Rin Institute, Inc.

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.

TMEM180, a novel colon cancer-specific protein with a 12-transmembrane topology, is upregulated at low oxygen. Previously, we established a humanized monoclonal antibody against TMEM180 aimed at clinical trials. Prior to such trials, it is necessary to clarify the function of TMEM180 in cancer. To compare SW480 human colon cancer cells and their TMEM180-knockdown derivatives, we analyzed proliferation and oxygen consumption, and also performed phosphorylation proteomics, metabolomics, and next-generation sequencing (NGS). The preliminary results revealed that TMEM180 appeared to promote the growth of colon cancer but had almost no effect on oxygen consumption or expression of phosphorylated proteins. By contrast, glycolysis differed dramatically between SW480 and TMEM180-knockdown cells. The NGS analysis revealed that TMEM180 promotes enzyme expression in nitric oxide (NO) synthesis system, suggesting that it promotes glucose and glutamine metabolism, thereby contributing to cancer growth. Overall, the results of this study warrant further basic studies of TMEM180 molecule.

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 antibodies that recognized cancer-specific antigens on the exosome. In this study, we performed immunoprecipitation 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. 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 has potential to 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, which may allow the antibody to advance to new antibody therapies.