{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,6,1]],"date-time":"2024-06-01T04:38:19Z","timestamp":1717216699305},"reference-count":44,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2012,8,28]],"date-time":"2012-08-28T00:00:00Z","timestamp":1346112000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2012,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy\u2010handling flow cytometric cell\u2010based assay to evaluate Ab\u2010binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)\/ErbB1\u2010directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1\/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium\u201090. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab\u2010binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of Cetuximab is neither affected by covalent modification with CHX\u2010A\u2033\u2010DTPA (N\u2010[(R)\u20102\u2010Amino\u20103\u2010(<jats:italic>p<\/jats:italic>\u2010isothiocyanato\u2010phenyl) propyl]\u2010<jats:italic>trans<\/jats:italic>\u2010(<jats:italic>S<\/jats:italic>,<jats:italic>S<\/jats:italic>)\u2010cyclohexane\u20101,2\u2010diamine\u2010<jats:italic>N<\/jats:italic>,<jats:italic>N<\/jats:italic>,<jats:italic>N<\/jats:italic>\u2032,<jats:italic>N<\/jats:italic>\u2033,<jats:italic>N<\/jats:italic>\u2033\u2010pentaacetic acid) with a final chelator\u2010to\u2010Ab ratio of 5 nor by subsequent radiolabeling. [<jats:sup>90<\/jats:sup>Y]Y\u2010CHX\u2010A\u2033\u2010DTPA\u2010Cetuximab thus qualifies for preclinical treatment testing as a prerequisite for therapeutic application. \u00a9 2012 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.22110","type":"journal-article","created":{"date-parts":[[2012,8,28]],"date-time":"2012-08-28T20:03:22Z","timestamp":1346184202000},"page":"865-873","source":"Crossref","is-referenced-by-count":7,"title":["Flow cytometric cell\u2010based assay to preselect antibody constructs for radionuclide conjugation"],"prefix":"10.1002","volume":"81A","author":[{"given":"M.","family":"Ingargiola","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"C.","family":"Dittfeld","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"R.","family":"Runge","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M.","family":"Zenker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J.\u2010M.","family":"Heldt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J.","family":"Steinbach","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"N.","family":"Cordes","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M.","family":"Baumann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J.","family":"Kotzerke","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"L. 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