In addition to many positive effects, immunotherapy with CAR-T cells also has some disadvantages, such as excessive immune response, treatment failure and disease recurrence. To avoid this, it is necessary to design CAR constructs that would have a lower activation threshold and thus trigger a strong antitumor response at a lower concentration of tumor antigen. The receptors on CAR-T cells are complex structures that enable many changes and consequently different responses to the tumor antigen. In this master's thesis, we prepared various genetic constructs that contained changes in costimulatory domains and combinations of response amplification approaches. The expression of CAR on the surface of electroporated Jurkat cells was checked by flow cytometry and the secretion of hIL-2, which was increased by the addition of LAT in combination with the 4-1BB costimulatory domain, was monitored by the ELISA method. The presence of the variable CD20 fragment in the CAR structure showed a stronger antitumor effect compared to the CD19 variant. Also, the addition of the alternative SLP-76 domain from the LAT microcluster to the CAR structure had a positive effect on hIL-2 secretion in the presence of the 4-1BB costimulatory domain. We also showed that CAR-T with a costimulatory domain from T-cell signaling is activated even in the absence of the CD3ζ domain. We have also successfully tested several approaches of reducing tonic signaling, such as mutations of different tyrosine residues in LAT, mutations of ITAM motifs, and combinations of these two approaches.
|
