Retraction: Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2

Abstract

<b>181</b> <h3><b>Objectives:</b></h3> Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on the surface of activated T cells and some tumor cells, and is the target of the clinically-approved monoclonal antibody ipilimumab (Yervoy®, Bristol-Myers Squibb). While this drug is quite effective in a subset of patients, there are many unanswered questions in the understanding of its mechanisms, predictions of response, and the exact biodistribution of CTLA-4. In this study, we investigate specific binding of radiolabeled ipilimumab to CTLA-4 expressed by human non-small cell lung cancer cells <i>in vivo</i> using positron emission tomography (PET), demonstrating a useful tool for future CTLA-4 visualization. <h3><b>Methods:</b></h3> Ipilimumab was radiolabeled with ⁶⁴Cu (t_1/2 = 12.7 h) through the use of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to formulate ⁶⁴Cu-DOTA-ipilimumab. CTLA-4 expression in three non-small cell lung cancer (NSCLC) cell lines (A549, H460, and H358) was verified and quantified by Western blot and enzyme-linked immunosorbent assays (ELISA). A receptor binding assay was utilized to monitor the binding and internalization of ⁶⁴Cu-DOTA-ipilimumab in the NSCLC cell lines. Next, the biodistribution of ⁶⁴Cu-DOTA-ipilimumab was mapped by longitudinal PET imaging up to 48 h after injection. <i>Ex vivo</i> biodistribution and histological studies were employed to verify PET results. <h3><b>Results:</b></h3> By <i>in vitro</i> analysis, CTLA-4 was found to be expressed on all three NSCLC cell lines with A549 and H358 showing the highest and lowest level of expression, respectively. Ipilimumab was also shown to specifically bind at higher levels to cell lines with higher expression of CTLA-4. PET imaging and quantification verified these findings as the tracer accumulated highest in the A549 tumor model (9.80 ± 0.22 %ID/g at 48 h after injection; n=4), followed by H460 and H358 tumors with uptakes of 9.37 ± 0.26 %ID/g and 7.43 ± 0.05 %ID/g, respectively at the last timepoint (n=4). The specificity of the tracer was verified by injecting excess ipilimumab in A549 tumor-bearing mice, which decreased tracer uptake to 6.90 ± 0.51 %ID/g at 48 after injection (n=4). <i>Ex vivo</i> analysis following the last imaging session also corroborated these findings. <h3><b>Conclusion:</b></h3> ⁶⁴Cu-DOTA-ipilimumab showed enhanced and persistent accumulation in CTLA-4 - expressing tissues, which will enable researchers further insight into CTLA-4-targeted therapies in the future.