JP3887154B2 - Monoclonal antibody against human MK - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to a monoclonal antibody that specifically binds to human midkine , and a method for producing the same .
[0002]
[Prior art]
Midkine (MK) is a growth / differentiation factor discovered as a product of a gene that is transiently expressed during the process of induction of differentiation of embryonic tumor cells (EC) by retinoic acid. A rich 13 kDa polypeptide (Kadomatsu, K. et al .: Biochem. Biophys. Res. Commun., 151: 1312-1318; Tomomura, M. et al .: J. Biol. Chem., 265: 10765 -10770, 1990). MK has been found from humans to Xenopus, but protein conservation between species is high, and human and mouse MKs have 87% homology in amino acid sequence. MK has various biological activities. That is, it is considered to be involved in the construction of the nervous system by promoting neurite outgrowth and nerve cell survival. It is also expressed in tissues at the time of injury, many human cancers, and senile plaques of Alzheimer's disease, and is also noted in relation to tissue repair, fibrinolytic system activation ability, and pathological conditions.
Development of a measurement system for measuring MK with high sensitivity is desired because of the biological activity of such human MK (sometimes simply referred to as “MK”) and the possibility of involvement of MK in the course of disease. As a method for quantitatively measuring an antigen such as MK, enzyme immunoassay (EIA) is considered to be most appropriate. Heterogeneous non-competitive method (sandwich method) is the most commonly used measurement system for antigen measurement by EIA. In this method, an immunocomplex consisting of an antibody insolubilized on a solid phase, an antigen to be measured, and an enzyme-labeled antibody is formed on the solid phase, and the enzyme activity on the solid phase is measured and measured. This is a method of knowing the amount of antigen.
[0003]
A 1-step sandwich method has been proposed as an EIA for measuring MK (Japanese Patent Laid-Open No. 10-160735). In this method, polyclonal antibodies derived from different animals (rabbit and chicken) are used for both the immobilized antibody and the labeled antibody. Polyclonal antibodies have high avidity, but cross-reaction with unintended biomolecules mixed in the immunogen often becomes a problem, and it is impossible to supply antibodies of a certain quality indefinitely. On the other hand, since a monoclonal antibody recognizes a specific epitope site of a part of an antigen, it has high specificity and can supply an infinite number of antibodies of a certain quality. Therefore, it is desirable to change the antibody combination used in the 1-step sandwich method for measuring human MK from a polyclonal antibody-polyclonal antibody combination to a polyclonal antibody-monoclonal antibody or a monoclonal antibody-monoclonal antibody. ing. However, preparation of a monoclonal antibody that specifically recognizes MK has been attempted so far, but no anti-human MK monoclonal antibody has been obtained.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a monoclonal antibody that specifically recognizes human MK.
[0005]
[Means for Solving the Problems]
If the immunogen is an animal-derived biologically active substance, the immunized animal will have the same biological activity, and there may be many parts with homology in the chemical structure and few parts in the species-specific structure. The selection of immunized animals is difficult.
In the case of MK, as described above, protein conservation between species is high, and human and mouse MK have 87% homology in amino acid sequence. For this reason, when a mouse is immunized with the human MK protein as an immunogen, the degree of recognition as a foreign heterologous protein in the mouse may be reduced. Actually, there has been no report that anti-human MK monoclonal antibodies have been produced using mice as immunized animals.
The present inventors used multiple lines of mice knocked out of the MK gene as immunized animals. Examining the sex and age of mice, examining the immunization method, immunization amount, injection site (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intralymphatic node, intrafootpad), administration interval, etc. A mouse anti-human MK monoclonal antibody having a high antibody titer could be prepared.
[0006]
That is, the present invention provides an anti-midkine monoclonal antibody that specifically binds to human midkine.
In addition, mice knocked out of the midkine gene are immunized with human midkine or a human midkine fragment having the same biological activity as the human midkine, and cell fusion is performed between the spleen cells obtained from the immunized mouse and myeloma cells. The present invention also provides a method for producing an anti-human midkine / mouse monoclonal antibody, comprising selecting a monoclonal antibody-producing hybridoma, proliferating the selected hybridoma, and collecting the produced monoclonal antibody.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
1. Generation of MK gene knockout mice The technology of gene disruption by homologous recombination has already been established and has been attempted for a large number of genes. In particular, a means of disrupting (knocking out) a specific gene of embryonic stem cells (ES cells) and then injecting the cells into a blastocyst to produce a chimeric mouse produces a mutant mouse of the targeted gene. It is very useful in that you can explore its functions. Today, when a new gene is isolated, it is a natural research method to first create a knockout mouse.
The gene for human MK has already been cloned (Uehara, K. et al .: J. Biochem., 111: 563-567, 1992). Regarding mice knocked out of the MK gene, knockout mice in which a part of exon 2 and part of exon 3 of 129 / Sv mice were disrupted have already been prepared (Nakamura, E. et al .: Genes to Cells). , 3: 811-822, 1998). This MK gene knockout mouse is not lethal during the embryonic period, and has a significantly lower body weight than the heterozygote or wild type. Depending on the strain of the mouse, there are strains in which ES cell lines can be easily established, and strains that are not. From the 129 strain, it can be easily established without feeder cells.
There are a number of reports (eg, Mansor, SL et al .: Nature, 336: 348-352, 1988; Joyner, A., ed .: Gene targeting, IRL Press) related to the generation of mice that knocked out various genes. , 1993; Shinichi Aizawa: Generating mutant mice using gene targeting ES cells. Yodosha, 1995) and human MK genes are well known in the art. MK gene knockout mice can be easily prepared with reference to.
[0008]
2. Preparation of mouse anti-human MK monoclonal antibody As for monoclonal antibodies, the production method was reported by Köhler and Milstein in 1975, and many literatures and experimental books have been published on the production of monoclonal antibodies. However, in general, obtaining a monoclonal antibody having high specificity and affinity for any antigen has not yet become an established technique.
[0009]
A monoclonal antibody is generally prepared by the following steps.
1. 1. Preparation of antigen 2. Immunization of animals with antigens 3. Cell fusion 4. Screening for monoclonal antibodies Proliferation and freezing of antibody-producing cells The production of the anti-human MK monoclonal antibody of the present invention is also produced according to this process.
[0010]
1. Preparation of immunogen The cDNA of human MK has been cloned (JP-A-5-91880). The immunogenic MK protein can be prepared as a recombinant MK. For example, recombinant MK in which this cDNA is expressed in Pichia yeast has been reported (Japanese Patent Laid-Open No. 9-95454). Chemically synthesized MK can also be tried as an immunogen. Full length MK has been chemically synthesized (Inui, T. et al .: J. Peptide Sci., 2: 28-39, 1996). In the present invention, an MK fragment having the same biological activity (heparin binding ability, neurite extension ability, fibrinolytic system activation ability) as human MK, for example, a truncated MK lacking the N domain (Kaname, T. et al .: Biochem Biophys. Res. Commun., 219: 256-260, 1996) Fragments may be attempted as immunogens. Such an MK fragment may be used by covalently binding to a carrier protein so as to have sufficient immunogenicity.
Examples of carrier proteins include albumin (bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin), serum globulin fraction (bovine gamma globulin, horse serum globulin, sheep gamma globulin), thyroglobulin (bovine thyroglobulin, butyrothyro Globulin), hemocyanin (KLH), synthetic polypeptides (poly-L-lysine, polyglutamic acid) and the like.
As a method for binding the MK fragment to the carrier protein, (1) a method using SH of cysteine residue (MBS method), (2) a method using amino group (bisimide ester method, glutaraldehyde method), ( 3) A method using a phenol group (bis-diazobenzene method), (4) a method using a carboxyl group (carbodiimide method), and the like.
It is necessary to try various combinations of carrier proteins and binding methods that bind to MK and to select the most immunogenic combination. Since such a selection can be easily performed by a person skilled in the art through routine experiments, the combinations thus selected are included in the present invention.
[0011]
2. Immunization of MK Knockout Mice with MK Antigen When immunizing MK knockout mice, the animal species and the ability to respond to the antigen are important points. Generally, when the animal species used for immunization and the animal species of myeloma cells are the same, a hybridoma is efficiently formed. In particular, since all myeloma cells derived from mice originate from BALB / c mice, it is considered that BALB / c mice are suitable for MK knockout mouse strains of immunized animals. In this case, both spleen cells and myeloma cells are derived from BALB / c, and the resulting hybridoma can grow in the intraperitoneal cavity of BALB / c, so that a high concentration of monoclonal antibody can be obtained from ascites without any special treatment. . However, it is difficult to establish ES cells from BALB / c mice.
[0012]
For immunization, for many protein antigens and polysaccharide antigens, an emulsion is prepared by mixing 1 to 100 μg of an antigen and an equal amount of Freund's complete adjuvant, and then administered intraperitoneally or subcutaneously. Although the action of adjuvants is not fully understood, it is believed that there are at least two important actions. One is a function of preventing the antigen from being processed quickly, and the other is a function of inducing a non-specific immune reaction to easily cause an immune reaction against the target antigen. In general, the former is widely used, and an emulsion with an aqueous solution is prepared and used. Recently, liposomes and synthetic surfactants have also been used as adjuvants. In the latter, Lipid A and killed bacteria are used. Currently used dead bacteria are Bordetalla pertussis and Mycobacterium tuberculosis. Mycobacterium tuberculosis is a dead bacterium added to Freund's complete adjuvant. The active site has been shown to be present in muramyl dipeptide (MDP), and various properties of MDP are commercially available. In this way, a substance that retains an antigen and a substance that non-specifically elicits an immune reaction are combined and used as an adjuvant (including dead bacteria). In general, Freund's complete adjuvant is often used for the initial immunization, and incomplete adjuvant (not including dead bacteria) is often used for the booster immunization. Since the optimal selection and combination of adjuvants can be easily carried out by those skilled in the art through routine experimentation, combinations thus selected are included in the present invention.
One to several booster immunizations are performed at intervals of 2 to 3 weeks after the initial immunization. Immediately before the booster immunization and one week after that, a test blood is collected to measure antibody titer. The mouse with the highest antibody titer is selected and boosted to provide for experiments.
[0013]
The above immunization methods can vary depending on the nature and amount of the antigen, and selection of the optimal method is a routine task for those skilled in the art.
[0014]
3. Cell fusion Cell fusion is basically performed according to the method of Milstetan et al. (Galfre, G. & Milstein, C., Methods Enzymol. 73: 3-46, 1981). Representative mouse line myeloma cells used for monoclonal antibody production are P3-X63-Ag8, P3-X63-Ag8U1, X63Ag8.653, SP2 / 0-Ag14, FO, NSI / 1-Ag4-1, NSO / 1. FOX-NY, but all of these cell lines are derived from the P3K strain. P3K is a cell derived from the myeloma strain MOPC21 derived from mineral oil-induced BALB / c mice, and P3 and MS-1 were obtained as cell lines without the P3K HGPRT gene.
[0015]
4). Monoclonal antibody screening When screening for the presence or absence of antibodies in the culture supernatant after cell fusion 7 to 14 days later, it is convenient to use EIA in which MK is bound to a solid phase. After screening with EIA, cells in positive wells are expanded and cloned.
5). Positive wells detected by growth and freeze screening of antibody-producing cells should contain the cells of interest. However, as the passage continues, the target cell may be lost due to loss of survival competition with other cells or loss of chromosomes. In preparation for this situation, it is important to keep the cells frozen while cloning.
In the present invention, the mouse anti-human MK monoclonal antibody thus obtained, and Fab, Fab ′, F obtained by treating the antibody with an enzyme such as trypsin, papain, pepsin, etc. (ab ') is included.
[0016]
The mouse anti-human MK monoclonal antibody obtained by the present invention can be used in place of the mouse anti-human polyclonal antibody of the 1-step sandwich method (Japanese Patent Laid-Open No. 10-160735). In this case, as an antibody imparting a label, an IgG fraction, or a specific Fab ′ obtained by reduction after pepsin digestion can be used.
[0017]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[0018]
[Example 1] Production of mouse anti-human MK An immunized animal was an MK knockout mouse derived from two lines (A and B) of ES cells (Nakamura, E. et al .: Genes to Cells, 3: 811-822, 1998). ), Males and females were used as immunized animals. Human MK protein (Japanese Patent Laid-Open No. 9-95454) was used as the antigen. Lines and antigen doses are as follows.
・ Line A (male, 3) ・ ・ ・ MK100μg / animal ・ Line A (female, 2) ... MK1μg / animal ・ Line B (male, 4) ・ MK1μg / animal ・ Line B (female) 5) MK 10 μg / animal for the first time, immunized several sites subcutaneously in the back, and immunized intraperitoneally for the second and subsequent times. Immunization was performed approximately once every 3 weeks. All mice were immunized 3 times, and one week later, blood was collected from the orbit of each mouse, and the antibody titer was measured by EIA using the serum.
A marked increase in antibody titer was observed for 5 females in line B. Therefore, the final booster immunization was performed at 10 μg / mouse from the fundus vein of the mouse of these five MK knockout mice.
Cell fusion of spleen cells and myeloma cells (P3U1) obtained from each mouse individual is basically performed by the method of Milstein et al. (Galfre, G. & Milstein, C., Methods Enzymol. 73: 3-46). , 1981). As a result of usual cloning, five clones (clone A, B, C, D, and E) were obtained from one specific mouse individual. These clones were stored frozen. Monoclonal antibodies were prepared from these clones by conventional methods. All five subclasses of these antibodies were IgG (κ).
Next, the antibody titer (dilution ratio: 100, 300, 900, 2700, 8100, 24300, and 72900) of each of these clones was examined. The results are shown in FIG.
[0019]
【The invention's effect】
According to the present invention, a monoclonal antibody against human MK was obtained. This mouse anti-human MK monoclonal antibody can be effectively applied to the 1-step sandwich method.
[0020]
[Brief description of the drawings]
FIG. 1 is a diagram showing mutant chromosomes of knockout mice in which exons 2 and 3 of 129 / Sv mice are partially destroyed.
FIG. 2 shows the antibody titers of monoclonal antibodies obtained from clones A, B, C, D, and E.

Claims (3)

A mouse knocked out of a midkine gene is immunized with human midkine or a human midkine fragment having heparin binding ability, neurite outgrowth ability, and / or fibrinolytic system activation ability equivalent to the human midkine, An anti-human midkine / mouse monoclonal antibody, characterized in that a spleen cell obtained from the above and a myeloma cell are fused, a monoclonal antibody-producing hybridoma is selected, the selected hybridoma is expanded, and the produced monoclonal antibody is collected. Antibody production method. An antibody produced by the method according to claim 1. 3. An antibody fragment obtained from the anti-midkine monoclonal antibody according to claim 2, which specifically binds to human midkine.

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