Engineered Synthetic STxB for Enhanced Cytosolic Delivery

• Published in: Cells (Basel, Switzerland) Vol. 12; no. 9; p. 1291
• Main Authors: Hadjerci, Justine, Billet, Anne, Kessler, Pascal, Mourier, Gilles, Ghazarian, Marine, Gonzalez, Anthony, Wunder, Christian, Mabrouk, Nesrine, Tartour, Eric, Servent, Denis, Johannes, Ludger
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 30.04.2023; MDPI
• Subjects: Amino acids; Analysis; Antibodies; Antigens; Cancer; Care and treatment; CD8 antigen; CD8-Positive T-Lymphocytes; Cell culture; Chemical synthesis; Chloride; Chromatography; Confocal microscopy; Cytosol; Cytosol - metabolism; Dendritic cells; endosomal escape; Endosomes - metabolism; engineered protein; Health aspects; Homeopathy; hydrophobic moieties; Hydrophobicity; Intracellular Membranes - metabolism; Life Sciences; Lymphocytes T; Macromolecules; Materia medica and therapeutics; organic synthesis; Penicillin; Peptides; Proteins; Shiga toxin; Shiga Toxin - chemistry; Shiga Toxin - metabolism; synthetic carrier; Therapeutic applications; Therapeutics; France; Japan; endosomal escape; engineered protein; hydrophobic moieties; organic synthesis; synthetic carrier; Organic synthesis; Engineered protein; Synthetic carrier; Hydrophobic moieties; Endosomal escape
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells12091291

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8 T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.