Nannini, Francesco;
(2021)
Development of novel anti-mouse CD19 single-chain variable-fragments and establishment of an immunocompetent B-cell malignancy mouse model for the investigation of CAR-T cell therapies.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The use of chimeric antigen receptor (CAR) modified T cells targeting human CD19 has demonstrated great success in treating relapsed/refractory (r/r) Diffuse Large B Cell Lymphoma (DLBCL) and B cell Acute Lymphoblastic Leukaemia (B-ALL). However, the injection of CD19-directed CAR T-cells can cause severe side effects, such as cytokine release syndrome (CRS), neurotoxicity, and tumour lysis syndrome. Moreover, around 50% of patients still relapse after CAR T-cell therapy. The preclinical development of anti-CD19 CAR therapies has been primarily carried out in immunodeficient mice, limiting the possibility of understanding the interaction with the host immune cells and the “on-target, off-tumour” effects of the therapy. The aim of this thesis was to develop an immunocompetent mouse model recapitulating the therapeutic and toxic effects reported in human CAR-T trials. The challenges in identifying the ideal setting for tumour and CAR T-cells engraftment has proven total body irradiation (TBI) effective for the establishment of tumour and engraftment of second-generation anti-mouse CD19 CAR T-cells. Through this project, I have developed a model that demonstrates both efficacy and toxicity related to tumour burden and different doses of engineered CAR T-cells. The observation of elevated cytokines, such as IL-6, interferon-γ (IFN- γ), and MCP-1 in animals with the most severe side effects is similar to what is reported in human CD19 CAR-T trials. Moreover, B cell aplasia and persistence of CAR T-cells confirms the successful engraftment and function of anti-mouse CD19 CAR T-cells. In addition to the available anti-mouse CD19 binding domain (1D3), I have isolated through hybridoma technology four additional antibodies with improved thermal stability and one antibody binding to a separate epitope compared to the reference clone 1D3. Biophysical characterization revealed a similar kinetic profile to the reference clone, which resulted in equivalent in vitro CAR T-cells performance. In summary, this work successfully developed an immunocompetent mouse model for systemic B cell malignancy, which can be used to study the effects of anti-mouse CD19 CAR T-cells and explore improved CAR T-cell structures. Ultimately, the novel anti-mCD19 scFv developed through this project could be useful to construct and test alternative CAR architectures for in vivo applications.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of novel anti-mouse CD19 single-chain variable-fragments and establishment of an immunocompetent B-cell malignancy mouse model for the investigation of CAR-T cell therapies |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10140103 |
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