{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T10:50:43Z","timestamp":1776941443738,"version":"3.51.4"},"reference-count":84,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T00:00:00Z","timestamp":1776384000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"SECIHTI","award":["CBF-2025-G-1738"],"award-info":[{"award-number":["CBF-2025-G-1738"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Precise intracellular delivery of chemotherapeutics remains a major challenge in HER2-positive breast cancer, where intratumoral heterogeneity and limited tissue penetration constrain efficacy. A key contributor is the tumor-restricted epidermal growth factor receptor variant III (EGFRvIII), a constitutively active, ligand-independent mutant generated by deletion of exons 2\u20137. Although classically associated with glioblastoma, lung (NSCLC), head\/neck, and prostate cancers, EGFRvIII is also present in subsets of HER2-positive breast cancers, where low-abundance subclones drive aggressive phenotypes and attenuate therapeutic responses. HER2\u2013EGFRvIII co-expression amplifies oncogenic signaling, supported by frequent co-expression in ErbB2-positive primary tumors and metastases, and by sustained receptor phosphorylation in the absence of EGFR gene amplification, depicting EGFRvIII as a compelling therapeutic target. Methods: We evaluated the shark-derived single-domain antibody vNAR R426 as a modular theranostic platform for receptor-mediated cisplatin delivery. Conjugation to cisplatin and fluorescein enabled simultaneous intracellular drug transport and immunofluorescence-based detection in EGFRvIII-positive SKBR3 cells and 3D spheroids. The compact vNAR-based immunoconjugates support efficient receptor recognition, internalization, and intracellular trafficking, features rarely achieved by conventional IgG antibodies. Results: vNARCDDP elicited robust, receptor-mediated cytotoxicity, achieving an IC50 of 2.68 \u00b5M\u2014approximately 50-fold lower than that of free cisplatin\u2014while unconjugated vNAR maintained scaffold biocompatibility. In three-dimensional spheroid models, the theranostic vNAR (vNARCDDP+FITC) exhibited deep and uniform penetration throughout tumor-like architectures, with immunofluorescence intensity closely correlating with regions of intracellular drug delivery and the initiation of cytotoxic responses. Notably, cisplatin conjugation did not impair tissue diffusion or receptor engagement, facilitating effective payload delivery to both peripheral and central cell populations. Conclusions: By integrating tumor-restricted targeting and efficient intracellular drug delivery within a modular single-domain scaffold, vNAR R426 represents a next-generation theranostic platform capable of addressing intratumoral heterogeneity. This approach combines potent cytotoxic activity with immunofluorescence-based detection, thereby advancing the rational design of precision therapeutics for HER2-positive breast cancer.<\/jats:p>","DOI":"10.3390\/ph19040633","type":"journal-article","created":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T11:53:46Z","timestamp":1776426826000},"page":"633","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Theranostic vNAR-Based Immunoconjugates Achieve Selective Intracellular Cisplatin Delivery in Embedded 3D HER2-Positive Breast Cancer In Vitro Model"],"prefix":"10.3390","volume":"19","author":[{"given":"Andrea C.","family":"Alfonseca-Ladr\u00f3n de Guevara","sequence":"first","affiliation":[{"name":"Unidad de Biotecnolog\u00eda M\u00e9dica y Farmac\u00e9utica, Centro de Investigaci\u00f3n y Asistencia en Tecnolog\u00eda y Dise\u00f1o del Estado de Jalisco (CIATEJ), Guadalajara 44270, Jalisco, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2933-4720","authenticated-orcid":false,"given":"Alejandro","family":"Manzanares-Guzm\u00e1n","sequence":"additional","affiliation":[{"name":"Unidad de Biotecnolog\u00eda M\u00e9dica y Farmac\u00e9utica, Centro de Investigaci\u00f3n y Asistencia en Tecnolog\u00eda y Dise\u00f1o del Estado de Jalisco (CIATEJ), Guadalajara 44270, Jalisco, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica A.","family":"Badillo-Mata","sequence":"additional","affiliation":[{"name":"Unidad de Biotecnolog\u00eda M\u00e9dica y Farmac\u00e9utica, Centro de Investigaci\u00f3n y Asistencia en Tecnolog\u00eda y Dise\u00f1o del Estado de Jalisco (CIATEJ), Guadalajara 44270, Jalisco, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9095-3563","authenticated-orcid":false,"given":"Mirna","family":"Burciaga-Flores","sequence":"additional","affiliation":[{"name":"SECIHTI\u2014Departamento de Modelaci\u00f3n de Nanomateriales, Centro de Nanociencias y Nanotecnolog\u00eda, Universidad Nacional Aut\u00f3noma de M\u00e9xico (CNYN-UNAM), Carretera Tijuana\u2014Ensenada km 107, Ensenada 22860, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8872-8335","authenticated-orcid":false,"given":"Pavel H.","family":"Lugo-Fabres","sequence":"additional","affiliation":[{"name":"SECIHTI\u2014Unidad de Biotecnolog\u00eda M\u00e9dica y Farmac\u00e9utica, Centro de Investigaci\u00f3n y Asistencia en Tecnolog\u00eda y Dise\u00f1o del Estado de Jalisco (CIATEJ), Guadalajara 44270, Jalisco, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0540-0287","authenticated-orcid":false,"given":"Tanya A.","family":"Camacho-Villegas","sequence":"additional","affiliation":[{"name":"Unidad de Biotecnolog\u00eda M\u00e9dica y Farmac\u00e9utica, Centro de Investigaci\u00f3n y Asistencia en Tecnolog\u00eda y Dise\u00f1o del Estado de Jalisco (CIATEJ), Guadalajara 44270, Jalisco, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,17]]},"reference":[{"key":"ref_1","unstructured":"American Cancer Society (2025, December 15). HER2-Positive Breast Cancer. Understanding a Breast Cancer Diagnosis. Available online: https:\/\/www.cancer.org\/es\/cancer\/tipos\/cancer-de-seno\/comprension-de-un-diagnostico-de-cancer-de-seno\/estado-de-her2-del-cancer-de-seno.html."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"38840","DOI":"10.18632\/oncotarget.6111","article-title":"Synergistic antitumor efficacy against EGFRvIII+ HER2+ breast cancers by combining trastuzumab with anti-EGFRvIII antibody CH12","volume":"6","author":"Xu","year":"2015","journal-title":"Oncotarget"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Liu, X., Luan, L., Liu, X., Jiang, D., Deng, J., Xu, J., Yuan, Y., Xing, J., Chen, B., and Xing, D. (2023). A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1292839"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2670","DOI":"10.1038\/onc.2012.280","article-title":"EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms","volume":"32","author":"Giacomini","year":"2013","journal-title":"Oncogene"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5350","DOI":"10.1111\/febs.12393","article-title":"The epidermal growth factor receptor variant III (EGFRvIII): Where wild things are altered","volume":"280","author":"Gan","year":"2013","journal-title":"FEBS J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3436","DOI":"10.1158\/0008-5472.CAN-15-0989","article-title":"Ligand-Independent EGFR Signaling","volume":"75","author":"Guo","year":"2015","journal-title":"Cancer Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1818","DOI":"10.4161\/cbt.7.11.6847","article-title":"Co-expression of EGFRvIII with ErbB-2 enhances tumorigenesis: EGFRvIII mediated constitutively activated and sustained signaling pathways, whereas EGF-induced a transient effect on EGFR-mediated signaling pathways","volume":"7","author":"Yu","year":"2008","journal-title":"Cancer Biol. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"100758","DOI":"10.1016\/j.jbior.2020.100758","article-title":"Therapeutic resistance in breast cancer cells can result from deregulated EGFR signaling","volume":"78","author":"Steelman","year":"2020","journal-title":"Adv. Biol. Regul."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2657","DOI":"10.1158\/0008-5472.CAN-11-2656","article-title":"Epidermal growth factor receptor variant III contributes to cancer stem cell phenotypes in invasive breast carcinoma","volume":"72","author":"Jensen","year":"2012","journal-title":"Cancer Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Camacho-Villegas, T.A., Mata-Gonz\u00e1lez, M.T., Garc\u00eda-Ubbelohd, W., N\u00fa\u00f1ez-Garc\u00eda, L., Elosua, C., Paniagua-Solis, J.F., and Licea-Navarro, A.F. (2018). Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization. Mar. Drugs, 16.","DOI":"10.3390\/md16040113"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.dci.2018.09.007","article-title":"Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents","volume":"90","author":"Matz","year":"2019","journal-title":"Dev. Comp. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1016\/j.ijbiomac.2020.01.039","article-title":"Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody","volume":"147","author":"Cheong","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Manzanares-Guzm\u00e1n, A., Lugo-Fabres, P.H., and Camacho-Villegas, T.A. (2024). vNARs as Neutralizing Intracellular Therapeutic Agents: Glioblastoma as a Target. Antibodies, 13.","DOI":"10.3390\/antib13010025"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"15","DOI":"10.4161\/19420862.2015.989032","article-title":"Structural insights and biomedical potential of IgNAR scaffolds from sharks","volume":"7","author":"Zielonka","year":"2015","journal-title":"mAbs"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1907","DOI":"10.1016\/j.bpj.2019.04.013","article-title":"Molecular origin of the stability difference in four shark IgNAR constant domains","volume":"116","author":"Zhou","year":"2019","journal-title":"Biophys. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1016\/j.fsi.2022.01.027","article-title":"IgNAR antibody: Structural features, diversity and applications","volume":"121","author":"Khalid","year":"2022","journal-title":"Fish Shellfish Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Manzanares-Guzm\u00e1n, A., Alfonseca-Ladr\u00f3n de Guevara, A.C., Reza-Escobar, E., Burciaga-Flores, M., Canales-Aguirre, A., Esquivel-Sol\u00eds, H., Lugo-Fabres, P.H., and Camacho-Villegas, T.A. (2025). Isolation and Characterization of the First Antigen-Specific EGFRvIII vNAR from Freshwater Stingray (Potamotrygon spp.) as a Drug Carrier in Glioblastoma Cancer Cells. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26030876"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"103426","DOI":"10.1016\/j.drudis.2022.103426","article-title":"Advances in 3D culture systems for therapeutic discovery and development in brain cancer","volume":"28","author":"Wanigasekara","year":"2023","journal-title":"Drug Discov. Today"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1016\/j.trecan.2021.06.009","article-title":"Tumour microenvironment 3D-modelling: Simplicity to complexity and back again","volume":"7","author":"Carter","year":"2021","journal-title":"Trends Cancer"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"117784","DOI":"10.1016\/j.lfs.2020.117784","article-title":"Methodological aspects and pharmacological applications of three-dimensional cancer cell cultures and organoids","volume":"254","author":"Foglietta","year":"2020","journal-title":"Life Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1039\/C9NH00628A","article-title":"3D cultures for modeling nanomaterial-based photothermal therapy","volume":"5","author":"Darrigues","year":"2020","journal-title":"Nanoscale Horiz."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"100753","DOI":"10.1016\/j.drup.2021.100753","article-title":"In vitro biomimetic models for glioblastoma-a promising tool for drug response studies","volume":"55","author":"Dragoj","year":"2021","journal-title":"Drug Resist. Updat."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Jensen, C., and Teng, Y. (2020). Is It Time to Start Transitioning from 2D to 3D Cell Culture?. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.00033"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Colombo, E., and Cattaneo, M.G. (2021). Multicellular 3D Models to Study Tumour-Stroma Interactions. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22041633"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"20598","DOI":"10.1039\/C9NR06535K","article-title":"Novel anti-HER2 peptide-conjugated theranostic nanoliposomes combining NaYF4:Yb,Er nanoparticles for NIR-activated bioimaging and chemo-photodynamic therapy against breast cancer","volume":"11","author":"Panikar","year":"2019","journal-title":"Nanoscale"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Aguilera-Marquez, J.d.R., Manzanares-Guzm\u00e1n, A., Garc\u00eda-Uriostegui, L., Canales-Aguirre, A.A., Camacho-Villegas, T.A., and Lugo-Fabres, P.H. (2025). Alginate-Gelatin Hydrogel Scaffold Model for Hypoxia Induction in Glioblastoma Embedded Spheroids. Gels, 11.","DOI":"10.3390\/gels11040263"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1038\/s41401-025-01634-3","article-title":"Novel anti-HER2 nanobody-drug conjugates with enhanced penetration of solid tumor and BBB, reduced systemic exposure and superior antitumor efficacy","volume":"47","author":"Wang","year":"2026","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Hansen, A.H., Andersen, K.I.H., Xin, L., Krigslund, O., Behrendt, N., Engelholm, L.H., Bang-Bertelsen, C.H., Schoffelen, S., and Qvortrup, K. (2025). A HER2 Specific Nanobody-Drug Conjugate: Site-Selective Bioconjugation and In Vitro Evaluation in Breast Cancer Models. Molecules, 30.","DOI":"10.3390\/molecules30020391"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/s41573-022-00495-3","article-title":"Antibodies to combat viral infections: Development strategies and progress","volume":"21","author":"Pantaleo","year":"2022","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12929-019-0592-z","article-title":"Development of therapeutic antibodies for the treatment of diseases","volume":"27","author":"Lu","year":"2020","journal-title":"J. Biomed. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3","DOI":"10.4161\/mabs.3.1.14110","article-title":"Targeting antibodies to the cytoplasm","volume":"3","author":"Marschall","year":"2011","journal-title":"mAbs"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1016\/j.tips.2013.04.007","article-title":"Targeting protein-protein interactions as an anticancer strategy","volume":"34","author":"Ivanov","year":"2013","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"414729","DOI":"10.1155\/2011\/414729","article-title":"Mechanisms of cellular uptake of cell-penetrating peptides","volume":"2011","author":"Madani","year":"2011","journal-title":"J. Biophys."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Dombrowsky, C.S., Happel, D., Habermann, J., Hofmann, S., Otmi, S., Cohen, B., and Kolmar, H. (2024). A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery. Antibodies, 13.","DOI":"10.3390\/antib13020037"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1972","DOI":"10.1021\/nn4057269","article-title":"Cell-penetrating peptides: Design, synthesis, and applications","volume":"8","author":"Copolovici","year":"2014","journal-title":"ACS Nano"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1016\/j.addr.2007.09.012","article-title":"Proline-rich, amphipathic cell-penetrating peptides","volume":"60","author":"Pujals","year":"2008","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1038\/nchem.2779","article-title":"Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide","volume":"9","author":"Akishiba","year":"2017","journal-title":"Nat. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1007\/s12033-023-00679-1","article-title":"Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections","volume":"65","author":"Khairkhah","year":"2023","journal-title":"Mol. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1158\/1535-7163.MCT-14-0697","article-title":"Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance","volume":"14","author":"Pedersen","year":"2015","journal-title":"Mol. Cancer Ther."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.molimm.2018.01.010","article-title":"Liposomal nanoparticle armed with bivalent bispecific single-domain antibodies, novel weapon in HER2 positive cancerous cell lines targeting","volume":"96","author":"Nikkhoi","year":"2018","journal-title":"Mol. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1016\/j.tranon.2018.01.024","article-title":"A Single Domain-Based Anti-Her2 Antibody Has Potent Antitumor Activities","volume":"11","author":"Wu","year":"2018","journal-title":"Transl. Oncol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3553","DOI":"10.1021\/acs.molpharmaceut.0c00580","article-title":"Therapeutic Efficacy of 213Bi-labeled sdAbs in a Preclinical Model of Ovarian Cancer","volume":"17","author":"Dekempeneer","year":"2020","journal-title":"Mol. Pharm."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.jconrel.2020.04.030","article-title":"Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session","volume":"323","author":"Deken","year":"2020","journal-title":"J. Control. Release"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5525","DOI":"10.7150\/thno.57510","article-title":"Homogeneous tumor targeting with a single dose of HER2-targeted albumin-binding domain-fused nanobody-drug conjugates results in long-lasting tumor remission in mice","volume":"11","author":"Xenaki","year":"2021","journal-title":"Theranostics"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Yan, Y., Cheng, X., Li, L., Zhang, R., Zhu, Y., Wu, Z., and Ding, K. (2021). A Novel Small Molecular Antibody, HER2Nanobody, Inhibits Tumor Proliferation in HER2Positive Breast Cancer Cells In Vitro and In Vivo. Front. Oncol., 11.","DOI":"10.3389\/fonc.2021.669393"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"gzab030","DOI":"10.1093\/protein\/gzab030","article-title":"Generation of a nanobody against HER2 tyrosine kinase using phage display library screening for HER2-positive breast cancer therapy development","volume":"34","author":"Lamtha","year":"2021","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1038\/modpathol.3800438","article-title":"EGFR gene amplification in breast cancer: Correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations","volume":"18","author":"Bhargava","year":"2005","journal-title":"Mod. Pathol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2617","DOI":"10.1016\/j.ejca.2006.03.033","article-title":"Molecular detection of EGFRvIII-positive cells in the peripheral blood of breast cancer patients","volume":"42","author":"Silva","year":"2006","journal-title":"Eur. J. Cancer"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1361","DOI":"10.4161\/cbt.5.10.3226","article-title":"Hypophosphorylation of residue Y1045 leads to defective downregulation of EGFRvIII","volume":"5","author":"Han","year":"2006","journal-title":"Cancer Biol. Ther."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Khoury, R., Saleh, K., Khalife, N., Saleh, M., Chahine, C., Ibrahim, R., and Lecesne, A. (2023). Mechanisms of Resistance to Antibody-Drug Conjugates. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24119674"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"114","DOI":"10.7150\/ijbs.28679","article-title":"COPI-Mediated Nuclear Translocation of EGFRvIII Promotes STAT3 Phosphorylation and PKM2 Nuclear Localization","volume":"15","author":"Zhang","year":"2019","journal-title":"Int. J. Biol. Sci."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1002\/cac2.12387","article-title":"Resistance to antibody-drug conjugates in breast cancer: Mechanisms and solutions","volume":"43","author":"Chen","year":"2023","journal-title":"Cancer Commun."},{"key":"ref_53","first-page":"114","article-title":"Reforming solid tumor treatment: The emerging potential of smaller format antibody-drug conjugate","volume":"7","author":"Ma","year":"2024","journal-title":"Antib. Ther."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1038\/nrd.2016.268","article-title":"Strategies and challenges for the next generation of antibody-drug conjugates","volume":"16","author":"Beck","year":"2017","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1166\/jnn.2014.9082","article-title":"Endosomal escape: A bottleneck in intracellular delivery","volume":"14","author":"Shete","year":"2014","journal-title":"J. Nanosci. Nanotechnol."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Medina P\u00e9rez, V.M., Baselga, M., and Schuhmacher, A.J. (2024). Single-Domain Antibodies as Antibody-Drug Conjugates: From Promise to Practice-A Systematic Review. Cancers, 16.","DOI":"10.3390\/cancers16152681"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"17588359251324889","DOI":"10.1177\/17588359251324889","article-title":"Toxicities and management strategies of emerging antibody-drug conjugates in breast cancer","volume":"17","author":"Kang","year":"2025","journal-title":"Ther. Adv. Med. Oncol."},{"key":"ref_58","first-page":"11","article-title":"Antibody-drug conjugates in breast cancer treatment: Resistance mechanisms and the role of therapeutic sequencing","volume":"8","author":"Larose","year":"2025","journal-title":"Cancer Drug Resist."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Hrynchak, I., Santos, L., Falc\u00e3o, A., Gomes, C.M., and Abrunhosa, A.J. (2021). NanobodyBased Theranostic Agents for HER2-Positive Breast Cancer: Radiolabeling Strategies. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms221910745"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"967","DOI":"10.2967\/jnumed.115.171306","article-title":"Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET","volume":"57","author":"Vaidyanathan","year":"2016","journal-title":"J. Nucl. Med."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"5074","DOI":"10.7150\/thno.42441","article-title":"3D approaches to model the tumor microenvironment of pancreatic cancer","volume":"10","author":"Kieler","year":"2020","journal-title":"Theranostics"},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Alzeeb, G., Metges, J.P., Corcos, L., and Le Jossic-Corcos, C. (2020). Three-Dimensional Culture Systems in Gastric Cancer Research. Cancers, 12.","DOI":"10.3390\/cancers12102800"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"2854","DOI":"10.1016\/j.apsb.2024.03.016","article-title":"Single-domain antibodies as therapeutics for solid tumor treatment","volume":"14","author":"Wang","year":"2024","journal-title":"Acta Pharm. Sin. B"},{"key":"ref_64","doi-asserted-by":"crossref","unstructured":"Pothin, E., Lesuisse, D., and Lafaye, P. (2020). Brain Delivery of Single-Domain Antibodies: A Focus on VHH and VNAR. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12100937"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"144694","DOI":"10.1016\/j.ijbiomac.2025.144694","article-title":"A shark VNAR with cross-species reactivity for serum albumin: Implications for long-term therapeutic applications","volume":"316","author":"Hao","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/j.mam.2011.12.002","article-title":"Human serum albumin: From bench to bedside","volume":"33","author":"Fanali","year":"2012","journal-title":"Mol. Asp. Med."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"6167","DOI":"10.2174\/1381612822666160720145328","article-title":"Carrier Mediated Systemic Delivery of Protein and Peptide Therapeutics","volume":"22","author":"Zaman","year":"2016","journal-title":"Curr. Pharm. Des."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1007\/s12195-017-0481-z","article-title":"Anti-HER2 VHH Targeted Magnetoliposome for Intelligent Magnetic Resonance Imaging of Breast Cancer Cells","volume":"10","author":"Khaleghi","year":"2017","journal-title":"Cell. Mol. Bioeng."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"552","DOI":"10.2174\/1574892816666210806150929","article-title":"Anti-HER2 VHH Targeted Fluorescent Liposome as Bimodal Nanoparticle for Drug Delivery and Optical Imaging","volume":"16","author":"Khaleghi","year":"2021","journal-title":"Recent Pat. Anticancer Drug Discov."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"14751","DOI":"10.1039\/D0NR02962A","article-title":"Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature","volume":"12","author":"Leach","year":"2020","journal-title":"Nanoscale"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/978-1-4939-9853-1_7","article-title":"In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties","volume":"2070","author":"Steven","year":"2020","journal-title":"Methods Mol. Biol."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"7283239","DOI":"10.1155\/2020\/7283239","article-title":"In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development","volume":"2020","author":"Ubah","year":"2020","journal-title":"J. Immunol. Res."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"4304","DOI":"10.1111\/febs.15809","article-title":"Immunogenicity and humanization of single-domain antibodies","volume":"289","author":"Rossotti","year":"2022","journal-title":"FEBS J."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"e630","DOI":"10.1002\/cpz1.630","article-title":"Humanization of the Shark VNAR Single Domain Antibody Using CDR Grafting","volume":"3","author":"Zhang","year":"2023","journal-title":"Curr. Protoc."},{"key":"ref_75","first-page":"100150","article-title":"Diffusion kinetics and perfusion times in tissue models obtained by bioorthogonal Raman \u03bc-spectroscopy","volume":"4","author":"Altmaier","year":"2024","journal-title":"Biophys. Rep."},{"key":"ref_76","doi-asserted-by":"crossref","unstructured":"de Abreu Costa, L., Henrique Fernandes Ottoni, M., Dos Santos, M.G., Meireles, A.B., Gomes de Almeida, V., de F\u00e1tima Pereira, W., Alves de Avelar-Freitas, B., and Eust\u00e1quio Alvim Brito-Melo, G. (2017). Dimethyl Sulfoxide (DMSO) Decreases Cell Proliferation and TNF-\u03b1, IFN-\u03b3, and IL-2 Cytokines Production in Cultures of Peripheral Blood Lymphocytes. Molecules, 22.","DOI":"10.3390\/molecules22111789"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"e246054","DOI":"10.1590\/0103-644020246054","article-title":"Dimethyl sulfoxide affects the viability and mineralization activity of apical papilla cells in vitro","volume":"35","author":"Santos","year":"2024","journal-title":"Braz. Dent. J."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"3993","DOI":"10.1111\/j.1582-4934.2009.00783.x","article-title":"Targeting a unique EGFR epitope with monoclonal antibody 806 activates NF-kappaB and initiates tumour vascular normalization","volume":"13","author":"Gan","year":"2009","journal-title":"J. Cell. Mol. Med."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"4071","DOI":"10.1073\/pnas.0611693104","article-title":"A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors","volume":"104","author":"Scott","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"2953","DOI":"10.1038\/onc.2011.465","article-title":"A novel epidermal growth factor receptor variant lacking multiple domains directly activates transcription and is overexpressed in tumors","volume":"31","author":"Piccione","year":"2012","journal-title":"Oncogene"},{"key":"ref_81","unstructured":"Hermanson, G.T. (2013). Chapter 10: Preparation of Antibody\u2013Drug Conjugates. Bioconjugate Techniques, Academic Press\/Elsevier. [3rd ed.]."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"D196","DOI":"10.1149\/2.0501805jes","article-title":"Electrochemical Deposition of Cisplatin on Pure Magnesium","volume":"165","author":"Lai","year":"2018","journal-title":"J. Electrochem. Soc."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.18632\/oncotarget.26613","article-title":"Therapeutic potential of nimotuzumab PEGylated-maytansine antibody drug conjugates against EGFR positive xenograft","volume":"10","author":"Hartimath","year":"2019","journal-title":"Oncotarget"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1007\/978-1-62703-541-5_16","article-title":"Drug-to-antibody ratio (DAR) by UV\/Vis spectroscopy","volume":"1045","author":"Chen","year":"2013","journal-title":"Methods Mol. Biol."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/4\/633\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T10:17:34Z","timestamp":1776939454000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/4\/633"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,17]]},"references-count":84,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ph19040633"],"URL":"https:\/\/doi.org\/10.3390\/ph19040633","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,17]]}}}