Biology subjects

Rozumalski, L.

Publications and source records attributed to Rozumalski, L..

4 recordsLinked to original sources

bioRxiv2026

T-cell Receptor (TCR) targeting with Multivalent T-cell Engagers

T-cell engagers (TCEs) for cancer immunotherapy have traditionally relied on high affinity single chain fragment variable (scFv) domains to target CD3, specifically the {varepsilon} chain, for the activation of T-cells. Despite their clinical success, there have been reports of TCEs driving systemic toxicity, non-specific T-cell activation, on-target off-tumor effects, and severe inflammation due to cytokine release. To address these limitations, we designed multivalent TCEs using Chemically Self-Assembled Nanorings (CSANs) that target the /{beta} constant region of the T-cell receptor (TCR) in the TCR/CD3 complex using a moderate affinity TCR nanobody (TCRVHH). Nanobodies offer superior physical and chemical properties over scFvs-including higher solubility, stability and lower production cost-making them increasingly popular as structural units of TCEs. We compared the efficacy and safety profile of this moderate affinity, nanobody-based TCR binder against high affinity CD3scFv based CSANs across EGFR and PSMA expressing solid tumor models. While the CD3scFv CSANs offered potent cytotoxicity, they also induced antigen independent T-cell activation bypassing the requirement of tumor crosslinking for cytotoxicity. In contrast the TCRVHH CSANs required strict antigen engagement to trigger cytotoxicity, significantly reducing non-specific T-cell activation and thus enhancing the safety profile. Although the initiation of cytotoxicity was kinetically slower than the CD3scFv counterpart, TCRVHH CSANs achieved comparable end point cytotoxicity across multiple antigen densities, as well as in 3D tumor spheroids. Through this study we demonstrate the applicability of nanobodies as T-cell targeting domains, enhanced specificity and safety of moderate affinity T-cells binders and the diversification of T-cell targeting epitopes without compromising the efficacy of TCEs. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/725710v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@9bcb83org.highwire.dtl.DTLVardef@4e30daorg.highwire.dtl.DTLVardef@1695931org.highwire.dtl.DTLVardef@15ec7ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Understanding heterogeneous responses to T cell engagers: Binding characteristics and dosing thresholds determine cytotoxic efficacy

Therapeutic efficacy of multivalent T cell engagers varies widely across individuals, but the basis for this heterogeneity remains poorly understood. Here, we integrate in vitro experiments of antitumor immune responses with a mechanistic modeling framework to investigate sources of response variability across T cell donors and TE constructs, focusing on a novel multivalent bispecific T cell engager currently in development. We identify parameter regimes that accurately recapitulate dose-response behaviors across T cell donors and doses, and perform cross-validation studies that demonstrate the models predictive accuracy. We find that variability in therapy efficacy is governed by the relationship between binding affinity and dose. When dose exceeds the binding affinity, responses are relatively robust across donors; when dose is below the binding affinity, responses are more donor dependent. At smaller doses, the TE-specific shape characteristics of the tumor-binding dose response, its steepness in particular, is a key marker of therapy efficacy. More generally, our integrated modeling and experimental framework offers insights and tools that are applicable to other bispecific T-cell engagers, and provides a quantitative foundation for the systematic, in silico optimization of TE design and dosing strategies.

immunology↗

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↗