2021 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) |
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 |
Our project is to develop a mRNA-based treatment for creating in situ CAR T cells to treat cancer. This will be achieved by using anti-CD8 Fab'-installed polymeric micelles loading mRNA encoding anti-CD19 CAR constructs. This year we have improve the preparation of highly pure anti-CD8 Fab', which were modified with DBCO for installing on the micelles. We have also prepared the CAR mRNA by in vitro transcription of the corresponding DNA. Thus, the micelles were prepared by mixing the azide-PEG-poly(glycidyl-phenylalanine) (azide-PEG-PGPhe). Because we found that the pKa of the amines in this polymer is around 6, we prepared the micelles in acetate buffer (pH 4). The micelles prepared in acetate buffer attained higher protection of the mRNA against enzymatic degradation than the micelles prepared in HEPES buffer (pH 7.4). The micelles achieve high level of transfection in vivo in mice after local administration. Also, we found that azide-PEG-PGPhe can break cellular membranes at pH 5, but not at 7.4, which is useful for safe endosomal escape. We confirmed that the polymeric micelles delivering mRNA encoding reporter molecules, such as GFP and luciferase, can translate proteins in activated CD8+ T cells co-incubated with IL-2, but not quiescent T cells. In in vivo studies, we have also confirmed the creation of the tumor models using lymphoma A20 cells in Balb-c mice by intravenous injection, as well as subcutaneous injection. These models will be used to evaluate the antitumor activity in the next fiscal year.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We have successfully prepared all the materials for the in vivo evaluation of therapeutic strategy (polymers, micelles, mRNA, mouse tumor model). The mRNA for preparing the CAR T cells was prepared from a plasmid DNA coming from U.S.A (the plasmid DNA was delayed for more than 6 months). The approach of using the micelles for modifying the T cells was validated in vitro. Also, the ability of the micelles to produce proteins in vivo was confirmed after local injection. Unfortunately, because of the to COVID-19 restrictions in the animal facilities, we were not able to perform more extensive in vivo evaluation this year.
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| Strategy for Future Research Activity |
The plan for the future includes the following points: 1. Determine the targeting ability to T cells in vivo: The in vivo selectivity of the micelles to T cells in vivo will be evaluated after intrathymus and systemic injection of fluorescent-labeled micelles by flow cytometry of peripheral blood or thymus tissue collected at different time points. We will also quantify the distribution of the micelles in the organs by whole body and ex vivo fluorescence imaging; 2. Determine the T cell programming ability: We will determine the time profile that the micelles to reprogram T cells with the anti-CD19 CAR mRNA in situ by using flow cytometry after marking the CAR construct with antibodies; 3. Determine the anticancer activity of the micelles in mice bearing established lymphomas prepared by subcutaneous or intravenous inoculation of A20 cells. We will compare the therapeutic effects of the micelles with that of CAR T-cells transduced ex vivo with the mRNA encoding the CAR. The efficacy will be assessed by following mice survival; 4. Evaluate the toxicity of the treatments by macroscopic examination and histopathology of the organs. This analysis will be performed at 24 and 48 h after the injection of the micelles to reveal treatment-related lesions, and 1 week later to determine long term side effects. Changes in blood markers of toxicity, such as ALT, AST, BUN and CRE, will also be studied at 24 h, 48 h, and 1 week after the treatment.
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