JPH06153985A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a new monoclonal antibody useful for the purification of LACI while keeping its anticoagulant activity. CONSTITUTION:A monoclonal antibody capable of recognizing and bonding a polypeptide having the amino acid sequence of formula, e.g. antibody 2G9E9. It can be obtained e.g. from a hybridoma cell produced by immunizing a mouse with a partially synthesized peptide of LACI (having the amino acid sequence of formula), fusing the spleen cell with a mouse myeloma cell and selecting by using an antibody reactive with the peptide of formula. The antibody has specific action to the K2-site of human LACI.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that binds to a specific site of a blood coagulation inhibitor. More specifically, tissue factor inhibitor (Tiss
ue Factor Inhibitor: TFI, Lipoprotein Associated Coagul
ation Inhibitor: LACI or Tissue Factor Pathway Inhibitor: TF
PI) to a monoclonal antibody that binds to a specific site.

[0002]

2. Description of the Related Art Extrinsic blood coagulation is initiated by the contact of blood with tissue thromboplastin (hereinafter referred to as tissue factor or TF). Tissue factor (TF) is
Membrane protein, especially in large amounts in the brain and placenta. Contact with TF causes Factor VII or its active form VIIa
The factor forms a complex with TF and then factor X is proteolytically activated to factor Xa.

Previous studies of TF-initiated regulation of extrinsic blood coagulation have revealed that incubation of TF with serum suppresses its in vitro activity. At least one of these factors is defined as tissue factor inhibitor (TFI) or lipoprotein-binding coagulation inhibitor (LACI).

According to a recent study, it has been immunochemically examined that TF is produced by macrophages (derived from monocytes) that accumulate in arteriosclerotic lesions. The produced TF activates the blood coagulation system and causes platelet aggregation /
It is considered that arteriosclerosis progresses due to adhesion to blood vessels and formation / organization of fibrin.

Therefore, a lipoprotein-binding coagulation system inhibitor (hereinafter referred to as LACI) which is an inhibitor of TF
Is thought to play an important role in the association with arteriosclerosis and thrombosis.

Therefore, if the action mechanism of LACI can be elucidated and the structure-activity relationship of proteins can be elucidated, it is considered to have a very important meaning in the fields of basic medicine and clinical medicine.

According to Broze et al., Hep G2 cells (human
Has been shown to secrete an inhibitory component with similar properties to TFI present in serum (plasma) (Broze et al; Blood, 69, 150-155 (1
987)). About LACI, Nature, vol, 338,518-
520 (1989) describes the amino acid sequence and secondary structure.

On the other hand, a monoclonal antibody is specific to a single antigenic determinant and can stably produce an antibody having the same specificity. Therefore, analysis of the function and structure of an antigen protein or immunoassay ( EIA, RIA)
In recent years, it has become widely used. In particular, for functional analysis and molecular analysis of a protein having physiological activity, finding an antibody that recognizes a site involved in the function of the protein or a special structural site can be a powerful means.

There has been no report on a monoclonal antibody which recognizes a specific site of LACI. It is considered that this is because LACI is considered to exist in a trace amount in blood, and because it has a binding property with lipoprotein in blood, it was extremely difficult to purify it.

[0010] Therefore, the present inventors have paid attention to the three structural units of LACI called Kunitz domains, and have conducted research using a partially synthetic peptide corresponding to each Kunitz domain as an antigen. A monoclonal antibody was found.

[0011]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an amino acid sequence of the second Kunitz site (hereinafter referred to as K2 site) from the N-terminus of LACI: Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Pyr. LACI, which is a monoclonal antibody that recognizes and binds to the polypeptide Asn Asn Gln ... [I], is an inhibitor of the blood coagulation system,
It is a monoclonal antibody that enables purification while maintaining anticoagulant activity.

In the present specification, the amino acid sequences are abbreviated by the method adopted by the IUPAC-IUB Biochemistry Committee (CBN), and the following abbreviations are used, for example.

Ala L-alanine Arg L-arginine Asn L-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine Glu L-glutamic acid Gly glycine His L-histidine Ile L-isoleucine Leu leu leucine Leu Leu Met L-methionine Phe L-phenylalanine Pro L-proline Ser L-serine Thr L-threonine Trp L-tryptophan Tyr L-tyrosine Val L-valine

LACI purified using the monoclonal antibody of the present invention has a molecular weight of 39,000 to 42,000 as shown in the examples below. When purified using an antibody that recognizes other Kunitz sites, LACI
Other contaminating proteins exist, but when the monoclonal antibody of the present invention is used, a single band is obtained.

The present invention is also characterized in that a human LACI-containing mixture is brought into contact with an adsorbent in which the above-mentioned monoclonal antibody against human LACI is bound to an insoluble carrier to bind human LACI to the adsorbent. It is a highly pure separation method and purification method of human LACI having high anticoagulant activity from a human LACI-containing mixture.

The hybridoma cell producing the monoclonal antibody of the present invention is the method of Kohler & Milstein [Kohler &Milstein; Nature: 256 , 495-497 (1975)].
Obtained by

That is, after immunizing a mouse with a partial synthetic peptide of LACI represented by the above amino acid sequence [I], the spleen cells of this mouse are fused with mouse myeloma cells, and the resulting hybridoma cells are placed in a microtiter plate. Antibodies that react with the immobilized peptide are systematically tested and selected.

The monoclonal antibody of the present invention is obtained from the product produced by such a novel hybridoma cell, and specifically acts on the K2 site of human LACI.

The monoclonal antibody of the present invention is human LA
Since it has a property of highly specifically binding to the K2 site of CI, it is very advantageous when human LACI is separated and purified while maintaining its anticoagulant activity. That is, one kind of the monoclonal antibody of the present invention is immobilized on an insoluble carrier, and blood or other human LACI-containing mixture,
Alternatively, human LACI is adsorbed and separated from the crude extract, the crude product, and the solution thereof, and the appropriate buffer (eg, 20
mM Tris-HCl, 0.5M NaCl, pH
After washing with 7.4), the solution can be replaced with a solution containing chaotropic ions or the like (eg, 3M NaSCN, pH 7.0) to elute highly active human LACI.

Next, the specific method for producing the monoclonal antibody of the present invention will be described in detail.

A. Antigen The antigen used in the present invention is represented by the following formula [I]: an amino acid sequence having the following formula [I] Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn Gln ... In particular, the peptide is labeled with KLH (Keyhole Limpet Hemocyanin
Those bound to a carrier protein such as) are preferred.

B. Immunization of mice with the above antigens Female Balb / c mice can be used, but other strains (st
rain) mouse may be used. In doing so, the immunization regimen and antigen concentration should be chosen so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, 50μ
After immunizing the mouse intraperitoneally with 3 g of the antigen every 2 weeks, 30 μg of the antigen is intravenously administered. Spleen cells are removed for fusion several days after the final immunization.

C. Cell fusion Aseptically remove the spleen of the mouse immunized as described above,
From there, a single cell suspension is prepared. The spleen cells are fused with mouse myeloma cells from an appropriate line by using an appropriate fusion promoter. The preferred ratio of spleen cells to myeloma cells is in the range of about 20: 1 to about 2: 1. The use of 0.5-1.5 ml of fusion medium for about 10 ⁸ spleen cells is suitable.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8-U is used.
1 cell (P3-U1) [Yelton, DF et al, Current.
Topics in Microbiology and Immunology, 81, 1 (1978)
Reference] is preferred.

As a preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in the art can also be used.

D. Selection of fused cells Supports unfused mouse myeloma cells with a fusion product containing unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells in another container (eg, microtiter plate) Dilute with selective medium and culture for a sufficient time (about 1 week) to kill unfused cells. A medium (eg, HAT medium) that is drug resistant (eg, 8-azaguanine resistant) and does not support unfused mouse myeloma cells is used. Unfused myeloma cells die in this selective medium. These unfused spleen cells are non-neoplastic cells, so after a certain period (1 week)
Die. The cells fused to these have the tumorigenicity of the parental cells of myeloma and the properties of the parental splenocytes, and thus can survive in the selective medium.

E. Confirmation of resistance in each container After the hybridoma cells were thus detected, the culture supernatant was collected, and an enzyme-linked immunosorbent assay (Enzyme Linked Immuno-Sor) was performed on the antibody against the synthetic peptide of the formula [I].
Bent Assay) for screening.

F. Hybridoma that produces the desired antibody
Cloning of cells After the hybridoma cells producing the desired antibody are cloned by a suitable method (eg, limiting dilution method), the antibody can be produced by two different methods. According to the first method, by culturing the hybridoma cells in an appropriate medium for a certain period of time, the monoclonal antibody produced by the hybridoma cells can be obtained from the culture supernatant. According to the second method, the hybridoma cells can be injected into the abdominal cavity of a mouse having an isogenic or semiisogenic gene. The monoclonal antibody produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.

G. Human L from a mixture containing human LACI
Separation / Purification of ACI The monoclonal antibody against human LACI of the present invention is immobilized or bound to an insoluble carrier to obtain an adsorbent. The insoluble carrier used at that time may be any insoluble carrier generally used as a base material of a measurement reagent or a measurement kit using a monoclonal antibody. For example, sepharose, polyacrylamide, cellulose, dextran, maleic acid polymer or a mixture thereof is preferably used as the material. The form of these insoluble carriers can be various forms such as powder, granules, pellets, beads, and fibers. Also generally plasma,
Alternatively, it is advantageous to use a plate (well) having a large number of concave depressions used for measuring and separating the fractional components.

When the adsorbent is used and brought into contact with a mixture containing human LACI, the monoclonal antibody fixed to the adsorbent is bound to human LACI, and as a result, human LACI is bound to the adsorbent. By doing so, it is possible to separate or remove human LACI.

Further, human LACI was bound to the adsorbent as described above, and if possible, the remaining mixture was removed by washing,
Then, when human LACI bound to the adsorbent is contacted with or washed with an appropriate solution containing chaotropic ions (SCN ⁻ ), human LACI is released from the adsorbent, and human HACI having anticoagulant activity is isolated. LACI
Can be isolated.

Thus, according to the aforementioned separation method of the present invention, removal of human LACI from a mixture containing human LACI, separation and purification of human LACI from the mixture, measurement of human LACI content in the mixture, etc. Is achieved with an extremely simple operation.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0034]

Example 1 Preparation of synthetic peptide-hemocyanin (KLH) complex 0.3 mg of KLH (keyhole limpet hemocyanin) and synthetic peptide [amino acid sequence; Phe Leu Glu G]
lu Asp Pro Gly Ile Cys Ar
g Gly Tyr Ile Thr Arg Tyr
1 mg of Phe Tyr Asn Asn Gln] was dissolved in 0.5 ml of distilled pure water. Add EDCI to this solution
(1-ethyl-3- (3-dimethylaminopropyl) carbod
30 mg of iimide-HCl) was added, and the mixture was allowed to react overnight at room temperature while protected from light. The reaction solution was thoroughly dialyzed against 2 liters of distilled water. Thus, a synthetic peptide-bonded KLH solution (about 0.
6 mg / ml concentration) was obtained. The solution was used as an antigen for immunization.

[0035]

Example 2 Immunization of Mouse Synthetic peptide-bonded KLH solution obtained in Example 1 (0.
(6 mg / ml) 0.5 ml and Complete Freund's Adjuvant 0.5 ml were mixed using two syringes, and the mixture was intraperitoneally injected into two mice of 0.5 ml. Each was administered.

Two weeks later, 0.5 ml each of the above mixture was similarly administered to the abdominal cavity of the mouse. Then, 4 weeks later, 0.5 ml of a synthetic peptide-bound KLH solution (0.6 mg / ml) and Incomplete Freund's A
0.5 ml of djuvant) was mixed, and 0.5 ml of this mixture was administered to the abdominal cavity of the mouse. Four weeks later, 50 μg / 100 μl of the synthetic peptide-bonded KLH solution was intravenously administered to the above two mice, and three days after that, the spleen was excised and cell fusion was performed as shown in Example 3 below.

[0037]

Example 3 Cell fusion and production of desired monoclonal antibody
Selection and acquisition of hybridoma cells Spleen cells of the excised mouse and myeloma cells (P3U1) of the syngeneic mouse were mixed at a ratio of about 6: 1, and cell fusion was performed using 50% polyethylene glycol 1540 as a fusion promoter. . The cells after fusion were RPMI 1 containing 10% bovine serum so that the cell concentration was 1 × 10 ⁶ cells / ml.
The cells were suspended in 640 medium and 100 μl was dispensed into each well of a 96-well microplate.

The hybridoma (fused cell) is cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.), and the medium is exchanged with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), and the medium is transformed into HAT medium. Were screened for hybridomas consisting of spleen cells and myeloma cells.

The antibody in the culture supernatant of the hybridoma was detected by the ELISA method using a microtiter plate on which the above synthetic peptide was adsorbed. Cloning by the limiting dilution method was repeated twice for 8 wells among the antibody-producing positive wells, which had high hybridoma growth ability and strong binding to the synthetic peptide. EL
By the ISA method, four monoclones with high antibody-producing ability and strong antigen-binding ability were selected. 10 clones were obtained.
Suspended in 90% bovine serum solution containing% DMSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was purified by growing the clone in the abdominal cavity of Balb / c mice, and purifying it from the ascites using a protein A-Sepharose 4B column.

[0040]

Example 4 Determination of Class of Purified Monoclonal Antibody Subclasses were selected for IgG of each clone purified from mouse ascites by the Ouchterlony method.

[0041]

[Table 1] 2G9F1 was an antibody showing strong binding to the K2 synthetic peptide. 2G9E9, 2G9F1 and 2G9G
8 recognized the same epitope. 2G9 series and 2C1B1
Recognized different epitopes.

[0042]

Example 5 Monochrome from human hepatocyte (Hep G2) serum-free culture supernatant
Purification of LACI by internal antibody column 5 mg of the monoclonal antibody 2G9F1 described in Example 4 was coupled to bromocyan (CNBr) activated sepharose (Pharmacia Co.) to prepare 2.5 ml of antibody sepharose column. Then, human hepatocytes (Hep G
1 liter of the serum-free culture supernatant (ITES-eRDF medium) of 2) was passed through the column at a flow rate of 40 ml / hour for contact. 20 mM Tris containing 0.5 M NaCl, 0.05% Tween 20 and 10 U / ml aprotinin
After washing the column with 200 ml of HCl (pH 7.4), 3M
The protein adsorbed on the column was eluted with 15 ml of NaSCN (pH 7.0). The total protein content of the eluted fractions is about 30μ
It was g.

0.3 μg of the protein was reduced with 2-mercaptoethanol, applied to SDS-polyacrylamide gel having a gel concentration of 10 to 20% (gradient), and electrophoresed. The protein in the gel after electrophoresis was detected by the silver staining method. As a result, the monoclonal antibody 2G9F1
LA isolated from human hepatocyte cell line (Hep G2) culture supernatant (serum-free) using (Kd = 0.8 × 10 ⁻⁹ M)
CI was a protein material with a molecular weight of 39,000-42,000. The results are summarized in Fig. 1.

[0044]

Comparative Example 1 For the production of a monoclonal antibody that recognizes the first and third Kunitz sites (K1 and K3), a synthetic peptide having the following amino acid sequence was used, and Examples 1 to 4 were used.
The procedure was followed.

Polypeptide for preparing K1 recognizing monoclonal antibody: Sequence: Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe

Polypeptide for the production of K3 recognizing monoclonal antibody: Sequence: Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Asn Ser Val The monoclonal antibody thus obtained was used, and the monoclonal antibody thus obtained was used. The result of electrophoresis after that is also shown in FIG. The characteristics of the antibody used are as follows.

Anti-K1 antibody: 2F2 D9, Kd = 1.0
× 10 ^-9 M (H chain: γ _2b , L chain: κ) Anti-K3 antibody: 2A1 H8, Kd = 1.0 × 10 ^-9 M
(H chain: γ _2b , L chain: κ)

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: Phe Leu Glu Glu Asp Pro Gly Ile Cys ^{15 5} Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr ^{10 15} Asn Asn Gln ²⁰

[Brief description of drawings]

FIG. 1 L purified by a monoclonal antibody column
2 shows the results of ADS SDS-polyacrylamide electrophoresis and the molecular weight.

[Explanation of symbols]

Lane molecular weight markers (from top, 97K, 66K, 6
2K, 30K, 20K, 14K) Lane LACI purified with K1-recognizing antibody (2 μg) Lane LACI purified with K2-recognizing antibody of the present invention
(2 μg) Lane LACI purified with K3-recognizing antibody (2 μg)
Pattern

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[Procedure amendment]

[Submission date] November 20, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Explanation of code

[Correction method] Change

[Correction content]

[Explanation of symbols] Lane Molecular weight markers (from top, 97K, 66K, 4
2K , 30K, 20K, 14K) Lane LACI purified with K1 recognizing antibody (2 μg) lane LACI purified with K2 recognizing antibody of the present invention
(2 μg) Lane LACI purified with K3-recognizing antibody (2 μg)
Pattern

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12N 15/06 G01N 33/53 L 8310-2J 33/577 B 9015-2J // (C12P 21 / 08 C12R 1:91) C07K 99:00 8318-4H

Claims (4)

[Claims] 1. A monoclonal antibody which recognizes and binds to a polypeptide of amino acid sequence Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln. 2. A Lipoprotein Associated Coagulation Inhibitor:
A monoclonal antibody that recognizes and binds to the second Kunitz site from the N-terminus of LACI). 3. The monoclonal antibody according to claim 1, which recognizes and binds a protein substance having a tissue factor inhibitory activity and a blood coagulation factor Xa inhibitory activity. 4. A human LACI-containing mixture is brought into contact with an adsorbent in which the monoclonal antibody according to any one of the above 1, 2, and 3 is bound to an insoluble carrier, and the adsorbent is adsorbed with human LCI.
A method for separating human LACI from a mixture containing human LACI, which comprises binding ACI.