IF a logic-gated, bispecific AND-gate CAR-T cell construct co-targeting Fibroblast Activation Protein (FAP) and Urokinase Plasminogen Activator Receptor (uPAR) — each encoded as tandem scFv domains linked to split-signaling architecture (anti-FAP scFv driving CD3ζ; anti-uPAR scFv driving 4-1BB co-stimulation, requiring simultaneous engagement of both receptors for full T-cell activation) at a dose of 5×10⁶ cells/mouse administered intravenously is administered to 10–12-week-old male C57BL/6J mice at peak CCl4-induced liver fibrosis (week 8 of CCl4 protocol),

THEN liver collagen content will decrease by ≥40% (measured by Sirius Red morphometry and hydroxyproline assay), histological fibrosis score (METAVIR/Ishak) will improve by ≥2 stages, and serum ALT/AST will normalize to within 1.5× upper reference limits within 21 days post-infusion, with significantly attenuated on-target off-tumor toxicity (cardiac fibroblast depletion, bone marrow stromal damage, and cytokine release syndrome severity) compared to single-target anti-FAP scFv-CD3ζ-4-1BB CAR-T cells,

BECAUSE the following causal chain operates:

1. Senescent hepatic stellate cells (HSCs), which are the principal drivers of fibrotic ECM deposition in CCl4-induced liver fibrosis, simultaneously overexpress both FAP (a serine protease and fibroblast activation marker upregulated during HSC activation and senescence) and uPAR (a GPI-anchored receptor robustly upregulated across multiple senescent cell types as part of the senescent cell surface secretome), creating a combinatorial antigen signature that distinguishes them from non-senescent FAP+ tissues. (FAP overexpression on activated/senescent HSCs noted in Research Context; uPAR as a pan-senescence surface marker: described in the research framing context of Amor et al. Nature 2020 as cited in Research Context)

2. Non-liver FAP+ tissues — including cardiac fibroblasts, bone marrow stromal cells, and smooth muscle-associated fibroblasts — express FAP in the absence of the senescence-associated uPAR upregulation, meaning they present only one of the two AND-gate antigens and are therefore subthreshold for CAR-T cytotoxic activation under the split-signaling architecture. [SPECULATIVE — requires validation of uPAR expression profiling across FAP+ off-target tissues]

3. The AND-gate CAR architecture (split CD3ζ / 4-1BB signaling across two receptor chains) ensures that neither anti-FAP engagement alone nor anti-uPAR engagement alone delivers sufficient intracellular signal to trigger T-cell degranulation and cytotoxicity; only co-ligation of both antigens on the same target cell activates the full effector program, suppressing fratricide and bystander killing. [SPECULATIVE as applied to this specific FAP/uPAR pairing; AND-gate CAR logic validated in oncology contexts noted in broader CAR-T literature]

4. Selective elimination of FAP+/uPAR+ senescent HSCs removes the primary source of TGF-β1, CTGF, and collagen I/III secretion fr...

SENS category: GlycoSENS