Targeted gene transfer to enhance safety and efficacy of immunotherapy

Abstract: The remarkable success of chimeric antigen receptor (CAR) T cell therapy in phase I/II trials resulted in market approval for the treatment of specific B cell malignancies. However, a considerable number of relapses were reported, for instance due to impaired T cell persistence and antigen escape. Recently, a new mechanism of antigen escape has been described caused by the unintentional transduction of a single leukemic B cell during CAR T manufacture. Upon transduction, malignant B cells expressing the anti-CD19 CAR showed CD19-antigen blocking and masking in cis on the cell surface. The aim of the first part of this thesis was to shed light on the mechanisms contributing to the unintentional transduction of malignant B cells. It was demonstrated that VSV-G pseudotyped lentiviral vectors (LV) very efficiently transduce malignant B cells. More detailed analysis showed that LVs incorporate CARs in their envelope resulting in binding of LV to CAR-target expressing cells. Consequently, it was confirmed that enhanced binding of CAR-displaying LV to CAR-target expressing cells augmented the transduction of malignant B cells in a setting remodeling clinical CAR manufacturing. In conclusion, the need for improved gene transfer technologies was demonstrated.
In a heterogenous cell sample, exclusive gene transfer to the cell type of interest can be achieved by applying retargeted LV resulting in increased safety by reducing off-target transduction. LV pseudotyped with measles virus envelope proteins (MV-LV) have been redirected by ablating binding to the natural receptors and fusing single-chain antibodies (scFV) specific for the antigen of choice to the envelope protein. However, for each specificity a novel type of LV has to be designed and manufactured, which is laborious and limited to available scFVs. In the second part of this thesis a second-generation MV-LV system is presented with superior flexibility and control that requires no additional engineering. Universally targetable MV-LV vectors were generated that bind to a tag present only on adapter molecules specific for the antigen of choice, providing an additional layer of safety.
Selective transduction of targeted cell populations was demonstrated with a 100-1000 fold on-target to off-target ratio in mixed-cell culture populations such as peripheral-blood-mononuclear cells (PBMC). CAR T-cells with varying CD4/CD8-ratios were generated in a single transduction step directly from PBMC, by modulating the selectivity and efficiency in response to altering the specificity and concentration of the adapter. Importantly, universal targeted LV encoding and displaying universal targeted CARs further reduced binding to off-target cells, thus improving the selectivity of the targeted LV.
In summary, the benefit and function of a novel viral vector system has been demonstrated. The new system offers a higher degree of flexibility and control over the gene transfer efficiency, opening up potential application beyond the field of immunotherapy

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2020

Klassifikation und Themen

• Schlagwort: Genetic transformation ; Immunotherapy ; Immuntherapie ; Gentherapie

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2020
• Urheber: Cordes, Nicole
• Beteiligte Personen und Organisationen: Cathomen, Anton ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie

Weitere Informationen

• DOI: 10.6094/UNIFR/169884
• URN: urn:nbn:de:bsz:25-freidok-1698849
• Rechteinformation: Kein Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:48 MEZ