PT - JOURNAL ARTICLE AU - Bhuiyan, Mohammed AU - Chen, Chin-Tu AU - Freifelder, Richard AU - Zhang, Hannah AU - Meier, Jason AU - Tsai, Hsiu-Ming AU - Kao, Chien-Min AU - Kucharski Raubic, Anna AU - Nolen, Jerry AU - Rotsch, David AU - Kankanamalage, Pavithra AU - Leoni, Lara AU - Yamashita, Nami AU - Brossard, Thomas AU - Weichselbaum, Ralph AU - Kufe, Donald TI - <strong>Targeting MUC1-C for Cancer Theranostics Using Humanized 47Sc-mAb 3D1</strong> DP - 2022 Aug 01 TA - Journal of Nuclear Medicine PG - 2357--2357 VI - 63 IP - supplement 2 4099 - http://jnm.snmjournals.org/content/63/supplement_2/2357.short 4100 - http://jnm.snmjournals.org/content/63/supplement_2/2357.full SO - J Nucl Med2022 Aug 01; 63 AB - 2357 Introduction: Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of many human carcinomas and is an attractive target for the development of mAb-based therapeutics. The humanized monoclonal antibody 3D1 (mAb 3D1) binds with low nM affinity to the MUC1 C-terminal (MUC1-C) transmembrane subunit extracellular domain. The reactivity of mAb 3D1 is selective for MUC1-C expressing human cancer cell lines and primary cancer cells. Therefore, radiolabeled mAb 3D1 probes could be novel radiotheranostics for cancer imaging and therapy. We demonstrate here the 47Sc-radiolabeling of mAb 3D1 for MUC1 binding (Figure 1).Methods: Scandium-47 is produced via the 48Ti(g,p)47Sc production route using Argonne National Laboratory’s 55 MeV electron linac at the LEAF (Linear Electron Accelerator Facility), and shipped to the Cyclotron Facility at The University of Chicago for radiolabeling. Purified mAb 3D1 was buffer exchanged to 0.2 M bicarbonate buffer at pH 9.2. The DOTA-NHS ester dissolved in DMSO was added to the antibody at 100:1 ratio and mixed at room temperature for 1 h and then at 4 ºC overnight. The DOTA conjugated mAb 3D1 was isolated and concentrated using the 0.5 mL capacity 30,000 MWCO centrifugal filter units. 47ScCl3 was reconstituted in 0.1 M HCl and pH adjusted to 4.25 by 1 M ammonium acetate buffer to a final buffer concentration of 0.5 M. The DOTA-mAb 3D1 was added and the reaction was mixed gently for 4 h at 40 ºC. The resulting 47Sc labeled mAb 3D1 was then filtered and washed using the 30K MWCO filter units, which produced a &gt;90% decay corrected yield (based on the radioactivity) and &gt;95% radiochemical yield. Cellular uptake of 47Sc-mAb 3D1 was performed using BT-549 triple-negative breast cancer (TNBC) cells. BT-549 cells with stably silenced MUC1-C using a MUC1shRNA (BT-549/MUC1shRNA) served as negative control. Cells (1x105; duplicates) were plated overnight in 24-well plates using RPMI1640 supplemented with 10% FBS and 0.023 U/mL insulin. Cells were incubated with 47Sc-mAb 3D1 at indicated radioactivity levels in the absence or presence of unlabeled mAb 3D1 (3 ug/well) for 1 h at 37 ºC. Cells were then washed with cold PBS and lysed with 1 M NaOH. The radioactivity levels of cell lysates were counted in a gamma counter (Wizard2; PerkinElmer). Radioactivity used for calculation was decay corrected to the start of the radioligand treatment and converted (counts to dose) based on a calibration curve generated using known concentrations of serial dilutions. The binding efficiency of the DOTA conjugated mAb 3D1was checked with 4ug of mAb 3D1/DOTA-mAb 3D1 on TNBC MDAMB468 cells. The DOTA conjugated antibody showed a merginal 7% decrease in binding efficacy, demonstrating the antibody was mostly intact over the reaction conditions (Figure 2A)Results: Preliminary data show that, after 0.8 and 0.4 µCi of 47Sc-mAb 3D1 incubation for 60 min, the mAb 3D1 uptake of BT-549 were 59.96% and 62.42% per 105 cells, respectively (Figure 2B). Blocking with unlabeled mAb 3D1 decreased the binding to 24.52% per 105 cells for the 0.8 µCi treatment. Therefore, the overall cellular uptake was 35.44%. However, the negative control BT-549/MUC1shRNA cells had only slightly less uptake results in the preliminary experiments.Considering these initial results and ways to improve potential outcomes, additional labeling studies are being conducted with an emphasis on optimizing reaction conditions for DOTA-NHS to the mAb conjugation and 47Sc radiolabeling of the DOTA-mAb at room temperature for 5 hours. These experiments are currently on-going and additional results will be reported.Conclusions: Our preliminary studies illustrated the usefulness of 47Sc-mAb 3D1 as an effective cancer theranostic agent. Improved approaches are being developed for more efficient radiolabeling and additional cellular and animal experiments are on-going to further validate 47Sc-mAb 3D1’s utilities in cancer theranostic applications.