Biology subjects

Wagner, C. R.

Publications and source records attributed to Wagner, C. R..

8 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↗

bioRxiv2024

HINT1 Inhibitors as Selective Modulators of MOR-NMDAR Cross Regulation and Non-Opioid Analgesia

The Human Histidine Triad Nucleotide Binding Protein 1 (HINT1) has recently become a protein of interest due to its involvement in several CNS processes, including neuroplasticity and the development of several neuropsychiatric disorders. Crucially, HINT1 behaves as a mediator for the cross-regulation of the mu opioid receptor (MOR) and N-methyl-D-aspartate receptor (NMDAR). Active site inhibition of HINT1 using small molecule inhibitors has been demonstrated to have a significant impact on this cross-regulatory relationship in vivo. Herein, we describe the development of a series of ethenoadenosine HINT1 inhibitors to further evaluate the effect of HINT1 inhibition on morphines blockade of NMDA-evoked behaviors, the development of acute endomorphin-2 tolerance and analgesia. X-ray crystallographic analysis and HINT1 binding experiments demonstrate that modifications to the inhibitor nucleobase greatly impact the inhibitor binding interactions with HINT1. Our results reveal a complex structural-activity relationship for HINT1 inhibitors in which minor modifications to the ethenoadenosine scaffold resulted in dramatic changes to their activity in these assays modeling MOR-NMDAR interaction. Specifically, we observed the ability of HINT1 inhibitors to selectively affect individual pathways of MOR-NMDAR crosstalk. Furthermore, we observed that a carbamate ethenoadenosine inhibitor of HINT1 can induce analgesia, while not affecting opioid tolerance. Additionally, although past studies have indicated that that loss of HINT1 expression can result in the downregulation of p53, we have shown that inhibition of HINT1 has no effect on either the expression of HINT1 or p53. These studies highlight the critical role of HINT1 in MOR-NMDAR crosstalk and demonstrate the intriguing potential of using HINT1 active-site inhibitors as tools to probe its role in these biochemical pathways and its potential as a novel pain target.

neuroscience↗

bioRxiv2022

Multivalent, Bispecific ????B7-H3-????CD3 Chemically Self-Assembled Nanorings Direct Potent T-cell Responses Against Medulloblastoma

Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific chemically self-assembling nanorings (CSANs) that target the T cell receptor, CD3{varepsilon}, and tumor associated antigen, B7-H3, derived from the humanized 8H9 single chain variable fragment (scFv). We show that the B7-H3-CD3 CSANs increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma spheroids and that activity is independent of target cell MHC class I expression. Importantly, non-specific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the CD3 targeted monomer in the oligomerized CSAN. Intraperitoneal injections of B7-H3-CD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant anti-tumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with B7-H3-CD3 CSANs, intratumoral CD4+ and CD8+ T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Collectively, these results demonstrate the ability of our multi-valent, bispecific CSANs to direct potent anti-tumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors. SignificanceThis study presents B7-H3-CD3 bispecific CSANs as a promising, new immunotherapeutic option for patients with established B7-H3+ medulloblastomas.

cancer biology↗

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↗

bioRxiv2020

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T cell Mediated Anti-Tumor Response and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression

Numerous approaches have targeted the Epidermal Growth Factor Receptor (EGFR) for the development of anti-cancer therapeutics, since it is over-expressed on a variety of cancers. Recently, EGFR chimeric antigen receptor (CAR)-T cells have shown potential promise for the immunological control of tumors. Our laboratory has recently demonstrated that bispecific chemically self-assembled nanorings (CSANs) can modify T cell surfaces and function as prosthetic antigen receptors (PARs). This technology allows selective targeting of tumor antigens due to high avidity of the multimeric rings, while incorporating a mechanism to dissociate the rings to prevent further T cell stimulation. Previously, PARs with single-chain variable fragments (scFvs) have been successful in vitro and in vivo, activating T cells selectively at the tumor site. Alternatively, here we report fibronectin (FN3)-based PARs with improved properties such as increased protein yield, rapid protein production, increased protein stability and predicted low immunogenicity due to the human origin of fibronectins. We examined the cytotoxicity of EGFR-targeting PARs in vitro in which the affinities of the EGFR fibronectins, the EGFR/ CD3 valency of the CSANs and the antigen expression levels were varied. Based on these selective in vitro cytotoxicity results, we conducted an in vivo study of FN3-PARs using an orthotopic breast cancer model. The FN3-PARs demonstrated potent tumor growth suppression with no adverse effects. Furthermore, these results demonstrated that FN3-PARs modulated the tumor microenvironment by downregulating EGFR signaling resulting in decreased PD-L1 expression. In addition, the expression of PD-1 was also found to be reduced. Collectively, these results demonstrate that FN3-PARs have the potential to direct selective T cell targeted tumor killing and that EGFR FN3-PARs may enhance anti-tumor T cell efficacy by modulating the tumor microenvironment.

bioengineering↗

bioRxiv2019

Deconstructing the individual steps of vertebrate translation initiation

Translation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control 1. Despite this centrality the series of steps involved in this process are poorly understood 2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5 UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.

molecular biology↗