Year: 2026
Type: conference-abstract
Abstract: Background Achieving tumor-specific targeting while minimizing off-tumor toxicity remains a critical challenge for CAR-T cell therapy in solid tumors, driving the development of selective tumor antigens and logic-gated CAR designs that enhance local activation, specificity, efficacy, and safety. Tumor Endothelial Marker 8 (TEM8) is a transmembrane protein that is highly expressed on tumor-associated stromal cells and vasculature, with limited expression in normal adult tissues. Here, we engineered optimized human TEM8-specific scFvs and incorporated them into a SynNotch logic-gated platform, enabling TEM8-dependent induction of a CD276-targeting CAR in pancreatic cancer models.Methods Human TEM8-specific scFvs were isolated by yeast-display bio-panning and characterized using biolayer interferometry and flow cytometry, followed by functional evaluation in a 24-hour PANC1 cytotoxicity assay. Selected binders were incorporated into a SynNotch-inducible CD276-targeting CAR and evaluated in a 4-day real-time xCELLigence killing assay. Constructs with basal SynNotch activity were optimized by in silico ALPP score-guided reduction of positively charged surface area, reformatted as SynNotch CARs, and assessed in 2D and 3D PANC1 spheroid models.Results Five human TEM8-specific scFvs were identified, demonstrating favorable characteristics, including reduced aggregation, enhanced folding efficiency, and optimized affinity and stability. All binders demonstrated specific, high-affinity binding to human TEM8. CAR-T cells expressing individual TEM8-targeting binders showed robust viability, expansion, and transduction efficiency, with three binders mediating potent cytotoxicity against PANC1 cells, comparable to the reference control across effector-to-target ratios. Incorporation of these binders into a SynNotch-inducible CD276-targeting CAR circuit maintained robust T-cell fitness and TEM8-sensor expression; however, constructs containing two TEM8 binders exhibited antigen-independent CD276 CAR expression. To mitigate ‘leakiness,’ the binders were modified through in silico engineering, resulting in binder electrostatics modified variants. Of the resulting variants, mut2 and mut3 demonstrated reduced basal activity while retaining potent and selective cytotoxicity against PANC1 cells in both 2D and 3D spheroid models, with minimal activity against antigen-negative ASPC1 cells, identifying them as lead candidates for further development.Conclusions Human TEM8-specific binders were successfully engineered with favorable biophysical and functional characteristics for logic-gated CAR-T cell therapy. In silico optimization of binder electrostatics reduced basal SynNotch activity while maintaining antitumor efficacy, identifying mut2 and mut3 as lead candidates for pancreatic cancer.
Institution: Lentigen Technology (United States)
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225183527