JP2002085058A - Monoclonal antibody against human mk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody that specifically recognizes the human MK. SOLUTION: As a result that the MK gene knock-out mouse is used as an immune animal, an antimouse human MK monoclonal antibody could be created.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically binds to human MK, and to the use of the antibody.

[0002]

2. Description of the Related Art Midkine (MK) is a growth / differentiation factor discovered as a product of a gene that is transiently expressed during the induction of differentiation of embryonic tumor cells (EC) by retinoic acid. 13 kDa polypeptide rich in neutral amino acids and cysteine (Kadomatsu, K. et a
l .: Biochem. Biophys. Res. Commun., 151: 1312-13
18; Tomomura, M. et al .: J. Biol. Chem., 265: 10
765-10770, 1990). MKs have been found from humans to Xenopus laevis, but protein conservation between species is high, and human and mouse MKs have 87% homology in amino acid sequence. MK has various biological activities. In other words, it is considered that they promote neurite outgrowth and survival of nerve cells, and are involved in the construction of the nervous system. 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 activation, and pathology. Given the biological activity of such human MK (sometimes simply referred to as "MK") and the possibility of MK involvement in the course of disease, development of a measurement system for measuring MK with high sensitivity is desired. The enzyme immunoassay (EIA) is considered to be the most appropriate method for quantitatively measuring an antigen such as MK. Among antigen measurement systems using EIA, the heterogeneous non-competitive method (sandwich method) is the most frequently used measurement system. In this method, an immune complex 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,
This is a method of measuring the amount of an antigen to be measured by measuring the enzyme activity on a solid phase.

A one-step sandwich method has been proposed as an EIA for measuring MK (JP-A-10-160).
735). This method uses polyclonal antibodies derived from different animals (rabbit and chicken) for both the immobilized antibody and the labeled antibody. Although polyclonal antibodies have high avidity, cross-reaction with unintended biomolecules mixed in the immunogen often poses a problem, and it is impossible to supply antibodies of constant quality indefinitely. On the other hand, since a monoclonal antibody recognizes a part of a specific epitope of an antigen, it can supply an antibody of high specificity and constant quality indefinitely. Therefore, it is desired to change the combination of antibodies used in the one-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, attempts have been made to produce a monoclonal antibody that specifically recognizes MK, but no anti-human MK monoclonal antibody has been obtained.

[0004]

An object of the present invention is to provide a monoclonal antibody that specifically recognizes human MK.

[0005]

When the immunogen is an animal-derived biologically active substance, the animal to be immunized has the same biological activity, has many homologous chemical structures, and has a species-specific structure. The selection of an immunized animal is difficult because the portion of the immunized animal may be small. In the case of MK, the degree of conservation of proteins between species is high as described above, and human and mouse MKs have 87% homology in amino acid sequences. Therefore, using human MK protein as an immunogen,
When a mouse is immunized, it is considered that the degree of recognition as a foreign heterologous protein is reduced in the mouse.
Actually, there is no report to date that an anti-human MK monoclonal antibody was produced using a mouse as an immunized animal. The present inventors used multiple lines of mice in which the MK gene was knocked out as immunized animals. The gender and age of the mouse were examined.
By examining the injection site (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intralymphatic, intra-footpad), administration interval, etc., we were able to produce a mouse anti-human MK monoclonal antibody with high antibody titer .

That is, the present invention relates to (1) a monoclonal antibody that specifically reacts with human MK and a fragment thereof having the same immunoreactivity as the antibody, and (2) MK or MK in a mouse in which the MK gene is knocked out. (1) obtained by immunizing a fragment thereof having the same biological activity as described above, and a fragment thereof having the same immunoreactivity as the antibody.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Production of MK Gene Knockout Mice The technique of gene disruption by homologous recombination has been established, and attempts have been made for a large number of genes. In particular, a means of disrupting (knocking out) a specific gene of an embryonic stem cell (ES cell) and then injecting the cell into a blastocyst to produce a chimeric mouse is to produce a mutant mouse of the targeted gene. Very useful in the sense that you can explore its functionality. Nowadays, once a new gene has been isolated, it is a natural research technique 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). And for mice knocking out the MK gene, 12
Knockout mice in which a part of exon 2 and a part of exon 3 of 9 / Sv mouse have been destroyed have already been produced (Na
kamura, E. et al .: Genes to Cells, 3: 811-822, 19
98). The MK gene knockout mouse is not lethal during the embryonic period, and has a significantly lower body weight than the heterozygote or the wild type. Depending on the mouse strain, there are strains for which ES cell lines are easily established and strains for which ES cell strains are not. From line 129, it can be easily established without feeder cells. Numerous reports have been made on techniques related to the production of mice in which various genes have been knocked out (eg, Mansor, SL et al .: Nature, 336: 348-35).
2, 1988; Joyner, A., ed .: Gene targeting, IRL Pres
1993, Shinichi Aizawa: Generation of mutant mice using gene-targeting ES cells. Yodosha, 1995), and the human MK gene is known in the literature. MK easily by referring to the literature
Gene knockout mice can be produced.

[0008] 2. Production of Mouse Anti-Human MK Monoclonal Antibody For the monoclonal antibody, Koehler and Milstein reported a method for producing it in 1975.
Since then, numerous publications 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 all antigens has not yet been established.

[0009] A monoclonal antibody is generally prepared by the following steps. 1. 1. Preparation of antigen 2. Immunization of animals with antigen Cell fusion 4. Screening of monoclonal antibody Propagation and Freezing of Antibody-Producing Cells The preparation of the anti-human MK monoclonal antibody of the present invention is also prepared according to this step.

[0010] 1. Preparation of immunogen The cDNA of human MK has been cloned (JP
5-91880). The immunogenic MK protein is a recombinant MK
Can be prepared as For example, a recombinant MK expressing this cDNA in Pichia yeast or the like has been reported (JP-A-9-95454). Also, chemically synthesized MKs can be tried as immunogens. 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 elongation ability, fibrinolytic activation ability) as human MK, for example, a truncated MK lacking the N domain (Kana
me, T. et al .: Biochem. Biophys. Res. Commun., 21
9: 256-260, 1996) Fragments may be tried as immunogens.
Such an MK fragment should have sufficient immunogenicity,
It is good to use by covalently bonding to a carrier protein. As a carrier protein, 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, butathyroglobulin), hemocyanin (KLH), synthetic polypeptide (poly-L-lysine, poly Glutamic acid) and the like. Methods for binding the MK fragment to the carrier protein include (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 the carrier protein that binds to MK and the binding method, and select the combination having the strongest immunogenicity. Such a selection can be easily made by a person skilled in the art by routine experimentation, and thus the combination thus selected is included in the present invention.

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. In general, when the animal species used for immunization and the myeloma cell are the same, hybridomas are efficiently formed. In particular, since all mouse-derived myeloma cells originate from BALB / c mice, it is considered that BALB / c mice are suitable as the immunized animal MK knockout mouse strain. In this case, both splenocytes and myeloma cells are BALB
/ c, and the resulting hybridomas can grow in the intraperitoneal cavity of BALB / c, so that a high concentration of monoclonal antibody can be obtained from ascites without special treatment. But BA
It is difficult to establish ES cells from LB / c mice.

For immunization, for many protein antigens and polysaccharide antigens, an emulsion is prepared by mixing 1 to 100 μg of the antigen with an equal amount of complete Freund's adjuvant, and administered intraperitoneally or subcutaneously. The function of adjuvants is not fully understood, but is believed to have at least two important functions. One is to prevent the antigen from being promptly processed, and the other is to cause a non-specific immune response to easily cause an immune response to the target antigen. The former is widely used in general,
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 dead bacteria are used. Currently the most used killed bacteria are Bo
rdetalla pertussis and Mycobacterium tuberculosis. Mycobacterium tuberculosis is a killed bacterium that has been 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 nonspecifically elicits an immune response are used in combination as an adjuvant (including dead bacteria). Generally, Freund's complete adjuvant is used for the first immunization, and incomplete adjuvant (not including killed bacteria) is used for the booster immunization in many cases. Combinations so selected are encompassed by the present invention, as the optimal selection and combination of adjuvants can be readily determined by one skilled in the art through routine experimentation. One or several booster immunizations are given at 2-3 week intervals after the first immunization. Immediately before and 1 week after the booster immunization, blood is collected experimentally and the antibody titer is measured. The mouse with the highest antibody titer is selected and boosted for the experiment.

The above immunization methods can vary depending on the nature and amount of the antigen, and selection of the most appropriate method is a routine task for those skilled in the art.

3. Cell fusion Basically, cell fusion is carried out by 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, NS
I / 1-Ag4-1, NSO / 1 and FOX-NY, all of which are derived from the P3K strain. P3K is a mineral oil-induced BALB
/ c Cells derived from mouse myeloma strain MOPC21, P3K
P3 was obtained as a cell line without the HGPRT gene.
And MS-1.

4. Screening of Monoclonal Antibody After cell fusion, when screening for the presence or absence of the antibody in the culture supernatant 7-14 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. Proliferation and freezing of antibody-producing cells Positive wells detected by screening should contain the cells of interest. However, as the passage continues, the target cell may be lost due to loss of competition for survival with another cell or loss of chromosomes. In preparation for such a situation, it is important to keep the cells frozen while cloning. In the present invention, the mouse anti-human M thus obtained
K monoclonal antibody, and Fab, Fab ', F (ab') obtained by treating the antibody with an enzyme such as trypsin, papain, pepsin, and optionally reducing it.

Mouse anti-human MK obtained according to the present invention
The monoclonal antibody can be used in place of the mouse anti-human polyclonal antibody of the one-step sandwich method (Japanese Patent Laid-Open No. 10-160735). In this case, as an antibody to be labeled, an IgG fraction and further a specific Fab ′ obtained by digestion with pepsin followed by reduction can be used.

[0017]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of mouse anti-human MK [0018] Two lines (A and B) of ES cell-derived M
K knockout mouse (Nakamura, E. et al .: Genes
to Cells, 3: 811-822, 1998), males and females were used as immunized animals. The antigen used was human MK protein (JP-A-9-95454). Lines and antigen doses are as follows.・ Line A (male, 3) ・ ・ ・ MK100μg / unit ・ Line A (female, 2) ・ ・ ・ MK1μg / unit ・ Line B (male, 4) ・ ・ ・ MK1μg / unit ・ Line B (female) , MK 10 μg / mouse First, immunization was performed at several sites subcutaneously on the back, and after the second time, immunization was performed intraperitoneally. Immunization was performed at an interval of about once every three weeks. One week after all mice were immunized three times, blood was collected from the orbit of each mouse, and the serum was used to measure the antibody titer by EIA. A remarkable increase in antibody titer was confirmed in the five females on line B. Therefore, MK was obtained from the fundus vein of these five MK knockout mice.
A final boost was performed at 10 μg / animal. 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 cloning by a conventional method, 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 a conventional method. The subclasses of these five antibodies were all IgG (κ). Next, the antibody titer of each of these clones (dilution ratio: 100, 300, 900, 2700, 8
100, 24300, and 72900) were considered. The results are shown in FIG.

[0019]

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 one-step sandwich method.

[0020]

[Brief description of the drawings]

FIG. 1 is a view showing mutant chromosomes of a knockout mouse in which exon 2 and a part of exon 3 of 129 / Sv strain mouse are disrupted.

FIG. 2 shows the antibody titers of monoclonal antibodies obtained from clones A, B, C, D, and E.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12R 1:91) C12N 15/00 C (C12P 21/08 5/00 B C12R 1:91) C12R 1:91) ( 72) Inventor Takashi Muramatsu 2845-161 Black Stone, Tenpaku-cho, Tenpaku-cho, Nagoya-shi, Aichi Prefecture (72) Inventor Shinya Ikematsu 540 Narita, Odawara-shi, Kanagawa Prefecture Pharmaceutical Division, Meiji Dairies Co., Ltd. (72) Inventor Munehiro Oda, Kanagawa 540 Narita, Odawara-shi, Japan Meiji Dairy Products Co., Ltd. (72) Inventor Sadatoshi Sakuma 540 Narita, Odawara-shi, Kanagawa Prefecture Meiji Dairy Products Co., Ltd.F-term (reference) 4B024 AA11 BA45 DA02 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA14 4B065 AA92X AB05 CA25 4H045 AA11 BA10 CA41 DA01 DA76 EA50 FA72 FA74

Claims (2)

[Claims] 1. A monoclonal antibody which specifically reacts with human MK and a fragment thereof having the same immunoreactivity as said antibody. 2. The monoclonal antibody according to claim 1, which is obtained by immunizing a mouse in which the MK gene has been knocked out with MK or a fragment thereof having the same biological activity as the MK, and a monoclonal antibody having the same immunoreactivity as the antibody. fragment.