19P Design and evaluation of nanobodies targeting the human tumor protein TCTP

penetration while maintaining high antigen-binding affinity, making them ideal candidates for targeted cancer theranostics.Methods: VNAR sequences targeting a cancer-associated antigen were generated through in-house immunization of nurse sharks followed by phage display selection.To modulate pharmacokinetics, targeted mutations were introduced within hypervariable domain 2 (HV2) to adjust surface charge and isoelectric point (pI).Rational design used structural modeling and electrostatic surface analysis to predict pI shifts without disrupting the antigen-binding interface.Engineered variants were expressed, purified, and characterized for structural integrity and antigen-binding affinity using biolayer interferometry (BLI).pI modifications were validated experimentally, and clearance impact was assessed through 89 Zr labeling and PET/CT imaging in nave mouse models.Results: VNAR HV2 variants engineered to have a lower pI were primarily cleared via renal pathways, whereas those with higher pI favored hepatic clearance.Importantly, these engineered VNARs retained structural integrity and antigen-binding affinity while demonstrating distinct in vivo clearance profiles.Conclusions: VNAR antibodies can be engineered via HV2 mutagenesis to modulate pI and direct clearance pathways without compromising stability or binding affinity.This strategy enables optimization of theranostic agents to minimize off-target radiation exposure, offering a promising approach for safer and more effective cancer imaging and therapy.