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(Circulation. 1997;95:455-462.)
© 1997 American Heart Association, Inc.

Articles

Recombinant Staphylokinase Variants With Altered Immunoreactivity

III: Species Variability of Antibody Binding Patterns

Desire Collen, MD, PhD; Frans De Cock; Eddy Demarsin; Stephane Jenne, MSc; Ignace Lasters, PhD; Yves Laroche, PhD; Petra Warmerdam, PhD; Laurent Jespers, PhD

the Center for Molecular and Vascular Biology, University of Leuven, and the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, 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

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Background The "charged cluster–to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%. Therefore, the effect of the reversal of one or more of these substituted amino acids on the ratio of activity to antigenicity was studied.

Methods and Results Fourteen mutants with one to four "alanine-to–wild-type" reversals were expressed in Escherichia coli and highly purified (>95%). In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodominant residue could be identified. In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epitopes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent. In pooled plasma from patients with peripheral arterial occlusion treated with wild-type SakSTAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R77 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible contribution toward antibody recognition.

Conclusions The recognition pattern by SakSTAR variants of antibodies induced with wild-type SakSTAR differs markedly among species. This implies that a systematic evaluation of reduced antigen recognition and antibody induction in humans will require the development of human or humanized systems. Surprisingly, SakSTAR(K₇₄), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies induced in patients by treatment with wild-type SakSTAR.

Key Words: thrombolysis • infarction • plasminogen activators

	Introduction

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Wild-type staphylokinase (SakSTAR variant¹ ) contains three nonoverlapping immunodominant epitopes, two of which can be eliminated by specific site-directed substitution of clusters of two (K35A,E38A or E80A,D82A) or three (K74A,E75A,R77A) charged amino acids with Ala.² The combination mutants SakSTAR(K35A,E38A,K74A,E75A,R77A), in which Lys³⁵, Glu³⁸, Lys⁷⁴, Glu⁷⁵, and Arg⁷⁷ were substituted with Ala, and SakSTAR(K74A,E75A,R77A,E80A,D82A), in which Lys⁷⁴, Glu⁷⁵, Arg⁷⁷, Glu⁸⁰, and Asp⁸² were substituted with Ala (previously identified as SakSTAR.38 and SakSTAR.M89, respectively² ), were found to have a reduced reactivity with murine MAbs against two of the three immunodominant epitopes and to absorb on average only two thirds of the neutralizing antibodies elicited in 16 patients by treatment with wild-type SakSTAR.² These mutants also induced less antibody formation than wild-type SakSTAR in rabbit and baboon models and in patients with peripheral arterial occlusion.^{3 4} However, their specific activities were significantly lower (reduced by 50%) than that of wild-type SakSTAR, and they displayed a reduced temperature stability,² which would be of concern with respect to the clinical utility of these compounds.

In an effort to improve the activity and stability without loss of the reduced antibody recognition, the effect of a systematic reversal of one or more of these substituted amino acids to the wild-type residues was studied. Fourteen mutants were constructed, purified, and characterized in terms of specific activity, reactivity with the panel of murine MAbs, and absorption of antibodies from plasma of rabbits and baboons immunized with SakSTAR and of patients treated with wild-type SakSTAR. On the basis of the results, four variants were selected for large-scale purification and conditioning for subsequent use in vivo.

The present study focused on reversal from alanine to the wild-type residue of one or more of the seven amino acids of SakSTAR listed above, ie, Lys³⁵, Glu³⁸, Lys⁷⁴, Glu⁷⁵, Arg⁷⁷, Glu⁸⁰, and Asp⁸². The mutants are identified by the substituted amino acids in the order of their sequence in the molecule, with addition in subscript of those sequence numbers required to avoid ambiguity. For example, SakSTAR(K35A,E38A,K74A,E75A,R77A) is identified as SakSTAR(KEKER) and SakSTAR(K74A,E75A,R77A) as SakSTAR(K₇₄ER).

	Methods

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Reagents
The source of all reagents used in the present study has been reported previously.² pMEX.SakB (ie, pMEX.SakSTAR) was constructed as described elsewhere.⁵ SakSTAR, SakSTAR(KE₃₈), SakSTAR(K₇₄ER), SakSTAR(E₈₀D), SakSTAR(KEKER), and SakSTAR(K₇₄ERED) were produced and purified as described elsewhere.^{2 6}

DNA Preparation, Manipulation, and Sequencing
Plasmid DNA was isolated by use of the Qiagen plasmid kit (Hilden). All other methods used in the present study have been described.^{2 7}

Construction, Production, and Purification of SakSTAR Variants
The plasmids encoding SakSTAR(KEKE₇₅), SakSTAR(EER), SakSTAR(EE₇₅), SakSTAR(K₃₅E₇₅R), SakSTAR(K₃₅E₇₅), SakSTAR(E₈₀), SakSTAR(D), SakSTAR(E₇₅D), SakSTAR(K₇₄), and SakSTAR(E₇₅) were constructed by the spliced overlap extension polymerase chain reaction⁸ with Vent DNA polymerase (New England Biolabs) and available or generated SakSTAR variants used as template. Two fragments were amplified by PCR, the first one starting from the 5' end of the staphylokinase gene with primer 5'-CAGGAAACAGAATTCAGGAG-3' to the region to be mutagenized (forward primer), the second one from the same region (backward primer) to the 3' end of the staphylokinase gene with primer 5'-CAAAACAGCCAAGCTTCATTCATTCAGC-3'. The forward and backward primers shared an overlap of around 24 bp (primers not shown). The two purified fragments were then assembled together in a new primerless PCR using Taq polymerase (Boehringer Mannheim). After 7 cycles (1 minute at 94°C, 1 minute at 70°C), the extended product was reamplified by adding the 5' and 3' end primers (see above) to the PCR reaction and cycling 25 times (1 minute at 94°C, 1 minute 55°C, 1 minute at 72°C). The final product was purified, digested with EcoRI and HindIII, and cloned into the corresponding sites of pMEX.SakB.

The plasmid encoding SakSTAR(EKER) was assembled by digestion of pMEX.SakB and pMEX.SakSTAR(KEKER) with Bpm I, which cuts between the codons for K35 and E38 of SakSTAR, and ligation of the required fragments. The plasmid encoding SakSTAR(KKER) was constructed by digestion of pMEX.SakSTAR(KEKER) and pMEX.SakSTAR(K₇₄ER) with Bpm I and relegation of the required fragments. The plasmids encoding SakSTAR(KEER) and SakSTAR(KEKR) were constructed by two PCRs with pMEX.SakSTAR(KEKER) as template, followed by restriction ligation and recloning into pMEXSakB.

Analytical Purification of SakSTAR Variants
The SakSTAR variants were expressed and purified as described below from transformed Escherichia coli WK6 grown either in LB medium [SakSTAR(EKER), SakSTAR(K₇₄), SakSTAR(E₇₅), and SakSTAR(E₇₅D)] or in TB⁹ medium [SakSTAR(KKER), SakSTAR(KEER), SakSTAR(KEKR), SakSTAR(KEKE₇₅), SakSTAR(EER), SakSTAR(EE₇₅), SakSTAR(K₃₅E₇₅R), SakSTAR(K₃₅E₇₅), SakSTAR(E₈₀), and SakSTAR(D)].

For derivatives produced in LB medium, a 20-mL aliquot of an overnight saturated culture was used to inoculate a 2-L volume (in a 5-L flask) of LB medium containing 100 µg/mL ampicillin. After 3 hours' incubation at 37°C, IPTG (200 µmol/L) was added to induce expression from the tac promoter. The production phase was allowed to proceed for 4 hours, after which the cells were pelleted by centrifugation at 4000 rpm for 20 minutes, resuspended in 1/20 vol (100 mL) of 0.01 mol/L phosphate buffer, pH 6.5, and disrupted by sonication at 0°C. Cell debris was removed by centrifugation for 20 minutes at 20 000 rpm, and the supernatant, containing the cytosolic soluble protein fraction, was stored at -20°C until purification.

For the derivatives produced in TB medium, a 4-mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 2-L culture (in a 5-L flask) in TB containing 100 µg/mL ampicillin. The culture was grown with vigorous aeration for 20 hours at 30°C. The cells were pelleted by centrifugation, resuspended in 1/10 vol (200 mL) of 0.01 mol/L phosphate buffer, pH 6.5, and disrupted by sonication at 0°C. The suspension was then centrifuged for 20 minutes at 20 000 rpm, and the supernatant was stored at -20°C until purification.

Cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6x6-cm column of SP-Sephadex, followed by chromatography on a 1.6x5-cm column of Q-Sepharose [variants SakSTAR(EKER), SakSTAR(KKER), SakSTAR(KEER), SakSTAR(KEKR), and SakSTAR(KEKE₇₅)] or by chromatography on a 1.6x6-cm column of phenyl-Sepharose [variants SakSTAR(EER), SakSTAR(EE₇₅), SakSTAR(K₃₅E₇₅R), SakSTAR(K₃₅E₇₅), SakSTAR(K₇₄), SakSTAR(E₇₅), SakSTAR(E₈₀), SakSTAR(D), and SakSTAR(E₇₅D)]. The SakSTAR-containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis.

Preparative Purification of SakSTAR Variants for Use In Vivo
A 12- to 24-L culture (in 2-L batches) of the variant SakSTAR(K₇₄), SakSTAR(E₇₅), or SakSTAR(K₇₄ER) was grown and IPTG-induced in LB medium supplemented with 100 µg/mL ampicillin, pelleted, resuspended, disrupted by sonication, and cleared as described above. A 30-L culture (in 2-L batches) of the variant SakSTAR(EER) was grown in TB supplemented with 100 µg/mL ampicillin at 30°C for 20 hours. The cells were pelleted, resuspended in 2500 mL 0.01 mol/L phosphate buffer, pH 6.0, disrupted by sonication, and cleared as described above.

The compounds were purified by chromatography on a 5x20-cm column of SP-Sephadex, a 5x10-cm column of Q-Sepharose, and/or a 5x13-cm column of phenyl-Sepharose with buffer systems described elsewhere.⁵ The materials were then gel-filtered on sterilized Superdex 75 to further reduce their endotoxin content. The SakSTAR variant–containing fractions 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.

Physicochemical Analysis of SakSTAR Variants
The methodology used to determine specific activity and temperature stability has been described in detail previously.²

Fibrinolytic Properties of SakSTAR Variants in Human Plasma In Vitro
The fibrinolytic and fibrinogenolytic properties of the SakSTAR variants was determined as described previously.³

Reactivity of SakSTAR Variants With a Panel of Murine MAbs
The epitope specificity of a panel of 15 murine MAbs raised against wild-type SakSTAR was determined by real-time biospecific interaction analysis (BIA) with the BIAcore instrument (Pharmacia, Biosensor AB).¹⁰ The MAbs were immobilized on the surface of Sensor Chip CM5 with the Amine Coupling kit (Pharmacia Biosensor AB) as recommended by the manufacturer.¹¹ Immobilization was performed from protein solutions at a concentration of 20 µg/mL in 10 mmol/L sodium acetate at pH 5.0 at a flow rate of 5 µL/min during 6 minutes. This resulted in covalent attachment of 5000 to 10 000 RU of antibody (corresponding to 0.035 to 0.07 pmol/mm²).¹² The SakSTAR solutions were passed by continuous flow at 20°C past the sensor surface. At least four concentrations of each analyte (range, 50 nmol/L to 50 µmol/L) in 10 mmol/L HEPES, 3.4 mmol/L EDTA, 0.15 mol/L NaCl, and 0.005% Surfactant P20, pH 7.2, were injected at a flow rate of 5 µL/min during 6 minutes in the association phase. Then sample was replaced by buffer, also at a flow rate of 5 µL/min during 6 minutes. After each cycle, the surface of the sensor chip was regenerated by injection of 5 µL of 15 mmol/L HCl. Apparent association (k_ass) and apparent dissociation (k_diss) rate constants were derived from the sensorgrams as described in detail elsewhere.¹³

Absorption With SakSTAR Variants of Antibodies Elicited in Rabbits and Baboons by Immunization and in Patients by Treatment With Wild-Type SakSTAR
Plasma samples from 6 rabbits and those from 6 baboons obtained at 6 weeks after immunization with wild-type SakSTAR^{3 14} were pooled separately. The immunization consisted of infusion of 400 or 65 µg/kg SakSTAR IV, followed by subcutaneous injections of 400 µg SakSTAR moiety in Freund's adjuvant at weeks 2, 3, and 5.^{3 4} Plasma samples from 10 patients with acute myocardial infarction obtained several weeks after treatment with SakSTAR^{5 15 16} were used. Three plasma pools were made, one from all 10 patients, one from the 3 patients that absorbed <50% of the antibodies with SakSTAR(KEKER) (subpool B), and one from the 3 patients that absorbed >90% of the antibodies with SakSTAR(KEKER) (subpool C).

These plasma pools were diluted (1/30 to 1/200) until their binding to SakSTAR-substituted chips in the BIAcore instrument amounted to 2000 RU. From this dilution, a calibration curve for antibody binding was constructed with further serial twofold dilutions. The plasma pools were absorbed for 10 minutes with 100 nmol/L of the SakSTAR variants, and residual binding to immobilized SakSTAR was determined. Residual binding was expressed in percentage of unabsorbed plasma by use of the calibration curve.

ELISA for Staphylokinase-Related Antigen
SakSTAR-related antigen was assayed with the ELISA described elsewhere.³ The ELISA was calibrated against each of the SakSTAR variants to be quantified.

Biological Analysis of SakSTAR Variants for Use In Vivo
Endotoxin contamination, bacterial sterility, and acute toxicity in mice of SakSTAR(K₇₄), SakSTAR(E₇₅), SakSTAR(EER), and SakSTAR(K₇₄ER) were evaluated as previously described.³ Purity of the preparation was evaluated by HPLC with a KW802.5 gel filtration column (Shodex, Showa Denko) in PBS and by SDS-PAGE on 10% gels to which 40 µg of compound per lane was applied.

Pharmacokinetics of SakSTAR Variants After Intravenous Bolus Injection in Hamsters
The pharmacokinetic parameters of the disposition of SakSTAR variants from blood were evaluated in groups of 4 to 6 hamsters after bolus injection of 100 µg/kg IV wild-type SakSTAR or SakSTAR(K₇₄), SakSTAR(E₇₅), SakSTAR(EER), or SakSTAR(K₇₄ER) variants, as previously described.³ Pharmacokinetic parameters 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); AUC (in ng·min^-1·mL^-1), AUC=A/+B/ß; and plasma clearance (in mL·min^-1), Cl_p=dose/AUC.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Purification and Characterization of SakSTAR Variants
The recovery of protein from batches of 2 to 4 L of E. coli–conditioned culture broth of the 14 variants specifically constructed for the present study was 12 to 120 mg, and the final materials were obtained at a concentration of 0.5 to 4 mg/mL, with a specific activity of 19 000 to 170 000 U/mg. SDS-PAGE under nonreducing or reducing conditions (Fig 1) revealed the presence of single components or doublets with molecular weights of 16 000. The doublet bands are characteristic for partial removal of the NH₂-terminal 6 or 10 amino acids, as described in detail elsewhere.¹⁴

View larger version (53K): [in this window] [in a new window]   Figure 1. SDS-PAGE of 1-µg samples of analytical preparations of SakSTAR moieties. Lanes 2, SakSTAR; 3, SakSTAR(KE38); 4, SakSTAR(K74ER); 5, SakSTAR(KEKER); 6, SakSTAR(EKER); 7, SakSTAR(KKER); 8, SakSTAR(KEER); 9, SakSTAR(KEKR); 10, SakSTAR(KEKE75); 11, SakSTAR(EER); 12, SakSTAR(EE75); 13, SakSTAR(K35E75R); 14, SakSTAR(K35E75); 15, SakSTAR(K74); 16, SakSTAR(E75); 17, SakSTAR(K74ERED); 18, SakSTAR(E80); 19, SakSTAR(D); and 20, SakSTAR(E75D). Lanes 1 and 21 represent a protein calibration mixture comprising phosphorylase b (Mr, 94 000), BSA (Mr, 67 000), ovalbumin (Mr, 43 000), carbonic anhydrase (Mr, 30 000), soybean trypsin inhibitor (Mr, 20 000), and -lactalbumin (Mr, 14 000).

Fibrinolytic Properties of SakSTAR Variants Toward Human Plasma In Vitro
Dose- and time-dependent lysis of ¹²⁵I-fibrin–labeled human plasma clots submerged in human plasma was obtained with all variants tested (Table 1). Spontaneous clot lysis during the experimental period was 5% (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 (not shown), ranged from 0.3 to 0.4 µg/mL for SakSTAR, SakSTAR(K₇₄), and SakSTAR(E₇₅) to 0.9 to 1.0 µg/mL for SakSTAR(KEKER) and SakSTAR(K₇₄ERED) (Table 1).

View this table: [in this window] [in a new window]   Table 1. Fibrinolytic Properties of SakSTAR Variants in Human Plasma In Vitro

The concentrations of compound causing 50% fibrinogen degradation in 2 hours in human plasma in the absence of fibrin were determined graphically from dose-response curves (not shown). These values (mean±SD of three independent experiments) ranged from 23 to 130 µg/mL (Table 1).

Reactivity of SakSTAR Variants With Murine MAbs
Determination of the equilibrium association constants for the binding of wild-type and variant SakSTAR to insolubilized MAbs (Table 2) yielded apparent association constants of 10⁷ to 10⁸ (mol/L)^-1, which are one to two orders of magnitude lower than the apparent association constants previously obtained for the binding of these MAbs to insolubilized wild-type SakSTAR.² If the MAbs instead of the SakSTAR variants are insolubilized, avidity effects of the bivalent MAbs are avoided. These values are indeed in better agreement with known association constants of MAbs.

View this table: [in this window] [in a new window]   Table 2. Apparent Equilibrium Association Constants (KAx107 [mol/L]-1) for the Binding of SakSTAR Variants to Insolubilized Murine MAbs

In agreement with previous observations,² SakSTAR(K₇₄ER) did not react with 4 of the 5 MAbs constituting epitope cluster I, whereas SakSTAR(KE₃₈) did not react with 3 of the 5 and SakSTAR(E₈₀D) not with 4 of the 5 MAbs constituting epitope cluster III. These reduced reactivities were additive in SakSTAR(KEKER) and in SakSTAR(K₇₄ERED).

The reduced reactivity of SakSTAR(K₇₄ER) was fully maintained in SakSTAR(KEKE₇₅) and in SakSTAR(K₃₅E₇₅R), largely in SakSTAR(KEER), SakSTAR(EER), SakSTAR(EE₇₅), and SakSTAR(E₇₅), but much less in SakSTAR(KEKR) and SakSTAR(K₇₄), indicating that Glu⁷⁵ is the main contributor to the binding of the 4 MAbs of cluster I to SakSTAR. However, surprisingly, binding of epitope cluster I antibodies to SakSTAR(E₇₅D) was normal in two independent preparations from expression plasmids with confirmed DNA sequences.

The reduced reactivity of the 3 MAbs of cluster III with SakSTAR(KE₃₈) required both Lys³⁵ and Glu³⁸, as demonstrated with SakSTAR(EKER) and SakSTAR(KKER), with SakSTAR(EE₇₅) and SakSTAR(K₃₅E₇₅), and with SakSTAR(EER) and SakSTAR(K₃₅E₇₅R). The reduced reactivity of the 4 MAbs of cluster III with SakSTAR(E₈₀D) was maintained in SakSTAR(D) but not in SakSTAR(E₈₀).

Absorption With SakSTAR Variants of Antibodies Elicited in Rabbits and Baboons
Pooled plasma from 6 rabbits immunized with SakSTAR contained antibodies that were absorbed for >65% by all SakSTAR variants studied (Table 3). The combination variants SakSTAR(KEKER) and SakSTAR(K₇₄ERED) recognized 71% and 67% of the antibodies, and this reduced recognition was essentially maintained in SakSTAR(EER) and SakSTAR(E₈₀D). However, no clear pattern of immunodominance of a specific amino acid or of any of the three charge clusters emerged.

View this table: [in this window] [in a new window]   Table 3. Absorption With SakSTAR Variants of Antibodies Elicited With Wild-Type SakSTAR in Rabbits and Baboons and in Patients With Acute Myocardial Infarction

Pooled plasma from 6 baboons immunized with SakSTAR contained antibodies that recognized each of the three charge clusters (on average, 30%), with nearly additive recognition in the combination variants (on average, 50%). However, no single immunodominant residue could be identified.

Absorption With SakSTAR Variants of Antibodies Elicited in Patients by Treatment With Wild-Type SakSTAR
Whereas wild-type SakSTAR absorbed >95% of the binding antibodies from pooled plasma of 10 patients, incomplete absorption (<60%) was observed with SakSTAR(K₇₄ER), SakSTAR(KEKER), SakSTAR(EKER), SakSTAR(KKER), SakSTAR(KEKR), SakSTAR(KEKE₇₅), SakSTAR(K₇₄), and SakSTAR(K₇₄ERED), but absorption was nearly complete with SakSTAR(KE₃₈), SakSTAR(KEER), SakSTAR(EER), SakSTAR(E₃₈E₇₅), SakSTAR(K₃₅E₇₅R), SakSTAR(E₈₀D), SakSTAR(E₈₀), and SakSTAR(D). These results, surprisingly, demonstrate that 40% of the antibodies elicited in patients by treatment with wild-type SakSTAR depend on K74 for their binding (Table 3). Absorption with pooled plasma from 3 patients from whom <50% of the antibodies were absorbed with SakSTAR(KEKER) (subpool B) confirmed the predominant role of Lys⁷⁴ for antibody recognition. As expected, absorption with pooled plasma from 3 patients from whom >95% of the antibodies were absorbed with SakSTAR(KEKER) (subpool C) was nearly complete with all variants tested.

Large-Scale Purification and Conditioning of SakSTAR Variants for Use In Vivo
Of culture volumes of 12 to 30 L SakSTAR(K₇₄), SakSTAR(E₇₅), SakSTAR(EER), and SakSTAR(K₇₄ER), 120 to 540 mg of purified material was obtained, with specific activities of 85 000 to 125 000 U/mg. Gel filtration on HPLC revealed a single main symmetrical peak in the chromatographic range of the column, representing >98% of the eluted material (total AUC) (not shown). SDS-PAGE of 40-µg samples (Fig 2) revealed single main components and small amounts of dimers and trimers, as confirmed by Western blotting (not shown). The endotoxin content ranged between 1.3 and 11 IU/mg, which represents a >10⁶-fold separation from the SakSTAR compound compared with the culture broth (containing >100 000 IU/mL). Preparations sterilized by filtration proved to be sterile on 3-day testing as described in "Methods." Intravenous bolus injection of SakSTAR variants in groups of 5 mice (3 mg/kg body wt) did not provoke any acute reaction or reduced weight gain within 8 days, in comparison with mice given an equal amount of saline (not shown).

View larger version (70K): [in this window] [in a new window]   Figure 2. SDS–10% PAGE of 40-µg samples of preparative batches of SakSTAR moieties conditioned for use in vivo. Lanes 1, SakSTAR; 2, SakSTAR(K74); 3, SakSTAR(K74ER); and 4, protein calibration mixture comprising BSA (Mr, 98 000), glutamic dehydrogenase (Mr, 64 000), alcohol dehydrogenase (Mr, 50 000), carbonic anhydrase (Mr, 36 000), myoglobin (Mr, 30 000), lysozyme (Mr, 16 000), and aprotinin (Mr, 6000).

The stability of SakSTAR(K₇₄), SakSTAR(E₇₅), SakSTAR(EER), and SakSTAR(K₇₄ER) solutions at various temperatures is illustrated in Fig 3. At temperatures up to 37°C, all variants remained fully active for at least 24 hours. At 56°C and 70°C, the SakSTAR variants were somewhat but not dramatically less stable than wild-type SakSTAR. For comparison, results previously obtained with SakSTAR(KEKER) (identified as SakSTAR.M38) are included in Fig 3.

View larger version (29K): [in this window] [in a new window]   Figure 3. Temperature stability of SakSTAR (A), SakSTAR(K74) (B), SakSTAR(E75) (C), SakSTAR(EER) (D), SakSTAR(K74ER) (E), and SakSTAR (KEKER) (F). indicates at 0°C; , 22°C; , 37°C; , 56°C; and , 70°C.

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 groups of 4 to 6 hamsters could be adequately described by a sum of two exponential terms by graphical curve peeling (results not shown), from which the pharmacokinetic parameters summarized in Table 4 were derived. The pharmacokinetic parameters of the mutants were not markedly different from those of wild-type SakSTAR. Initial plasma half-lives [t_1/2()] ranged between 1.9 and 2.8 minutes and plasma clearances between 0.76 and 1.8 mL/min.

View this table: [in this window] [in a new window]   Table 4. Pharmacokinetic Parameters of the Disposition of Staphylokinase-Related Antigen From Plasma After Bolus Injection of SakSTAR Variants (100 µg/kg) in Hamsters

	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,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89), which induced fewer antibodies than wild-type SakSTAR in patients with peripheral arterial occlusion, had a significantly reduced specific activity.^{2 3} In an effort to restore the activity and temperature stability without loss of the reduced antibody recognition, the effect of reversal of one or more of these substituted residues to the original amino acids was studied.

Reversal of any of the five substituted amino acids of SakSTAR(KEKER) to the original residue, yielding SakSTAR(EKER), SakSTAR(KKER), SakSTAR(KEER), SakSTAR(KEKR), and SakSTAR(KEKE₇₅), did not restore the specific activity. Reversal of two or three amino acids in SakSTAR(EER), SakSTAR(K₃₅E₇₅R), and SakSTAR(EE₇₅) partially restored the specific activity, whereas specific activities similar to that of wild-type SakSTAR (100 000 U/mg) were obtained with SakSTAR(KE₃₈), SakSTAR(K₇₄ER), SakSTAR(K₃₅E₇₅), SakSTAR(K₇₄), and SakSTAR(E₇₅). Similar results were obtained with respect to fibrinolytic potency in human plasma in vitro. Evaluation of the binding of these variants to the panel of murine MAbs (Table 2) revealed that the binding of SakSTAR(K₇₄ER) with the antibody cluster of epitope I was dependent primarily on the presence of Glu⁷⁵. The reactivity of SakSTAR(KE₃₈) with the antibody panel representing epitope III required the presence of both amino acids, as revealed from the comparative binding of the pairs SakSTAR(EKER) and SakSTAR(KKER), SakSTAR(EER) and SakSTAR(K₃₅E₇₅R), and SakSTAR(EE₇₅) and SakSTAR(K₃₅E₇₅). The reduced binding of SakSTAR(E₈₀D) with the antibody cluster against epitope III appeared to be largely maintained in SakSTAR(D). Surprisingly, however, the altered reactivities of SakSTAR(E₇₅) to epitope cluster I and of SakSTAR(D) to cluster III could not be combined in SakSTAR(E₇₅D). In aggregate, these data suggest that the amino acid responsible for binding of murine MAbs to SakSTAR(K₇₄ER) is Glu⁷⁵; to SakSTAR(KE₃₈), both Lys³⁵ and Glu³⁸; and to SakSTAR(E₈₀D), Asp⁸².

The capacity of SakSTAR(KEKER) and SakSTAR(K₇₄ERED) relative to that of SakSTAR to absorb antibodies induced by immunization with wild-type SakSTAR was reduced to 70% in pooled plasma of 6 rabbits. The reduced recognition pattern of SakSTAR(KEKER) was largely maintained with SakSTAR(EER), but no clear immunodominant residue could be identified. From this analysis, SakSTAR(EER) emerged as the variant with the best ratio of activity to antigenicity relative to SakSTAR(KEKER) in the rabbit. Alternatively, the reduced reactivity with SakSTAR(KERED) was largely maintained in SakSTAR(E₈₀D) and partially in SakSTAR(D).

When pooled plasma from 6 baboons immunized with SakSTAR was used, both SakSTAR(KEKER) and SakSTAR(K₇₄ERED) were not recognized by nearly 50% of the antibodies. The contributions of the K35,E38 and the K74,E75,R77 epitopes to the reduced absorption with SakSTAR(KEKER) and of the K74,E75,R77 and E80,D82 epitopes to the reduced absorption with SakSTAR(K₇₄ERED) were comparable and nearly additive. The reactivity of the K74,E75,R77 epitope was largely conserved in SakSTAR(K₇₄), but no clear immunodominant residue could be identified in the reactivity of the K35,E38 and E80,D82 epitopes.

In the plasma pool from 10 patients treated with SakSTAR for acute myocardial infarction, the elicited antibodies were absorbed for only 50% by SakSTAR(KEKER) and SakSTAR(K₇₄ERED). Unlike the observation in the baboon, this reduced reactivity could be fully ascribed to the K74,E75,R77 epitope, with little contribution by the K35,E38 or E80,D82 epitope. Furthermore, the reduced antibody recognition was largely maintained in SakSTAR(K₇₄), which had an intact specific activity and clearly the best ratio of activity to antigenicity in humans of all variants studied. In subpool B, containing plasma from 3 patients from whom SakSTAR(KEKER) absorbed <50% of the antibodies elicited by SakSTAR treatment, similar although more pronounced altered antibody recognition patterns were observed with the SakSTAR variants. As expected, subpool C, prepared from plasma of 3 patients from whom SakSTAR(KEKER) absorbed >95%, similar antibody recognition patterns were observed with all SakSTAR variants studied.

In aggregate, the results of the present study, which focused on the seven amino acids Lys³⁵, Asp³⁸, Lys⁷⁴, Glu⁷⁵, Arg⁷⁷, Glu⁸⁰, and Asp⁸², which had previously been identified as playing a role in the altered reactivity of SakSTAR with a panel of murine MAbs² and demonstrated to be less immunogenic in humans,³ reveal that recognition patterns by SakSTAR variants of antibodies elicited with wild-type SakSTAR vary markedly across species. The most surprising observation in humans, which could not have been extrapolated from results obtained in mice, rabbits, and baboons, is that only one of these seven amino acids, namely Lys⁷⁴, is responsible for most of the reduction in antibody recognition of the SakSTAR(KEKER) and SakSTAR(K₇₄ERED) variants, although a subgroup of patients (subpool C) did not develop significant levels of antibodies against these epitopes. The markedly different patterns of antibody recognition in mice, rabbits, baboons, and humans suggest that a systematic evaluation of the reduction of antigen recognition and antibody induction in humans probably will eventually require the development of panels of human MAbs for analysis of variants.

On the basis of the present results, four variants were selected for large-scale purification and conditioning for use in vivo. SakSTAR(K₇₄), with a single substitution of Lys⁷⁴ with Ala, and SakSTAR(K₇₄ER), with substitution of Lys⁷⁴, Glu⁷⁵, and Arg⁷⁷ with Ala, were selected because of their reduced reactivity with human and baboon antibodies; SakSTAR(E₇₅), with a single substitution of Glu⁷⁵ with Ala, because of its reduced reactivity with murine MAbs; and SakSTAR(EER), with Glu³⁸, Glu⁷⁵, and Arg⁷⁷ substituted with Ala, because of its reduced reactivity with rabbit antibodies. All variants had a significantly higher specific activity than SakSTAR(KEKER), from which they were derived; they were found to contain low endotoxin levels, to be devoid of acute toxicity in mice after intravenous bolus injection at a dose of 3 mg/kg, and to have pharmacokinetic properties similar to those of wild-type SakSTAR. Their relative capacities to induce antibodies were then evaluated by immunization in rabbits and baboons and by treatment of patients with peripheral arterial occlusion, as reported elsewhere.¹⁷

	Selected Abbreviations and Acronyms

 

AUC = area under the curve LB = Luria-Bertani MAb = monoclonal antibody RU = resonance unit TB = terrific broth

	Acknowledgments

 
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 D. Collen has an equity interest.

	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 D. Collen has an equity interest.

Received June 16, 1996; revision received August 16, 1996; accepted August 31, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Collen D, Zhao ZA, Holvoet P, Marijnen P. Primary structure and gene structure of staphylokinase. Fibrinolysis. 1992;6:226-231.

2. Collen D, Bernaerts R, Declerck P, De Cock F, Demarsin E, Jenne S, Laroche Y, Lijnen HR, Silence K, Verstreken M. Recombinant staphylokinase variants with altered immunoreactivity, I: construction and characterization. Circulation. 1996;94:197-206.[Abstract/Free Full Text]

3. Collen D, Moreau H, Stockx L, Vanderschueren S. Recombinant staphylokinase variants with altered immunoreactivity, II: thrombolytic properties and antibody induction. Circulation. 1996;94:207-216.[Abstract/Free Full Text]

4. Vanderschueren S, Stassen JM, Collen D. Comparative antigenicity of recombinant wild-type staphylokinase (SakSTAR) and a selected mutant (SakSTAR.M38) in a baboon thrombolysis model. J Cardiovasc Pharmacol. 1996;27:809-815.[Medline] [Order article via Infotrieve]

5. Schlott B, Hartmann M, Guhrs KH, Birch-Hirschfeld E, Pohl HD, Vanderschueren S, Van de Werf F, Michoel A, Collen D, Behnke D. High yield production and purification of recombinant staphylokinase for thrombolytic therapy. Biotechnology. 1994;12:185-189.[Medline] [Order article via Infotrieve]

6. Silence K, Hartmann M, Guhrs K-H, Gase A, Schlott B, Collen D, Lijnen HR. Structure-function relationships in staphylokinase as revealed by "clustered charge-to-alanine" mutagenesis. J Biol Chem. 1995;270:27192-27198.[Abstract/Free Full Text]

7. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.

8. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989;77:61-68.[Medline] [Order article via Infotrieve]

9. Tartof KD, Hobbs CA. Improved media for growing plasmid and cosmid clones. Bethesda Res Lab Focus. 1987;9:12.

10. Jonsson U, Malmqvist M. Real time biospecific interaction analysis: the integration of surface plasmon resonance detection, general biospecific interface chemistry and microfluidics into one analytical system. Adv Biosensors. 1992;2:291-336.

11. Johnsson B, Lofas S, Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem. 1991;198:268-277.[Medline] [Order article via Infotrieve]

12. BIAcore System Manual, 5-2. Uppsala, Sweden: Pharmacia Biosensor AB.

13. Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991;145:229-240.[Medline] [Order article via Infotrieve]

14. Collen D, Silence K, Demarsin E, De Mol M, Lijnen HR. Isolation and characterization of natural and recombinant staphylokinase. Fibrinolysis. 1992;6:203-213.

15. Collen D, Van de Werf F. Coronary thrombolysis with recombinant staphylokinase in patients with evolving myocardial infarction. Circulation. 1993;87:1850-1853.[Abstract/Free Full Text]

16. Vanderschueren S, Barrios L, Kerdsinchai P, Van den Heuvel P, Hermans L, Vrolix M, De Man F, Benit E, Muyldermans L, Collen D, Van de Werf F. A randomized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction. Circulation. 1995;92:2044-2049.[Abstract/Free Full Text]

17. Collen D, Stockx L, Lacroix H, Suy R, Vanderschueren S. Recombinant staphylokinase variants with altered immunoreactivity, IV: identification of variants with reduced antibody induction but intact potency. Circulation. 1997;95:463-472.[Abstract/Free Full Text]

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