{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T18:55:02Z","timestamp":1777488902872,"version":"3.51.4"},"reference-count":63,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T00:00:00Z","timestamp":1737072000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioinform."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The development of nanobodies targeting Programmed Cell Death Protein-1 (PD-1) offers a promising approach in cancer immunotherapy. This study aims to design and characterize a PD-1-specific nanobody using an integrated computational and experimental approach.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>An <jats:italic>in silico<\/jats:italic> design strategy was employed, involving Complementarity-Determining Region (CDR) grafting to construct the nanobody sequence. The three-dimensional structure of the nanobody was predicted using AlphaFold2, and molecular docking simulations via ClusPro were conducted to evaluate binding interactions with PD-1. Physicochemical properties, including stability and solubility, were analyzed using web-based tools, while molecular dynamics (MD) simulations assessed stability under physiological conditions. The nanobody was produced and purified using Ni-NTA chromatography, and experimental validation was performed through Western blotting, ELISA, and dot blot analysis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Computational findings demonstrated favorable binding interactions, stability, and physicochemical properties of the nanobody. Experimental results confirmed the nanobody\u2019s specific binding affinity to PD-1, with ELISA and dot blot analyses providing evidence of robust interaction.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>This study highlights the potential of combining computational and experimental approaches for engineering nanobodies. The engineered PD-1 nanobody exhibits promising characteristics, making it a strong candidate for further testing in cancer immunotherapy applications.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fbinf.2024.1488331","type":"journal-article","created":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T06:53:37Z","timestamp":1737096817000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Innovative CDR grafting and computational methods for PD-1 specific nanobody design"],"prefix":"10.3389","volume":"4","author":[{"given":"Jagadeeswara Reddy","family":"Devasani","sequence":"first","affiliation":[]},{"given":"Girijasankar","family":"Guntuku","sequence":"additional","affiliation":[]},{"given":"Nalini","family":"Panatula","sequence":"additional","affiliation":[]},{"given":"Murali Krishna Kumar","family":"Muthyala","sequence":"additional","affiliation":[]},{"given":"Mary Sulakshana","family":"Palla","sequence":"additional","affiliation":[]},{"given":"Teruna J.","family":"Siahaan","sequence":"additional","affiliation":[]}],"member":"1965","published-online":{"date-parts":[[2025,1,17]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"4301","DOI":"10.3390\/jcm12134301","article-title":"Review of the immune checkpoint inhibitors in the context of cancer treatment","volume":"12","author":"Alturki","year":"2023","journal-title":"J. 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