{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:41:23Z","timestamp":1785436883595,"version":"3.56.0"},"reference-count":28,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T00:00:00Z","timestamp":1776038400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"the National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["22477128"],"award-info":[{"award-number":["22477128"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["22477128"],"award-info":[{"award-number":["22477128"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (KD = 214 \u00b1 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, KD = 18.5 \u00b1 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems.<\/jats:p>","DOI":"10.3390\/antib15020034","type":"journal-article","created":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T11:38:26Z","timestamp":1776080306000},"page":"34","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Human IgG1 Monoclonal Antibody Targeting Transferrin Receptor 1 for Antitumor Drug Delivery"],"prefix":"10.3390","volume":"15","author":[{"given":"Tingting","family":"Ji","sequence":"first","affiliation":[{"name":"Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoyun","family":"Zong","sequence":"additional","affiliation":[{"name":"Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ningyuan","family":"Gong","sequence":"additional","affiliation":[{"name":"Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghui","family":"Yan","sequence":"additional","affiliation":[{"name":"Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiyu","family":"Chen","sequence":"additional","affiliation":[{"name":"Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.clim.2006.06.010","article-title":"The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer","volume":"121","author":"Daniels","year":"2006","journal-title":"Clin. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Candelaria, P.V., Leoh, L.S., Penichet, M.L., and Daniels-Wells, T.R. (2021). Antibodies targeting the transferrin receptor 1 (TfR1) as direct anti-cancer agents. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.607692"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1007\/978-1-4939-2095-2_10","article-title":"A systems biology approach to iron metabolism","volume":"844","author":"Chifman","year":"2014","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"102427","DOI":"10.1016\/j.sbi.2022.102427","article-title":"Capturing the mechanics of clathrin-mediated endocytosis","volume":"75","author":"Smith","year":"2022","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1038\/7727","article-title":"Transferrin receptor is necessary for development of erythrocytes and the nervous system","volume":"21","author":"Levy","year":"1999","journal-title":"Nat. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1097\/CJI.0000000000000304","article-title":"An IgG1 version of the anti-transferrin receptor 1 antibody ch128.1 shows significant anti-tumor activity against different xenograft models of multiple myeloma: A brief communication","volume":"43","author":"Candelaria","year":"2020","journal-title":"J. Immunother."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Daniels-Wells, T.R., Candelaria, P.V., Kranz, E., Wen, J., Wang, L., Kamata, M., Almagro, J.C., Mart\u00ednez-Maza, O., and Penichet, M.L. (2023). Efficacy of antibodies targeting TfR1 in xenograft mouse models of AIDS-related non-Hodgkin lymphoma. Cancers, 15.","DOI":"10.3390\/cancers15061816"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1159","DOI":"10.1158\/1535-7163.MCT-23-0548","article-title":"INA03: A potent transferrin-competitive antibody\u2013drug conjugate against CD71 for safer acute leukemia treatment","volume":"23","author":"Bratti","year":"2024","journal-title":"Mol. Cancer Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"eadi2245","DOI":"10.1126\/scitranslmed.adi2245","article-title":"Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier","volume":"16","author":"Barker","year":"2024","journal-title":"Sci. Transl. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5901","DOI":"10.1093\/nar\/gkad415","article-title":"Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs)","volume":"51","author":"Malecova","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1186\/s12987-025-00737-7","article-title":"Transferrin receptor-binding blood-brain barrier shuttle enhances brain delivery and plaque-clearing efficacy of a therapeutic anti-A\u03b2 antibody","volume":"22","author":"Vega","year":"2025","journal-title":"Fluids Barriers CNS"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"600","DOI":"10.1016\/j.bbrc.2014.10.133","article-title":"Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells","volume":"454","author":"Yamaguchi","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1186\/s12987-025-00693-2","article-title":"High-affinity transferrin receptor binding improves brain delivery of bispecific antibodies at tracer dose","volume":"22","author":"Bonvicini","year":"2025","journal-title":"Fluids Barriers CNS"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1326","DOI":"10.1158\/1535-7163.MCT-21-0193","article-title":"Nonclinical efficacy and safety of CX-2029, an anti-CD71 probody-drug conjugate","volume":"21","author":"Singh","year":"2022","journal-title":"Mol. Cancer Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1016\/S1357-2725(99)00070-9","article-title":"The transferrin receptor: Role in health and disease","volume":"31","author":"Ponka","year":"1999","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.bbagen.2011.09.009","article-title":"The intracellular trafficking pathway of transferrin","volume":"1820","author":"Mayle","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.bbagen.2011.07.016","article-title":"The transferrin receptor and the targeted delivery of therapeutic agents against cancer","volume":"1820","author":"Daniels","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"10706","DOI":"10.1073\/pnas.162362999","article-title":"An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells","volume":"99","author":"Ng","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2995","DOI":"10.1158\/1535-7163.MCT-07-0330","article-title":"Conjugation of an anti-transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells","volume":"6","author":"Daniels","year":"2007","journal-title":"Mol. Cancer Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.tiv.2012.10.006","article-title":"Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1","volume":"27","author":"Helguera","year":"2013","journal-title":"Toxicol. In Vitro"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1002\/jgm.1345","article-title":"A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide","volume":"11","author":"Morizono","year":"2009","journal-title":"J. Gene Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1002\/jgm.2754","article-title":"Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter","volume":"16","author":"Leoh","year":"2014","journal-title":"J. Gene Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"204","DOI":"10.4161\/mabs.27227","article-title":"The influence of antibody fragment format on phage display based affinity maturation of IgG","volume":"6","author":"Steinwand","year":"2014","journal-title":"mAbs"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1016\/S0014-5793(03)00602-1","article-title":"Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries","volume":"546","author":"Pieters","year":"2003","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1080\/19420862.2018.1426421","article-title":"Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGF\u03b21 antibody","volume":"10","author":"Lord","year":"2018","journal-title":"mAbs"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"828","DOI":"10.2174\/1871520615666150302125448","article-title":"Advances in the development of site-specific antibody-drug conjugation","volume":"15","author":"Zhou","year":"2015","journal-title":"Anti-Cancer Agents Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Dan, N., Setua, S., Kashyap, V.K., Khan, S., Jaggi, M., Yallapu, M.M., and Chauhan, S.C. (2018). Antibody-drug conjugates for cancer therapy: Chemistry to clinical implications. Pharmaceuticals, 11.","DOI":"10.3390\/ph11020032"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zhou, Q. (2017). Site-specific antibody conjugation for ADC and beyond. Biomedicines, 5.","DOI":"10.3390\/biomedicines5040064"}],"container-title":["Antibodies"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4468\/15\/2\/34\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T04:18:25Z","timestamp":1776226705000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4468\/15\/2\/34"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,13]]},"references-count":28,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["antib15020034"],"URL":"https:\/\/doi.org\/10.3390\/antib15020034","relation":{},"ISSN":["2073-4468"],"issn-type":[{"value":"2073-4468","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,13]]}}}