Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and 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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Production and characterization of a bacterial single-chain Fv fragment specific to human truncated midkine

Cancer Letters ◽

10.1016/s0304-3835(01)00376-7 ◽

2001 ◽

Vol 164 (2) ◽

pp. 169-176 ◽

Cited By ~ 2

Author(s):

Warunee Dansithong ◽

Sharan Paul ◽

Tomohiro Mitsumoto ◽

Satoshi Saruhashi ◽

Takao Shinozawa

Keyword(s):

Single Chain ◽

Single Chain Fv ◽

Single Chain Fv Fragment

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Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis

Molecular Immunology ◽

10.1016/s0161-5890(99)00057-7 ◽

1999 ◽

Vol 36 (7) ◽

pp. 433-446 ◽

Cited By ~ 56

Author(s):

A McCall

Keyword(s):

Single Chain ◽

Isolation And Characterization ◽

Single Chain Fv ◽

Single Chain Fv Fragment

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Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

FEBS Letters ◽

10.1016/j.febslet.2013.07.057 ◽

2013 ◽

Vol 587 (20) ◽

pp. 3335-3340

Author(s):

Catherine Fallecker ◽

Nicolas Tarbouriech ◽

Mohammed Habib ◽

Marie-Anne Petit ◽

Emmanuel Drouet

Keyword(s):

Monoclonal Antibody ◽

Functional Characterization ◽

Specific Monoclonal Antibody ◽

Single Chain ◽

Single Chain Fv ◽

Single Chain Fv Fragment

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Characterization of the linker peptide of the single-chain Fv fragment of an antibody by NMR spectroscopy

FEBS Letters ◽

10.1016/0014-5793(93)80070-b ◽

1993 ◽

Vol 320 (2) ◽

pp. 97-100 ◽

Cited By ~ 61

Author(s):

Christian Freund ◽

Alfred Ross ◽

Birgith Guth ◽

Andreas Plückthun ◽

Tad A. Holak

Keyword(s):

Nmr Spectroscopy ◽

Single Chain ◽

Linker Peptide ◽

Single Chain Fv ◽

Single Chain Fv Fragment

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Sequence analysis, expression, and paratope characterization of a single-chain Fv fragment for the eukaryote ribosomal P proteins

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(02)03074-7 ◽

2003 ◽

Vol 301 (4) ◽

pp. 819-824 ◽

Cited By ~ 3

Author(s):

Pablo López Bergami ◽

Pablo Mateos ◽

Johan Hoebeke ◽

Mariano Jorge Levin ◽

Alberto Baldi

Keyword(s):

Sequence Analysis ◽

Single Chain ◽

Single Chain Fv ◽

P Proteins ◽

Ribosomal P ◽

Single Chain Fv Fragment

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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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Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα

Thrombosis Research ◽

10.1016/s0049-3848(03)00152-x ◽

2003 ◽

Vol 109 (2-3) ◽

pp. 137-144 ◽

Cited By ~ 6

Author(s):

Kesheng Dai ◽

Huaiping Zhu ◽

Changgeng Ruan

Keyword(s):

Platelet Glycoprotein ◽

Single Chain ◽

Single Chain Fv ◽

Fv Antibody ◽

Single Chain Fv Antibody

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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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