Abstract
242030
Introduction: Mucins are high molecular weight glycoproteins secreted by a variety of epithelial cells in the body. MUC1 in the Mucins family is a highly glycosylated transmembrane protein whose overexpression is commonly associated with colon, breast, ovarian, lung, and pancreatic cancers. In addition, there are differences in glycosylation of MUC1 between tumor and normal tissues. These two are the basis for the development of broad-spectrum tumor-specific drugs targeting MUC1.
Methods: The radiochemical purity of 89Zr-16A was analyzed by radio-TLC. In vitro stability tests were performed in 0.01 M PBS and 5%HSA. Cellular uptake and affinity assays were performed in adherent cultured A549, 293T-MUC1 and 293T-COSMCKO-MUC1, B16-MUC1 and B16-COSMCKO-MUC1 cells. Cellular internalization of 89Zr-16A was measured at 4℃ and 37℃ in B16-OVA-MUC1 and COSMC knockout cells. Female KM mice (n=3) were sacrificed 2 h, 24 h, 48 h, 72 h, 96 h and 120 h after radiotracer injection to determine the bio-distribution of 89Zr-16A in normal mice. In order to verify the tumor specificity of 89Zr-16A, micro-PET scans were performed in 293T-MUC1/COSMC knockout group, B16-OVA-MUC1/COSMC knockout group, A549, SW780, BGC823 and BxPC3 models, and images were gain at the same time-point with biodistribution.
Results: The radiochemical yield of the manually prepared 89Zr-16A without attenuation correction was 73.75%, the purified radiochemical purity was greater than 95%, and it was stable in vitro within 120 hours. The uptake of 89Zr-16A in B16-OVA-COSMCKO-MUC1 cells with COSMC gene knockout was significantly higher than wild-type B16-OVA-MUC1 cells (20.78±2.79% vs. 2.99±0.09%, 4 h), but the affinity (expressed as equilibrium dissociation constant KD) was opposite (21.52 nM vs. 0.27 nM). The internalization rate of 89Zr-16A in COSMC knockout cells was higher than that in wild-type cells, and it decreased with time, but the difference between different temperatures was small (82.23-60.64% vs. 46.61-28.65% for 4℃, 75.21-56.27% vs. 44.96-24.82% for 37℃). Results of both bio-distribution and micro-PET imaging showed that 89Zr-16A was largely cleared from the blood within 24 h and metabolized mainly by the liver. Maximum standard uptake value (SUVmax) of micro-PET images showed that 89Zr-16A had high tumor uptake in B16-OVA-MUC1 (4.48)/B16-OVA-COSMCKO-MUC1 (5.67), A549 (6.47), SW780 (3.42), BGC823 (3.86) and BxPC3 (3.12) models, and lower tumor uptake in 293T-MUC1 (1.55)/293T-COSMCKO-MUC1 (1.97) models. 89Zr-IgG PET imaging as a control initially demonstrated the specificity of 89Zr-16 in tumor uptake in the B16-OVA-COSMCKO-MUC1 model.
Conclusions: The tumor specificity of the original mab 16A radiolabeled with 89Zr was validated in preclinical experiments in lung, bladder, pancreatic, and gastric cancer models. Moreover, COSMC knockout cells (mimic tumor cells) showed higher selectivity to 89Zr-16A than wild-type cells, which might be due to the abnormal expression of MUC1. 89Zr-16A has shown potential for clinical translation as a PET probe targeting MUC1 in tumors.
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