Open AccessA Novel and Potent Thrombolytic Fusion Protein Consisting of Anti-Insoluble Fibrin Antibody and Mutated Urokinase
Author(s) -
Shingo Hanaoka,
Shinji Saijou,
Yasuhiro Matsumura
Publication year - 2021
Publication title -
thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.97
H-Index - 188
eISSN - 2567-689X
pISSN - 0340-6245
DOI - 10.1055/a-1488-3723
Subject(s) - plasmin , urokinase , fibrin , fibrinolysis , fusion protein , tissue plasminogen activator , chemistry , in vivo , antibody , plasminogen activator , cleavage (geology) , monoclonal antibody , medicine , thrombus , pharmacology , microbiology and biotechnology , immunology , biochemistry , recombinant dna , biology , enzyme , paleontology , fracture (geology) , gene
Tissue plasminogen activator (tPA) is used clinically because it has a higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity against substrates other than IF. UK has the advantage that it is specifically activated on IF; however, it binds IF weakly. Previously, we established a monoclonal antibody (mAb) that recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the reduction of plasma plasminogen levels in vivo relative to UK. In a photochemically induced mouse model of thrombus, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA treatment group, and 90% (9/10) in the AMU1114 treatment group. Although no death was observed 1 hour after administration of each thrombolytic agent, some mice died within 24 hours in all treatment groups, including control. These data indicate the need for further basic studies of AMU1114.