Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase

In this study, we used restriction and modification enzymes according to the suppliers' recommendations (New England Biolabs, Leusden, The Netherlands; Boehringer Mannheim, Mannheim, Germany; or Pharmacia Biotech, Uppsala, Sweden) and mutagenic oligonucleotides and primers (Eurogentec, Seraing, Belgium). Plasmid DNA was isolated, as recommended, using a purification kit (Qiagen, Hilden, Germany). Transformation of E coli TG1 or WK6 cells were performed following the calcium chloride procedure. Nucleotide sequences were determined on double-strand plasmid DNA with the dideoxy chain termination method by using the T7 sequencing kit (Pharmacia Biotech). Polymerase chain reaction (PCR) procedures were performed using Thermus aquaticus (Taq) polymerase (Boehringer Mannheim). Protein concentrations were determined according to Bradford17 or by absorption at 280 nm, using 19 200 molL^-1cm^-1 as molar extinction coefficient.18 Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed (mini-Protean-II system; Biorad, The Netherlands) using 12% or 15% gels and Coomassie brillant blue R-250 staining.

The specific activities of SakSTAR solutions were determined with a chromogenic substrate assay carried out in microtiter plates using a mixture of 80 μL SakSTAR solution and 100 μL glutamic acid (Glu) plasminogen solution (final concentration 0.5 μmol/L). After incubation for 30 minutes at 37°C, generated plasmin was quantitated by the addition of 20 μL S2403 (Chromogenix, Mölndahl, Sweden) at a final concentration of 1 mmol/L, and the absorption was measured at 405 nm. The activity was expressed in home units (HU) by comparison with an in-house standard (lot STAN5), which was assigned an activity of 100 000 HU (100 kHU) per mg protein.

The variants SakSTAR(K96C), SakSTAR(K102C), and SakSTAR(K109C) were constructed by the spliced overlap extension PCR (SOE-PCR)19 usingpMEX.SakSTAR7 encoding SakSTAR as a template. We amplified 2 fragments by PCR for 30 cycles, 30 seconds at each temperature: 94°C, 50°C, and 72°C. The first fragment began from the 5′ end (sense external primer 818A) of the staphylokinase gene to the region to be mutagenized (antisense internal primer), and the second fragment began from this same region (sense internal primer) to the 3′ end of the gene with the antisense external primer 818D. The sense and antisense primers, which shared an overlap of around 24 base pair (bp) are summarized in Table 1.

The 2 purified fragments were then assembled together in a second PCR reaction with the external primers 818A and 818D for 30 cycles, 30 seconds at each temperature: 94°C, 50°C, and 72°C. The mutants SakSTAR(K135C) and SakSTAR(K96C, K135C) were directly obtained by a single PCR for 30 cycles, 30 seconds at each of the same temperatures described for external primers 818A and 818D, using pMEX.SakSTAR or pMEX.SakSTAR(K96C), respectively, as a template. LJ522 was used as an antisense primer, and either LJ337 or 818A was used as a sense primer. For each mutant, the amplified product was purified, digested withE coli RY13 (EcoRI) andHaemophilus influenzae Rd (HindIII), and ligated into the corresponding sites of pMEX.SakSTAR. All the constructions were confirmed by sequencing the entireSakSTAR coding region.

The mutants SakSTAR(S3C) and SakSTAR(S2C, S3C) were constructed by PCR using LY459 or LY461 as sense primer and 819D as an antisense primer. PCR products were purified, 3′-end cut with HindIII and cloned inpMEX-SakSTAR digested by StuI andHindIII.

The SakSTAR variants were expressed and purified from transformed E coli TG1 or WK6 cells grown in terrific broth (TB) medium.20 A 2 mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 1 L 7B culture supplemented with 100 μg/mL ampicillin. The culture was incubated with vigorous aeration at 30°C. After about 16 hours incubation, 200 μmol/L IPTG (isopropylthiogalactoside) was added to the culture to boost expression from the tac promoter. After 3 hours of induction, the cells were pelleted by centrifugation at 4 000 rpm for 20 minutes, resuspended in 100 mL of 0.01 mol/L phosphate buffer (pH 5.5-6.0) and disrupted by sonication at 0°C. The suspension was cleared by 20 minutes of centrifugation at 20 000 rpm, and the supernatant was stored at -20°C until purification. The material was purified as follows: Using experimental conditions previously described,7,21 cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6 × 6-cm column of SP-Sephadex. This was followed by chromatography on a 1.6 × 8-cm column of phenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gel electrophoresis, were pooled for further derivatization.

The thiol groups of the cysteine mutants SakSTAR(K96C), SakSTAR(K102C), SakSTAR(K109C), SakSTAR(K135C), SakSTAR(K96C, K135C), SakSTAR(S3C), and SakSTAR(S2C, S3C) were derivatized with ortho-pyridyl-disulfide-PEG (OPSS-PEG) of molecular weight 5 kd (Shearwater Polymers Europe, Enschede, The Netherlands) to yield C-SP5 derivatives of SakSTAR. With the exception of the SakSTAR(K102C) variant, which was obtained exclusively in monomeric form after purification, the Cys substituted variants of SakSTAR (100 μmol/L solution) were subjected to reduction by a 90-minute incubation at 37°C with a 30-fold molar excess of dithiothreitol (DTT) prior to modification. Excess DTT was removed by applying the sample on a PD-10 column (Pharmacia) in a 10 mmol/L phosphate buffer containing 1 mmol/L EDTA (ethylene-diamine tetraacetic acid), pH 8.0, and PEGylation was then directly achieved by reacting the molecule with a 3-fold molar excess of OPSS-PEG at room temperature. The extent of the reaction was monitored by following the release of 2-thiopyridone from OPSS-PEG at 343 nm. After completion of the reaction (<15 minutes), the excess of unreacted OPSS-PEG was removed by purifying the derivatized SakSTAR variants on a 1.6 × 5-cm column of SP-Sephadex.

Alternatively, the thiol group of the cysteine mutant SakSTAR(S3C) was derivatized with maleimide-PEG (MAL-PEG) (Shearwater Polymers Europe, Enschede, The Netherlands). Linear MAL-PEG (MP) of 5 kd, 10 kd, or 20 kd reacted specifically with thiol groups under mild conditions to yield C-MP5, C-MP10, or C-MP20 derivatives of SakSTAR. Modification of the variants was achieved by reduction with DTT, desalting on Sephadex G25, and incubating the molecule (100 μmol/L) with a 3-fold excess of MAL-PEG in 15 mmol/L phosphate buffer, pH 7.9, at room temperature. After reaction (about 60 minutes), excess MAL-PEG was removed by desalting the derivatized SakSTAR variant on Sephadex G25, concentration on a 1 × 6-cm column of SP-Sephadex and gel filtration on a 6 × 60 column of Superdex G75. The fractions containing PEGylated SakSTAR variant, localized by absorbance at 280 nm, were pooled; the protein concentration was adjusted to 1 mg/mL; and the material was sterilized by filtration through a 0.22 μm millipore filter. Homogeneity of the product was assessed by SDS-PAGE analysis.

Equimolar plasminogen-staphylokinase complexes (final concentration 1.1 μmol/L plasminogen/1μmol/L staphylokinase) were prepared by incubation of human plasminogen with SakSTAR or variants at 37°C for 15 minutes in 0.1 mol/L phosphate buffer, pH 7.4. Reaction mixtures were supplemented with 15% glycerol and stored on ice. For kinetic analysis, plasminogen-staphylokinase complexes (final concentration 2 nmol/L) were incubated with various final concentrations of plasminogen (125, 250, or 500 nmol/L) at 37°C in 0.1 mol/L phosphate buffer, pH 7.4. Generated plasmin was measured at different time intervals (0 to 10 minutes) with 0.3 mmol/L S2403 (Chromogenix) after 10-fold dilution of the sample. Initial activation rates were obtained from plots of the concentration of generated plasmin versus time.

Purified preparations of SakSTAR variants were diluted to a concentration of 1 mg/mL in 0.15 mol/L sodium chloride, 0.05 mol/L Tris-HCl (tris(hydroxymethyl) aminomethane hydrochloride) buffer, pH 7.5, and incubated at 37°C, 56°C, and 70°C. Aliquots were removed at different time intervals (1 hour to 5 days), and the residual activity was determined by the plasminogen-coupled chromogenic substrate assay as described previously.21Parallel samples were submitted to a SDS-PAGE analysis to check the integrity of the PEGylated variants.

The fibrinolytic and fibrinogenolytic properties of the SakSTAR variants were determined in human plasma in which a clot, fibrin labeled with 125 iodine (¹²⁵I-fibrin clot), was submerged as previously described.12 

The turnover of the derivatized SakSTAR variants was evaluated in groups of 4 hamsters following intravenous bolus injection over 2 minutes of 100 μg/kg variant. SakSTAR-related antigen in plasma was assayed using the enzyme-linked immunosorbent assay (ELISA) described elsewhere12 and calibrated against the SakSTAR variant to be quantitated. Pharmacokinetic parameters included: initial half-life (in minutes), t1/2α = ln2/α; terminal half-life (in minutes), t1/2β = ln2/β; volume of the central (plasma) compartment (in mL), V_C = dose/(A + B); area under the curve (in μg · min⁻¹ · mL^-1), AUC = A/α + B/β; and plasma clearance (in mL/minutes^-1), ClP = dose/AUC.22 

The PEGylated SakSTAR variants were evaluated in the pulmonary embolism model in hamsters as previously described.23A ¹²⁵I-fibrin platelet-poor human plasma clot corresponding to 50 μL original plasma was injected into the jugular vein; the thrombolytic agents were injected as a bolus over the course of 2 minutes; and 90 minutes after injection, lysis was measured as the difference between the radioactivity initially incorporated into the clot and the residual radioactivity in the lungs and the heart. Fibrinogen and α₂-antiplasmin levels in plasma from blood samples taken before and after the experiment were determined as previously described. Staphylokinase-related antigen concentrations in plasma were determined in blood samples taken at 30 minutes and 90 minutes using a specific ELISA.2 

The immunogenicity of the core-substitution derivatives SakSTAR(K109C-SP5), SakSTAR(K135C-SP5), and SakSTAR(K96C-SP5, K135C-SP5) was studied following intravenous injection in rabbits. Groups of 8 rabbits were allocated to each agent. The thrombolytic potency was measured using 0.3 mL of ¹²⁵I-fibrin platelet-poor rabbit plasma clots, inserted into an extracorporeal arteriovenous loop, as described elsewhere.12 Using a bolus injection over the course of 2 minutes, the rabbits were immunized with 400 μg/kg wild type or variant SakSTAR at week 0 (to determine the baseline clot lysis capacity). This was followed by intravenous administration of 400 μg/kg of the same agent at weeks 2, 3, and 5. At week 6, the humoral antibody response was quantitated by determination of the staphylokinase variant-neutralizing activity in plasma. The rabbits were again treated with the same SakSTAR variant as the one used for their immunization (400 μg/kg, bolus injection over 2 minutes), and during the next 2 hours, the time course of clot lysis was monitored continuously by external gamma counting. 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 following the guiding principles of the American Physiological Society and the International Committee on Thrombosis and Hemostasis.24 

Data are expressed as mean values ± SEM (standard error of the mean) or as median and ranges. Significance levels were determined by paired or unpaired Student's t test, in the case of Gaussian distributions, or by the Mann-Whitney U test, in the case of non-Gaussian distributions. Statistical significance was indicated with 2-tailed P < .05.

Site-directed mutagenesis was used to substitute 4 exposed lysine residues in the core of staphylokinase (Lys 96, Lys 102, Lys 109, and Lys 135) with cysteines and to generate 4 mono-PEGylated SakSTAR variants derivatized with OPSS-PEG, a 5-kd PEG molecule that carries a single activated thiol group at one end. This thiol group reacts specifically at slightly alkaline pH with free thiols, thereby yielding SakSTAR(K96C-SP5), SakSTAR(K102C-SP5), SakSTAR(K109C-SP5), and SakSTAR(K135C-SP5) and 1 bis-PEGylated variant, SakSTAR(K96C-SP5, K135C-SP5). In addition, 1 or 2 serine residues in the NH₂-terminal region of the staphylokinase molecule (Ser2 and/or Ser3), which are released during activation, were substituted with cysteines and derivatized with OPSS-PEG to generate SakSTAR(S3C-SP5) and SakSTAR(S2C-SP5, S3C-SP5). The purified cysteine variants were reduced to monomers with DTT prior to derivation with OPSS-PEG. Alternatively, purified SakSTAR(S3C) was derivatized with linear MAL-PEG molecules with molecular weights of 5 000, 10 000, or 20 000. The MAL-PEG molecules carried a reactive maleimide group, which yielded SakSTAR(S3C-MP5), SakSTAR(S3C-MP10), and SakSTAR(S3C-MP20).

The specific activities of the purified PEGylated SakSTAR variants are summarized in Table 2. SDS-PAGE (15% nonreducing gel) confirmed that PEG modification was nearly quantitative (Figure 1). The apparent molecular weights were about 25 000 for the derivatives with P5 and about 36 000 for the bis-PEGylated derivatives. MAL-PEG substituted variants migrated at apparent approximate molecular weights of 30 000 (MP5), 36 000 (MP10), and 50 000 (MP20), respectively. The relatively lower mobility of PEGylated proteins is well-known and is probably due to hydration of the polymer.25Additionally, minor amounts of unPEGylated monomers (molecular weight of 16 000) or SakSTAR homodimers (molecular weight of 32 000) can be observed for all variants and is probably due to the presence of copurified unPEGylated SakSTAR variant.

The catalytic efficiencies for plasminogen activation (k_cat/K_M) of the variants were similar to that of wild type SakSTAR (0.13 to 0.29 μmol/L^-1s^-1 as compared to 0.21 μmol/L^-1s^-1), except for SakSTAR(K96C-SP5, K135C-SP5) and SakSTAR(K135C-SP5) , which had a catalytic efficiency that was 7 to 10 times lower (Table 2). The reduced catalytic efficiency of the K135 mutants was not due to reduced binding to human plasmin, as determined by ELISA (not shown), but probably to steric hindrance of the binding of plasminogen substrate to the active SakSTAR:plasmin complex.

Dose- and time-dependent lysis of ¹²⁵I-fibrin human clots submerged in human plasma were obtained for all derivatives; spontaneous clot lysis during the experimental period was ≤5% (not shown). Equally effective concentrations of test compound (C₅₀, causing 50% clot lysis in 2 hours), determined graphically from plots of clot lysis at 2 hours versus the concentration of plasminogen activator, were 0.26 μg/mL for wild type SakSTAR and between 0.18 and 0.45 μg/mL for the variants. The exceptions to this range were SakSTAR(K96C-SP5, K135C-SP5) and SakSTAR(K135C-SP5), both of which had a 10-fold lower clot lysis activity (Table 2).

PEGylation only marginally affected the thermal stability of the variants because all compounds remained fully active for up to 3 days at 37°C and for more than 5 hours at 56°C (data not shown). SDS-PAGE revealed that the disulfide bonds introduced with OPSS-PEG (SP5 variants) were, however, less stable than the thioether bonds introduced with MAL-PEG (MP variants) (Figure2).

The disposition rate of staphylokinase-related antigen from blood following bolus injection of 100 μg/kg of the SakSTAR variants in groups of 4 hamsters could adequately be described by a sum of 2 exponential terms by graphical curve peeling (not shown). The derived pharmacokinetic parameters for the PEGylated compounds are summarized in Table 3. The plasma clearances (Clp) of the variants substituted with P5 were reduced 2-to 5-fold, whereas the variants with 2 P5 or a P10 molecule had a 10-to 30-fold lower plasma clearance. The clearances of the NH₂-terminal substitution variants derivatized with MAL-PEG molecules were inversely proportional to the molecular weight of the PEG molecules, with a reduction of 2.3-fold with P5, 11-fold with P10, and 37-fold with P20 (Table 3).

The thrombolytic properties of the PEGylated staphylokinase variants were evaluated following bolus injection in a hamster pulmonary embolism model and compared to SakSTAR. Saline bolus injection yielded, at 90 minutes, a value for spontaneous lysis of 20 ± 3% (mean ± SEM, n = 9), as depicted in Table4. Fibrinogen levels at the end of the experiment were 110 ± 6% of the baseline value, and α₂-antiplasmin levels were 120 ± 6%. All SakSTAR variants induced dose-related pulmonary clot lysis. Ninety minutes after the SakSTAR bolus, lysis increased from 34 ± 6% with 27 μg/kg to 86 ± 4% with 240 μg/kg, which corresponded to a C₅₀ value of 110 μg/kg. SakSTAR(K135C-SP5) was nearly equipotent to wild type SakSTAR; SakSTAR(K96C-SP5), SakSTAR(K102C-SP5), and SakSTAR(K109C-SP5) had 2-fold lower C₅₀ values; and SakSTAR(K96C-SP5, K135C-SP5) had a nearly 4-fold higher thrombolytic potency. SakSTAR(S3C-SP5) had a 5.2-fold lower C₅₀ than wild type SakSTAR, and the C₅₀ of SakSTAR(S2C-SP5, S3C-SP5) was 18-fold lower. Finally, relatively higher thrombolytic potencies were observed with NH₂-terminal substitution variants derivatized with MAL-PEG, and C₅₀ values were 4.6-fold, 6.5-fold, and 16-fold lower for the P5, P10, and P20 derivatives, respectively. Fibrinogen and α₂-antiplasmin levels did not decrease following bolus administration of any of the agents, and at 90 minutes, the residual plasma staphylokinase-related antigen level increased proportionally to the dose and inversely proportionally to the plasma clearance (Table 4).

The immunogenic properties of the core-substitution variants SakSTAR(K109C-SP5), SakSTAR(K135C-SP5), and SakSTAR(K96C-SP5, K135C-SP5) were tested in groups of 8 rabbits and compared to wild type SakSTAR. For each agent, the baseline thrombolytic potency was assessed by monitoring the course of clot lysis following a bolus intravenous administration of 400 μg/kg compound over 2 minutes. The results, summarized in Table 5, show that in this rabbit model, covalent attachment of 1 PEG molecule on staphylokinase at position 109, yielding SakSTAR(K109C-SP5), did not affect its thrombolytic properties, while the extent of lysis reached with SakSTAR(K135C-SP5) and SakSTAR(K96C-SP5, K135C-SP5) was slightly lower (36% and 40%, respectively) than with SakSTAR wild type (53%).

The immunogenicity of the compounds was estimated by monitoring the neutralizing activity and residual thrombolytic potency at week 6. High neutralizing antibody titers were elicited in all 8 rabbits treated with wild type SakSTAR, and no residual thrombolytic potency was obtained at 6 weeks. None of the mono-PEGylated variants induced significantly less neutralizing antibodies or were associated with a significantly higher residual thrombolytic potency at 6 weeks. With SakSTAR(K135C-SP5) the median (range) neutralizing activity at 6 weeks was 9 (0 to 27) μg compound neutralized per mL plasma and the residual thrombolytic potency was 5 (3 to 47) percent as compared to 20 (5 to 53) μg/mL and 3 (2 to 6) percent for wild type SakSTAR (P = .08 each by Mann Whitney U test). With bis-PEGylated SakSTAR(K96C-SP5, K135C-SP5), no significant reduction of the immunogenicity was observed either.

Covalent attachment of PEG molecules or derivatives to a variety of proteins has been shown to prolong their circulatory half-life and to reduce their immunological reactivity. PEGylated adenosine deaminase (PEG-ADA)26 has been used since 1991 to treat children with severe combined immunodeficiency, while several other PEGylated proteins are currently under development for therapeutic use.14 Since the initial work of Abuchowski et al,15,16 PEG molecules are usually covalently attached to proteins via the ε-amino group of lysine residues.

In the present study, PEG derivatives of staphylokinase, a highly fibrin-selective thrombolytic agent, were obtained by chemical coupling at a genetically introduced cysteine(s). Indeed, since wild type staphylokinase does not contain cysteine but as many as 20 lysines, this strategy was preferred over the more common amine-directed conjugation. This allowed better control of the extent of modification and maintenance of activity. Specifically, 1 or 2 exposed lysine residues, previously shown to be antigenic in man,27 were substituted with cysteines and modified with a 5-kd thiol-specific OPSS-PEG molecule to yield 4 mono-PEGylated variants, SakSTAR(K96C-SP5), SakSTAR(K102C-SP5), SakSTAR- (K109C-SP5), and SakSTAR(K135C-SP5), and 1 bis-PEGylated protein, SakSTAR(K96C-SP5, K135C-SP5). The substitution, derivation, and purification strategies yielded homogeneous end products with essentially intact enzymatic properties (except for the 2 K135C mutants). The PEGylated variants were, however, 2-to 10-fold less potent in an in vitro clot lysis assay. Attachment of a polymer molecule to SakSTAR thus appears to attenuate the fibrinolytic potency, possibly because of its reduced diffusion rate within the fibrin clot. This is in agreement with the decreased electrophoretic mobility of the variants observed in SDS-PAGE. Alternatively, 1 or 2 amino acids (Ser2 and/or Ser3) that are located in the NH₂terminal region, which is removed during activation of staphylokinase,28 were substituted by cysteine and coupled with a 5-kd PEG molecule to yield mono-PEGylated SakSTAR(S3C-SP5) and bis-PEGylated SakSTAR(S2C-SP5, S3C-SP5). These PEGylated variants were nearly equipotent to SakSTAR in the in vitro clot lysis assay, which is possibly a result of the PEG removal during activation at the clot surface.

The plasma clearance in hamsters of variants with 1 P5 molecule was reduced 2-to 6-fold and associated with up to 2-fold increased thrombolytic potencies following bolus injection. These apparent discrepancies between in vivo and in vitro potencies are probably due to the fact that increasing the Stokes radius reduces the diffusion rate within the clot; in vivo, this is overcompensated for by the much longer circulatory half-life of the agent. A similar mechanism might explain the several-fold higher potency of the bis-PEGylated variants. The contradictory results obtained for the K135C mutants, which show a very poor activity in vitro (Table 2) while exerting relatively good thrombolytic activity in animals (Tables 4 and 5), could also be explained by an overcompensatory effect of the prolonged plasma half-life induced by PEGylation. Again, the NH₂-terminal substitution variants appeared to have a somewhat higher specific thrombolytic potency, possibly due to the removal of the PEG during processing at the clot surface. Upon prolonged incubation at 37°C, however, the PEG molecules were released (Figure 2), presumably by disruption of the disulfide bridge or of the PEGylated NH₂ terminal decapeptide; the result was active but unPEGylated staphylokinase variants.

Derivatization of the NH₂-terminal substitution variant SakSTAR(S3C) with MAL-PEG yielded compounds with clearances that were inversely proportional and thrombolytic potencies that were directly proportional to the molecular weight of the PEG substituent. These derivatives were more resistant against release of the PEG substituent upon incubation at 37°C.

Derivatization of SakSTAR with 1 or 2 PEG molecules does not significantly decrease the immunoreactivity of staphylokinase in rabbits, although a trend toward a higher mean lysis and a lower mean antibody titer at week 6 was obtained with SakSTAR(K135C-SP5) (Table5). These results suggest that the introduction of a polyethylene glycol at position 135 might slightly reduce the immunogenicity. This is in line with the results of the epitope mapping.27 The difference in immunogenic properties between SakSTAR(K135C-SP5) and SakSTAR(K96C-SP5, K135C-SP5) might then be due to the longer circulatory half-life of the latter compound, eliciting an increased humoral response. In view of the rapidly decreasing clearance with increasing molecular weight or number of PEG molecules added, it appears that not enough linear PEG molecules have been added to staphylokinase to significantly alter its immunogenicity without excessive clearance reduction. Possibly, branched PEG molecules might have different relative effects on clearance versus immunogenicity.

In conclusion, the present study suggests that introduction of a single PEG molecule at selected positions may alter the pharmacokinetic properties of staphylokinase without affecting its activity. In the preferred embodiment, the PEG molecules are linked by “maleylation” to cysteines introduced in the NH₂-terminal part of the molecule to yield stable, fully active molecules with a clearance inversely proportional to the molecular weight of the PEG substitutent. Such PEGylated proteins would appear to be suitable for treatment of acute myocardial infarction by single intravenous bolus administration.

The authors thank Frans De Cock, Berthe Van Hoef, and Huberte Moreau for their skillful technical assistance.