RT info:eu-repo/semantics/doctoralThesis
T1 Novel allogeneic CAR-T cell therapy targeting NKG2D ligands: universal immunotherapy against solid and haematological tumours
A1 Aparicio Fernández, Cristina
A2 Universidad de Valladolid. Escuela de Doctorado
K1 Genética
K1 Immunotherapy
K1 Inmunoterapia
K1 Allogeneic CAR-T cells
K1 Células CAR-T alogénicas
K1 Oncology
K1 Oncología
K1 Genetic engineering
K1 Ingeniería genética
K1 2409 Genética
AB Chimeric Antigen Receptor (CAR)-T cell therapy has demonstrated impressive clinical outcomes, even in case of relapsed or refractory haematological malignancies. However, all currently commercialized therapies are autologous and target a single cancer-specific antigen. This approach entails some limitations, including high costs, manufacturing delays, and the risk of antigen-negative tumour relapses. To address these challenges, a novel CAR-T cell therapy was developed for allogeneic use by combining CRISPR/Cas9 technology to eliminate the expression of T-cell receptor (TCR) and HLA class I complexes in healthy donor T cells, thereby reducing the risk of graft-versus-host disease and immune rejection, respectively, with lentiviral transduction to introduce an atypical CAR, NKG2D-CAR, which targets eight different ligands commonly upregulated in both solid and haematological tumours, theoretically being less prone to tumour resistance and relapse.The novel CAR-T cell prototype was optimized through testing different interleukin (IL) culture supplementations (IL-2; IL-7/IL-15; IL-7/IL-15/IL-21) to promote an early memory T cell phenotype, which is associated with improved in vivo performance, identifying the IL-7/IL-15/IL-21 supplementation as the most effective combination.In vitro assays demonstrated their strong antitumour efficacy against both solid tumours (cervicouterine and colorectal) and haematological malignancies (multiple myeloma), accompanied by high pro-inflammatory cytokine secretion. An alternative T cell source (bone marrow) was also assessed and found to be equally viable compared to buffy coat for CAR-T cell production. Strategies to promote CAR-T cell expansion were evaluated, including activation of CAR signalling through soluble or overexpressed NKG2D ligands. To boost therapeutic potential, PD-1 knockout was performed to increase efficacy against solid tumours, while base editing technologies were applied to minimize off-target effects and enhance safety. Finally, manufacturing process improvements, including time reduction and large-scale electroporation, were implemented to support scalable and efficient production.In summary, this study presents the development of a novel, universal NKG2D CAR-T cell prototype for allogeneic use, representing a promising advancement in cancer immunotherapy.
YR 2025
FD 2025
LK https://uvadoc.uva.es/handle/10324/80035
UL https://uvadoc.uva.es/handle/10324/80035
LA eng
NO Escuela de Doctorado
DS UVaDOC
RD 29-nov-2025