Abstract 3616: Towards intracellular targeting: Cytosolic delivery using TAT-trimers
Authors: Tietz Ole , Chalk Rod , Vallis Katherine A. 
Published: August 2019

Large therapeutic biomolecules, particularly antibodies, are generally directed against cancer cell surface antigens to facilitate antibody-antigen interaction. This limitation excludes a rich array of potential intracellular cancer targets, which has prompted research to solve the problem of transport of large molecules across the cell membrane and their release into the cytoplasm. However, current efforts suffer from endosomal entrapment and lack of efficacy due to low intracellular concentration ranges. Using the archetypal cell penetrating peptide (CPP) TAT, we report the design, synthesis and evaluation of two novel trimeric TAT clusters with enhanced efficiency in the low micromolar range. In addition, we demonstrate that geometry and conformation of TAT clusters affects internalization properties and mechanism. TAT-trimers were synthesized using copper catalyzed 2+3 cycloaddition between tetrakis core structures and azide / alkyne modified TAT (49-57) peptide. The trimeric clusters were furnished with AlexaFluor488 (AF488) for in vitro detection. Tri-TAT A is designed around a smaller, more strained core structure than tri-TAT B, which is designed around a larger core structure and more flexible. Both trimers, as well as monomeric AF488 labelled TAT (mono-TAT), were evaluated for their cell penetrating properties using live-cell fluorescence confocal microscopy in HeLa and CHO cells. The uptake of tri-TAT A and tri-TAT B into HeLa and CHO was found to be significantly higher than that of mono-TAT (1 μM; 60 min); in addition, the uptake of the trimers (1 μM) was found to be significantly higher than that of 10 μM mono-TAT, indicating that clustering TAT peptides improves their efficacy. Interestingly, we found that tri-TAT A shows homogenous cytosolic uptake and nucleolar staining at 1 μM, while tri-TAT B shows no evidence of cytosolic uptake or nucleolar staining in either HeLa or CHO cells. The fluorescence signal in cells treated with tri-TAT B was focal in nature, which is typical of endosomal entrapment. These results suggest that the geometry of the core structure and the conformational flexibility of TAT trimers is of decisive importance to the efficacy of cytosolic uptake. Time course experiments with tri-TAT A indicate that the homogenous cytosolic signal spreads evenly from the membrane into the cytosol, suggesting that the trimer translocates directly across the membrane, rather than or in addition to entry into the cytosol via endosomal escape. TAT-trimers were successfully conjugated to antibodies and antibody fragments (Fab). These constructs showed favourable internalisation properties in preliminary cell uptake studies in comparison single TAT constructs. Trimeric arrangement of CPPs on a rigid scaffold represents a promising approach to promoting cell uptake of anticancer drugs directed against intracellular targets and a significant improvement over recently reported CPP approaches.

Citation Format: Ole Tietz, Rod Chalk, Katherine A. Vallis. Towards intracellular targeting: Cytosolic delivery using TAT-trimers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3616.