[Screening and characterization of two nanobodies with different CDR3 sequences against porcine CD205]

The Complementarity-determining region (CDR) is the core region that determines the functions and characteristics of nanobodies. To analyze the impact of the CDR3 region on the developability of nanobodies and antigen-binding activity, this study used two porcine CD205-specific nanobodies (Nb205-1 and Nb205-10) with different CDR3 regions as models and compared them in terms of prokaryotic expression, Ni column purification, and antigen binding, providing references for the screening and evaluation of nanobodies. Phage display technology was employed to screen porcine CD205-specific nanobodies. Positive clones were subjected to sequence alignment, and Swiss-Model was utilized for homology modeling and structural alignment. The Escherichia coli system was employed to evaluate the strain proliferation capacity, in vitro protein expression form, and Ni-NTA affinity purification efficiency. ELISA was used to detect their binding activity with the target protein of porcine CD205 and their antigenic epitopes. Phage-ELISA results demonstrated that two nanobodies, Nb205-1 and Nb205-10, were screened out with high binding activity to the target protein of porcine CD205. Sequence alignment analysis revealed that the framework regions of the two nanobodies were highly conserved, with only several amino acid residue differences in the CDR3 region. Strain Nb205-1 exhibited superior developability, with high protein expression in E. coli, strong binding affinity to Ni-NTA resin, and ease of purification. In contrast, Nb205-10 existed in the inclusion body form, showed weak binding affinity to Ni-NTA resin, and had extremely low purification efficiency. The 3D structural modeling and electrostatic potential analysis revealed that the CDR3 loop of Nb205-1 presented a convex conformation and was rich in positive charges, while Nb205-10 exhibited a relatively flat concave conformation and neutral charge, suggesting that the two may recognize similar antigenic epitopes through different molecular mechanisms. Epitope analysis indicated that the antigenic epitope recognized by Nb205-1 was located in the region ¹MITPLNDWIVAKDCDKTK¹⁸. This study identified two porcine CD205 nanobodies (Nb205-1 and Nb205-10) with similar binding activity, while their developability (expression and purification efficiency) was significantly influenced by sequence differences in the CDR3 region. In future therapeutic nanobody screening, comprehensive evaluation of their developability should be conducted to provide theoretical and practical bases for rational design.