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Biology subjects

CHAMES, P.

Publications and source records attributed to CHAMES, P..

3 recordsLinked to original sources
bioRxiv2026

Tuning T-cell immunological synapse by modular DNA-Nanobody engagers for precision immunotherapy

Bispecific T-cell engagers (TCEs) are a promising class of cancer immunotherapies, but their clinical use is limited by toxicity and insufficient specificity. Tuning the T cell- tumor interface through engager architecture may address these drawbacks. To this end, we engineered hybrid constructs composed of two nanobodies targeting CD3 and the model tumor antigen HER2, respectively, connected by rigid DNA linkers of variable length. Using cytotoxicity assays and hybrid biophysical platforms, we demonstrate a linker-length dependence of cell spreading on antigen, target killing and cytokine release, revealing a functional decoupling between killing and cytokine secretion, and implicating the glycocalyx as a key player. Through the addition of EGFR targeting, we also generate trispecific constructs implementing an "OR-gate" logic to address tumor heterogeneity and reduce resistance due to antigen loss. Overall, these versatile constructs show great therapeutic promise, and at the same time serve as platforms to test hypotheses on biophysical mechanisms.

synthetic biology↗
bioRxiv2026

A syndecan-based genetic approach to coat the surface of small extracellular vesicles with Nanobodies

Small extracellular vesicles (sEVs) are promising vehicles for targeted therapeutic delivery, but strategies for their surface functionalization remain limited. Here, we present a reliable and simple genetic approach that enables customized modification of sEV surfaces and supports enhanced sEV uptake by recipient cells. This strategy is based on the fusion of targeting moieties to the C-terminal fragment of syndecan-1 (SDC1-CTF), a peptide naturally enriched in sEVs. Combining various analytical approaches including single-vesicle analysis, we establish that this strategy enables decoration of up to 20% of secreted sEVs with nanobodies (Nbs). In quantitative bioluminescence assays, using concentrated conditioned media, we demonstrate that sEV-coating with anti-EGFR Nb supports enhanced sEV uptake by EGFR-expressing cells. This new strategy thus offers a robust and modular solution for endowing sEV surfaces with defined targeting properties to support further sEV-based therapeutic applications.

cell biology↗
bioRxiv2025

Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

The heparan sulphate proteoglycan, Glypican-4 (GPC-4), is an integral component of cell surfaces that fulfils key functions as a modulator of cell communication. Over time, human GPC-4 (hGPC4) has gained recognition as a valuable target for enhancing the therapeutic potential of human pluripotent stem cells (hPSCs). hGPC-4 is also a promising diagnostic and therapeutic target for a range of developmental and neurological disorders, as well as cancer. Its involvement in multiple biological processes and its impact on cellular signaling pathways make it a compelling candidate for future research and clinical applications. Here, we report RB1 and RB3 as the first hGPC-4-specific nanobodies, exhibiting high affinity for both recombinant and cell surface-associated hGPC-4 molecules. Notably, the bivalent nanobody Fc-fusion form of RB1, termed RB1-Fc, demonstrates a significant [~]14-fold increase in apparent binding affinity on cells when compared to the monovalent RB1. Furthermore, binding of RB1-Fc to hGPC-4 is dependent on the native conformation of hGPC-4, demonstrating that RB1-Fc is a conformational nanobody. Notably, RB1-Fc neutralizes the activity of GPC-4, as shown by our functional studies in hPSCs. These studies demonstrate the potent efficacy of the lead hGPC4 nanobodies, RB1-Fc and RB3. They also provide a solid rationale for using these nanobodies in the detection and characterization of physiologically and clinically relevant hGPC-4. Additionally, their potential as agents for therapeutic targeting of hGPC-4 opens new avenues for treating disorders associated with dysregulated hGPC-4 activity. HighlightsO_LIDiscovery and generation of nanobodies targeting human Glypican-4. C_LIO_LIRB1-Fc, a bivalent Fc-fusion nanobody, selectively binds to the native human Glypican-4 with high affinity. C_LIO_LIRB1-Fc binding to human Glypican-4 enhances human induced pluripotent stem cells differentiation ability, mimicking human Glypican-4 downregulation C_LIO_LIRB1-Fc is a promissing tool for detecting, characterizing, and therapeutically targeting human Glypican-4 in developmental, neurological, and cancer-related contexts. C_LI Graphical abstractSchematic illustration of this study. Nanobodies (Nbs) specific for native human Glypican-4 (hGPC4) were isolated from a phage-display library generated after immunizing a llama with membrane extracts from hGPC4-transfected HEK 293 cells. Two nanobodies, RB3 and RB1, bind recombinant and cell-expressed hGPC4 with nanomolar affinity. A bivalent RB1-Fc fusion was engineered and showed enhanced binding to endogenous hGPC4 via a conformational epitope. The specificity of the RB1-Fc Nb for hGPC4 is further supported by functional studies in hiPSCs, which demonstrated its blocking activity. Differentiation analyses revealed that RB1-Fc- treated hiPSCs exhibited a significantly enhanced capacity to differentiate into endoderm, closely resembling the phenotype observed in hiPSCs with downregulate hGPC4. These results suggest that RB1-Fc binding functionally inhibits hGPC4, potentially acting as an orthosteric competitor or an allosteric negative modulator.

bioengineering↗