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. 2020 May 7;21(9):3310.
doi: 10.3390/ijms21093310.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1

Anzhelika Vorobyeva  1   2 , Elena Konovalova  3 , Tianqi Xu  1 , Alexey Schulga  2   3 , Mohamed Altai  1 , Javad Garousi  1 , Sara S Rinne  4 , Anna Orlova  2   4   5 , Vladimir Tolmachev  1   2 , Sergey Deyev  2   3   6   7
Affiliations

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1

Anzhelika Vorobyeva et al. Int J Mol Sci. .

Abstract

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

Keywords: EpCAM; PIB; SPECT; cancer; iodine; molecular imaging; ovarian; radionuclide.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Radioiodination of designed ankyrin repeats protein (DARPin) Ec1 is a one-pot reaction achieved in two steps without intermediate purification. First, N-succinimidyl-para-(trimethylstannyl)benzoate is iodinated using Chloramine-T (CAT); in the second step, [125I]-para-iodobenzoate ([125I]I-PIB) is attached to lysine residues in DARPin Ec1.
Figure 2
Figure 2
Binding specificity of [125I]I-PIB-Ec1 to epithelial cell adhesion molecule (EpCAM)-expressing OVCAR-3 and SKOV-3 ovarian cancer cells in vitro. For blocking, 100-fold molar excess of non-labeled Ec1 DARPin was added to blocked groups. Final concentration of radiolabeled compound was 2 nM. Data are presented as mean from three samples ± SD.
Figure 3
Figure 3
LigandTracer sensorgrams of [125I]I-PIB-Ec1 binding to (A) OVCAR-3 cells and to (B) SKOV-3 cells. The association was measured at 3 and 9 nM concentrations.
Figure 4
Figure 4
Cellular processing of [125I]I-PIB-Ec1 by OVCAR-3, SKOV-3 ovarian cancer cells, and BxPC-3 pancreatic cancer cells over 24 h continuous incubation. Data are shown as mean ± SD (n = 3); when error bars are smaller than symbols, they might not be visible.
Figure 5
Figure 5
Comparative biodistribution of [125I]I-PIB-Ec1 at 6 h pi in Balb/c nu/nu mice bearing EpCAM-expressing OVCAR-3 and SKOV-3 xenografts and EpCAM-negative Ramos xenografts. Data are presented as the mean ± SD from three to six mice. Letters indicate significant differences (p < 0.05, one-way ANOVA with Bonferroni’s multiple comparisons test) between: a tumor uptake in SKOV-3 and Ramos xenografts; b tumor uptake in OVCAR-3 and Ramos xenografts.
Figure 6
Figure 6
Tumor uptake of [125I]I-PIB-Ec1 in Balb/c nu/nu mice bearing SKOV-3 xenografts injected with 0.8, 4, 40, or 640 µg (corresponding to 0.044, 0.22, 22, and 35 nmol) total protein amount 6 h pi. Asterisks show significant differences (p < 0.05, one-way ANOVA with Bonferroni’s multiple comparisons test) between the groups.
Figure 7
Figure 7
Micro-single photon emission computed tomography (SPECT)/CT imaging of EpCAM expression in Balb/c nu/nu mice bearing (A) OVCAR-3 and (B) SKOV-3 xenografts using [125I]I-PIB-Ec1 6 h pi. K—kidneys, T—tumor.
See this image and copyright information in PMC

References

    1. Narod S. Can advanced-stage ovarian cancer be cured? Nat. Rev. Clin. Oncol. 2016;13:255–261. doi: 10.1038/nrclinonc.2015.224. - DOI - PubMed
    1. Went P.T., Lugli A., Meier S., Bundi M., Mirlacher M., Sauter G., Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum. Pathol. 2004;35:122–128. doi: 10.1016/j.humpath.2003.08.026. - DOI - PubMed
    1. Spizzo G., Fong D., Wurm M., Ensinger C., Obrist P., Hofer C., Mazzoleni G., Gastl G., Went P. EpCAM expression in primary tumour tissues and metastases: An immunohistochemical analysis. J. Clin. Pathol. 2011;64:415–420. doi: 10.1136/jcp.2011.090274. - DOI - PMC - PubMed
    1. Spizzo G., Went P., Dirnhofer S., Obrist P., Moch H., Baeuerle P.A., Mueller-Holzner E., Marth C., Gastl G., Zeimet A.G. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol. Oncol. 2006;103:483–488. doi: 10.1016/j.ygyno.2006.03.035. - DOI - PubMed
    1. Bellone S., Siegel E.R., Cocco E., Cargnelutti M., Silasi D.A., Azodi M., Schwartz P.E., Rutherford T.J., Pecorelli S., Santin A.D. Overexpression of epithelial cell adhesion molecule in primary, metastatic, and recurrent/chemotherapy-resistant epithelial ovarian cancer: Implications for epithelial cell adhesion molecule-specific immunotherapy. Int. J. Gynecol. Cancer. 2009;19:860–866. doi: 10.1111/IGC.0b013e3181a8331f. - DOI - PubMed

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