1 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-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
(<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-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 <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