Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies

Journal of hnmunological Methods, 130 (1990) 81-90 Elsevier

81

JIM 05587

Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies Ute Zacharias, Wilhelm Handschack, Frank Schneider, Klemens L~Sster, Christel Kleitke, Franz Noll and Horst Will Central Institute of Molecular Biology, Academy of Sciences of the GDR, Berlin-Buch 1115, G.D.R.

(Received 30 August 1989, revised received 14 November 1989, accepted 12 February 1990)

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass=2.1 × 107 M-I). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-pnitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass= 1.0× 10 7 M - l ) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function. Key words: Urokinase-type plasminogen activator; Urokinase receptor; Monoclonal antibody

Introduction

Urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) convert the zymogen plasminogen to active plasmin, a protease of broad substrate specificity. Plasminogen activators and plasmin are involved in diverse physiological and pathological processes including

Correspondence to: U. Zacharias, Central Institute of Molecular Biology, Roben-RSssle-Strasse 10, Berlin-Buch 1115, G.D.R. Abbreviations: u-PA, urokinase-type plasminogen activator; t-PA, tissue-type plasminogen activator; mAB, monoclonal antibody; HRP, horseradish peroxidase; ATF, aminoterminal fragment; pNA, p-nitroanilide; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

fibrinolysis, ovulation, trophoblast invasion, nervous system morphogenesis, inflammation, tumor invasion and metastasis (Will, 1988). Plasminogen activators are also increasingly being successfully used for thrombolytic therapy (Collen et al., 1988). Although great advances have been made in elucidating the structure and function of human plasminogen activators, many details require clarification. Of the particular interest is the specificity exhibited by plasminogen activators in their interactions with the substrate, plasminogen, and with other potential protein substrates (Sullivan et al., 1986), with the fibrin polymer (Higgins et al., 1987), with protein inhibitors (Gurewich et al., 1987) and with high affinity receptors on cell membranes (Vassalli et al., 1985). Urokinase-type PA and t-PA differ from one another and from

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

82 other serine proteases in their macromolecular specificities. Dissimilarities in the functional behaviour of u-PA and t-PA are likely to be determined by peculiarities of their interactions with macromolecular substrates and effector substances as well as with their receptors. Monoclonal antibodies are valuable probes for characterizing the recognition and reactivity sites of proteins. Although a number of monoclonal antibodies against u-PA have been generated (Herion et al., 1983; Wojta et al., 1989), only a small number have been characterized in detail. The present paper describes two monoclonal antibodies against human u-PA and their interaction with the enzyme. One of the antibodies inhibits plasminogen activation by u-PA, while the other prevents u-PA binding to cell membrane receptors. Neither antibody reacts with t-PA.

Materials and methods

Materials Glu-plasminogen was purified from human plasma by chromatography on lysine-Sepharose and on aprotinin-Sepharose. Fibrinogen I-O was purchased from the Bezirksinstitut fiir Blutspendewesen Gera, G.D.R. Urokinase was obtained from Serono. The preparation consisted almost exclusively of high M r u-PA (Mr55,000) as judged by SDS-PAGE. Urokinase was labelled with N-succinimidyl-3(4-hydroxy, 5-125I-iodo phenyl) propionate (Bolton-Hunter reagent) from Amersham International. The specific activity of 125I-u-PA was 10 #Ci//~g u-PA. Gelafusal, a plasma substitute, was purchased from VEB Serum-Werk Bernburg, G.D.R. Visotrast is a product of VEB Fahlberg List Magdeburg, G.D.R. The synthetic substrates D-Val-Leu-Lys-pNA and DVal-Leu-Arg-pNA were obtained from Serva. Ficoll, dextran and CNBr-activated Sepharose were from Pharmacia, hydroxylapatite from BioRad, Freund's adjuvant from Difco and 0.45 /xm pore size nitrocellulose paper from Sartorius. Preparation of polyclonal and monoclonal antibodies against u-PA Polyclonal antibodies were raised in rabbits by subcutaneous immunization with 100 ~tg u-PA in

complete Freund's adjuvant followed by another 100/zg u-PA 4 weeks later. The IgG fraction was isolated from rabbit antiserum by precipitation with ammonium sulfate and was further purified by immune affinity chromatography on u-PA-Sepharose. Monoclonal antibodies were prepared as described by KiShler and Milstein (1975). Briefly, BALB/c mice were immunized twice intraperitoneally with 50 ~g u-PA in Freund's adjuvant. 4 days after a final boost with 50 ~g u-PA intravenously, spleen lymphocytes were collected and fused with SP2/0 myeloma cells. Supernatants of hybridomas grown in selective hypoxanthine-azaserine-thymidine medium were screened for specific antibody production with an immunodot-ELISA. Antibody binding to u-PA adsorbed on nitrocellulose was detected with HRP-conjugated sheep anti-mouse IgG and 2bromo-l-naphthol as chromogen. Positive hybridomas were cloned by limiting dilution. The class specificity of monoclonal antibodies was determined by an immunodot-ELISA using rabbit antisera to murine immunoglobulin subtypes from ICN Biomedicals. Milligram amounts of antibodies were produced by injection of hybridoma cells into pristane-primed BALB/c mice. Immunoglobulins were purified from ascitic fluid by chromatography on hydroxylapatite and conjugated with HRP by the periodate-oxidation method (Nakane et al., 1974).

Immunoenzymometric assay Polystyrene microtiter plates were incubated overnight at 37°C with 100 /xl/well of coating solution containing 0.015 M Na2CO 3, 0.035 M NaHCO 3, pH 9.6, and 2-5 ~g/ml of the first monoclonal anti-u-PA IgG. The following incubations were performed at room temperature with gentle shaking. Non-specific binding sites were blocked by a 1 h incubation with 200 /~l/well of 0.05 M Tris-HCl, 0.2 M NaC1, 0.1% Tween 20, 10% Gelafusal, pH 7.4 (TBS, Tw, Gelafusal). The plates were emptied and a 100 ~1 aliquot of a dilution of u-PA standard was added per well. The plates were incubated for 2.5 h and then washed with TBS. Following the addition of 50/xl/well of the second HRP-conjugated monoclonal antibody diluted 1/500 in TBS, Tw, Gelafusal the plate was

83

incubated for a further period of 1 h. and then washed as before. H R P reactions were started by adding 200 #l/well of a solution containing 0.1 M citrate, 0.2 M sodium phosphate, pH 5.0, 0.4 m g / m l o-phenylenediamine and 0.015% H202. Reactions were stopped after 15 min with 50 #1 2.5 N H2SO 4 and the absorbance was read at 492 nm in a plate reader.

Determination of 'Antibody association constants' Urokinase, ranging in concentration from 0.04 to 0.5/~M, was incubated overnight at 4 ° C in 40 /~1 50 mM sodium phosphate, 80 mM NaCI, 0.4% BSA, 0.01% Tween 80, pH 7.4, in the absence or presence of either 0.067/~M antibody H6 or 0.043 ~tM antibody E2. After 16-18 h, 5 ~1 of mouse serum, diluted 1 / 2 0 with PBS, 5/~1 of 1.2 m g / m l anti-mouse IgG-antibody and 10 /~1 of 18% PEG 6000 were added. Incubation was continued for 1 h at 37 o C. The mixtures were then centrifuged for 10 min at 10,000 × g using a bench centrifuge. Supernatants were diluted appropriately and assayed spectrophotometrically for u-PA activity. Quadruplicate samples of 6 - 8 u-PA concentrations were run in parallel in order to obtain a full binding isotherm. Experimental data were analysed in Scatchard plots and binding parameters were calculated by least square fit linear regression.

Immunoblotting of proteolytic fragments of u-PA Autolysis of u-PA and the separation of the aminoterminal fragment (ATF, M r 17,000) were carried out as described by Stoppelli et al. (1985). Urokinase fragments and purified ATF were subjected to SDS-PAGE (Laemmli, 1970) under reducing (150 mM dithiothreitol) and non-reducing conditions. Following electrophoresis proteins were transferred to a nitrocellulose membrane according to Towbin et al. (1979). For the immunodetection of u-PA fragments nonspecific binding sites on the nitrocellulose membrane were first saturated by incubation of the membrane with TBS, Tw, Gelafusal for 30 min. Membrane strips were then incubated for 1 h in the same buffer containing 10 /~g/ml polyclonal anti-u-PA IgG. This was followed by 1 h incubation with HRP-conjugated sheep anti-rabbit IgG. Alternatively, membrane strips were incubated with

HRP-labelled monoclonal antibodies. Peroxidase activity was visualized with 2-bromo-l-naphthol and H202.

Influence of antibodies on plasminogen activation and on amidolytic activity of u-PA Plasminogen activation by u-PA was analysed with a fibrin plate assay (Jespersen et al., 1983) and with a spectrophotometric assay (Verheijen et al., 1985). In the first method, areas of lysis produced by u-PA in fibrin-agarose-layers composed of 1% agarose, 0.4 t t g / m l plasminogen and fibrin formed from 1 m g / m l fibrinogen by 0.75 NIH U / m l thrombin were measured. The layers were buffered with 50 mM Tris-HCl, 140 mM NaCI, 12.5 mM CaCI 2, 6.25 mM MgCI 2, pH 7.4. In the spectrophotometric method, plasmin generated from plasminogen by u-PA was determined with a chromogenic substrate. Various concentrations of u-PA were incubated with 0.2 /~M plasminogen, 1 mM D-Val-Leu-Lys-pNA, 50 mM sodium phosphate, 80 mM NaC1, 0.2% BSA, 0.01% Tween 80, pH 7.4, in a total volume of 100 #1. The mixtures were incubated in 96-well microtiter plates at 37 o C and activities calculated from plots of absorbance change at 405 nm vs. (time) 2. In order to determine the effects of antibodies on plasminogen activation u-PA samples were preincubated for 1 h at 3 7 ° C in the absence and presence of antibodies, respectively. The amidolytic activity of u-PA, preincubated with or without antibodies, was measured spectrophotometrically with the substrate D-VaI-Leu-ArgpNA. Evaluation of the kinetic data on plasminogen activation as measured in the spectrophotometric assay was done according to Drapier et al. (1979). For analysis of the inhibition of u-PA activity by mAB E2 it was assumed that the two antigen binding sites on the antibody acted independently and that the total concentration of u-PA binding s i t e s , [B]total, was twice the concentration of antibody. Binding of u-PA to MAB E2 was described by the equations: u-PA+ B~ u-PA.B [u-PA" B] Kass= [u_PA]r,ee. [B]rr~ [u-PA]k,ta, = [u-PA]tree " [u-PA-B] [B],o,~, = 2[E2],,,,~, = [B]t~, + [u-PA-B]

84 From these equations the following formula can be derived:

[u-PAIrr~vs. 2[E2l,o,a~-([u-PAlto,a,- [u-PA]f,~)

finally suspended in the same solution containing in addition 0.1% BSA. Binding of u-PA to 2 - 4 × 10 7 granulocytes/ml was studied in the presence o f 10 - 9 M 125I-u-PA and different concentrations of non-labelled u-PA. Urokinase was first incubated for 40 min at 3 7 ° C with or without antibodies in Hanks' solution supplemented with 0.1% BSA. 60 /zl of the preincubation mixture were then added to 180 ~1 granulocytes suspension, and incubation was continued at 3 7 ° C for 30 min. Thereafter 100 ~tl aliquots of the suspension were centrifuged through a 0.8 ml layer of 20% sucrose in polypropylene snap-cap tubes. 1251u-PA radioactivity associated with the cell pellets was counted in a G a m m a counter.

yields a straight line with coordination axis intercepts equal to

Results

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• {2[E21 - ([u-PAhot.,- [u-PA].~) } where 2[E2ltot.j-([u-PAltot~j- [u-PAI.~) = [B].~ At constant concentrations of u-PAtotal and variable concentrations of [E2] total, a plot of 1

[u-PAl,oral and 1 Kas$

respectively. If this type of analysis is extended to several concentrations of [u-PA]total, a number of straight lines focussing on the abscissa point 1 ga~

is obtained.

Isolation of human granulocytes and effects of antibodies on the binding of u-PA to granulocytes The first step in the isolation of granulocytes consisted of mixing 8 ml samples of heparinized blood with 2 ml 5% dextran T 500 in PBS. Red blood cells were allowed to settle for 45 rain at 37 ° C and the top layers subsequently centrifuged for 30 min at 700 x g on 6.2% Ficoll-Visostrast cushions (d = 1.076 g/ml). Cell pellets containing granulocytes were suspended in Hanks' balanced salt solution and were centrifuged for 10 rain at 400 × g. After brief hypotonic lysis of residual red blood cells, granulocytes were washed three times with Hanks' balanced salt solution. They were

Production and characterization of anti-u-PA monoclonal antibodies A single fusion of S P 2 / 0 myeloma cells with the spleen cells of an immunized mouse resulted in seven hybridomas producing antibodies against u-PA. From the antibody secreting hybridomas two stable clones were selected. The clones were designated E2 and H6 and the secreted antibodies mAB E2 and mAB H6, correspondingly. Both antibodies belong to the IgG1 subclass. Neither mAB E2 nor mAB H6 react with t-PA in an immunodot-ELISA. A two-site binding assay was used to decide whether mAB E2 and mAB H6 recognize separate or overlapping epitopes. In the assay, one of the antibodies was adsorbed to the wells of a microtiter plate. After incubation with u-PA the second antibody, labelled with HRP, was allowed to interact with bound u-PA. Fig. 1 demonstrates colour development by the immunobound H R P in relation to the u-PA concentration when mAB H6 is used as the capture antibody and mAB E2 as the indicator antibody. Similar results were obtained when the order of the antibodies in the assay was reversed (data not shown). The experiments demonstrate that mAB H6 and mAB E2 were able to bind simultaneously to the u-PA molecule and hence recognized separate epitopes. The sandwich assay permitted the quantification

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[u-PAl, nglrn[ Fig. I. Immunoenzymometric assay for u-PA. Monoclonal antibody H6 was adsorbed to polystyrene plates. Binding of u-PA to fixed mAB H6 was detected with m A B E2 conjugated to HRP. Values represent the means of triplicate measurements. For further details see the materials and methods section.

of u-PA antigen in a buffer system down to a detection limit of 1 n g / m l .

Determination of antibody association constants The affinity of the monoclonal antibodies towards the two-chain u-PA ( M r 55,000) was determined at constant concentrations of either mAB H6 or mAB E2 and differing concentrations of

enzyme. The method used for the determination of binding affinities permitted the estimation of interaction parameters between non-modified u-PA and non-modified antibodies. Free u-PA was determined by enzyme activity measurements after precipitation of u-PA-antibody complexes. From Scatchard plots (Fig. 2), association constants of Kass = 1.0 × 107 M - l and Kass = 2.1 × 1 0 7 M - I were calculated for complexes of u-PA with mAB H6 and mAB E2, respectively. The maximal amount of u-PA bound to either mAB H6 or mAB E2 was found to be about twice the concentration of antibody. This would be predicted from the fact that IgG molecules contain two identical antigen binding sites.

Reactivity of monoclonal antibodies towards proteolytic fragments of u-PA When two-chain u-PA (Mr 55,000) and u-PA fragments are separated by S D S - P A G E and blotted onto a nitrocellulose membrane, they are readily recognized by polyclonal anti-u-PA antibodies (Fig. 3). Analysis of the reactivity of mAB E2 towards u-PA fragments revealed strong binding of this antibody to low M r u-PA ( M r 33,000) and no binding to the aminoterminal fragment of u-PA ( M r 17,000). In contrast, mAB H6 bound to the aminoterminal fragment but did not interact with low M r u-PA (Fig. 3).

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86

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partial inhibition. When plasminogen activation was estimated in a spectrophotometric assay, the inhibition by mAB E2 was confirmed, but there was no inhibition - not even partial - by mAB H6 (Fig. 5A). It is suggested that the partial inhibition of plasminogen activation by mAB H6 observed in the fibrin plate assay was not true inhibition of u-PA activity, but rather the effect of retarded diffusion of the u-PA-mAB H6 complex as compared to free u-PA in the fibrin-agarose gel. The M r of the u-PA-antibody complex is almost four-fold higher than the M r of u-PA alone. The diffusion coefficient of the complex should therefore be considerably smaller than that of the free enzyme. Kinetic data for the inhibition of u-PA by mAB E2 measured spectrophotometrically were evaluated according to a simple kinetic scheme. The scheme supposes that binding of u-PA to mAB E2 completely inhibits u-PA activity and that there is no interaction between two identical antigen binding sites on each antibody molecule. In a plot relating

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[u-PA]free Fig. 3. Immunoblotting of electrophoretically separated u-PA and u-PA fragments. Partially autolysed u-PA and purified aminoterminal fragment (ATF) were subjected to SDS-PAGE and blotting onto nitrocellulose membrane, u-PA-derived polypeptides were detected with mAB H6-HRP-conjugate (lane 1), mAB E2-HRP-conjugate(lane 2), and rabbit polyclonal anti-uPA antibody in combination with HRP labelled anti-rabbit IgG (lane 3).

Reduction of the disulfide bonds in u-PA before electrophoresis leads to separation of the u-PA heavy chain ( M r 32,000) and the u-PA light chain ( M r 20,000). The former was shown to interact with mAB E2 and the latter with mAB H6. It is noteworthy that mAB H6 also recognized the reduced form of the aminoterminal fragment (data not shown).

Influence of antibodies on u-PA activity Fig. 4 demonstrates that polyclonal antibodies and mAB E2 were able to strongly inhibit plasminogen activation in the fibrin plate assay. Under the same conditions, mAB H6 resulted in only

to the concentration of free u-PA binding sites on mAB E2, variations in the concentration of mAB E2 yielded a straight line for each of three different concentrations of [u-PA]total (Fig. 5B). From the intercepts of the lines with the abscissa an association constant of Ka.~.~ = 3.3 × 10 7 M - 1 was derived for the u-PA-mAB E2 complex. This value is close to the value of Ka.,~ = 2.1 × 10 7 M - 1 determined in the binding experiments. Neither mAB H6 nor mAB E2 significantly inhibited the amidolytic activity of u-PA. As shown in Fig. 6, cleavage of a low molecular weight substrate was little affected by preincubation of the u-PA even with a 100-fold molar excess of antibody.

Effect of antibodies on binding of u-PA to granulocytes An important functional site in u-PA - apart from the active centre - is the site involved in u-PA receptor binding. To study the effect of antibodies on the interaction of u-PA with u-PA receptors on cell membranes, intact human granuIocytes from peripheral blood were used. Fig. 7

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Fig. 4. Effects of polyclonal (A) and monoclonal antibodies (B) on u-PA-catalyzed activation of plasminogen in a fibrin plate assay. Aliquots containing 8 pg u - P A / m l were incubated for 1 h at 37 ° C with different concentrations of polyclonal antibody (o), mAB H6 (O) and mAB E2 (o). 5 pl samples were then applied to holes punched into the fibrin plate. Lysis zones were measured after 18 h incubation at 37 o C.

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-0.05 0 0.05 01 015 02 [2 E2]total- ([u - PA ],o,(~L - [u - PA ]f~ee),,UM Fig. 5. Inhibition of u-PA-catalysed plasminogen activation by mAB E2. A: various concentrations of mAB E2 ( o ) and of mAB H6 (O) were incubated for 16 h at 4 ° C with 0.041 #M u-PA. Plasminogen activating activity of the mixtures was estimated spectrophotometrically with the plasmin substrate D-Val-Leu-Lys-pNA. The abscissa values represent the concentration of active u-PA determined from calibration curves established in the absence of antibody. B: dependence of 1 [u-PA]free

on the concentration of free u-PA binding sites on mAB E2 at [u-PA]to~at equal to 0.018 #M (o), 0.041/~M ((l)), and 0.082/~M (O), respectively.

88

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Fig. 6. Amidolytic activity of u-PA preincubated with monoclonal antibodies. Aliquots of 0.5 ~tg/ml u-PA were incubated for 1 h at 37°C without (.) and with either 150/~g/ml mAB H6 (Q) or 150 /tg/ml mAB E2 (O). Amidolytic activity of u-PA was determined spectrophotometrically using D-Val-LeuArg-pNA as substrate.

illustrates high affinity binding of u-PA to isolated granulocytes and the effect of monoclonal antibodies on u-PA binding. It is evident from Fig. 7A that granulocytes exhibit a single type of high affinity u-PA binding site. Interaction of u-PA

with these sites is characterized by an association constant of Kass = 6.7 x 108 M - t. A similar value has been reported recently for the same system by Miles and Plow (1987). Preincubation of u-PA with mAB E2 did not affect high affinity u-PA binding (Fig. 7B). Similarly there was no effect when anti-t-PA antibodies were used (data not shown). However, preincubation of u-PA with mAB H6 strongly reduced u-PA binding to granulocytes. The amount of tracer 12SI-u-PA bound to granulocytes in the presence of mAB H6 was less than the amount of ~2SI-u-PA bound in the presence of a 500-fold molar excess of nonlabelled u-PA. This indicates that binding of u-PA to mAB H6 prevents u-PA-binding to high affinity sites.

Discussion

The present paper describes two monoclonal anti-u-PA antibodies which recognize distinct epitopes on the u-PA molecule. Both antibodies are

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Fig. 7. Binding of u-PA to granulocytes (A) and effect of monoclonal antibodies on u-PA binding (B). A: Scatchard plot of u-PA binding to granulocytes. Granulocytes at a concentration of 3.2 x 107 cells/ml were incubated with 0.9 nM 12SI-u-PA and increasing concentrations of non-labelled u-PA. The figure represents specific binding calculated by subtracting non-specific binding measured in the presence of 0.5/AM non-labelled u-PA from total binding. B: dependence of 12~l-u-PA-radioactivity bound to granulocytes on the concentration of mAB E2 and mAB H6. The final incubation mixtures contained 2."/x 107 granulocytes/ml and 1 nM 12Sl-u-PA. The figure shows the total radioactivity associated with the granulocytes. Non-specifically bound radioactivity determined in the presence of 0.5 pM non-labelled u-PA amounted to 525 cpm.

89 potentially useful for the characterization of u-PA structure and function. One of the antibodies, mAB E2, binds to the heavy chain of u-PA which comprises the protease moiety of this plasminogen activator. Such antibody binding clearly inhibits plasminogen activation. A kinetic analysis of the inhibitory effect demonstrates that u-PA-mAB E2 complexes are completely devoid of plasminogen activating activity. At variance with plasminogen activation, binding of mAB E2 does not affect the amidolytic activity of u-PA. The latter result indicates that the antibody does not directly interact with amino acid residues involved in the catalytic steps. It appears rather that mAB E2 interferes with binding and proper positioning for peptide cleavage of the macromolecular substrate plasminogen. As pointed out by Liebmann (1986), the specificity of regulatory proteases for their respective macromolecular substrates is determined primarily by interactions of the substrates with regions of the enzyme other than the active sites. Of particular importance are loop structures located in the vicinity of the active site and surrounding it. In binding to such structures mAB E2 may prevent binding of plasminogen, leaving at the same time the active site accessible to low M r peptide substrates. While the precise identification of the epitope needs further elucidation, the present characterization should enable mAB E2 to be used as a specific inhibitor of u-PA-catalyzed plasminogen activation in analytical and other experimental studies. The second antibody, mAB H6, was shown to bind to the light chain of u-PA and, more precisely, to the aminoterminal fragment of the enzyme. This fragment consists of 135 amino acid residues (Stoppelli et al., 1985) and contains a growth factor homologous domain and a kringle domain. The growth factor domain is involved in binding of u-PA to high affinity receptors on cell membranes. Peptide inhibition studies revealed that binding is determined primarily by the second loop of the growth factor region comprising u-PA amino acid positions 20-30 (Appella et al., 1987). Monoclonal antibody H6 prevents interaction of u-PA with high affinity u-PA receptors on cell membranes, as demonstrated here for u-PA receptors on human granulocytes. The antibody does not affect the hydrolytic activity of u-PA.

An anti-u-PA monoclonal antibody interacting with the aminoterminal fragment has been described previously by Nolli et al. (1986). This antibody resembled mAB H6 in that it interfered with u-PA receptor binding. However, unlike mAB H6, the antibody had no appreciable binding affinity for the aminoterminal fragment when the disulfide bridges cross-linking the fragment were reduced. Monoclonal antibody H6 reacted with both the non-reduced and the reduced fragments. The ability of mAB H6 to prevent u-PA receptor binding is of potential value when investigating the role of this process in cellular systems. In addition to granulocytes, u-PA receptors are also present on monocytes, fibroblasts and diverse tumor cells (Blasi et al. 1987). Urokinase bound to high affinity membrane receptors remains enzymatically active. It is not readily degraded or endocytosed. It has been postulated therefore that binding of u-PA to cell membranes creates high local proteolytic activity which may be important in cell-cell and cell-matrix interactions and in facilitating cell migration and cell invasion. Using mAB H6 it should be possible to distinguish the functional significance of cell membrane localized u-PA activity as opposed to u-PA activity per se. The association constants of the complexes of u-PA with mAB E2 and mAB H6 are 2.1 X 1 0 7 M -1 and 1.0 x 107 M -1, respectively. The antibodies are thus appropiate reagents for immunohistochemical detection and immunometric quantitation of u-PA antigen.

References Appella, E., Robinson, E.A., Ullrich, S.J., Stoppelli, M.P., Corti, A., Cassani, G. and Blasi, F. (1987) The receptorbinding sequence of urokinase. J. Biol. Chem. 262, 4437. Blasi, F., Vassalli, J.D. Dano, K. (1987) Urokinase-typeplasminogen activator: proenzyme, receptor and inhibitors. J. Cell. Biol. 104, 801. Collen, D., Stump, D.C. and Gold, H.K. (1988) Thrombolytic therapy. Ann. Rev. Med. 39, 405. Drapier, J.C., Tenu, J.P., Lemaire, G. and Petit, J.F. (1979) Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. Role of serum studied by a new spectrophotometric assay for plasminogen activators. Biochimie 61,463. Gurewich, V. and Pannell, R. (1987) The zymogenic and

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