


The C-Trap® provides the world’s first dynamic single-molecule microscope to allow simultaneous manipulation and visualization of single-molecule interactions in real time.

Unlike conventional drugs, whose activity is shaped by binding to a single target, CAR T-cell (CART) engagement with tumor cells involves multiple receptors, adhesion molecules, and signaling proteins. These components are partially organized within cholesterol-rich membrane microdomains called lipid rafts, forming the CAR immune synapse (CARIS)—the interface through which a CART recognizes, attaches to, and kills a tumor cell.
To evaluate CARIS as a pharmacological interface, we compared CD28- and 4-1BB-CARTs, which exhibit distinct therapeutic profiles. The rapid antitumor activity of CD28-CARTs was associated with brief, dynamic synapses supporting rapid signaling and serial killing. Conversely, 4-1BB-CARTs formed longer, mechanically stable synapses consistent with their slower but sustained activity. Synapse strength was measured using z-Movi acoustic force microscopy.
Lipid rafts were enriched at CARIS. Attenuating membrane lipid raft cholesterol preferentially compromised 4-1BB CARIS mechanical stability, while predominantly impairing CD28 CARIS activation and cytolytic output. The molecular networks underlying these differences were mapped by integrating lipid-raft lipidomics, differential SILAC proteomics to distinguish CART-derived from tumor-derived components, and T-cell transcriptomics.
CD28-associated lipid rafts were enriched in diacylglycerol signaling components, PKCθ, small GTPases, Src-family kinases, and AKT/PI3K-proximal modules, supporting rapid signal-to-function coupling. Stomatin-family and flotillin scaffolds favored dynamic remodeling, while CD8-dominant transcriptional programs supported cytolytic degranulation. Engaged tumor cells displayed membrane-stress signatures consistent with late-stage senescence and apoptotic commitment.
In contrast, 4-1BB-associated lipid rafts were enriched in lysophosphatidic acid, an inhibitory lipid known to suppress CD8 effector functions while promoting integrin-dependent adhesion, consistent with the marked enrichment of integrins at CARIS, NF-κB and cytokine signaling components, and ERM scaffolds that couple lipid rafts to cortical actin, sustaining CARIS stability. Engaged tumor cells exhibited signatures of early apoptotic resistance, including damage-response and repair markers.
Together, these findings establish CARIS as a central determinant of CART function and therapeutic behavior. Mapping and manipulating its lipid–protein networks provide a platform for understanding CART pharmacodynamics and developing strategies to improve therapeutic efficacy.
T cells play a pivotal role in tumor immunosurveillance. Multispecific cell engagers (CEs) have been adopted in the field of immuno-oncology to redirect T cells toward cancer cells, thereby unleashing the anti-tumor potential of the patient’s immune system. CE-mediated cell binding induces T cell activation and the formation of an immunological synapse, which is a prerequisite for effective tumor cell lysis.
The strength of the initial binding events between a T cell and a tumor cell dictates the efficiency of the anti-tumor response. Assessing cell avidity, i.e. the total intercellular interaction strength between two cells, gives crucial insights into the efficacy of CEs as anti-tumor therapeutic agents.
Here, we deploy LUMICKS’ high throughput avidity measurement (HTAM) technology to measure CE-induced cell avidity in a high throughput manner. We demonstrate the assay performance characteristics, i.e. specificity, precision, and range, via CE titration experiments in the context of a Jurkat T cell model system. We find that the HTAM CA assay is suitable for candidate screening in high throughput, with high sensitivity and precision.