Abstract A007: Early Signaling Networks of Anti-CD19 CAR-T Cells Revealed by Quantitative Proteomic and Phosphoproteomic Profiling

Abstract Chimeric antigen receptor (CAR) T-cell therapy has revolutionized immunotherapy for hematologic malignancies, yet its broader efficacy remains limited by toxicity, antigen escape, and incomplete mechanistic understanding. To dissect the molecular events underlying CAR-T activation, we established an anti-CD19 CAR-Jurkat T-cell model using a locally engineered second-generation CAR construct and co-cultured it with SILAC-labeled Raji B cells for 10 minutes to capture early effector–target interactions. Proteomic and phosphoproteomic analyses were conducted using data-independent acquisition (DIA) on an Orbitrap Astral LC-MS/MS platform, identifying >8,800 proteins and 5,600 phosphorylation sites (1% FDR, six replicates). SILAC-based differentiation enabled precise profiling of effector versus target cell responses. CAR-T cells exhibited rapid modulation of proteins associated with antigen presentation (CD74, MHC molecules) and key signaling kinases, including LCK, MAPK14 (p38), CASP3, CD247, and DAP. Phosphosite enrichment highlighted activation of canonical pathways involving pERK1/2 (T202/Y204), pAKT (S473/T308), and pNF-κB (S932). Validation via immunoblotting and pharmacologic inhibition confirmed the critical role of LCK in early CAR-T signaling. Notably, PRKCD (S302/S304) and PRKD2 (S212/S214/T714) emerged as novel dual regulators of cell fate and cytokine signaling, influencing NFAT-driven transcription of IL-2 and IFN-γ. Integrative literature and pathway analyses identified additional effectors (IFN-γ, IL-5, granzyme B, TNF-α, JAK2, B-Raf) central to CAR-T cytotoxicity. Together, these findings delineate an integrated phosphoprotein network coordinating apoptosis and immune activation during early CAR-T engagement. This systems-level characterization advances mechanistic understanding of CAR-T function and highlights actionable signaling nodes to enhance next-generation CAR-T efficacy and safety. Citation Format: John Oluwafemi. Teibo, Roberta Maraninchi . Silveira, Virginia Campos . Silvestrini, Izadora Archiolli, Ana Paula . Masson, Beatriz Pereira . de Morais, Dayane Schmidt, Matheus Henrique . dos Santos, Germano Aguiar . Ferreira, Carolina Hassibe . Thomé, Dominic Helm, Raja Sekhar . Nirujogi, Dairo Renato . Alessi, Virginia Picanço-Castro, Lucas Eduardo Botelho . de Souza, Vitor Marcel . Faça. Early Signaling Networks of Anti-CD19 CAR-T Cells Revealed by Quantitative Proteomic and Phosphoproteomic Profiling [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A007.