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(Circulation. 1996;94:207-216.)
© 1996 American Heart Association, Inc.

Articles

Recombinant Staphylokinase Variants With Altered Immunoreactivity

II: Thrombolytic Properties and Antibody Induction

Desire Collen, MD, PhD; Huberte Moreau; Luc Stockx, MD; Steven Vanderschueren, MD

the Center for Molecular and Vascular Biology (D.C., H.M., S.V.), and the Department of Radiology, University Hospitals Gasthuisberg (L.S.), University of Leuven, Belgium.

Correspondence to D. Collen, MD, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg O & N, Herestr 49, B-3000 Leuven, Belgium. E-mail desire.collen@med.kuleuven.ac.be.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The substitution variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) of recombinant staphylokinase (SakSTAR) with reduced antibody reactivity were assayed for thrombolytic potency and antibody induction in animal models and in patients.

Methods and Results In a ¹²⁵I-fibrin–labeled pulmonary embolism model in the hamster, the doses giving 50% clot lysis in 90 minutes were 25 µg/kg for SakSTAR, 85 µg/kg for SakSTAR.M38, and 90 µg/kg for SakSTAR.M89. In rabbits with ¹²⁵I-fibrin–labeled plasma clots incorporated into extracorporeal arteriovenous loops, lysis within 2 hours was 76±18% (mean±SD, n=28) with 400 µg/kg SakSTAR, 53±13% (n=8) with 1000 µg/kg SakSTAR.M38, and 39±13% (n=6) with 800 µg/kg SakSTAR.M89. When groups of eight rabbits were immunized by intravenous administration of 0.2 to 1.0 mg/kg compound followed by subcutaneous injection of 0.4 mg in Freund's adjuvant at 2, 3, and 5 weeks, SakSTAR.M38 and SakSTAR.M89 elicited markedly less circulating neutralizing activity, compared with SakSTAR, when determined at 6 weeks (neutralizing 6.1±3.0 and 4.9±1.3 µg compound/mL plasma, respectively, versus 20±17 µg/mL; P=.02 and P=.01, respectively) and induced significantly less resistance to thrombolysis (residual thrombolytic potency producing 59±25% and 39±12% lysis, respectively, versus 8.5±5.7%; P=.008 and P=.006, respectively). In patients with peripheral arterial occlusion, intra-arterial administration of SakSTAR.M38 (n=4) or SakSTAR.M89 (n=4) induced significantly fewer circulating neutralizing antibodies (P=.03) and specific IgG (P=.01) at 2 to 3 weeks than SakSTAR (n=8).

Conclusions SakSTAR.M38 and SakSTAR.M89 induce less antibody formation and might constitute preferred agents for thrombolytic therapy in humans.

Key Words: staphylokinase • immunology • antibodies

	Introduction

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Thrombotic complications of cardiovascular diseases are a main cause of death and disability, and consequently, thrombolysis (ie, pharmacological dissolution of the blood clot) favorably influences the outcome of such life-threatening diseases as myocardial infarction. Thrombolytic agents are plasminogen activators that convert plasminogen, the inactive proenzyme of the fibrinolytic system in blood, to the proteolytic enzyme plasmin, which dissolves the fibrin of a blood clot.¹ Currently, six thrombolytic agents are used in patients with acute myocardial infarction. These include streptokinase, urokinase, rTPA or derivatives of it, APSAC, rscuPA, and recombinant staphylokinase.^{1 2} Reduction of infarct size, preservation of ventricular function, and reduction in mortality have been reported with streptokinase, rTPA, and APSAC.¹

Wild-type staphylokinase (SakSTAR variant³ ) was initially found to induce fewer antibodies than streptokinase in dogs⁴ and baboons,⁵ but this could not be extended to patients given an intravenous infusion of up to 40 mg SakSTAR.^{6 7 8 9} Thus, like streptokinase, administration of wild-type staphylokinase would be restricted to single use. However, SakSTAR contains three nonoverlapping immunodominant epitopes.¹⁰ Two of these could be eliminated, without extensive inactivation of the molecule, by specific site-directed mutagenesis of clusters of two or three charged amino acids to alanine. The combination mutants SakSTAR.M38, in which Lys35, Glu38, Lys74, Glu75, and Arg77 were substituted with Ala, and SakSTAR.M89, in which Lys74, Glu75, Arg77, Glu80, and Asp82 were substituted with Ala, were found to combine the reduced reactivity of their parent molecules with a panel of monoclonal antibodies and to incompletely absorb neutralizing antibodies elicited in patients by treatment with wild-type SakSTAR.¹⁰

In the present study, we report that these combination mutants have a significant residual thrombolytic potency in hamster and rabbit models and that they induce fewer antibodies than wild-type SakSTAR after repeated administration in rabbits. SakSTAR.M38 and SakSTAR.M89 also induced less neutralizing antibody formation within 2 to 3 weeks than SakSTAR after intra-arterial administration of 5.5 to 13 mg in patients with PAO.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
Recombinant SakSTAR was prepared and characterized as described elsewhere.⁷ The recombinant staphylokinase mutants with Lys35 and Glu38 substituted with Ala (SakSTAR.M3); with Lys74, Glu75, and Arg77 substituted with Ala (SakSTAR.M8); and with Glu80 and Asp82 substituted with Ala (SakSTAR.M9) were obtained as described elsewhere.¹¹ The combination mutants SakSTAR.M38 and SakSTAR.M39 were obtained as described in the accompanying article.¹⁰

Staphylokinase-Neutralizing Activity in Plasma
Staphylokinase-neutralizing activity in plasma was determined by addition of increasing concentrations of wild-type or variant SakSTAR (50-µL volumes containing 0.2 to 1000 µg/mL) to a mixture of 300 µL citrated pooled human plasma and 50 µL buffer or test plasma (rabbit or human), followed immediately by addition of 100 µL of a mixture containing thrombin (50 NIH U/mL) and CaCl₂ (25 mmol/L). The plasma clot lysis time was measured and plotted against the concentration of the staphylokinase moiety. From this curve, the concentration of plasminogen activator that produced clot lysis in 20 minutes was determined. The neutralizing activity titer was determined as the difference between the test plasma and buffer values and was expressed in micrograms of staphylokinase moiety neutralized per milliliter of test plasma.

ELISA for Staphylokinase-Related Antigen
The Ig fraction of a polyclonal antiserum obtained from rabbits immunized with STAN5¹² was diluted to a final concentration of 4 µg/mL in 0.14 mol/L NaCl, 0.04 mol/L PBS, pH 7.4, and 200-µL samples were incubated for 48 hours at 4°C in the wells of polystyrene microtiter plates (Costar). The plates were emptied, and the wells were treated for 2 hours at room temperature with 200 µL PBS containing 10 g/L BSA. Then the wells were washed with 200 µL PBS and finally with a solution containing 100 g mannitol and 20 g saccharose per liter. The plates were stored at -20°C. Immediately before use, the plates were washed once with PBS containing 0.002% Tween 80. Plasma samples were diluted in PBS containing Tween 80 (0.002%), EDTA (5 mmol/L), and BSA (1 g/L) (dilution buffer), and 180-µL samples were added to the wells. After incubation for 18 hours at 4°C in a moist chamber, the wells were emptied and washed with PBS containing 0.002% Tween 80. Tagging was performed with HRP-conjugated rabbit anti-human IgG. The Ig-HRP conjugate was prepared as described by Nakane and Kawaoi.¹³ Ig-HRP, 70 µL, diluted to 0.4 µg/mL in PBS-Tween was applied to the wells and incubated for 2 hours at room temperature. After washing of the plates, 160-µL aliquots of a 0.1 mol/L citrate/0.2 mol/L sodium phosphate buffer, pH 5.0, containing 200 µg/mL o-phenylenediamine and 0.003% hydrogen peroxide were added. After 15 minutes at room temperature, the peroxidase reaction was arrested with 50 µL of 4 mol/L H₂SO₄. The absorbance was measured at 492 nm with a multiscan spectrophotometer, EAR 400AT (SLT-Lab Instruments).

Under these conditions, a linear dose-response in buffer was obtained between 0.1 and 1.2 ng/mL staphylokinase. When SakSTAR or its variants were diluted 1:10 in plasma, no interference of plasma proteins with the antigen assay was observed. Thus, the lower limit of sensitivity in plasma was 1.0 ng/mL. The ELISA was calibrated against the SakSTAR variants to be quantified.

Fibrinolytic Properties of SakSTAR Variants in Human Plasma In Vitro
¹²⁵I-fibrin–labeled plasma clots were prepared from pooled normal human plasma after addition of 500 000 cpm/mL of ¹²⁵I-fibrinogen and coagulation with CaCl₂ (final concentration, 35 mmol/L) and thrombin (final concentration, 2 NIH U/mL). Lysis of ¹²⁵I-fibrin–labeled plasma clots (0.12-mL volume) by addition of different concentrations of SakSTAR, SakSTAR.M38, or SakSTAR.M89 (final concentration, 0.05 to 1.6 µg/mL) in 0.5 mL normal human plasma was measured over 2 hours as previously described.¹⁴ The concentration of fibrinolytic agent required to obtain 50% clot lysis in 2 hours (C₅₀) was determined from plots of percent lysis versus the concentration of test compound.

Systemic activation of the fibrinolytic system by SakSTAR, SakSTAR.M38, or SakSTAR.M89 (final concentration, 0 to 200 µg/mL) in normal human plasma in the absence of fibrin was determined at 2 hours by measurement of residual fibrinogen levels. The concentration of plasminogen activator required to obtain 50% fibrinogen degradation within 2 hours was determined graphically from dose-response curves representing the concentration of test compound versus residual reactive protein (expressed as a percentage of the baseline value).

Thrombolytic Properties of SakSTAR Variants in a Hamster Pulmonary Embolism Model
The thrombolytic potency of SakSTAR, SakSTAR.M3, SakSTAR.M8, SakSTAR.M9, SakSTAR.M38, and SakSTAR.M89 was evaluated by use of the pulmonary embolism model in heparinized hamsters as described previously.¹⁵ A ¹²⁵I-fibrin–labeled platelet-poor plasma clot corresponding to 50 µL original plasma was injected into the jugular vein, the thrombolytic agents were infused intravenously over a 60-minute period, and lysis was measured 30 minutes after the end of the infusion as the difference between the radioactivity initially incorporated into the clot and the residual radioactivity in the lungs and the heart. Fibrinogen and ₂-antiplasmin levels in plasma from blood samples taken before and at the end of the experiment were determined as described elsewhere.¹⁵ The aPTT was assayed with a routine laboratory assay. Steady-state staphylokinase-related antigen concentrations in plasma during infusion of the agents were determined on blood samples taken at 30 and 60 minutes by the specific ELISA described above.

Pharmacokinetics of SakSTAR Variants After Intravenous Bolus Injection in Hamsters
The pharmacokinetic parameters of the disposition of staphylokinase from blood were evaluated in groups of at least four hamsters after intravenous bolus injection of 100 µg/kg SakSTAR, SakSTAR.M3, SakSTAR.M8, SakSTAR.M9, SakSTAR.M38, or SakSTAR.M89. Concentrations of staphylokinase-related antigen in plasma were measured at different time intervals after bolus injection by the ELISA described above. The results were plotted on semilogarithmic paper and fitted with a sum of two exponential terms, C(t)=Ae^-t+Be^-ßt, by graphical curve peeling.¹⁶ Pharmacokinetic parameters, calculated from the coefficients (A and B) and exponents ( and ß) by use of standard formulas derived by Gibaldi and Perrier,¹⁶ included initial half-life (in minutes), t_1/2=ln2/; terminal half-life (in minutes), t_1/2ß=ln2/ß; volume of the central (plasma) compartment (in mL), V_C=dose/(A+B); area under the curve (in ng·min^-1·mL^-1), AUC=A/+B/ß; and plasma clearance (in mL·min^-1), Cl_p=dose/AUC.

Alternatively, the clearance of the SakSTAR variants during their infusion in hamsters was estimated from the infusion rate (in µg/min) and the steady-state plasma staphylokinase-related antigen level (in µg/mL).

Antigenicity of SakSTAR Variants After Repeated Administration in Rabbits
The comparative antigenicity of SakSTAR versus each of the SakSTAR variants, SakSTAR.M3, SakSTAR.M8, SakSTAR.M9, SakSTAR.M38, and SakSTAR.M89, was studied after subcutaneous immunization in groups of 4 or 8 rabbits allocated to SakSTAR and 8 rabbits allocated to the variant. The humoral response was quantified at 6 weeks by determination of the staphylokinase-neutralizing activity in plasma as described above and of the residual thrombolytic potency as described below. Immunization was carried out by intravenous infusion of 400 µg/kg SakSTAR and of 200 to 1000 µg/kg of the SakSTAR variants at week 0 (to determine the baseline clot lysis capacity) followed by subcutaneous injection of 400 µg of the same agent in complete Freund's adjuvant at week 2 and in incomplete Freund's adjuvant at weeks 3 and 5.

The thrombolytic properties were studied by use of 0.3 mL ¹²⁵I-fibrin–labeled platelet-poor rabbit plasma clots inserted into an extracorporeal arteriovenous loop. An exposed femoral artery was catheterized with a 4F catheter (Portex White) and connected via two hypodermic syringes to a catheterized ear vein. The blood flow through the extracorporeal loop was maintained at 10 mL/min with a peristaltic pump. ¹²⁵I-fibrin–labeled plasma clots were introduced into each of two syringes inserted into the loop. The plasma clots were prepared by mixing of 0.3 mL platelet-poor plasma with a trace amount (1.5 µCi) ¹²⁵I-labeled human fibrinogen solution (Amersham) and 0.07 mL of a mixture of bovine thrombin (15 NIH U/mL) and 0.5 mol/L CaCl₂, followed by incubation for 30 minutes at 37°C. Thirty minutes before the start of the infusion, 7.5 mg/kg ridogrel (a combined thromboxane synthase inhibitor and prostaglandin endoperoxide receptor antagonist)¹⁷ was administered as an intravenous bolus to prevent platelet deposition in the extracorporeal loop. The animals were anticoagulated with heparin (Novo Nordisk) (300 U/kg followed by a continuous infusion of 200 U·kg^-1·h^-1 throughout the experiment) and randomly allocated at baseline to infusion with 400 µg/kg wild-type SakSTAR (4 or 8 rabbits) or 200 to 1000 µg/kg variant SakSTAR (8 rabbits). At 6 weeks, half of the rabbits allocated to a SakSTAR variant were again treated with the same SakSTAR variant and the other half with wild-type SakSTAR, while the rabbits immunized with SakSTAR were treated with the SakSTAR variant (when this control group consisted of 4 rabbits) or randomized either to SakSTAR or to the SakSTAR variant (when this control group contained 8 rabbits). The thrombolytic agents were given intravenously as a 10% bolus and a 90% infusion over 1 hour. The time course of clot lysis was monitored continuously during 2 hours by external gamma counting with two 3x0.5-in sodium iodide/thallium crystals (Bicron) positioned over the extracorporeal thrombi. The scintillation crystals were connected to a dedicated Canberra-S100 system (Canberra-Packard), and the data were analyzed as described elsewhere.¹⁸ At the end of the experiment, the residual clots were recovered from the syringes for determination of their radioisotope content. The animal experiments were conducted according to the guiding principles of the American Physiological Society and the International Committee on Thrombosis and Haemostasis.¹⁹

Biological Analysis of SakSTAR.M38 and SakSTAR.M89 for Use in Patients
Endotoxin contamination of the sterile-filtered SakSTAR.M38 and SakSTAR.M89 preparations was determined with the limulus amoebocyte lysate assay of Chromogenix according to the instructions of the manufacturer. Preparations of SakSTAR.M38 and SakSTAR.M89 were tested for bacterial sterility on blood agar plates, which were incubated under aerobic and anaerobic conditions, on McConkie and chocolate-agar media, and in thioglycolate and tryptic soy broth media.

The acute toxicity of the purified sterilized SakSTAR.M38 and SakSTAR.M89 preparations versus saline was evaluated by intravenous bolus injection of 200 µL of the purified sterilized material at a dose of 3 mg/kg in groups of 6 mice weighing 15 g. The animals were observed for acute reactions (tachypnea, restlessness, stupor) and were weighed daily during an 8-day observation period.

Comparative Thrombolytic Efficacy and Immunogenicity of SakSTAR.M38 and SakSTAR.M89 Versus SakSTAR in Patients With PAO
SakSTAR (n=8), SakSTAR.M38 (n=4), and SakSTAR.M89 (n=4) were administered intra-arterially at or in the proximal end of the occlusive thrombus as a bolus of 2 mg followed by an infusion of 1 mg/h in patients with angiographically documented arterial occlusion of a peripheral artery or bypass graft. Patients were studied after they had given informed consent, and the protocol was approved by the Human Studies Committee of the University of Leuven. Inclusion and exclusion criteria were essentially as previously described,⁹ except that retreatment with the recombinant staphylokinase moiety within 48 hours was allowed. Conjunctive antithrombotic treatment with heparin, aspirin, and oral anticoagulants was as previously described.⁹

The patency status of the occluded peripheral artery or bypass graft was serially evaluated before, at least every 4 hours during, and at the end of the intra-arterial infusion of wild-type or variant SakSTAR. The angiographic patency status of the target vessel at the end of the infusion constituted the main study end point. The administration of thrombolytic agent was terminated when adequate vessel patency was achieved, when complications required its cessation, or when two consecutive angiograms failed to demonstrate progression of clot lysis. Recanalization was defined as clot lysis sufficient to restore brisk anterograde flow throughout the previously occluded segment. Complementary intravascular procedures such as PTA were allowed when the investigators judged that the thrombus was sufficiently lysed or that no further thrombolysis was to be expected.

Blood pressure and heart rate were monitored before, during, and after infusion of SakSTAR, SakSTAR.M38, or SakSTAR.M89. Blood samples were collected before, at the end of, and 6 hours after the angiographic procedure. Measurements included peripheral blood count, prothrombin time, aPTT, fibrinogen, ₂-antiplasmin, plasminogen, and biochemical hepatic and renal function tests. SakSTAR-neutralizing, SakSTAR.M38-neutralizing, and SakSTAR.M89-neutralizing activities and anti-SakSTAR, anti-SakSTAR.M38, and anti-SakSTAR.M89 IgG and IgM were serially determined, essentially as described in detail elsewhere,²⁰ on blood samples drawn during hospitalization and after discharge. Clinical follow-up focused on recurrence of thrombosis and on adverse events such as allergic reactions and major bleeding (ie, need for blood transfusion or surgical control, a drop in hematocrit of >10%, or intracranial bleeding).

Statistical Analysis
To analyze the evolution of neutralizing activities and thrombolytic potencies at baseline versus at 6 weeks within the same group of rabbits, paired Student's t test was used. For comparison of wild-type versus variant SakSTAR, the unpaired t test or Mann-Whitney rank-sum test was used for gaussian and nongaussian distributed data, respectively.

Student's t test was used to compare coagulation parameters in patients with PAO before and after thrombolytic therapy. Neutralizing activities and specific IgG against wild-type and variant SakSTARs at 2 and 3 weeks after treatment were compared by Mann-Whitney rank-sum test. A value of P<.05 was considered to indicate statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Fibrinolytic Properties of SakSTAR Variants in Human Plasma In Vitro
Dose- and time-dependent lysis of ¹²⁵I-fibrin–labeled human plasma clots submerged in human plasma was obtained with SakSTAR, SakSTAR.M38, and SakSTAR.M89. Spontaneous clot lysis during the experimental period was 5% (data not shown). Equieffective concentrations of test compound (causing 50% clot lysis in 2 hours; C₅₀) determined graphically from plots of clot lysis at 2 hours versus the concentration of plasminogen activator (data not shown) were 0.29±0.01 µg/mL for SakSTAR, 1.0±0.06 µg/mL for SakSTAR.M38, and 0.91±0.19 µg/mL for SakSTAR.M89 (mean±SD, n=6).

The concentrations of compound causing 50% fibrinogen degradation in 2 hours in normal human plasma in the absence of fibrin were determined graphically from dose-response curves (data not shown). These values were (mean±SD of three independent experiments) 23±1.0 µg/mL for SakSTAR, 110±8.7 µg/mL for SakSTAR.M38, and 100±6.9 µg/mL for SakSTAR.M89.

Thrombolytic Properties of SakSTAR Variants in a Hamster Pulmonary Embolism Model
The thrombolytic potencies of SakSTAR, SakSTAR.M3, SakSTAR.M8, SakSTAR.M9, SakSTAR.M38, and SakSTAR.M89 were compared in a hamster pulmonary embolism model (Table 1). Saline infusion yielded a value for spontaneous lysis at 90 minutes of 22±4% (mean±SEM, n=7). Fibrinogen levels at the end of the experiment were 150±18% of the baseline value, and ₂-antiplasmin levels were 140±17% (data not shown). With SakSTAR, lysis at 90 minutes after the start of the infusion in groups of 4 to 8 hamsters increased from 29±4% with 9 µg/kg to 77±5% with 81 µg/kg. With SakSTAR.M38, clot lysis was 31±3% with 27 µg/kg and 66±6% with 250 µg/kg. With SakSTAR.M89, clot lysis was 40±6% with 27 µg/kg and 86±7% with 250 µg/kg. Fibrinogen and ₂-antiplasmin levels did not drop after infusion of SakSTAR, SakSTAR.M38, or SakSTAR.M89, whereas the steady-state staphylokinase-related antigen level in plasma increased proportionally with the infusion rate to between 6.0±1.0 and 130±26 ng/mL for SakSTAR, to between 13±1.0 and 97±19 ng/mL for SakSTAR.M38, and to between 31±2.4 and 410±53 ng/mL for SakSTAR.M89 (Table 1). Corresponding antigen values for SakSTAR.M3, SakSTAR.M18, and SakSTAR.M19 and their plasma clearances are summarized in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clot Lysis Parameters of SakSTAR and SakSTAR Mutants in a Hamster Pulmonary Embolism Model

Pharmacokinetic Properties of SakSTAR Variants After Bolus Injection in Hamsters
The disposition rate of staphylokinase-related antigen from blood after bolus injection of 100 µg/kg of the SakSTAR variants in 4 to 7 hamsters could be adequately described by a sum of two exponential terms by graphical curve peeling (results not shown). This yielded the following equation: C(t)=1.4 exp(-0.36t)+0.14 exp(-0.069t) for SakSTAR, C(t)=1.4 exp(-0.38t)+0.19 exp(-0.086t) for SakSTAR.M38, and C(t)=0.97 exp(-0.38t)+0.11 exp(-0.078t) for SakSTAR.M89, from which the following pharmacokinetic parameters were derived: t_1/2=1.9 minutes, t_1/2ß=10 minutes, V_c=6.6±0.2 mL, and Cl_p=1.8±0.1 mL/min for SakSTAR; t_1/2=1.8 minutes, t_1/2ß=8.0 minutes, V_c=6.3±0.5 mL, and Cl_p=1.7±0.1 mL/min for SakSTAR.M38; and t_1/2=1.8 minutes, t_1/2ß=9.0 minutes, V_c=9.6±0.9 mL, and Cl_p=2.6±0.2 mL/min for SakSTAR.M89 (Table 2). The clearance, determined from the steady-state plasma antigen level and the infusion rate, ranged between 1.3±0.4 and 3.0±0.5 mL/min for SakSTAR, between 3.3±0.3 and 5.4±0.6 mL/min for SakSTAR.M38, and between 1.1±0.2 and 1.5±0.1 mL/min for SakSTAR.M89 (Table 1).

View this table: [in this window] [in a new window]   Table 2. Pharmacokinetic Parameters of the Disposition of Staphylokinase-Related Antigen From Plasma After Bolus Injection (100 µg/kg) in Hamsters

Antigenicity of SakSTAR Variants After Repeated Administration to Rabbits
The antigenicity of wild-type SakSTAR versus the respective single mutants (SakSTAR.M3, SakSTAR.M8, and SakSTAR.M9) is compared in Table 3, top, and that of wild-type SakSTAR versus the combination mutants (SakSTAR.M38 and SakSTAR.M89) in Table 3, bottom. Results are expressed as mean±SD. In 8 rabbits assigned to the SakSTAR.M38 group, the baseline neutralizing activity in plasma was 0.6±0.3 µg/mL against SakSTAR and 3.5±2.0 µg/mL against SakSTAR.M38. Intravenous infusion of 1000 µg/kg SakSTAR.M38 produced 53±13% lysis. These rabbits were then immunized with 400 µg SakSTAR.M38 suspended in complete Freund's adjuvant at week 2 and with the same amount in incomplete Freund's adjuvant at weeks 3 and 5. At week 6, the plasma neutralizing activity was increased only to 1.7±0.7 µg/mL against SakSTAR and to 6.1±3.0 µg/mL against SakSTAR.M38. Infusion of 400 µg/kg SakSTAR at week 6 in 4 of these rabbits produced 77±18% clot lysis, whereas infusion of 1000 µg/kg SakSTAR.M38 in the other 4 rabbits produced 59±25% lysis. In 8 rabbits assigned to the SakSTAR group, the baseline neutralizing activity in plasma was 0.6±0.4 µg/mL against SakSTAR and 2.0±2.0 µg/mL against SakSTAR.M38. Intravenous infusion of 400 µg/kg SakSTAR produced 80±10% lysis. These rabbits were then immunized subcutaneously with 400 µg SakSTAR suspended in complete Freund's adjuvant at week 2 and with the same amount in incomplete Freund's adjuvant at weeks 3 and 5. At week 6, the plasma neutralizing activity was increased to 20±15 µg/mL against SakSTAR and to 21±22 µg/mL against SakSTAR.M38, whereas the residual thrombolytic potency of 400 µg/kg SakSTAR had decreased to 8.5±5.7% and of 1000 µg/kg SakSTAR.M38 to 30±29% (Table 3, bottom).

View this table: [in this window] [in a new window]   Table 3. Immunogenicity of Staphylokinase Variants in Rabbits

In 8 rabbits assigned to the SakSTAR.M89 group, the baseline neutralizing activity in plasma was 0.3±0.2 µg/mL against SakSTAR and 1.6±0.5 µg/mL against SakSTAR.M89. Intravenous infusion of 800 µg/kg SakSTAR.M89 produced 39±13% clot lysis at baseline. These 8 rabbits were then immunized with 400 µg SakSTAR.M89 suspended in complete (week 2) or incomplete (weeks 3 and 5) Freund's adjuvant. At 6 weeks, the plasma neutralizing activity was increased only to 2.5±1.5 µg/mL against SakSTAR and to 4.9±1.3 µg/mL against SakSTAR.M89. At week 6, infusion of 400 µg/kg SakSTAR in 4 of these rabbits produced 51±35% clot lysis, and infusion of 800 µg/kg SakSTAR.M89 in the other 4 rabbits produced 39±12% lysis. In 4 control rabbits assigned to SakSTAR, the pretreatment neutralizing activity was 0.2±0.1 µg/mL against SakSTAR and 0.7±0.3 µg/L against SakSTAR.M89. Intravenous infusion of 400 µg/kg SakSTAR induced 67±19% clot lysis. These 4 rabbits were then immunized with 400 µg SakSTAR suspended in complete (week 2) and incomplete (weeks 3 and 5) Freund's adjuvants, respectively. At week 6, the plasma neutralizing activity was increased to 20±15 µg/mL against SakSTAR and to 18±15 µg/mL against SakSTAR.M89, whereas the residual thrombolytic efficacy of 800 µg/kg SakSTAR.M89 had decreased only to 31±30% lysis (Table 3, bottom). The comparative values obtained with wild-type SakSTAR versus the single mutants (SakSTAR.M3, SakSTAR.M8, and SakSTAR.M9) are summarized in Table 3, top.

These results show that in this directly comparative study of SakSTAR and selected variants, the double mutants (SakSTAR.M38 and SakSTAR.M89) in particular induce significantly less antibody-related neutralizing activity and resistance to lysis than wild-type SakSTAR.

Biological Analysis of SakSTAR.M38 and SakSTAR.M89 for Use in Patients
The endotoxin content of the purified preparations was 3.3±2.4 U/mg for SakSTAR.M38 and 12±3.0 U/mg for SakSTAR.M89, which represented a >10⁶-fold separation from SakSTAR compared with the culture broth (containing >100 000 U/mL). Preparations sterilized by filtration proved to be sterile on 3-day testing, as described in "Methods." Intravenous bolus injection of either SakSTAR.M38 or SakSTAR.M89 in groups of 6 mice (3 mg/kg body wt) did not provoke any acute reaction or reduced weight gain within 8 days in comparison with the mice given an equal amount of saline (Fig 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of intravenous bolus injection of 3 mg/kg body wt of SakSTAR.M38 (O) and SakSTAR.M89 (), compared with saline (), on body weight in mice.

Comparative Thrombolytic Efficacy and Antigenicity of SakSTAR.M38 and SakSTAR.M89 Versus SakSTAR After Intra-arterial Administration in Patients With PAO
Groups of 4 to 8 patients (41 to 73 years old) with angiographically documented PAO with an estimated duration of 1 to 120 days and a length of 8 to 50 cm were treated with SakSTAR.M38, SakSTAR.M89, or SakSTAR. One patient (W.A.L.) given wild-type SakSTAR developed anaphylactic shock within 5 minutes after the 2-mg bolus administration. The infusion was immediately interrupted, and the blood pressure returned to normal within 20 minutes during infusion of plasma expanders. This patient was not included for calculation of mean±SEM in Tables 4 through 6. One patient (L.A.N.) given SakSTAR.M89 developed reocclusion after 30 hours, which was treated with 6.5 mg of the variant. This patient developed 7.8 µg/mL SakSTAR.M89-neutralizing activity and 270 µg/mL specific anti-SakSTAR.M89 IgG after 2 to 3 weeks and was not included for calculation of the median and range in Fig 2.

View this table: [in this window] [in a new window]   Table 4. Characteristics of Patients With PAO Treated With SakSTAR.M38, SakSTAR.M89, or SakSTAR

View this table: [in this window] [in a new window]   Table 5. Treatment and Outcome in Patients With PAO, Treated With SakSTAR.M38, SakSTAR.M89, or SakSTAR

View this table: [in this window] [in a new window]   Table 6. Coagulation Parameters Before and After Administration of SakSTAR.M38, SakSTAR.M89, or SakSTAR in Patients With PAO

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course of circulating staphylokinase-neutralizing activity (left) and anti-staphylokinase IgG (right) after intra-arterial infusion of SakSTAR in 8 patients (solid lines) or of SakSTAR.M38 or SakSTAR.M89 (dotted lines) in 7 patients with peripheral arterial occlusion. The data represent median values and 15 to 85 percentile range.

Relevant baseline characteristics of the individual patients are shown in Table 4. The majority of PAOs were at the femoropopliteal level. All were due to in situ thrombosis. Two grafts and two iliac stent occlusions were included. Nine patients presented with incapacitating claudication, 2 with chronic ischemic rest pain, 4 with subacute ischemia, and 1 with acute ischemia.

Table 5 summarizes the individual results of treatment and outcome. Intra-arterial infusion, at a dose of 5.5 to 13 mg over a period of 3.5 to 11 hours, induced complete recanalization in 13 patients, partial recanalization in 1 patient, and no improvement in 1 patient. Complementary endovascular procedures (mainly PTA) were performed in 12 patients and complementary surgery immediately after thrombolysis in 1 patient. Recurrence of thrombosis after the end of the angiographic procedure occurred in 4 patients: the first was successfully retreated after 30 hours with 6.5 mg SakSTAR.M89, the second underwent aortobiiliac bypass grafting, the third was successfully recanalized after 20 hours with 40 mg rTPA, and in the fourth the thrombus was aspirated transluminally. Bleeding complications were absent or limited to mild to moderate hematoma formation at the angiographic puncture sites except for one patient who developed a hematoma within the right quadriceps muscle. Other complications related to endovascular manipulations included distal embolization and arterial dissection, which necessitated premature cessation of thrombolytic infusion in 1 patient. One superficial femoral arterial occlusion proved to be resistant to 8.0 mg SakSTAR infused over 6.0 hours and was subsequently managed successfully by PTA (Table 5).

Circulating fibrinogen, plasminogen, and ₂-antiplasmin levels remained unchanged during infusion of the SakSTAR moieties (Table 6), reflecting absolute fibrin specificity of these agents at the dosages used. Substantial in vivo fibrin digestion occurred, as evidenced by elevation of D-dimer levels. Intra-arterial heparin therapy prolonged the aPTT (Table 6).

Antibody-related SakSTAR-, SakSTAR.M38-, and SakSTAR.M89-neutralizing activity and anti–SakSTAR-, anti–SakSTAR.M38-, and anti–SakSTAR.M89-specific IgG were low at baseline and during the first week after the infusion (Fig 2). From the second week on, neutralizing activity levels increased to median values of 2.9 and 3.3 µg SakSTAR variant neutralized per milliliter of plasma in the patients treated with SakSTAR.M38 and SakSTAR.M89, respectively, which is significantly lower than the median value of 9.1 µg wild-type SakSTAR neutralized per milliliter in the patients treated with SakSTAR (P=.03 for variants versus wild-type by Mann-Whitney rank-sum test). The levels of SakSTAR-specific IgG increased to median values of 51 and 31 µg/mL plasma in patients treated with SakSTAR.M38 and SakSTAR.M89, respectively, which is significantly lower than the median value of 240 µg/mL in the patients treated with SakSTAR (P=.01 for variants versus wild-type by Mann-Whitney rank-sum test).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study was initiated by the observation that the "clustered charge–to-alanine" substitution variants K35A,E38A (SakSTAR.M3), K74A,E75A,R77A (SakSTAR.M8), E80A,D82A (SakSTAR.M9), K35A,E38A,K74A,E75A,R77A (SakSTAR.M38), and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) had an altered epitope expression toward a panel of 17 murine monoclonal antibodies and a reduced reactivity with antibodies elicited in patients by treatment with SakSTAR.¹⁰ Therefore, SakSTAR.M3, SakSTAR.M8, SakSTAR.M9, and the combination variants SakSTAR.M38 and SakSTAR.M89 were subjected to evaluation of their antigenicity in vivo. A rabbit model developed previously for the evaluation of the antigenicity of SakSTAR was used.²¹ The immunization scheme was intensified to include the use of subcutaneous injections with Freund's adjuvant, although this entailed the risk of exposure of neoantigens after denaturation of the molecules. Immunization with SakSTAR induced high titers of neutralizing antibodies and refractoriness to clot lysis within 6 weeks, whereas immunization with SakSTAR.M3, SakSTAR.M8, and SakSTAR.M9 and, more so, with the combination variants SakSTAR.M38 and SakSTAR.M89 induced significantly less circulating neutralizing antibodies, resulting in a higher residual thrombolytic response on repeated administration either of the variant or of wild-type SakSTAR after 6 weeks.

Therefore, SakSTAR.M38 and SakSTAR.M89 were selected for evaluation of their antigenicity after single intra-arterial administration in patients with PAO. The highly purified materials, after sterilization by filtration and conditioning for use in humans, had a thrombolytic potency in human plasma in vitro and in a hamster pulmonary embolism model of approximately one third of that of wild-type SakSTAR. Their pharmacokinetics after bolus injection were indistinguishable from that of SakSTAR. At a dose of 3 mg/kg, they did not produce acute reactions or affect body weight in mice.

Intra-arterial administration in groups of 4 to 8 patients of a bolus of 2 mg SakSTAR.M38, SakSTAR.M89, or SakSTAR followed by an infusion of 1 mg/h during a 3.5- to 11-hour period produced complete recanalization in 13 of 16 patients. One patient given wild-type SakSTAR developed an anaphylactic reaction after the bolus injection, leading to immediate termination of the infusion. Of a total of more than 100 patients treated with recombinant staphylokinase to date, this patient is the first to have a major systemic reaction against SakSTAR.^{6 7 8 9} His anti-SakSTAR antibody titer was low at baseline (0.3 µg staphylokinase-neutralizing activity and 7.3 µg specific IgG per milliliter of plasma) and did not markedly increase within 4 weeks after the bolus injection (peak values of 6.5 µg neutralizing activity and 190 µg specific IgG per milliliter plasma).

After administration of wild-type or variant SakSTAR, neutralizing antibody titers and specific IgG levels remained low for 1 week. From the second or the third week onward, marked increases of SakSTAR-neutralizing activity and anti-SakSTAR IgG levels were observed in the patients given SakSTAR, whereas these values were significantly lower for the variants. These observations indicate that the combination variants SakSTAR.M38 and SakSTAR.M89 not only have a reduced immunoreactivity with murine monoclonal antibodies and with antibodies induced in patients by treatment with SakSTAR¹⁰ but also have a reduced antigenicity, both on repeated administration in rabbits and after intra-arterial infusion in patients with PAO.

A significant limitation of the present study is that it does not allow us to extrapolate that the observed reduced induction of antibody with the SakSTAR variants would be sufficient to protect against potential immune-mediated disease after repeated treatment or after subsequent exposure to staphylokinase-producing staphylococci. This will require more extensive prospective studies.

The present study does not provide information on the mechanism of the observed reduced humoral response. One interesting hypothesis is the shift from a secondary immune response to a primary one. This implies that the immune system of the studied patients must have met SakSTAR before treatment, possibly via Staphylococcus aureus infections. The SakSTAR mutants lack two of the three immunodominant B-cell epitopes of wild-type SakSTAR. Consequently, the putative SakSTAR specific-memory B cells present in the treated patients would not recognize the mutants as well, and therefore proliferation and production of antibodies would be reduced. Another important aspect to generate humoral response is the absolute necessity for T-cell help. It is possible that one or more T-cell epitopes have been mutated as well. However, since the majority of T-cell epitopes are major histocompatibility complex–restricted, one would not expect abrogation of T-cell help in all patients included in this study.

In aggregate, the present observations constitute proof of the principle that both the immunoreactivity of and the antibody induction with staphylokinase can be reduced by protein engineering. To the best of our knowledge, this constitutes the first report of such reduction in which a heterologous protein with potential therapeutic application was used in humans. Further work aiming at the elucidation of the mechanism of the reduced antigenicity and at the optimization of the ratio of antigenicity to specific activity would seem to be warranted. Indeed, the development of nonimmunogenic staphylokinase variants with intact specific activity and fibrin specificity would represent a significant step toward optimization of agents for rapid, safe, and affordable thrombolytic therapy in patients with acute myocardial infarction.

	Acknowledgments

 
Dr Vanderschueren is a research assistant of the National Fund for Scientific Research in Belgium. The authors are grateful to Dr P.A.M. Warmerdam for helpful discussions on mechanisms of immunogenicity.

	Selected Abbreviations and Acronyms

 

APSAC = anisoylated plasminogen streptokinase activator complex aPTT = activated partial thromboplastin time HRP = horseradish peroxidase PAO = peripheral arterial occlusion PTA = percutaneous transluminal angioplasty rscuPA = recombinant single-chain urokinase-type plasminogen activator, recombinant prourokinase rTPA = recombinant tissue-type plasminogen activator

	Footnotes

 
This study was supported in part by a sponsored research agreement between the University of Leuven (Leuven Research and Development VZW) and Thromb-X NV, a spin-off company of the University of Leuven, in which Dr Collen has an equity interest.

Received October 9, 1995; revision received January 8, 1996; accepted January 22, 1996.

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