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.