Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin- specific antibody fused to single-chain urokinase

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single- chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady- state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody- targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.

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Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin- specific antibody fused to single-chain urokinase

Blood ◽

10.1182/blood.v81.3.696.696 ◽

1993 ◽

Vol 81 (3) ◽

pp. 696-703 ◽

Cited By ~ 30

Author(s):

P Holvoet ◽

Y Laroche ◽

JM Stassen ◽

HR Lijnen ◽

B Van Hoef ◽

...

Keyword(s):

Steady State ◽

Fold Increase ◽

Plasminogen Activators ◽

Clot Lysis ◽

Chimeric Antibody ◽

Maximal Rate ◽

Single Chain ◽

Thrombolytic Activity ◽

Single Chain Fv ◽

Single Chain Fv Fragment

Abstract The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single- chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady- state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody- targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.

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Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb17499.x ◽

1992 ◽

Vol 210 (3) ◽

pp. 945-952 ◽

Cited By ~ 7

Author(s):

Paul HOLVOET ◽

Yves LAROCHE ◽

Henri Roger LIJNEN ◽

Berthe HOEF ◽

Els BROUWERS ◽

...

Keyword(s):

Specific Antibody ◽

Biochemical Characterization ◽

Plasminogen Activators ◽

Single Chain ◽

Single Chain Fv ◽

Single Chain Fv Fragment

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Computational and Rational Design of Single-Chain Antibody against Tick-Borne Encephalitis Virus for Modifying Its Specificity

Viruses ◽

10.3390/v13081494 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1494

Author(s):

Ivan K. Baykov ◽

Pavel Y. Desyukevich ◽

Ekaterina E. Mikhaylova ◽

Olga M. Kurchenko ◽

Nina V. Tikunova

Keyword(s):

Rational Design ◽

Fold Increase ◽

Encephalitis Virus ◽

Chimeric Antibody ◽

Single Chain ◽

Single Chain Antibody ◽

Glycoprotein E ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Far Eastern

Tick-borne encephalitis virus (TBEV) causes 5−7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

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Anti-Human Epidermal Growth Factor Receptor 2 Single-Chain Fv Fragment-Decorated DM1 Nanoparticles for Specific Targeting of Human Epidermal Growth Factor Receptor 2-Positive Breast Tumor Cells

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3043 ◽

2021 ◽

Vol 17 (3) ◽

pp. 447-455

Author(s):

Ling Ni ◽

You-Xin Li

Keyword(s):

Breast Cancer ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Single Chain ◽

Human Epidermal Growth Factor ◽

Single Chain Fv ◽

Epidermal Growth ◽

Single Chain Fv Fragment

Purpose: Although monoclonal antibodies are used to decorate nanoparticles to target specific cells, penetration of tumor tissues by monoclonal antibodies is limited by their large size. Therefore, we prepared DM1 nanoparticles decorated with the small anti-HER2 single-chain Fv fragment (scFvHER2) of trastuzumab (TMAB) for targeting to human epidermal growth factor receptor 2 (HER2) overexpressing in breast cancer effectively. Methods: ScFvHER2 fragment was coupled with DM1 nanoparticles (NPs) via covalent thiol-maleimide linkages. Their physicochemical properties, uptake by cells, and toxicity to tumor cells were investigated. Their vivo biodistribution was assessed employing liquid chromatographytandem mass spectrometry, while their antitumor activity was investigated in nude mice burdened with BT-474 tumor. Results: Viability of BT-474 cells incubated with scFvHER2-DM1-Nanoparticles (scFv-DM1-NPs) was significantly lower than that of BT-474 cell treated with TMAB-DM1-Nanoparticles (TMAB-DM1-NPs) (P < 0 05). Uptake by cells of scFvDM1-NPs was significantly higher than TMAB-DM1-NPs (P < 0 01). Accumulation of scFv-DM1-NPs in tumor tissue was notably higher than TMAB-DM1-NPs (P < 0 05). scFv-DM1-NPs exhibited improved antitumor effects compared to TMABDM1-NPs (P < 0 05), showing a tumor inhibition rate of more than 70%. Conclusions: ScFvHER2 fragment could serve as a more effective targeting ligand than TMAB, and scFv-DM1-NPs could be developed as a possible drug delivery system to target HER2-positive breast cancer.

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Thrombolytic and pharmacokinetic properties of an immunoconjugate of single-chain urokinase-type plasminogen activator (u-PA) and a bispecific monoclonal antibody against fibrin and against u-PA in baboons

Blood ◽

10.1182/blood.v79.9.2322.2322 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2322-2329 ◽

Cited By ~ 19

Author(s):

Y Imura ◽

JM Stassen ◽

T Kurokawa ◽

S Iwasa ◽

HR Lijnen ◽

...

Keyword(s):

Body Weight ◽

Plasminogen Activator ◽

Autologous Blood ◽

Clot Lysis ◽

Maximal Rate ◽

Antigen Level ◽

Single Chain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Pharmacokinetic Properties

Abstract Targeting of plasminogen activators to the fibrin component of a thrombus with the use of monoclonal antibodies (MA) directed against human fibrin may enhance their thrombolytic potency and fibrin- specificity. The thrombolytic and pharmacokinetic properties of rscu- PA/MA-FU1–74, an immunoconjugate of recombinant single-chain urokinase- type plasminogen activator (rscu-PA) and a bispecific MA directed against u-PA and against the beta-chain of human fibrin (MA-FU1–74), were investigated in baboons with a [125I]fibrin-labeled autologous blood clot in the femoral vein. Continuous intravenous infusion of rscu- PA/MA-FU1–74 (1:1.2 molar ratio) over 2 hours showed a fivefold increased thrombolytic potency (lysis per unit dose) over that of unconjugated rscu-PA, as evidenced both by a higher maximal rate of lysis (380% +/- 68% v 78% +/- 25% lysis per mg u-PA equivalent of compound administered per kg body weight, P less than .001), and by a lower dose at which the maximal rate of lysis occurs (0.19 +/- 0.03 v 0.82 +/- 0.10 mg compound per kg body weight, P less than .001). The specific thrombolytic activity (percent lysis per unit steady-state plasma u-PA antigen level) was lower for rscu-PA/MA-FU1–74 than for rscu-PA, as shown by both a lower maximal rate of lysis (60% +/- 13% v 220% +/- 22% lysis per microgram/mL u-PA antigen level in plasma, P less than .001) and a higher plasma antigen level at which maximal lysis is achieved (1.2 +/- 0.17 v 0.20 +/- 0.01 microgram/mL, P less than .001). The thrombolytic potency of rscu-PA/MA-UK1–3, an immunoconjugate of rscu-PA with the parental anti-u-PA antibody was similar to that of unconjugated rscu-PA. Clot lysis was achieved without systemic fibrinogen or alpha 2-antiplasmin consumption, and with a minor transient prolongation of the bleeding time. After the end of the infusions, u-PA-related antigen disappeared from plasma in a biphasic manner, with an initial half-life of 3.3 +/- 0.4 minutes for rscu-PA, 13 +/- 1 minutes for rscu-PA/MA-FU1–74, and 13 +/- 1 minutes for rscu-PA/MA-UK1–3, with corresponding plasma clearances of 340 +/- 28, 10 +/- 1, and 37 +/- 4 mL/min, respectively (mean +/- SEM). rscu- PA/MA-FU1–74 has a fivefold higher thrombolytic potency than unconjugated rscu-PA, as a result both of fibrin targeting by the specific idiotype of the antibody and of a slower plasma clearance.

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