Immuno-PET\nImaging of CD93 Expression with <sup>64</sup>Cu-Radiolabeled NOTA-mCD93\n([<sup>64</sup>Cu]Cu-NOTA-mCD93) and Insulin-Like\nGrowth Factor Binding Protein 7 ([<sup>64</sup>Cu]Cu-NOTA-IGFBP7)

CD93\nis overexpressed in multiple solid tumor types,\nserving as\na novel target for antiangiogenic therapy. The goal of this study\nwas to develop a <sup>64</sup>Cu-based positron emission tomography\n(PET) tracer for noninvasive imaging of CD93 expression. Antimouse-CD93\nmAb (mCD93) and the CD93 ligand IGFBP7 were conjugated to a bifunctional\nchelator, <i>p</i>-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic\nacid (<i>p</i>-SCN-NOTA) and labeled with <sup>64</sup>Cu.\nTo evaluate the pharmacokinetic properties and tumor-targeting efficacy\nof [<sup>64</sup>Cu]Cu-NOTA-mCD93 and [<sup>64</sup>Cu]Cu-NOTA-IGFBP7,\nPET imaging and biodistribution were performed on both 4T1 murine\nbreast tumor-bearing mice and MDA-MB-231 human breast tumor-bearing\nmice. The tumor model HT1080-FAP, which does not overexpress CD93,\nwas used as a negative control. Fluorescent immunostaining was conducted\non different tissues to correlate radiotracer uptake with CD93 expression. <sup>64</sup>Cu-labeling was achieved with high yield and specific activity.\nSerial PET imaging revealed that the <i>in vivo</i> performance\nof [<sup>64</sup>Cu]Cu-NOTA-IGFBP7 was superior to that of [<sup>64</sup>Cu]Cu-NOTA-mCD93, and that the tracer [<sup>64</sup>Cu]Cu-NOTA-IGFBP7\nexhibited elevated tumor uptake values and excellent tumor retention\nin MDA-MB-231 mice, rather than in 4T1 murine mice. The MDA-MB-231\ntumor uptake of [<sup>64</sup>Cu]Cu-NOTA-IGFBP7 was 2.85 ± 0.15,\n3.69 ± 0.60, 6.91 ± 0.88, and 6.35 ± 0.55%ID/g at 1,\n4, 24, and 48 h p.i., respectively, which were significantly higher\nthan that in the CD93-negative HT1080-FAP tumor (0.73 ± 0.15,\n0.97 ± 0.31, 1.00 ± 0.07, and 1.02 ± 0.11%ID/g, respectively).\nThe significant difference between positive and negative tumors indicated\n[<sup>64</sup>Cu]Cu-NOTA-IGFBP7 was specifically binding to CD93.\nBiodistribution data as measured by gamma counting were consistent\nwith the PET analysis. <i>Ex vivo</i> histology further\nconfirmed the high CD93 expression on MDA-MB-231 tumor tissues. Herein,\nwe prepared two novel radiotracers, [<sup>64</sup>Cu]Cu-NOTA-mCD93\nand [<sup>64</sup>Cu]Cu-NOTA-IGFBP7, for the first immune-PET imaging\nof CD93 expression. Our results suggest that [<sup>64</sup>Cu]Cu-NOTA-IGFBP7\nis a more potential radiotracer for visualizing angiogenesis due to\nits sensitive, persistent, and CD93-specific characteristics.