Dokument: Adoptive T-cell Therapy via Chimeric Antigen Receptors (CARs) against Leukemia in Combination with a human Suicide Gene

Titel:	Adoptive T-cell Therapy via Chimeric Antigen Receptors (CARs) against Leukemia in Combination with a human Suicide Gene
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=53084
URN (NBN):	urn:nbn:de:hbz:061-20200430-113629-9
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Ibach, Tabea [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 3,12 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 23.04.2020 / geändert 23.04.2020
Beitragende:	PD Dr. Hanenberg, Helmut [Gutachter] Prof. Dr. Gattermann, Norbert [Gutachter]
Stichwörter:	CAR, T-cell, suicide gene, Suizidgen, T-Zelle
Dewey Dezimal-Klassifikation:	600 Technik, Medizin, angewandte Wissenschaften » 610 Medizin und Gesundheit
Beschreibung:	Autologous T-cells equipped with a chimeric antigen receptor (CAR) represent a novel and highly successful approach in the fight against B-cell lineage leukemia and lymphoma. However, using CARs for the treatment of myeloid and T-cell lineage malignancies and especially solid cancers has proven to be much more difficult and will require several modifications for increased specificity and safety. One obstacle in the search for an ideal CAR construct is the design of an optimal region that connects the single chain fragment with the transmembrane region of the CAR, the so-called hinge region or spacer. Therefore, a major focus of this thesis was to develop a new universal hinge region of human origin that can replace the commonly used CH2CH3 fragment of human IgG and ideally contains epitopes recognized by monoclonal antibodies that facilitate the detection and selection of CAR-expressing T-cells under GMP-compliant conditions. To this end, different hinges derived from the human surface molecules CD34 or CD271 were introduced into a standard CD19 CAR construct and expressed in primary human T-cells using lentiviral vectors. The results demonstrated that two hinge regions (#C3 and #C6) from human CD34 allowed to detect and also enrich CAR-expressing human T-cells via the MACS microbeads system and did not affect the cytotoxic activities of the genetically modified T- cells against CD19+ leukemic cells. The inclusion of a safety switch that facilitates in vivo control of the engineered T-cells is also important to control potentially life-threatening adverse events in the patient. Therefore, the cDNA of a novel human suicide gene system, a human modified CYP4B1 enzyme which has been developed by our laboratory to activate the prodrug 4-Ipomeanol, was introduced into the CD19 CAR constructs. The new hinges enabled selection of highly CAR expressing human T-cells that were efficiently eliminated after killing their CD19+ target cells by incubation with 4-Ipomeanol. I also verified that a naturally occuring similar prodrug, Perilla ketone, could be used to kill the selected CD19 CAR positive T-cells that coexpressed the suicide gene. Finally, I tested whether the two prodrugs, 4-Ipomeanol and Perilla ketone, can also be activated by the most important cytochromes in human hepatocytes. To this end, I stably expressed the human cytochromes 1A2, 3A4, 3A5 and 2E1 in the human liver cell line HepG2 and demonstrated that only 1A2 expression led to cellular toxicity at high concentrations. In summary, I successfully established a new hinge region derived from human CD34 that can be used in standard CAR constructs in combination with a suicide gene for improved safety of human CAR T-cells.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Medizinische Fakultät
Dokument erstellt am:	30.04.2020
Dateien geändert am:	30.04.2020
Promotionsantrag am:	15.04.2019
Datum der Promotion:	07.02.2020