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. Access through your institution Buy or subscribe This is a

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TRANSPORT OF A CATALYTIC TOXIN Article Open access 17 January 2023 PEPTIDES AS MULTIFUNCTIONAL PLAYERS IN CANCER THERAPY Article Open access 01 June 2023 COMPARISON OF ANTIBODY-SCTRAIL FC

FUSION PROTEINS WITH VARYING VALENCY FOR EGFR AND TRAIL RECEPTORS Article Open access 06 May 2025 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.

REFERENCES * Carter, P. J. & Lazar, G. A. Next generation antibody drugs: pursuit of the ‘high-hanging fruit’. _Nat. Rev. Drug Discov._ 17, 197–223 (2018). Article  CAS  PubMed  Google

Scholar  * Verdine, G. L. & Walensky, L. D. The challenge of drugging undruggable targets in cancer: lessons learned from targeting BCL-2 family members. _Clin. Cancer Res._ 13,

7264–7270 (2007). Article  CAS  PubMed  Google Scholar  * Stewart, M. P. et al. In vitro and ex vivo strategies for intracellular delivery. _Nature_ 538, 183–192 (2016). Article  CAS  PubMed

  Google Scholar  * Samal, S. K. et al. Cationic polymers and their therapeutic potential. _Chem. Soc. Rev._ 41, 7147–7194 (2012). Article  CAS  PubMed  Google Scholar  * Brooks, H., Lebleu,

B. & Vivès, E. Tat peptide-mediated cellular delivery: back to basics. _Adv. Drug Del. Rev._ 57, 559–577 (2005). Article  CAS  Google Scholar  * Peraro, L. & Kritzer, J. A. Emerging

methods and design principles for cell-penetrant peptides. _Angew. Chem. Int. Ed._ 57, 11868–11881 (2018). Article  CAS  Google Scholar  * Pei, D. & Buyanova, M. Overcoming endosomal

entrapment in drug delivery. _Bioconjug. Chem._ 30, 273–283 (2019). Article  CAS  PubMed  Google Scholar  * Fu, A., Tang, R., Hardie, J., Farkas, M. E. & Rotello, V. M. Promises and

pitfalls of intracellular delivery of proteins. _Bioconjug. Chem._ 25, 1602–1608 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fawell, S. et al. Tat-mediated delivery of

heterologous proteins into cells. _Proc. Natl Acad. Sci. USA_ 91, 664–668 (1994). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cornelissen, B. et al. Imaging DNA damage in vivo

using γH2AX-targeted immunoconjugates. _Cancer Res._ 71, 4539–4549 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Singh, K., Ejaz, W., Dutta, K. & Thayumanavan, S.

Antibody delivery for intracellular targets: emergent therapeutic potential. _Bioconjug. Chem_ 30, 1028–1041 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Brock, R. The

uptake of arginine-rich cell-penetrating peptides: putting the puzzle together. _Bioconj. Chem._ 25, 863–868 (2014). Article  CAS  Google Scholar  * Madani, F., Lindberg, S., Langel, U.,

Futaki, S. & Gräslund, A. Mechanisms of cellular uptake of cell-penetrating peptides. _J. Biophys._ 2011, 414729 (2011). Article  PubMed  PubMed Central  Google Scholar  * Dougherty, P.

G., Sahni, A. & Pei, D. Understanding cell penetration of cyclic peptides. _Chem. Rev._ 119, 10241–10287 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Milletti, F.

Cell-penetrating peptides: classes, origin and current landscape. _Drug Discov. Today_ 17, 850–860 (2012). Article  CAS  PubMed  Google Scholar  * Nischan, N. et al. Covalent attachment of

cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. _Angew. Chem. Int. Ed._ 54, 1950–1953 (2015). Article  CAS  Google Scholar  * Herce, H.

D. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. _Nat. Chem._ 9, 762–771 (2017). Article  CAS  PubMed  Google Scholar  * Schneider,

A. F. L., Kithil, M., Cardoso, M. C., Lehmann, M. & Hackenberger, C. P. R. Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives.

_Nat. Chem._ 13, 530–539 (2021). Article  CAS  PubMed  Google Scholar  * Akishiba, M. et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. _Nat. Chem._ 9, 751–761

(2017). Article  CAS  PubMed  Google Scholar  * Ovacik, M. & Lin, K. Tutorial on monoclonal antibody pharmacokinetics and its considerations in early development. _Clin. Transl. Sci._

11, 540–552 (2018). Article  PubMed  PubMed Central  Google Scholar  * Kauffman, W. B., Fuselier, T., He, J. & Wimley, W. C. Mechanism matters: a taxonomy of cell penetrating peptides.

_Trends Biochem. Sci._ 40, 749–764 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Herce, H. D. & Garcia, A. E. Molecular dynamics simulations suggest a mechanism for

translocation of the HIV-1 TAT peptide across lipid membranes. _Proc. Natl Acad. Sci. USA_ 104, 20805–20810 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lawrence, M. S.,

Phillips, K. J. & Liu, D. R. Supercharging proteins can impart unusual resilience. _J. Am. Chem. Soc._ 129, 10110–10112 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Cronican, J. J. et al. Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein. _ACS Chem. Biol._ 5, 747–752 (2010). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Freire, J. M., Almeida Dias, S., Flores, L., Veiga, A. S. & Castanho, M. A. R. B. Mining viral proteins for antimicrobial and cell-penetrating

drug delivery peptides. _Bioinformatics_ 31, 2252–2256 (2015). Article  CAS  PubMed  Google Scholar  * Tung, C.-H., Mueller, S. & Weissleder, R. Novel branching membrane translocational

peptide as gene delivery vector. _Biorg. Med. Chem._ 10, 3609–3614 (2002). Article  CAS  Google Scholar  * Angeles-Boza, A. M., Erazo-Oliveras, A., Lee, Y.-J. & Pellois, J.-P. Generation

of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in _cis_ or _trans_. _Bioconjug. Chem._ 21, 2164–2167

(2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fu, J., Yu, C., Li, L. & Yao, S. Q. Intracellular delivery of functional proteins and native drugs by cell-penetrating

poly(disulfide)s. _J. Am. Chem. Soc._ 137, 12153–12160 (2015). Article  CAS  PubMed  Google Scholar  * Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an

endosomolytic agent. _Nat. Methods_ 11, 861–867 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Erazo-Oliveras, A. et al. The late endosome and its lipid BMP act as gateways

for efficient cytosolic access of the delivery agent dfTAT and its macromolecular cargos. _Cell Chem. Biol._ 23, 598–607 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Najjar, K. et al. Unlocking endosomal entrapment with supercharged arginine-rich peptides. _Bioconj. Chem._ 28, 2932–2941 (2017). Article  CAS  Google Scholar  * Kez, C., Lin, H., Peter, D.

W. & Arwyn, T. J. Endocytosis, intracellular traffic and fate of cell penetrating peptide based conjugates and nanoparticles. _Curr. Pharm. Des._ 19, 2878–2894 (2013). Article  Google

Scholar  * Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I. & North, A. J. Tutorial: guidance for quantitative confocal microscopy. _Nat. Protoc._ 15, 1585–1611 (2020). Article

  CAS  PubMed  Google Scholar  * Herbert, A. GDSC colocalisation plugins http://www.sussex.ac.uk/gdsc/intranet/microscopy/UserSupport/AnalysisProtocol/imagej/colocalisation (2013). * Costes,

S. V. et al. Automatic and quantitative measurement of protein-protein colocalization in live cells. _Biophys. J._ 86, 3993–4003 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar

  * Ramirez, O., García, A., Rojas, R., Couve, A. & Härtel, S. Confined displacement algorithm determines true and random colocalization in fluorescence microscopy. _J. Microsc._ 239,

173–183 (2010). Article  CAS  PubMed  Google Scholar  * Söderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. _Nat. Methods_ 3,

995–1000 (2006). Article  PubMed  Google Scholar  * Rouet, R. et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. _J. Am. Chem. Soc._ 140,

6596–6603 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Qian, Z. et al. Discovery and mechanism of highly efficient cyclic cell-penetrating peptides. _Biochemistry_ 55,

2601–2612 (2016). Article  CAS  PubMed  Google Scholar  * Liu, H., Gaza-Bulseco, G., Faldu, D., Chumsae, C. & Sun, J. Heterogeneity of monoclonal antibodies. _J. Pharm. Sci._ 97,

2426–2447 (2008). Article  CAS  PubMed  Google Scholar  * Delavoie, F., Soldan, V., Rinaldi, D., Dauxois, J. Y. & Gleizes, P. E. The path of pre-ribosomes through the nuclear pore

complex revealed by electron tomography. _Nat. Commun._ 10, 497 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Paci, G., Zheng, T., Caria, J., Zilman, A. & Lemke, E. A.

Molecular determinants of large cargo transport into the nucleus. _eLife_ 9, e55963 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Avrameas, A., Ternynck, T., Nato, F.,

Buttin, G. & Avrameas, S. Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules. _Proc.

Natl Acad. Sci. USA_ 95, 5601–5606 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gordon, R. E., Nemeth, J. F., Singh, S., Lingham, R. B. & Grewal, I. S. Harnessing SLE

autoantibodies for intracellular delivery of biologic therapeutics. _Trends Biotechnol._ 39, 298–310 (2021). Article  CAS  PubMed  Google Scholar  * Bernardes, N. E. & Chook, Y. M.

Nuclear import of histones. _Biochem. Soc. Trans._ 48, 2753–2767 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Placek, B. J., Harrison, L. N., Villers, B. M. & Gloss,

L. M. The H2A.Z/H2B dimer is unstable compared to the dimer containing the major H2A isoform. _Protein Sci._ 14, 514–522 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references 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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK

Ole Tietz, Fernando Cortezon-Tamarit, Sarah Able & Katherine A. Vallis * Centre for Medicines Discovery, University of Oxford, Oxford, UK Rod Chalk Authors * Ole Tietz View author

publications You can also search for this author inPubMed Google Scholar * Fernando Cortezon-Tamarit View author publications You can also search for this author inPubMed Google Scholar *

Rod Chalk View author publications You can also search for this author inPubMed Google Scholar * Sarah Able View author publications You can also search for this author inPubMed Google

Scholar * Katherine A. Vallis View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Chemistry_ thanks Wouter Verdurmen and the other, anonymous, reviewer(s) for their contribution to the peer review of this

work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supporting Information. REPORTING SUMMARY 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. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 16 September 2019 * Accepted: 19 November

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