Biology subjects

Hanaoka, S.

Publications and source records attributed to Hanaoka, S..

3 recordsLinked to original sources

bioRxiv2021

Identification of CD73 as the antigen of an antigen-unknown monoclonal antibody established by exosome immunization, and its antibody-drug conjugate exerts an antitumor effect on glioblastoma cell lines

Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibod-ies that recognized cancer-specific antigens on the exosome. In this study, we performed im-munoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5 and found that the humanized antibody did not inhibit CD73 enzymatic activity. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody-drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC could be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization.

biophysics↗

bioRxiv2020

A novel and potent thrombolytic fusion protein consisting of anti-insoluble fibrin antibody and mutated urokinase

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.

pharmacology and toxicology↗

bioRxiv2018

Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

The present state of therapy for colorectal cancer (CRC) is far from satisfactory, highlighting the need for new targets for this disease. We identified a new colorectal cancer (CRC)-specific molecule, TMEM180, a predicted eleven-pass transmembrane protein that apparently functions as a cation symporter. Our anti-TMEM180 monoclonal antibody (mAb) eradicated SW480 CRC xenografts in mice. The TMEM180 promoter region contains ten hypoxia-responsive element consensus sequences; accordingly SW480 cells upregulated TMEM180 under low-oxygen conditions. TMEM180 expression in SW480 cells was positively correlated with anchorage-independent colony formation and tumourigenesis. TMEM180-positive SW480 cells resided at the tumour-stroma interface manifested by SMA-positive fibroblasts, also known as the tumour niche. Some clusters of TMEM180-positive cells adjacent to the niche were integrin 6-positive. These data indicate that TMEM180 represents a possible cancer stem cell marker and that a mAb against this protein could be used as antibody-based therapeutic against CRC.

cancer biology↗