Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin

Abstract

With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

Dendrimer‐Like Supramolecular Assembly of Proteins with a Tunable Size and Valency Through Stepwise Iterative Growth

“…For this, a protein delivery system was constructed by combining the protein dendrimers with our previous work. [ 41 , 42 ] As depicted in Figure 3a , the protein delivery system is composed of three protein modules which are genetically fused using a flexible linker: a translocation module comprised of a cell‐targeting moiety and a translocation domain (TDP) of Pseudomonas aeruginosa exotoxin, a cargo module and a conjugation module. [ 43 ] As a model cargo, eGFP was employed.…”

“…Gelonin is a plant‐derived N‐glycosidase, which inactivates the 60S ribosomal subunit and is known to be unable to cross the cell membrane alone. [ 41 , 46 ] For this, eGFP was replaced with gelonin in the previous construct, yielding G mono (indicating a monomeric cargo with gelonin) ( Figure 4a ). The resulting construct with a size of 75.7 kDa was expressed as a soluble form in E. coli and used for functionalization of the protein dendrimers.…”

Highly Efficient Method for Intracellular Delivery of Proteins Mediated by Cholera Toxin-Induced Protein Internalization

“…In addition to CtxB-mediated endocytosis, a handful of other methods to enhance the uptake of macromolecules via receptor-mediated endocytosis have been developed in past years. Some of these methods utilize other bacterial toxins such as anthrax, − diphtheria, − and Pseudomonas exotoxin A. , Some other methods rely on the use of inherent ligand–receptor pairs found on the surface of mammalian cells, such as transferrin with the transferrin receptor, − somatostatin analogues with the somatostatin receptor, , integrin-binding peptide with integrin, − LRP1-binding peptide with LRP1, and LHRH with the LHRH receptor. − While bacterial toxins are generally very potent at inducing endocytosis, an advantage of using the inherent ligand–receptor pair is that it is unlikely to trigger an immune response. For all of these methods, the delivery efficiency counts on the abundance of the receptors on the cell surface.…”

“…Some of these methods utilize other bacterial toxins such as anthrax, 45−50 diphtheria, 51−53 and Pseudomonas exotoxin A. 54,55 Some other methods rely on the use of inherent ligand− receptor pairs found on the surface of mammalian cells, such as transferrin with the transferrin receptor, 56−61 somatostatin analogues with the somatostatin receptor, 62,63 integrin-binding peptide with integrin, 64−67 LRP1-binding peptide with LRP1, 68 and LHRH with the LHRH receptor. 69−72 While bacterial toxins are generally very potent at inducing endocytosis, an advantage of using the inherent ligand− receptor pair is that it is unlikely to trigger an immune response.…”

Molecularly engineered tumor acidity-responsive plant toxin gelonin for safe and efficient cancer therapy

“…[ 7 ], and seven of the top ten best-selling drugs worldwide in 2018 belong to protein [ 8 , 9 ]. Albeit protein agents display prominent advantages over small-molecule drugs, such as unmatched potency, high specificity to their intracellular targets and low toxicity [ 4 , [10] , [11] , [12] , [13] ], their clinical translation has been undermined by several unfavorable intrinsic characteristics including large size, hydrophilicity, membrane impermeability and susceptibility to enzymatic degradation [ 10 , 11 ]. Thus, a variety of delivery approaches have been exploited for intracellular and cytosolic delivery of proteins [ [14] , [15] , [16] , [17] , [18] , [19] ].…”