CA2123874A1 - Methods for detecting and isolating upa-r and inhibiting the binding of upa to upa-r - Google Patents
Methods for detecting and isolating upa-r and inhibiting the binding of upa to upa-rInfo
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
Antibodies specific for Mo3 are capable of binding to uPA-R and may be used to detect or isolate uPA-R or to inhibit the binding of uPA to uPA-R.
Description
W093/~9X0~ 2 1 2 3 ~ 7 4 PCT/US92/0~977 Description METHODS FOR DETECTING AND ISOLATING uPA-R AND
INHIBITING THE BINDING OF uPA TO uPA-R
BACKGROUND OF THE INVENTION
_echnical Fiel_ The present invention relates to methods for detecting and isolating uPA-R utilizing monoclon~l antibodies. The present invention also relates to methods for inhibiting the bindir.g of uPA to uP~-R.
~ac~ro~nd Art Urokinase-type plasminogen activa~or ~uPA3 is an enzym~
responsible for the generatlon of ~he pro~ease, pla~min. uPA is thought to be ir~volved in the generation of ex~racellular proteolytic activity in physiological and pathological processes such as cell migration, tissue remodeling, and tumor in~asion and metastasis ~Ad~. Cancer ResO, vol. 44, pp. 139266 (lg85). The evidence that uPA plays a specific role in these pro~ess~s includes the observation that uPA binds to a specific cellular receptor (uPA R) on a wide variety of cell types of s~ormal and malignant origin (-J. C 11 Biol. ~ vol" 100, pp. ~6-96 (1985); Pro, Natl . Acad. Sci . _U. S.A., vol . 82 , pp. 4939 a~943 tl985~; and ;L.
Bi~h~~ vol. 263, pp. 2358-2363 tl988). uPA bound to this receptor, which ha~ been characterized (J. Biol. Chem., vol. 2~5, pp. 6453-6460 (1990); and EMES0 J., vol. 9, pp. 467-474 (1990) ), has been shown t~ retain its proteolytic ac~tivity (J. Cell Biol., vol~ 100, pp. 86-92 (1985); Proc Nail. Acadt Sci~ U.S.P.., vol.
82 , pp. 49394943 ~ 1985~ ; J. Biol:. Chem., vol. . 264 , pp. 2185~2188 (1989); and J._ Cell Biol., voï. las, pp. 1987-1995 (1989) ~ .
Plasminogen is also able to bind to many r,ell types (J. Biol.
Chem., vol. 260, pp. 4303-4311 (1985) ) and it has been demonstrated that the conconaitant binding o:E uPA and plasminogen results in the generation of plasmin activity on the cell surface W~93~09808 PCT/US9~/~$977 2123~74 (J. Biol._Chem., vol. 264, pp. 2185-2188 (1989); and J. Cell Biol., vol. 108, pp. 1987-1995 (198~)) and also that this system constitutes a mechanism for accelerating the activation of the pro-enzyme form o~ uPA (_. Biol. Chem., vol. 264, pp. 2185-218 (lg~9) ) -Mo3 is a glycoprotein whose expression on human monocytes and myel~monocytic cell lines is induced by bacterial LPS and muramyl dipeptide (~DP), as well as certain cytokines and pharmacologic agonists of protein kinase C and CAMP. Mo3 was originally idenki~ied as a monocyte surface antigen recogniæed by ~a panel of monoclonal antibodies that were generated aga.inst antigens selectively expressed on activaked macrophages ~.
Immunol., vol. 135, p. 3869 (1985); Blood Cells, vol. 16, p. 167 (1990) ; and J. I mmunol., vol. 137, p. 448 (1986)). By immunofluorescence flow cytometry, Mo3 is barely detectable on freshly isolated monocytes, but is prominent on the surfac~ of monocytes activated by culture in media containing soluble inflamma~ory factors such as LCP, MDP, and cytokines including TNF, M-CSF, GM-C5F, and IL-3. In vivo, ~o3 expression is seen predominantly in inflammatory or malignant tissues upon examination of human ti55U8S (Blood Cells, vol 16, p. 167 (1990~.
Macrophages in normal ti~sues are negative ~or Mo3 stai~ing except for a variable degree of expression by pulmonary macrophages. Among non-phagocytic cells, Mo3 expression appears to be more constitutive: normal tonsillar epithelium, hepatocytes, and derm~l collagen were all positive for Mo3 staining. Mo3 expression in endothelial cells, as in monocytes, is'more speci~ic for inflammatory tissues.
Xncreased plasminogen activator has been reported in metast~tic tumors and is implicated in tumor invasion into other tis~ues ~ dv. Cancer Res., vol. 44, p~ :L39 (1985)). More recently, uPA receptors have been described t.o be associated with adhesion (J. Cell Biol., vol. 104, p. 1085 (1987)) and W093~09X08 P~T/US92/0~77 2123~74 cytoskeletal proteins such as vinculin (J. Cell Biol., vol~
106,, vol. 1241 (1988)), alpha-actinin and actin in contact sites on metastatic tumor cells (Thromb. Res. Sup~, vol. 10, p. 55 (1990)). This presumably enables the tumor cell to stimulate the degradation of basement membrane and extracellular matrix proteins, allowin~ it to invade other tissues and become metastatic. In a previous survey of surface Mo3 expression, Mo3 on tumorassociated macrophages in squamous cell carcinoma of the lung has been detected. Interestingly, the tumor cells surrounding these macrophages were negative for Mo3 staining (Blood C~lls, vol. 16, p. 167 (1990)). .
"
Monoclonal antibodies specific for Mo3 have b2en report~d (J Immunol~., vol 137~ pp. 448-455 (1986); Blood, ~ol. 59, p.p.
775 (1982); and J. Immunol., vol. 144, pp. 1841 1~48 ~l9~0~).
In addition, a monoclonal antibody speci~ic for uPA-~ has recently been reported (F~S Letters, vol 288, pp. 233-236 ~199~)). However, there remains a need for methods for detecting and isolating uPA-R. There al50 remains a need for a method ~or inhibiting the binding of uPA to uPA-R and treating diseases mediated by the bind of uP~ to uPA-R.
pisclosure p~ the Iny~ntion :
Accordingly, one object of the present invention is to provide a method f~r isolating UPA-R.
It is another object of the present invention to provide method for~detecting uPA-R.
~ t is another object o~ th~ present invention ~o provide ~ethod for detecting uPA-R bound on the surfaces oE cells.
It is another object of the present invention to provide method for detec~ing free uPA-R.
W093/09~0~ ~ ~ 2 3 ~ 7 4 PCT/US~2/0~977 It is an~ther o~j~ct of the present invention to provide method for inhibiting the binding of uPA to uPA-R.
It is another object of the present i.nvention to provide a method for treating diseases which are mediate~ by ~he binding of uPA to uPA~R.
It is another object of the present invention to provide a method for inhibiting tissue destruction by inflammatory macrophages. .
It is another object of the present inventiQn to provide a method for inhibiting t~e metastatic invasion of tumor cells.
These and other objects, which will become apparent during the course of the following detailed descrip~ion, have been achieved by the inventors' disco~ery that antibodies which bind to Mo3 also bind to uPA-R.
Brief D~cripti ~ of the Dr~winq~.
A more complete appreclation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the foll~wing detailed description when considered in connection with the accom~anying drawings, wherein:
Figure 1 illustrates the competitive blocking of uPA-FITC by murine ~onoclonal and rabbit polyc~onal antibodies specific for Mo3. Acid washed, PMA-stimulated ~-937 cell~ were preincubated in buf~er sontaining the indicated dilutions of murin~e monoclonal antibodies: IgG2a anti-Mo3f ( + ), IgM anti-Mo3e ~ , or isotype~identical negative control antibodies, IgG~a SB7 (~
or ~g~ anti-CD14 (~ ); or polyclonal rabbit anti-Mo3 ( ~ -) or normal rabbit serum ( ~ ), or no antibody (X)~ After 60 W093/~9808 PCT/U~2/~8~77 2123~7~
minutes at 4C, uPA-FITC was added, and the cell suspension was incubated for an additional 60 min at 4C. ~fter washing and fixation, the cells were subjected to flow cytometric analysis in which 5000 cells per determination were assessed fcr uPA-FITC
binding~ The mean channel fluorescence of each stained population is indicated on the y-axis.
Best Mod~ for Carryinq Ou~ th¢ Invent _n The cDNA encoding Mo.~, an activation antigen expressed on the sur~ace of monocy~es and U-937 cells, has been clon~d.
~Consistent with previous observations tha~ Mo3 i5 a highly glycosylated protein in which 40-50% of the molecular weight is attributable to N-linked glycosylation, ~he Mo3 cDNA sequence predicts 5 potential N-linked glycosylation sites (Asn-X-SerlThr)O A signal peptide of 22 amino acids is predicted by the method of von Heijne ~Nucleic Acids ~es~, vol. 14, p. 4683 (1986)) to give an N-~erminal sequence of ~RCMQ .~. in the mature form of the Mo3 proteinO It has been previQusly shown that Mo3 is a cell surface protein that is anchQred to the plasma membrane by a GPI linkage (J. Immunol., vol~ , p. 1841 (1990~).
Supporting this conclusion is the fact that in addition to bein~
susceptible to cleavage from the cell surface by PI-PLC, Mo3 surface expression is deficient in a patient with paroxysmal nocturnal hemoglobinuria (J Immunol., vol. 144, p. 1841 (19~0)), a disease that is characterized by the absence of GPI-linked determinants on l~ukocyte~ (J. Ex~ Med., vol. 166, p. 1011 ~ 87)). At the C-terminal end, a stretch of about 20 ! hydrophobi~ amino acids is present and may represent the plasma membrane anchor which would be expected to ~e clipped off during the formation of the GPX linkage ~Ann, Rev,:Biochem~, vol. 57, p.
285 (19~8))- The expected size c~ the ma~ure Mo3 surface-expressed protein after cleavage of. the putatiYe signal peptide and the C-terminal anchor, about 2'30 amino acids, is in close agreement with the 29 kD molecular weight observed for W093/~9808 2 1 2 3 ~ 7 ~ P~T/U~92/08977 deylycosylated Mo3 (J. Immunol., vol. 1~7, p. 1331 (1991)).
The most direct evidence that the CDNA clone isolated does indeed encode Mo3 protein comes ~rom the kransf2ction of Cos cells with M03 CDN~. After transfection, ~he surface expression of ~o3 on the Cos cells was determined by ~L~S~ using various anti-Mo3 monoclonal A~s and a polyclonal rabbit anti-Mo3 antiserum. Only the cells that were trans~ected with Mo3 CDNA
were immunoreactive with anti-MO3 antibodies; the mock-~ran~fected cells ~ere negative for surface expression of Mo3. Isotype-matched control mabs and normal rabbit serum ~ailed ~to react, with both Mo3- and mock-tr~nsfected Cos cells. In another experiment, cells transfected with ICAM-1 CDN~ did not react with anti-MO3 monoclonal antibodies, although they showed positive staininy with anti~ICAM-1 antibodies. It can be . concluded from the transfection experimen~s that the Mo3 cDNA
clone isolated does in fact encode the cell surface antigen previou~ly defined as Mo3.
As a result of the clonin~ and sequencing of the gene encoding Mo3, it has now been discovered that ~o3 is the same as uPA-R. A computer search of the NBRF database suggested ~hat Mo3 is identical to the human receptor for uro~inase plasminogen activator ~uPA-R), and this was conf irmed by comparison of the comple~e sequence for uPA~R (EMBQ J0, vol. 9, pp. 4~7-474 (1390)~
with khat of ~o3.
It has now been discovered that antibodies specific for Mo3 i aiso bind ~o uPA~ Thus, in one embodiment, the present invention relates to the detection of uPA-R by observing the binding of monoclonal anti-Mo3 antibo~ies: to uPA-R~ Suitabl~ monoclona~
anti-Mo3 antîbodies include anti-~o3a~f. The pxoduction of anti-~o3f is described in J. Immunol., vol.. 144, pp. 1841-1848 (1990), and the generation of anti Mo3a-e is described in J
Immunol., vol 137l ppO 448-455 (1986) and Blood, vol. 59, p. 775 W~93/09808 PCT/U~92/0~977 2123~74 (19~2). The hybridoma strain which produces anti-Mo3f has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA 29852, on November 16, 1991 .
The present detection method may be carried out on free or membrane bound uP~-R. In the case of detecting membrane bound uPA-R, it is pre~erred to use an~i-Mo3e or anti-Mo3f.
Particularly preferred is the use of anti-Mo3f.
The uPA-R may be detected by the use of an ELISA. For ~example, a cell suspec~ed of expressing uPA-R may be incubated with one of the present antibodies. After washing, the cells may be incubated with another antibody, which binds to the present ~ntibody, and which is conjugated with an enzyme suitable ~or - ELISA, such as horse ra~ish peroxidase, HRP. After another washing, the c~ll may be incubated with a substrate for the enzyme and the activity of the enzyme may be determined. The activity of the enzyme will be related ko the amount of uPA-R on the surface of the cell. Such an ELXSA may also be used to detect soluble uPA-R in body fluids, (e.g., plasma, urine, a~d inflammatory exudates) as a marker of inflammation and/or cancer.
Alternatively, ~he cells may be incubated with one of khe present antibodies whi~h has ~een modified to carry a label. For example, the anti~ody may be treated with periodate to generate carbonyl groups on the sugar ~roups o~ the antibody. Then a suitable label may be covalently linked to the antibody via a difunctional linking group. Suitable labels and li~king groups are disclo~ed in auqland, Handbook of Flou~ y~ Probes and Rese3rch Chemicals, Molecular Probes, Inc., E~gen~, Oxagan (1989) and Pierce _Immun technoloq~ Cataloq and Handboo_, Pierce, Rockford, I~ (l990).
The present invention also relates to the isolation of uPA-R, by use of an antibody specific for ~o3. Thus, uPA-R may be isolated by immunoprecipita~ion with an anti~ody specific for Mo3. It is preferred to use anti-Mo3f for the immunoprecipitation. The antibody may be bound on a suitable support, e.g., protein A-Sephorose~ (Pharmacia-LKB ~iotechnology, Inc., Piscataway, NJ) as described in J. Immunol., vol. 147, pp.
1331-1337 (1991), and the bound antibody may be used to isolate uP~-R obtained from lysed cells or from the supernatants of uPA-R
bearing cells exposed to phosphatidylino~itol-specific phospholipase~ The uPA-R may be labelled before lysing o~ 'he cells by either ~reatment with ~he N-hydroxysuccinimide ester of biotin or incubation of the cell with a nutrient containing a radio label, e.g~ 35s-methionine or 3H-mannose, The present invention also relates to a method for inhibiting the binding of uPA to uPA-R by treating uPA-R with an antibody specific for Mo3. I~ is preferred that th~ antibody be an~i-Mo3~ or anti-~o3~. It is especially preferred that the antibody be anti-Mo3f. ~he inhibition o~ the binding of uPA to uPA-R may be carried out in ~itro (extracorporeal) or _n vivo.
Thus, the inhibition of khe binding uPA to uPA-R may comprise one aspect of an in vitro assay ~or the pr~sence of uPA-R.
The pra~ent method of inhibiting the binding of uPA to uPA-R
may also be carried out in the body. By administration of an antibody the ~L~iYe binding of uPA to uPA-R may be inhibitedO
Thus, the present method encompasses preventing or reducing tissue damage by inflammatory phagocytic cells (macrophages and neutrophils) ~nd metastatic invasion by tumor cells. In this method, the antibody is administered sys~ematically (~.g. by intravenous injectiQn). Examples o~ infl~mmatory processes in which the method may be applied include the ~ollowing: rheumatoid arthritis, immune vasculitis, glomerulonephritis, inflammatory ~owel disease, and adult respiratory distress syndrome. The method may also be applied to inhibit tumor cell invasion 21 23~7 1 (metastases invasion) by various human tumors expressing uPA-R, e.g., malignant melanoma, breast cancer, and sarcoma.
The actual dosage and regimen of antibody administration will of coursé depend on the health of the patient and the condition being treated. However, good results may be achie~ed with dosages o~ O.Ol to 5 mg/kg of body weight, preferably 0.1 to 1 mg/kg o~ body weight. This schedule of administration may be ~arried out for a few (1 ko 10) days. In this cas2 of in~lammation, the antibody may be adminis~ered early in the course of inflammation, and in ~he case o~ cancer, a~ ~he time of 'surgical resection to prevent metas~asis ~ormation from surgically-dislsdged tumor cells.
The a~tibody may be administered in various pharmaceutical compositions in the form of an injectable solution or ~uspen~ion.
Thus, the present invention provides a method for inhibiting the binding of uPA to uPA-R. As described in the examples gi~en below, antibodies specific for Mo3 are capable of inhibiti~g the binding o~ uPA to uPA-R. Figure 1 ~hows the results of ~he competitive blocking of uPA-FITC tfluorescein isothiocyanate~
with a variety ~f antibodies speci~ic for Mo3. The results in Figure 1 clearly demonstrate that the antibodies specific for Mo3 (anti-Mo3f, ~ ~ ); anti-~o3e ~ ~ ); and polyclonal rabbit an~i-Mo3 ( - ~- ) are capable of blocking the binding of uPA-FITC
to uPA-R.
It is ~o be understood that while the exemplified antibodies of the invention are of murine origin, the pre~ent methods include the use of humanized version~ ~f these antibodies (N~ture, vol. 351, pp. 501V502 (1991~).
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments W093/098~ 2 1 2 3 8 7 4 PCT/~S92/~8977 which are given for i.llustration of the invention and are not intended to be limiting thereof.
EXAMPLES
MATERIALS AND METHODS
High molecular weight urokinase (uPA; 80/000 IU/mgr catalog #124) (Ho~pe Sev~er's Z. PhYsiol. chem., vol. 363, p. 133 (1982)) was purchased from ~merican Diagnostics, Inc. (Greenwich, CT).
FITC conjugated uPA ~uPA-FIT~; 25 ~g/ml) was a generous gift of American Diagnostics, Inc.
Acid Washed,_PMA-Stimulated U-937 Cells A~ter culture for 2 days in medium containing 10 nM PMA as pre~iously described (J. Immunol., vol. 144, p. 1841 (1990)), U-937 cells were washed twice in PBS, then resuspended in glycine-NaCl buffer (50 mM glycine, 100 mM NaCl, pH 3.0~ and incubated at 4C for 3 min with gentle agitation. A 4~% volume of quench solution (500 mm ~EPES, 100 m~ NaCi, 1 mg/ml BSA, pH
7.5) was then added. The cells were pelleted and resuspended in wash buffer (PBS supplemented with 1 ~gtml glucose and 1 mg/ml human Ig ~J. _Immunol.~ vol. 137, P. ~48 ~ 6~) at a concentration of 1 X 107 cells/ml.
om~etitive~ glL~rgLs~ ~gPA-FITC bY Monoc Anti-Mo3 ~ntibodies x 105 acid-washed PMA stimulated U-937 cells were preincubated for 60 min at 4C in 150 ~l wash bu~`fer (same as above) containing various dilutions of murine monoclonal anti~o3 mabs (ascites; or isotype-identical control reagents) or with W093/~9808 P~T/US9~/0~977 2123~7~
varying dilutions of rabbit polyclonal anti~Mo3 antiserum (or normal rabbit serum). 50 ~1 o~ uPA-FITC in wash buffer was then added (125 mg per 5 x 105 cells) and the mixture was incubated for an additional 60 min at 4C. The cells were then centrifuged at 1000 x g for 5 min at 4C, washed with 100 ~1 wash buffer, and fixed in 0.5 ml PBS containing 1% formaldehyde.
Immunofluorescence Flow Cytomet~ic Analysis Binding of uPA-FITC to khe surface of U-937 cells was quantitated by flow cytometric analysis on a Coulter ELITE flow ~cytometer (Coulter Electronics, Inc., Hial~ah, FL). The mean channel fluorescence ~linear scale) of 5000 cells/determination was calculated and used as a quantitative measure o~ relative uPA FITC binding. The results are shown graphicall~ in Figure 1.
Obviously, numerous modifications and variations of the present invention are possible in ligh~ of the above teachings.
It is therefore to be understood that, within the scope of the appended claims, the invention may be pr~cticed otherwise khan as specifically described herein.
INHIBITING THE BINDING OF uPA TO uPA-R
BACKGROUND OF THE INVENTION
_echnical Fiel_ The present invention relates to methods for detecting and isolating uPA-R utilizing monoclon~l antibodies. The present invention also relates to methods for inhibiting the bindir.g of uPA to uP~-R.
~ac~ro~nd Art Urokinase-type plasminogen activa~or ~uPA3 is an enzym~
responsible for the generatlon of ~he pro~ease, pla~min. uPA is thought to be ir~volved in the generation of ex~racellular proteolytic activity in physiological and pathological processes such as cell migration, tissue remodeling, and tumor in~asion and metastasis ~Ad~. Cancer ResO, vol. 44, pp. 139266 (lg85). The evidence that uPA plays a specific role in these pro~ess~s includes the observation that uPA binds to a specific cellular receptor (uPA R) on a wide variety of cell types of s~ormal and malignant origin (-J. C 11 Biol. ~ vol" 100, pp. ~6-96 (1985); Pro, Natl . Acad. Sci . _U. S.A., vol . 82 , pp. 4939 a~943 tl985~; and ;L.
Bi~h~~ vol. 263, pp. 2358-2363 tl988). uPA bound to this receptor, which ha~ been characterized (J. Biol. Chem., vol. 2~5, pp. 6453-6460 (1990); and EMES0 J., vol. 9, pp. 467-474 (1990) ), has been shown t~ retain its proteolytic ac~tivity (J. Cell Biol., vol~ 100, pp. 86-92 (1985); Proc Nail. Acadt Sci~ U.S.P.., vol.
82 , pp. 49394943 ~ 1985~ ; J. Biol:. Chem., vol. . 264 , pp. 2185~2188 (1989); and J._ Cell Biol., voï. las, pp. 1987-1995 (1989) ~ .
Plasminogen is also able to bind to many r,ell types (J. Biol.
Chem., vol. 260, pp. 4303-4311 (1985) ) and it has been demonstrated that the conconaitant binding o:E uPA and plasminogen results in the generation of plasmin activity on the cell surface W~93~09808 PCT/US9~/~$977 2123~74 (J. Biol._Chem., vol. 264, pp. 2185-2188 (1989); and J. Cell Biol., vol. 108, pp. 1987-1995 (198~)) and also that this system constitutes a mechanism for accelerating the activation of the pro-enzyme form o~ uPA (_. Biol. Chem., vol. 264, pp. 2185-218 (lg~9) ) -Mo3 is a glycoprotein whose expression on human monocytes and myel~monocytic cell lines is induced by bacterial LPS and muramyl dipeptide (~DP), as well as certain cytokines and pharmacologic agonists of protein kinase C and CAMP. Mo3 was originally idenki~ied as a monocyte surface antigen recogniæed by ~a panel of monoclonal antibodies that were generated aga.inst antigens selectively expressed on activaked macrophages ~.
Immunol., vol. 135, p. 3869 (1985); Blood Cells, vol. 16, p. 167 (1990) ; and J. I mmunol., vol. 137, p. 448 (1986)). By immunofluorescence flow cytometry, Mo3 is barely detectable on freshly isolated monocytes, but is prominent on the surfac~ of monocytes activated by culture in media containing soluble inflamma~ory factors such as LCP, MDP, and cytokines including TNF, M-CSF, GM-C5F, and IL-3. In vivo, ~o3 expression is seen predominantly in inflammatory or malignant tissues upon examination of human ti55U8S (Blood Cells, vol 16, p. 167 (1990~.
Macrophages in normal ti~sues are negative ~or Mo3 stai~ing except for a variable degree of expression by pulmonary macrophages. Among non-phagocytic cells, Mo3 expression appears to be more constitutive: normal tonsillar epithelium, hepatocytes, and derm~l collagen were all positive for Mo3 staining. Mo3 expression in endothelial cells, as in monocytes, is'more speci~ic for inflammatory tissues.
Xncreased plasminogen activator has been reported in metast~tic tumors and is implicated in tumor invasion into other tis~ues ~ dv. Cancer Res., vol. 44, p~ :L39 (1985)). More recently, uPA receptors have been described t.o be associated with adhesion (J. Cell Biol., vol. 104, p. 1085 (1987)) and W093~09X08 P~T/US92/0~77 2123~74 cytoskeletal proteins such as vinculin (J. Cell Biol., vol~
106,, vol. 1241 (1988)), alpha-actinin and actin in contact sites on metastatic tumor cells (Thromb. Res. Sup~, vol. 10, p. 55 (1990)). This presumably enables the tumor cell to stimulate the degradation of basement membrane and extracellular matrix proteins, allowin~ it to invade other tissues and become metastatic. In a previous survey of surface Mo3 expression, Mo3 on tumorassociated macrophages in squamous cell carcinoma of the lung has been detected. Interestingly, the tumor cells surrounding these macrophages were negative for Mo3 staining (Blood C~lls, vol. 16, p. 167 (1990)). .
"
Monoclonal antibodies specific for Mo3 have b2en report~d (J Immunol~., vol 137~ pp. 448-455 (1986); Blood, ~ol. 59, p.p.
775 (1982); and J. Immunol., vol. 144, pp. 1841 1~48 ~l9~0~).
In addition, a monoclonal antibody speci~ic for uPA-~ has recently been reported (F~S Letters, vol 288, pp. 233-236 ~199~)). However, there remains a need for methods for detecting and isolating uPA-R. There al50 remains a need for a method ~or inhibiting the binding of uPA to uPA-R and treating diseases mediated by the bind of uP~ to uPA-R.
pisclosure p~ the Iny~ntion :
Accordingly, one object of the present invention is to provide a method f~r isolating UPA-R.
It is another object of the present invention to provide method for~detecting uPA-R.
~ t is another object o~ th~ present invention ~o provide ~ethod for detecting uPA-R bound on the surfaces oE cells.
It is another object of the present invention to provide method for detec~ing free uPA-R.
W093/09~0~ ~ ~ 2 3 ~ 7 4 PCT/US~2/0~977 It is an~ther o~j~ct of the present invention to provide method for inhibiting the binding of uPA to uPA-R.
It is another object of the present i.nvention to provide a method for treating diseases which are mediate~ by ~he binding of uPA to uPA~R.
It is another object of the present invention to provide a method for inhibiting tissue destruction by inflammatory macrophages. .
It is another object of the present inventiQn to provide a method for inhibiting t~e metastatic invasion of tumor cells.
These and other objects, which will become apparent during the course of the following detailed descrip~ion, have been achieved by the inventors' disco~ery that antibodies which bind to Mo3 also bind to uPA-R.
Brief D~cripti ~ of the Dr~winq~.
A more complete appreclation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the foll~wing detailed description when considered in connection with the accom~anying drawings, wherein:
Figure 1 illustrates the competitive blocking of uPA-FITC by murine ~onoclonal and rabbit polyc~onal antibodies specific for Mo3. Acid washed, PMA-stimulated ~-937 cell~ were preincubated in buf~er sontaining the indicated dilutions of murin~e monoclonal antibodies: IgG2a anti-Mo3f ( + ), IgM anti-Mo3e ~ , or isotype~identical negative control antibodies, IgG~a SB7 (~
or ~g~ anti-CD14 (~ ); or polyclonal rabbit anti-Mo3 ( ~ -) or normal rabbit serum ( ~ ), or no antibody (X)~ After 60 W093/~9808 PCT/U~2/~8~77 2123~7~
minutes at 4C, uPA-FITC was added, and the cell suspension was incubated for an additional 60 min at 4C. ~fter washing and fixation, the cells were subjected to flow cytometric analysis in which 5000 cells per determination were assessed fcr uPA-FITC
binding~ The mean channel fluorescence of each stained population is indicated on the y-axis.
Best Mod~ for Carryinq Ou~ th¢ Invent _n The cDNA encoding Mo.~, an activation antigen expressed on the sur~ace of monocy~es and U-937 cells, has been clon~d.
~Consistent with previous observations tha~ Mo3 i5 a highly glycosylated protein in which 40-50% of the molecular weight is attributable to N-linked glycosylation, ~he Mo3 cDNA sequence predicts 5 potential N-linked glycosylation sites (Asn-X-SerlThr)O A signal peptide of 22 amino acids is predicted by the method of von Heijne ~Nucleic Acids ~es~, vol. 14, p. 4683 (1986)) to give an N-~erminal sequence of ~RCMQ .~. in the mature form of the Mo3 proteinO It has been previQusly shown that Mo3 is a cell surface protein that is anchQred to the plasma membrane by a GPI linkage (J. Immunol., vol~ , p. 1841 (1990~).
Supporting this conclusion is the fact that in addition to bein~
susceptible to cleavage from the cell surface by PI-PLC, Mo3 surface expression is deficient in a patient with paroxysmal nocturnal hemoglobinuria (J Immunol., vol. 144, p. 1841 (19~0)), a disease that is characterized by the absence of GPI-linked determinants on l~ukocyte~ (J. Ex~ Med., vol. 166, p. 1011 ~ 87)). At the C-terminal end, a stretch of about 20 ! hydrophobi~ amino acids is present and may represent the plasma membrane anchor which would be expected to ~e clipped off during the formation of the GPX linkage ~Ann, Rev,:Biochem~, vol. 57, p.
285 (19~8))- The expected size c~ the ma~ure Mo3 surface-expressed protein after cleavage of. the putatiYe signal peptide and the C-terminal anchor, about 2'30 amino acids, is in close agreement with the 29 kD molecular weight observed for W093/~9808 2 1 2 3 ~ 7 ~ P~T/U~92/08977 deylycosylated Mo3 (J. Immunol., vol. 1~7, p. 1331 (1991)).
The most direct evidence that the CDNA clone isolated does indeed encode Mo3 protein comes ~rom the kransf2ction of Cos cells with M03 CDN~. After transfection, ~he surface expression of ~o3 on the Cos cells was determined by ~L~S~ using various anti-Mo3 monoclonal A~s and a polyclonal rabbit anti-Mo3 antiserum. Only the cells that were trans~ected with Mo3 CDNA
were immunoreactive with anti-MO3 antibodies; the mock-~ran~fected cells ~ere negative for surface expression of Mo3. Isotype-matched control mabs and normal rabbit serum ~ailed ~to react, with both Mo3- and mock-tr~nsfected Cos cells. In another experiment, cells transfected with ICAM-1 CDN~ did not react with anti-MO3 monoclonal antibodies, although they showed positive staininy with anti~ICAM-1 antibodies. It can be . concluded from the transfection experimen~s that the Mo3 cDNA
clone isolated does in fact encode the cell surface antigen previou~ly defined as Mo3.
As a result of the clonin~ and sequencing of the gene encoding Mo3, it has now been discovered that ~o3 is the same as uPA-R. A computer search of the NBRF database suggested ~hat Mo3 is identical to the human receptor for uro~inase plasminogen activator ~uPA-R), and this was conf irmed by comparison of the comple~e sequence for uPA~R (EMBQ J0, vol. 9, pp. 4~7-474 (1390)~
with khat of ~o3.
It has now been discovered that antibodies specific for Mo3 i aiso bind ~o uPA~ Thus, in one embodiment, the present invention relates to the detection of uPA-R by observing the binding of monoclonal anti-Mo3 antibo~ies: to uPA-R~ Suitabl~ monoclona~
anti-Mo3 antîbodies include anti-~o3a~f. The pxoduction of anti-~o3f is described in J. Immunol., vol.. 144, pp. 1841-1848 (1990), and the generation of anti Mo3a-e is described in J
Immunol., vol 137l ppO 448-455 (1986) and Blood, vol. 59, p. 775 W~93/09808 PCT/U~92/0~977 2123~74 (19~2). The hybridoma strain which produces anti-Mo3f has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA 29852, on November 16, 1991 .
The present detection method may be carried out on free or membrane bound uP~-R. In the case of detecting membrane bound uPA-R, it is pre~erred to use an~i-Mo3e or anti-Mo3f.
Particularly preferred is the use of anti-Mo3f.
The uPA-R may be detected by the use of an ELISA. For ~example, a cell suspec~ed of expressing uPA-R may be incubated with one of the present antibodies. After washing, the cells may be incubated with another antibody, which binds to the present ~ntibody, and which is conjugated with an enzyme suitable ~or - ELISA, such as horse ra~ish peroxidase, HRP. After another washing, the c~ll may be incubated with a substrate for the enzyme and the activity of the enzyme may be determined. The activity of the enzyme will be related ko the amount of uPA-R on the surface of the cell. Such an ELXSA may also be used to detect soluble uPA-R in body fluids, (e.g., plasma, urine, a~d inflammatory exudates) as a marker of inflammation and/or cancer.
Alternatively, ~he cells may be incubated with one of khe present antibodies whi~h has ~een modified to carry a label. For example, the anti~ody may be treated with periodate to generate carbonyl groups on the sugar ~roups o~ the antibody. Then a suitable label may be covalently linked to the antibody via a difunctional linking group. Suitable labels and li~king groups are disclo~ed in auqland, Handbook of Flou~ y~ Probes and Rese3rch Chemicals, Molecular Probes, Inc., E~gen~, Oxagan (1989) and Pierce _Immun technoloq~ Cataloq and Handboo_, Pierce, Rockford, I~ (l990).
The present invention also relates to the isolation of uPA-R, by use of an antibody specific for ~o3. Thus, uPA-R may be isolated by immunoprecipita~ion with an anti~ody specific for Mo3. It is preferred to use anti-Mo3f for the immunoprecipitation. The antibody may be bound on a suitable support, e.g., protein A-Sephorose~ (Pharmacia-LKB ~iotechnology, Inc., Piscataway, NJ) as described in J. Immunol., vol. 147, pp.
1331-1337 (1991), and the bound antibody may be used to isolate uP~-R obtained from lysed cells or from the supernatants of uPA-R
bearing cells exposed to phosphatidylino~itol-specific phospholipase~ The uPA-R may be labelled before lysing o~ 'he cells by either ~reatment with ~he N-hydroxysuccinimide ester of biotin or incubation of the cell with a nutrient containing a radio label, e.g~ 35s-methionine or 3H-mannose, The present invention also relates to a method for inhibiting the binding of uPA to uPA-R by treating uPA-R with an antibody specific for Mo3. I~ is preferred that th~ antibody be an~i-Mo3~ or anti-~o3~. It is especially preferred that the antibody be anti-Mo3f. ~he inhibition o~ the binding of uPA to uPA-R may be carried out in ~itro (extracorporeal) or _n vivo.
Thus, the inhibition of khe binding uPA to uPA-R may comprise one aspect of an in vitro assay ~or the pr~sence of uPA-R.
The pra~ent method of inhibiting the binding of uPA to uPA-R
may also be carried out in the body. By administration of an antibody the ~L~iYe binding of uPA to uPA-R may be inhibitedO
Thus, the present method encompasses preventing or reducing tissue damage by inflammatory phagocytic cells (macrophages and neutrophils) ~nd metastatic invasion by tumor cells. In this method, the antibody is administered sys~ematically (~.g. by intravenous injectiQn). Examples o~ infl~mmatory processes in which the method may be applied include the ~ollowing: rheumatoid arthritis, immune vasculitis, glomerulonephritis, inflammatory ~owel disease, and adult respiratory distress syndrome. The method may also be applied to inhibit tumor cell invasion 21 23~7 1 (metastases invasion) by various human tumors expressing uPA-R, e.g., malignant melanoma, breast cancer, and sarcoma.
The actual dosage and regimen of antibody administration will of coursé depend on the health of the patient and the condition being treated. However, good results may be achie~ed with dosages o~ O.Ol to 5 mg/kg of body weight, preferably 0.1 to 1 mg/kg o~ body weight. This schedule of administration may be ~arried out for a few (1 ko 10) days. In this cas2 of in~lammation, the antibody may be adminis~ered early in the course of inflammation, and in ~he case o~ cancer, a~ ~he time of 'surgical resection to prevent metas~asis ~ormation from surgically-dislsdged tumor cells.
The a~tibody may be administered in various pharmaceutical compositions in the form of an injectable solution or ~uspen~ion.
Thus, the present invention provides a method for inhibiting the binding of uPA to uPA-R. As described in the examples gi~en below, antibodies specific for Mo3 are capable of inhibiti~g the binding o~ uPA to uPA-R. Figure 1 ~hows the results of ~he competitive blocking of uPA-FITC tfluorescein isothiocyanate~
with a variety ~f antibodies speci~ic for Mo3. The results in Figure 1 clearly demonstrate that the antibodies specific for Mo3 (anti-Mo3f, ~ ~ ); anti-~o3e ~ ~ ); and polyclonal rabbit an~i-Mo3 ( - ~- ) are capable of blocking the binding of uPA-FITC
to uPA-R.
It is ~o be understood that while the exemplified antibodies of the invention are of murine origin, the pre~ent methods include the use of humanized version~ ~f these antibodies (N~ture, vol. 351, pp. 501V502 (1991~).
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments W093/098~ 2 1 2 3 8 7 4 PCT/~S92/~8977 which are given for i.llustration of the invention and are not intended to be limiting thereof.
EXAMPLES
MATERIALS AND METHODS
High molecular weight urokinase (uPA; 80/000 IU/mgr catalog #124) (Ho~pe Sev~er's Z. PhYsiol. chem., vol. 363, p. 133 (1982)) was purchased from ~merican Diagnostics, Inc. (Greenwich, CT).
FITC conjugated uPA ~uPA-FIT~; 25 ~g/ml) was a generous gift of American Diagnostics, Inc.
Acid Washed,_PMA-Stimulated U-937 Cells A~ter culture for 2 days in medium containing 10 nM PMA as pre~iously described (J. Immunol., vol. 144, p. 1841 (1990)), U-937 cells were washed twice in PBS, then resuspended in glycine-NaCl buffer (50 mM glycine, 100 mM NaCl, pH 3.0~ and incubated at 4C for 3 min with gentle agitation. A 4~% volume of quench solution (500 mm ~EPES, 100 m~ NaCi, 1 mg/ml BSA, pH
7.5) was then added. The cells were pelleted and resuspended in wash buffer (PBS supplemented with 1 ~gtml glucose and 1 mg/ml human Ig ~J. _Immunol.~ vol. 137, P. ~48 ~ 6~) at a concentration of 1 X 107 cells/ml.
om~etitive~ glL~rgLs~ ~gPA-FITC bY Monoc Anti-Mo3 ~ntibodies x 105 acid-washed PMA stimulated U-937 cells were preincubated for 60 min at 4C in 150 ~l wash bu~`fer (same as above) containing various dilutions of murine monoclonal anti~o3 mabs (ascites; or isotype-identical control reagents) or with W093/~9808 P~T/US9~/0~977 2123~7~
varying dilutions of rabbit polyclonal anti~Mo3 antiserum (or normal rabbit serum). 50 ~1 o~ uPA-FITC in wash buffer was then added (125 mg per 5 x 105 cells) and the mixture was incubated for an additional 60 min at 4C. The cells were then centrifuged at 1000 x g for 5 min at 4C, washed with 100 ~1 wash buffer, and fixed in 0.5 ml PBS containing 1% formaldehyde.
Immunofluorescence Flow Cytomet~ic Analysis Binding of uPA-FITC to khe surface of U-937 cells was quantitated by flow cytometric analysis on a Coulter ELITE flow ~cytometer (Coulter Electronics, Inc., Hial~ah, FL). The mean channel fluorescence ~linear scale) of 5000 cells/determination was calculated and used as a quantitative measure o~ relative uPA FITC binding. The results are shown graphicall~ in Figure 1.
Obviously, numerous modifications and variations of the present invention are possible in ligh~ of the above teachings.
It is therefore to be understood that, within the scope of the appended claims, the invention may be pr~cticed otherwise khan as specifically described herein.
Claims (10)
1. In a method for detecting uPA-R, comprising contacting a sample which may contain uPA-R, with an antibody or a labelled antibody and determining the amount of antibody or labelled antibody bound to uPA-R, the improvement comprising said antibody or labelled antibody being specific for Mo3.
2. The method of Claim 1, wherein said antibody is anti-Mo3f.
3. In a method for isolating uPA-R, comprising immunoprecipitating uPA-R with an antibody, the improvement comprising said antibody being specific for Mo3.
4. The method of Claim 3, wherein said antibody is anti-Mo3f.
5. A method for inhibiting the binding of uPA to uPA-R, comprising contacting uPA-R with an antibody specific for MO3.
6. The method of Claim 5, wherein said antibody is anti-Mo3f.
7 . A method f or inhibiting tissue destruction by inflammatory microphages, comprising administering an effective amount of an antibody specific for Mo3 to a patient in need thereof.
8. The method of Claim 7, wherein said antibody is anti-Mo3f.
9. A method for inhibiting the metastatic invasion of tumor cells, comprising administering an effective amount of an antibody specific for Mo3 to a patient in need thereof.
10. The method of Claim 9, wherein said antibody is anti-Mo3f.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79320891A | 1991-11-18 | 1991-11-18 | |
| US793,208 | 1991-11-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2123874A1 true CA2123874A1 (en) | 1993-05-27 |
Family
ID=25159386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002123874A Abandoned CA2123874A1 (en) | 1991-11-18 | 1992-10-27 | Methods for detecting and isolating upa-r and inhibiting the binding of upa to upa-r |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0642354A4 (en) |
| JP (1) | JPH07501524A (en) |
| CA (1) | CA2123874A1 (en) |
| WO (1) | WO1993009808A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE182336T1 (en) * | 1993-05-28 | 1999-08-15 | Chiron Corp | PEPTIDE INHIBITORS OF UROKINASE RECEPTOR ACTIVITY |
| JP2852192B2 (en) * | 1994-07-08 | 1999-01-27 | カンセアフォースクニングスフォンデン・アフ・１９８９（フォンデン・チル・フレメ・アフ・エクスペリメンテル・カンセアフォースクニング） | uPAR binding site of domain 2 + 3 of uPAR and antibody |
| US6077508A (en) * | 1998-03-23 | 2000-06-20 | American Diagnostica Inc. | Urokinase plasminogen activator receptor as a target for diagnosis of metastases |
| EP0982036A1 (en) * | 1998-08-28 | 2000-03-01 | Wilex Biotechnology GmbH | Modulation of Beta2 -integrin-mediated cell adhesion |
| TW200813091A (en) * | 2006-04-10 | 2008-03-16 | Amgen Fremont Inc | Targeted binding agents directed to uPAR and uses thereof |
| US8105602B2 (en) | 2006-12-08 | 2012-01-31 | Tactic Pharma, Llc | Urokinase-type plasminogen activator receptor epitope, monoclonal antibodies derived therefrom and methods of use thereof |
-
1992
- 1992-10-27 JP JP5509252A patent/JPH07501524A/en active Pending
- 1992-10-27 EP EP92922134A patent/EP0642354A4/en not_active Withdrawn
- 1992-10-27 CA CA002123874A patent/CA2123874A1/en not_active Abandoned
- 1992-10-27 WO PCT/US1992/008977 patent/WO1993009808A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP0642354A4 (en) | 1995-07-12 |
| EP0642354A1 (en) | 1995-03-15 |
| WO1993009808A1 (en) | 1993-05-27 |
| JPH07501524A (en) | 1995-02-16 |
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