| Summary: | Glioblastoma is the most malignant form of gliomas and is a highly infiltrative while non-metastatic tumor of the central nervous system. Patients with glioblastoma have a poor prognosis of 15 months median survival after diagnosis. Promising results were reported in recent clinical trial regarding glioblastoma treatment with chimeric antigen receptor (CAR) T therapy. The lab has previously developed five novel scFvs targeting IL13R⍺2, a tumor-associated antigen in glioblastoma, and integrated them into the second-generation CAR. We named them, 10CAR, 27CAR, 55CAR, 75CAR and 117CAR. The ex vivo cytotoxicity and proliferation assay demonstrated that the 117CAR T construct has the best functionality, while 27CAR T construct has a poor functionality compared to the rest of the constructs. FACS analysis was performed to check the CAR expression in different constructs. 27CAR T cells showed the lowest surface CAR expression and 117CAR T cells displayed the highest out of five constructs. 27CAR T cells were also activated more without stimulation compared to other constructs. We selected out 27CAR and 117CAR T cells for the further investigation to understand the attribution of the discrepancy between 27CAR and 117CAR T cells. We observed a larger cellular size for 27CAR T cells compared to the rest constructs in flowcytometry analysis, which is usually associated with activation. IFN-γ production of all constructs without target cells stimulation were detected to examine the activation state of different constructs. We observed the highest IFN-γ production in 27CAR T cells without stimulation. These results together indicate that a potent antigen-independent activation or, in other words, tonic signaling is present in 27CAR T cell. The tonic signaling further leads to an early exhaustive phenotype of 27CAR T cells, that is not present in 117CAR T cells. Removing the endodomain of CAR rescued the antigen-independent activation and early exhaustion of 27CAR T cells. The surface and total CAR expression of 27CAR and 117CAR T cells were determined by flowcytometry. 27CAR T cells presented a lower expression of both surface and total CAR. A significantly lower percentage of total CAR on the surface indicates the internalization of CARs in 27CAR T cells. Removing the intracellular domain of 27CAR did not restore the surface expression of CAR. 27CAR and 117CAR differ in four CDRs of scFv, CDR1, 2,3 in the heavy chain and CDR3 in the light chain. We replaced all the amino acids differing between these two constructs with alanine in a CDR-by-CDR manner and obtained five alanine substitution constructs. We then analyzed the CAR expression in Jurkat cells, and we found that the trafficking of CAR to the surface was significantly improved by mutating the CDR2 in the heavy chain or CDR3 in the light chain. Moreover, when the two CDRs were replaced simultaneously, almost all transduced cells expressed CAR, as was the case of cells transduced with 117CAR. To summarize, the tonic signaling induced by higher tendency of clustering of 27scFv results in the antigen-independent activation and early exhaustion of 27CAR T cells. By removing the endodomain of 27CAR, we abrogated the phenomenon. Further, CDR2 in heavy and CDR3 in light chain in 27scFv are responsible for the impaired trafficking of CAR to the surface.
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