| 研究課題/領域番号 |
22F22024
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| 配分区分 | 補助金 |
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| 研究機関 | 東京大学 |
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研究代表者 |
菅 裕明 東京大学, 大学院理学系研究科(理学部), 教授 (00361668)
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| 研究分担者 |
COLAS KILIAN 東京大学, 大学院理学系研究科, 外国人特別研究員
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| 研究期間 (年度) |
2022-04-22 – 2024-03-31
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| キーワード | KRas / lasso-graft / macrocylic peptides / RaPID |
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| 研究実績の概要 |
Two anti-KRas peptides (KD2 and KWT9) have been grafted to Ubiquitin to provide two initial generations of hybrid constructs called “Ubodies” with different selectivity profiles. KD2-derived Ubodies are selective for the KRas “on” vs “off” state. KWT9 is newly identified, and displays (together with its Ubody derivatives) selectivity towards the mutant KRas over the healthy protein. A further generation of de novo Ubodies was also developed. Initial evaluation shows several hit compounds featuring excellent activity on the malignant target protein and selectivity over the healthy protein and/or the “off” state. Early results from collaborators validate the potential of the peptides, although further experimental work is needed on the Ubodies. The overall workflow for Ubody development has now been streamlined, from peptide hit identification, lasso-graft tether optimization, expression and purification to binding kinetics estimation. This experimental scheme can now be applied to different targets. The main challenge remaining to this project is now the delivery of the Ubodies into the cell by viral vector transfection.
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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
Using previously published peptide KD2, an overall workflow has been optimized for the discovery of lasso-grafted peptides onto Ubiquitin (“Ubodies”). The grafting tethers are first optimized using RaPID selection. Next-generation sequencing and computer modelling using Alphafold allow the identification of lead sequences. The selected Ubodies are then encoded as plasmids and expressed by Ecoli bacteria, followed by two different purification methods. Binding kinetics are then estimated by Surface Plasmon Resonance (SPR).
This led to a first generation of KD2-derived Ubodies that have been shipped to our collaborators in the University of San Francisco (UCSF) for in vivo tests. Furthermore, new peptide KWT9 was identified and shows excellent affinity/selectivity for the mutant KRas over the healthy protein, which was confirmed by our collaborators. Similarly to KD2, KWT9 does not cross the cell membrane. Thus, it was grafted onto Ubiquitin following the above-described workflow, providing a second generation of anti-KRas Ubodies that will be shipped to UCSF imminently. A third generation of de novo Ubodies will also be evaluated.
Once Ubody leads emerge, the main focus will turn to optimizing the drug delivery methodology. Their corresponding plasmids will thus be delivered to the cell by viral vector infection, and the cell can then express the Ubody directly, thus greatly limiting potential side-effects as well as optimal drug bioavailability and efficiency.
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| 今後の研究の推進方策 |
The main focus this year will be the optimization of the drug delivery system. As soon as lead Ubodies are identified in vivo, we will begin work with our collaborators to encode the corresponding genetic sequence into engineered viral vectors. Viral delivery and intracellular efficacy will then be determined. We anticipate this to be technically challenging, as this topic is further from our field of specialty, and it is the first time Ubodies will be used for an intracellular target. Regular feedback from UCSF will allow fine-tuning of the design of the plasmids, viral vector and/or Ubodies. We expect this should result in potential clinical candidates by 2024, as well as a first proof-of-concept publication.
In the meantime, additional targets will be pursued using the now-optimized Ubody identification platform described above. We will focus on currently undruggable cancer-related intracellular targets associated with poor prognosis. This type of targets is in urgent need of novel approaches, as their associated cancers are typically uncurable and/or fully resistant to traditional chemo- and radiotherapies. Currently, worldwide research on such topics focuses largely on immunotherapy, which shows great potential but remains effective in only a small fraction of patients. Alternative approaches are clearly need and Ubodies could be one such path forward.
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