table 1] Reactivity of human monoclonal antibody produced by HB4C5 to lung cancer tissue ──────────────────────────────── Human Monochrome Squamous cell carcinoma Adenocarcinoma Large cell carcinoma Small cell carcinoma Narrow antibody ──────────────────────────────── HB4C5 5/6 5/5 2/3 2/2 ──────────────────────────────── Number in the table: Number of positive specimens / Number of test samples

As is clear from the results shown in Table 1, HB4C5
The human monoclonal antibody produced by S. cerevisiae reacted with various lung cancers with a high probability (88%). Human serum IgM used as a control did not react with all lung cancer tissues examined.

Example 4 Preparation of mRNA The HB4C5 cells obtained in Example 3 were treated with 10% FCS-E.
The cells were seeded in RDF medium up to 5 × 10 ⁵ cells / ml. After culturing for 2 days, 5 × 10 ⁸ cells were obtained.

The cells were washed 3 times with PBS (previously ice-cooled) at 4 ° C. (2000 × g 5 min.). First, Guan the 2-Mercaptoethanol to a final concentration of 1%
Added to idium Thiocyanate Buffer. Add 5 volumes of 2-Mercaptoethanol-Guanidium Thiocyanate Buffer
The mixture was suspended in, and a syringe with a 23G needle attached was suctioned and discharged to crush it until the viscosity disappeared.
Sodium Lauryl Sarcosinate was added at a final concentration of 0.5% and stirred well. The insoluble fraction was removed by centrifugation at 5,000 xg for 10 min at room temperature. 23 of supernatant
Suction with a syringe equipped with a G needle, 5.7M CsCl, 0.01M ED in a transparent ultracentrifuge tube.
It was gently overlaid on a cushion of TA (pH 7.5). The top of the cushion was marked on the outside of the tube. Weight correction was performed with Guanidium Thiocyanate. SW5
Using a 0.1 Swing rotor, 20 ℃, 40,000rpm
Ultracentrifugation was performed under the conditions of 24 hours. After centrifugation, 0.
A line was drawn at a position of 5 cm. The fluid was carefully removed with a Pasteur pipette to above the level of the cushion. The liquid was removed to the bottom line with a new pipette, and the tube was cut with a razor just above the remaining liquid. The bottom of the tube was placed on a Kimwipe pad and the remaining liquid was carefully drained with an automatic pipettor. The tube was inverted and the remaining liquid was completely removed. Check that the RNA pellet did not fall outside the tube with the tube facing up. RNA to remove CsCl
The pellet was washed with Ethanol. Fill the bottom of the tube with 70% Ethanol (room temperature) and invert the tube.
Discarded Ethanol. The presence of RNA was confirmed again. RN
A was dried at room temperature. TE containing 0.1% SDS (pH
At 7.6), the RNA was lysed by pipetting with an automatic pipettor fitted with a tip of Tespo. R
The NA solution was taken in an Eppendorf tube. The bottom of the centrifuge tube was washed with 25 μl of TE, and the washing solution was also transferred to an Eppendorf tube. 150 μl TE, 30 μl 3M Sodium Acetate (pH 5.2), 0.9 ml ice-cold Ethano
l was added. After mixing, at least 30min, 0 ℃
saved. The pellet was washed with Ethanol and centrifuged again briefly. Then, it was dried in air. 1 ml of RNA
It was dissolved in TE, added with 3 volumes of Ethanol, stored in small aliquots at -70 ° C until needed. As a result of the above,
It was possible to extract 4 mg of RNA.

Spu filled with oligo (dT) -cellulose
The resin was well suspended by shaking the n column well. The bottom cap of the column was removed and then the top cap was removed. The column was set in a 15 ml centrifuge tube and the storage buffer was drained. 1 ml of High-salt buffer was added and drained by gravity (twice). The liquid accumulated at the bottom of the centrifuge tube was discarded, and the column was set in the centrifuge tube.

The RNA sample was treated with 1.0 ml TE (pH 7.
It was dissolved in 4) and treated at 65 ° C. for 5 minutes. At this time, RN
It was confirmed that A was completely dissolved. Also Eluti
The on buffer was kept warm at 65 ° C. The sample was ice-cooled, 0.2 ml of sample buffer was added, and the mixture was gently stirred. The sample was loaded onto the prepared column and eluted by gravity. It was centrifuged at 1400 rpm (r = 165) for 2 minutes. Add High-salt buffer to the 0.25 ml column,
It was centrifuged (twice). The column was washed with 0.25 ml of Low-salt buffer (3 times). Buffer accumulated at the bottom of the centrifuge tube
Then, the sterilized Eppendorf tube was set in the centrifuge tube, and the column was set in the centrifuge tube so that the eluent of the column entered the Eppendorf tube.
In order to elute the poly (A) ⁺ RNA, the column was washed 4 times with 0.25 ml of Elution buffer kept at 65 ° C. The column was removed from the centrifuge tube, and the Eppendorf tube was taken out with dry tweezers and immediately cooled with ice. 1/10 vol. Of 3M Sodium Acetate
Was added, 2.5 to 3 vol. Of ethanol was added, and the mixture was stored at -80 ° C.

As a result, 200 μg of mRNA could be obtained. When this mRNA was electrophoresed, r
RNA was not degraded, and mRNA of 5 kb or more was also confirmed. As a result of in vitro translation, it was confirmed that the heavy chain gene was contained. It was decided to perform cDNA synthesis using this mRNA.

Example 5 Synthesis of First-Stranded cDNA Prepare a Water Bath (42 ° C. and 12 ° C.) and use Kit.
Reagents other than the enzyme (cDNA synthesis system plus (Amersham)) and mRNA were dissolved and ice-cooled. The enzyme was stored at -20 ° C until just before use. The following reagents were added in order.

[0030]

[Table 2] 5 × buffer for first strand synthesis reaction 10.0 μl sodium pyrophosphate solution 2.5 μl human placental ribonucleose inhibitor 2.5 μl deoxynucleotide triphosphate mixture 5.0 μl oligo (dT) primer 2.5 μl mRNA (In water) 17.5 μl Total 40.0 μl Reverse transcriptase 10.0 μl Total 50.0 μl

Mix gently and collect the mixture by centrifugation at the bottom of the centrifuge tube. 20 units of reverse transcriptase was added per 1 μg of mRNA, mixed, and incubated at 42 ° C. for 40 min. The temperature should be strictly 42 ° C. and the time should not be longer than 40 min. Immediately after the incubation, it was cooled on ice. Then, it preserve | saved at -20 degreeC. For long-term storage, it was stored at -80 ° C.

Example 6 Amplification of Antibody Variable Region cDNA by PCR Method Reference (James W. Larrick et al., Biochem. Biop.
hys. Res. Commun., Vol. 160, p.1250-1256 (1989); En
mes W. Larrick et al., Bio / Technology, Vol. 7, p.9
34-938 (1989)), primers (HS-1, 2, 3) corresponding to the leader sequence of human antibody heavy chain and human Ig
Create a primer corresponding to the constant region of the M antibody heavy chain,
By PCR with these three combinations, HB
An attempt was made to amplify the heavy chain variable region of the human monoclonal antibody produced by 4C5 cells. Similarly for the light chain variable region, 2 corresponding to the leader sequence and constant region of human lambda chain.
The primers of the book were prepared and amplification was tried by PCR using these. The PCR reaction was performed by adding the following reagents in order.

[0033]

[Table 3] Water 10.6 μl Buffer for 10 × PCR 2.0 μl Deoxynucleotide triphosphate mixed solution 3.3 μl Primer (+) 1.0 μl Primer (−) 1.0 μl First strand cDNA 2.0 μl Taq Polymerase 0 1 μl Total 20.0 μl

PCR is carried out at 94 ° C. for 1 min, 55 ° C. for 2 min
30 cycles at a reaction temperature of 72 ° C. for 3 min.
As a result, the size of the cDNA of the variable region of the IgM antibody heavy chain predicted by PCR using HS-3 (about 490 b
An amplified fragment of p) was observed. From this, it was considered that the cDNA of the antibody heavy chain variable region was specifically amplified.
It was decided to determine the base sequence of this amplified fragment.

Example 7 Subcloning of Amplified Fragment Using the restriction enzyme sites inserted in the amplified fragment, pUC
119 of the sequence vector Eco RI- Hin d I
Subcloned into II. The amplified fragment of 1.5% agarose gel electrophoresis, separated, extracted with glass beads, and Eco RI- Hin d III process.
On the other hand, the vector, pUC119, is also Eco RI- H.
in d III treated, amplified fragment with that restriction enzyme treatment, after deproteinization, were ligated. Transformation was performed using MV1184 as the host.

As a result, about 50% of them contained the insert fragment.
Obtained. When the size of each of these clones was confirmed using the alkaline rapid method, 30 clones out of 32 clones had a plasmid that was considered to be the target size. Of the 12 clones among these, the size of the inserted fragment was confirmed by the alkaline SDS method. As a result, all 12 clones contained fragments of the desired size. For these 4 clones, single-stranded DNA was prepared and the nucleotide sequence was determined.

Example 8 After confirming the size of the single-stranded DNA sequence insert for nucleotide sequencing , the helper phage M1
Single strands were prepared with 3KO7.

MV1184 (pUC119-Insert) is 2 × Y
The cells were precultured with T (containing 150 μg / ml Ampicillin).
30 μl of helper phage M13K07 solution (> 1010 pfu / ml) was added to 30 μl of pre-cultured solution, and 30 mi at 37 ° C.
n Let stand. 2 x YT broth warmed to 37 ° C
h (150 μg / ml Ampicillin, 70 μg / ml Kanamyci
n, containing 0.01% thiamine. ) Was added. Three
It was cultured at 7 ° C for 14 to 18 hours. (At this time, like the preparation of the helper phage solution, it was cultivated under conditions with good air permeability.) The culture solution was transferred to a microcentrifuge tube, the bacterial cells were removed by centrifugation, and the supernatant was put into another microcentrifuge tube. I took it.
200 μl of PEG-NaCl solution was added to 1 ml of the culture supernatant, mixed well, and left at room temperature for 15 minutes. The centrifugation supernatant was removed. The PEG-NaCl solution was completely removed with a filter paper or the like. The precipitate was completely dissolved with 100 µl of TE-buffer. Add 50 μl of TE-saturated Phenol to about 1
It was shaken for 0 sec and left for about 10 min. 50 μl of chloroform: isoamyl alcohol was added, and the mixture was shaken for about 10 seconds. After centrifugation for 5 minutes, the aqueous layer (upper layer) was transferred to another microcentrifuge tube. Add 10 μl of 3M Sodium Acetate and add 2
Add 50 μl cold Ethanol and mix, then mix at -70 ° C for 5
min left. It was left in this state for long-term storage. After centrifugation for 5 minutes, the precipitate was washed with 70% Ethanol. The solution was re-centrifuged, the supernatant was removed, and the residue was dried under reduced pressure. TE-buffer
The precipitate was dissolved in 50 µl and stored at -20 ° C.

Example 9 Nucleotide Sequence Determination Nucleotide sequence determination was carried out using Sequenase. less than,
The method for determining the nucleotide sequence is shown.

Sequencing reaction Annealing and labeling were performed in a 0.5 ml tube. 1. Annealing Primer 1 μl 5 × Sequenase Buffer 2 μl DNA (about 1 μg) 7 μl A tube was capped, incubated at 65 ° C. for 2 min, and then returned to room temperature over 30 min. 2. Labeling DTT 0.1M 1.0 μl Diluted Labeling Mix 2.0 μl [α- ³² P] dCTP 0.5 μl Diluted Sequenase 2.0 μl 3. Termination G. 4 tubes. A. T. Enter C and add 2.5 μl of Termination Mix to each tube.

1. After annealing is complete, add 2. After adding to the reaction solution of 3 and gently stirring, 3.5 μl of each 3. The tube was attached to the edge of the tube, being careful not to mix the liquid. After incubating at room temperature for 5 minutes, the solution was mixed by centrifugation. Mix the liquid again,
Incubated at 37 ° C for 5 min. Add 4 μl of Stop Solution to each Termination reaction mixture and mix well.
Cooled on ice until ready to load onto a Sequencing Gel. When ready, the sample was heated at 75-80 ° C. for 2 min and then loaded in 4 μl aliquots on the Gel. Electrophoresis was performed at 50 V / cm. After migration, using a gel dryer,
The gel was dried well and autoradiographed. As a result, a nucleotide sequence of cDNA encoding the antibody heavy chain of the present invention as shown in FIG. 1 was obtained.
By translating this nucleotide sequence, the amino acid sequence of the variable region of the antibody heavy chain was elucidated and shown in FIG.

The amino acid sequence shown in FIG. 3 is a list of amino acid sequence data of known human antibodies (Elvin A. Kabat
et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL IN
TEREST, FIFTH EDITION, US DEPARTMENT OF HEALTH A
ND HUMAN SERVICES, PublicHealth Service, National
1) and the database of DNA and protein (EMBL-GDB (Release 41)).
, LASL-GDB (Release 86), NBRF-PD
B (Release 43) and SWISS-PROT (Release
30)) was subjected to a homology search to determine that the antibody heavy chain belongs to subgroup IV of the subgroups I to VI. Furthermore, the sequences of the amino acids of the four FRs located at the positions sandwiching the three CDRs are generally conserved in each subgroup.
1, the amino acid sequences shown in FIG. 12 and FIG. 13 are CDR-1, CDR-2 and CDR- of the antibody heavy chain, respectively.
It was determined to be 3. This gives the CDR- of the antibody heavy chain.
It became clear that the nucleotide sequences encoding 1, CDR-2 and CDR-3 are represented in Figures 5, 6 and 7, respectively.

The above operation was carried out for the light chain of the antibody of the present invention, and the respective sequences were determined.

[Brief description of drawings]

FIG. 1 is a nucleotide sequence encoding the variable region of the heavy chain of the antibody of the present invention.

FIG. 2 is a nucleotide sequence encoding the variable region of the light chain of the antibody of the present invention.

FIG. 3 is an amino acid sequence constituting the variable region of the heavy chain of the antibody of the present invention.

FIG. 4 is an amino acid sequence constituting the variable region of the light chain of the antibody of the present invention.

FIG. 5 is a nucleotide sequence encoding CDR-1 of the heavy chain of the antibody of the present invention.

FIG. 6 is a nucleotide sequence encoding CDR-2 of the heavy chain of the antibody of the present invention.

FIG. 7 is a nucleotide sequence encoding CDR-3 of the heavy chain of the antibody of the present invention.

FIG. 8 is a nucleotide sequence encoding CDR-1 of the light chain of the antibody of the present invention.

FIG. 9 is a nucleotide sequence encoding CDR-2 of the light chain of the antibody of the present invention.

FIG. 10 is a nucleotide sequence encoding CDR-3 of the light chain of the antibody of the present invention.

FIG. 11 is an amino acid sequence constituting CDR-1 of the heavy chain of the antibody of the present invention.

FIG. 12 is an amino acid sequence constituting CDR-2 of the heavy chain of the antibody of the present invention.

FIG. 13 is an amino acid sequence constituting CDR-3 of the heavy chain of the antibody of the present invention.

FIG. 14 is an amino acid sequence constituting CDR-1 of the light chain of the antibody of the present invention.

FIG. 15 is an amino acid sequence constituting CDR-2 of the light chain of the antibody of the present invention.

FIG. 16 is an amino acid sequence constituting CDR-3 of the light chain of the antibody of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 16/30 9162-4B C12N 15/00 C C12N 5/10 9281-4B 5/00 B (C12P 21/08 C12R 1:91)

Claims (12)

[Claims] 1. A cDNA encoding the variable region of an antibody heavy chain.
A cDNA encoding a human monoclonal antibody, wherein A comprises the nucleotide sequence shown in FIG. 2. A cDNA encoding the variable region of an antibody light chain
A cDNA encoding a human monoclonal antibody, wherein A comprises the nucleotide sequence shown in FIG. 3. An antibody, wherein the variable region of the antibody heavy chain is represented by the amino acid sequence shown in FIG. 4. An antibody, wherein the variable region of the antibody light chain is represented by the amino acid sequence shown in FIG. 5. A DNA which encodes the variable region of an antibody heavy chain consisting of the amino acid sequence shown in FIG. 6. A DNA encoding the variable region of an antibody light chain consisting of the amino acid sequence shown in FIG. 7. The cDNA according to claim 1, wherein CDR-1, CDR-2 and CDR-3 of the antibody heavy chain are encoded by the nucleotide sequences shown in FIGS. 5, 6 and 7, respectively. . 8. The cDNA according to claim 2, wherein the antibody light chain CDR-1, CDR-2 and CDR-3 are encoded by the nucleotide sequences shown in FIGS. 8, 9 and 10, respectively. . 9. The antibody heavy chain CDR-1, CDR-2 and CDR-3 are shown in FIG. 11, FIG. 12 and FIG. 1, respectively.
An antibody having the amino acid sequence of 3. 10. An antibody, which has the amino acid sequences shown in FIGS. 14, 15 and 16 as CDR-1, CDR-2 and CDR-3 of the antibody light chain, respectively. 11. FIGS. 11, 12 and 13 respectively.
CDR-1 of the antibody heavy chain consisting of the amino acid sequence of
A DNA characterized in that it encodes CDR-2 and CDR-3. 12. FIGS. 14, 15 and 16 respectively.
CDR-1 of the antibody light chain consisting of the amino acid sequence described in
A DNA characterized in that it encodes CDR-2 and CDR-3.