{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T21:48:56Z","timestamp":1792619336946,"version":"4.5.0"},"reference-count":101,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2022,10,18]],"date-time":"2022-10-18T00:00:00Z","timestamp":1666051200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"publisher","award":["Intramural Research Program, CAN 8029883"],"award-info":[{"award-number":["Intramural Research Program, CAN 8029883"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007188","name":"Uniformed Services University of the Health Sciences","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007188","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and\n                    <jats:italic>in vitro<\/jats:italic>\n                    characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (A\u03b2) VHH dimer and labeled with fluorescent dye for direct assessment of\n                    <jats:italic>in vivo<\/jats:italic>\n                    target engagement in a mouse model of AD-related A\u03b2 plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of &lt;1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol\/kg body weight, ranging from undetectable for nonbinding mutants to -6\u00b0C for the best mutants. The anti-mTfR VHH variant M1\n                    <jats:sub>P96H<\/jats:sub>\n                    with the strongest hypothermia effect was fused to the anti-A\u03b2 VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and A\u03b2 plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol\/kg body weight into APP\/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce\n                    <jats:italic>in situ<\/jats:italic>\n                    labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with A\u03b2-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0276107","type":"journal-article","created":{"date-parts":[[2022,10,18]],"date-time":"2022-10-18T13:32:21Z","timestamp":1666099941000},"page":"e0276107","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":13,"title":["Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer\u2019s related amyloid-beta pathology"],"prefix":"10.1371","volume":"17","author":[{"given":"Shiran","family":"Su","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas J.","family":"Esparza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4864-3262","authenticated-orcid":true,"given":"David L.","family":"Brody","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,10,18]]},"reference":[{"issue":"4","key":"pone.0276107.ref001","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.molmed.2005.02.009","article-title":"Alzheimer\u2019s disease: Abeta, tau and synaptic dysfunction","volume":"11","author":"FM LaFerla","year":"2005","journal-title":"Trends Mol Med"},{"issue":"3","key":"pone.0276107.ref002","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1002\/alz.12328","article-title":"2021 Alzheimer\u2019s disease facts and figures","volume":"17","author":"The Alzheimer\u2019s Association","year":"2021","journal-title":"Alzheimers Dement"},{"key":"pone.0276107.ref003","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1146\/annurev-med-042915-103753","article-title":"Update on Alzheimer\u2019s Disease Therapy and Prevention Strategies","volume":"68","author":"WV Graham","year":"2017","journal-title":"Annu Rev Med"},{"issue":"4","key":"pone.0276107.ref004","doi-asserted-by":"crossref","first-page":"1271","DOI":"10.3233\/JAD-141134","article-title":"Epidemiological and economic burden of Alzheimer\u2019s disease: a systematic literature review of data across Europe and the United States of America","volume":"43","author":"C Takizawa","year":"2015","journal-title":"J Alzheimers Dis"},{"key":"pone.0276107.ref005","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1146\/annurev-clinpsy-050212-185535","article-title":"Neuroimaging and other biomarkers for Alzheimer\u2019s disease: the changing landscape of early detection","volume":"9","author":"SL Risacher","year":"2013","journal-title":"Annu Rev Clin Psychol"},{"issue":"1","key":"pone.0276107.ref006","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1159\/000109754","article-title":"Biomarkers for early detection of Alzheimer pathology","volume":"16","author":"CM Clark","year":"2008","journal-title":"Neurosignals"},{"issue":"4","key":"pone.0276107.ref007","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1097\/NEN.0b013e31824b211b","article-title":"Accuracy of the clinical diagnosis of Alzheimer disease at National Institute on Aging Alzheimer Disease Centers, 2005\u20132010","volume":"71","author":"TG Beach","year":"2012","journal-title":"J Neuropathol Exp Neurol"},{"issue":"3 Suppl 1","key":"pone.0276107.ref008","doi-asserted-by":"crossref","first-page":"S47","DOI":"10.1016\/j.metabol.2014.10.034","article-title":"Biomarkers in the diagnosis and management of Alzheimer\u2019s disease","volume":"64","author":"R. 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