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Tricyclic cell-penetrating peptides for efficient delivery of functional antibodies into cancer cells

Abstract

The intracellular environment hosts a large number of cancer- and other disease-relevant human proteins. Targeting these with internalized antibodies would allow therapeutic modulation of hitherto undruggable pathways, such as those mediated by protein–protein interactions. However, one of the major obstacles in intracellular targeting is the entrapment of biomacromolecules in the endosome. Here we report an approach to delivering antibodies and antibody fragments into the cytosol and nucleus of cells using trimeric cell-penetrating peptides (CPPs). Four trimers, based on linear and cyclic sequences of the archetypal CPP Tat, are significantly more potent than monomers and can be tuned to function by direct interaction with the plasma membrane or escape from vesicle-like bodies. These studies identify a tricyclic Tat construct that enables intracellular delivery of functional immunoglobulin-G antibodies and Fab fragments that bind intracellular targets in the cytosol and nuclei of live cells at effective concentrations as low as 1 μM.

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Fig. 1: Synthesis of trimer Tat constructs.
Fig. 2: Live-cell confocal microscopy of linear and cyclic Tat trimers in HeLa and CHO cells.
Fig. 3: Continuous live-cell confocal microscopy of trimers in HeLa cells.
Fig. 4: Co-delivery of antibodies and antibody fragments in live HeLa cells using tri-cTat B.
Fig. 5: Co-delivery of functional antibodies and antibody fragments in live HeLa cells.
Fig. 6: Proximity ligation assays.

Data availability

All the data supporting the findings of this study are available within the Article, the Supplementary Information or the source data. The data are also available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank R. S. Wilson and C. Lang at the Department of Physiology, Anatomy and Genetics, Oxford University, for assistance with microscopy and L. Ittner and M. Gill for helpful discussions. We acknowledge funding support from Cancer Research UK (CRUK, C5255/A15935), a CRUK grant (C5255/A18085) through the CRUK Oxford Centre, the Medical Research Council (MC_PC_12004) and the Engineering and Physical Sciences Research Council (EPSRC) Oxford Centre for Drug Delivery Devices (EP/L024012/1). This work has also received support from the Wellcome Trust (grant no. 106169).

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Authors and Affiliations

Authors

Contributions

O.T. designed, conceived and synthesized the Tat trimers, designed, conceived and acquired microscopy studies, performed data analysis and wrote the manuscript. F.C.-T. acquired and analysed microscopy data and performed the PLA assay. S.A. synthesized the IgG and Fab conjugates. R.C. carried out mass spectrometry of the Tat trimers. K.A.V. contributed to conception and design, data analysis and acquired funding and supervised the study. All authors reviewed and revised the final manuscript.

Corresponding author

Correspondence to Katherine A. Vallis.

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Nature Chemistry thanks Wouter Verdurmen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Membrane porosity following treatment with Tat-trimer.

(a, b) Addition of 40 μM propidium iodide (PI) 20 min after addition of 1 μM trimer; image at 30 min after start of experiment. Cells treated with tri-Tat A (a) co-stain with PI; cells treated with tri-cTat B (b) are PI negative. (c) Average fluorescence intensity of PI per cell, 45 min after the start of the experiment (n = 25). Cells treated with tri-Tat A show significantly higher PI uptake, indicative of pore formation. (d) Cells treated with tri-Tat A (solid line) or tri-cTat B (dotted line) for 60 min and metabolic activity as an indicator of cell viability assessed using MTT assay after 1 h, 2 h, 4 h, 3 days (n = 3 biologically independent experiments). Data presented as mean ± standard deviation. Scale bar: 20 μm.

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Source data

Source Data Fig. 1

Statistical source data for the main figures and Extended Data figures.

Source Data Fig. 2

Statistical source data for the main figures and Extended Data figures.

Source Data Fig. 3

Statistical source data for the main figures and Extended Data figures.

Source Data Fig. 4

Statistical source data for the main figures and Extended Data figures.

Source Data Fig. 6

Statistical Source Data for main Figures and Extended Data Figures

Source Data Extended Data Fig. 1

Statistical source data for the main figures and Extended Data figures.

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Tietz, O., Cortezon-Tamarit, F., Chalk, R. et al. Tricyclic cell-penetrating peptides for efficient delivery of functional antibodies into cancer cells. Nat. Chem. 14, 284–293 (2022). https://doi.org/10.1038/s41557-021-00866-0

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