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Suppression of plasminogen activator inhibitor 1 (PAI-1) activity levels in rats by monoclonal antibodies
Fibrinolysis & Proteolysis (1998) 12 (6), 335-339 © HarcourtBrace & Co. Ltd 1998
Suppression of plasminogen activator inhibitor 1 ( P A l - l ) activity levels in rats by monoclonal antibodies T.-H. Ngo, P. J. Declerck Laboratory for Pharmaceutical Biology and Phytopharmacolegy Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
Summary Background: Elevated levels of plasminogen activator inhibitor-1 (PAl-l) are associated with several important thrombotic diseases. Rat models have been shown to be suitable for in vivo investigations on thrombolysis and fibrinolysis. In the present study, monoclonal antibodies (MA) against rat PAl-1 are used to reduce PAl-1 levels in endotoxin-treated rat plasma. Results: A large panel of murine monoclonal antibodies was raised against recombinant rat PAl-1. Out of 237 monoclonal antibodies, six antibodies showed a strong inhibition towards recombinant rat PAl-1 activity in vitro resulting in 70-100% inhibition of PAl-1 activity at a 16-fold molar excess of MA over PAI-I. Relative epitope analysis revealed that this panel represented at least two different epitopes. Subsequent selection of two monoclonal antibodies (MA-124K1 and MA-159M12) and evaluation of their PAl-1 neutralizing properties in vitro in rat plasma, supplemented with recombinant rat PAl-1 and in plasma from endotoxin-treated rats, revealed that MA-124K1 was capable of inhibiting native rat PAl-1 in a plasma environment. Based on these data, MA-124K1 was selected for evaluation in in vivo experiments. Rats were pre-treated with endotoxin (0.5 mg/kg, ip, 90 min prior to MA administration) to obtain high levels of native rat PAl-1 in vivo. Levels of PAl-1 antigen and activity in plasma were measured by specific ELISAs. Injection of a single dose of MA-124K1 (3 mg/kg body weight) resulted in a ~60% inhibition of PAl-1 activity without affecting PAl-1 antigen levels. No changes were observed upon administration of MA32K3, a control monoclonal antibody. Conclusion: MA-124K1 may serve as a useful tool in studies on the evaluation of the role of PAl-1 in cardiovascular disease using rat models.
INTRODUCTION
In recent years, plasminogen activator inhibitor-1 (PAI-1) has gained a lot of attention as a unique member of the superfamily of serine proteinase inhibitors (serpin). Elevated levels of plasminogen activator inhibitor 1 (PAI-1) are associated with several important thrombotic diseases. 1,2Agents aimed at the inhibition of PAI-1 activity could offer a useful strategy for the therapy or prevention of thrombotic events. Enhancement of fibrinolysis, as well as antithrombotic effects both in vivo and in vitro Received: 5 June 1998 Accepted after resubmission: 29 September 1998 Correspondence to: Paul J. Declerck, Laboratory for Pharmaceutica) Biology and Phytopharmacolegy, Van Evenstraat 4, B-3000 Leuven, Belgium. Tel.: 32 16 323431 ; Fax: 32 16 323460; E-mail: paut,[email protected]; website: http://www.farm.kuleuven,ac.be/biotech
through the inhibition of PAI-1 activity or PAI-1 synthesis, has recently been shown in a variety of studies. 3-z Strategies to interfere with PAI-1 activity are based on the use of polyclonal antibody preparations, 3 monoclonal antibodies 4,5 or low molecular weight compounds? An attractive approach for the rational design of PM-1 neutralizing compounds is the generation and characterization of monodonal antibodies (Mr ~ 150 kDa) with PAI-1 neutralizing properties, the subsequent generation of singlechain Fv derivatives (Mr - 25 kDa) thereof,s possibly followed by a further minimization of this antibody fragment. Subsequent studies on the molecular mechanism of inactivation of PAI-I by cloned antibody fragments (e.g. epitope analysis) will allow the use of such an antibody fragment as a starting point for the rational design of PAI-1 irflaibitors (e.g. development of peptidomimetics). A large number of studies have shown that experimental rat models are suitable for in vivo investigations on 335
336
Ngo, Declerck
thrombolysis and fibrinolysis?-13 Even though in such rat models polyclonal antibody preparations have been used, 3,~4 these cannot be used in the application of the above-mentioned rational design. Therefore, in the present study, monoclonal antibodies, raised against recombinant rat PAI-1,15,~6are investigated with respect to their inhibitory properties towards PAI-1 activity in vitro and in vivo. To demonstrate that the observed effects in vivo, i.e. a decrease in PAI-1 activity, were indeed due to a specific interference with PAI-1 activity and not the consequence of, e.g. an accelerated clearance of PAI-1, we have evaluated both rat PAI-1 activity and rat PAI-1 antigen levels.
with different concentrations of monoclonal antibodies. Briefly, 100 gl recombinant rat PAI-1 (50 ng/ml in TBS) were incubated either with 100 gl buffer (TBS) or with 100 gl of a solution containing purified monoclonal antibodies at the indicated concentration. After incubation of this mixture for 25 min at 37°C, residual PAI-1 activity was quantitated with the PAI-1 activity assay as described above. The percentage inhibition (i.e. neutralization of PAI-1 activity) was then calculated based on the ratio of the PAI-1 activity in the presence versus the absence of antibodies.
MATERIALS AND METHODS
The levels of rat PAI-1 antigen and activity were measured using two ELISAs as described by Ngo et al. 16 In brief, 96-wells polystyrene microtiter plates (Costa, Cambrige, MA) were filled with 200~tl of 4gg/ml MA-51L2 (for activity assay) or MA-42S4 (for antigen assay) diluted in phosphate-buffered saline (0.04 M phosphate, 0.14 M NaC1, pH Z4; PBS) and incubated for 48 h at 4°C. The plates were emptied by shaking and the wells were treated for 2 h at room temperature with 200 ~tl PBS containing 10 g/1 bovine serum albumin. Then the wells were washed with PBS containing 0.002% Tween 80 (PBSTween), and finally with 200 gl of a solution containing 100g mannitol and 20g saccharose/L. The plates were stored at -20°C. Immediately before use, plates were washed once with PBS-Tween. Samples were diluted in PBS containing Tween 80 (0.002%), bovine serum albumin (1 g/l) (PTA) and EDTA (5 raM) (dilution buffer), and 180 gl samples were applied to the wells (in the activity assay, samples were incubated with an excess of t-PA for 25 rain at 37°C prior to analysis). After incubation for about 18h at 4°C in a moist chamber, the wells were emptied and washed with PBS-Tween. The plates were subsequently filled with 170 gl samples of HRP-conjugated monoclonal antibodies (MA-62E8-HRP for activity assay and MA-8M7-HRP for antigen assay) diluted 1:4000 in PTA, and incubated for 2 h at room temperature. After washing the plates, the peroxidase reaction was performed by addition of 160 gl aliquots of a 0.1 M citrate-0.2 M sodium phosphate buffer pH 5.0, containing 400 gg/ml o-phenylenediamine and 0.003% hydrogen peroxide to the wells. After 10-30 min at room temperature, the reaction was stopped with 50 gl of 4 M H2SO4. The absorbance was measured at 492 nm with a SLT Spectra (Gr6dig/ Salzburg, Austria). Calibration was performed with purified recombinant rat PAI-1 as described. 1~
Materials
Chromogenic substrate S-2403 was obtained from Kabi Vitrum (Stockholm, Sweden). Human t-PA (predominantly in the single-chain form) was a kind gift from Boehringer Ingelheim (Brussels, Belgium). Recombinant rat PAI-1 was produced as described. 15Endotoxin-treated rat plasma, obtained after injection (ip, 3 h) of rats with endotoxin (lipopolysaccharide from E.coli serotype 0127:B8, L-3129, Sigma, MO, USA) at 500 gg/kg weight was used as a source of native rat PAI-1 for in vitro experiments. Nembutal came from Sanofi, Brussels, Belgium. Wistar rats were purchased from the Animal Center of the Katholieke Universiteit Leuven. Monoclonal antibodies
Monoclonal antibodies against rat PAI-1 were produced essentially as described by Galfr6 and Milstein] 7 and the details were described previously26 Determination of overlapping epitopes was carried out using a two-site ELISA as decribed previously. ~s PAl-1 activity assay with purified proteins in buffer
PAI-1 activity was determined using the method as described by Verheijen et al.~9 In brief, PAI-1 containing samples (50 gl) in Tris-buffered saline (0.1 M NaC1, 0.05 M Tris/HC1, pH Z4, containing 0.01% Tween 80, TBS) were mixed with an equal volume of t-PA (20 U/ml in TBS) and incubated for 15 min at 37°C. Subsequently, 100gl of a solution containing plasminogen (1 gM), CNBr-digested fibrinogen (1 gM) and S-2403 (0.6 raM) were added. Residual t-PA activity was then quanfitated by measuring the absorbance at 405 nm. One unit of PAI-1 is defined as the amount of PAI-1 required to neutralize one international unit of t-PA. PAI-1 neutralization assays were based on quantitation of residual PAI-1 activity after preincubation of PAI-1 Fibrinolysis & Proteolysis (1998) 12(6), 335-339
PAl-1 antigen and activity assays in rat plasma
Animals and sample collection
Rats (Wistm, 300-400 g) were injected intraperitoneally with 500 gg/kg of endotoxin and anaesthetized by © Harcourt Brace & Co, Ltd 1998
Suppression of PAl-1 activity levels by monoclonal antibodies
intraperitoneal injection of 90 mg/kg Nembutal. A tracheal cannula was inserted and the rats were allowed to breath spontaneously. Blood samples were taken from the carotid artery using an Insyte-w catheter (0.7 x 19 mm, Becton Dickinson, Milano, Italy). A blood sample was taken (T = 0) 90 min after endotoxin injection, and monoclonal antibodies (3 mg/kg weight) were immediately administered as a bolus via the carotid cannula. Blood samples were then collected at the indicated time points into tubes containing the proper a m o u n t of anticoagulant (ACD Solution A, Becton Dickinson, NJ, USA) and immediately put on ice. Then, plasma was obtained by centrifugation at 2000 g for 25 rain at 4°C and stored at -20°C until use. Statistical analysis
The statistical significance of differences was evaluated using Student's t-test; p-values >0.05 were considered to be not significant. RESULTS AND DISCUSSION Selection of monoclonal antibodies
Out of 237 mufine monoclonal antibodies raised against rat PAI-1 and evaluated for their PAI-1 neutralizing properties in vitro, six showed pronounced inhibitory effects towards PAI-1 activity in a dose-dependent manner (Fig. 1). For three monoclonal antibodies (MA-124K1, MA147N10 and MA-154S8) a steep dose response curve was observed resulting in a ~ 50% inhibition at an equimolar concentration of MA and PAI-1 and a virtual m a x i m u m effect (- 80%) at a two to four fold molar excess of MA over PAI-1. For all six antibodies 70-100% PAI-1 inhibition was achieved at a 16-fold molar excess. Addition of the control antibody MA-32K3 (one of the antibodies raised against rat PAL1) did not affect the inhibitory activity of PAI-1. From the data obtained with a two-site ELISA (cfr methods), MA-124K1 and MA-159M12 were shown to represent two different epitopes and were, therefore, chosen for further experiments.
337
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fold molar excess of MA Fig. 1 Inhibitory effects of monoclonal antibodies towards recombinant rat PAl-1 activity in vitro: (II)MA-124K1; (A)MA147N10; (~)MA-154S8; (o)MA-167P3; (C])MA-177K4; (~{~)MA159M12; and (V)MA-32K3. The results represent mean values+SD (n = 3) and were obtained by the method of Verheijen et al. ~ (see Materials and Methods)
Since the antibodies were raised against and selected on recombinant rat PAI-1 in buffer, the inhibitory effects of the selected antibodies was further evaluated on recombinant rat PAI-1 in plasma and on native rat PAI-1 in plasma in vitro. From the data with recombinant rat PAI-1 (Table 1) it can be concluded that both antibodies exert their effects also in a plasma environment. Importantly, whereas MA-159M 12 has virtually no effect on native rat PAI-1, MA-124K1 also exerts its PAI-1 neutralizing properties on native rat PAI-1 (14+6.4% vs 42_+10% PAI-1 inhibition, see Table 1). After considering all data, MA-124K1 was selected for in vivo experiment in rats. In vivo experiments in endotoxin-treated rats
Because PAI-1 levels in rat plasma are very low, 1-2 ng/ml, ~6 endotoxin-treated rats were used to provide a high level of PAL1 in plasma. After endotoxin injection, b o t h PAI-1 antigen and activity levels in rat plasma i n c r e a s e y 3,1<2° thereby reaching a m a x i m u m
Table 1 Inhibitory effects of monoclonal antibodies towards recombinant and native rat PAl-1 activity in vitro in different conditions Recombinant rat PAl-l(*)
Native rat PAl-l(*)
Antibodies(**) MA
buffer
normal plasma undiluted
normal plasma diluted (1/5-1/40)
endotoxin plasma diluted(I/20-1/40)
MA-124K1 MA-159M 12
72+1.9 79+_2.6
83+_4.6 79_+2.5
81+3.5 76+2.5
42+10.0 14+6.4
The data (obtained by the immunofunctional assay,le see Materials and Methods) are expressed as percentage inhibition of PAl-1 activity and represent mean_+SD(n = 3). *at 5 ng/ml **at 5 btg/ml Endotexin-treated rat plasma was taken 3 h after endotoxin injection and contained about 200 ng/mU 6
© Harcourt Brace & Co. Ltd 1998
Fibrinolysis & Proteolysis (1998) 12(6), 335-339
338
Ngo, Declerck
800-
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600~
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p < 0.001, see Fig. 2 & Table 2). The extent of PAI-1 inhibition is comparable to that observed in vitro (Table 1), i.e. MA-124K1 can reduce the activity of native PAI-1 to ~ 50% of its original activity. Thus, this study demonstrates that with a single dose of 3 m g / k g of MA-124K1, an effect is observed at _>30 rain, lasting at least up to 3.5 h after administration. At this time point the antibody can still reduce PAI-1 activity levels from ~ 800 ng/ml to ~ 300 ng/ml, i.e. under these conditions up to 500 ng active PAI-1 per ml is completely neutralized. Even though this antibody exerts a highly potent effect on PAI-1 activity, 40% of the PAI-1 still remains active. The reason for this discrepancy is not clear. The possible presence of a heat-stable form (distinct from PAI-1) as reported previously ~4,2° can be excluded, since we have used a highly PAI-1 specific immunoassay. This is also further illustrated by the data involving recombinant rat PAI-1 in plasma (Table 2), where an inhibition of over 80% was observed. One could speculate that the lack of full inhibition of native PAI-1 might be related to the differences in glycosylation between recombinant and native PAI-1. In this respect, it is important to realize that the current experiments were carried out under extreme conditions, i.e. resulting in extremely high PAI-1 levels, and without optimizing the administration of the PAI-1 neutralizing compound, i.e. single dose i.v. administration of MA-124K1. This taken into account, and based on the properties of other PAI-1 neutralizing compounds, it is clear that the currently described monoclonal antibody MA-124K1 is a promising agent for in vivo interference with PAI-1 activity in rats. It may, therefore, also play an important role for the development of smaller, recombinant, antibody fragments (scFv, CDRs) to be evaluated further in vivo and to be used in the rational design of PAI-1 neutralizing drugs. In contrast to all previous approaches, 3-6,~4the current rat PAI-1 inhibitor was generated against, and selected on, rat PAI-1. Development of 'small' rat PAI-1 inhibitors using the antigen-binding region of MA-124K1 as a lead compound, should resuk in a pharmacologically-active compound that can be fully evaluated for its efficacy in a wide variety of rat models. Such a compound, its development and
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Fig. 2 Inhibitory effects of monoclonal antibodies on PAl-1 activity in vivo. (A)MA-124K1; (II)MA-32K3, closed symbol: PAl-1 antigen; open symbol: PAl-1 activity. The graph shows the data of a representative experiment (cfr methods, sample analysis by ELISA. 16Time indicates the time starting from the MA administration (i.e. 90 min after endotoxin injection)
concentration followed by a decrease. 21 In addition, the rate of increase of PAI-1 levels depends on the dose of endotoxin used, on the route of administration and on the particular rat itself. Therefore, to define unambiguously the inhibitory effect of monoclonal antibodies on PAI-1 activity in vivo, both PAI-1 antigen and activity levels need to be measured. Upon endotoxin treatment, levels of PAI-1 antigen and activity increased from 1 to 800 ng/ml (Fig. 2). In three rats injected with MA-32K3, PAI-1 activity levels continuously increased in parallel with PAI-1 antigen levels (Fig. 2) and are indicative of the release of fully active PAI- 1 as observed previously. ~6 Administration of MA-124K1 (n = 4) did not affect the endotoxin-induced increase in PAI-1 antigen levels. However, under those conditions PAI-1 activity levels were decreased significantly in comparison to MA-32K3 administration. Whereas upon MA-32K3 administration, the PAI-1 activity/antigen ratios remained nearly constant (Table 2), administration of MA-124K1 resulted in a decrease of the specific activity within 30 mins eventually resulting in a specific activity of 40_+11% after 3.5 h (compared to 94_+2% in the presence of MA-32K3,
Table 2 The ratio between PAl-1 activity and PAl-1 antigen levels after injection of antibodies Time (min)
MA-124K1 MA-32K3 p value (*)
0
0.71+0.21 0.73+_0.11 NS
30
0.56_+0.22 0.78+_0.10 NS
60
0.62_+0.26 0.79+_0.04 NS
90
0.59_+0.15 0.73+_0.22 NS
120
0.51+0.08 1.05+_0.24 <0.005
150
180
0.36+0.05 0.93_+0.23 <0.005
0.47+0.11 0.86_+0.05 <0.002
210
0.40+0.11 0.94+_0.02 <0.001
Mean+SD (n = 3-4) (*) MA-124K1 vs MA-32K3 NS: non significant T = 0: samples were taken immediately before administration of MA Fibrinolysis & Proteolysis (1998) 12(6), 335-339
© Harcourt Brace & Co. Ltd 1998
Suppression of PAl- 1 activity levels by monoclonal antibodies
p r o p e r t i e s , m a y c o n t r i b u t e d i r e c t l y or i n d i r e c t l y t o t h e d e s i g n a n d d e v e l o p m e n t o f PAI-1 n e u t r a l i z i n g c o m p o u n d s for t h e p r e v e n t i o n of t h r o m b o t i c e v e n t s i n humans.
ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e F u n d for scientific R e s e a r c h F.W.O. V l a a n d e r e n , p r o j e c t G . 0 2 6 6 . 9 7 a n d t h e N a g a i F o u n d a t i o n T o k y o (Japan). N.T.H. is a r e c i p i e n t of a K . U . L e u v e n D o c t o r a l Scholarship.
REFERENCES 1. Declerck PJ, Juhan-Vague I, Felez J, Wiman B. Pathophysiology of fibrinolysis. J Int Med 1994; 236: 425-432. 2. Van Meijer M, Pannekoek H. Structure of plasminogen activator inhibitor 1 (PAI-1) and its function in fibrinolysis: an update. Fibrinolysis 1995; 9: 263-276. 3. Abrahamsson T, Nerme V, Str6mqvist M, Akerblom B, Legnehed ,4,, Pettersson K, Eriksson WA. Anti-thrombotic effect of a PAI-1 inhibitor in rats given endotoxin. Thromb Haemostas 1996; 75: 118-126. 4. Levi M, Biemond BJ, van Zonneveld AJ, ten Cate JW0, Pannekoek H. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation 1992; 85:305-312. 5. Biemond BJ, Levi M, Coronel R, Janse MJ, ten Cate JW, Pannekoek H. Thrombolysis and reocclusion in experimental jugular vein and coronary artery thrombosis. Effects of a plasminogen activator inhibitor type 1 neutralizing monocloual antibody. Circulation 1995; 91 : 1175-1181. 6. Charlton PA, Faint RW, Bent F et al. Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. Thromb Haemostas 1996; 75: 808-815. 7 Pawlowska Z, Pluskota E, Chabielska E et al. Phosphorothioate oligodeoxyribonucleotides antisense to PAI-1 mRNA increase fibrinolysis and modify experimental thrombosis in rats. ~21romb Haemostas 1998; 79: 348-353. 8. Debrock S, Sironi L, Declerck PJ. Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate. Gene 1997; 189: 83-88.
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9. Emeis JJ. Perfused rat hindlegs. A model to study plasminogen activator release. Thromb Res 1983; 30: 195-203. 10. Emeis JJ, Brouwer A, Barelds RJ, Horan MA, Durham SK, Kooistra T. On the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytoMnes. Thromb Haemostas 1992; 67: 679-701. 11. Emeis JJ, Van den Hoogen, Diglio CA. The RHE rat heart endothelial cell line: a model system for studying the synthesis, storage, and acute release of tissue-type plasminogen activator. Thromb Haemostas 1993; 69: 1233. 12. Gallimore MJ, Tyle HM, Shaw JTB. The measurement of fibrinolysis in the rat. Thromb Diath Haemorrh 1971; 26: 279-309. 13. Van Giezen JJJ. Modulation of fibrinolytic activity in rats and other species. Phi) thesis. Universiteit Utrecht, The Netherlands, 1993. 14. Van Giezen JJJ, Nerme V, Abrahamsson T. PAI-1 inhibition enhances the lysis of the platelet-rich part of arterial-like thrombi formed in vitro. A comparative study using thrombi prepared from rat and human blood. Blood Coag Fibrinol 1998; 9:11-18. 15. Ngo TH, Bijnens AP, Knockaert I, Declerck pJ. Expression, purification and characterization of recombinant rat plasminogen activator inhibitor-1. Fibrinolysis 1997; 11 : 37-43. 16. Ngo TH, Verheyen S, Knockaert I, Declerck PJ. Monoclonal antibody-based immunoassays for the specific quantitation of rat PAI-1 antigen and activity in biological samples. Thromb Haemostas 1998; 79: 808-812. 1Z Galfr6 E Milstein C. Preparation of monodonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3-46. 18. Debrock S, Declerck PJ. Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor-I after cleavage of the reactive center loop or after complex formation with various serine proteinases. FEBS Letters 1995; 376: 243-246. 19. Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51 : 392-395. 20. Emeis JJ, Van Den Hoogen CM. Pharmacological modulation of the endotoxin-induced increase in plasminogen activator inhibitor activity in rats. Blood Coag Fibrinolysis 1992; 3: 575-581. 21. Quax PHA, vanden Hoogen CM, Verheijen JH et al. Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo. J Biol Chem 1990; 265: 15560-15563.
Fibrinolysis & Proteolysis (1998) 12(6), 335-339
Suppression of plasminogen activator inhibitor 1 ( P A l - l ) activity levels in rats by monoclonal antibodies T.-H. Ngo, P. J. Declerck Laboratory for Pharmaceutical Biology and Phytopharmacolegy Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
Summary Background: Elevated levels of plasminogen activator inhibitor-1 (PAl-l) are associated with several important thrombotic diseases. Rat models have been shown to be suitable for in vivo investigations on thrombolysis and fibrinolysis. In the present study, monoclonal antibodies (MA) against rat PAl-1 are used to reduce PAl-1 levels in endotoxin-treated rat plasma. Results: A large panel of murine monoclonal antibodies was raised against recombinant rat PAl-1. Out of 237 monoclonal antibodies, six antibodies showed a strong inhibition towards recombinant rat PAl-1 activity in vitro resulting in 70-100% inhibition of PAl-1 activity at a 16-fold molar excess of MA over PAI-I. Relative epitope analysis revealed that this panel represented at least two different epitopes. Subsequent selection of two monoclonal antibodies (MA-124K1 and MA-159M12) and evaluation of their PAl-1 neutralizing properties in vitro in rat plasma, supplemented with recombinant rat PAl-1 and in plasma from endotoxin-treated rats, revealed that MA-124K1 was capable of inhibiting native rat PAl-1 in a plasma environment. Based on these data, MA-124K1 was selected for evaluation in in vivo experiments. Rats were pre-treated with endotoxin (0.5 mg/kg, ip, 90 min prior to MA administration) to obtain high levels of native rat PAl-1 in vivo. Levels of PAl-1 antigen and activity in plasma were measured by specific ELISAs. Injection of a single dose of MA-124K1 (3 mg/kg body weight) resulted in a ~60% inhibition of PAl-1 activity without affecting PAl-1 antigen levels. No changes were observed upon administration of MA32K3, a control monoclonal antibody. Conclusion: MA-124K1 may serve as a useful tool in studies on the evaluation of the role of PAl-1 in cardiovascular disease using rat models.
INTRODUCTION
In recent years, plasminogen activator inhibitor-1 (PAI-1) has gained a lot of attention as a unique member of the superfamily of serine proteinase inhibitors (serpin). Elevated levels of plasminogen activator inhibitor 1 (PAI-1) are associated with several important thrombotic diseases. 1,2Agents aimed at the inhibition of PAI-1 activity could offer a useful strategy for the therapy or prevention of thrombotic events. Enhancement of fibrinolysis, as well as antithrombotic effects both in vivo and in vitro Received: 5 June 1998 Accepted after resubmission: 29 September 1998 Correspondence to: Paul J. Declerck, Laboratory for Pharmaceutica) Biology and Phytopharmacolegy, Van Evenstraat 4, B-3000 Leuven, Belgium. Tel.: 32 16 323431 ; Fax: 32 16 323460; E-mail: paut,[email protected]; website: http://www.farm.kuleuven,ac.be/biotech
through the inhibition of PAI-1 activity or PAI-1 synthesis, has recently been shown in a variety of studies. 3-z Strategies to interfere with PAI-1 activity are based on the use of polyclonal antibody preparations, 3 monoclonal antibodies 4,5 or low molecular weight compounds? An attractive approach for the rational design of PM-1 neutralizing compounds is the generation and characterization of monodonal antibodies (Mr ~ 150 kDa) with PAI-1 neutralizing properties, the subsequent generation of singlechain Fv derivatives (Mr - 25 kDa) thereof,s possibly followed by a further minimization of this antibody fragment. Subsequent studies on the molecular mechanism of inactivation of PAI-I by cloned antibody fragments (e.g. epitope analysis) will allow the use of such an antibody fragment as a starting point for the rational design of PAI-1 irflaibitors (e.g. development of peptidomimetics). A large number of studies have shown that experimental rat models are suitable for in vivo investigations on 335
336
Ngo, Declerck
thrombolysis and fibrinolysis?-13 Even though in such rat models polyclonal antibody preparations have been used, 3,~4 these cannot be used in the application of the above-mentioned rational design. Therefore, in the present study, monoclonal antibodies, raised against recombinant rat PAI-1,15,~6are investigated with respect to their inhibitory properties towards PAI-1 activity in vitro and in vivo. To demonstrate that the observed effects in vivo, i.e. a decrease in PAI-1 activity, were indeed due to a specific interference with PAI-1 activity and not the consequence of, e.g. an accelerated clearance of PAI-1, we have evaluated both rat PAI-1 activity and rat PAI-1 antigen levels.
with different concentrations of monoclonal antibodies. Briefly, 100 gl recombinant rat PAI-1 (50 ng/ml in TBS) were incubated either with 100 gl buffer (TBS) or with 100 gl of a solution containing purified monoclonal antibodies at the indicated concentration. After incubation of this mixture for 25 min at 37°C, residual PAI-1 activity was quantitated with the PAI-1 activity assay as described above. The percentage inhibition (i.e. neutralization of PAI-1 activity) was then calculated based on the ratio of the PAI-1 activity in the presence versus the absence of antibodies.
MATERIALS AND METHODS
The levels of rat PAI-1 antigen and activity were measured using two ELISAs as described by Ngo et al. 16 In brief, 96-wells polystyrene microtiter plates (Costa, Cambrige, MA) were filled with 200~tl of 4gg/ml MA-51L2 (for activity assay) or MA-42S4 (for antigen assay) diluted in phosphate-buffered saline (0.04 M phosphate, 0.14 M NaC1, pH Z4; PBS) and incubated for 48 h at 4°C. The plates were emptied by shaking and the wells were treated for 2 h at room temperature with 200 ~tl PBS containing 10 g/1 bovine serum albumin. Then the wells were washed with PBS containing 0.002% Tween 80 (PBSTween), and finally with 200 gl of a solution containing 100g mannitol and 20g saccharose/L. The plates were stored at -20°C. Immediately before use, plates were washed once with PBS-Tween. Samples were diluted in PBS containing Tween 80 (0.002%), bovine serum albumin (1 g/l) (PTA) and EDTA (5 raM) (dilution buffer), and 180 gl samples were applied to the wells (in the activity assay, samples were incubated with an excess of t-PA for 25 rain at 37°C prior to analysis). After incubation for about 18h at 4°C in a moist chamber, the wells were emptied and washed with PBS-Tween. The plates were subsequently filled with 170 gl samples of HRP-conjugated monoclonal antibodies (MA-62E8-HRP for activity assay and MA-8M7-HRP for antigen assay) diluted 1:4000 in PTA, and incubated for 2 h at room temperature. After washing the plates, the peroxidase reaction was performed by addition of 160 gl aliquots of a 0.1 M citrate-0.2 M sodium phosphate buffer pH 5.0, containing 400 gg/ml o-phenylenediamine and 0.003% hydrogen peroxide to the wells. After 10-30 min at room temperature, the reaction was stopped with 50 gl of 4 M H2SO4. The absorbance was measured at 492 nm with a SLT Spectra (Gr6dig/ Salzburg, Austria). Calibration was performed with purified recombinant rat PAI-1 as described. 1~
Materials
Chromogenic substrate S-2403 was obtained from Kabi Vitrum (Stockholm, Sweden). Human t-PA (predominantly in the single-chain form) was a kind gift from Boehringer Ingelheim (Brussels, Belgium). Recombinant rat PAI-1 was produced as described. 15Endotoxin-treated rat plasma, obtained after injection (ip, 3 h) of rats with endotoxin (lipopolysaccharide from E.coli serotype 0127:B8, L-3129, Sigma, MO, USA) at 500 gg/kg weight was used as a source of native rat PAI-1 for in vitro experiments. Nembutal came from Sanofi, Brussels, Belgium. Wistar rats were purchased from the Animal Center of the Katholieke Universiteit Leuven. Monoclonal antibodies
Monoclonal antibodies against rat PAI-1 were produced essentially as described by Galfr6 and Milstein] 7 and the details were described previously26 Determination of overlapping epitopes was carried out using a two-site ELISA as decribed previously. ~s PAl-1 activity assay with purified proteins in buffer
PAI-1 activity was determined using the method as described by Verheijen et al.~9 In brief, PAI-1 containing samples (50 gl) in Tris-buffered saline (0.1 M NaC1, 0.05 M Tris/HC1, pH Z4, containing 0.01% Tween 80, TBS) were mixed with an equal volume of t-PA (20 U/ml in TBS) and incubated for 15 min at 37°C. Subsequently, 100gl of a solution containing plasminogen (1 gM), CNBr-digested fibrinogen (1 gM) and S-2403 (0.6 raM) were added. Residual t-PA activity was then quanfitated by measuring the absorbance at 405 nm. One unit of PAI-1 is defined as the amount of PAI-1 required to neutralize one international unit of t-PA. PAI-1 neutralization assays were based on quantitation of residual PAI-1 activity after preincubation of PAI-1 Fibrinolysis & Proteolysis (1998) 12(6), 335-339
PAl-1 antigen and activity assays in rat plasma
Animals and sample collection
Rats (Wistm, 300-400 g) were injected intraperitoneally with 500 gg/kg of endotoxin and anaesthetized by © Harcourt Brace & Co, Ltd 1998
Suppression of PAl-1 activity levels by monoclonal antibodies
intraperitoneal injection of 90 mg/kg Nembutal. A tracheal cannula was inserted and the rats were allowed to breath spontaneously. Blood samples were taken from the carotid artery using an Insyte-w catheter (0.7 x 19 mm, Becton Dickinson, Milano, Italy). A blood sample was taken (T = 0) 90 min after endotoxin injection, and monoclonal antibodies (3 mg/kg weight) were immediately administered as a bolus via the carotid cannula. Blood samples were then collected at the indicated time points into tubes containing the proper a m o u n t of anticoagulant (ACD Solution A, Becton Dickinson, NJ, USA) and immediately put on ice. Then, plasma was obtained by centrifugation at 2000 g for 25 rain at 4°C and stored at -20°C until use. Statistical analysis
The statistical significance of differences was evaluated using Student's t-test; p-values >0.05 were considered to be not significant. RESULTS AND DISCUSSION Selection of monoclonal antibodies
Out of 237 mufine monoclonal antibodies raised against rat PAI-1 and evaluated for their PAI-1 neutralizing properties in vitro, six showed pronounced inhibitory effects towards PAI-1 activity in a dose-dependent manner (Fig. 1). For three monoclonal antibodies (MA-124K1, MA147N10 and MA-154S8) a steep dose response curve was observed resulting in a ~ 50% inhibition at an equimolar concentration of MA and PAI-1 and a virtual m a x i m u m effect (- 80%) at a two to four fold molar excess of MA over PAI-1. For all six antibodies 70-100% PAI-1 inhibition was achieved at a 16-fold molar excess. Addition of the control antibody MA-32K3 (one of the antibodies raised against rat PAL1) did not affect the inhibitory activity of PAI-1. From the data obtained with a two-site ELISA (cfr methods), MA-124K1 and MA-159M12 were shown to represent two different epitopes and were, therefore, chosen for further experiments.
337
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4
6
8
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fold molar excess of MA Fig. 1 Inhibitory effects of monoclonal antibodies towards recombinant rat PAl-1 activity in vitro: (II)MA-124K1; (A)MA147N10; (~)MA-154S8; (o)MA-167P3; (C])MA-177K4; (~{~)MA159M12; and (V)MA-32K3. The results represent mean values+SD (n = 3) and were obtained by the method of Verheijen et al. ~ (see Materials and Methods)
Since the antibodies were raised against and selected on recombinant rat PAI-1 in buffer, the inhibitory effects of the selected antibodies was further evaluated on recombinant rat PAI-1 in plasma and on native rat PAI-1 in plasma in vitro. From the data with recombinant rat PAI-1 (Table 1) it can be concluded that both antibodies exert their effects also in a plasma environment. Importantly, whereas MA-159M 12 has virtually no effect on native rat PAI-1, MA-124K1 also exerts its PAI-1 neutralizing properties on native rat PAI-1 (14+6.4% vs 42_+10% PAI-1 inhibition, see Table 1). After considering all data, MA-124K1 was selected for in vivo experiment in rats. In vivo experiments in endotoxin-treated rats
Because PAI-1 levels in rat plasma are very low, 1-2 ng/ml, ~6 endotoxin-treated rats were used to provide a high level of PAL1 in plasma. After endotoxin injection, b o t h PAI-1 antigen and activity levels in rat plasma i n c r e a s e y 3,1<2° thereby reaching a m a x i m u m
Table 1 Inhibitory effects of monoclonal antibodies towards recombinant and native rat PAl-1 activity in vitro in different conditions Recombinant rat PAl-l(*)
Native rat PAl-l(*)
Antibodies(**) MA
buffer
normal plasma undiluted
normal plasma diluted (1/5-1/40)
endotoxin plasma diluted(I/20-1/40)
MA-124K1 MA-159M 12
72+1.9 79+_2.6
83+_4.6 79_+2.5
81+3.5 76+2.5
42+10.0 14+6.4
The data (obtained by the immunofunctional assay,le see Materials and Methods) are expressed as percentage inhibition of PAl-1 activity and represent mean_+SD(n = 3). *at 5 ng/ml **at 5 btg/ml Endotexin-treated rat plasma was taken 3 h after endotoxin injection and contained about 200 ng/mU 6
© Harcourt Brace & Co. Ltd 1998
Fibrinolysis & Proteolysis (1998) 12(6), 335-339
338
Ngo, Declerck
800-
~,, , /
600~
//"
400-
p < 0.001, see Fig. 2 & Table 2). The extent of PAI-1 inhibition is comparable to that observed in vitro (Table 1), i.e. MA-124K1 can reduce the activity of native PAI-1 to ~ 50% of its original activity. Thus, this study demonstrates that with a single dose of 3 m g / k g of MA-124K1, an effect is observed at _>30 rain, lasting at least up to 3.5 h after administration. At this time point the antibody can still reduce PAI-1 activity levels from ~ 800 ng/ml to ~ 300 ng/ml, i.e. under these conditions up to 500 ng active PAI-1 per ml is completely neutralized. Even though this antibody exerts a highly potent effect on PAI-1 activity, 40% of the PAI-1 still remains active. The reason for this discrepancy is not clear. The possible presence of a heat-stable form (distinct from PAI-1) as reported previously ~4,2° can be excluded, since we have used a highly PAI-1 specific immunoassay. This is also further illustrated by the data involving recombinant rat PAI-1 in plasma (Table 2), where an inhibition of over 80% was observed. One could speculate that the lack of full inhibition of native PAI-1 might be related to the differences in glycosylation between recombinant and native PAI-1. In this respect, it is important to realize that the current experiments were carried out under extreme conditions, i.e. resulting in extremely high PAI-1 levels, and without optimizing the administration of the PAI-1 neutralizing compound, i.e. single dose i.v. administration of MA-124K1. This taken into account, and based on the properties of other PAI-1 neutralizing compounds, it is clear that the currently described monoclonal antibody MA-124K1 is a promising agent for in vivo interference with PAI-1 activity in rats. It may, therefore, also play an important role for the development of smaller, recombinant, antibody fragments (scFv, CDRs) to be evaluated further in vivo and to be used in the rational design of PAI-1 neutralizing drugs. In contrast to all previous approaches, 3-6,~4the current rat PAI-1 inhibitor was generated against, and selected on, rat PAI-1. Development of 'small' rat PAI-1 inhibitors using the antigen-binding region of MA-124K1 as a lead compound, should resuk in a pharmacologically-active compound that can be fully evaluated for its efficacy in a wide variety of rat models. Such a compound, its development and
//;---°"
:
........
50
100 150 Time(rain)
200
Fig. 2 Inhibitory effects of monoclonal antibodies on PAl-1 activity in vivo. (A)MA-124K1; (II)MA-32K3, closed symbol: PAl-1 antigen; open symbol: PAl-1 activity. The graph shows the data of a representative experiment (cfr methods, sample analysis by ELISA. 16Time indicates the time starting from the MA administration (i.e. 90 min after endotoxin injection)
concentration followed by a decrease. 21 In addition, the rate of increase of PAI-1 levels depends on the dose of endotoxin used, on the route of administration and on the particular rat itself. Therefore, to define unambiguously the inhibitory effect of monoclonal antibodies on PAI-1 activity in vivo, both PAI-1 antigen and activity levels need to be measured. Upon endotoxin treatment, levels of PAI-1 antigen and activity increased from 1 to 800 ng/ml (Fig. 2). In three rats injected with MA-32K3, PAI-1 activity levels continuously increased in parallel with PAI-1 antigen levels (Fig. 2) and are indicative of the release of fully active PAI- 1 as observed previously. ~6 Administration of MA-124K1 (n = 4) did not affect the endotoxin-induced increase in PAI-1 antigen levels. However, under those conditions PAI-1 activity levels were decreased significantly in comparison to MA-32K3 administration. Whereas upon MA-32K3 administration, the PAI-1 activity/antigen ratios remained nearly constant (Table 2), administration of MA-124K1 resulted in a decrease of the specific activity within 30 mins eventually resulting in a specific activity of 40_+11% after 3.5 h (compared to 94_+2% in the presence of MA-32K3,
Table 2 The ratio between PAl-1 activity and PAl-1 antigen levels after injection of antibodies Time (min)
MA-124K1 MA-32K3 p value (*)
0
0.71+0.21 0.73+_0.11 NS
30
0.56_+0.22 0.78+_0.10 NS
60
0.62_+0.26 0.79+_0.04 NS
90
0.59_+0.15 0.73+_0.22 NS
120
0.51+0.08 1.05+_0.24 <0.005
150
180
0.36+0.05 0.93_+0.23 <0.005
0.47+0.11 0.86_+0.05 <0.002
210
0.40+0.11 0.94+_0.02 <0.001
Mean+SD (n = 3-4) (*) MA-124K1 vs MA-32K3 NS: non significant T = 0: samples were taken immediately before administration of MA Fibrinolysis & Proteolysis (1998) 12(6), 335-339
© Harcourt Brace & Co. Ltd 1998
Suppression of PAl- 1 activity levels by monoclonal antibodies
p r o p e r t i e s , m a y c o n t r i b u t e d i r e c t l y or i n d i r e c t l y t o t h e d e s i g n a n d d e v e l o p m e n t o f PAI-1 n e u t r a l i z i n g c o m p o u n d s for t h e p r e v e n t i o n of t h r o m b o t i c e v e n t s i n humans.
ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e F u n d for scientific R e s e a r c h F.W.O. V l a a n d e r e n , p r o j e c t G . 0 2 6 6 . 9 7 a n d t h e N a g a i F o u n d a t i o n T o k y o (Japan). N.T.H. is a r e c i p i e n t of a K . U . L e u v e n D o c t o r a l Scholarship.
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