Unraveling the proteomic and phosphoproteomics early events molecular effectors and signaling pathways of the Anti-CD19 CAR-T cell therapy

Chimeric Antigen Receptor T-cell (CAR-T) therapy is a leading therapy in the field of cell immunotherapy, which utilizes genetically engineered T-lymphocyte cells expressing receptors against specific targets on tumor cells, such as CD-19, to induce cytotoxicity and the death of malignant cells. Using a locally-developed third-generation CAR-CD19, we generated an Anti-CD19 CAR-Jurkat, a T-cell line, which we used to characterize early events of proteomic and phosphoproteomic alterations important for understanding the signaling pathways and mechanism of action for this CAR. Anti-CD19 CAR-Jurkat and SILAC-heavy labeled RAJI B-cells were co-cultured for 10 minutes. The proteomic and phosphoproteomic profiles were obtained using the DIA approach on the Orbitrap Astral LC-MS/MS platform. The data were processed by DIA-NN and organized to discriminate the proteins and phosphopeptides from the effector CAR-Jurkat and the target RAJI cells based on the SILAC isotopically labeled peptides. The analysis of six biological replicates provided very deep coverage of the proteome and phosphoproteome, resulting in the identification and quantification of approximately 8,800 proteins and 20,000 phosphorylation sites, respectively. As expected, there were minor early proteomic alterations in the effector CAR-Jurkat cells, but few statistically significant alterations indicated special processes of antigen presentation by CD74 and other MHC molecules. On the other hand, the target RAJI B-cells had more statistically significant changes, including alterations in adhesion molecules and CD28, which are responsible for T-cell activation and survival. Additionally, other proteins with undisclosed direct connections with T-cell engagement were observed, which might represent interesting molecules that modulate the CAR response. Regarding the phosphoproteomic alterations, a large number of regulated phosphosites were observed for both the effector CAR-Jurkat and target RAJI cells. Interestingly, for both cells, a network analysis indicated a very significant alteration in proteins associated with splicing and the spliceosome. While the association of splicing and T-cell activation has been recently described, it is striking how early these events take place in CAR-T cell function. Based on our study, we uncovered proteins and phosphosites that play roles as molecular effectors of Anti-CD19 CAR-T cell therapy at early stages of CAR-T-target cell engagement and are expanding the understanding of the mechanism and signaling pathways that will aid the further development of CARs and improve the efficacy of such a revolutionary cancer therapy strategy.

Keywords: CAR-T cell, Proteome, Phosphoproteome, Signaling pathways, Molecular Effectors.

Support: CNPq (316934/2021-6) FAPESP (2020/13463-2, 2013/08135-2), RSC (C23-6685539336).