PT  - JOURNAL ARTICLE
AU  - Shingo Hanaoka
AU  - Shinji Saijou
AU  - Yasuhiro Matsumura
TI  - A novel and potent thrombolytic fusion protein consisting of anti-insoluble fibrin antibody and mutated urokinase
AID  - 10.1101/2020.09.06.284596
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.09.06.284596
4099  - http://biorxiv.org/content/early/2020/09/08/2020.09.06.284596.short
4100  - http://biorxiv.org/content/early/2020/09/08/2020.09.06.284596.full
AB  - Because the risk of thromboembolism increases with age, as well as due to infectious diseases, safer and more effective thrombolytic agents are in greater demand. Tissue plasminogen activator (tPA) is currently used clinically because it has higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity in places other than IF. Meanwhile, UK has the advantage that it is specifically activated on IF, but it only binds IF weakly. Unlike the anti-IF monoclonal antibody (mAb) established in the past, our anti-IF mAb 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 systemic side effects of UK in vivo. In a mouse thrombus formation experiment, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA, and 90% (9/10) in the AMU1114 treatment group. These data support future clinical development of AMU1114.Competing Interest StatementY.M. is co-founder, shareholder, and Board Member of RIN Institute, Inc., a venture company spun out from the National Cancer Center, Japan. S.H. and S.S. are employees of RIN Institute, Inc.