Mapping the functional protein nanoscale organization in T Cell bispecific antibody-induced immunological synapses using LUX-MS

T cell bispecific antibodies (TCBs) are potent cancer immunotherapies that redirect T cells to attack tumors. Recent studies reveal that TCBs can induce the formation of immunological synapses between T cells and cancer cells, mimicking physiological immune synapses. However, these TCB-induced synapses exhibit distinct spatial and temporal patterns, suggesting unique receptor-ligand interactions govern their formation. To identify new target cell surface proteins sustaining TCB-induced synapses, we employed the optoproteomic LUX-MS technology. LUX-MS enables the discovery of proximal protein interaction networks within (cis) and between (trans) living cells using light-controlled singlet oxygen generators (SOG). We established a co-culture model of naive T cells and OCI-LY18 lymphoma B cells, treated with either Glofitamab (CD20-TCB) or Gazyva (CD20-Antibody) as a negative control. After confirming the functionality of SOG-conjugated Glofitamab, we identified 1 hour as an optimal time point for synapse formation analysis through FACS-based time-course experiments. Our spatial proteotyping analysis revealed cell surface protein target candidates involved in the acute organization of the TCB-induced synapse between B lymphoma cells and primary T cells. Integrative network analysis showed that most identified proteins belonged to integrin-mediated signaling networks, heterotypic cell-cell adhesion networks, and extracellular structure organization pathways. Validation of the functional roles of these targets in sustaining TCB-mediated effects using CRISPR-Cas9 depleted knock-out cells is currently ongoing. Ultimately, our spatial proteotyping strategy enables time-resolved analysis of the TCB-induced surfaceome synapse at the molecular nanoscale level. The generated data provides a rational path forward to target validated surfaceome hits synergizing with TCB efficacy and decrease the threshold of TCB-induced T cell activation for therapeutic application.