ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 (⁶⁴Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of ⁶⁴Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking

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

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 ⁶⁴Cu-A14-ChAcNLS, ⁶⁴Cu-A14-NLS, and ⁶⁴Cu-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 ⁶⁴Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per ⁶⁴Cu-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. ⁶⁴Cu-A14-ChAcNLS increased ⁶⁴Cu cellular accumulation in HT-1376 and HT-B9 cells relative to ⁶⁴Cu-A14 and ⁶⁴Cu-A14-NLS. In addition, we tested ⁶⁴Cu-A14-ChAcNLS <i>in vivo</i> 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 ⁶⁴Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to ⁶⁴Cu-A14. However, ⁶⁴Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to ⁶⁴Cu-A14. This resulted in ⁶⁴Cu-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 ⁶⁴Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to ⁶⁴Cu-A14. In addition, ⁶⁴Cu-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