JPH07501524A - Method for detecting and separating uPA-R and method for inhibiting binding of uPA to uPA-R - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for detecting and separating uPA-R and method for inhibiting binding of uPA to uPA-R The present invention , relates to a method for detecting and separating uPA-R using a monoclonal antibody. Also The invention also relates to methods of inhibiting the binding of uPA to uPA-R.

Background technology Urokinase-type plasminogen activator (uPA) is a protein component This is an enzyme that produces plasmin, which is a degrading enzyme. uPA promotes cell migration and tissue remodeling. extracellular agents in physiological or pathological processes such as tumor invasion and metastasis. It is thought to be involved in the expression of proteolytic activity (Adv, Cance rRes,.

vol, 44.139266, (1985)). uPA is normal and malignant binds to specific cellular receptors (uPA-Rs) on various types of cells It is known that uPA plays a special role in these processes. It is clear that (J, Ce1l Biol, vol, 100.8 pp. 6-96 (1985); Pro, Natl, Acad, Sci, U A.S.A.

vol, 82.4939-4943 (1985), and J. Biol. Chem, vol. 263.2358-2363 (1988)). child has been characterized by binding to the receptors of .

vol, 265.6453-6460, (1990); and EMBOJ, , vol. 9, pp. 467-474 (1990)) uPA has proteolytic activity. Q, Ce1l Biol, which has been shown to retain its properties.

vol, 100.86-92 pages (+985); Proc, Natl, A cad, Sci, U, S, A,, vol, 82.4X39- 4943 pages (1985): J, Biol, Chem, vol, 26 4. pp. 2185-2188 (1989) and J. Ce11Bio1. , vo I, 108. pp. 1987-1995 (1989)). Also has a lot of plasminogen J, Biol, Chem, vol, 260 ．． 4303-4311, (1985)), the relationship between uPA and plasminogen. It has become clear that plasmin activity occurs on the cell surface through cell binding. Q, Biol, Chem, vol, 264.2185-218 8 (1989) and J.

Ce1l Biol, vol, 108.1987-1995 pages (1989 )). This system also promotes the activation of the uPA zymogen. I know (J, Biol, Chem,, vol.

264, pp. 2185-2188 (1989)).

Mo3 is a glycoprotein, and its expression in human mononuclear cells and bone marrow outstanding cell lines is Pharmacological agonists of certain cytokinins, protein kinase C and CAMP Similarly, it is induced by bacterial LPS and muramyl dipeptide (MDP).

Mo3 is originally produced in response to antigens selectively expressed on activated macrophages. Identified as a mononuclear cell surface antigen recognized by a group of monoclonal antibodies (J, Immunol, vol. 135.3869 (1985) ; Blood Ce1ls, vol, 16.167 (1990), and J , Immunol, vol. 137.448 (1986)), Immunofluorescence In flow cytometry, Mo3 was detected on freshly isolated mononuclear cells. TNF, M-C3F, GM-CSF and and cytokines such as IL-3 and soluble inflammatory factors such as LCPSMDP. clearly visible on the surface of mononuclear cells activated by the culture in medium It will be done. In vivo expression of Mo3 was found to be inflammatory or is prominent in malignant tissues (Blood Cells, vol. 16.167 (1990)). Macrophages in normal tissues are lung macrophages. Negative for Mo3 staining, except for variable expression depending on the stage. Non-kura food details Within the cyst, Mo3 expression is more essential: normal tonsillar epithelium, hepatocytes, and All dermal collagens were positive for Mo3 staining. Mo in endothelial cells Expression of 3 is more specific to inflamed tissues as well as in mononuclear cells.

It has been reported that plasminogen activator levels are increased in metastatic tumors. has been associated with tumor invasion into other tissues (Adv, Canc er　Res,.

vol. 44.139 (1985)). More recently, the uPA receptor is adhesions (Cel Biol,.

vol, p. 104.1085 (+987)), cytoskeleton like hyculin Protein (J, Ce1lBio1., vol, 106. vol, +24 1. (1988)) and alpha-activation at the contact site of metastatic tumor cells. nin and actin (Thromb, Res, 5upp1., vol. 10.55 (1990)). this The tumor cells probably invade other tissues, become metastatic, and invade the basement membrane and extracellular areas. It becomes possible to stimulate and promote the deterioration of matrix proteins. Surface Mo3 expression Previous studies have shown that tumor-associated macrophage in squamous cell rutinoma of the lung Mo3 was detected on the phage. Interestingly, these macrophages The surrounding tumor cells were negative for Mo3 staining (Blood Ce1 ls, vol, 16.167 (1990)).

Monoclonal antibodies specific for Mo3 have been reported (J.

1iwnuno1. vol, 137.448-455 (1986): B food, vol, 59.775 pages (1982); and i.

Immunol, vol. 144.1841-1848 (1990)).

Furthermore, monoclonal antibodies specific for uPA-R have recently been reported. (FEBS Letters, vol, 288゜pages 233-236 ( 1991)). However, the development of methods to detect and isolate uPA-R is difficult. desired. It also blocks the binding of uPA to uPA-R, and It is also desired to develop methods for treating diseases caused by binding to R.

Disclosure of invention Therefore, it is an object of the present invention to provide a method for isolating uPA-R.

Another object of the invention is to provide a method for detecting uPA-R.

Still another object of the present invention is to provide a method for detecting uPA-R bound to the surface of cells. It is to provide.

Yet another object of the present invention is to provide a method for detecting free uPA-R. Ru.

Yet another object of the invention is to provide a method of inhibiting the binding of uPA to uPA-R. It is to be.

Still another object of the present invention is to prevent diseases caused by binding of uPA to uPA-R. The purpose is to provide a treatment method.

Still another object of the present invention is a method for preventing tissue destruction by inflammatory macrophages. The goal is to provide the following.

Yet another object of the invention is to provide a method for preventing metastatic invasion of tumor cells. It is.

These and other objects of the invention will be made clear by the following description. However, these antibodies that bind to Mo3 also bind to uPA-R. This was achieved by the inventors' discovery.

Brief description of the drawing A more complete understanding of the invention may be obtained by reading the following detailed description in conjunction with the accompanying drawings. Once a full understanding is reached, the many advantages of the present invention will be appreciated. Attached diagram In terms of: Figure 1 shows murine monoclonal and rabbit polyclonal clones specific for Mo3. Figure 2 shows competitive blocking of uPA-F ITC by the antibody. Pickling and PMA Stimulated U-937 cells containing murine monoclonal antibodies at various dilutions IgG2a anti-Mo3f (-・ -), TgM anti-Mo3e (-11), isotype-equivalent negative control Antibody/IgG2a 5B7 (-〇-) or IgM anti-CD+4 (-mouth-) : Polyclonal rabbit anti-Mo3 (-Mo3) or normal rabbit serum (-△-) , or without antibody (X), after 60 min at 4°C, add uPA-FITC, The cell suspension was incubated for an additional 60 minutes at 4°C. After washing and fixation, Cells were subjected to flow cytometry to measure binding to uPA-F ITC in a single time. 5000 cells per sample were collected. Average of each stained population Channel fluorescence is shown on the Y-axis.

BEST MODE FOR CARRYING OUT THE INVENTION Mo3 is an active antibody expressed on the surface of mononuclear cells and U-937 cells; A cDNA encoding o3 was cloned. The Mo3 cDNA sequence has five It is predicted that it has an N-linked glycosylation site (Asn-X-3er/Thr). Mo3 is a highly glycosylated protein and 40-50% of its molecular weight is This is consistent with previous research showing that N-links contribute to the formation of sugar chains. Von He1jne (Nucleic Acid Res,, vol, 14.46 According to the method of 83 (+986)), a signal peptide consisting of 22 amino acids The N-terminus of LRCMQ in the mature form of Mo3 protein Expected to give an array. So far, Mo3 has been It is known that it is a cell surface protein attached to the plasma membrane (J, Immunol, vol. 144.1841 (1990)). PI-PL In addition to being easily separated from the cell surface by C, the expression of Mo3 on the surface The fact that it is absent in patients with nocturnal hemoglobinuria supports this conclusion. (J.

Immunol, vol, 144.1841 pages (+990)). Seizure here Nocturnal hemoglobinuria is a disease characterized by the absence of GPI-linked determinants on white blood cells. It is a disease characterized by symptoms (J, Exp.

Med, vol, 1661011 (1987). Approximately 2 There is a part consisting of 0 hydrophobic amino acids, which is cut during the link formation process with GPT. It may be a plasma membrane anchor that is expected to be dropped (A nn, Rev, Biochem,, vol.

57, 285 (+988)). The putative signal peptide and approximately 290 of the mature Mo3 surface expressed protein after cleavage of the C-terminal anchor, which is an amino acid. The expected size is 29kD molecular weight, which is determined for Mo3 without sugar chains. (J, [nwnunol, vol, 147.133 1 page (1991)).

The most direct possibility is that the isolated cDNA clone actually encodes the Mo3 protein. Direct evidence is provided by transfection of Cos cells with Mo3 cDNA. can be obtained. After transfection, surface expression of Mo3 on Cos cells was Polyclonal rabbit anti-Mo3 antiserum and various anti-Mo3 monoclonal antibodies Confirmed by ELISA using Transfected by Mo3 cDNA Only transfected cells were immunoreactive with anti-Mo3 antibodies: truly transfected cells. Cells that were not infected were negative for surface expression of Mo3. isotype -Matched control monoclonal antibody and normal rabbit serum Cos cells transfected with o3 were also not truly transfected. It also did not react with other Cos cells. In other experiments, we used ICAM-] cDNA to Although transfected cells showed positive staining with anti-ICAM-1 antibody, However, it did not react with the anti-Mo3 monoclonal antibody. These transfers From the injection experiment, the isolated Mo3 cDNA clone was actually previously It can be seen that it encodes a cell surface antigen that has been identified as o3.

As a result of cloning and sequencing of the gene encoding Mo3, Mo3 is uPA- It turned out to be the same as R. NBRF data base computer server When I performed a test, Mo3 was found to be urokinase plasminogen activator. It has been suggested that the receptor for uPA-R is the same as the receptor for u Confirmed by comparing the complete sequence of PA-R with that of Mo3 (EMB O.J., vol. 9.467-474 (1990)).

It was found that antibodies specific for Mo3 also bind to uPA. Okay In one embodiment of the invention, anti-Mo3 monoclonal antibodies are used to detect uPA-R. The binding of the antibody to uPA-R is investigated. Suitable anti-Mo3 monoclonal anti- Examples of antibodies include anti-Mo3a-f. J. for preparation of anti-Mo3f.

Immunol, vol, 144. pages 1841-1848 (+990)) J, Inwnunol, for the generation of anti-Mo3a-e. vol, 137.448-455 (1986) and Blood, vol. .

59, p. 775 (1982). Hype that produces anti-Mo3f The ridoma strain is ATCC (American Type Culture Co1). 1ection), +2301 Parklawn Drive, Rotskwil, Meri Deposited on November 16, 1991 in State of Land, United States of America 29852. There is.

The detection method of the present invention is based on the detection method of free uPA-R or membrane-bound uPA-R. It is also used in When detecting membrane-bound uPA-R, anti-Mo3 Preferably, anti-Mo3f is used. Especially using anti-Mo3f is preferred.

uPA-R can be detected by ELISA method. For example, if uPA-R is expressed Cells suspected of being infected are incubated with any of the antibodies of the invention. After washing , which binds to the antibody of the present invention and which also binds to horseradish peroxidase (H These cells were incubated with other antibodies that bind to enzymes suitable for ELTSA (RP). Incubate. After another wash, cells were incubated with enzyme substrate. and measure the enzyme activity. Enzyme activity is related to the amount of uPA-R on the cell surface.

Such ELISA methods allow detection of body fluids (e.g. plasma, urine, exudates from inflammation). uPA-R dissolved in can also be detected as a marker of inflammation and/or cancer. I'll come.

Alternatively, one of the antibodies of the invention may be labeled and incubated with cells. good. For example, treating antibodies with periodate creates carbonyl groups on their sugar groups. make it happen A suitable label is covalently attached to the antibody via a bifunctional linking group. Ru. For suitable labels and linking groups, see Haugland, Handbook. of Flourescent Probes and Re5arch C chemical, Molecular Probes, Inc., Euge ne, Ore■≠■ (+989) and Pierce [mmunotechnology Cat alog and Handbook, Pierce.

Rockford, IL (1990).

The present invention also relates to the isolation of uPA-R using antibodies specific for N403. Ru. uPA-R is produced by immunoprecipitation reaction with Mo3-specific antibody. isolated. It is preferable to use anti-Mo3f for the immunoprecipitation reaction. Antibody J .

Immunol, vol. 147.1331-1337 (199+). Protein A-Sepharose (registered trademark, Pharmasoar) LKB Biotechnology Co., Ltd., Biscataway, N.Y.) on a suitable support.

This conjugated antibody was used to develop a phosphaticylinositol-specific phosphatide. from the supernatant of uPA-R-containing cells exposed to olipase or lysed. uPA-R is isolated from the cells. uPA-R is treated with biotin prior to cell lysis. treatment with N-hydroxysuccinimide ester or 36S-methio Incubate the cells in a nutrient source containing a radioactive label such as nin or 3H-mannose. It can be labeled by

The present invention shows that uPA is reduced by treating uPA-R with an antibody specific to Mo3. Also relates to a method of inhibiting binding to uPA-R. In this case, the preferred antibody is Anti-Mo3e or anti-Mo3f. Particularly preferred is anti-Mo3f.

Inhibition of the binding of uPA to uPA-R can be done in vitro. ・You can go to Bipo. Therefore, inhibition of uPA binding to uPA-R It encompasses one embodiment of an in vitro assay for the presence of PA-R.

The method of the present invention for inhibiting the binding of uPA to uPA-R can also be carried out in the body. can. In vivo binding of uPA to uPA-R by administering antibodies is inhibited. Therefore, the method of the present invention is effective against inflammatory macrophages (macrophages and neutrophils). prevent tissue damage caused by tumor cells) and metastatic invasion of tumor cells, or It also includes methods of reducing In this method, antibodies are administered systemically (immediately). Administer (intravenous injection). Diseases with an inflammatory course to which this method is applicable include the following: Rheumatoid arthritis, immune vasculitis, glomerulonephritis, inflammatory enteritis and and respiratory distress in adults. The method of the invention can be applied to various human tumors that express uPA-R, e.g. For example, invasion of tumor cells (metastatic invasion) by malignant melanoma, breast cancer, sarcoma, etc. It can be applied to prevent attacks.

The dose and schedule for administering antibodies will naturally depend on the patient's health status and treatment. It depends on the state of the disease. However, 0.0% per kg of body weight. 5mg from Ol, preferred A dosage of 0.1 to 1 mg may give good results. This dosing schedule Continue the routine for several days (1 to 10 days). In cases of inflammation, antibodies It is administered at an early stage, and in cancer cases, when surgical resection is performed. to prevent tumor cells from surgically relocating and forming metastatic tumors.

Antibodies are administered in the form of various pharmaceutical compositions, such as injections and suspensions.

Thus, the present invention provides a method for preventing uPA from binding to uPA-R. It is something that As described in the Examples below, antibodies specific for Mo3 PA can be prevented from binding to uPA-R. Figure 1 shows uPA-FITC (fluorescein isothiocyanate) is competed with various antibodies specific for Mo3. Shows the result of blocking. The results in Figure 1 clearly show that Mo3-specific antibodies (anti- -Mo3f (-・-): anti-Mo3e (-11) and polyclonal rabbit anti -Mo3 (-Mu-) can prevent uPA-FITC from binding to uPA-R. Which indicates that.

As an example of the antibody of the present invention, an antibody obtained from a mouse was shown as an example, but the method of the present invention This includes the use of modified versions of these antibodies that are compatible with the human body ( Nature, vol. 351.501-502 (1991)).

Other features of the present invention will become clear from the following examples; The examples are presented for illustrative purposes and are not intended to limit the invention in any way. It's not something that's done.

Example Materials and methods reagent High molecular weight urokinase (uPA; 80.0001U/mg, catalog # 124) (HoppeSeyer's Z, Physiol, Che m,, vol, 363.133 pages (1982)) American Diagnosis Purchased from Stix Inc. (Greenwich, CT).

uPA combined with FITC (uPA-FITC; 25 phyclogg/ml ) was a kind gift from American Diagnostics. used. Acid-washed PMA-stimulated U-937 cells 10 nM of PM as described above. A (J, [mmunol, vol, 144.1841 pages (containing 199) After culturing for 2 days in a medium containing PBS, the U-937 cells were washed twice with PBS and -NaCI buffer (50mM glycine, 100mM NaCL pH 3, 0) and incubated for 3 minutes at 4°C with gentle agitation.

Then add 40% cooling solution (500mm HEPES, 100mM NaC! , Img/ml BSA, pH 7,5) was added. Sediment cells and wash buffer (1mg/ml glucose and Img/ml human Ig (J, 1mmuno1 ．． vol, 137°, p. 448 (1986)) in PBS). The cells were resuspended to a concentration of 0' cells/ml.

of uPA-FITC with monoclonal or polyclonal anti-Mo3 antibodies. 5X10' pickling, PMA-stimulated U-937 cells were washed with 150 microliters. Breincubated in clean buffer (same as above) for 60 minutes at 4°C. .

This wash buffer was prepared using murine anti-Mo3 monoclonal antibody (ascites or mouse). isotype-equivalent control reagent) at various dilution concentrations; rabbit polyclonal anti-Mo3 antiserum (or healthy rabbit serum) at different concentrations. The content was changed. Contains 50 microliters of uPA-FITC Wash buffer was added to this (125 mg 15X1O' cells) and the mixture was Incubated for an additional 60 minutes at °C. Centrifuge cells at 11,000X for 5 minutes at 4°C. After separation, wash with 100 microliters of washing buffer and add 1% formaldehyde. It was fixed with 0.5 ml of PBS containing aldehyde.

Table of U-937 in uPA-FITC analyzed by immunofluorescence flow cytometry Binding to the surface was performed using a Coulter ELITE flow cytometer (Coulter ELITE flow cytometer). Flow cytometry (Electronics, Inc., Hiary, FL) It was quantified by Average channel fluorescence (linear scale) measured for 5000 cells/cells This was used as a quantitative value of the relative uPA-FITC binding amount. The results are shown in Figure 1. Show it in a graph.

Obviously, many modifications and variations may be made to the present invention in light of the above teachings. It is clear. Therefore, within the scope stated in the attached claims, other than the above The invention can be practiced.

RELATIVE FLUORESCENT INTENSITY

Claims (10)

[Claims] 1. Treat samples that may contain uPA-R with antibodies or labeled antibodies. to measure the amount of antibody bound to uPA-R or labeled antibody. In the uPA-R detection method, the antibody or labeled antibody is attached to Mo3. A detection method characterized by being specific. 2. 2. The method according to claim 1, wherein said antibody is anti-Mo3f. 3. Method for isolating uPA-R comprising immunoprecipitating uPA-R with an antibody An isolation method characterized in that the antibody is specific to Mo3. 4. 4. The method of claim 3, wherein the antibody is anti-Mo3f. 5. characterized by contacting uPA-R with an antibody specific for Mo3, A method of preventing uPA from binding to uPA-R. 6. 6. The method of claim 5, wherein said antibody is anti-Mo3f. 7. Administering an effective amount of an antibody specific to Mo3 to a patient in need of treatment. A method for inhibiting tissue destruction by inflammatory macrophages, characterized by: 8. 8. The method of claim 7, wherein said antibody is anti-Mo3f. 9. Administering an effective amount of an antibody specific to Mo3 to a patient in need of treatment. A method for inhibiting metastatic invasion of tumor cells, characterized in that: 10. 10. The method of claim 9, wherein said antibody is anti-Mo3f.