JPH07109300A - Antibody capable of binding to human ii type phospholipase a2 - Google Patents

Abstract

PURPOSE:To obtain the antibody, capable of promoting physiological actions such as anticoagulant or antimicrobial actions or ameliorating actions on the bloodstream circulation and expected as a therapeutic agent or a diagnostic agent for diseases such as coagulation and fibrinolysis abnormality, septicemia, asthma, arthritis or dermatitis. CONSTITUTION:This antibody is capable of binding to a human II type phospholipase A2 (II type PLA2) and has the activity in promoting the binding of the II type PLA2 to a polysaccharide sulfate such as a heparin. The antibody is obtained by carrying out the cell fusion of B cell of an animal immunized by repetitively injecting a purified II type PLA2 to a cell of a myeloma, culturing the resultant fused cell, sorting out a hybridoma producing the antibody capable of binding to the II type PLA2 from the survived cell according to the enzyme-linked immunosorbent assay (ELISA) method, etc., cloning the resultant hybridoma capable of producing a monoclonal antibody serveral times, then sorting out the hybridoma cell strain capable of producing the monoclonal antibody having the activity in promoting the binding of the II type PLA2 to the polysaccharide sulfate and proliferating the hybridoma cell strain according to a conventional method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody which binds to human type II phospholipase A2. More specifically, it relates to a monoclonal antibody having an activity of promoting the binding between human type II phospholipase A2 and a sulfated polysaccharide such as heparin.

[0002]

2. Description of the Related Art In recent years, phospholipase A2 was purified from human inflammatory diseases and inflammatory localities of animal models of inflammation, and its properties were clarified (Ichiro Kudo, Keizo Inoue, Biochemistry, 6).
4 , 3330-3344, 1992). Human type II phospholipase A2 (hereinafter referred to as "type II PLA2")
Has a molecular weight of 14 KDa, which is almost the same as phospholipase A2 isolated from the pancreas up to that point, but the primary sequence is different from that. Further, unlike the pancreas-derived phospholipase A2, it has a high affinity for sulfated polysaccharides. This property is important physiologically, and the type II P expression-induced by inflammatory cytokines (IL-1, TNF, etc.)
LA2 has been shown to exist in a form bound to heparan sulfate on the cell surface (Murakami, M. et al .: J.
Biol. Chem., 268: 839-844, 1993). However, so far no substance is known to promote such binding.

On the contrary, an antibody that recognizes the heparin-binding domain of type II PLA2 is known as a substance that inhibits this binding (Murakami, M. et al., Methods in Enzymolo).
gy, 197, 223-233, 1991).

On the other hand, it is considered that type II PLA2 acts on vascular endothelial cells to induce PGI ₂ production and exert anticoagulant action and blood circulation improvement action (Murakami, M.).
et al .: J. Biol. Chem., 268: 839-844,1.
993). In addition, it has been reported that it acts on the in vitro blood coagulation system and exhibits an anticoagulant effect (G. Cirin
o, C. Cicala, L. Sorrentino, JL Browning, Thromb
osis Research, 70, 337-342, 1993). Furthermore, it has been reported that type II PLA2 disrupts the membrane of Gram-negative bacteria in the presence of neutrophil-produced cell membrane permeability enhancer (Elsbach, P., Weiss, J., Biochem. Bio
phys. Acta, 947, 29-52, 1988). It is considered that the heparin binding site is also involved in these reactions.

[0005]

The object of the present invention is to find a substance having an activity of promoting the binding between the above-mentioned type II PLA2 and a sulfated polysaccharide such as heparin. Such substances can be expected to promote physiological effects of type II PLA2 such as anticoagulant action, blood circulation improvement action, and antibacterial action. And new therapeutic agents for coagulation / fibrinolysis, ischemia, asthma, arthritis, sepsis and inflammatory diseases of the skin,
It can be a diagnostic agent.

[0006]

[Means for Solving the Problems] The present inventors have investigated the type II PLA.
We obtained 54 kinds of monoclonal antibodies that bind to 2, and found that 10 kinds of them have an activity of promoting the binding between type II PLA2 and sulfated polysaccharides, and completed the present invention.

That is, the present invention is an antibody that binds to human type II phospholipase A2 and is a monoclonal antibody having an activity of promoting the binding between human type II phospholipase A2 and sulfated polysaccharides. The present invention also includes animal cells such as hybridoma cells that produce such monoclonal antibodies.

[0008] Generally, a monoclonal antibody has a property of recognizing a specific site of an antigen molecule and binding to it. Here, when the site in such an antigen molecule is a site that participates in the binding between the antigen molecule and another molecule, the other molecule and the monoclonal antibody compete for the site in the antigen molecule. , The binding of antigen molecules to other molecules will be inhibited. Further, even when the antigen recognition site of the monoclonal antibody is in the vicinity of the binding site with another molecule, when the monoclonal antibody binds, the steric hindrance caused by it also inhibits the binding with other molecules. Become. When the antigen recognition site of the monoclonal antibody is other than these sites, the binding to other molecules may not be inhibited, but in any case, the binding of the monoclonal antibody promotes the binding to other molecules. Sometimes it was unexpected.

In fact, the present inventor also investigated the monoclonal antibody against type II PLA2 and found that it was type II PLA2.
We have found many antibodies that do not affect the binding of sulphated polysaccharides and antibodies that inhibit this binding. However, the present inventor has newly found an antibody having an activity of promoting this binding in addition to these antibodies.

The monoclonal antibody of the present invention can be prepared, for example, by the method described below. That is, basically, Galfre and Milstein, Methods in Enzymolog
y, vol. 73, Academic Press, New York (1981); James
W. Goding, Monoclonal Antibodies: Principles and P
practice, Academic Press, Orlando, Florida (1986);
Current Protocols in Molecular Biology, FM Ausub
The method described in Wiley Interscience, New York (1987), co-edited by el et al.

To prepare a monoclonal antibody,
It is better to purify the antigen protein. This purified protein is also used for antibody titer assays. The method for purifying type II PLA2 used in the preparation of the monoclonal antibody of the present invention will be described in detail in Examples.

Then, the purified type II PLA2 is injected into the animal together with an adjuvant intraperitoneally or subcutaneously to cause an immune response in the animal. The animals used are preferably mammals, and more preferably rats and mice. Most preferred are balb / c mice.

Subsequently, B lymphocytes are taken out from the immunized animal and subjected to cell fusion with animal cells having infinite proliferative ability by the method described in the above literature. The preferred cell line for this fusion is a mouse myeloma cell line, P3-X63
-Ag8-U1 myeloma cells (ATCC CRL15
97) is particularly preferred.

After cell fusion, culturing is continued in an appropriate selective medium according to a conventional method, and cell lines producing an antibody that binds to type II PLA2 are selected from the surviving cells by the ELISA method or the like. Further, it is desirable that the obtained monoclonal antibody-producing cells be cloned several times to select a strain having monoclonality and stable antibody-producing ability. As the cloning method, a limiting dilution method, a soft agarose method, a selection method using a cell sorter, or the like can be used.

Among the thus obtained monoclonal antibodies against type II PLA2, those having an activity of promoting the binding between type II PLA2 and sulfated polysaccharide are selected by, for example, the following method. That is, EL bound with heparin
Biotinylated type II P on 96-well plate for ISA
By adding LA2 and subsequently reacting with alkaline phosphatase-conjugated streptavidin, heparin and type II P
The monoclonal antibody to be evaluated is appropriately diluted and added to the system for detecting the binding to LA2, and the inhibitory or promoting activity of the binding is examined. Thus, from the monoclonal antibodies that bind to type II PLA2, those having an activity of promoting the binding between type II PLA2 and heparin can be selected.

When the monoclonal antibody of the present invention is used for treating humans, it is desirable to use a human type monoclonal antibody. Human type monoclonal antibodies include
In addition to human monoclonal antibodies, so-called chimeric antibodies, that is, those in which only the variable region consists of an amino acid sequence derived from an animal such as a mouse and the constant region uses a human amino acid sequence are also included. In addition, a monoclonal antibody according to the so-called CDR grafting technique, that is, a complementarity determining region consisting of an amino acid sequence of animal origin only and all other sequences of human origin is also included in the human-type monoclonal antibody. Be done.

According to the present gene recombination technology, the chimeric antibody or the monoclonal antibody by the CDR grafting technique is a human antibody gene obtained by extracting the corresponding gene from the mouse hybridoma producing the monoclonal antibody of the present invention. It can also be easily prepared by recombination with, introducing into animal cells and expressing. For example, Int. J. Cancer, 44, 424-
433 (1989), Pro Natl Acad Sci USA, 87, 8
095-8099 (1990).
It is also possible to express the gene corresponding to the antibody of the present invention extracted from the hybridoma in other animal cells for the purpose of improving the production efficiency of the monoclonal antibody without changing the amino acid sequence. The monoclonal antibody of the present invention also includes the monoclonal antibody obtained by these gene recombination techniques and animal cells producing the same.

In addition to the above, there is also a method for producing human monoclonal antibodies by cell fusion of human B lymphocytes with human or mouse myeloma or heteromyeloma. Examples of the method for cell fusion with human myeloma include, for example, J. Immunol Methods, 61, 17 to 32.
(1983), a method of cell fusion with mouse myeloma is described in, for example, Proc Natl Acad Sci USA, 77,6.
841 to 6845 (1980), cell fusion with heteromyeloma is exemplified by Proc Natl Acad Sci.
USA, 81, 3214 (1984) and the like. In carrying out these methods, it is better to stimulate human B lymphocytes in vitro before cell fusion. Examples of the method include Biochem Biop
The method described in hys Rec Commun, 135, 495 (1986) may be used.

In addition, as a method for producing human monoclonal antibodies, transformation of human B lymphocytes by EB virus can be mentioned.

That is, the monoclonal antibody of the present invention is characterized by having an unprecedented activity of having a binding promoting activity between type II PLA2 and sulfated polysaccharides. It does not matter whether or not it is produced. One skilled in the art would have no difficulty in changing species or changing the method of preparation, provided that the monoclonal antibody having such activity exists.

The monoclonal antibody of the present invention is a type II PLA.
It can also be used to measure 2. That is, E
The II type PLA2 concentration can be measured by an immunochemical measuring means known as the LISA method or the RIA method. For example, type II PLA2 can be measured with high sensitivity by using a plate having heparin immobilized on the solid phase side.

Furthermore, the monoclonal antibody of the present invention can also be used to measure type II PLA2 forming a bond with a sulfated polysaccharide. This complex with a sulfated polysaccharide can serve as a Mercumar for the progression of pathological conditions such as inflammation. Examples of such sulfated polysaccharides include heparins. It is known that the type II PLA2 has an increased enzymatic activity in the presence of heparin, whereas the type I PLA2 decreases in the opposite. Therefore, in addition to the blood concentration of type II PLA2 itself, sulfated polysaccharide There is significance in measuring the blood concentration of the complex with.

[0023]

According to the monoclonal antibody of the present invention,
By using it as a therapeutic agent, it can be expected to promote physiological effects of type II PLA2 such as anticoagulant activity, blood circulation improving effect, and antibacterial effect.

Further, according to the diagnostic agent and diagnostic kit using the monoclonal antibody of the present invention, for example, type II PL in blood
The A2 concentration and the concentration of the complex of type II PLA2 and sulfated polysaccharide can be measured.

[0025]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[0026]

Example 1 Preparation of Type II PLA2 The type II PLA2 cDNA was reported by JJ Seilhamer et al.
Biol. Chem., 264, 5335 (1989)). That is, RN derived from human placenta
Reverse transcriptase RA from A (purchased from Clontech)
A cDNA library was prepared by using V-2 (Takara Shuzo) according to the attached protocol.

Next, (5 ') AACTCTAGACCA
TGAAGACCCTCC (3 ') and (5') AG
AAGATCTCCTGCCTGGGCCTCTA
The type II PLA2 cDNA was amplified by the PCR method using a single-stranded synthetic DNA primer of the sequence (3 '). The reaction was performed by using Taq polymerase manufactured by Perkin Elmer Co., Ltd. according to the attached protocol. About 560
The bp type II PLA2 cDNA fragment was purified by agarose gel electrophoresis.

Subsequently, type II PLA2 cDNA was synthesized with synthetic D
Baculovirus recombinant vector pAC YM1 (Matsuura, Y. et al, T. Gen. Viro
L., 68, 1233-1250 (1987); National Institute of Health, Granted by Dr. Zenji Matsuura) BamH
Incorporated into I site.

[0029] The baculovirus (Ac
Recombinant virus was obtained by transfecting insect cells Sf-9 with NPV) DNA. Sf
-9 cells were purchased from Invitrogen and the attached protocol was followed for the procedure of transfection and purification of recombinant virus.

Next, Sf-9 cells were statically cultured on a scale of 20 ml / flask (manufactured by Falcon), and MOI =
1 was infected with the above recombinant virus and cultured at 25 ° C. for 4 days, and the culture supernatant was recovered.

From this culture supernatant, a heparin sepharose column (Pharmacia CL-6B) was used.
Type II PLA2 was purified. That is, φ1.5 × 5 cm
After passing 1.1 liters of culture supernatant using the column of 10 ml, 50 ml of 50 mM Tris.HCl buffer (p
H7.4), 50 ml of 0.3 M NaCl / 50 mM
Tris.HCl (pH 7.4) buffer, 50 ml of 0.6 M NaCl / 50 mM Tris.HCl (p
H7.4) buffer solution was sequentially passed through to obtain 0.6 M Na.
Type II PLA2 was eluted in the Cl fraction.

Next, this eluate was added to Waters 600E.
Reversed phase column (Vyd combined with HPLC system
It was further purified by applying it to an ac ^™ C-4 column, φ2.2 × 25 cm). The buffer used was 0.1% TFA / H ₂ O.
A linear gradient (60 minutes) of 0% to 50% of acetonitrile was applied at. The purification method described here and the antibody production method described below are described in Methods in Enzymology, 197,
223-233 was used as a reference. Thus, type II PLA2
Was purified and used for immunization as described below. The purified type II PLA2 has a single peak in HPLC, and Laemli has a single peak.
There was a single band in PAGE of the system.

[0033]

Example 2 Preparation of Monoclonal Antibody Reacting with Type II PLA2 10 μg of Type II PLA2 prepared in Example 1 was treated with Freund's complete adjuvant (manufactured by BACTO, 1:
In addition to 1), Balb / c mice (6 weeks old, male) were administered i. p. Was administered. After this was done 4 times, the 5th time, i. v. Was administered.

Immediately before cell fusion, the mouse was killed, splenocytes were homogenized in PBS, the residue was filtered through a nylon mesh, and then washed once with PBS. 2 × 10 ^{7 of} these splenocytes were converted into mouse myeloma (P3-X63-Ag
8-U1) 4 × 10 ⁷ and common method (Kohler, Milstein; Natu
re, 256, 495-497 (1975)).

That is, both cells were mixed, spun down, and then Cel
After 1 pack, 1 ml of 50% polyethylene glycol (Wako Pure Chemical Industries, 2000) HAT medium (GIT medium, Wako Pure Chemical Industries) was added dropwise over 2 minutes, and polyethylene glycol was diluted with 10 ml of HAT medium over 5 minutes. After making up to 100 ml with the same medium,
It was dispensed into 5 96-well plates at 00 μl / well. After culturing at 37 ° C. in 5% CO ₂ for 1 week, half the amount (100
μl / well) was removed, and HT medium (GIT medium, Wako Pure Chemical Industries, Ltd.) was added (100 μl / well). Two weeks later, screening with a plate coated with type II PLA2 described below was performed.

That is, 50 mM Na of type II PLA2
CO ₃ buffer solution (pH 9.5) solution (1 μg / ml) was added to 9
50 μl of each well was dispensed into a 6-well plate (Falcon, PVC) and left at 37 ° C. for 1 hour. After washing, add 3% BSA / PBS to each well to 200
μl was added and blocking was performed at 37 ° C. for 1 hour. After washing again, add 50 μl of culture supernatant to each well and
It was left to stand for 3 hours and washed 3 times with 0.05% Tween / PBS.

Next, a goat anti-mouse IgG-alkaline phosphatase conjugate (Ta) diluted 2000-fold with PBS containing 3% BSA and 0.2% skim milk was prepared.
50 μl was added to each well, and left at room temperature for 1 hour. It was washed again, and a 1 mg / ml solution of p-nitrophenyl phosphate disodium salt (Wako Pure Chemical Industries, Ltd.) dissolved in 1 M diethanolamine buffer (pH 9.8) containing 0.25 mM magnesium chloride was added to each well at 100 μm / well.
1 was added and reacted at room temperature for 30 minutes. The absorbance at 405 nm was examined using an ELISA reader measuring instrument for 96-well plates (Vmax, Molecular Davice), and hybridomas secreting a monoclonal antibody that binds to type II PLA2 were selected.

Regarding the hybridoma selected in this way,
And cloned twice by limiting dilution method
did. Specifically, 10 cells of mouse peritoneal exudate were placed in HT medium.
⁶Dispense into each well what was prepared at a rate of
The cells were suspended in HT medium at a rate of 0.5 per well.
Hybridoma cells were seeded. 5% CO₂CO ₂
Incubate at 37 ℃ for 2 weeks in an incubator and culture
The nutrient supernatant was screened by the above-mentioned ELISA method,
Establish a strain by picking up a single colony
It was

[0039]

Example 3 Acquisition of Monoclonal Antibody that Promotes Binding of Type II PLA2 to Heparin Biotinylation of Type II PLA2 1 mg of Type II PLA2 obtained in Example 1
NHS-Lc B manufactured by PIERCE
10-fold equivalent of iotin was added, and the mixture was reacted at 37 ° C. for 1 hour and adsorbed on a 2 ml heparin sepharose CL6B column. Add this to 10 ml of 0.3 M NaCl / 50
Wash with mM Tris.HCl (pH 7.4), and then add 10 ml of 0.6 M NaCl / 50 mM Tris
Elution with HCl (pH 7.4), which was used in the following experiments.

Preparation of heparin binding plate 96 well plate for ELISA (Falcon, P
50 μl of PBS containing 20 μg / ml of poly-L-lysine was added to each well of VC) and left at room temperature for 1 hour. Next, each well was washed twice with PBS, 50 μl of heparin PBS solution (20 μg / ml) was added to each well, and further reacted at room temperature for 1 hour. Then each well is PBS
The plate was washed twice with, and 200 μl of PBS solution of BSA (3 mg / ml) was added to each well and reacted at 37 ° C. for 1 hour to prepare a heparin-binding plate. The plate was stored at 4 ° C.

Promotes the binding between type II PLA2 and heparin
Of the monoclonal antibody having the following activity: The hybridoma cells obtained in Example 2 were treated with 10% FCS / R.
The cells were cultured in a 300 ml flask using PMI 1640 medium. The culture supernatant was collected at the end of logarithm. 50 μl of this culture supernatant was added with 0.1 μg / ml of biotinylated type II PLA2.
50 μl of a 3% BSA / PBS solution containing the above was added, left at room temperature for 1 hour, and then placed on a heparin-bound 96-well plate. After reacting for 1 hour, P containing 0.05% Tween 20
Wash each well with BS, followed by 3% BSA and 0.
Alkaline phosphatase-conjugated streptavidin (T) diluted 2000-fold with PBS containing 2% skim milk (T
100 μl per well was added and the reaction was carried out at room temperature for 1 hour. Next, in the same manner as the ELISA method described in Example 2, the activity of promoting the binding between type II PLA2 and heparin was examined by measuring the enzyme activity of alkaline phosphatase. Thus, a hybridoma cell line producing a monoclonal antibody having an activity of promoting the binding between type II PLA2 and heparin was selected. As shown in Table 1, three types were found: one that does not affect the binding between type II PLA2 and heparin, one that inhibits binding, and one that promotes the present invention.

[0042]

[Table 1]

FIG. 1 shows the concentration dependence of the activity of the four strains having the activity of promoting the binding between type II PLA2 and heparin.

The horizontal axis of the graph represents the amount of hybridoma culture supernatant added, and the vertical axis represents the amount of biotinylated type II PLA2 reacted with the heparin-bound plate.

[Brief description of drawings]

FIG. 1 Activity promoting the binding between type II PLA2 and heparin

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 39/395 ADZ C12N 5/10 C12P 21/08 9161-4B G01N 33/573 A 33/577 B // C12N 15/02 (C12P 21/08 C12R 1:91)

Claims (6)

[Claims] 1. A monoclonal antibody which binds to human type II phospholipase A2 and has an activity of promoting the binding between human type II phospholipase A2 and sulfated polysaccharide. 2. The monoclonal antibody according to claim 1, wherein the sulfated polysaccharide is heparin. 3. The monoclonal antibody according to claim 1 or 2, which is produced by the cell line K3-1 (Accession No. FERMP-13849, National Institute of Advanced Industrial Science and Technology). 4. An animal cell that produces the monoclonal antibody according to claim 1 or 2. 5. The cell according to claim 4, wherein the animal cell is a hybridoma cell or a cell derived from the hybridoma cell. 6. A cell line K3-1 (Biotechnology Institute of Technology, Accession No. FERMP-13849).