{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T16:32:33Z","timestamp":1793291553094,"version":"4.5.0"},"reference-count":46,"publisher":"Oxford University Press (OUP)","license":[{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,1,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complementarity-determining region 2 (CDR2) of a soluble, clinically validated HER2 nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered the CALR nanobody 02 by adapting the core of Cal3 to a consensus framework sequence for stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a soluble protein with an increased thermal stability and a five-fold higher calreticulin binding affinity (KD: 25\u00a0nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.<\/jats:p>","DOI":"10.1093\/protein\/gzag030","type":"journal-article","created":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T11:45:38Z","timestamp":1792583138000},"source":"Crossref","is-referenced-by-count":0,"title":["Improved calreticulin nanobody by framework engineering"],"prefix":"10.1093","volume":"39","author":[{"given":"Logan","family":"Mavar","sequence":"first","affiliation":[{"name":"Department of Microbiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikhail","family":"Pavlenok","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avraneel","family":"Paul","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3582-6462","authenticated-orcid":false,"given":"Lucinda","family":"Hall","sequence":"additional","affiliation":[{"name":"Department of Radiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]},{"name":"Department of Surgery, University of Alabama at Birmingham , Birmingham, AL 35233 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin M","family":"Larimer","sequence":"additional","affiliation":[{"name":"Department of Radiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]},{"name":"O\u2019Neal Comprehensive Cancer Center, University of Alabama at Birmingham , Birmingham, AL 35233 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4068-8092","authenticated-orcid":false,"given":"Michael","family":"Niederweis","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Alabama at Birmingham , Birmingham, AL 35294 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,10,21]]},"reference":[{"key":"2026102911424160600_ref1","doi-asserted-by":"publisher","DOI":"10.1152\/function.107.2025","article-title":"Tiny tools closing the gap: Nanobodies in research and therapy","volume":"7","author":"Abdellatif","year":"2026","journal-title":"Function"},{"key":"2026102911424160600_ref2","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"2026102911424160600_ref3","doi-asserted-by":"publisher","DOI":"10.1177\/18758592241301690","article-title":"The relationship between serum levels of GRP78, GRP94, calnexin, and calreticulin and prognosis in patients with lung cancer","volume":"42","author":"Ak","year":"2025","journal-title":"Cancer Biomark"},{"key":"2026102911424160600_ref4","doi-asserted-by":"publisher","first-page":"19074","DOI":"10.1038\/s41598-024-70101-6","article-title":"TEMPRO: Nanobody melting temperature estimation model using protein embeddings","volume":"14","author":"Alvarez","year":"2024","journal-title":"Sci Rep"},{"key":"2026102911424160600_ref5","doi-asserted-by":"publisher","first-page":"1589","DOI":"10.3389\/fimmu.2017.01589","article-title":"Camelid single-domain antibodies: Historical perspective and future outlook","volume":"8","author":"Arbabi-Ghahroudi","year":"2017","journal-title":"Front Immunol"},{"key":"2026102911424160600_ref6","doi-asserted-by":"publisher","first-page":"2128","DOI":"10.1016\/j.cell.2026.01.017","article-title":"Large-scale proteomics across neurological disorders uncovers biomarker panel and targets in multiple sclerosis","volume":"189","author":"Bader","year":"2026","journal-title":"Cell"},{"key":"2026102911424160600_ref7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2426644122","article-title":"Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal","volume":"122","author":"Banuelos","year":"2025","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026102911424160600_ref8","doi-asserted-by":"publisher","first-page":"13703","DOI":"10.1073\/pnas.96.24.13703","article-title":"Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm","volume":"96","author":"Bessette","year":"1999","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2026102911424160600_ref9","doi-asserted-by":"publisher","first-page":"8897","DOI":"10.1074\/jbc.273.15.8897","article-title":"Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli","volume":"273","author":"Betton","year":"1998","journal-title":"J Biol Chem"},{"key":"2026102911424160600_ref10","doi-asserted-by":"publisher","first-page":"3608","DOI":"10.1021\/acssynbio.3c00409","article-title":"Hyperstable synthetic mini-proteins as effective ligand scaffolds","volume":"12","author":"Blanchard","year":"2023","journal-title":"ACS Synth Biol"},{"key":"2026102911424160600_ref11","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1126\/science.1225416","article-title":"Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses","volume":"337","author":"Burton","year":"2012","journal-title":"Science"},{"key":"2026102911424160600_ref13","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1097\/CJI.0000000000000553","article-title":"High expression of calreticulin affected the tumor microenvironment and correlated with worse prognosis in patients with triple-negative breast cancer","volume":"48","author":"Cao","year":"2025","journal-title":"J Immunother"},{"key":"2026102911424160600_ref14","doi-asserted-by":"publisher","first-page":"547","DOI":"10.1038\/s41591-019-0412-8","article-title":"Broadly neutralizing anti-HIV-1 monoclonal antibodies in the clinic","volume":"25","author":"Caskey","year":"2019","journal-title":"Nat Med"},{"key":"2026102911424160600_ref15","doi-asserted-by":"publisher","first-page":"3086","DOI":"10.1016\/j.cell.2021.05.005","article-title":"Tackling COVID-19 with neutralizing monoclonal antibodies","volume":"184","author":"Corti","year":"2021","journal-title":"Cell"},{"key":"2026102911424160600_ref16","doi-asserted-by":"publisher","first-page":"332","DOI":"10.1016\/j.tibs.2019.12.008","article-title":"Miniproteins as a powerful modality in drug development","volume":"45","author":"Crook","year":"2020","journal-title":"Trends Biochem Sci"},{"key":"2026102911424160600_ref17","doi-asserted-by":"publisher","first-page":"6616","DOI":"10.1158\/1078-0432.CCR-17-0310","article-title":"(131)I-labeled anti-HER2 camelid sdAb as a Theranostic tool in cancer treatment","volume":"23","author":"D'Huyvetter","year":"2017","journal-title":"Clin Cancer Res"},{"key":"2026102911424160600_ref18","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.68253","article-title":"A general approach for stabilizing nanobodies for intracellular expression","volume":"11","author":"Dingus","year":"2022","journal-title":"Elife"},{"key":"2026102911424160600_ref19","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1038\/s41422-020-0383-9","article-title":"Calreticulin and cancer","volume":"31","author":"Fucikova","year":"2021","journal-title":"Cell Res"},{"key":"2026102911424160600_ref20","doi-asserted-by":"publisher","first-page":"73","DOI":"10.4236\/abb.2013.44A011","article-title":"Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments","volume":"04","author":"Gil","year":"2013","journal-title":"Adv Biosci Biotechnol"},{"key":"2026102911424160600_ref21","doi-asserted-by":"publisher","article-title":"Generation of a synthetic single domain antibody library for radiopharmaceutical ligand discovery","author":"Hall","DOI":"10.1101\/2025.05.14.654066"},{"key":"2026102911424160600_ref22","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202500316","article-title":"Pathological Disulfide bond crosslinking: Molecular insights into Amyloidogenesis and diseases progression","volume":"26","author":"Kang","year":"2025","journal-title":"Chembiochem"},{"key":"2026102911424160600_ref23","doi-asserted-by":"publisher","DOI":"10.1080\/2162402X.2020.1792037","article-title":"Surface-exposed and soluble calreticulin: Conflicting biomarkers for cancer prognosis","volume":"9","author":"Kepp","year":"2020","journal-title":"Oncoimmunology"},{"key":"2026102911424160600_ref24","doi-asserted-by":"publisher","DOI":"10.3390\/cells10010130","article-title":"Calreticulin-multifunctional chaperone in immunogenic cell death: Potential significance as a prognostic biomarker in ovarian cancer patients","volume":"10","author":"Kielbik","year":"2021","journal-title":"Cells"},{"key":"2026102911424160600_ref25","doi-asserted-by":"publisher","first-page":"956","DOI":"10.2967\/jnumed.120.245290","article-title":"Imaging calreticulin for early detection of immunogenic cell death during anticancer treatment","volume":"62","author":"Kim","year":"2021","journal-title":"J Nucl Med"},{"key":"2026102911424160600_ref26","doi-asserted-by":"publisher","first-page":"756","DOI":"10.1158\/1078-0432.CCR-25-3297","article-title":"Improving a plasma biomarker panel for early detection of pancreatic ductal adenocarcinoma with aminopeptidase N (ANPEP) and polymeric immunoglobulin receptor (PIGR)","volume":"32","author":"Krusen","year":"2026","journal-title":"Clin Cancer Res"},{"key":"2026102911424160600_ref27","doi-asserted-by":"publisher","first-page":"2196","DOI":"10.1016\/j.bbagen.2017.06.014","article-title":"Exploiting sequence and stability information for directing nanobody stability engineering","volume":"1861","author":"Kunz","year":"2017","journal-title":"Biochim Biophys Acta Gen Subj"},{"key":"2026102911424160600_ref28","doi-asserted-by":"publisher","first-page":"241","DOI":"10.1093\/protein\/gzz017","article-title":"Nanobody stability engineering by employing the DeltaTm shift; a comparison with apparent rate constants of heat-induced aggregation","volume":"32","author":"Kunz","year":"2019","journal-title":"Protein Eng Des Sel"},{"key":"2026102911424160600_ref29","doi-asserted-by":"publisher","first-page":"7934","DOI":"10.1038\/s41598-018-26338-z","article-title":"The structural basis of nanobody unfolding reversibility and thermoresistance","volume":"8","author":"Kunz","year":"2018","journal-title":"Sci Rep"},{"key":"2026102911424160600_ref30","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1186\/s12934-022-01803-7","article-title":"Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus origami strain","volume":"21","author":"Lopez-Cano","year":"2022","journal-title":"Microb Cell Factories"},{"key":"2026102911424160600_ref31","doi-asserted-by":"publisher","first-page":"1004","DOI":"10.1002\/pro.3831","article-title":"Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the listeria virulence factor InlB","volume":"29","author":"Mendoza","year":"2020","journal-title":"Protein Sci"},{"key":"2026102911424160600_ref32","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural single-domain antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu Rev Biochem"},{"key":"2026102911424160600_ref33","doi-asserted-by":"publisher","first-page":"401","DOI":"10.1146\/annurev-animal-021419-083831","article-title":"Applications of nanobodies","volume":"9","author":"Muyldermans","year":"2021","journal-title":"Annu Rev Anim Biosci"},{"key":"2026102911424160600_ref34","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1038\/nm1523","article-title":"Calreticulin exposure dictates the immunogenicity of cancer cell death","volume":"13","author":"Obeid","year":"2007","journal-title":"Nat Med"},{"key":"2026102911424160600_ref35","doi-asserted-by":"publisher","first-page":"496","DOI":"10.3390\/bios11120496","article-title":"Native llama nanobody library panning performed by phage and yeast display provides binders suitable for C-reactive protein detection","volume":"11","author":"Oloketuyi","year":"2021","journal-title":"Biosensors (Basel)"},{"key":"2026102911424160600_ref36","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1016\/j.nbt.2023.09.003","article-title":"Approaches for high-throughput quantification of periplasmic recombinant proteins","volume":"77","author":"Osgerby","year":"2023","journal-title":"New Biotechnol"},{"key":"2026102911424160600_ref37","doi-asserted-by":"publisher","first-page":"674","DOI":"10.1038\/nprot.2014.039","article-title":"A general protocol for the generation of nanobodies for structural biology","volume":"9","author":"Pardon","year":"2014","journal-title":"Nat Protoc"},{"key":"2026102911424160600_ref38","doi-asserted-by":"publisher","first-page":"1617","DOI":"10.1016\/j.cell.2024.02.041","article-title":"Cancer biomarkers: Emerging trends and clinical implications for personalized treatment","volume":"187","author":"Passaro","year":"2024","journal-title":"Cell"},{"key":"2026102911424160600_ref39","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1146\/annurev-chembioeng-062011-081052","article-title":"Engineering aggregation-resistant antibodies","volume":"3","author":"Perchiacca","year":"2012","journal-title":"Annu Rev Chem Biomol Eng"},{"key":"2026102911424160600_ref40","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1002\/pro.3943","article-title":"UCSF ChimeraX: Structure visualization for researchers, educators, and developers","volume":"30","author":"Pettersen","year":"2021","journal-title":"Protein Sci"},{"key":"2026102911424160600_ref41","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1186\/1475-2859-12-24","article-title":"Optimizing heterologous protein production in the periplasm of E. Coli by regulating gene expression levels","volume":"12","author":"Schlegel","year":"2013","journal-title":"Microb Cell Factories"},{"key":"2026102911424160600_ref42","doi-asserted-by":"publisher","first-page":"2314","DOI":"10.1002\/anie.201708459","article-title":"Nanobodies: Chemical functionalization strategies and intracellular applications","volume":"57","author":"Schumacher","year":"2018","journal-title":"Angew Chem Int Ed Engl"},{"key":"2026102911424160600_ref43","doi-asserted-by":"publisher","first-page":"2141973","DOI":"10.1080\/2162402X.2022.2141973","article-title":"Ecto-calreticulin expression in multiple myeloma correlates with a failed anti-tumoral immune response and bad prognosis","volume":"11","author":"Serrano Del Valle","year":"2022","journal-title":"Oncoimmunology"},{"key":"2026102911424160600_ref44","doi-asserted-by":"publisher","DOI":"10.3390\/cells10040930","article-title":"Biomarkers of radiotherapy-induced immunogenic cell death","volume":"10","author":"Vaes","year":"2021","journal-title":"Cells"},{"key":"2026102911424160600_ref45","doi-asserted-by":"publisher","first-page":"baad021","DOI":"10.1093\/database\/baad021","article-title":"NbThermo: A new thermostability database for nanobodies","volume":"2023","author":"Valdes-Tresanco","year":"2023","journal-title":"Database (Oxford)"},{"key":"2026102911424160600_ref46","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1038\/s41586-025-10018-w","article-title":"Biofluid biomarkers in Alzheimer's disease and other neurodegenerative dementias","volume":"650","author":"Zetterberg","year":"2026","journal-title":"Nature"},{"key":"2026102911424160600_ref47","doi-asserted-by":"publisher","DOI":"10.3390\/cancers13112801","article-title":"Engineering calreticulin-targeting Monobodies to detect immunogenic cell death in cancer chemotherapy","volume":"13","author":"Zhang","year":"2021","journal-title":"Cancers (Basel)"}],"container-title":["Protein Engineering, Design and Selection"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/peds\/advance-article-pdf\/doi\/10.1093\/protein\/gzag030\/72251224\/gzag030.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/peds\/article-pdf\/doi\/10.1093\/protein\/gzag030\/72251224\/gzag030.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/peds\/article-pdf\/doi\/10.1093\/protein\/gzag030\/72251224\/gzag030.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T15:42:49Z","timestamp":1793288569000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/peds\/article\/doi\/10.1093\/protein\/gzag030\/8964913"}},"subtitle":[],"editor":[{"given":"Feng","family":"Ni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026]]},"references-count":46,"URL":"https:\/\/doi.org\/10.1093\/protein\/gzag030","relation":{},"ISSN":["1741-0126","1741-0134"],"issn-type":[{"value":"1741-0126","type":"print"},{"value":"1741-0134","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026]]},"published":{"date-parts":[[2026]]},"article-number":"gzag030"}}