JP2779193B2 - Anti-human tissue factor monoclonal antibody - Google Patents

Description

The present invention relates to a monoclonal antibody against human tissue factor (Tissue Factor), in particular, a monoclonal antibody that recognizes and binds to human placenta-derived tissue factor apoprotein, and the monoclonal antibody. And a method for producing the monoclonal antibody.

(B) Conventional Technique Tissue Factor is also called tissue thromboplastin and is known to play an important role as a starting substance for extrinsic coagulation. That is, it is a substance that forms a complex with Factor VII and activates Factor X and Factor IX. Tissue factor is a glycolipid protein composed of a lipid part and a protein part (apoprotein), and both must be present for its activity expression. Apoprotein is a glycoprotein having a molecular weight of around 50,000 and is considered to be a kind of membrane protein. In intact cells, tissue factor is considered to be present in a state where the surface is covered in the cell membrane. In particular, tissue or blood vessel damage in cancer or the like exposes the tissue factor to the cell surface, and intravascular coagulation is likely to occur. TNF (Tumor Necrosis Facto)
r), IL (Inter Leukin) and cytokines have been reported to stimulate certain cells to express tissue factor activity on the cell surface [PRConkling, CSGreenberg, and J.
B. Weinberg; Blood, vol 72, No. 1, 128-133 (1988)].

Recently, a cDNA clone encoding human tissue factor apoprotein has been isolated, and the primary structure of the protein has been elucidated [EK
Spicer, et al; Proc. Natl. Acad. Sci. USA, vol. 84, 5148-5.
152 (1987)].

It has also been shown that human tissue factor apoprotein is insoluble, but solubilizes when digested by enzymes such as trypsin.

Human tissue factor is present in various organs, especially in the lungs,
It is known that vascular endothelial cells also produce tissue factor, abundant in the brain and placenta [Colucci M, et al; J, Clin. Invest. 7
1 , 1893-1896 (1983)].

Human brain-derived tissue factor apoprotein has been purified and found to have a molecular weight of 44,000 [GJ
Broze, Jr, et al, J. Biol. Chem. Vol. 260, No. 20, 10917-10.
920 (1985)]. A monoclonal antibody against tissue factor apoprotein from human brain has also been reported [SDCars
on, et al, Bloodr, vol 70, No. 7, p490-493 (1987)].
This monoclonal antibody is an antibody that binds to human brain-derived tissue factor apoprotein and suppresses the activity of causing coagulation.

This is because the monoclonal antibody that binds to human brain-derived tissue factor apoprotein recognizes the binding site of human tissue factor to factor VII, and competition inhibition between factor VII and the antibody occurs. It is considered that human tissue factor to which the antibody is bound cannot form a complex with factor VII, and cannot activate blood coagulation ability of factor VII, thereby suppressing blood coagulation.

Therefore, it is not possible to detect a complex between human tissue factor and factor VII using this monoclonal antibody.

On the other hand, tissue factor apoprotein derived from human placenta is also purified,
Its molecular weight is reported to be 48,000-58,000 [Gonmori H, et al; Thromb. Haemostas. 36, 90-103, 197].
6]. It is considered that tissue factor apoprotein derived from human brain and tissue factor apoprotein derived from human placenta have different molecular weights and are different substances. In addition, a monoclonal antibody against tissue factor apoprotein derived from human placenta has not yet been reported.

(C) Object of the Invention Accordingly, the present inventors provide an anti-human tissue factor monoclonal antibody that does not inhibit the blood coagulation activity of human tissue factor and binds to two different sites of human tissue factor apoprotein. If these methods can be used, detection of human tissue factor, a complex of human tissue factor and factor VII, and degradation products of human tissue factor can be performed by using them.
The inventors of the present invention have thought that purification and quantification can be performed, and as a result of intensive research, have reached the present invention.

(D) Configuration of the Invention That is, the present invention is an anti-human tissue factor monoclonal antibody that binds to human tissue factor and does not inhibit the blood coagulation activity of the human tissue factor, and binds to human placenta-derived tissue factor. An anti-human tissue factor monoclonal antibody that does not inhibit the blood coagulation activity of human tissue factor, binds to human placenta-derived tissue factor apoprotein, and does not inhibit the blood coagulation activity of the human tissue factor. The human placenta-derived tissue factor apoprotein derived from human placenta is not bound to the carboxyl-terminal fragment containing the phospholipid-binding domain of human placenta-derived tissue factor apoprotein. Binds to the amino-terminal fragment not containing the phospholipid-binding domain of tissue factor apoprotein and does not inhibit the blood coagulation activity of the human tissue factor A human tissue factor monoclonal antibody, the following general formula (I) Does not bind to the fragment of human tissue factor apoprotein represented by
The following general formula (II) [Where the abbreviations for amino acids are the same as in formula (I). An anti-human tissue factor monoclonal antibody that binds to a fragment of human tissue factor apoprotein represented by the formula: and does not inhibit the blood coagulation activity of said human tissue factor.

 Hereinafter, the present invention will be described in detail.

As described above, human tissue factor is a glycolipid protein composed of a lipid portion and a protein portion (apoprotein), and forms a complex with factor VII to activate factor X and factor IX. Starting material. Human tissue factor is present in intact cells with its surface covered in the cell membrane. In recent years, human tissue factor apoprotein, and human tissue factor apoprotein fragments obtained by digesting human tissue factor apoprotein with enzymes such as trypsin or decomposing by CNBr or the like have been obtained. The presence of both a lipid part and a protein part (apoprotein) is required.

The origin of the human tissue factor used in the present invention is not particularly limited, but is preferably placenta, urine and brain, particularly preferably placenta and urine, and more preferably placenta.

The anti-human tissue factor monoclonal antibody used in the present invention is a monoclonal antibody that binds to human tissue factor and does not inhibit the blood coagulation activity of the human tissue factor. More specifically, FERM P-10505 (producing GX3), FERM P-10506 (producing GX4), FERM P-10
507 (producing EX6) and an anti-human tissue factor monoclonal antibody having binding properties equivalent to those produced by the hybridoma cells.

Among these antibodies, preferred are those that bind human tissue factor apoprotein, and particularly preferred are those that bind human tissue factor apoprotein to fragments treated with CNBr, and more preferred are those that bind to human tissue factor apoprotein. Of the human tissue factor apoprotein treated with CNBr does not bind to the carboxyl-terminal fragment containing the domain that binds the phospholipid of human tissue factor apoprotein. A human tissue factor apoprotein represented by the formula (II), which does not bind to a fragment of the amino group terminal side which does not contain a lipid binding domain, and does not bind to a fragment of the human tissue factor apoprotein represented by the formula (I) It binds to protein fragments.

These antibodies can be used as they are in their intact form, as well as antibody fragments whose intrinsic binding ability is maintained, for example, antibodies of the universal type, Fab, Fab ', (Fa
b ') It can also be used as ₂ etc.

The features of the GX3, GX4 and EX6 anti-human tissue factor monoclonal antibodies produced by the hybridoma cells deposited at the Institute of Fine Technology in connection with the present invention will be described.

These both bind to human tissue factor and do not inhibit the blood coagulation activity of said human tissue factor, and GX3 recognizes formula (II), GX4 recognizes formula (II), and formula (I)
It also recognizes the higher-order structure of I), and EX6 has the formula (I)
It recognizes.

The method for producing the anti-human tissue factor monoclonal antibody used in the present invention is not particularly limited as long as it has the above-described characteristics. As a specific method, spleen cells of a mouse immunized with human tissue factor or the like as an antigen are fused with myeloma cells of the mouse, and the obtained hybridoma cells [Khler &Milstein; Nature: 256 , 49
5-497 (1975)].

A. Antigens The antigens used in the present invention include phospholipids obtained by decomposing human placenta-derived tissue factor, human placenta-derived tissue factor apoprotein, and human placenta-derived tissue factor apoprotein with CNBr. And a fragment of the human tissue factor apoprotein represented by the formula (II), which include a human placenta-derived tissue factor apoprotein and a human placenta-derived tissue factor apoprotein.
It is preferably a fragment on the amino group terminal side which does not contain a phospholipid-binding domain obtained by decomposing with CNBr, and a fragment of human tissue factor apoprotein represented by the formula (II). Tissue factor apoprotein to CN
An amino-terminal fragment not containing a phospholipid-binding domain obtained by digestion with Br and a compound of the formula (I
Particularly preferred is a fragment of the human tissue factor apoprotein represented by I).

Tissue factor apoprotein derived from human placenta used for antigen is Go
nmori et al. [Gonmor H, et al; Thromb. Haemostas. 36,
90-103 (1976)] and purified from human placenta, and its amino acid sequence is represented by the following formula (III): [Where the abbreviations for amino acids are the same as in formula (I). ] Is represented.

B. Immunization of mice with the above antigens Female Balb / C mice can be used, but other strains (st
rain) mice may be used. At that time, the immunization plan,
And the concentration of antigen should be chosen such that a sufficient amount of antigen-stimulated, lymphocytes are formed. For example, mice are immunized intraperitoneally three times at intervals of two weeks with 50 μg of antigen, and then 30 μg is administered intravenously. Several days after the final immunization, spleen cells are removed for fusion.

C. Cell fusion The spleen of the mouse immunized as described above is aseptically removed, and a single cell suspension is prepared therefrom. The spleen cells are fused with mouse myeloma cells from an appropriate line by using an appropriate fusion promoter. Preferred ratios of spleen cells to myeloma cells range from about 20: 1 to about 2: 1. Is appropriate use of a fusion medium 0.5~1.5ml for about 10 ⁸ splenocytes.

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

Preferred fusion promoters include, for example, an average molecular weight of 10
00-4000 polyethylene glycol can be used advantageously, but other fusion promoters known in the art can also be used.

D. Selection of fused cells Selection of a mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells in a separate container (eg, microtiter plate) that does not support unfused mouse myeloma cells Dilute with medium and incubate for a time sufficient to kill unfused cells (about 1 week). The medium may be drug resistant (eg, 8-azaguanine resistant) and does not support unfused mouse myeloma cells (eg, HA
T medium) is used. In this selective medium, unfused myeloma cells die. Since the unfused spleen cells are non-neoplastic cells, they die after a certain period of time (one week later).
Cells fused to these cells can survive in a selective medium because they have the neoplastic properties of parental cells of myeloma and the properties of parental spleen cells.

E. Confirmation of human tissue factor antibody in each container After the detection of the hybridoma cells in this manner, the culture supernatant was collected, and an antibody to human tissue factor was subjected to an enzyme immunoassay (enzyme linked immuno-sorbent assay).
Screen according to y).

F. Cloning of Hybridoma Cells Producing the Antibody of Interest Cloning the hybridoma cells producing the antibody of interest by an appropriate method (for example, limiting dilution method) results in 2 antibodies.
Produced in two different ways. According to the first method, a monoclonal antibody produced by the hybridoma cell can be obtained from the culture supernatant by culturing the hybridoma cell for a certain period of time in an appropriate medium. According to the second method, the hybridoma cells can be injected into the peritoneal cavity of a mouse that has an isogenic or semi-isogenic gene. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.

(E) Effects of the Invention The anti-human tissue factor monoclonal antibody obtained in the present invention is a novel monoclonal antibody using a tissue factor derived from human placenta as an antigen, and does not inhibit the blood coagulation activity of human tissue factor. Further, the use of the monoclonal antibody of the present invention enables extraction, purification, and quantification of human tissue factor, a complex of human tissue factor and factor VII, and a degradation product of human tissue factor from living tissue. It becomes possible to carry out immunoassay (EIA, RIA) of human tissue factor apoprotein in a state (for example, human plasma, human urine), and a degradation product thereof. Furthermore, the monoclonal antibody can be used for tissue staining, diagnosis, and imaging in various cancers based on the specificity and binding strength of the monoclonal antibody.

(F) Example Hereinafter, the present invention will be described with reference to examples.

Example 1 Obtaining Hybridoma (Fused Cell) Producing Monoclonal Antibody That Binds to Human Tissue Factor (TF) Human tissue factor apoprotein (hereinafter abbreviated as antigen 1) purified from human placenta extract and human placenta-derived By decomposing tissue factor apoprotein with CNBr, amino-terminal fragments (hereinafter abbreviated as antigen 2) that do not contain a phospholipid-binding domain are each a female Balb / c mouse (4 weeks old) for a total of 3 The animals were immunized four times at 14 day intervals.
For the first immunization, 50 μg of antigen dissolved in physiological saline was mixed with an equal volume of complete Freund's adjuvant, and the emulsion was administered intraperitoneally. For the second and third immunizations, 50 μg of the antigen was also mixed with Freund's incomplete adjuvant and administered intraperitoneally. Final immunity (the fourth)
In mice, 30 μg of the antigen was additionally administered to mice intravenously. Three days after the final immunization, spleen cells of the immunized mouse were used for cell fusion.

The extracted mouse spleen cells and syngeneic mouse myeloma cells (P3U1) were mixed at a ratio of about 5: 1, and fusion was carried out using 50% polyethylene glycol 1540 as a fusion promoter according to the method of Kohler and Milstein. Was.

The cells after the fusion were suspended in RPMI 1640 medium containing 10% bovine serum so as to have a cell concentration of 1 × 10 ⁶ cells / ml, and 100 μl per well was dispensed into a 96-well microplate.

Hybridomas (fused cells) are cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.), exchanged with a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), and grown in a HAT medium. A hybridoma consisting of spleen cells and myeloma cells was screened.

The antibody in the culture supernatant of the hybridoma was detected by ELISA using a microtiter plate on which human tissue factor apoprotein purified from human placenta was adsorbed. Colonies were formed in 669 wells out of a total of 1022 wells seeded with hybridomas, of which 35 were antibody-producing positive wells binding to human placenta-derived tissue factor apoprotein.
6 wells. (Table 1) Cloning by limiting dilution was repeated twice for four of the antibody production positive wells to obtain three monoclones. The resulting clone is
The cells were suspended in a 90% bovine serum solution containing 10% DMSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was obtained by growing the clone in the abdominal cavity of a Balb / c mouse, and purifying the ascites fluid using a protein A-Sepharose 4B column.

Example 2 Class of Purified Monoclonal Antibody The class of IgG of each clone purified from mouse ascites was determined by the Ouchterlony method.

Example 3 Binding to Human Placenta-Derived Tissue Factor Apoprotein Human tissue factor apoprotein extracted and purified from human placenta was adsorbed to a microtiter plate at a concentration of 5 μg / ml, blocked with 1% BSA, and adjusted to an appropriate concentration. The monoclonal antibody was reacted with a monoclonal antibody solution (0.16 to 5.0 μg / ml) diluted with an anti-mouse antibody labeled with alkaline phosphatase, and the binding of the three types of monoclonal antibodies to human tissue factor apoprotein was performed. Was detected and examined.

The three types of monoclonal antibodies obtained showed strong binding to human tissue factor apoprotein (FIG. 1). Example 4 Isomorphism of the antigen-recognition site of the monoclonal antibody. Adsorb to microtiter plate at a concentration of ml and blockin with 1% BSA.
After g, various monoclonal antibody solutions (0.16-5.0 μg / ml) diluted to an appropriate concentration and an EX6 antibody labeled with a peroxidase enzyme were simultaneously reacted with the antigen.

The results suggested that EX6 and GE4 can bind to human tissue factor apoprotein simultaneously and have different antigen labeling sites. EX
6 and GX3 inhibit the competition when binding to human tissue factor apoprotein, but since the inhibition of EX6 and peroxidase-labeled EX6 is about 50% less than that of the same antibody, the binding of EX6 and GX3 The antigen recognition sites were different and suggested to be sterically close sites (FIG. 2).

Example 5 Binding of Monoclonal Antibody to CNBr-Treated Human Tissue Factor Apoprotein HCOOH was added to a human tissue factor apoprotein (40 μg) solution to prepare a 70% HCOOH solution. Add CNBr powder to this solution, dissolve and react at room temperature for 18 hours, then dry HCOOH
up. After adding 40μH ₂ O to dissolve the protein, 2μg
The equivalent was reduced in the presence of 2-mercaptoethanol, and
SDS-polyacrylamide electrophoresis was performed using a 20% concentration gradient gel (MW 31,000 and 27,000 fragments). For comparison, tissue factor apoprotein (MW58,000) not treated with CNBr was similarly reduced and electrophoresed.

After the electrophoresis, the proteins in the gel were electrically transferred to a nitrocellulose membrane using a blotting device (manufactured by Marisol Co., Ltd.).

TBS containing 3% gelatin (20 mM Tris solution-0.15 M NaC
l After blocking the nitrocellulose membrane with pH 7.4), apply various monoclonal antibodies (GX3, GX4 and EX6) at 2 μg / ml.
It was allowed to react overnight at room temperature with a 1% gelatin-TBS solution containing the same at a concentration. The nitrocellulose membrane was washed three times with 0.05% Tween 20-RTBS, and reacted with a 1% gelatin-TBS solution of a peroxidase-labeled anti-mouse Ig antibody at room temperature for 4 hours. After washing, 4-chloro-1-naphthal substrate solution was added,
The enzyme-labeled antibody bound on the nitrocellulose membrane was developed. The protein bound by the monoclonal antibody was detected as a dark blue band.

Table 3 shows the binding characteristics of the three monoclonal antibodies obtained.

Example 6 Effect of monoclonal antibody on tissue factor activity Each of the obtained monoclonal antibodies (GX3, GX4, EX6) 50
μg and human placenta-derived tissue factor 100 μg were mixed to form a 1 ml solution, reacted at 37 ° C. for 1 hour, and allowed to stand at 4 ° C. overnight. Of these, 200 μm was added to 100 μm of human plasma and allowed to react, and the coagulation time (PT) was measured. As a control, rabbit serum against tissue factor apoprotein was used instead of the monoclonal antibody. Measurement was performed by Sysmex Blood Coagulation Anal.
Performed using yzer CA-100. FIG. 3 is a graph showing the solidification time of each sample.

Three types of monoclonal antibodies of the present invention (GX3, GX4, EX6)
Did not affect the activity of human placenta-derived tissue factor in causing coagulation.

Example 7 Detection of Human Tissue Factor Apoprotein in Test Liquid Monoclonal antibody GX3 was diluted with PBS (10 mM phosphate buffer-0.15 M NaCl pH 7.4) to a concentration of 20 μg / ml, and microtiter rate was determined. 100 μl was added to the wells and left overnight to allow the antibody to adsorb to the solid phase. 1% BSA
PBS containing (bovine serum albumin) was added at 150 μ / well, and left at room temperature for 2 hours. Then 0.05% Tween 20 and 0.1
The plate was washed with PBS (washing buffer) containing% BSA. Next, the human placenta-derived tissue factor apoprotein was washed with a washing buffer for 25 minutes.
The resultant was diluted to concentrations of ng / ml, 50 ng / ml, and 100 ng / ml, and human urine was diluted 80-fold and 1-fold, added to 100 μ / well, respectively, and reacted at 37 ° C. for 1 hour. After washing three times with the washing buffer, the peroxidase-labeled monoclonal antibody GX4 was diluted with the washing buffer to a concentration of 300 ng / ml, added at 100 μ / well, and reacted at 37 ° C. for 1 hour. After washing three times with the washing buffer, the substrate solution (ABTS) was added at 100 μ / well, and the absorbance at a wavelength of 415 nm was measured. Table 4 shows the measurement results.

The monoclonal antibody of the present invention was found to be useful as a means for binding to and detecting tissue factor apoprotein contained in human urine.

[Brief description of the drawings]

FIG. 1 shows the binding strength of the monoclonal antibody of the present invention to human tissue factor apoprotein.
FIG. 2 shows the isomerism of three types of antigen recognition sites (epitope) of the monoclonal antibody of the present invention. FIG.
Shows the effect of the monoclonal antibody of the present invention on tissue factor activity.

──────────────────────────────────────────────────続 き Continuation of front page (51) Int.Cl. ⁶ identification code FI G01N 33/577 C12N 15/00 C (C12P 21/08 C12R 1:91) (58) Fields surveyed (Int.Cl. ⁶ , DB name) C12P 21/08 C12N 15/06-15/08 BIOSIS (DERWENT) WPI (DERWENT)

Claims (7)

(57) [Claims] An anti-human tissue factor monoclonal antibody which binds to human tissue factor and does not inhibit the blood coagulation activity of said human tissue factor. 2. The anti-human tissue factor monoclonal antibody according to claim 1, which binds to human placenta-derived tissue factor and does not inhibit the blood coagulation activity of said human tissue factor. 3. The anti-human tissue factor monoclonal antibody according to claim 2, which binds to human placenta-derived tissue factor apoprotein and does not inhibit the blood coagulation activity of said human tissue factor. 4. A method in which a human placenta-derived tissue factor apoprotein obtained by treating with CNBr does not bind to a carboxyl-terminal fragment containing a phospholipid-binding domain of a human placenta-derived tissue factor apoprotein. The anti-human tissue factor monoclonal according to claim 3, which binds to a fragment on the amino-terminal side of the tissue factor apoprotein derived from human placenta which does not contain a phospholipid-binding domain and does not inhibit the blood coagulation activity of said human tissue factor. antibody. 5. The following general formula (I) Does not bind to the fragment of human tissue factor apoprotein represented by
The following general formula (II) The anti-human tissue factor monoclonal antibody according to claim 3, which binds to a fragment of the human tissue factor apoprotein represented by the formula: and does not inhibit the blood coagulation activity of the human tissue factor. 6. A hybridoma cell that produces the anti-human tissue factor monoclonal antibody according to claim 1. 7. A method for producing an anti-human tissue factor monoclonal antibody, which comprises a step of culturing the hybridoma producing the anti-human tissue factor monoclonal antibody according to claim 1.