NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and <i>In Vivo</i> Tumor-Targeting Properties in a Bladder Cancer Model (2018)

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed <sup>64</sup>Cu-A14-ChAcNLS, <sup>64</sup>Cu-A14-NLS, and <sup>64</sup>Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved <sup>64</sup>Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per <sup>64</sup>Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. <sup>64</sup>Cu-A14-ChAcNLS increased <sup>64</sup>Cu cellular accumulation in HT-1376 and HT-B9 cells relative to <sup>64</sup>Cu-A14 and <sup>64</sup>Cu-A14-NLS. In addition, we tested <sup>64</sup>Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that <sup>64</sup>Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to <sup>64</sup>Cu-A14. However, <sup>64</sup>Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to <sup>64</sup>Cu-A14. This resulted in <sup>64</sup>Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that <sup>64</sup>Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to <sup>64</sup>Cu-A14. In addition, <sup>64</sup>Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.