JAGADEESWARA REDDY DEVASANI ^1* Girija Sankar Guntuku ^1* Mary Sulakshana Palla ^2* Murali Krishna M ^1*

• ¹ Andhra University, Visakhapatnam, India
• ² Gandhi Institute of Technology and Management (GITAM), Visakhapatnam, Andhra Pradesh, India

The development of nanobodies targeting Programmed Cell Death Protein-1 (PD-1) offers a promising approach in cancer immunotherapy. This study combines computational design with experimental validation to engineer and characterize a PD-1-specific nanobody using an integrated in silico and in vitro approach. Initially, an in-silico design strategy was employed, where Complementarity-Determining Region (CDR) grafting was used to construct the nanobody sequence. The three-dimensional structure of the nanobody was predicted using AlphaFold2, followed by molecular docking simulations via ClusPro to assess binding interactions with PD-1.Physicochemical analyses, performed using web-based tools, provided insights into the stability, solubility, and other properties critical to nanobody efficacy. Molecular dynamics (MD) simulations further evaluated the stability and conformational dynamics under physiological conditions. To validate these computational findings, the nanobody was produced and purified using Ni-NTA chromatography. Characterization was carried out through Western blotting and ELISA, which gives nanobody specific binding affinity for PD-1. Dot blot analysis provided additional evidence of binding specificity and interaction robustness. Our combined computational and experimental results indicate that the engineered PD-1 nanobody is a strong candidate for further testing in cancer immunotherapy applications.