Novel 64Cu-labeled antibody-conjugates designed to evade the endosomal-lysosomal pathway for increased intracellular radio-accumulation in target IL-5Rα-positive muscle invasive bladder cancer cells

Objectives Research into the field of targeted drug delivery by antibody-conjugates (ACs) continues to focus on methods of increasing intracellular accumulation to improve specific targeting. A novel technology, described as cholic acid linked to a 13-mer peptide harboring the nuclear localization sequence from SV-40 Large T-antigen (ChAcNLS) previously demonstrated effective endosome escape coupled to nuclear trafficking. This resulted in enhanced intracellular accumulation of attached molecular payloads. The aim of this study was to evaluate if the functionalization of the 64Cu-labeled, Interleukin-5-receptor (IL-5Rα)-targeting monoclonal antibody (mAb) A14 with ChAcNLS moieties will also increase intracellular accumulation in IL-5R-positive human bladder cancer cells.

Methods NOTA-NHS was added in 25-fold molar excess to A14 and incubated for 1 h at room temperature in 0.1M Na2CO3 pH 9.0. NOTA-A14 was then reacted with a 50-fold molar excess of the crosslinker sulfo-SMCC for conjugations to ChAcNLS moieties. Radiolabeling was performed by mixing excess 64Cu with NOTA-A14-ChAcNLS at 37°C for 1 h, then purified by serial centrifugation on a 50 kDa-cutoff filtered column. Two muscle invasive bladder cancer (MIBC) cell lines, HT1376 and HTB9, each high and low expression levels of IL-5Rα, respectively, were grown in vitro, and then exposed to [64Cu]-A14 or [64Cu]-A14-ChAcNLS for various times. Assays were also done at 4°C to assess internalization inhibition, and at 37°C with co-addition of 400 nM unlabeled A14 to block specific binding. Cellular fractionation was performed and counted in a gamma-counter. Saturation curves were obtained to assess affinity of both antibodies.

Results A14 was conjugated with an average of 5 NOTA according to an Arsenazo III colorimetric assay. Approximately 20 ChAcNLS moieties were added per antibody according to SDS-PAGE molecular weight shift analysis. Radiolabeling reached a specific activity of 250 MBq/mg. Dissociation constant (KD) were similar for both antibodies, at 2.4±1.2 nM for [64Cu]-A14 and at 3.0±2.0 nM for [64Cu]-A14-ChAcNLS. In vitro, there was respectively a 2- and 5-fold increase in nuclei accumulation in HT1376 and HTB9 cells treated with [64Cu]-A14-ChAcNLS at 24 h, compared to [64Cu]-A14. Intracellular uptake was also increased by ≍20% at 24 h in MIBC cells treated with [64Cu]-A14-ChAcNLS relative to [64Cu]-A14. Internalization and nuclear localization were inhibited at 4°C, or by addition of a blocking dose of unlabeled A14.

Conclusions Addition of ChAcNLS moieties to a 64Cu-labeled IL-5Rα-targeting antibody greatly improved the subcellular localisation to the nuclei of IL-5Rα+ bladder cancer cell lines, without affecting its affinity. Further in vivo tests will be performed to confirm its properties as an improved PET tracer.