Proteomics analysis reveal early event molecular effectors of anti-CD19 CAR-T cell therapy in hematological cancer.

Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of the field of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation anti-CD19 CAR construct, which allowed us to analyse early proteomic changes that are crucial for comprehending the signalling pathways and mechanism of action of this CAR-T cell. SILAC-heavy tagged Raji B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were acquired via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in about 8800 protein identifications at 1 % FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target Raji B-cells exhibited more significant alterations. Effector proteins, namely CD247, CD28, DAP, LCK, p38 MAPK, and CASP3, were validated, as they have critical roles in antigen presentation, T-cell activation, and apoptosis. Pharmacological inhibition of LCK using Dasatinib further suggested its pivotal role in early CAR-T signalling. This study led us to identify proteins that function as molecular effectors of anti-CD19 CAR-T cell therapy during the initial phases of CAR-T-target cell engagement, advancing our knowledge of the mechanism and signalling pathways that will support CAR-T cell development. SIGNIFICANCE: Chimeric antigen receptor T-cell (CAR-T cell) therapy is state-of-the-art in cell and immunotherapy. Determining important players in cellular communication and signalling mediated by membranes and intracellular proteins requires understanding the connection between tumours and modified cells. We employed global proteomics in this study to better grasp the functional protein networks using a high-sensitivity mass spectrometric platform for protein identification and quantification. We identified proteins as molecular effectors of anti-CD19 CAR-T cell treatment during the early stages of CAR-T-target cell interaction. Our understanding of the mechanism and signalling pathways will promote the development of new CAR constructs and improve the efficacy and ability to overcome the resistance of this innovative cancer treatment strategy, which will advance the identification of adjuvant molecules for the regulation of CAR-T responses.