2 research outputs found
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-Ī± (IL-3RĪ±). Our aim was to determine
whether 7G3-ChAcNLS increased intracellular accumulation while retaining
nanomolar affinity and IL-3RĪ±-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
(<sup>64</sup>Cu) 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-3RĪ±-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 <sup>64</sup>Cu 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-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