Impact of glycosylation on the CAR T response
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of patients with relapse or refractory diffuse large B cell lymphoma (DLBCL). However, still 40-50% of the patients do not respond, warranting improvement of CAR T cell therapy. An important but commonly neglected factor of cellular signaling is glycosylation, i.e. the ‘sugar decoration’ of cells. This study aims to investigate how glycosylation impacts on CAR T cell responses, with the eventual aim to develop a glycan-modified CAR T cell with improved activity.
Glycosylation patterns on CAR T cells and DLBCL cells were modulated using glycosylation inhibitors and neuraminidase, an enzyme that cleaves-off sialic acids (sialidase). The impact of the glycosylation modifications on the CAR T cell response was evaluated using Jurkat reporter CAR T cells (Jurkat cells expressing a CD19-CAR and NFAT-luc2 response element). The neuraminidase CAR T cell (NEU-CAR) was generated by expressing a CD19-CAR construct and human NEU into Jurkat- and T cells. NEU production and activity was validated using RTqPCR, western blot, and sialidase activity assays. Activity of the NEU-CAR was assessed using Jurkat reporter CAR T cells and cytotoxicity assays.
The most prominent increase in the CAR T cell response was observed upon removal of sialic acids on either DLBCL or CAR T cells. Reversely, elevating sialic acid levels reduced the CAR T cell response. Based on these results, a neuraminidase producing CAR T cell was designed (NEU-CAR). The NEU-CAR was successfully generated in both Jurkat reporter cells and T cells as evidenced by the increased NEU mRNA/ protein expression, and sialidase activity. Also, NEU-CAR T cells demonstrated improved activity compared to regular CAR T cells.
Sialic acids on cancer and CAR T cells inhibit the CAR T cell response. Correspondingly, the NEU-CAR, that produces enzymatically active sialidase, has improved activity. Thus, glycosylation seems to be an important determinant of the CAR T response, and may be exploited to improve CAR T cell therapy.