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Imunoterapija raka je skupina sodobnih terapevtskih pristopov za tarčno zdravljenje malignih obolenj. Zelo uspešna imunoterapevtska strategija je zdravljenje s celicami CAR-T. Zdravljenje poteka z avtolognimi limfociti T, v katere se vstavi genski zapis za receptor CAR, celice pa se nato znova injicira v pacienta. Celice CAR-T prepoznajo tumorski antigen, kar povzroči T-celično aktivacijo in uničenje rakavih celic. CAR-T celične terapije so odobrene za klinično rabo, vendar so uspehi zaenkrat omejeni le na nekatere vrste krvnih rakov. V okviru magistrskega dela smo razvili nov dizajn receptorjev CAR, ki temeljijo na uporabi verige CD3ε, s čimer smo želeli doseči kooperativnost med CAR in nativnim TCR. Pripravili smo pet različnih genskih konstruktov CAR in potrdili njihovo izražanje na površini limfocitov T. Novi receptorji CAR so ob stimulaciji s tarčnim antigenom sprožili T-celično aktivacijo. Interakcija med receptorjem CAR in nativnim TCR je bila bolj izrazita pri inovativnih receptorjih CAR kot pri konvencionalnih CAR. Za preučevanje mehanizma interakcije med CAR in TCR smo z uporabo metode CRISPR-Cas9 začeli razvijati celično linijo z izbitim genom za CD3ε. Dizajn receptorjev CAR na osnovi verige CD3ε bo služil za nadaljnji razvoj in optimizacijo CAR-T celične terapije. Cancer immunotherapy is a group of modern therapeutic approaches for targeted treatment of malignancies. CAR-T cell therapy is a very successful immunotherapeutic strategy where a genetic material for the CAR receptor is inserted into the patient's lymphocytes and then reinfused back into the body. CAR-T cells are able to recognize tumour antigen, trigger T-cell activation, and subsequently kill cancer cells. While CAR-T cells are already approved for clinical use, their success is limited to certain blood cancers. We developed a new design of CAR receptor that is based on the CD3ε chain, through which we tried to achieve cooperation between CAR and TCR. We designed and prepared five CAR constructs and showed their surface expression. We confirmed that the receptors were able to trigger T-cell activation upon stimulation with the target antigen. Interaction between CAR and TCR was more pronounced in new designs of CARs than in conventional CARs. We have also started developing a cell line with knocked-out CD3ε gene for studying the mechanism of TCR-CAR interaction. CARs based on the CD3ε chain will serve as a platform for further development and optimisation of CAR-T cell therapies. |
