1 research outputs found
NLS-Cholic Acid Conjugation to IL-5RĪ±-Specific Antibody Improves Cellular Accumulation and <i>In Vivo</i> Tumor-Targeting Properties in a Bladder Cancer Model
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 Ī±-subunit (IL-5RĪ±)-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 ā¼20 ChAcNLS or NLS moieties
per <sup>64</sup>Cu-A14 were prepared in good yield, high monomer
content, and maintaining high affinity for IL-5RĪ±. 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 <i>in vivo</i> to evaluate
its tissue distribution properties and, ultimately, tumor uptake and
targeting. A model of human IL-5RĪ± MIBC was developed by implanting
NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which
grow containing high and low IL-5RĪ±-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-5RĪ±-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting