2020 Fiscal Year Annual Research Report
Cancer immunotherapy by in vivo generation of CAR T cells based on targeted mRNA delivery
| Project/Area Number |
20H04524
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Cabral Horacio 東京大学, 大学院工学系研究科(工学部), 准教授 (10533911)
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| Co-Investigator(Kenkyū-buntansha) |
内田 智士 東京大学, 大学院工学系研究科(工学部), 特任助教 (20710726)
垣見 和宏 東京大学, 医学部附属病院, 特任教授 (80273358)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Nanomedicine / mRNA / CAR T cells / Cancer / Immunotherapy / Polymeric micelles / antibody fragments |
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| Outline of Annual Research Achievements |
In this project we aim to develop CAR T cells in situ after injection of mRNA-loaded polymeric micelles conjugated with antibody fragments (Fab'). To prepare this micelles, we have constructed a series of block copolymers based of poly(ethylene glycol)-poly(glycidol) (PEG-PG) modified with amino acids on the side chain. We have tested the stability of the micelles and found that the polymers having tryptophan, tyrosine and phenylalanine on the side chain promote the stability against dissociation from polyion exchange and against enzymatic attack. The micelles can also load small and large mRNA of more 10,000 bases mRNA. Moreover, we have synthesized azide-terminated PEG-PG (N3-PEG-PG) for installing the Fab' by click chemistry and modify it with phenylalanine for further micelle preparation. The mRNA-loaded micelles prepared from PEG-PG(tryptophan)and PEG-PG(phenylalanine) were tested in vitro and in vivo. Since the micelles delivered mRNA encoding luciferase, we tested the resulting bioluminescent signal. In vitro, the micelles gave bioluminescence in cancer cells, kidney cells and CD8+ T cells in a comparable manner. In in vivo experiments, we tested for subcutaneous, intramuscular, intrathymic and intravenous injection. At a dose of 5 ug of mRNA, both micelles showed high levels of luminescence after local administration (skin, muscle, thymus). However, the bioluminescent signal in the organs was much weaker after intravenous injection than after local injection. These results suggest that the strategy could work after thymus injection at a relatively low mRNA dose.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The development micelles showed enhanced ability to protect mRNA, which is necessary for in vivo applications. Unfortunately, because of the limited laboratory use during COVID-19, the biological animal experiments were less than expected. However, we could confirm the production of proteins in vivo after administering the micelles through different administration routes.
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| Strategy for Future Research Activity |
The next fiscal year we will digest whole anti-CD8 antibodies to prepare highly pure Fab'. The anti-CD8 FAB' will be modified with maleimide-PEG-DBCO. The binding ability of the anti-CD8 Fab' will be determined by comparing with the parent whole antibody. The anti-CD8 Fab'-DBCO will be installed on the surface of the micelles by click chemistry. We will also prepare the mRNA encoding the CAR construct by doing in vitro transcription of the plasmid DNA publicly available. We will prepare CAR mRNA-loaded micelles from the azide-PEG-PG(phenylalanine) polymers, which were found to give good protection of mRNA and efficient delivery. If possible, we will study the mechanism of this polymer for the enhanced intracellular delivery of mRNA. We will also evaluate the ability of the Fab'-installed micelles to transfect CD8+ T cells in vitro and in vivo. Moreover, we will start to establish the tumor models for testing the efficacy of our approach. Thus, we will inject murine lymphoma A20 cells in Balb-c mice by intravenous and subcutaneous injection, and check their grafting ability and histology.
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