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Background: The inability of biologics to pass the plasma membrane prevents their development as therapeutics for intracellular targets. To address the lack of methods for cytosolic protein delivery, we used the type III secretion system (T3SS) of Y. enterocolitica, which naturally injects bacterial proteins into eukaryotic host cells, to deliver monobody proteins into cancer cells. Monobodies are small synthetic binding proteins that can inhibit oncogene signaling in cancer cells with high selectivity upon intracellular expression. Here, we engineered monobodies targeting the BCR::ABL1 tyrosine kinase for efficient delivery by the T3SS, quantified cytosolic delivery and target engagement in cancer cells and monitored inhibition of BCR::ABL1 signaling., Methods: In vitro assays were performed to characterize destabilized monobodies (thermal shift assay and isothermal titration calorimetry) and to assess their secretion by the T3SS. Immunoblot assays were used to study the translocation of monobodies into different cell lines and to determine the intracellular concentration after translocation. Split-Nanoluc assays were performed to understand translocation and degradation kinetics and to evaluate target engagement after translocation. Phospho flow cytometry and apoptosis assays were performed to assess the functional effects of monobody translocation into BCR:ABL1-expressing leukemia cells., Results: To enable efficient translocation of the stable monobody proteins by the T3SS, we engineered destabilized mutant monobodies that retained high affinity target binding and were efficiently injected into different cell lines. After injection, the cytosolic monobody concentrations reached mid-micromolar concentrations considerably exceeding their binding affinity. We found that injected monobodies targeting the BCR::ABL1 tyrosine kinase selectively engaged their target in the cytosol. The translocation resulted in inhibition of oncogenic signaling and specifically induced apoptosis in BCR::ABL1-dependent cells, consistent with the phenotype when the same monobody was intracellularly expressed., Conclusion: Hence, we establish the T3SS of Y. enterocolitica as a highly efficient protein translocation method for monobody delivery, enabling the selective targeting of different oncogenic signaling pathways and providing a foundation for future therapeutic application against intracellular targets., (© 2024. The Author(s).)

Carbon nanotubes (CNTs) are increasingly being used in industrial applications, but their toxicological data in animals and humans are still sparse. To assess the toxicological dose-response of CNTs and to evaluate their pulmonary biopersistence, their quantification in tissues, especially lungs, is crucial. There are currently no reference methods or reference materials for low levels of CNTs in organic matter. Among existing analytical methods, few have been fully and properly validated. To remedy this, we undertook an inter-laboratory comparison on samples of freeze-dried pig lung, ground and doped with CNTs. Eight laboratories were enrolled to analyze 3 types of CNTs at 2 concentration levels each in this organic matrix. Associated with the different analysis techniques used (specific to each laboratory), sample preparation may or may not have involved prior digestion of the matrix, depending on the analysis technique and the material being analyzed. Overall, even challenging, laboratories' ability to quantify CNT levels in organic matter is demonstrated. However, CNT quantification is often overestimated. Trueness analysis identified effective methods, but systematic errors persisted for some. Choosing the assigned value proved complex. Indirect analysis methods, despite added steps, outperform direct methods. The study emphasizes the need for reference materials, enhanced precision, and organized comparisons.