Rozumalski, L. | bioRxiv Science

bioRxiv2025

Multivalent Tri-Functional T-cell Engagers by Chemically Induced Protein Self-Assembly

T-cell engagers (TCEs) show promise in cancer immunotherapy but face challenges in solid tumors due to heterogeneity, antigen escape, and limited T-cell infiltration. To address this, we developed a modular platform using chemically self-assembled nanorings (CSANs). We engineered a bifunctional fusion protein, E1-DHFR2-CD3, with an EGFR-binding fibronectin (E1) and an anti-CD3 scFv on a DHFR2 scaffold. With bis-methotrexate, the monomers formed multivalent cis-CSANs. Both monomers and CSANs bound EGFR+ tumor cells and T-cells, were internalized, and induced dose-dependent, EGFR- and T-cell-dependent cytotoxicity in co-culture assays. The system was reproducible across T-cell donors. To expand targeting, E1-DHFR2-CD3 co-assembled with other DHFR2 monomers targeting GFP or EpCAM, forming trispecific CSANs capable of binding multiple antigens and mediating cytotoxicity. This platform enables the discovery of potent multispecific TCEs to address antigen escape and solid tumor heterogeneity. Future work will focus on optimizing antigen combinations to enhance efficacy across breast cancer subtypes.

immunology↗

bioRxiv2022

Macro-Chemical Biology: Engineering Biomimetic Trogocytosis with Farnesylated Chemically Self-Assembled Nanorings

With the recent success of cell-based therapies, there has been a rapidly emerging interest in the engineering of cell-cell interactions and communications. Inspired by the natural intercellular material transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (farnesyl-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells; thus, allowing cell-cell interactions to be monitored, as well as facilitating communication between the cells by delivery of bioactive species. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted farnesyl-CSANs and to be efficiently transferred to receiver cells expressing the appropriate receptor by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor-specific and dependent on direct cell-cell interactions, the rate of receptor internalization and the amount of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides (ASO). Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be used to monitor cell-cell interactions, as well as the engineering of cell-cell communications, such as cell-based drug delivery.

bioengineering↗