Engineering Quiescent Viral Entry Pathways for in Vivo CAR-T Generation via Binder-Fusogen Combinatorics

Abstract Background: In vivo delivery of CAR constructs via lentiviral vectors is hindered by a critical limitation: conventional viral envelopes trigger premature T-cell activation, leading to excessive cytokine release and functional exhaustion that compromise therapeutic efficacy and safety. Precise control of T-cell activation during in vivo lentiviral delivery is therefore essential to balance efficacy with toxicity. We hypothesize that the activation threshold can be fine-tuned through rational engineering of synergistic interactions between viral binders and fusogens, enabling efficient transduction while maintaining T cells in a functionally quiescent state. Methods: We systematically screened binders targeting multiple T-cell surface markers (e.g., CD3, CD7, CD8) in diverse formats (scFv, VHH), paired with a library of engineered fusogen variants. Each binder-fusogen combination was evaluated for activation dynamics using CD69/CD25 surface expression, cytokine secretion (IL-2, IFN-γ, IL-6), and transcriptomic profiling in primary human T cells. Transduction specificity was assessed by comparative tropism analysis of resting versus activated T-cell subsets, while off-target potential was mapped through transduction profiling across primary human non-immune cell lineages. Lead candidates were validated in humanized mouse models, assessing antigen-independent activation, tumor-specific cytotoxicity, and systemic inflammatory responses. Results: Screening identified distinct binder-fusogen combinations capable of achieving high-titer transduction while inducing minimal early activation markers and cytokine release. Optimal pairs demonstrated rapid receptor internalization without sustained TCR signaling complex formation, thereby preserving resting T-cell metabolic profiles and preventing exhaustion marker upregulation. These “functionally silent” configurations maintained baseline cytokine levels during transduction, yet enabled robust CAR-driven expansion upon subsequent antigen encounter. In vivo validation confirmed that lead candidates mediated complete tumor clearance without measurable cytokine release syndrome or off-target toxicity, whereas control combinations with identical binders but suboptimal fusogens triggered premature T-cell activation and pronounced interleukin cascades. Conclusion: We established a combinatorial entry engineering platform that effectively decouples viral transduction from T-cell activation. Systematic screening of binder-fusogen pairs across activation thresholds identified configurations enabling “silent” CAR delivery with high specificity and built-in safety features. This approach expands the therapeutic window for in vivo CAR-T therapies by exerting precise control over early T-cell stimuli and is broadly applicable to next-generation in vivo adoptive therapies.