Generation of Nano‐Antibodies Targeting the SNMP1 Membrane Receptor of <i>Spodoptera littoralis</i> Using Extracellular Vesicles

Handling membrane proteins (MPs) in their native environment is crucial for ensuring proper folding and stability. Extracellular vesicles (EVs) offer a superior alternative to detergents and artificial lipid environments. Expressing recombinant MPs on the surface of EVs allows for their display in a native context, making them ideal for llama immunization to generate nano-antibodies (Nabs) targeting MPs. In this study, we focused on the sensory neuron membrane protein 1 (SNMP1) from <i>Spodoptera littoralis</i>, a key player in transferring hydrophobic pheromones from pheromone-binding proteins to olfactory receptors, thereby triggering behavioral responses in moths. Targeting this protein with Nabs could provide a potential biocontrol strategy by limiting moth reproduction. Llama immunization was carried out using EVs displaying SNMP1 on their surface. A multi-layer approach was then employed, incorporating an anchor protein co-displayed with the target protein to specifically expose the EVs of interest for biopanning through phage display and selection via ELISA. Nabs against SNMP1 were successfully isolated, and their interactions with the membrane protein were characterized using bio-layer interferometry (BLI) with the same multi-layer approach. Furthermore, epitope binning by BLI aligned with predictions made by AlphaFold3.