Abstract
ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motif 13)-related bleeding disorder has been frequently observed as a life-threatening clinical complication in patients carrying a circulatory assist device. Currently, treatment modalities for the bleeding disorder are very limited and not always successful. To address the unmet medical need, we constructed humanized antibodies of mouse anti-ADAMTS13 antibody A10 (mA10) by using complementarity-determining region (CDR) grafting techniques with human antibody frameworks, 8A7 and 16E8. The characteristics of the two humanized A10 antibodies, namely A10/8A7 and A10/16E8, were assessed in vitro and in silico. Among the two humanized A10 antibodies, the binding affinity of A10/16E8 to ADAMTS13 was comparable to that of mA10 and human-mouse chimeric A10. In addition, A10/16E8 largely inhibited the ADAMTS13 activity in vitro. The results indicated that A10/16E8 retained the binding affinity and inhibitory activity of mA10. To compare the antibody structures, we performed antibody structure modeling and structural similarity analysis in silico. As a result, A10/16E8 showed higher structural similarity to mA10, compared with A10/8A7, suggesting that A10/16E8 retains a native structure of mA10 as well as its antigen binding affinity and activity. A10/16E8 has great potential as a therapeutic agent for ADAMTS13-related bleeding disorder.
Highlights
ADAMTS13-related bleeding disorder has been frequently observed as a life-threatening clinical complication in patients carrying a circulatory assist device
Overactivation of ADAMTS13 by increased fluid shear stress causes excessive cleavage of large von Willebrand factor (VWF), resulting in VWF depletion and a bleeding disorder called acquired von Willebrand syndrome3. aVWS-like bleeding episodes have been frequently observed in patients with a mechanical circulatory assist d evice[4,5,6], and attempts have been made to prevent and cure the bleeding disorder with mainly chemotherapy, surgery, and pump speed modulation[6]
After determining the complementarity-determining region (CDR) and framework regions (FRs) in the VH and VL of mouse anti-ADAMTS13 antibody A10 (mA10), 8A7, and 16E8 in accordance with IMGT numbering scheme[17], a global alignment was manually conducted by adjusting the positions and sequence motifs around CDRs between the amino acid sequences (Figs. 1,2)
Summary
ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motif 13)-related bleeding disorder has been frequently observed as a life-threatening clinical complication in patients carrying a circulatory assist device. AVWS-like bleeding episodes have been frequently observed in patients with a mechanical circulatory assist d evice[4,5,6], and attempts have been made to prevent and cure the bleeding disorder with mainly chemotherapy, surgery, and pump speed modulation[6]. These conventional treatment options have been frequently unsuccessful and raise the risks of thrombosis[7]. We constructed humanized A10 antibodies by CDR grafting with two human antibody frameworks, 8A7 and 16E816, to develop an applicable therapeutic agent for the treatment of patients with ADAMTS13-related bleeding disorder. The characteristics of the two humanized A10 antibodies, namely A10/8A7 and A10/16E8, were assessed in vitro as well as in silico
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