JPH02203795A - Anti-human tissue factor monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody which is useful in detection, purification, or determination of human tissue factor, its complex with seventh factor and their decomposition products, because it specifically binds to human tissue factor, but does not inhibit the blood coagulation activity. CONSTITUTION:The subject monoclonal antibody specifically binds to human tissue factor without inhibition of its blood coagulation activity. The antibody preferably originates from human placenta among human tissues. This antibody is produced by hybridoma cells of FERM P-10505, P-10506, and P-10507.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to human tissue factor (Tissue Factor).
The present invention relates to a monoclonal antibody that specifically recognizes and binds to tissue factor apoprotein derived from human placenta, a hybridoma cell that produces the monoclonal antibody, and a method for producing the monoclonal antibody.

(b) Conventional technology Tissue factor is also called tissue thromboplastin, and is known to play an important role as an initiator of extrinsic coagulation. That is, Chapter V
It is a substance that forms a complex with factor I and activates factor X and factor 1X. Tissue factor is a glycolipid protein (lycol 1popr) consisting of a lipid part and a protein part (apoprotein).
otein), and the presence of both is required for the expression of its activity. Apoprotein is a glycoprotein with a molecular weight of around 50,000 and is considered to be a type of membrane protein. In 1ntaCt cells, tissue factor is thought to exist with the surface covered by the cell membrane, and tissue factor is exposed on the cell surface due to damage to tissues and blood vessels, particularly in cancer, etc., making it easier for intracanal coagulation to occur. Also TNF (Tumor Necrosis)
Factor) and I L (InterLeukin)
It has been reported that cytokines stimulate certain cells to express tissue factor activity on the cell surface [P, R
, Conkling, C.S., Greenberg
and J, B, Weinberg; Blood;
vol 72. No. 1.128-133 (1988
)reference].

Recently, a C[)NA clone encoding the human tissue factor apoprotein was isolated, and the primary structure of the protein was revealed [
E., 5picer, et al; Proc.
Natl, 8cad, sci, tlsA, vo! ,
84. 5148-5152 (19B?) Reference]
.

It has also been revealed that human tissue factor apoprotein is insoluble, but becomes soluble when digested with enzymes such as trypsin.

Human tissue factor is present in various organs throughout the body, especially in the lungs, brain,
It is abundant in the placenta, and vascular endothelial cells are also known to produce tissue factor [Colucci M, et al; J
, Cl1n, Invest, 71.1893-1
896 (1983)].

Tissue factor apoprotein derived from human brain has already been purified and its molecular weight has been revealed to be 44,000 [
G, J, Broze, Jr, et at, J,
Biol, Chem.

vol, 260. No, 20.10917-1092
0 (1985)], and monoclonal antibodies against tissue factor apoprotein derived from human brain have also been reported [S
, D., Carson, et al.

Blood, vol 70. No, 2. p490
-493 (1987)]. This monoclonal antibody is an antibody that binds to human brain-derived tissue factor apoprotein and suppresses its activity to cause coagulation.

This is because the monoclonal antibody that binds to the human brain-derived tissue factor apoprotein recognizes the binding site for human tissue factor factor V][factor], and competitive inhibition occurs between factor V and the antibody. occurs, and the human tissue factor to which the antibody binds cannot form a complex with factor V and cannot activate the blood coagulation ability of factor V, thereby suppressing blood coagulation. Conceivable.

Therefore, the complex between human tissue factor and factor VII cannot be detected using this monoclonal antibody.

On the other hand, tissue factor apoprotein derived from human placenta has also been purified, and its molecular weight is reported to be 48,000 to 58,000 [Gonmori Het am Throm
b.

Haemostas, 36.90-103.1976
]. Tissue factor apoprotein derived from human brain and tissue factor apoprotein derived from human placenta have different molecular weights and are considered to be different substances. Furthermore, no monoclonal antibody against tissue factor apoprotein derived from human placenta has yet been reported.

(C) Purpose of the Invention Therefore, the present inventors provided 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 possible, we believe that by using these, it will be possible to detect, purify, quantify, etc. human tissue factor, the complex of human tissue factor and factor Vl, and the degradation products of human tissue factor. As a result of research, we have arrived at the present invention.

(d) Structure of the invention, that is, the present invention specifically binds to human tissue factor,
An anti-human tissue factor monoclonal antibody that does not inhibit the blood coagulation activity of the human tissue factor, which specifically binds to tissue factor derived from human placenta, and which does not inhibit the blood coagulation activity of the human tissue factor. and
This is an anti-human tissue factor monoclonal antibody that specifically binds to tissue factor apoprotein derived from human placenta and does not inhibit the blood coagulation activity of the human tissue factor. Does not bind to the carboxyl-terminal fragment containing the phospholipid-binding domain of tissue factor apoprotein derived from human placenta, and does not contain the phospholipid-binding domain of tissue factor apoprotein derived from human placenta. It is an anti-human tissue factor monoclonal antibody that binds to the amino group terminal fragment and does not inhibit the blood coagulation activity of the human tissue factor, and has the following general formula (I) H2N -GQEKGEFREIFYIIGAVVFV
VIILVIILAISLHKCRKAGVGQSWK
It does not bind to the human tissue factor apoprotein fragment represented by ENSPLNVS-COON-(I), but binds to the human tissue factor apoprotein fragment represented by the following general formula (I), and increases the blood coagulation activity of the human tissue factor. It is an anti-human tissue factor monoclonal antibody that does not inhibit.

The present invention will be explained in detail below.

As mentioned above, human tissue factor is a glycolipid protein consisting of a lipid part and a protein part (apoprotein), and it forms a complex with factor V and activates factor X and factor 1X. It is an initiator of coagulation. Human tissue factor is 1ntaC
In T cells, the surface is covered with a cell membrane. In recent years, human tissue factor apoprotein and further human tissue factor apoprotein have been digested with enzymes such as trypsin or CN8.
Although fragments of the human tissue factor apoprotein degraded by eg R and the like have been obtained, the presence of both a lipid moiety and a protein moiety (apoprotein) is required for the expression of blood coagulation activity.

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

The anti-human tissue factor monoclonal antibody used in the present invention is a monoclonal antibody that specifically binds to human tissue factor and does not inhibit the blood coagulation activity of the human tissue factor. Specifically, the microtechnical research trust number FERN P-1050
5 (produces G X 3), FERNP-1050
6 (produces GX4), FERN P-105
07 (which produces EX6) and an anti-human tissue factor monoclonal antibody having binding properties equivalent to the antibody produced by the hybridoma cell.

Among these antibodies, those that specifically bind to human tissue factor apoprotein are preferred, and particularly preferred are those that specifically bind to a fragment of human tissue factor apoprotein treated with CNBr. Among the fragments obtained by treating human tissue factor apoprotein with CNBr, it is more preferable to use a fragment derived from human placenta that does not bind to the carboxyl-terminus fragment containing the phospholipid-binding domain of human tissue factor apoprotein. One that binds to the amino group-terminus fragment of tissue factor apoprotein that does not contain the phospholipid-binding domain, and one that does not bind to the fragment of human tissue factor apoprotein represented by formula (I> and has the formula (n) It binds to a fragment of the expressed human tissue factor apoprotein.

These antibodies can be used in their entirety, as well as antibody fragments that maintain their essential binding ability, such as universal antibodies.

It can also be used as Fab, Fab', (Fab')2, etc.

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

Both of these bind specifically to human tissue factor and do not inhibit the blood coagulation activity of the human tissue factor, and furthermore, GX3 recognizes formula (II>), GX4 recognizes formula (II), and It also recognizes the higher-order structure of formula (II), and EX6 recognizes formula (I).

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 characteristics. A specific method involves fusing mouse lung cells immunized with human tissue factor or the like with mouse myeloma cells, and then using the resulting hybridoma cells [K6hler &Hilstein;
Nature: 256.495-497 (1975
) ] is produced by.

A. Antigen The antigens used in the present invention include tissue factor derived from human placenta, tissue factor apoprotein derived from human placenta, and phospholipids obtained by decomposing tissue factor apoprotein derived from human placenta with CN Br. Fragments of the amino terminal side that do not contain a binding domain and fragments of human tissue factor apoprotein represented by the formula (II>) are listed, including tissue factor apoprotein derived from human placenta and tissue factor apoprotein derived from human placenta. It is preferable to use a fragment on the amino terminal side that does not contain the phospholipid-binding domain obtained by decomposing the phospholipid-binding domain with CN Br, and a fragment of human tissue factor apoprotein represented by the formula (IF>). The fragment of the human tissue factor apoprotein expressed by the formula (II> and the amino group-terminal fragment that does not contain the phospholipid-binding domain obtained by decomposing the derived tissue factor apoprotein with CNBr) Particularly preferred.

The tissue factor apoprotein derived from human placenta used as an antigen is Go
The method of nmori et al.
et at dinghromb, uaemostas, 3
6.90-103 (1976)] from human placenta and its amino acid sequence is represented by the following formula (III).

B. Immunization of mice with the above antigens 1Balb/C mice can be used, but other systems (
A mouse (train) may also be used. In this case, the immunization regimen and the concentration of antigen should be chosen such that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, after immunizing a mouse with 50 μQ of the antigen three times intraperitoneally at two-week intervals, 30 μQ is administered intravenously. A few days after the final immunization, lung cells are removed for fusion.

C0 cell fusion The lungs of mice immunized as above were removed aseptically.
From there, prepare a single cell suspension. The lung cells are fused with mouse myeloma cells from an appropriate line using an appropriate fusion promoter. A preferred ratio of splenic cells to myeloma cells ranges from about 20:1 to about 2:1. It is appropriate to use 0.5 to 1.5 - of fusion media for about 108 lung cells.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8-U1 cells (P3-U1) [Yelton, D, F.
et atCurrent, Topics in H
icrobiology and immunology
, 81.1 (1978)] is preferred.

As a preferable fusion promoter, for example, an average molecular weight of 10
00-4000 polyethylene glycol can be advantageously used, although other fusion promoters known in the art can also be used.

D. Selection of fused cells. Mixture of unfused spleen cells, unfused mouse myeloma cells, and fused hybridoma cells in a separate container (e.g., microtiter plate) that does not support unfused mouse myeloma cells. Dilute with selective medium and culture for a sufficient time (about 1 week) to kill unfused cells. The medium should be one that is drug resistant (e.g. 8-azaguanine resistant) and does not support unfused mouse myeloma cells (e.g. HA
T medium) is used. In this selective medium, unfused myeloma cells die. Since these unfused lung cells are non-tumor cells, they die after a certain period of time (one week).

Cells fused to these cells can survive in the selective medium because they have the tumor properties of the parent cells of myeloma and the properties of the parent tile cells.

E. Confirmation of human tissue factor antibodies in each container After hybridoma cells have been detected, their culture supernatants are collected and analyzed for antibodies to human tissue factor by enzyme immunoassay (
Enzyme Linked Immuno-3orb
ENT As5ay).

F. Cloning of hybridoma cells producing the antibody of interest Once the hybridoma cells producing the antibody of interest are cloned by an appropriate method (eg, limiting dilution), the antibody can be produced in two different ways. According to the first method, monoclonal antibodies produced by the hybridoma cells can be obtained from the culture supernatant by beating hybridoma cells for a certain period of time and culturing them in an appropriate medium.

According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. 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 that uses tissue factor derived from human placenta as an antigen, and furthermore does not inhibit the blood coagulation activity of human tissue factor.

Furthermore, by using the monoclonal antibody of the present invention, human tissue factor, complexes of human tissue factor and factor V, and decomposition products of human tissue factor can be extracted from adult tissues.
Purification and quantification are possible, and human tissue factor apoprotein in solution state (e.g., human plasma, human urine), as well as immunoassay of their decomposition products (EIA, R'lA>
becomes possible.

Furthermore, due to the specificity and binding strength of the monoclonal antibody, it can be used for tissue staining and diagnosis of various cancers and imaging.

(f) Examples Hereinafter, the present invention will be explained by giving examples.

Example 1 Preparation of a bilidoma (syncytia) that produces a monoclonal antibody that binds to human tissue factor (TF) Human tissue factor apoprotein (hereinafter abbreviated as antigen 1) purified from a human placenta extract and human placenta-derived tissue By decomposing the factor apoprotein with CN Br, an amino terminal fragment (
Hereinafter, it is abbreviated as antigen 2), respectively, of female Ba I b
A total of three /c mice (4 weeks old) were immunized four times at 14-day intervals. The first immunization was with 50 mg dissolved in physiological saline.
The μQ antigen was mixed with an equal volume of Freund's complete adjuvant, and the emulsion was administered intraperitoneally. Second time,
For the third immunization, 50 μQ of the same antigen was mixed with Freund's incomplete adjuvant and administered intraperitoneally. For the final immunization (fourth time), 30 μg of antigen was additionally administered to the mouse intravenously. Lung cells from mice immunized 3 days after the final immunization were used for cell fusion.

The extracted mouse lung cells and syngeneic mouse myeloma cells (P3tJ1) were mixed at a ratio of approximately 5:1, and cell fusion was performed using 50% polyethylene glycol 1540 as a fusion promoter according to the method of Kohler and Milstein. I did it.

Cells after fusion are 1 x 106 cells/rail
RPMI containing 10% bovine serum to give a cell concentration of
The suspension was suspended in 1640 medium, and 100 μl was dispensed per well into a 96-well microplate.

Hybridomas (fused cells) are CO2 incubators.
(5% CO2, 37°C), hypoxanthine,
The medium was exchanged with a medium containing aminopterin and thymidine (HAT medium), and the cells were grown in HAT medium to screen for bilidoma consisting of lung cells and myeloma cells.

Antibodies in the hybridoma culture supernatant were detected by ELISA using a microtiter plate adsorbed with human tissue factor apoprotein purified from human placenta. 669 out of a total of 1022 wells seeded with bipridoma
Colony formation was observed in the wells, and among these, 356 wells were positive for producing antibodies showing binding to tissue factor apoprotein derived from human placenta. (Table 1) Table 1 Cell Fusion Four of these antibody acidic positive wells were cloned twice by limiting dilution method to obtain three monoclones.

The obtained clones were suspended in a 90% bovine serum solution containing 10% DMSO and stored in liquid nitrogen. The monoclonal antibodies produced by each clone are Ba I
b/c was grown intraperitoneally in mice and purified from the ascites using a protein A-Sepharose 4B column.

Example 2 Class of Purified Monoclonal Antibodies The IQG class of each clone purified from mouse ascites was determined by the Ophthalony method.

Table 2 Monoclonal antibody class Example 3 Binding to human placenta-derived tissue factor apoprotein Human tissue factor apoprotein extracted and purified from human placenta at 5 μO/d
Adsorb onto a microtiter plate at a concentration of 1% B.
After blocking with SA, add a monoclonal antibody solution diluted to an appropriate concentration (0.16 to 5.0 μg).
/d) was reacted. Next, an anti-mouse antibody labeled with alkaline phosphatase was added to detect and examine the binding properties of the three monoclonal antibodies to human tissue factor apoprotein.

The three types of monoclonal antibodies obtained showed strong binding to human tissue factor apoprotein (Figure 1). ] Adsorb onto a microtiter plate at a concentration of /d and block with 1% BSA.
ing, various monoclonal antibody solutions diluted to appropriate concentrations (0.16 to 5.0μ (1/mA)) and EX6 antibody labeled with peroxidase enzyme were simultaneously reacted with the antigen.

The results showed that EX6 and GX4 can bind simultaneously within human tissue factor apocans, suggesting that the antigen labeling sites are different. EX6 and GX3 are competitively inhibited when binding to tissue factor apoprotein, but the inhibition is approximately 50% compared to the inhibition of EX6 and peroxidase-labeled EX6 by the same antibody. It was suggested that the antigen recognition sites were different and sterically close (Figure 2).

Example 5 HCOOH was added to a human tissue factor apoprotein (40Hg) solution to make a 70% HCOOH solution. C in this solution
After adding and dissolving NBr powder and reacting at room temperature for 18 hours, HCOOH was dried up.

After adding 40 μl of HzO to dissolve the protein, the equivalent of 2 μg was reduced in the presence of 2-mercaptoethanol to give a concentration of 4 to 20%.
5DS-polyacrylamide electrophoresis was performed using a gradient gel of
000 and 27,000 fragments). CN for comparison
Tissue factor apoprotein (H, W, 58
,000) was similarly reduced and electrophoresed.

After electrophoresis, the proteins in the gel were electrically transferred to a nitrocellulose membrane using a plotting device (manufactured by Marizuru Aji).

TBS containing 3% gelatin (20 mH Tris solution 0.
After blocking the nitrocellulose membrane with 15HNaC2 pH 7,4), various monoclonal antibodies (GX3゜GX
4 and EX6) at a concentration of 2 μg/− in 1% gelatin-TBS at room temperature overnight.

The nitrocellulose membrane was washed three times with 0.05% Tween 20-TBS and reacted with a 1% gelatin-TBS solution of peroxidase-labeled anti-mouse I antibody for 4 hours at room temperature. After washing, A 4-chloro-1-naphthal substrate solution was added to develop the enzyme-labeled antibody bound to the nitrocellulose membrane.The protein bound to the monoclonal antibody could be detected as a dark blue band.

The binding characteristics of the three monoclonal antibodies obtained are shown in Table 3.

Table O: Strongly binds △: Weakly binds ×: Does not bind Example 6 Effect of monoclonal antibodies on tissue factor activity Obtained monoclonal antibodies (GX3.GX4.EX6>
50μ9 and human placenta-derived tissue factor 100μq were mixed to make a 1r11tl solution, and after reacting at 37°C for 1 hour,
Leave at 1°C. Of this, 200 μl was added to human plasma 1
00 μl was added to react, and the clotting time (P'T-) was measured. As a control, a rabbit antiserum against tissue factor apoprotein was used instead of the monoclonal antibody. Measurement was performed using Blood Coagulat manufactured by Sysmex.
The analysis was performed using ion Analyzer CA-100. Figure 3 shows the clotting time of each sample.

Three types of monoclonal antibodies of the present invention (GX3°GX4.
EX6) had no effect on the coagulation activity of human placenta-derived tissue factor.

Example 7 Detection of human tissue factor apoprotein in test solution Monoclonal antibody GX3 was added to PBS (10mM phosphate buffer -〇, 158
It was diluted with NaαI)H7,4), 100 μl was added to the wells of a microtiter plate, and the mixture was incubated overnight to allow the antibody to be adsorbed in the same phase. 1% BSA (bovine serum albumin)
15 () μi/well of PBS containing PBS was added and left at room temperature for 2 hours. Next, 0.05% Dingween 20 and 0.05%.
It was washed with PBS (washing buffer) containing 1% BSA. Next, human placenta-derived tissue factor apoprotein was added to the washing buffer at 25n! II/d, 50ri(]/d, 1
It was diluted to a concentration of 00n(]/d, and human urine was diluted 80 times and 40 times and added at 100 μi/well each, and reacted for 1 hour at 37°C. Washed 3 times with washing buffer. After that, the peroxiderpi-labeled monoclonal antibody GX4 was washed with washing buffer at 300n(]/m
The solution was diluted to a concentration of i, added 100μ, +2/well, and reacted at 37°C for 1 hour. After washing three times with washing buffer, the substrate solution (ABTS>) was added at 100 μm/
The absorbance was measured at a wavelength of 415 nm. The measurement results are shown in Table 4.

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

Table 4

[Brief explanation of the drawing]

Attached Figure 1 shows the binding strength of the monoclonal antibody of the present invention to human tissue factor apoprotein.
FIG. 2 shows the homology of the antigen recognition sites (epitopes) of three types of monoclonal antibodies of the present invention. FIG. 3 shows the effect of the monoclonal antibody of the present invention on tissue factor activity. Figure 1゜Monochrome - At 171 years old, I am a 17-year-old man and I am a 17-year-old man. Seven clones, nine bodies; 71
& (Me 6 Procedural Supplementary Letter to the Commissioner of the Special Fraud Agency, 1989, Patent Application for Indication of the Case, 1998, Name of the Invention, Anti-Human Tissue Factor Monoclonal Antibody, March 1st, 1989, Page 6, Bottom of the Specification) (2) In the fifth line from the bottom of page 10 of the specification, the phrase "tryprophane" is corrected to "tryptophan." (3) ) rtrain J in the first line from the bottom of page 17 of the specification.
Correct it to rstrainJ. (4) On page 21, lines 6 and 7 of the specification, "r adult tissue" is corrected to "biological tissue." (5) In the seventh line from the bottom of page 24 of the specification, "antibody acidic positive wells" should be corrected to "antibody production positive wells." Teijin Ltd. and above

Claims (7)

[Claims] (1) An anti-human tissue factor monoclonal antibody that specifically binds to human tissue factor and does not inhibit the blood coagulation activity of the human tissue factor. (2) The anti-human tissue factor monoclonal antibody according to claim 1, which specifically binds to tissue factor derived from human placenta and does not inhibit the blood coagulation activity of the human tissue factor. (3) The anti-human tissue factor monoclonal antibody according to claim 2, which specifically binds to tissue factor apoprotein derived from human placenta and does not inhibit the blood coagulation activity of the human tissue factor. (4) The tissue factor apoprotein derived from human placenta is obtained by treating the tissue factor apoprotein derived from human placenta with CNBr. 4. The anti-human tissue factor monoclonal antibody according to claim 3, which binds to the amino-terminal fragment of the placenta-derived tissue factor apoprotein that does not contain the phospholipid-binding domain, and does not inhibit the blood coagulation activity of the human tissue factor. (5) The following general formula (I) [There is a gene sequence. ]...(I) Here, the abbreviations of amino acids are as follows: N: Asparagine D: Aspartic acid A: Alanine R: Arginine I: Isoleucine G: Glycine Q: Glutamine E: Glutamic acid C: Cysteine S: Serine Y: Tyrosine W: Tryptophan T: Threonine V: Valine H: Histidine F: Phenylalanine P: Proline M: Methionine K: Lysine L: Does not bind to the fragment of human tissue factor apoprotein expressed by leucine,
General formula (II) below [There is a gene sequence. ]... [II] [Here, the abbreviations of amino acids are the same as in formula (I). The anti-human tissue factor monoclonal antibody according to claim 3, which binds to a fragment of human tissue factor apoprotein represented by the following formula and does not inhibit the blood coagulation activity of the human tissue factor. (6) A hybridoma cell producing the anti-human tissue factor monoclonal antibody according to claim 1. (7) A method for producing the anti-human tissue factor monoclonal antibody according to claim 1.