| Project/Area Number |
17570120
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Tokai University |
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Principal Investigator |
FUJITA Yoko Tokai University, School of Engineering, Professor (10328106)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAYANAGI Atsushi Keio University, School of Medicine, Junior Associate Professor (80245464)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,750,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Anti-IGF-I receptor / single chain antibody / cancer molecular thera / downregulation / Anti-tumor erowth / 抗IGF-I 受容体 / ヒト単鎖抗体 / Phage display法 / downregulation |
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| Research Abstract |
The aims of this study were to produce more effective therapeutic antihodies against the IGF-I receptor which can inhibit cancer growth, and to understand mechanisms by which anti-IGFIR induce down-regulation of IGFIR.
1. Preparation of human anti-IGFIR with higher affinities than that of the 1H7 single chain antibody (scFv).
We obtained 20 clones from a phage library displaying human scFvs. After extensive analyses of those clones, however, we found that they were cross-reactive to the insulin receptor as well as some other non-related proteins. In contrast, scFvs prepared from hybridoma producing 1H7 or 3B7 anti-IGFIR monoclonal antibody (mAb) showed strict specificity for IGFIR. These results suggested that rigorous screening is required to obtain scFvs of our interest in vitro antibody production using phage display technologies. Construction, expression, purification, and characterization of the two scFvs derived from hybridomas resulted in valuable information on their affinity and epitopes. The results have been published.
2. Understanding mechanism by which anti-IGFIR antibodies induce down-regulation of IGFIR
We found that all five mAbs used, despite showing different effects on IGFIR signaling, down-regulated IGFIR. In contrast, IGF-I apparently internalized the receptor, but did not lead to its degradation as evidenced by intact IGFIR subunits detected by immunoblotting. Pretreatment of MCF-7 cells with methylamine substantially reduced the antibody-mediated IGFIR down-regulation whereas MG115 did not. No ubiquitination was detected with IGFIR in MCF-7 cells treated by IGFIR mAb nor IGF-I. These results suggest that anti-IGFIR antibodies with different epitope-specificities can cause IGFIR down-regulation via a lysosome-dependent pathway in an IGFIR activation-independent manner. The results were presented at the Annual Meeting of ASBMB, 2007 (manuscript in preparation).
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