Publications by authors named "Shingo Hanaoka"

4 Publications

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

Thromb Haemost 2021 Apr 21. Epub 2021 Apr 21.

National Cancer Center Research Institute, Immune Medicine, Tokyo, Japan.

Tissue plasminogen activator (tPA) is 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. UK has the advantage that it is specifically activated on IF, but 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 as compared to UK. In the photo-chemically induced thrombus mouse model, 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. Although no death was observed 1 hour after administration of each thrombolytic agent, some dead mice were identified within 24 hours in all treatment groups including control. These data indicate the need for further basic studies of AMU1114.
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http://dx.doi.org/10.1055/a-1488-3723DOI Listing
April 2021

Significant antitumor effect of an antibody against TMEM180, a new colorectal cancer-specific molecule.

Cancer Sci 2019 Feb 4;110(2):761-770. Epub 2019 Jan 4.

Division of Developmental Therapeutics, Exploratory Oncology Research & Clinical Trial Centre, National Cancer Centre, Kashiwa, Japan.

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 CRC-specific molecule, TMEM180, a predicted 11-pass transmembrane protein that apparently functions as a cation symporter. We developed an anti-TMEM180 mAb and then succeeded in humanizing the mAb. Immunohistochemistry (IHC) in CRC with the mAb showed a similar positivity rate as compared with anti-epidermal growth factor receptor mAb, and IHC with anti-TMEM180 mAb did not show staining in major organs used in this study. Immune electron microscopy clearly indicated that TMEM180 was present on the tumor exosome. The TMEM180 promoter region contains 10 hypoxia-responsive element consensus sequences; accordingly, SW480 cells upregulated TMEM180 under low-oxygen conditions. Anti-TMEM180 mAb has in vitro antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity, and SW480 CRC xenografts were eradicated by the mAb. These data indicate that TMEM180 may be a new CRC marker and that a mAb against this protein could be used as antibody-based therapy against CRC.
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http://dx.doi.org/10.1111/cas.13907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361608PMC
February 2019

Discovery of an uncovered region in fibrin clots and its clinical significance.

Sci Rep 2013 ;3:2604

Division of Developmental Therapeutics, Research Centre for Innovative Oncology, National Cancer Centre Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, Japan.

Despite the pathological importance of fibrin clot formation, little is known about the structure of these clots because X-ray and nuclear magnetic resonance (NMR) analyses are not applicable to insoluble proteins. In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognises an uncovered region that is exposed only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ chain that interacted closely with a counterpart region on the γ chain in a soluble state. New anti-Bβ and anti-γ mAbs specific to peptides lining the discovered region appeared to bind exclusively to fibrin clots. Furthermore, the radiolabelled 102-10 mAb selectively accumulated in mouse spontaneous tumours, and immunohistochemistry using this mAb revealed greater fibrin deposition in World Health Organization (WHO) grade 4 glioma than in lower-grade gliomas. Because erosive tumours are apt to cause micro-haemorrhages, even early asymptomatic tumours detected with a radiolabelled 102-10 mAb may be aggressively malignant.
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http://dx.doi.org/10.1038/srep02604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764439PMC
February 2014

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities.

Protein Sci 2005 Jan;14(1):119-30

Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.
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http://dx.doi.org/10.1110/ps.04983705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253311PMC
January 2005