JPH0771510B2 - Method for producing monoclonal antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to preparation of a complex protein in which a compound of interest is bound to the carboxyl terminal of dihydrofolate reductase (DHFR) and used as an immunizing antigen to prepare a monoclonal antibody. It is about the method. Industrial fields of application of the present invention include the fields of clinical testing and pharmaceutical manufacturing.

[0002]

2. Description of the Related Art As a method for specifically detecting and quantifying a trace amount of physiologically active substance present in a living body, high sensitivity detection of immunological molecular recognition property in an antigen-antibody reaction and radioisotope radioactivity or enzyme activity. Radioimmunoassay (RIA) and Enzyme Immunoassay (EI)
A) is known. Since these immunoassays can quantify target substances in samples such as serum with high sensitivity and relatively easily, the number of target substances is steadily increasing year by year. It is forming an industry. In particular, EIA has the inconvenience of using a radioisotope that is legally regulated by RIA, but it is easy to handle because it uses enzyme activity as a detection means. Recently, the measuring method by EIA has become widespread. is there.

A monoclonal antibody is an antibody produced by a uniform cell population produced by division of one B cell or one cell, and recognizes only a specific antigenic determinant of an antigen molecule. In addition, not only the antibody specificity, but also the class, subclass, affinity, and other properties are completely uniform. Therefore, the effectiveness of a monoclonal antibody can be determined by the homogeneity of the antibody. By utilizing this property, various obstacles derived from the heterogeneity of the antibody can be theoretically analyzed when performing various immunoassays. It has the feature that it can be removed.

When establishing an immunoassay using a monoclonal antibody, it is necessary to prepare a monoclonal antibody that specifically binds to the physiologically active molecule to be measured. Generally, when preparing a monoclonal antibody, an immunized animal is immunized, and spleen cells of the animal that has become capable of producing antibodies are taken out and fused with cells that can be cultured in vitro such as myeloma cells ( A hybridoma) is prepared and screened to select a hybridoma which produces the desired monoclonal antibody, and the hybridoma thus obtained is used to prepare the desired monoclonal antibody.

[0005]

[Problems to be Solved by the Invention] Various problems have been raised in producing a monoclonal antibody.

First, compounds having a molecular weight of several thousand or less have extremely low antigenic activity. Therefore, generally, a target compound is chemically bound to a polymer carrier such as bovine serum albumin (BSA) to prepare a complex having enhanced antigenic activity, and the complex is used for immunization. However, a divalent cross-linking reagent such as glutaraldehyde is used for binding the target compound to the polymer carrier, and the efficiency of the cross-linking reaction is low. In principle, there is a problem that it is difficult to avoid, such as the possibility that its own structure may differ from the intended structure, and that the binding site of the polymer carrier cannot be specified.

The second problem is that screening for a specific hybridoma producing the desired monoclonal is complicated. In particular, when many types of antibodies are produced against a polymer carrier that binds the target compound, when screening for production of a monoclonal antibody against the target compound, high levels of the target compound used for immunization are detected. No molecular complex can be used and two different antigens must be prepared for immunization and antibody detection.

[0008]

[Means for Solving the Problems] The present inventors have conducted earnest research on means for essentially eliminating the above problems, and have already found that the present inventors have two types of DHFR, Bacillus subtilis and Escherichia coli.
We focused on the fact that we have developed various methods for effectively binding the target compound to each carboxy terminus using. That is, a complex protein in which the target compound is bound to the carboxyl terminal side of DHFR is prepared, and an immunized animal is immunized with the complex protein. It was shown that the above-mentioned problems can be solved by devising a method for screening a hybridoma using a molecular carrier.

That is, the present invention uses one of the two kinds of complex proteins obtained by binding the compound of interest to the carboxyl terminal of each of the two kinds of dihydrofolate reductases having different antigenicity. The resulting immune cells are fused with proliferative cells, and then the fused cells that produce the desired monoclonal antibody are selected from the fused cells using the other complex protein. A method for producing a monoclonal antibody, which comprises producing a monoclonal antibody from Examples of the two types of dihydrofolate reductase include those derived from Escherichia coli and Bacillus subtilis, and examples of cells that can proliferate include mouse myeloma cells. Hereinafter, the present invention will be described in detail.

The present invention comprises (1) preparation of a composite DHFR having a target compound on the carboxyl terminal side, and (2) preparation of a monoclonal antibody using the composite DHFR.

The composite DHFR having the target compound on the carboxyl terminal side can be prepared by various methods.

When the target compound is a peptide, it can be prepared by genetic engineering. A method for preparing a DHFR fusion protein having a target peptide on the carboxy terminal side by a genetic engineering method (Patent Publication No. 3-64112).
No. 3, Patent Publication No. 3-49559, Patent Publication No. 3-64111) and a method for purifying and separating a target fusion protein from a host bacterium (Patent Publication No. 3-33315, Patent Publication No. 1-555108). Gazette, Patent Publication No. 3
No. 646517) has been developed by the present inventors, and a highly purified target fusion protein can be obtained by following the method. The method for producing a fusion protein with DHFR is known for the method using DHFR derived from Bacillus subtilis and Escherichia coli, but it can be easily analogized that it is also possible to use DHFR derived from various organisms. As it is possible, the present invention is not limited to the origin of DHFR.

The fusion protein is produced by synthesizing or isolating the DNA encoding the peptide of interest and incorporating it into a vector for producing a fusion protein which the present inventors have already developed. Express in host cells. The host cells are cultured, and the expressed fusion protein is highly purified using the DHFR activity as a guide. Regarding the target peptide, when DHFR of Escherichia coli was used, it was successful in expressing and producing a fusion protein with prolactin of about 200 amino acids (Patent Publication No. 2-142479). Can be used.

When the target compound cannot be prepared by the above-mentioned method, DHFR in which a cystine residue is introduced at the carboxyl terminal of DHFR (patent application on Nov. 26, 1991, "Novel dihydrofolate reductase and modified dihydrofolate This can be achieved by utilizing a reductase gene ").

The DHFR used for this purpose has a structure in which an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus is introduced at the carboxy terminus of the enzyme protein. The reactivity of the carboxy terminal mercapto group in this DHFR is high, and the neutrality (pH
7.0), thiol-disulfide exchange reaction with 5,5'-dithiobis (2-nitrobenzoic acid) at room temperature (15-30 ° C) reacts rapidly and completes in about several minutes. It has already been shown (November 26, 1991)
Japanese patent application "Novel dihydrofolate reductase and modified dihydrofolate reductase genes"). Therefore, when a thiol derivative of the target compound is prepared, the target compound can be obtained by utilizing the thiol-disulfide exchange reaction.
It is possible to bind to the carboxyl terminus of.

Further, as specific reactions of thiol (SH group), other than this, reaction with (I) metal ion and organic mercury compound, (II) alkylation reaction, etc. are known (for reference, For example, “Chemical modification of SH group”, Masatsune Ishiguro, Academic Society Publishing Center, 1978) By utilizing such a reaction, it is possible to bind each reaction derivative of the target compound to the carboxyl terminal of DHFR. .

The immunization of an immunized animal using the composite protein prepared by the above-mentioned method can be carried out by a conventional immunization method. That is,
It can be carried out by mixing the fusion protein with an adjuvant, injecting it in several divided doses at intervals, collecting blood, separating the antibody from the blood, and examining the antibody titer.

The advantage of using the complex DHFR having the target compound on the carboxy terminal side as an immunizing antigen is that it can be highly purified by utilizing the DHFR enzyme activity, and thus it can be used to eliminate unnecessary antibodies derived from impurities. Production induction can be suppressed. The complex protein is homogeneous. The other structures are the structures of interest except some of the compounds are attached to the carboxy terminus of DHFR. Since DHFR is derived from a bacterium and is closely related to an immunized animal, it can be expected to have an enhancing effect on antibody production. And so on.

In the examples of the present invention, leucine enkephalin (LEK), which is composed of 5 amino acid residues and has no specificity among species and thus has extremely weak antigenic activity, is effectively immunized with a very small amount of 1.4 μg as LEK. It is shown that sensitization is performed, showing that the present invention is excellent.
The procedure for preparing the monoclonal antibody of the present invention will be described below by taking LEK as an example. When DHFR in which a compound other than LEK is bound to the carboxyl terminus is used, the same preparation procedure can be applied.

Immunization was carried out by using a fusion protein (bs) in which LEK was linked to the carboxy terminus of Bacillus subtilis-derived DHFR.
DHFR-LEK) was prepared and bsDHF was added to BALB / c female mice which were used as experimental animals for immunization.
After diluting R-LEK with physiological saline to a concentration of 0.5 mg / ml, an equal volume of complete Freund's adjuvant (first time) or incomplete Freund's adjuvant (2
And 3rd time) and intraperitoneally administered to the mouse 3 times every 3 weeks. Efficient antibody production is observed 14 days after the final injection.

For antibody production, blood was collected from the tail vein of mouse, serum was separated, and this was analyzed by solid phase enzyme immunoassay (ELISA).
Can be performed by measuring the titer. In this example, the following method was used, but the present invention is not limited by the method of ELISA and the protein immobilized with LEK used therein.

A fusion protein (ecDHFR-LEK) in which LEK is linked to the carboxyl terminal of DHFR derived from Escherichia coli can be used as the polymer-bound antigen for titer measurement. The antigenicity of bs-DHFR and ec-DHFR are different from each other, and the antibody produced by immunizing bs-DHFR cannot recognize ec-DHFR.
An antibody only to the EK part can be specifically detected.

The titer of LEK antibody when immunized with bsDHFR-LEK is measured by the following method. ecDHF
R-LEK at 1 M in 0.1 M sodium carbonate buffer pH 9.5
Dilute to a concentration of -10 μg / ml. 50 μl of this is dispensed into a commercially available 96-well microplate for ELISA per well, and the mixture is allowed to stand at 4 ° C. for 2 hours or more and adsorbed to the well.
After washing 3 times with water, 0.5% BSA-0.025M EDTA-0.15M NaCl-10m
M Fill the wells with phosphate buffer (pH 7.5) (BEPBS) and leave at room temperature for at least 1 hour. Then 0.05% Tween 20-0.15 M NaC
Wash the plate 3 times with l-10mM phosphate buffer pH7.5 (TPBS) and water, drain well and store at -20 ℃ until just before use. Dispense 50 μl of the serum to be assayed onto the protein-immobilized plate per well, incubate for 1 hour at room temperature, wash 3 times with TPBS and water, and increase by 50 μl to 400 times.
Dispense a horseradish peroxidase-labeled goat anti-mouse IgG + IgM antibody solution diluted with 0.05% Tween 20-BE PBS (TBPBS). Then, after allowing to stand at room temperature for 1 hour, wash the plate in the same manner as above and drain it well.
l ABTS (2,2'-Azino-bis (3-ethylbenzothiatholin-6-sulfonic acid) -0.
0.25% H ₂ O ₂ -0.1M citrate buffer (pH 4.2) is added and color reaction is performed. The reaction is stopped by adding 150 μl of 0.5% SDS aqueous solution, and the increase in absorption at 405 nm is measured with a microplate reader (Corona) to quantify the mouse gammaglobulin remaining on the plate. By this method, only the antibody against LEK can be detected, and it can be known whether the animal is immunized with the LEK part. When immunizing with ecDHFR-LEK,
The same can be done using bsDHFR-LEK.

Hybridomas are produced by cell fusion by Kohl.
It can be done according to the method of er and Milstein. In the example, the ELIS among the mice immunized for the third time
In one animal in which anti-LEK activity was strongly detected in A, 17 μg of DHFR-L was injected into the tail vein 3 weeks after the third sensitization.
The spleen was removed 4 days after stimulating the immune system by injecting 0.1 ml of physiological saline containing EK. For cell fusion, dispersed spleen cells and mouse myeloma cells x63 / Ag8 / 653 strain were mixed at a ratio of 5: 1, and cell fusion was performed in 1 ml of 50% PEG-RPMI.
After fusion, dispense 100 μl of the cell dispersion into 695 wells.
Hybridomas were selected in RPMI-HAT-15% FCS medium. Hybridoma cloning was performed by the dilution method.

The cloned hybridoma is LEK
EcDHFR
-Perform according to the above ELISA with LEK immobilized.
The ecDHFR-LEK is adsorbed to the well, washed three times, and the cell culture medium of the hybridoma is used as a measurement.

The ELISA used to identify the globulin class of a monoclonal antibody can be carried out according to the prescription using an antibody kit for immunoglobulin class identification from Zymed. In addition, LE of anti-LEK monoclonal antibody
The ELISA for measuring the immune cross-activity against various peptides, which was carried out to examine the K recognition site, is carried out as follows. bsDHFR-LEK fusion protein 100 μg / ml
20μ in each well of the microplate immobilized by
50 μl of g / ml anti-LEK monoclonal antibody and 50 μl of peptide solution with a concentration of serial dilution with TBPBS were dispensed, reacted at room temperature for 2 hours, washed with TPBS and water, and washed with horseradish peroxidase-labeled goat anti-mouse IgG +. Using the IgM antibody solution, the amount of anti-LEK monoclonal antibody remaining on the plate is color-determined as described above. In the embodiment of the present invention,
Finally, two types of monoclonal antibodies against LEK were obtained, and the method of the present invention was confirmed to be effective.

[0027]

EXAMPLES Next, examples of the present invention will be described. Example 1 Preparation of DHFR-LEK Fusion Protein Escherichia coli containing a plasmid having a DHFR-LEK gene prepared by ligating a gene encoding LEK to a DHFR gene of Bacillus subtilis or Escherichia coli was prepared by genetic engineering (Patent Publication No. 1-48754, Patent Publication No. 3-55108). Liquid culture was carried out for each of these Escherichia coli, and the obtained bacterial cells were subjected to ultrasonic treatment and DEAE-
DHFR by Toyopearl 650M ion exchange chromatography and Toyopearl HW55 gel chromatography
-LEK protein fraction was purified and purified, and Bacillus subtilis and Escherichia coli DHFR-LE showing uniform bands on SDS electrophoresis
A K fusion protein was obtained.

Example 2 Immunization with bsDHFR-LEK fusion protein [0028] Serum after three times of immunization of three bsDHFR-LEK 50 µg-administered groups and three 10 µg-administered groups per mouse was separated and the antibody titer was measured. . In the ELISA performed by immobilizing ecDHFR-LEK on the plate, there was no difference in serum antibody titer between the two groups, but for BSA-LEK prepared by binding LEK to BSA by the glutaraldehyde method. The presence of LEK binding activity was observed only in the sera of two mice in the 50 μg administration group. This is 1) bsDHF
In order to express anti-LEK antibody by three times administration of R-LEK, 50 μg of bsDHFR-LEK (LEK content:
If about 1.4 μg) is administered to mice, the production of antibodies against LEK can be induced even with a fusion protein in which one molecule of DHFR is bound to one molecule of LEK. 2) It is suggested that DHFR can be a useful carrier that can be imparted with antigenicity by binding a low molecular weight peptide originally having no antigenic activity to the carboxyl terminus by genetic engineering.

Example 3 Preparation of Antibody-Producing Hybridoma Cells One mouse of which the anti-LEK activity was strongly detected by ELISA among the mice immunized for the third time was tail vein 3 weeks after the third sensitization. 17 μg of bcDHFR-LE in
The spleen was extracted 4 days after stimulating the immune system by injecting 0.1 ml of physiological saline containing K. For cell fusion, dispersed spleen cells and mouse myeloma cells x63 / Ag8 / 653 strain were mixed at a ratio of 5: 1, and cell fusion was performed in 1 ml of 50% PEG-RPMI. After fusion, disperse the cell dispersion into 695 wells in 100 μl aliquots.
Hybridomas were selected in MI-HAT-15% FCS medium.
Hybridoma cloning was performed by the dilution method. 695
From the cell dispersion liquid dispersed in each well, formation of hybridoma colonies was observed in 146 wells by HAT selection. As a result of ELISA performed by immobilizing bsDHFR-LEK on a 96-well microplate, bsDHFR- was added to a culture medium of 11 of 146 wells.
LEK binding activity was observed. About 10 strains obtained by purifying hybridomas by cloning these wells, 3 strains (NO.36, NO.74, NO.11 by ELISA performed by fixing the plate with ecDHFR-LEK) were used.
It was revealed that 3) has antibody binding activity to LEK. This supports that DHFR is useful as a carrier for preparing anti-LEK antibody.

Example 4 Identification of the subclass of the obtained monoclonal antibody. 10 strains (ec) having binding activity of bsDHFR-LEK
(Including 3 strains showing a binding activity to DHFR-LEK) were identified by ELISA for identifying the gamma-globin class producing 7 strains out of 10 strains were Ig.
It was found that the strain was G ₁ κ type and that the remaining 3 strains produced IgM.

Example 5 Obtained Monoclonal Antibody, bsDHFR
-LEK-36 and -74 antibodies, immunocross-activity ELIS for various peptides and proteins to examine the immuno-cross-reactivity of monoclonal antibodies against LEK
A was performed. As a result, the bsDHFR-LEK-36 and -74 antibodies showed 10 ^-7 M to 10 ^-4 against LEK.
A dose-response curve was shown over a concentration range of M, with IC _50's of 3.74 × 10 ⁻⁶ M and 4.66 × 10 ⁻⁶ M, respectively. In addition, bsDHFR-LEK-No. We have not examined the 113 antibody. This is bsDHFR-L
The possibility of quantitative ELISA using EK is suggested. The major LEK recognition sites of both antibodies showed weak immunocross activity against methionine enkephalin, which has methionine at the carboxyl terminus instead of leucine, while LEK-NH ₂ and the amino acid sequence of LEK at the amino terminus. Since it did not show any cross-activity with respect to α-neoendrophin having, it was shown to be the side chain portion of the leucine residue on the carboxyl terminal side and the carboxyl group. Also, both antibodies D-, L- leucine and ^{D-Ala 2, D-Leu} 5 -enkeph
The fact that it did not show cross-activity to alin also suggests that it recognizes the asymmetry of the leucine residue and that there is a recognition site at other sites of LEK.
This means that Tyr which is a sulfonated tyrosine residue at position 1
This is also supported by the fact that both antibodies differ in cross-activity to (Sul) ^1- enkephalin. Furthermore, while it showed immunocross activity against ecDHFR-LEK, it did not bind LEK to the carboxyl terminus.
Since it did not react with sDHFR or ecDHFR, both antibodies were revealed to be antibodies that specifically recognize the LEK portion of bsDHFR-LEK.

According to the present invention, it is possible to easily prepare a monoclonal antibody against a compound having extremely low antigenic activity. That is, by changing the complex protein used for immunization of animals and the complex protein used for screening of fused cells, many kinds of antibodies against DHFR binding to the target compound used for immunization are produced. It is possible to eliminate the complexity of the screening due to. Further, this largely contributes to various analyzes using the monoclonal antibody.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // C07K 16/44 8318-4H C12N 15/02 (C12P 21/08 C12R 1:91)

Claims (3)

[Claims] 1. Immunization of an animal with one of two kinds of complex proteins obtained by binding a compound of interest to the carboxyl terminal of each of two kinds of dihydrofolate reductases having different antigenicity. Then, the obtained immune cells and proliferative cells are fused, and then, using the other complex protein, a fused cell that produces the desired monoclonal antibody is selected from the fused cells, and the monoclonal antibody is obtained from the obtained fused cells. A method for producing a monoclonal antibody, which comprises producing the monoclonal antibody. 2. The method for producing a monoclonal antibody according to claim 1, wherein the two types of dihydrofolate reductases are derived from Escherichia coli and Bacillus subtilis. 3. The method for producing a monoclonal antibody according to claim 1, wherein the proliferative cells are mouse myeloma cells.