The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates

Antibody-based agents are increasingly used as therapeutics and imaging agents, yet are generally restricted to cell surface targets due to inefficient cellular internalisation and endosomal entrapment. Enhanced cell membrane translocation of antibodies can be achieved by the covalent attachment of cell-penetrating peptides, including the HIV-1-derived transactivator of transcription (TAT) peptide. This study evaluated the cellular internalisation properties of five anti-HER2 Herceptin–TAT conjugates with degrees of TAT labelling (DOL(TAT)) ranging from one to five. Herceptin–TAT conjugates were synthesised via a strain-promoted alkyne–azide cycloaddition reaction, characterised by UV-vis spectroscopy, MALDI-TOF, and gel electrophoresis, then radiolabelled with zirconium-89 to permit measurement of cellular internalisation by gamma counting. [(89)Zr]Zr–DFO–Her–TAT((0–5)) conjugates were isolated in high radiochemical purity (>99%) and exhibited high stability in murine and human serum over 7 days at 37 °C. Significant increases in cellular internalisation were observed for [(89)Zr]Zr–DFO–Her–TAT conjugates with DOL(TAT) values of 2 and above in SKBR3 (high HER2) cells over 48 h, in contrast to low-level non-specific uptake in MDA-MB-468 (low HER2) cells that did not increase over time. Notably, [(89)Zr]Zr–DFO–Her–TAT conjugates with DOL(TAT) of 3, 4, and 5 reached uptake values in SKBR3 cells of 5, 6, and 8% of the applied dose at 48 h respectively, representing 9, 10, 14-fold increases relative to the TAT-free control conjugate, [(89)Zr]Zr–DFO–Her–TAT((0)).