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Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1 ) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes. Glypican-1 (GPC1) expression is elevated in pancreatic cancer and has been exploited as a therapeutic target. Here the authors report the development of V H H nanobody-based CAR-T cells targeting GPC1, showing anti-tumor activity in pancreatic cancer preclinical models.

BackgroundMultiplexed protein analysis in native cellular contexts holds great promise for uncovering the composition, interactions, and functions of distinct cell types in complex biological systems. However, current multiplexed protein imaging technologies often face limitations in target flexibility, detection sensitivity and sample integrity that involve technically demanding protocols. To address these issues, we have developed an ultra-sensitive, multiplexed in situ protein profiling approach that combines off-the-shelf antibodies and lectins with the newly developed Cleavable Fluorescent Probe (CFP™) cleavable TSA fluorophores.MethodsTarget proteins are recognized by primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP), which catalyzes the covalent deposition of CFP fluorophores on or close to the target proteins. After imaging, the fluorophores are chemically cleaved, and antibodies stripped, enabling subsequent rounds of staining, imaging, cleavage, and stripping. This approach allows for the interrogation of many targets in individual cells while maintaining spatial resolution and sample integrity.In addition to protein analysis, glycan modifications can also be profiled using this approach. Glycan modifications also play critical roles in cell function in both healthy and diseased states, from controlling proliferation to modulating the immune response. Lectins are glycan-binding proteins that recognize specific glycan structures. Their glycan-specific nature makes them important tools to profile, characterize, and capture the complexity of glycans in biological systems.We demonstrated the compatibility of CFP cleavable TSA fluorophores with lectins developed by Vector Laboratories. This assay facilitates the exploration of major glycan motifs including sialylation, fucosylation, and galactosylation, for characterizing glycan distributions in target specimens.We also utilized a novel proximity-based detection technology to investigate changes in protein glycosylation from healthy to diseased brain tissues. This tool enriches biomarker discovery and therapeutic targeting, thus emphasizing the transformative potential of glycosylation-focused research in advancing precision medicine. Results and Conclusions Our novel CFP technology enables a simplified and flexible workflow using off-the-shelf antibodies and lectins for investigating numerous targets on the same tissue section. Our results enabled us to simultaneously explore the altered protein expression and glycosylation states in specimens across various disease indications. In addition, we utilized in situ Proximity Ligation Assay to explore the glycosylation changes of specific neural cell markers in Alzheimer’s Disease brain tissues. This new data will add to the growing understanding of the impact of protein glycosylation on brain pathology.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1 ) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V H nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of V H-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Information systems are essential to global management and marketing operations. In order to meet competitive and efficiency challenges, global managers rely on information systems' support. As such, it is important to appropriately evaluate their interaction with the information systems. A study assesses the applicability of a user-information-satisfaction scale, touted to have wide applicability, cross-culturally and across disciplines. In the process, potential applications of the scale in management and marketing user environments are explored.