(251d) Phospho-Proteomic Analysis of CAR-T Cell Signaling Following Activation By Antigen-Presenting Cancer Cells

Chimeric antigen receptors (CARs) are synthetic biomolecules comprised of an extracellular antigen recognition domain and intracellular signaling domains. Immune cells engineered to express CARs are one of the most promising forms of immunotherapy because CARs direct their host cells to kill diseased cells expressing the antigen recognized by the CAR. Although signaling pathways downstream of CAR activation control the cytotoxic function of CAR-expressing cells, studies of the phosphorylation-based signaling pathways initiated by CARs have been limited. For comprehensive, unbiased, and quantitative characterization of protein phosphorylation, liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics is the technology of choice. However, phospho-proteomic studies of CAR signaling have been limited by the challenge of stimulating CAR-expressing cells with their in vivo stimulus, antigen-presenting cancer cells. Here, we demonstrate an efficient and cost-effective label-free phospho-proteomic approach to analyze CAR signaling in immune cells stimulated with antigen-presenting cancer cells (Fig. 1). Following co-culture of CAR-T cells with cancer cells, we first preserve phospho-signaling by cross-linking proteins with formalin. Then, we use magnet-activated cell sorting (MACS) to isolate CAR-T cells from the co-culture. Validation experiments demonstrated that formalin fixation did not alter the phospho-proteome and that MACS achieved >90% CAR-T cell purity. Next, we compared the phospho-proteome in CAR-T cells stimulated with either CD19-expressing or non-CD19-expressing SKOV3 ovarian cancer cells. This analysis revealed that CAR signaling activated known pathways including the mitogen-activated protein kinases (MAPKs) ERK1/2. Bioinformatic approaches further showed that CAR activation induced other signaling pathways including the MAPK p38α, protein kinase A, and checkpoint kinase 1 (CHK1). Taken together, our work represents the first label-free phospho-proteomic analysis of CAR signaling in CAR-T cells stimulated with antigen-presenting cancer cells. Our results provide insight into the signaling mechanisms downstream of CAR activation and suggest how CARs direct T cells to kill target cells. By coupling phospho-proteomic profiling with functional analysis of CAR-T cell cytotoxicity, we envision that an improved understanding of CAR signaling will enable engineering of next-generation CARs with increased potency, improved persistence, and reduced toxic side effects.