| 研究課題/領域番号 |
22KJ1057
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| 補助金の研究課題番号 |
22J21384 (2022)
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| 研究種目 |
特別研究員奨励費
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| 配分区分 | 基金 (2023)
補助金 (2022) |
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| 応募区分 | 国内 |
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| 審査区分 |
小区分37030:ケミカルバイオロジー関連
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| 研究機関 | 東京大学 |
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研究代表者 |
黄 薇恩 東京大学, 理学系研究科, 特別研究員(DC1)
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| 研究期間 (年度) |
2023-03-08 – 2025-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
2,500千円 (直接経費: 2,500千円)
2024年度: 800千円 (直接経費: 800千円)
2023年度: 800千円 (直接経費: 800千円)
2022年度: 900千円 (直接経費: 900千円)
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| キーワード | Mirabody / Sialidase / Breast Cancer / Breast cancer / TAM / Macrophage polarization / RaPID |
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| 研究開始時の研究の概要 |
My research is to develop a tri-functional immune checkpoint inhibitor and immune activator for triple negative breast cancer. I will use Sialidase, a sialic acid-cleaving enzyme, as scaffold and insert two cyclic peptides into its loop regions. This bi-grafted Sialidase will first recognize EpCAM, a biomarker on breast cancer cell, and then cleave the recognition sugar on CD24 to stop the interaction with Siglec-10. The second target, CSF1-R on tumor-associated macrophage, would be inhibited by another grafted peptide, inducing polarization and restoring its phagocytotic ability.
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| 研究実績の概要 |
To develop a lasso-grafted mirabody conjugated sialidase to the breast cancer tumor microenvironment,
I tried to express grafted mirabody with SHuffle T7 Competent E. coli. The scaffold mirabody were successfully expressed with the wild-type and variant binding to FcγRI receptor. The following expression trail with grafted-mirabody in both scaffold were also successful. Then, the binding affinity of E.coli-expressed grafted-miraboy was measured with surface plasma resonance. The dissociation constant of variants showed no difference compared to the traditional mammalian cell-expressed mirabody. More, those grafted-mirabody variants’ binding affinity to the breast cancer cell was tested with flow cytometry analysis, showing 50% increased binding intensity compared to the wild-type.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The new expression system of Mirabody by SHuffle T7 Competent E. coli was successfully implemented.
With the optimization of codon for scaffold and grafted peptide sequence insertion by in-fusion cloning, the two chosen Fc scaffold, wildtype and variant binding to FcγRI receptor, were suitable for large expression of grafted-mirabody. Moreover, it also maintained the similar binding affinity to either immobilized protein of interest or cell-expressed protein of interest. With the new expression system of E. coli, the production procedure would be simpler and production cost could decrease greatly.
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| 今後の研究の推進方策 |
With this stable production of grafted-mirabody, their inhibitory effect to the protein of interest will be examined by western blot. After co-incubation of grafted-mirabody and breast cancer cells expressing protein of interest (POI), the phosphorylation level of POI will be stained by phosphorylation-sensitivity antibody. If the phosphorylation level decrease, it could suggest the inhibitory effect of grafted-mirabody.
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