The CAR Immune Synapse Interactome: How Membrane Organization Shapes CAR T–Tumor Cell Interactions

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.

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CD19-directed CAR-T therapy has achieved remarkable clinical outcomes in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Nevertheless, a significant proportion of patients develop primary resistance or relapse, often associated with CD19 antigen loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual antigen targeting could improve therapeutic durability and mitigate antigen escape. To address this, we developed a panel of CD19/BCMA dual-targeting CAR-T cells building on our academic platforms targeting CD19 (varnimcabtagene autoleucel, ARI0001) and BCMA (cesnicabtagene autoleucel, ARI0002h). Multiple strategies were explored, including pooled mono-targeted products, co-transduction with two lentiviral vectors, bicistronic constructs, and tandem/loop CAR designs incorporating dual binding domains within a single receptor. Functional activity and avidity were assessed across varying antigen expression conditions. We found that dual CAR-T cells generated through co-transduction consistently showed superior performance relative to single-target CD19 CAR-T cells and other dual-targeting formats, particularly in models with low CD19 expression. A first-in-human phase I clinical trial (CARTDBG-01; NCT06097455) is currently underway to evaluate the safety and efficacy of ARI0003 in NHL.

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Related publication: Bachiller, M., Barceló-Genestar, N., Rodriguez-Garcia, A., Alserawan, L., Dobaño-López, C., Giménez-Alejandre, M., ... & Guedan, S. (2025). ARI0003: Co-transduced CD19/BCMA dual-targeting CAR-T cells for the treatment of non-Hodgkin lymphoma. Molecular Therapy, 33(1), 317-335. https://doi.org/10.1016/j.ymthe.2024.11.028

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90% of immuno-oncology drugs entering Phase I clinical trials fail, due to a poor predictive value of current preclinical assays. To solve this, scientists and drug developers are turning to Cell Avidity, the total binding strength between cells, to better predict therapeutic efficacy.​

​Join us for an exclusive virtual demonstration of Avidion, the next generation of Cell Avidity technology. This session isdesigned for researchers and groups who understand the "why" of avidity but want to see the "how." ​

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Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, but its efficacy remains limited in solid tumors due to antigen heterogeneity, an immunosuppressive microenvironment, and the glycocalyx barrier. The glycocalyx, composed of dense glycoproteins such as MUC1, is markedly expanded in cancers, where it impedes immune cell access and antigen engagement, thereby reducing therapeutic efficacy. In most adenocarcinomas, the Tn antigen, comprising N-acetylgalactosamine linked to serine or threonine, is overexpressed. Tn-MUC1, a truncated form of MUC1 decorated with Tn antigen, is frequently overexpressed in pancreatic cancer. Here, we incorporate a non-signaling glyco-bridge binder recognizing Tn-MUC1 into mesothelin-directed CAR-T cells. This bridge enhances tumor recognition and cytotoxicity by increasing avidity and facilitating CAR activation in a density- and affinity-dependent manner. To directly validate these effects at the cell interaction level, we used Lumicks z-Movi to quantify CAR-T binding strength to tumor targets. CAR-T cells equipped with the Tn-MUC1 glyco-bridge exhibited higher cell avidity toward Tn-MUC1-expressing tumor cells compared to a CD19 bridge control. To broaden its applicability, we design a tandem Helix pomatia agglutinin (HPA) lectin-based bridge that recognizes Tn antigens across cancer types. CAR-T cells with the HPA-bridge exhibit superior cytotoxicity in pancreatic cancer models.