(Circulation. 1997;95:463-472.)
© 1997 American Heart Association, Inc.
Articles |
Recombinant Staphylokinase Variants With Altered Immunoreactivity
IV: Identification of Variants With Reduced Antibody Induction but Intact Potency
the Center for Molecular and Vascular Biology (D.C., S.V.), the Department of Radiology (L.S.), and the Department of Vascular Surgery (H.L., R.S.), University of Leuven, and the Center for Transgene Technology and Gene Therapy (D.C.), 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{at}med.kuleuven.ac.be
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Abstract |
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Background The thrombolytic potency and antibody induction of selected variants of recombinant staphylokinase (SakSTAR), including SakSTAR(K74) with Lys74, SakSTAR(E75) with Glu75, SakSTAR(EER) with Glu38, Glu75, and Arg77, and SakSTAR(K74ER) with Lys74, Glu75, and Arg77 replaced by Ala, were studied.
Methods and Results In rabbits, SakSTAR(K74) and SakSTAR(EER) elicited significantly less circulating neutralizing activity than SakSTAR and SakSTAR(E75) (P=.005 and P=.0002 versus SakSTAR, respectively). In baboons, SakSTAR(K74) induced significantly fewer antibodies than wild-type SakSTAR (P<.05). Intra-arterial administration in patients with peripheral arterial occlusion of SakSTAR(K74) (n=11) or SakSTAR(K74ER) (n=6) induced significantly fewer circulating neutralizing antibodies [median values and interquartile ranges, 20 (3.8 to 26) and 2.4 (0.2 to 59) µg compound neutralized/mL plasma after 3 to 4 weeks (P=.01 and P=.035 versus SakSTAR, respectively)] than SakSTAR (n=9) [median value and interquartile range, 93 (24 to 110) µg compound neutralized/mL plasma]. Overt neutralizing antibody induction (>10 µg compound neutralized/mL plasma) occurred in all 9 patients given wild-type SakSTAR, in 6 of the 11 SakSTAR(K74) patients (P=.038 versus SakSTAR), and in 2 of the 6 SakSTAR(K74ER) patients (P=.011 versus SakSTAR).
Conclusions SakSTAR(K74), a variant of recombinant staphylokinase with a single substitution of Lys74 with Ala, and SakSTAR(K74ER), with Lys74, Glu75, and Arg77 substituted with Ala, have intact thrombolytic potencies but induce significantly less antibody formation in patients.
Key Words: thrombolysis • infarction • plasminogen activators
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In a previous study, two mutants of recombinant staphylokinase (SakSTAR variant) were constructed, SakSTAR(K35A,E38A,K74A,E75A,R77A) (previously called SakSTAR.M38), in which Lys35, Glu38, Lys74, Glu75, and Arg77 were substituted with Ala, and SakSTAR(K74A,E75A,R77A,E80A,D82A) (previously called SakSTAR.M89), in which Lys35, Glu38, Lys34, Glu75, Arg77, Glu80, and Asp82 were substituted with Ala.1 These mutants were found to induce less antibody formation than wild-type SakSTAR in patients with peripheral arterial occlusion.2 However, their specific activities were significantly lower than that of wild-type SakSTAR, and they displayed a reduced temperature stability.1
A systematic study of the reversal of one or more of these substituted amino acids to the wild-type residues revealed that the antibody recognition patterns by these variants were markedly different between mice, rabbits, baboons, and humans.3 Because of these marked interspecies differences in recognition patterns by SakSTAR variants of antibodies induced with wild-type SakSTAR, selected variants were generated for the evaluation of their thrombolytic potency and immunogenicity in rabbits and baboons and in patients with peripheral arterial occlusion.
The present study deals with four selected variants derived from SakSTAR(K35A,E38A,K74A,E75A,R77A) by reversal of some of the substituted amino acids to the wild-type residue. The variants are identified by the substituted amino acids in the order of their sequence in the molecule, with addition of those sequence numbers required to avoid ambiguity. The selected variants were SakSTAR(K74), with a single substitution of Lys74 with Ala, and SakSTAR(K74ER) (previously identified as SakSTAR.M8), with substitution of Lys74, Glu75, and Arg77 with Ala, 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 monoclonal antibodies; and SakSTAR(EER), with Glu38, Glu75, and Arg77 substituted with Ala, because of its reduced reactivity with rabbit antibodies.3
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Reagents
SakSTAR, SakSTAR(K74), SakSTAR(E75), SakSTAR(EER), and SakSTAR(K74ER) were prepared and conditioned for use in vivo as described elsewhere.1
Staphylokinase-Neutralizing Activity in Plasma
Staphylokinase-neutralizing activity in plasma was determined as previously described.2
Specific Staphylokinase-Neutralizing Antibodies in Plasma
Antigen-specific IgG and IgM antibodies were quantified with ELISAs in polystyrene microtiter plates essentially as described previously.4
In the IgG assays, dilution curves of affinospecific anti-SakSTAR IgG antibodies were included on each plate. These antibodies were isolated from plasma obtained from three patients, after thrombolytic therapy with wild-type SakSTAR, by chromatography on protein A–Sepharose and insolubilized SakSTAR and elution of bound antibodies with 0.1 mol/L glycine HCl, pH 2.8. The purity of the IgG preparation was confirmed by SDS-PAGE.
In the IgM assays, titers defined as the plasma dilution giving an absorbancy at 492 nm equivalent to that of a 1/640 dilution of pooled plasma were determined and compared with the titer of baseline samples before treatment (median value, 1/410; interquartile range, 1/120 to 1/700).
Antibody Induction With SakSTAR Variants After Immunization in Rabbits
The comparative antigenicity of SakSTAR versus each of the variants SakSTAR(K74), SakSTAR(E75), and SakSTAR(EER) was studied after subcutaneous immunization in groups of 8 rabbits allocated to SakSTAR and 8 or 9 rabbits allocated to the variant as previously described.1 The antibody response and the residual thrombolytic potency were quantified at 0, 2, 3, 5, and 6 weeks as described elsewhere.1
Antibody Induction With SakSTAR(K74) After Immunization in Baboons
The antigenicity of SakSTAR(K74) was studied after subcutaneous immunization in three adult male baboons (Papio hamadryas) as described elsewhere.5
Comparative Thrombolytic Efficacy and Antibody Induction of SakSTAR(K74) and SakSTAR(K74ER) Versus SakSTAR in Patients With Peripheral Arterial Occlusion
Wild-type SakSTAR or the variant SakSTAR(K74) or SakSTAR(K74ER) was 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 (reduced overnight in some patients to 0.5 mg/h) in groups of 6 to 8 patients with angiographically documented occlusion of a peripheral artery or bypass graft <120 days previously. Patients were studied after giving informed consent, and the protocol was approved by the Human Studies Committee of the University of Leuven. Inclusion and exclusion criteria, conjunctive antithrombotic treatment (including continuous intravenous heparin), and the study protocol were essentially as previously described.1
Absorption With SakSTAR Variants of Antibodies Elicited in Patients by Treatment With SakSTAR, SakSTAR(K74), or SakSTAR(K74ER)
Plasma samples obtained 3 weeks after treatment with SakSTAR, SakSTAR(K74), or SakSTAR(K74ER) in patients with peripheral arterial occlusion in whom the staphylokinase-neutralizing activity exceeded 10 µg/mL [7 patients given SakSTAR, 3 patients treated with SakSTAR(K74), and 2 patients given SakSTAR(K74ER)] were used.
The plasma samples were serially diluted (50- to 400-fold) for the construction of individual calibration curves for antibody binding to SakSTAR, SakSTAR(K74), SakSTAR(K74ER), or SakSTAR(KEKER). The plasma samples were then absorbed for 10 minutes with 250 nmol/L of the SakSTAR variants, and residual binding to immobilized SakSTAR, SakSTAR(K74), SakSTAR(K74ER), and SakSTAR(KEKER) was determined by biospecific interaction analysis as described elsewhere.3 Residual binding was expressed in percentage of unabsorbed plasma with the individual calibration curves.
Statistical Analysis
Data are expressed as mean±SEM or median and (interquartile) range. Significance levels were determined by paired or unpaired Student's t test in case of Gaussian distributions and by the Kruskal-Wallis or Mann-Whitney rank sum test in case of non-Gaussian distributions. 
2 analysis and Fisher's exact test were carried out as indicated in the text. A two-tailed value of P<.05 was considered to indicate statistical significance.
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Comparative Thrombolytic Efficacy and Antibody Induction With SakSTAR Variants After Immunization in Rabbits
The relative antigenicity of wild-type SakSTAR versus the respective mutants is compared in Table 1
. In the 8 rabbits assigned to the SakSTAR group in each of the comparative studies versus the SakSTAR variants, the median baseline neutralizing activity in plasma was 0.4 to 0.7 µg/mL against SakSTAR and 0.5 to 0.9 µg/mL against the respective variants. Intravenous infusion of 400 µg/kg SakSTAR produced 59% to 79% lysis (mean values). These rabbits were then immunized 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 median plasma neutralizing activity was increased to 19 to 50 µg/mL against SakSTAR and to 22 to 33 µg/mL against the variants. Infusion of 400 µg/kg SakSTAR in 4 of these rabbits produced 3.6% to 9.4% clot lysis, whereas infusion of 400 µg/kg SakSTAR(K74) or SakSTAR(E75) or of 800 µg/kg of SakSTAR(EER) in the 4 other rabbits produced 7.9% to 16% lysis. In the 8 or 9 rabbits assigned to the SakSTAR variant group, the median baseline neutralizing activity in plasma was 0.5 to 0.8 µg/mL against SakSTAR and 0.6 to 1.1 µg/mL against the respective variants. Intravenous infusion of 400 µg/kg SakSTAR(K74) or SakSTAR(E75) or of 800 µg/kg of SakSTAR(EER) produced 61% to 80% lysis. These rabbits were then immunized subcutaneously with 400 µg of the respective SakSTAR variant 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 median plasma neutralizing activity was increased to 3.5 to 20 µg/mL against SakSTAR and to 7.0 to 22 µg/mL against the respective variants, whereas the residual thrombolytic potency of 400 µg/kg SakSTAR had decreased to 20% to 38% and that of 400 µg/kg SakSTAR(K74) or SakSTAR(E75) or of 800 µg/kg of SakSTAR(EER) to 15% to 59% of the theoretical maximum (100%) (Table 1). Statistical analysis revealed that SakSTAR(K74) and SakSTAR(EER) but not SakSTAR(E75) induced less neutralizing activity than SakSTAR. In addition, SakSTAR(EER), and to a lesser extent SakSTAR(K74), displayed significantly more residual thrombolytic potency than SakSTAR at 6 weeks. For comparison, the results previously obtained with SakSTAR(K74ER)2 are included in Table 1.
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Thrombolytic Efficacy and Antibody Induction With SakSTAR(K74) After Immunization in Baboons
At baseline, 50 µg/kg SakSTAR(K74) given intravenously over 1 hour induced 71±3.8% (mean±SEM) clot lysis over 2 hours in 3 baboons. In a previous study,5 the baseline clot lysis rates with 50 µg/kg SakSTAR and SakSTAR(KEKER) in 6 baboons each were comparable: 77±2.9% and 83±3.6%, respectively (all P=NS). Subsequent subcutaneous immunization with SakSTAR(K74) at weeks 2, 3, and 5 in the 3 baboons induced a gradual increase of neutralizing activity from a baseline titer (median and range) of 0.6 (0.0 to 0.8) µg/mL to 22 (14 to 24) µg/mL SakSTAR(K74) neutralized/mL plasma at week 6 (P<.05 versus baboons immunized with SakSTAR). These neutralizing titers were sufficiently high to neutralize the thrombolytic effect of 50 µg/kg intravenous SakSTAR(K74): clot lysis rate at 6 weeks was only 6.8±1.4%. The SakSTAR(K74)-neutralizing activity titers in plasma rose to 39 (29 to 49) µg/mL at 8 weeks and subsequently declined to 9.0 (7.0 to 12) µg/mL at 18 weeks (Fig 1 and Table 2). Bolus injection of 250 µg/kg at week 18 induced 47±2.6% lysis, compared with 39±5.3% with SakSTAR (P=.2). For comparison, the results previously obtained with SakSTAR and SakSTAR(KEKER)5 are included in Fig 1 and Table 2.
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, n=6), SakSTAR(K74) (
, n=3), or SakSTAR(KEKER) (
, n=6). The data represent median values and ranges in µg/mL. *P<.05 versus SakSTAR by Mann-Whitney rank-sum test.
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Comparative Thrombolytic Efficacy and Antibody Induction of SakSTAR(K74) and SakSTAR(K74ER) Versus SakSTAR After Intra-arterial Administration in Patients With Peripheral Arterial Occlusion
Groups of 6 to 11 patients (39 to 74 years old) with angiographically documented peripheral arterial occlusions 
0.3 to 120 days old and 5 to 78 cm long were treated with SakSTAR, SakSTAR(K74), or SakSTAR(K74ER). Relevant baseline characteristics of the individual patients are shown in Table 3. The majority of peripheral arterial occlusions were at the femoropopliteal level. Two iliac stent and 8 graft occlusions were included. Eight patients presented with incapacitating claudication, 5 with chronic ischemic rest pain, 7 with subacute ischemia, and 7 with acute ischemia. One patient (P.O.E.) who had been treated with SakSTAR 2 years previously was included in the SakSTAR(K74) group. This patient was not included in the statistical analyses in Tables 4 and 5 and Fig 2.
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Table 4
summarizes the individual treatments and outcomes. Intra-arterial infusion at a dose of 6.0 to 25 mg and a duration of 4.0 to 23 hours induced complete recanalization in 24 patients and partial recanalization in 3. Complementary endovascular procedures (mainly percutaneous transluminal angioplasty) were performed in 17 patients and complementary reconstructive vascular surgery after thrombolysis in 3. No patient underwent major amputation. Early recurrence of thrombosis after the end of the angiographic procedure occurred in 4 patients. Bleeding complications were absent or limited to mild to moderate hematoma formation at the angiographic puncture sites, except for 5 patients who required transfusion (Table 4). Intracranial or visceral hemorrhage was not observed.
Circulating fibrinogen, plasminogen, and 
2-antiplasmin levels remained essentially unchanged during infusion of the SakSTAR moieties (Table 5), reflecting absolute fibrin specificity of these agents at the dosages used. Significant in vivo fibrin digestion occurred, as evidenced by elevation of fibrin fragment D-dimer levels. Intra-arterial heparin therapy prolonged activated partial thromboplastin time levels to a variable extent (Table 5).
Antibody-related SakSTAR-, SakSTAR(K74)-, and SakSTAR(K74ER)-neutralizing activity and anti-SakSTAR, anti-SakSTAR(K74), and anti-SakSTAR(K74ER) 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 reach median values at 3 to 4 weeks of 20 µg SakSTAR(K74) and 2.4 µg SakSTAR(K74ER) neutralized/mL plasma in the patients treated with SakSTAR(K74) and SakSTAR(K74ER), respectively, which is significantly lower than the median value of 93 µg wild-type SakSTAR neutralized/mL plasma in the patients treated with SakSTAR (P=.024 for differences between the three groups by Kruskal-Wallis analysis and P=.01 and P=.036, respectively, for variants versus wild-type by Mann-Whitney rank-sum test). The levels of anti-SakSTAR(K74) and of anti-SakSTAR(K74ER) IgG increased to median values at 3 to 4 weeks of 270 and 82 µg/mL plasma in patients treated with SakSTAR(K74) and SakSTAR(K74ER), respectively, which is significantly lower than the median value of 1800 µg anti-SakSTAR/mL plasma in the patients treated with SakSTAR (P=.024 for differences between the three groups by Kruskal-Wallis analysis and P=.007 and P=.05, respectively, for variants versus wild-type by Mann-Whitney rank-sum test).
The titers of anti-SakSTAR(K74) and of anti-SakSTAR(K74ER) IgM increased from median baseline values of 1/460 and 1/410 to median values at 1 week of 1/510 and 1/450 in patients treated with SakSTAR(K74) and SakSTAR(K74ER), respectively, which was not significantly different from the median values of 1/320 at baseline and 1/640 at week 1 in patients treated with SakSTAR. Corresponding values at 2 weeks were 1/590 and 1/550 in patients given SakSTAR(K74) and SakSTAR(K74ER), not significantly different from 1/930 with SakSTAR (data not shown).
Absorption With SakSTAR Variants of Antibodies Elicited in Patients by Treatment With SakSTAR, SakSTAR(K74), or SakSTAR(K74ER)
Overt immunization (neutralizing activity at 3 to 4 weeks of 
10 µg compound/mL plasma) was observed in all of the 9 patients treated with SakSTAR, in 6 of the 11 patients given SakSTAR(K74) (P=.038 versus SakSTAR by Fisher's exact test), and in 2 of the 6 patients exposed to SakSTAR(K74ER) (P=.011 versus SakSTAR). The absorption and binding patterns of the antibodies in 12 of these patients were evaluated as described in "Methods" (Table 6). Wild-type SakSTAR absorbed virtually all the antibodies (median value, >95%) binding to insolubilized SakSTAR, SakSTAR(K74), and SakSTAR(K74ER) irrespective of the compound given. SakSTAR(K74) and SakSTAR(K74ER) absorbed all the antibodies (median value, >95%) binding to insolubilized SakSTAR, SakSTAR(K74), or SakSTAR(K74ER) from patients treated with SakSTAR(K74) but not from patients treated with SakSTAR. In the latter patients, SakSTAR(K74) absorbed a median 89% (range, 63% to 93%) of antibodies binding to SakSTAR (P=.02 versus SakSTAR by paired nonparametric test), SakSTAR(K74ER) absorbed a median 88% (range, 62% to 95%) [P=.5 versus SakSTAR(K74ER)], and SakSTAR(KEKER) absorbed a median 79% (range, 59% to 93%) [P=.03 versus SakSTAR(K74ER)].
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Discussion |
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The present study was initiated by the observation that "clustered charge–to-alanine" substitution variants derived from wild-type SakSTAR by substitution of one or more of the amino acids Lys35, Glu38, Lys74, Glu75, Arg77, Glu80, and Asp82 had a reduced reactivity with a panel of murine monoclonal antibodies and with antibodies elicited in rabbits and baboons by immunization with SakSTAR and in patients by treatment with SakSTAR.1 2 The antibody recognition patterns by these variants, however, were markedly different among these species.3 A panel of murine monoclonal antibodies directed against the K74,E75,R77 epitope reacted mainly with E75, one against the K35,E38 epitope reacted comparably with both amino acids, and one against the E80,D82 epitope reacted primarily with Asp82.3 In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 and K74,E75,R77,E80,E82 epitopes accounted for recognition of only 30% of circulating antibodies, and no clear immunodominant residue could be identified. In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes on the one hand and the K74,E75,R77 and E80,D82 epitopes on the other contributed equally to account for 50% of total antibody recognition, but no single immunodominant residue was apparent. Surprisingly, nearly half of the antibodies elicited by treatment with wild-type SakSTAR in patients recognized K74 of epitope K74,E75,R77, whereas epitopes K35,E38 and E80,D82 had a negligible contribution to antibody recognition.3 To further detail the correlation between reduced antibody recognition and reduced antibody induction, four variants were selected for further study in vivo. SakSTAR(K74), with a single substitution of Lys74 with Ala, and SakSTAR(K74ER), with Lys74, Glu75, and Arg77 substituted with Ala, were selected because of their reduced in vitro reactivity with human and baboon antibodies; SakSTAR(E75), with a single substitution of Glu75 with Ala, because of its dominance in the binding of murine monoclonal antibodies directed against the K74,E75,R77 epitope; and SakSTAR(EER), with Glu38, Glu75, and Arg77 substituted with Ala, because of its reduced reactivity with rabbit antibodies elicited with wild-type SakSTAR. All variants had a significantly higher specific activity and fibrinolytic potency in an in vitro human plasma system than SakSTAR(KEKER), with Lys35, Glu38, Lys74, Glu75, and Arg77 substituted with Ala, from which they were derived. The highly purified, sterilized preparations of these variants 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 after bolus intravenous injection similar to those of SakSTAR and SakSTAR(KEKER).3
The variants were evaluated against SakSTAR in terms of thrombolytic potency and of antibody induction in groups of 8 or 9 rabbits with 125I-labeled clots incorporated into an extracorporeal arteriovenous shunt, as previously described.2 In this model, the thrombolytic potencies of SakSTAR(K74) and SakSTAR(E75) were indistinguishable from that of wild-type SakSTAR (a dose of 400 µg/kg inducing 60% to 65% clot lysis within 90 minutes), and SakSTAR(EER) was about equipotent (800 µg/kg inducing 80% clot lysis). SakSTAR(K74), and to an even greater degree SakSTAR(EER), induced significantly less antibody formation after 6 weeks than SakSTAR, whereas SakSTAR(E75) was comparable to SakSTAR. Likewise, the residual thrombolytic potency of SakSTAR(K74), and to an even greater degree that of SakSTAR(EER), was higher at 6 weeks than that of wild-type SakSTAR or SakSTAR(E75). These data indicate that the degree of immunogenicity of these variants does not strictly parallel their pattern of antibody recognition in serum of rabbits immunized with SakSTAR.3 Indeed, such parallelisms would have suggested comparable immunogenicities of SakSTAR(K74) and SakSTAR(E75). Furthermore, the immunodominance of Glu75 in the recognition of the murine monoclonal antibody cluster reacting with the K74,E75,R77 epitope does not translate into a significant role in antibody recognition or in antibody induction in rabbits.
Immunization of 3 baboons with SakSTAR(K74) according to exactly the same scheme that previously demonstrated a significantly lower induction of IgG-related neutralizing activity with SakSTAR(KEKER) than with SakSTAR yielded intermediary results. Antibody titers increased to 50 (23 to 110), 22 (14 to 24), and 16 (2.0 to 45) µg compound neutralized/mL plasma for SakSTAR, SakSTAR(K74), and SakSTAR(KEKER) at week 6, respectively [P<.05 for SakSTAR(K74) versus SakSTAR and P=.01 for SakSTAR(KEKER) versus SakSTAR]. In agreement with the results in rabbits, SakSTAR(K74)- and SakSTAR(KEKER)-neutralizing activities never exceeded SakSTAR-neutralizing activities, suggesting that no immunodominant epitopes were created that would be recognized by SakSTAR(K74) or by SakSTAR (KEKER) but not by SakSTAR.
Intra-arterial administration of wild-type SakSTAR, SakSTAR(K74), or SakSTAR(K74ER) as a bolus of 2 mg followed by an infusion of 1 mg/h in 6 to 11 patients with angiographically documented occlusion of a peripheral artery or bypass graft surgery resulted in complete recanalization in 24 patients and partial recanalization in 3, without measurable systemic plasminogen activation. After administration of wild-type or variant SakSTAR, neutralizing antibody titers and specific IgG levels remained low for 1 week. From the second or third week onward, an increase of SakSTAR-neutralizing activity to 10 µg/mL plasma was observed in the 9 patients given SakSTAR but only in 6 of 11 patients given SakSTAR(K74) (P=.038 versus SakSTAR) and in 2 of 6 patients given SakSTAR(K74ER) (P=.011 versus SakSTAR). These neutralizing activities were most likely due to specific IgG, as demonstrated by the increase of staphylokinase-specific IgG in the absence of increased IgM titers. The antibodies induced by treatment with SakSTAR were completely absorbed by SakSTAR but incompletely by SakSTAR(K74) and by SakSTAR(K74ER), confirming the immunogenicity of the K74,E75,R77 epitope and the dominant role of Lys74 in the binding of antibodies directed against this epitope. The antibodies induced by treatment with SakSTAR(K74) or SakSTAR(K74ER) were completely absorbed by SakSTAR, by SakSTAR(K74), and by SakSTAR(K74ER), indicating that immunization was not due to neoepitopes generated by substitution of Lys74 with Ala but rather to epitopes different from the K74,E75,R77 epitope.
The present experience in 26 patients treated with SakSTAR (n=9), SakSTAR(K74) (n=11), and SakSTAR(K74ER) (n=6) combined with previous experience in 14 patients with SakSTAR (n=7) and SakSTAR(KEKER) (n=7)1 and in 24 patients with SakSTAR6 allows an initial estimation of the prevalence of immunization by intra-arterial treatment with SakSTAR or variants with an altered K74,E75,R77 epitope [SakSTAR(K74), SakSTAR(K74ER), and SakSTAR(KEKER)]. Neutralizing activity data after 2 to 4 weeks, available in 40 patients with peripheral arterial occlusion given intra-arterial SakSTAR, revealed that 33 patients (83%; 95% CI, 70% to 95%) had levels of >5 µg compound neutralized/mL plasma. Of the patients given SakSTAR(K74), SakSTAR(K74ER), or SakSTAR(KEKER), 9 of the 20 (45%; 95% CI, 21% to 69%) had neutralizing activity levels of >5 µg compound/mL plasma. This difference is statistically highly significant (P=.006 by Fisher's exact test), indicating that the K74,E75,R77 epitope is a major determinant of antibody induction. However, to adequately estimate the relative prevalence of specific immunocompetence against SakSTAR(K74) compared with that against wild-type SakSTAR, more extensive randomized treatment with evaluation of the associated immunization is required.
In summary, the present experience illustrates that staphylokinase variants with reduced antibody induction but intact thrombolytic potency can be generated. This observation represents a small but significant step toward the development of nonimmunogenic staphylokinase variants for fibrin-selective thrombolytic therapy in patients with thromboembolic disease. To the best of our knowledge, the present study constitutes the first case in which a heterologous protein, by use of protein engineering techniques, was rendered less immunogenic without reduction of its biological activity.
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Acknowledgments |
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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. Dr Vanderschueren is a research assistant for the National Fund for Scientific Research in Belgium. The authors thank Frans De Cock, Huberte Moreau, Maria Verstreken, Zhang Zhi-yong, and Zhu Ping for their skillful technical assistance.
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Footnotes |
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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 May 13, 1996; revision received August 5, 1996; accepted September 1, 1996.
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References |
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1. 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.
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Vaughan DE, Van Houtte E, Declerck PJ, Collen D. Streptokinase-induced platelet aggregation: prevalence and mechanism. Circulation. 1991;84:84-91.
5. 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]
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Vanderschueren S, Stockx L, Wilms G, Lacroix H, Verhaeghe R, Vermylen J, Collen D. Thrombolytic therapy of peripheral arterial occlusion with recombinant staphylokinase. Circulation. 1995;92:2050-2057.
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