Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR::ABL1 signaling

Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR::ABL1 signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR::ABL1 signaling Chiara Lebon, Sebastian Grossmann, Greg Mann, Florian Lindner, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4705983/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted 8 You are reading this latest preprint version Abstract 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Targeted cancer therapeutics specifically inhibit oncoproteins and oncogenic pathways and are thus being used as a personalized therapy option with fewer side effects compared to chemotherapy and other conventional cancer treatments. Currently available targeted therapeutics can be categorized into small molecule inhibitors 1 , 2 , often binding protein kinases and few other intracellular enzymes, and biologics, mostly therapeutic antibodies 3 – 6 , which target extracellular and membrane-bound proteins. While their clinical application has led to therapeutic breakthroughs in recent years, several limitations of these drugs have arisen 7 . Small molecule inhibitors often lack high selectivity, leading to off-target binding and resulting in adverse effects, leaving many potential targets “undruggable”. Therapeutic antibodies, while highly specific, are complex structures with large sizes and limited tissue/tumor penetration. Importantly, antibodies are precluded from inhibiting intracellular targets, as they cannot cross cellular membranes. These drawbacks highlight the need for alternative targeted therapeutics and efficient approaches for the intracellular delivery of biologics. Synthetic binding proteins are a recent development in the field of targeted therapeutics. These binding proteins are engineered from stable scaffold proteins, using molecular display techniques. The obtained binders can target the protein of interest with high affinity and selectivity and often result in preventing protein-protein interactions or inhibiting enzymatic activity of the target. Commonly used engineered binding proteins include derivatives of immunoglobulin scaffolds (scFvs, Fabs, nanobodies) and non-immunoglobulin scaffolds (monobodies, DARPins, affibodies, anticalins) 8 – 11 . Due to their small size (~ 6–20 kDa) and their ability to bind with high affinity and selectivity, they overcome limitations of current targeted therapeutics and thus have substantial therapeutic potential 7 . Among the most commonly used synthetic binder classes are monobodies (Mb), which are developed based on the protein scaffold derived from a human fibronectin type III domain 12 . We have engineered and characterized several monobodies as potent antagonists of oncoproteins, including kinases (BCR::ABL1 13–15 , LCK 16 ), phosphatases (SHP2 17 ), transcription factors (STAT3 18 ) and small GTPases (H-/K-RAS 19–21 ), demonstrating that it is possible to develop selective monobodies to challenging intracellular targets. These monobodies were introduced into cells as genetically encoded reagents using DNA transfection and viral gene delivery, where they inhibit the function of their targets. Monobodies lack endogenous disulfides, and consequently they readily fold into the fully functional form in the reducing environment of the cytoplasm. A number of studies have demonstrated the effectiveness of monobodies against intracellular targets for discovering and validating therapeutic approaches and elucidating the structural basis for specific recognition of challenging targets 22 , 23 . Additionally, recent advances have substantially improved the plasma stability and pharmacokinetics of monobodies, providing a solid groundwork for future therapeutic translation 24 . The limited availability of efficient intracellular drug delivery systems poses a major roadblock for macromolecular therapeutics like peptides and nucleic acids, but in particular for proteins. Although monobodies and other synthetic binding proteins can achieve high selectivity and potency against the most challenging targets, the inability of monobodies to readily pass the plasma membrane barrier has so far limited their use as protein therapeutics against cytoplasmic and nuclear targets. Several protein delivery strategies have been explored, ranging from physical methods (e.g. electroporation, microinjection) and viral delivery to nanoparticles 25 – 28 . In particular, various fusion strategies have been studied for the delivery of proteins such as bacterial toxin subunits 28 , 29 and cell-penetrating peptides (CPPs) 30 , 31 . Often these delivery strategies were tested with model cargoes, such as fluorescent proteins or highly active enzymes, where cytosolic delivery of very low amounts is already sufficient for a measurable readout. By contrast, few studies have shown an effect on oncogenic signaling after delivery of protein-based inhibitors. We have already demonstrated the cytosolic delivery of monobodies by fusing them to a chimeric bacterial toxin subunit 32 , 33 . Further modification even allowed target degradation after uptake 33 , but we also experienced difficulties during recombinant production and also assume high immunogenicity using this system due to the large size of the toxin. Most cellular delivery methods rely on uptake of the cargo protein through endocytosis, which in turn requires efficient endosomal escape afterwards to prevent cargo degradation in lysosomes. Inefficient endosomal escape and thus insufficient cytosolic amounts of binders is a common challenge that still has not been fully overcome 29 . Different endosomal escape strategies have been proposed 34 – 36 , but their efficiency is highly cargo-, cell- and delivery strategy-dependent and thus no universal strategy can promise cytosolic delivery of a wide variety of cargos. Hence, delivery tools that can circumvent endocytosis and directly deliver functional binders into the cytosol are of particular interest. The bacterial type III secretion system (T3SS) is used by many bacteria to directly inject proteins into eukaryotic host cells 37 , using a hollow needle attached to an export machinery in the bacterial membranes and cytosol (Fig. 1 a). As a system evolutionary optimized for the efficient delivery of proteins into the cytosol, the T3SS has been used to deliver different cargo proteins 38 , 39 into eukaryotic target cells, including cell lines difficult to manipulate by transfection or other means 40 . Cargo proteins are targeted to the T3SS by a short (15–150 amino acids) unstructured N-terminal secretion signal 41 , which can be removed by site-specific proteases or cleavage at the C-terminus of a ubiquitin domain by the native host cell machinery in the target cell 42 , 43 . While the properties of cargo proteins can influence translocation rates, and very large or stably folded proteins are exported at a lower rate, most proteins, including molecular weights above 60 kDa, can be exported by the T3SS and delivered to eukaryotic cells at rates of up to 100 proteins per second, allowing the specific delivery of hundreds of thousands of cargo proteins per host cell 42 , 44 – 48 . Cargo proteins pass the needle unfolded with the N-terminus first, facilitating their native folding, and consequently function, in the target cell. The amount of injection into host cells can be titrated by adjusting the expression level and multiplicity of infection (MOI; ratio of bacteria to host cells). Taken together, these properties make the T3SS an efficient and versatile tool for protein delivery into eukaryotic cells 40 . In this study, we use the T3SS of an avirulent Yersinia enterocolitica strain, ΔHOPEMTasd 49 . Yersinia features a well-characterized and remarkably efficient T3SS, which can secrete large concentrations of effectors within short time (> 90% of all extracellular proteins are T3SS export substrates 50 ). Y. enterocolitica has an unusually low number of native effector proteins, which can easily be deleted for increased biosafety and possibly increased export of heterologous cargo proteins. Given that Y. enterocolitica actively targets tumors 51 – 53 , the Yersinia T3SS is a highly promising carrier for monobodies, as evidenced by an ongoing clinical trial for cancer therapy 54 . To establish the T3SS of Y. enterocolitica as a monobody delivery tool, we focus on the well-characterized AS25 monobody and its target, the Abelson tyrosine kinase 1 (Abl1). The oncogenic counterpart of Abl1 is BCR::ABL1, the product of the Philadelphia chromosomal translocation, which results in the fusion of the breakpoint cluster region (BCR) and ABL1 genes 55 . The fusion protein BCR::ABL1 is a constitutively active kinase that is a central driver of chronic myeloid leukemia (CML) 56 . When expressed intracellularly, AS25 inhibits BCR::ABL1 kinase activity by targeting an intramolecular allosteric interface formed by the Src Homology 2 (SH2) domain and the kinase domain. AS25 thus disrupts BCR::ABL1-mediated signaling in CML cells, inhibiting their proliferation and survival 13 . Here, we show the efficient direct cytosolic delivery of the AS25 monobody to different human cell lines using the T3SS of Y. enterocolitica . Concentrations in the cytosol reached mid-micromolar, ~ 100-fold higher than in previous studies and well above the binding affinity. The delivered monobodies readily refold and are able to engage their targets in cells. We demonstrate specific inhibition of BCR::ABL1 signaling and induction of apoptosis in CML cells by T3SS-mediated delivery of AS25. Materials and Methods Antibodies Antibodies were purchased from Promega (Mouse anti-HiBiT (N7200)), ThermoFisher Scientific (Mouse anti-beta tubulin-DyLight™ 680 (MA5-16308-D680)), Rockland (Rabbit anti-FLAG® (600-401-383S)), Sigma (Goat anti-Rabbit IgG Peroxidase antibody (A8275)) and LI-COR (IRDye®800CW Goat anti-Mouse IgG Secondary Antibody (926-32210)). Plasmids and cloning Gene fragments encoding monobodies for protein purification were cloned into a pHFT2 vector, a modified pET vector 57 . Destabilizing and non-binding mutation were introduced through site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (200523, Agilent) according to manufacturer instructions. Retroviral transduction was performed using pRV vector constructs with an IRES site followed by a GFP gene for selection. LgBiT gene, Abl-SH2-LgBiT fusion and Lck-SH2-LgBiT fusions were inserted into the pRV vector using Gibson Assembly®. Retroviral expression system encoding the VSV-G envelope (pCMB-VSV-G) was obtained from the Worzfeld lab. For bacterial expression plasmids, a pBAD/His B-based plasmid with SycE-YopE 1− 138 -insert (pAD722) was constructed by PCR-based restriction cloning. This plasmid served as backbone for the insertion of monobody variants by restriction enzymes; SmBiT and nonbinding monobody variants were constructed using the Q5® Site-Directed Mutagenesis Kit (E0554, New England Biolabs) according to manufacturer instructions. All DNA constructs were confirmed by Sanger sequencing (Microsynth). Plasmids, primers and bacterial strains used in this study are listed in Supplementary Tables 5–7. Cultivation of bacteria Y. enterocolitica strains were cultivated in BHI (Brain Heart Infusion Broth) medium (3.7% w/v), complemented with nalidixic acid (35 µg/ml), 2,6-diaminopimelic acid (DAP, 60 µg/ml), and ampicillin (200 µg/ml) (cultivation medium). For overnight cultures, 5 ml of cultivation medium was inoculated and cultivated overnight at 28°C in a shaking incubator. Cell culture All cell lines were cultured in 5% CO 2 at 37°C. K562, Jurkat, HeLa and HEK293 cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Cat# ACC 10, ACC 282, ACC 57 and ACC 305, respectively). K562 and Jurkat cells were grown in Roswell Park Memorial Institute (RPMI) 1640 GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 50 U/ml Penicillin and 50 µg/ml Streptomycin (Gibco). HeLa Kyoto and HEK293 cells were grown in high glucose DMEM GlutaMAX medium (Gibco) supplemented with 10% FBS and 50 U/ml Penicillin and 50 µg/ml Streptomycin. Antibiotics (Penicillin and Streptomycin) were removed a day prior to infections. Hela LgBiT cells for Fig. 2 b and 3 c were kindly gifted by Samuel Wagner (Tübingen). These cells were grown in Roswell Park Memorial Institute (RPMI) 1640 (Gibco™, 11875093), supplemented with 10% FBS (Gibco). Cell lines used in this study are listed in Supplementary Table 8. Generation of stable cell lines As previously described 18 , LgBiT with a N-terminal FLAG, Abl-SH2-LgBiT fusion with a N-terminal 6 x myc and Lck-SH2-LgBiT fusion with a N-terminal 6 x myc cloned into the retroviral pRV vector were used to establish the following stable cell lines: K562 LgBiT, Jurkat LgBiT, HeLa Abl-SH2-LgBiT and HeLa Lck-SH2-LgBiT. Cells expressing IRES-GFP were selected and sorted using FACS. Expression and functionality were assessed via immunoblotting and in functional assays. Protein expression and purification The monobodies were produced with an N-terminal His 10 , FLAG and tobacco etch virus (TEV) protease recognition site using the pHFT2 vector 57 . Abl SH2 was produced with an N-terminal His 6 , GST and a TEV protease cleavage site using a pETM30 vector. All proteins were expressed in BL21 (DE3) E.coli cells at 16°C for 16 h in auto induction LB medium. Protein purification was done by nickel-affinity chromatography (column: 1 ml or 5 ml His-Trap FF crude) and subsequent size exclusion chromatography (column: HiLoad 16/600 Superdex 75 pg) on an Äkta Avant system (Cytiva). The His 6 -GST tag of Abl-SH2 was cleaved off using TEV protease before size exclusion chromatography. Purity of all purified proteins was assessed via SDS-PAGE. Amino acid sequences of the monobodies are listed in Supplementary Table 1. Thermal Shift™ Assay (TSA) Protein Thermal Shift™ Assay was performed to determine the thermal stability of the monobody mutants. The measurements were done using the Protein Thermal Shift™ Dye Kit (ThermoFisher Scientific, 4461146) and performed on a StepOne™ Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). Samples were measured in triplicates and contained 3 µg of protein in 1x DPBS. A thermal profile from 25°C to 95°C with a ramp rate of 1% was acquired using StepOnePlus Software (Applied Biosystems, ThermoFisher Scientific) and analyzed using Protein Thermal Shift Software (Applied Biosystems, ThermoFisher Scientific). Isothermal titration calorimetry (ITC) Proteins were dialyzed overnight at 4°C against 50 mM Tris (pH 7.0), 250 mM NaCl and 5% glycerol. The protein concentration was determined by measuring UV absorbance at 280 nm on a NanoDrop 2000c. ITC measurements were acquired on a MicroCal PEAQ-ITC instrument (Malvern Panalytical) and thermodynamic parameters were determined with the MicroCal PEAQ-ITC analysis software. The protein in the syringe (Abl SH2, 200 µM) was titrated to the monobody solution (AS25 or AS25 A57G , 20 µM) in 19 steps with 0.5 µl for the first and 2 µl each for the other steps. The titration of Abl SH2 (300 µM) to AS25 Y45A − A57G (30 µM) was done in 13 steps with 0.5 µl for the first and 3 µl for the subsequent steps. The duration of each injection was 4 s with 150 s spacing in between injections for all measurements. The reference power was set to 10 µcal/s, the stir speed to 750 rpm and feedback to high. All measurements were performed at 25°C. In vitro secretion assay Bacteria day cultures were inoculated from stationary overnight cultures to an OD 600 of 0.15 in cultivation medium complemented with MgCl 2 (20 mM), glycerol (0.4% w/v), and EGTA (5 mM). The cultures were cultivated shaking for 90 min at 28°C and then shifted to a 37°C and incubated for 3 h. Protein expression from plasmids was induced with 0.2% L-arabinose (w/v), before shifting to 37°C. 2 ml of bacterial culture were collected by centrifugation (10 min at 16,000 x g), and proteins from 1800 µl supernatant were precipitated with 200 µl trichloroacetic acid (100% w/v) overnight at 4°C. The precipitated proteins were collected by centrifugation (15 min at 16,000 x g) and washed with ice-cold acetone. Samples were resuspended in SDS-PAGE loading buffer (SDS (2% w/v), Tris (0.1 M), glycerol (10% w/v), dithiothreitol (0.05 M), pH = 6.8) and heated at 99°C for 5 min. Unless stated differently, proteins expressed by 1.2 × 10 8 bacteria or secreted by an equivalent of 2.4 × 10 8 bacteria were loaded onto SDS-PAGE gels. The gels were run for 1.5 h (135 V, 500 mA), using BlueClassic Prestained Marker [Jena Biosciences (PS-107)] or Precision Plus Dual Color Protein Standard [Bio-Rad (1610374)] as size standards. Immunoblotting of secretion assays SDS-PAGE gels were blotted on a Amersham™ Protran® Western Blotting nitrocellulose membrane (0.2 µm) [Cytiva (10600001)] using a Trans-Blot Turbo Transfer System [Bio-Rad (1704150)] with the settings: 1.3 A, 25 V, 7 min. Immunoblots were carried out using primary rabbit antibodies against the FLAG peptide [Rockland (600-401-383), 1:5,000] in combination with a secondary goat anti-rabbit antibody conjugated to a peroxidase [Sigma (A8275) 1:10,000] and visualized with Immobilon Forte Western HRP substrate [Merck (WBLUF0500)] on a LAS-4000 Luminescence Image Analyzer. Unprocessed blots can be found in Supplementary Figs. 4 and 8. Infection of adherent eukaryotic cells (HEK293 and HeLa cells) A day prior to infections, HeLa and HEK293 were seeded at 30% confluency in cell culture medium (DMEM GlutaMAX with 10% FBS) without antibiotics. Bacteria day cultures were inoculated from stationary overnight cultures to an OD 600 of 0.12 in cultivation medium complemented with MgCl 2 (20 mM), glycerol (0.4% w/v), and 200 µg/ml ampicillin. CaCl 2 (5 mM) was added for non-secreting conditions. The cultures were cultivated shaking for 90 min at 28°C. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with 0.2% L-arabinose (w/v) and cultures were shifted to 37°C for 120 min to induce T3SS formation. After that, bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v). Medium of the eukaryotic cell culture was changed to colorless RPMI, supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v). For the infection, Yersinia were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated in 5% CO 2 at 37°C (non-shaking). After 2 h incubation, the bacteria were removed and the eukaryotic cells were further incubated for 1 h in cell culture medium (DMEM GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 µg/ml Streptomycin) supplemented with 200 µg/ml gentamicin. Cells were washed twice with 1 x phosphate buffered saline (PBS) and maintained in normal cell culture medium (DMEM GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 µg/ml Streptomycin) until further analysis. Infection of non-adherent eukaryotic cells (K562 and Jurkat cells) K562 and Jurkat cells were seeded at 3 x 10 6 cells/ml in cell culture medium (RPMI 1640 GlutaMAX with 10% FBS) without antibiotic supplementation. Bacterial cells were prepared as described for the infection of adherent eukaryotic cells. For the infection, Yersinia were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated at 37°C (non-shaking) at 5% CO 2 . After 2 h incubation, Jurkat and K562 cells were diluted in cell culture medium (RPMI 1640 GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 µg/ml Streptomycin) supplemented with 200 µg/ml gentamicin or 100 µg/ml gentamicin, respectively, and further incubated for 1 h. Cells were centrifuged (5 min at 500 x g) and washed twice with 1 x PBS. After the wash, the cells were further diluted to a confluency of 0.5 x 10 6 cells/ml and maintained in normal cell culture medium (RPMI 1640 GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 µg/ml Streptomycin) until further analysis. Measurement of injection kinetics into HeLa LgBiT, K562 LgBiT and Jurkat LgBiT cells For the kinetics measurements displayed in Fig. 2bc and Fig. 5 a, Hela LgBiT cells were seeded at 20,000 cells/well in RPMI medium without antibiotic supplementation into a black 96-well microtitration plate [BRAND (781668)] on the day prior to infections. Suspension cell lines, Jurkat LgBiT cells and K562 LgBiT cells, were seeded at 360,000 cells/well in Opti-MEM™ (31985070, Gibco), supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v), without antibiotic supplementation into a black 96-well microtitration plate [BRAND (781668)] on the day of infection. Bacteria day cultures were inoculated from stationary overnight cultures to an OD 600 of 0.12 in cultivation medium complemented with MgCl 2 (20 mM), glycerol (0.4% w/v), and CaCl 2 (5 mM). The cultures were cultivated shaking for 90 min at 28°C and then shifted to a 37°C and incubated for 120 min to induce T3SS formation. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with 0.2% L-arabinose (w/v). Bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v). For the infection, Yersinia cells were added to the eukaryotic cells at a multiplicity of infection (MOI) of 20. The enzymatic Nano-Glo® Luciferase Assay System [Promega (N1110)] was used according to manufacturer instructions. 30 µl of Nano-Glo® Luciferase Assay Reagent (substrate:buffer 1:50) was added to each sample. Bioluminescence was detected every 3 min in a microplate reader [Tecan Infinite 200 PRO], for 2 h at 37°C with an integration and settle time of 200 ms, each. The background signal was subtracted from the obtained values. Immunoblot analysis of monobody levels after translocation Monobody levels after translocation were assessed by taking samples directly after (0 h) and 24 h after gentamicin treatment. Total protein extraction was done in lysis buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), 5 mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% NP-40) supplemented with 50 mM NaF, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µl/ml Tosyl-L-phenylalaninchloromethylketon (TPCK) and protease inhibitors (cOmplete™, CO-RO, Roche) and cleared by centrifugation (20 min at 20,000 x g). Protein concentrations were determined using a Bradford assay (Bio-Rad). Equal amounts of proteins (50 µg cell lysate) were separated on a SDS-polyacrylamide electrophoresis (PAGE) gel and transferred to a nitrocellulose membrane (0.2 µm) by wet transfer. Membranes were blocked for 1 h at room temperature in blocking buffer (2.5% BSA and 2.5% milk powder in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20; pH7.6)). Subsequently, the membranes were probed with mouse anti-HiBiT (1:1000) diluted in blocking buffer at 4°C overnight. This was followed by incubation with secondary antibodies, IRDye®800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000 in TBS-T) and anti-beta tubulin-DyLight™ 680 (1:500 in TBS-T). Fluorescent detection was performed using the LI-COR Imaging system. Protein levels were quantified using the Empiria Studio® software and normalized to tubulin and control protein. 24 h sample was normalized to the 0 h sample of the respective protein. All blots were performed in three independent experiments. Unprocessed blots can be found in Supplementary Figs. 5 and 7. Quantitative immunoblotting for intracellular concentration Intracellular monobody concentrations in HeLa and K562 cells were determined with quantitative immunoblotting. Samples were taken after gentamicin treatment and total protein extraction was done as described above. Equal amounts of proteins (30 or 50 µg cell lysate) were loaded on a SDS-PAGE gel along with various amounts of purified HiBiT-Monobody protein. After transfer to nitrocellulose membrane, the membrane was probed with anti-HiBiT and anti-beta tubulin antibodies, as described above. Fluorescent detection was performed using the LI-COR Imaging system. Protein levels were quantified using the Empiria Studio® software. The purified protein samples were used to obtain a standard curve. Then, the protein amount in the cell lysate samples was determined using the standard curve. The final concentration in HeLa and K562 cells was calculated with the cell number used for the blot and a single cell volume of 4.2 pL 39 and 1.65 pL 58 , respectively. All blots were performed in three independent experiments. Unprocessed blots can be found in Supplementary Fig. 6. Intracellular stability of the monobody Eukaryotic cell culture was prepared as described above. Bacteria day cultures were inoculated from stationary overnight cultures to an OD 600 of 0.12 in cultivation medium complemented with MgCl 2 (20 mM), glycerol (0.4% w/v), and 200 µg/ml ampicillin. CaCl 2 (5 mM) was added for non-secreting conditions. The cultures were cultivated shaking for 90 min at 28°C and shifted to 37°C for 60 min to induce T3SS formation. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with L-arabinose (w/v) and cultures were incubated for another 60 min at 37°C. After that, bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v). Medium of the eukaryotic cell culture was changed to colorless RPMI, supplemented with DAP (60 µg/ml) and 0.2% L-arabinose (w/v). For the infection, Yersinia cells were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated at 37°C (non-shaking). After 2 h incubation, 150 µg/ml gentamicin was added prior to a further incubation for 1 h. Finally, the eukaryotic cells were washed with cell culture medium (RPMI) supplemented with 150 µg/ml gentamicin, 10% FCS (v/v) and endurazine (1:100), as stated in the manufacturer’s instructions [Promega Nano-Glo® Endurazine™ Live Cell Substrate (N2570)]. Bioluminescence was detected every 3 min in a microplate reader [Tecan Infinite 200 PRO], for 24 h and 37°C with an integration and settle time of 200 ms, each. The background signal was subtracted from the obtained values. NanoBiT Protein-Protein Interaction for determining monobody and target interaction A day prior to infection, HeLa cells stably expressing Abl-SH2-LgBit or Lck-SH2-LgBiT were seeded at 25,000 per well into a white 96 well LUMITRAC microplate (655074, Greiner Bio-One) in cell culture medium (DMEM GlutaMAX with 10% FBS) without antibiotics. Cells were infected and treated as described above. After gentamicin treatment, cells were washed and kept in 100 µl Opti-MEM (31985070, Gibco) supplemented with 10% FBS. Nano-Glo Live Cell Reagent (N2011, Promega) was prepared by combining 1 volume of Nano-Glo Live Cell Substrate with 19 volumes of Nano-Glo LCS Dilution buffer. 25 µl of Nano-Glo Live Cell Reagent was added to each well and the plate was gently mixed on an orbital shaker (30 seconds for 300 rpm). Luminescence was immediately measured on a SpectraMax M5 (Molecular Devices) with an exposure time of 500 ms. Phospho-STAT5 (pY694-STAT5) STAT5 phosphorylation (pY694) in K562 cells was assessed upon monobody translocation. Cells were infected and treated as described above. K562 cells continuously treated with 1 µM imatinib or 10 µM imatinib served as positive controls. Samples were taken 5 h and 24 h after the start of the infection or imatinib treatment. 1 x 10 6 cells were spun down (5 min at 500 x g) and resuspended in 1 x PBS. Then, the cells were fixed in 3.2% paraformaldehyde (PFA, E15710, Science Services) for 10 min at room temperature. After fixation, the cells were spun down (5 min at 300 x g) and stored in 95% ice-cold methanol at -20°C overnight. On the next day, the cells were washed with 1 x PBS, spun down (5 min at 400 x g), resuspended in 1 x PBS with 4% FBS (FACS buffer) and incubated at 4°C for 2 h. Cells were spun down (5 min at 500 x g) and resuspended in Human SeroBlock (1:20 in FACS buffer; BUF070A, Bio-Rad). After blocking for 15 min, the cells were stained with BD Phosflow™ Alexa Fluor® 647 Mouse Anti-Stat5 (pY694) (1:5 in FACS buffer; 612599, BD Biosciences) for 45 min on ice. Lastly, cells were spun down (5 min at 400 x g), resuspended in 1 x PBS and analyzed on a Guava easyCyte™ (Luminex) using the 642 nm laser and a 661/15 nm bandpass filter. Data was analyzed using FlowJo (v10). Gating strategy is shown in Supplementary Fig. 10. Analysis of STAT5 phosphorylation was done in three independent experiments. In each experiment, the mean fluorescence intensity (MFI) of untreated cells was set to 1 and the relative MFI of all samples was calculated. Apoptosis Assay (Activated Caspase 3/7 and Dead Cell Stain) CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (C10427, Molecular Probes) was used to study initiation of apoptosis in K562 upon monobody translocation. Cells were infected and treated as described above. K562 cells continuously treated with 1 µM imatinib or 10 µM imatinib served as positive controls. Samples were taken 24 h and 48 h after the start of the infection or imatinib treatment. 0.5 x 10 6 cells were centrifuged (5 min at 500 x g) and resuspended in 1 x PBS with 2% FBS. Then, the cells were stained with the CellEvent™ Caspase-3/7 Green Detection Reagent (1:1000) and incubated for 30 min at 37°C. Afterwards, cells were stained with SYTOX™ AADvanced™ Dead Cell Stain (1:1000) for 5 min at 37°C. After staining, samples were directly analyzed on a Guava easyCyte™ (Luminex) using the 488 nm laser and a 525/30 nm bandpass filter and 642 nm laser and a 695/50 nm bandpass filter. Single stained samples were used for compensation. Data was analyzed using FlowJo (v10). Gating strategy is shown in Supplementary Fig. 10. Induction of apoptosis was analyzed in three to five independent experiments. Quantification and statistical analysis Quantification and statistical analysis were performed using GraphPad Prism 10 and data are presented as mean ± standard deviation, as specified in the figure legends. Statistical analyses were performed with an ordinary one-way ANOVA followed by Šidák multiple comparisons tests. P values below 0.05 were considered statistically significant. Sample sizes ( n ) are provided in the respective figure legend. Asterisks represent statistical significance (ns denotes p > 0.05, * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001). Results Engineering of monobody variants with reduced stability We wanted to exploit the T3SS of Y. enterocolitica for the direct cytosolic delivery of monobodies that bind intracellular oncoproteins. Besides an N-terminal T3SS secretion signal 41 for recognition by the system, the translocation through the needle requires unfolding of the cargo protein (Fig. 1 a). Less stable proteins are more efficiently translocated 59 , whereas very stable proteins like GFP can block the needle 60 . Monobodies, including AS25, have high thermodynamic stability (Supplementary Fig. 1). Therefore, we attempted to engineer less stably folded monobody variants that would translocate more efficiently whilst retaining target binding. The D7K mutation in monobodies had been introduced to enhance its stability based on the identification of electrostatic repulsion involving Asp7 in the 10th FN3 domain (10FN3) of human fibronectin, the scaffolding domain for monobody engineering 61 , and thus we reverted it. Sequence comparison of 10FN3 with the 3rd FN3 domain (3FN3), as well structural modeling pointed us towards another mutation, A57G, that might decrease monobody stability without affecting target binding. AS25 with the A57G mutation was selected for further characterization, as much higher soluble expression of this variant in E. coli was observed than for AS25 with the K7D reversion (data not shown). Additionally, analysis of the co-crystal structure of AS25 with its target identified Y45 as a possible critical residue for Abl1 SH2 domain binding, and we therefore included a Y45A mutation as a negative control with possible decreased binding to Abl1 SH2 (Fig. 1 b, Supplementary Fig. 2). We assessed thermodynamic stability of the AS25 variants with a thermal shift assay (Fig. 1 c). While the wildtype AS25 monobody had a high melting temperature of ~ 74°C, the inclusion of the A57G mutation decreased the melting temperature by more than 13°C. The addition of the Y45A mutation only led to a minor decrease of further 4°C (Fig. 1 c). Next, to monitor effects of the mutations on target binding, we determined thermodynamic binding parameters of these monobody mutants using isothermal titration calorimetry (ITC) measurements (Fig. 1 d-f, Supplementary Fig. 3). All measurements suggested an Abl1 SH2:AS25 monobody binding stoichiometry of 1:1. The AS25 A57G variant showed no decreased binding affinity ( K d = 156 nM; Fig. 1 e) when compared to the wildtype AS25 ( K d = 180 nM; Fig. 1 d). In contrast, the AS25 Y45A − A57G variant resulted in a ~ 15-fold decreased binding affinity ( K d = 2690 nM, Fig. 1 f). Also, the binding enthalpy of AS25 Y45A − A57G was half of the binding enthalpy of the other variants (Supplementary Table 4). In summary, we engineered a destabilized variant of AS25 (AS25 A57G ) that retained binding affinity to its target, as well as a variant with a strongly reduced binding affinity (AS25 Y45A − A57G ), for testing secretion specificity and efficiency with the T3SS system. This low-affinity AS25 variant (AS25 Y45A − A57G ) will be used as negative control for all experiments and termed ‘non-binding’ for simplicity from here onwards. In vitro secretion of monobody variants using the Y. enterocolitica T3SS Using the N-terminal secretion signal of a native T3SS effector, YopE 1 − 138 , all three variants of AS25 were expressed by the bacteria and efficiently secreted in an in vitro secretion assay (Fig. 1 g-h). Notably, the destabilized variants allowed for a stronger concurrent secretion of SctA, a protein that is essential for the formation of the translocon in the host cell membrane (Fig. 1 g, left), indicating that indeed, the destabilized variants of the monobody prevented the blocking of the needle. Importantly, we verified that a strain lacking SctQ ( ΔsctQ ; non-secreting strain) showed expression (Fig. 1 h), but no detectable secretion of the monobody into the culture supernatant (Fig. 1 g, right). Translocation of monobodies into eukaryotic cells In order to monitor translocation of monobodies into eukaryotic cells in real-time, we employed a live cell split-NanoLuc luciferase system 62 – 64 . We used HeLa and Jurkat cell lines stably expressing the large domain of the NanoLuc luciferase (LgBiT) in the cytoplasm. Upon addition of bacteria expressing monobodies tagged with the HiBiT peptide, only successful cytoplasmic delivery would result in high affinity binding of HiBiT to LgBiT ( K d = 0.7 nM), leading to a reconstitution of a functional NanoLuc enzyme (Fig. 2 a). Starting measurements immediately after the addition of the bacteria, we observed a strong luciferase signal upon translocation of AS25 A57G and AS25 Y45A − A57G into HeLa cells, which increased over 120 min (Fig. 2 b). AS25, without destabilizing mutation, resulted in much weaker translocation. The secretion-deficient strain (Δ sctQ ) expressing AS25 A57G showed weak luminescence signal (Fig. 2 b). In Jurkat cells, also AS25 A57G was translocated strongest, while AS25 Y45A − A57G showed similar translocation kinetics as AS25 (Fig. 2 c). We next tested whether the monobodies are translocated into different human cell lines and analyzed monobody levels by immunoblotting after infection. Besides HeLa, we used human embryonic kidney (HEK293) as a second adherent cell line. In addition, we chose two hematopoietic, non-adherent cell lines: Jurkat, the most commonly used T lymphocyte cell line, and K562, the most commonly used cell line expressing BCR::ABL1. All cell lines were incubated with bacteria expressing AS25 A57G or AS25 Y45A − A57G for 2 h. A non-secreting bacterial strain (Δ sctQ ), expressing AS25 A57G , was used as a negative control. After incubation, the cells were treated with gentamicin to kill bacteria before preparing cellular extracts for immunoblotting (Fig. 2 d). To obtain a high translocation efficiency with non-adherent cell lines, we used higher cell densities of the target cells while maintaining a MOI of 100. Translocation of monobodies was achieved in all four cell lines and robustly detected by immunoblotting of a 29 kDa band, which is in line with the expected molecular weight of the monobody with secretion signal (Fig. 2 e-h). In the two adherent cell lines, HEK293 and HeLa, the non-binding AS25 variant (AS25 Y45A − A57G ) was detected at slightly higher concentrations than the binding counterpart (AS25 A57G ; Fig. 2 g-h). In contrast, in the two suspension cell lines, Jurkat and K562, less of the non-binding AS25 was detected (Fig. 2 e-f). Due to the higher abundance of BCR::ABL1 and Abl1 in K562 and Jurkat, respectively, it is possible that the binding of AS25 to its target leads to a longer half-life, whereas the non-binding AS25 could be less stable in these cells, leading to a lower abundance even though the efficiency of translocation is comparable. In the experiment using adherent cell lines, we observed some signal from cells incubated with a non-secreting strain ( ΔsctQ) . As this strain was clearly deficient in cytosolic delivery of monobodies (Fig. 1 g, 2 b-c), we suspected that this signal could stem from adherent bacteria that were not efficiently cleared by the gentamicin treatment. In contrast, the non-secreting strain only resulted in less than 10% of the signal in the suspension cell lines (Fig. 2 e-f). Cytosolic concentrations substantially higher than the binding affinity of the AS25 monobody ( K d = 156 nM, Fig. 1 e) are required for significant target inhibition. Therefore, we assessed the intracellular concentrations of monobodies that were translocated after monobody translocation by the T3SS of Y. enterocolitica in HeLa cells (Fig. 2 i). For absolute quantifications, we used serial dilutions of known amounts of recombinant AS25 monobody (without secretion signal) as a reference in immunoblots next to cell lysates after translocation. Taking the cell numbers and cell volume into account, we estimate the intracellular concentrations of the binding and non-binding AS25 in HeLa cells to be ~ 40 µM (Fig. 2 i). This indicated that sufficient amounts of monobodies were translocated to enable intracellular target binding and inhibition. Intracellular stability of monobodies after translocation To assess the duration of the inhibitory effect on the target protein, we next analyzed monobody half-life after translocation in HeLa and Jurkat cells (Fig. 3 ). To obtain first insights, we compared samples directly after gentamicin treatment (0 h) to samples taken 24 h later. We observed that initial monobody amounts decreased to ~ 10% of the initial amounts for both binding and non-binding variants in HeLa cells (Fig. 3 a). Also, in Jurkat cells, a strong decrease was observed (Fig. 3 b) but less pronounced for the binding AS25 as compared to its non-binding counterpart, in line with the observations from above (see Fig. 2 e). To study the degradation kinetics of monobodies after translocation in more detail, we translocated HiBiT-tagged monobodies into HeLa and Jurkat cells stably expressing LgBiT and then treated the cells with gentamicin, which allowed us to monitor the change of luminescence over time as a readout for monobody degradation. Luminescence intensity, corresponding to LgBit complementation by the HiBiT-tagged monobody, peaked after 2–4 hours and then gradually decreased in HeLa cells (Fig. 3 c). Wildtype AS25 showed both the slowest increase and the weakest decrease over time ( 60% degradation after 24 h, compared to the 2 h peak concentration). The Δ sctQ strain shows a very weak translocation (Fig. 3 c), supporting the interpretation that the signal obtained in the immunoblot analysis for this strain with the adherent cell lines (Fig. 2gh, 3a) is due to unspecific binding of non-lysed bacteria and not due to translocation. Translocation into Jurkat cells showed similar kinetics (Fig. 3 d), albeit with a stronger signal for the wild-type AS25 monobody. Target binding after monobody translocation We so far detected the cytosolic delivery of AS25 monobody variants in a split-NanoLuc assay and by immunoblotting. The translocated monobodies were detectable for several hours. Still, although monobodies and their parental 10FN3 are known to rapidly and reversibly refold 61 these experiments do not allow to conclude if the monobodies refold after translocation and are thus functional binders. Thus, we next set up an assay to measure the monobody-target interaction in the cytoplasm. For this purpose, we employed HeLa cell lines stably expressing LgBiT fused to the Abl1 SH2 domain (AS25 monobody target) or the Lck SH2 domain (negative control) and delivered monobodies tagged with SmBiT using T3SS. As the binding affinity of the LgBiT-SmBiT interaction is in the high micromolar range ( K d = 190 µM), only the interaction of monobody and target will result in reconstituted, functional luciferase 65 . Therefore, this system can be used to assess translocation of functional refolded monobody and target engagement in living cells (Fig. 4 a). Similar to the untagged variants of the monobody (Fig. 1 g), AS25-SmBiT was secreted in an in vitro secretion assay, with an increased secretion of monobody and translocator SctA for the destabilized AS25 A57G variants (Fig. 4 b, left). Again, we verified that all monobody variants are expressed (Fig. 4 c), while the strain lacking SctQ ( ΔsctQ ) showed no detectable secretion into the culture supernatant (Fig. 4 b, right). AS25 A57G -SmBiT resulted in a very strong increase in luminescence showing interaction of AS25 with the Abl1 SH2 domain (Fig. 4 d). The high specificity of this interaction was underlined by the low signal of the non-binding AS25, as well as the non-secreting strain. The difference between the wildtype AS25 and the destabilized AS25 A57G again shows the significantly enhanced translocation of the destabilized variant. Importantly, in the control cell line expressing LgBiT fused to the Lck-SH2 domain, we could only detect background signal for all AS25 variants/strains, underlining the high selectivity of AS25 and the highly specific readout of this assay system (Fig. 4 d). In summary, these results show refolding and specific target interaction of AS25 after translocation into HeLa cells. Monobody translocation leads to inhibition of BCR::ABL1 signaling We next assessed if the translocated monobodies exert an inhibitory effect on target signaling. Therefore, we chose the BCR::ABL1-expressing cell line K562, which is dependent on active BCR::ABL1 signaling for growth and survival. We first determined AS25 translocation and degradation kinetics, as well as the intracellular monobody concentration (Fig. 5 a-c). As for HeLa cells (see Fig. 2 b), both AS25 A57G and AS25 Y45A − A57G showed efficient translocation into K562 cells, which plateaued after ~ 75 minutes, while the wildtype AS25 showed slower kinetics (Fig. 5 a). The intracellular monobody stability was similar to the other cell lines (Fig. 5 b, see Fig. 3 c-d for comparison). The intracellular concentration of the binding AS25 A57G was ~ 30 µM, which surpasses the binding affinity of AS25-Abl SH2 interaction by far (see Fig. 1 e), while the non-binding AS25 Y45A − A57G accumulated to around half the concentration (~ 13 µM; Fig. 5 c). We then studied the functional consequences of AS25 monobody binding on BCR::ABL1 signaling in K562 cells after translocation into the cytoplasm. Inhibition of BCR::ABL1 kinase activity results in inhibition of STAT5 phosphorylation on Tyr-694 (pY-694), a critical BCR::ABL1 substrate and signaling mediator 66 , which can be conveniently and reliably measured by intracellular FACS staining. We observed a strong reduction of STAT5 phosphorylation 5 h after translocation of AS25 A57G (Fig. 5 d). A similar degree of reduction was obtained by treating the cells with the BCR::ABL1 tyrosine kinase inhibiting drug imatinib. In contrast, no reduction was observed with the non-binding variant or a non-secreting strain ( ΔsctQ ). Also, translocation of an unrelated destabilized monobody targeting the SHP1 tyrosine phosphatase (MbC A57G ) showed no reduced STAT5 phosphorylation (Fig. 5 d). These results show that the observed inhibition of BCR::ABL1 signaling by AS25 A57G was dependent on functional translocation and monobody-target binding. In line with the monobody degradation kinetics, the inhibitory effect of AS25 A57G after 24 h was weaker as compared to 5 h, but still significant in comparison to the negative controls (Fig. 5 e). Induction of apoptosis after monobody translocation Finally, we wanted to monitor if the pronounced inhibition of STAT5 phosphorylation in K562 cells resulted in apoptosis induction and cell death as monitored by caspase 3/7 activation and staining for dead cells, respectively, using FACS. The translocation of AS25 A57G led to a strong increase in apoptotic cells after 24 h (Fig. 6 a and c). After 48 h, the effect was still significant, albeit weaker, possibly due to degradation of the translocated monobody and continued cell proliferation (Fig. 6 b and c). As expected, imatinib treatment showed a more pronounced effect after 48 h than after 24 h. The non-binding AS25 variant and the unrelated monobody (MbC A57G ) did not result in a significant increase of apoptotic cells (Fig. 6 a-c), showing that induction of apoptosis was a direct consequence of target binding. Infection with the non-secreting strain (Δ sctQ ) expressing AS25 A57G led to a weaker, but detectable response at 24 h, but not at 48 h (Fig. 7a-c). As the strain is deficient in monobody delivery (see Fig. 1 g, 2 b-c, 3 d and 4 b) and did not affect BCR::ABL1 signaling (Fig. 5 d-e), we suspect this to be an unspecific effect at the earlier timepoint of this particular strain. In summary, delivery of binding AS25 into K562 cells resulted in selective inhibition of BCR::ABL1 signaling and led to induction of apoptosis in CML cells. Discussion We demonstrated that the T3SS of an avirulent Yersinia enterocolitica strain can be re-engineered to serve as a versatile and highly efficient system for protein delivery of a functional BCR::ABL1-targeting monobody. The delivered monobodies are able to engage and inhibit their target in cells, which results in perturbation of BCR:ABL1 signaling. We further demonstrate that this selective inhibition after delivery leads to induction of apoptosis in BCR:ABL1-dependent cells. To improve translocation efficiency, we created a destabilized monobody variant by mutating Ala-57 that faces the hydrophobic core of the monobody. As this position is not used for making a combinatorial library and is located on the opposite side relative to the intended target binding interfaces 67 , we believe to have established a general strategy that can be adopted for efficient delivery of any monobody. This view is supported by the efficient delivery of the A57G mutant SHP1 SH2-targeting monobody MbC (see Supplementary Fig. 9). Using the T3SS of Y. enterocolitica for the delivery of monobody proteins has several positive features: Firstly, we observed that T3SS-mediated delivery of the destabilized cargo into different cell lines resulted in high cytosolic concentrations in the mid-micromolar concentration range already shortly after initiation of delivery (Fig. 2 i, 5 c). Such concentrations exceed the binding affinities of monobody-target interaction by > 10-fold and hence enable efficient target inhibition in cells. Additionally, high concentrations of translocated cargo are desirable as this lowers the dosage required to elicit a functional effect. So far, only few studies have determined the amount of translocated cargo. In HeLa cells, we were able to translocate around 10 8 molecules per cell, which amounts to a concentration of 40 µM. Previous studies using the T3SS of Salmonella Typhimurium SPI-1 or Escherichia coli to translocate binding proteins into HeLa showed concentrations around 200 nM 39 or around 10 5 -10 6 translocated molecules per cell 43 . Delivery of AS25 with the T3SS of Y. enterocolitica , therefore, appears to be at least 100-fold more efficient. Secondly, delivery to the cytoplasm of target cells is ensured given the direct injection of the cargo through the plasma membrane without the need to cross other membranes or requirement for specific receptors. In contrast, other protein delivery approaches depend on specific receptors and endocytic uptake pathways 33 , 68 . T3SS-mediated delivery circumvents the challenge to enable escape from the endo-/lysosomal compartments, which can result in entrapment of cargo and subsequent degradation 29 , 34 , 36 , 69 . Thirdly, the intrinsic ability of Yersinia to target tumors 51 – 53 makes it an ideal candidate for monobody delivery and inhibition of oncogenic signalling. Furthermore, this system can be engineered to specifically target cancer cells 70 or to respond to external stimuli – an engineered version of the Y. enterocolitica T3SS incorporating an optogenetic switch can be activated by illumination with high temporal and spatial resolution 64 . Controlled delivery combined with high intracellular concentrations of translocated monobody would thus further lower the toxicity of bacterial application and safer for future clinical application. On the other hand, T3SS-mediated monobody delivery has also limitations. One restraint is the limitation to genetically encoded cargos: Approaches to enhance stability and half-life using in vitro synthesized mirror-image monobodies composed of D-amino acids (Hantschel lab, unpublished observations) cannot be combined with the T3SS. Similarly, cargo that is labeled with small-molecule fluorescent dyes cannot be easily translocated by the T3SS to follow translocation kinetics and intracellular fate. The use of self-labeling tags, e.g. Halo, SNAP or CLIP, may circumvent this problem 48 , but requires additional experimental steps and may perturb translocation kinetics 71 , whereas the split-NanoLuc luciferase employed by us only required minor modifications of the cargo. Our kinetics experiments showed a fast injection of monobodies, with peak concentrations 1–4 h after addition of the bacteria, followed by a decrease to 25–50% 24 h after T3SS-mediated delivery (see Fig. 2 ). It is important to understand the pathways that led to this reduction in addition to dilution effects due to cell growth and division. Proteasomal degradation via K48-polyubiquitination is a possible candidate and removal of Lys residues in the monobody sequence and/or the N-terminal secretion signal could therefore result in increased intracellular half-life. Another important factor influencing cytosolic monobody stability appears to be target binding. We observed faster kinetics and higher levels of binding AS25 when compared to its non-binding counterpart in Jurkat and K562 cells that have high expression of the AS25 target proteins ABL1 and BCR::ABL1. This was despite very similar in vitro secretion of the two AS25 variants. A possible explanation could be that target binding may stabilize monobody levels, whereas non-binding monobodies or cells that express only low levels of target result in faster turn-over and degradation. This is in line with a previous study showing that the expression of the target in the cells led to a higher accumulation of the translocated binder 43 . Notably, both translocation and degradation kinetics varied between cell lines, indicating potential variations in refolding and proteolysis kinetics or different amounts of stabilizing target proteins. A related question concerns the observation why the relatively mild (~ 15-fold) reduction in target binding affinity in vitro caused by the Y45A mutation in AS25 (Fig. 1 f) was sufficient to abolish cytosolic target engagement (Fig. 4 d), to prevent inhibition of STAT5 phosphorylation (Fig. 5 d-e) and to abolish induction of apoptosis (Fig. 6 c). As the mechanism of inhibition of AS25 requires competition with the intramolecular SH2-kinase domain interface of BCR::ABL1 15 , a higher AS25 concentration may be required than what would be predicted from the binding affinity to the isolated ABL1 SH2 domain, as previously observed 13 . Hence, even a mild mutation, such as Y45A, can result in a loss-of-function in cells. Future applications of T3SS-mediated monobody delivery may include targeted protein degradation approaches, such as AdPROMs or bioPROTACs. 72 , 73 For these approaches, monobodies were fused to different E3 ubiquitin ligases to induce the degradation of monobody target protein after transfection or viral transduction in cancer cell lines. While not unique to T3SS-mediated delivery, there is a high flexibility in terms of delivered cargo. For example, subcellular targeting moieties can be fused to delivered monobodies to enable e.g. nuclear or membrane localization 42 . Also, two monobodies targeting different domains in a target protein can be delivered as a tandem fusion to enhance target selectivity and efficacy of inhibition, as previously demonstrated 15 . Alternatively, one could also envision tandem fusion monobodies with specificity for two different targets, which can induce de novo protein-protein interactions. Still, all these approaches may need further optimization as the larger size of the cargo might decrease translocation efficiency. The delivery of immunomodulating proteins to cancer cells and the tumour microenvironment by the Y. enterocolitica T3SS is currently evaluated in a clinical trial 54 and hence indicates a clear path to clinical translation. While type I interferons and certain natural pro-apoptotic proteins have been delivered before 52 , our work provides the groundwork for the delivery selective protein-based signaling inhibitors to cancer cells by the Y. enterocolitica T3SS, which resulted in inhibition of a central oncogene, its downstream pathways and induction of apoptosis. For the future in vivo applications of T3SS-mediated delivery, enhancement of anti-tumor immunity by the immunogenic properties of most bacteria can be advantageous and is realized by different biotech start-up companies 74 . On the other hand, a fine balance needs to be struck to prevent an overactivation of the immune system resulting in acute inflammation and cytokine storm. Some of these limitations may be addressed by bioengineering, which is increasingly used to improve the characteristics of bacteria as drug delivery vehicles, including improvements of their safety profiles, and modification of their immunogenicity and targeting specificity 75 , 76 . Recent advances for the specific use of the T3SS for drug delivery include the engineering of carrier bacteria for lower immunogenicity 77 , 78 , the modification of the T3SS for further increased translocation speed 79 , transfer or even synthetic de novo assembly of the T3SS in selected carrier bacteria 80 – 82 . In addition, the development of a light-controlled T3SS using optogenetic switches allows for control of protein delivery with high temporal and spatial precision 64 . Conclusion Overall, we showed that the T3SS of Y. enterocolitica can serve as an efficient system for the delivery of monobody proteins to cancer cells, which resulted in oncogene-dependent perturbation of signaling and cell proliferation. This delivery approach holds great promise for future therapeutic use without the need to genetically manipulate target cells. Abbreviations 10FN3: 10 th FN3 domain 3FN3: 3 rd FN3 domain Abl: Abelson tyrosine kinase BCR: breakpoint cluster region CML: chronic myeloid leukemia CPP: cell-penetrating peptides FACS: fluorescence activated cell sorting HEK293: human embryonic kidney ITC: isothermal titration calorimetry LgBiT: large domain of the NanoLuc luciferase Mb: Monobodies MOI: multiplicity of infection SH2: Src Homology 2 STAT5: signal transducer and activator of transcription 5 T3SS: Type III secretion system T m : melting temperature TSA: thermal shift assay Declarations Authors information Chiara Lebon and Sebastian Grossmann contributed equally to this work. Authors and Affiliations Institute of Physiological Chemistry, Faculty of Medicine, Philipps-University of Marburg, 35043 Marburg, Germany Chiara Lebon & Oliver Hantschel Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany Sebastian Grossmann, Florian Lindner & Andreas Diepold Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne, 1015 Lausanne, Switzerland Greg Mann Department of Medicine, New York University School of Medicine, Laura and Isaac Perlmutter Cancer Center, New York University Langone Health, Department of Biochemistry and Molecular Pharmacology, New York, NY 10016, USA Akiko Koide & Shohei Koide Contributions CL and SG planned, conducted and analyzed most experiments. GM performed work on engineering and characterization of destabilized monobodies. FL conducted and analyzed initial experiments. AK and SK provided critical advice on the project and design of destabilized monobodies. AD and OH designed and coordinated the study, planned the experiments and analyzed data. AD, OH, CL and SG wrote the manuscript. All authors edited the manuscript. Corresponding authors Correpondence to Oliver Hantschel or Andreas Diepold Competing Interests AK and SK are listed as inventors on issued and pending patents on the monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications). SK is a co-founder, receives consulting fees and hold equity in Aethon Therapeutics; is a co-founder and holds equity in Revalia Bio; has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health, all outside of the current work. AD and FL are listed as inventors on an issued patent on the light-controlled T3SS filed by Max Planck Innovation (WO/2020/201115). The other authors declare no competing interests. Electronic supplementary material Supplementary material includes ten figures and eight tables. Funding We acknowledge support by the European Research Council (Consolidator Grant; ERC-2016-CoG 682311) to O.H., C.L. and G.M., and by the Max Planck Society to A.D., S.G. and F.L. Author Contribution CL and SG planned, conducted and analyzed most experiments. GM performed work on engineering and characterization of destabilized monobodies. FL conducted and analyzed initial experiments. AK and SK provided critical advice on the project and design of destabilized monobodies. AD and OH designed and coordinated the study, planned the experiments and analyzed data. AD, OH, CL and SG wrote the manuscript. All authors edited the manuscript. Acknowledgement We thank all members of the Diepold and Hantschel labs for input and discussions. 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ACS Synth Biol. 2015;4:644–54. 10.1021/acssynbio.5b00002 . Song M, et al. Control of type III protein secretion using a minimal genetic system. Nat Commun. 2017;8:14737. 10.1038/ncomms14737 . Hansen-Wester I, Chakravortty D, Hensel M. Functional Transfer of Salmonella Pathogenicity Island 2 to Salmonella bongori and Escherichia coli. Infect Immun. 2004;72:2879–88. 10.1128/iai.72.5.2879-2888.2004 . Additional Declarations Competing interest reported. AK and SK are listed as inventors on issued and pending patents on the monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications). SK is a co-founder, receives consulting fees and hold equity in Aethon Therapeutics; is a co-founder and holds equity in Revalia Bio; has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health, all outside of the current work. AD and FL are listed as inventors on an issued patent on the light-controlled T3SS filed by Max Planck Innovation (WO/2020/201115). The other authors declare no competing interests. Supplementary Files SIwuncroppedgelsIBs.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Oct, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 15 Aug, 2024 Reviews received at journal 19 Jul, 2024 Reviewers agreed at journal 15 Jul, 2024 Reviewers agreed at journal 14 Jul, 2024 Reviewers invited by journal 13 Jul, 2024 Editor assigned by journal 10 Jul, 2024 Submission checks completed at journal 10 Jul, 2024 First submitted to journal 08 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4705983","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332145919,"identity":"a8d04380-d62c-40d2-a54d-f1fda61b1c7f","order_by":0,"name":"Chiara Lebon","email":"","orcid":"","institution":"Philipps University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Lebon","suffix":""},{"id":332145920,"identity":"8c325fe2-7633-4eae-9950-0df04e40063c","order_by":1,"name":"Sebastian Grossmann","email":"","orcid":"","institution":"Max Planck Institute for Terrestrial Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Grossmann","suffix":""},{"id":332145921,"identity":"0f487ff7-82bf-4d08-89dd-6183fab24a71","order_by":2,"name":"Greg Mann","email":"","orcid":"","institution":"École Polytechnique Fédérale de Lausanne","correspondingAuthor":false,"prefix":"","firstName":"Greg","middleName":"","lastName":"Mann","suffix":""},{"id":332145922,"identity":"218bd7d3-4569-4423-9d38-8fad32c7389c","order_by":3,"name":"Florian Lindner","email":"","orcid":"","institution":"Max Planck Institute for Terrestrial Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Lindner","suffix":""},{"id":332145923,"identity":"be74329e-bb73-4d03-bc82-79aea222d558","order_by":4,"name":"Akiko Koide","email":"","orcid":"","institution":"New York University","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Koide","suffix":""},{"id":332145924,"identity":"6ba8d647-f591-4a3d-9c61-8260a3bca157","order_by":5,"name":"Shohei Koide","email":"","orcid":"","institution":"New York University Langone Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Shohei","middleName":"","lastName":"Koide","suffix":""},{"id":332145925,"identity":"e497e05b-4768-4801-839b-6ad582c65f97","order_by":6,"name":"Andreas Diepold","email":"","orcid":"","institution":"Max Planck Institute for Terrestrial Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Diepold","suffix":""},{"id":332145926,"identity":"860adf38-cc04-48ed-a126-942c916800aa","order_by":7,"name":"Oliver Hantschel","email":"data:image/png;base64,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","orcid":"","institution":"Philipps University of Marburg","correspondingAuthor":true,"prefix":"","firstName":"Oliver","middleName":"","lastName":"Hantschel","suffix":""}],"badges":[],"createdAt":"2024-07-08 13:40:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4705983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4705983/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-024-01874-6","type":"published","date":"2024-10-16T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61769226,"identity":"62d27d2d-9679-483c-8e64-75107f3672ce","added_by":"auto","created_at":"2024-08-05 11:03:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":390835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineering and characterization of destabilized monobody variants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic overview of monobody delivery by the T3SS. Monobodies expressed with a secretion signal are translocated through the T3SS needle, which requires unfolding. After refolding, the delivered monobody can interact with the target protein in the cytoplasm and affect target activity and signaling. \u003cstrong\u003eb \u003c/strong\u003eCartoon representation of monobody AS25 (yellow):Abl-SH2 domain (grey) complex (PDB: 5DC4). Diversified residues of monobody scaffold are shown in green. Y45 (blue), a key residue for target binding was mutated to alanine to obtain a low affinity variant. For the destabilization, the A57 residue (red) was mutated to glycine. \u003cstrong\u003ec \u003c/strong\u003eThermodynamic stability of the AS25 variants assessed by thermal shift assay. Derivative fluorescence of one representative was plotted over temperature. Melting temperatures of triplicates were averaged and are shown as mean\u0026nbsp;±\u0026nbsp;SD. \u003cstrong\u003ed-f\u003c/strong\u003e Isothermal calorimetric titration (ITC) of AS25 (panel d), AS25\u003csub\u003eA57G\u003c/sub\u003e (panel e)\u003csub\u003e \u003c/sub\u003eand AS25\u003csub\u003eY45A-A57G\u003c/sub\u003e (panel f) to Abl-SH2. Upper panels: Raw heat signal; lower panels: Integrated calorimetric data of the area for each peak. The continuous line represents the best fit of the data and the binding parameters \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e and stoichiometry (N) are calculated from the fit. A representative measurement (n=2) for each monobody is shown. Thermodynamic parameters are listed in Supplementary Tables 2-3. \u003cstrong\u003eg \u003c/strong\u003e\u003cem\u003eIn vitro\u003c/em\u003e secretion assay (n=3) showing export of YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT variants and native T3SS substrates (the translocator proteins SctA and SctB contribute to formation of a pore in the eukaryotic membrane and the regulatory protein SctW) by \u003cem\u003eY.\u0026nbsp;enterocolitica\u003c/em\u003e. Proteins secreted over 180 min were precipitated and analyzed by SDS-PAGE. Left, Coomassie staining (native substrates indicated on right side); right, Western blot anti-FLAG. Expected size: YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT: 28.7 kDa (marked with *). \u003cstrong\u003eh \u003c/strong\u003eImmunoblot analysis of YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT expression levels in the indicated strains used in panel g.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/87ed8e58280aa055e4aadef3.png"},{"id":61769228,"identity":"ca355199-89b2-4516-9bfd-c43b3dff1eea","added_by":"auto","created_at":"2024-08-05 11:03:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslocation of AS25 monobodies into eukaryotic cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic representation of the live cell split-NanoLuc system for the detection of monobody translocation. Monobodies with secretion signal and HiBiT peptide are expressed in \u003cem\u003eY.\u0026nbsp;enterocolitica\u003c/em\u003e. The large domain of the Nano-Luc luciferase (LgBiT) is stably expressed in\u003c/p\u003e\n\u003cp\u003ehost cells. Upon infection, translocation of monobody-HiBiT leads to complementation and reconstitution of a functional Nano-Luc enzyme, which can be read out by measuring luminescence. \u003cstrong\u003eb-c\u003c/strong\u003e Luminescence signal of YopE\u003csub\u003e1-138\u003c/sub\u003e-Monobody-FLAG-HiBiT variants translocated into LgBiT-expressing HeLa (b) or Jurkat (c) cells\u003cem\u003e.\u003c/em\u003e The secretion deficient ∆\u003cem\u003esctQ\u003c/em\u003e mutant and empty plasmid (pBAD) served as negative controls. At time point zero, HeLa or Jurkat cells were infected with \u003cem\u003eY. enterocolitica \u003c/em\u003eand incubated in the presence of NanoLuc substrate Furimazine. Luminescence was followed in 3\u0026nbsp;min intervals over 2\u0026nbsp;h. Error area represents mean ± SD of three independent measurements (n = 3). \u003cstrong\u003ed\u003c/strong\u003e Experimental outline for testing monobody translocation into eukaryotic host cells. Bacteria expressing HiBiT-tagged monobodies are added to eukaryotic cells at MOI of 100. After a 2-hour incubation, cells are treated with gentamicin and washed with PBS. Cell lysates are analyzed by immunoblotting to assess translocated monobody levels. \u003cstrong\u003ee-h \u003c/strong\u003eImmunoblot analysis of monobody-HiBiT levels in Jurkat (e), K562 (f), HEK293 (g) and HeLa (h) after infection with the indicated bacterial strains. Quantification of monobody-HiBiT levels, normalized to tubulin and AS25\u003csub\u003eA57G\u003c/sub\u003e, from three independent experiments are shown below and plotted as mean ± SD (n = 3). \u003cstrong\u003ei\u003c/strong\u003e Immunoblot analysis to determine intracellular monobody concentrations in HeLa cells after infection with indicated bacterial strains. Total cell lysates and serial dilutions of recombinant monobody (without secretion signal) were analyzed. Intracellular concentration was calculated based on cell number and cell volume from three independent experiments (n = 3) and is denoted as mean ± SD.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/80a95e8175abf39d522da843.png"},{"id":61769231,"identity":"3c62b092-ff13-41d0-92c5-df60e50739c6","added_by":"auto","created_at":"2024-08-05 11:03:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular stability of translocated AS25 monobodies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b\u003c/strong\u003e Immunoblot analysis of monobody-HiBiT levels in HeLa (panel a) and Jurkat (panel b) cells at 0 h and 24 h after infection with the indicated bacterial strains and gentamicin treatment. Quantification of monobody-HiBiT levels, normalized to tubulin and respective protein, from three independent experiments (n = 3) are shown below and plotted as mean\u0026nbsp;±\u0026nbsp;SD. \u003cstrong\u003ec-d\u003c/strong\u003e Degradation kinetics of YopE\u003csub\u003e1-138\u003c/sub\u003e-Monobody-FLAG-HiBiT variants in LgBiT-expressing HeLa (panel c) or Jurkat (panel d) cells after delivery via the \u003cem\u003eY.\u0026nbsp;enterocolitica\u003c/em\u003e T3SS\u003cem\u003e.\u003c/em\u003e The secretion deficient ∆\u003cem\u003esctQ\u003c/em\u003e mutant served as negative control. HeLa or Jurkat cells were infected with monobody-expressing \u003cem\u003eY. enterocolitica \u003c/em\u003eand incubated at 37°C (w/o CO\u003csub\u003e2\u003c/sub\u003e) for 2 h. After gentamicin treatment, cell culture medium was changed and the long-lasting NanoLuc substrate Endurazine was added. From this point (T=0), the luminescence signal was followed in 3\u0026nbsp;min intervals over 24\u0026nbsp;h. Error area represents mean ± SD of three independent measurements (n\u0026nbsp;=\u0026nbsp;3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/9f2014954fa0bcf0e1a64624.png"},{"id":61769230,"identity":"c0e33e01-6087-4d70-b894-ee50bce35165","added_by":"auto","created_at":"2024-08-05 11:03:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":305071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular target engagement of translocated AS25 monobodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic representation of a live cell protein-protein interaction assay with a split-NanoLuc system. Monobodies with secretion signal and SmBiT-peptide are expressed in \u003cem\u003eY.\u0026nbsp;enterocolitica\u003c/em\u003e. The large domain of the Nano-Luciferase (LgBiT) fused to the target protein is stably expressed in the cytosol of eukaryotic cells. Upon infection, the translocation of monobody-SmBiT and interaction of the monobody with its target brings the SmBiT-peptide and the LgBiT domain in close proximity. This leads to complementation and reconstitution of a functional Nano-Luc enzyme, which can be read out by measuring luminescence. \u003cstrong\u003eb \u003c/strong\u003e\u003cem\u003eIn vitro\u003c/em\u003e secretion assay (n = 3) showing export of YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT monobody variants and indicated native T3SS substrates by \u003cem\u003eY.\u0026nbsp;enterocolitica\u003c/em\u003e. Proteins secreted over 180 min were precipitated and analyzed by SDS-PAGE. The secretion deficient strain Δ\u003cem\u003esctQ\u003c/em\u003e was used as control. Left, Coomassie staining of all exported proteins; right, Western blot anti-FLAG. Molecular weight indicated in kDa, expected size of YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT (Mb): 28.7 kDa (marked with *). \u003cstrong\u003ec \u003c/strong\u003eExpression levels of YopE\u003csub\u003e1-138\u003c/sub\u003e-AS25-FLAG-HiBiT in the indicated strains used in panel b. Western blot anti-FLAG for cellular proteins. \u003cstrong\u003ed\u003c/strong\u003e Luminescence measurement of HeLa cells expressing either Abl-SH2-LgBiT (left) or Lck-SH2-LgBiT (right) after infection with the indicated bacterial strains secreting the indicated monobody variants and gentamicin treatment. Results from three independent experiments (n\u0026nbsp;=\u0026nbsp;3) performed in triplicates are shown and presented as mean ±\u0026nbsp;SD. Ordinary one-way ANOVA followed with Šidák multiple comparisons tests was performed for the Abl-LgBiT samples against the untreated sample. Additional comparisons were made between AS25 and AS25\u003csub\u003eA57G \u003c/sub\u003eand AS25\u003csub\u003eA57G\u003c/sub\u003e and AS25\u003csub\u003eY45A-A57G\u003c/sub\u003e. \u003cem\u003eP \u003c/em\u003evalues below 0.05 were considered statistically significant and asterisks represent statistical significance (\u003csup\u003e*\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003csup\u003e**\u003c/sup\u003e\u0026nbsp;denotes \u003cem\u003ep\u003c/em\u003e ≤ 0.01, \u003csup\u003e***\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e ≤ 0.001). Only significant results are denoted.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/9d8c87fb8feca015be95685d.png"},{"id":61769725,"identity":"3874852f-fd4b-459c-8cad-ef11d55abc69","added_by":"auto","created_at":"2024-08-05 11:11:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":270740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of BCR::ABL1 signaling in CML cells after AS25 translocation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eLuminescence signal of YopE\u003csub\u003e1-138\u003c/sub\u003e-Monobody-FLAG-HiBiT variants translocated into LgBiT-expressing K562 cells\u003cem\u003e.\u003c/em\u003e At time point zero, K562 cells were infected with indicated strains\u003cem\u003e \u003c/em\u003eand incubated with NanoLuc substrate Furimazine. Luminescence was followed in 3\u0026nbsp;min intervals over 2\u0026nbsp;h. Error area represent mean ± SD of three independent measurements (n = 3). \u003cstrong\u003eb \u003c/strong\u003eDegradation kinetics of YopE\u003csub\u003e1-138\u003c/sub\u003e-Monobody-FLAG-HiBiT variants in LgBiT-expressing K562 cells after delivery. K562 cells were infected with indicated strains\u003cem\u003e \u003c/em\u003eand incubated for 2h. After gentamicin treatment, the long lasting NanoLuc substrate Endurazine was added. Luminescence signal was followed in 3\u0026nbsp;min intervals over 24\u0026nbsp;h. Error area represents mean ± SD of three independent measurements (n = \u003cem\u003e3\u003c/em\u003e). \u003cstrong\u003ec\u003c/strong\u003e Immunoblot analysis to determine intracellular monobody concentrations in K562 cells after infection with the indicated strains. Cell lysates and serial dilutions of recombinant monobody (without secretion signal) were analyzed. Intracellular monobody concentration was calculated based on cell number and cell volume from three independent experiments (n = 3) and is indicated below as mean ± SD. \u003cstrong\u003ed-e\u003c/strong\u003e Flow cytometric analysis of STAT5 phosphorylation (pY694) in K562 cells 5 h (d) and 24\u0026nbsp;h (e) after infection with indicated strains or treatment with BCR::ABL1 inhibitor imatinib. Left panel: Signal intensities (MFI) of cells stained with anti-phospho-STAT5 antibody. Right panel: Quantification of pSTAT5 levels (relative MFI), normalized to untreated, from three independent experiments (n\u0026nbsp;= 3) plotted as mean ± SD. Ordinary one-way ANOVA followed by Šidák multiple comparisons tests was performed by comparing against the untreated sample. Additional comparison was made between AS25\u003csub\u003eA57G\u003c/sub\u003e and AS25\u003csub\u003eY45A-A57G\u003c/sub\u003e. \u003cem\u003eP \u003c/em\u003evalues below 0.05 were considered statistically significant and asterisks represent statistical significance (\u003csup\u003e*\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e\u0026nbsp;≤\u0026nbsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u0026nbsp;denotes \u003cem\u003ep\u003c/em\u003e\u0026nbsp;≤\u0026nbsp;0.01, \u003csup\u003e***\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e ≤ 0.001). Only significant results are denoted.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/e8de627eb456a0941ce788fd.png"},{"id":61769229,"identity":"74649e09-e26d-49d2-9c91-0bdea332f813","added_by":"auto","created_at":"2024-08-05 11:03:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":248746,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptosis induction in CML cells after AS25 translocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b \u003c/strong\u003eFlow cytometric analysis of apoptosis in K562 cells 24 h (panel a) and\u003cstrong\u003e \u003c/strong\u003e48\u0026nbsp;h (panel b) after infection with the indicated bacterial strains or treatment with the BCR::ABL1 inhibitor imatinib at indicated concentrations. Representative dot plots depict cells stained for activated Caspase 3/7 and stained with a dead cell stain. \u003cstrong\u003ec \u003c/strong\u003ePercentage of apoptotic K562 cells after 24 h (left panel) and 48 h (right panel) after infection/treatment. The percentage of apoptotic cells was defined as the sum of activated Caspase 3/7 positive cells (Q3) and both activated Caspase3/7 and dead cell stain (double) positive cells (Q2). Data from at least three independent experiments (n = 3-5) are shown and depicted as mean ± SD. Ordinary one-way ANOVA followed by Šidák multiple comparisons tests was performed by comparing against the untreated sample. Additional comparison was made between AS25\u003csub\u003eA57G\u003c/sub\u003e and AS25\u003csub\u003eY45A-A57G\u003c/sub\u003e. \u003cem\u003eP \u003c/em\u003evalues below 0.05 were considered statistically significant and asterisks represent statistical significance (\u003csup\u003e*\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e\u0026nbsp;≤\u0026nbsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u0026nbsp;denotes \u003cem\u003ep\u003c/em\u003e\u0026nbsp;≤\u0026nbsp;0.01, \u003csup\u003e***\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e ≤ 0.001). Only significant results are denoted. F values and degrees of freedom for 24 h analysis were 30.82 and 25, respectively. F values and degrees of freedom for 48 h analysis were 8.479 and 30, respectively.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/1607f7030204770961a0b317.png"},{"id":67149542,"identity":"2850007c-b1f5-458c-a4b1-ed1b8d1b486b","added_by":"auto","created_at":"2024-10-21 16:13:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2499812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/7dd87254-a3a9-4499-9905-cd902be3aaa3.pdf"},{"id":61769232,"identity":"ac5019bd-dc5b-40e1-83b4-b347aed4948a","added_by":"auto","created_at":"2024-08-05 11:03:16","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2619162,"visible":true,"origin":"","legend":"","description":"","filename":"SIwuncroppedgelsIBs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4705983/v1/e747cb8c91188aae8fab8270.pdf"}],"financialInterests":"Competing interest reported. AK and SK are listed as inventors on issued and pending patents on the monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications). SK is a co-founder, receives consulting fees and hold equity in Aethon Therapeutics; is a co-founder and holds equity in Revalia Bio; has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health, all outside of the current work. AD and FL are listed as inventors on an issued patent on the light-controlled T3SS filed by Max Planck Innovation (WO/2020/201115). The other authors declare no competing interests.","formattedTitle":"Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR::ABL1 signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTargeted cancer therapeutics specifically inhibit oncoproteins and oncogenic pathways and are thus being used as a personalized therapy option with fewer side effects compared to chemotherapy and other conventional cancer treatments. Currently available targeted therapeutics can be categorized into small molecule inhibitors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, often binding protein kinases and few other intracellular enzymes, and biologics, mostly therapeutic antibodies\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, which target extracellular and membrane-bound proteins. While their clinical application has led to therapeutic breakthroughs in recent years, several limitations of these drugs have arisen\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Small molecule inhibitors often lack high selectivity, leading to off-target binding and resulting in adverse effects, leaving many potential targets \u0026ldquo;undruggable\u0026rdquo;. Therapeutic antibodies, while highly specific, are complex structures with large sizes and limited tissue/tumor penetration. Importantly, antibodies are precluded from inhibiting intracellular targets, as they cannot cross cellular membranes. These drawbacks highlight the need for alternative targeted therapeutics and efficient approaches for the intracellular delivery of biologics.\u003c/p\u003e \u003cp\u003eSynthetic binding proteins are a recent development in the field of targeted therapeutics. These binding proteins are engineered from stable scaffold proteins, using molecular display techniques. The obtained binders can target the protein of interest with high affinity and selectivity and often result in preventing protein-protein interactions or inhibiting enzymatic activity of the target. Commonly used engineered binding proteins include derivatives of immunoglobulin scaffolds (scFvs, Fabs, nanobodies) and non-immunoglobulin scaffolds (monobodies, DARPins, affibodies, anticalins)\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Due to their small size (~\u0026thinsp;6\u0026ndash;20 kDa) and their ability to bind with high affinity and selectivity, they overcome limitations of current targeted therapeutics and thus have substantial therapeutic potential\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the most commonly used synthetic binder classes are monobodies (Mb), which are developed based on the protein scaffold derived from a human fibronectin type III domain\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We have engineered and characterized several monobodies as potent antagonists of oncoproteins, including kinases (BCR::ABL1\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e, LCK\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e), phosphatases (SHP2\u003csup\u003e17\u003c/sup\u003e), transcription factors (STAT3\u003csup\u003e18\u003c/sup\u003e) and small GTPases (H-/K-RAS\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e), demonstrating that it is possible to develop selective monobodies to challenging intracellular targets. These monobodies were introduced into cells as genetically encoded reagents using DNA transfection and viral gene delivery, where they inhibit the function of their targets. Monobodies lack endogenous disulfides, and consequently they readily fold into the fully functional form in the reducing environment of the cytoplasm. A number of studies have demonstrated the effectiveness of monobodies against intracellular targets for discovering and validating therapeutic approaches and elucidating the structural basis for specific recognition of challenging targets\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, recent advances have substantially improved the plasma stability and pharmacokinetics of monobodies, providing a solid groundwork for future therapeutic translation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe limited availability of efficient intracellular drug delivery systems poses a major roadblock for macromolecular therapeutics like peptides and nucleic acids, but in particular for proteins. Although monobodies and other synthetic binding proteins can achieve high selectivity and potency against the most challenging targets, the inability of monobodies to readily pass the plasma membrane barrier has so far limited their use as protein therapeutics against cytoplasmic and nuclear targets.\u003c/p\u003e \u003cp\u003eSeveral protein delivery strategies have been explored, ranging from physical methods (e.g. electroporation, microinjection) and viral delivery to nanoparticles\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In particular, various fusion strategies have been studied for the delivery of proteins such as bacterial toxin subunits\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and cell-penetrating peptides (CPPs)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Often these delivery strategies were tested with model cargoes, such as fluorescent proteins or highly active enzymes, where cytosolic delivery of very low amounts is already sufficient for a measurable readout. By contrast, few studies have shown an effect on oncogenic signaling after delivery of protein-based inhibitors. We have already demonstrated the cytosolic delivery of monobodies by fusing them to a chimeric bacterial toxin subunit\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Further modification even allowed target degradation after uptake\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, but we also experienced difficulties during recombinant production and also assume high immunogenicity using this system due to the large size of the toxin.\u003c/p\u003e \u003cp\u003eMost cellular delivery methods rely on uptake of the cargo protein through endocytosis, which in turn requires efficient endosomal escape afterwards to prevent cargo degradation in lysosomes. Inefficient endosomal escape and thus insufficient cytosolic amounts of binders is a common challenge that still has not been fully overcome\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Different endosomal escape strategies have been proposed\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, but their efficiency is highly cargo-, cell- and delivery strategy-dependent and thus no universal strategy can promise cytosolic delivery of a wide variety of cargos. Hence, delivery tools that can circumvent endocytosis and directly deliver functional binders into the cytosol are of particular interest.\u003c/p\u003e \u003cp\u003eThe bacterial type III secretion system (T3SS) is used by many bacteria to directly inject proteins into eukaryotic host cells\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, using a hollow needle attached to an export machinery in the bacterial membranes and cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). As a system evolutionary optimized for the efficient delivery of proteins into the cytosol, the T3SS has been used to deliver different cargo proteins\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e into eukaryotic target cells, including cell lines difficult to manipulate by transfection or other means\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCargo proteins are targeted to the T3SS by a short (15\u0026ndash;150 amino acids) unstructured N-terminal secretion signal\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, which can be removed by site-specific proteases or cleavage at the C-terminus of a ubiquitin domain by the native host cell machinery in the target cell\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. While the properties of cargo proteins can influence translocation rates, and very large or stably folded proteins are exported at a lower rate, most proteins, including molecular weights above 60 kDa, can be exported by the T3SS and delivered to eukaryotic cells at rates of up to 100 proteins per second, allowing the specific delivery of hundreds of thousands of cargo proteins per host cell\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Cargo proteins pass the needle unfolded with the N-terminus first, facilitating their native folding, and consequently function, in the target cell. The amount of injection into host cells can be titrated by adjusting the expression level and multiplicity of infection (MOI; ratio of bacteria to host cells). Taken together, these properties make the T3SS an efficient and versatile tool for protein delivery into eukaryotic cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we use the T3SS of an avirulent \u003cem\u003eYersinia enterocolitica\u003c/em\u003e strain, ΔHOPEMTasd\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eYersinia\u003c/em\u003e features a well-characterized and remarkably efficient T3SS, which can secrete large concentrations of effectors within short time (\u0026gt;\u0026thinsp;90% of all extracellular proteins are T3SS export substrates\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e). \u003cem\u003eY. enterocolitica\u003c/em\u003e has an unusually low number of native effector proteins, which can easily be deleted for increased biosafety and possibly increased export of heterologous cargo proteins. Given that \u003cem\u003eY. enterocolitica\u003c/em\u003e actively targets tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, the \u003cem\u003eYersinia\u003c/em\u003e T3SS is a highly promising carrier for monobodies, as evidenced by an ongoing clinical trial for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo establish the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e as a monobody delivery tool, we focus on the well-characterized AS25 monobody and its target, the Abelson tyrosine kinase 1 (Abl1). The oncogenic counterpart of Abl1 is BCR::ABL1, the product of the Philadelphia chromosomal translocation, which results in the fusion of the breakpoint cluster region (BCR) and ABL1 genes\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The fusion protein BCR::ABL1 is a constitutively active kinase that is a central driver of chronic myeloid leukemia (CML)\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. When expressed intracellularly, AS25 inhibits BCR::ABL1 kinase activity by targeting an intramolecular allosteric interface formed by the Src Homology 2 (SH2) domain and the kinase domain. AS25 thus disrupts BCR::ABL1-mediated signaling in CML cells, inhibiting their proliferation and survival\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we show the efficient direct cytosolic delivery of the AS25 monobody to different human cell lines using the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e. Concentrations in the cytosol reached mid-micromolar, ~\u0026thinsp;100-fold higher than in previous studies and well above the binding affinity. The delivered monobodies readily refold and are able to engage their targets in cells. We demonstrate specific inhibition of BCR::ABL1 signaling and induction of apoptosis in CML cells by T3SS-mediated delivery of AS25.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eAntibodies were purchased from Promega (Mouse anti-HiBiT (N7200)), ThermoFisher Scientific (Mouse anti-beta tubulin-DyLight\u0026trade; 680 (MA5-16308-D680)), Rockland (Rabbit anti-FLAG\u0026reg; (600-401-383S)), Sigma (Goat anti-Rabbit IgG Peroxidase antibody (A8275)) and LI-COR (IRDye\u0026reg;800CW Goat anti-Mouse IgG Secondary Antibody (926-32210)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and cloning\u003c/h2\u003e \u003cp\u003eGene fragments encoding monobodies for protein purification were cloned into a pHFT2 vector, a modified pET vector\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Destabilizing and non-binding mutation were introduced through site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (200523, Agilent) according to manufacturer instructions. Retroviral transduction was performed using pRV vector constructs with an IRES site followed by a GFP gene for selection. LgBiT gene, Abl-SH2-LgBiT fusion and Lck-SH2-LgBiT fusions were inserted into the pRV vector using Gibson Assembly\u0026reg;. Retroviral expression system encoding the VSV-G envelope (pCMB-VSV-G) was obtained from the Worzfeld lab. For bacterial expression plasmids, a pBAD/His B-based plasmid with SycE-YopE\u003csub\u003e1\u0026minus;\u0026thinsp;138\u003c/sub\u003e-insert (pAD722) was constructed by PCR-based restriction cloning. This plasmid served as backbone for the insertion of monobody variants by restriction enzymes; SmBiT and nonbinding monobody variants were constructed using the Q5\u0026reg; Site-Directed Mutagenesis Kit (E0554, New England Biolabs) according to manufacturer instructions. All DNA constructs were confirmed by Sanger sequencing (Microsynth).\u003c/p\u003e \u003cp\u003ePlasmids, primers and bacterial strains used in this study are listed in Supplementary Tables\u0026nbsp;5\u0026ndash;7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCultivation of bacteria\u003c/h2\u003e \u003cp\u003e \u003cem\u003eY. enterocolitica\u003c/em\u003e strains were cultivated in BHI (Brain Heart Infusion Broth) medium (3.7% w/v), complemented with nalidixic acid (35 \u0026micro;g/ml), 2,6-diaminopimelic acid (DAP, 60 \u0026micro;g/ml), and ampicillin (200 \u0026micro;g/ml) (cultivation medium). For overnight cultures, 5 ml of cultivation medium was inoculated and cultivated overnight at 28\u0026deg;C in a shaking incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eAll cell lines were cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. K562, Jurkat, HeLa and HEK293 cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Cat# ACC 10, ACC 282, ACC 57 and ACC 305, respectively). K562 and Jurkat cells were grown in Roswell Park Memorial Institute (RPMI) 1640 GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin (Gibco). HeLa Kyoto and HEK293 cells were grown in high glucose DMEM GlutaMAX medium (Gibco) supplemented with 10% FBS and 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin. Antibiotics (Penicillin and Streptomycin) were removed a day prior to infections. Hela LgBiT cells for Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec were kindly gifted by Samuel Wagner (T\u0026uuml;bingen). These cells were grown in Roswell Park Memorial Institute (RPMI) 1640 (Gibco\u0026trade;, 11875093), supplemented with 10% FBS (Gibco). Cell lines used in this study are listed in Supplementary Table\u0026nbsp;8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of stable cell lines\u003c/h2\u003e \u003cp\u003eAs previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, LgBiT with a N-terminal FLAG, Abl-SH2-LgBiT fusion with a N-terminal 6 x myc and Lck-SH2-LgBiT fusion with a N-terminal 6 x myc cloned into the retroviral pRV vector were used to establish the following stable cell lines: K562 LgBiT, Jurkat LgBiT, HeLa Abl-SH2-LgBiT and HeLa Lck-SH2-LgBiT. Cells expressing IRES-GFP were selected and sorted using FACS. Expression and functionality were assessed via immunoblotting and in functional assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eThe monobodies were produced with an N-terminal His\u003csub\u003e10\u003c/sub\u003e, FLAG and tobacco etch virus (TEV) protease recognition site using the pHFT2 vector\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Abl SH2 was produced with an N-terminal His\u003csub\u003e6\u003c/sub\u003e, GST and a TEV protease cleavage site using a pETM30 vector. All proteins were expressed in BL21 (DE3) \u003cem\u003eE.coli\u003c/em\u003e cells at 16\u0026deg;C for 16 h in auto induction LB medium. Protein purification was done by nickel-affinity chromatography (column: 1 ml or 5 ml His-Trap FF crude) and subsequent size exclusion chromatography (column: HiLoad 16/600 Superdex 75 pg) on an \u0026Auml;kta Avant system (Cytiva). The His\u003csub\u003e6\u003c/sub\u003e-GST tag of Abl-SH2 was cleaved off using TEV protease before size exclusion chromatography. Purity of all purified proteins was assessed via SDS-PAGE. Amino acid sequences of the monobodies are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThermal Shift\u0026trade; Assay (TSA)\u003c/h2\u003e \u003cp\u003eProtein Thermal Shift\u0026trade; Assay was performed to determine the thermal stability of the monobody mutants. The measurements were done using the Protein Thermal Shift\u0026trade; Dye Kit (ThermoFisher Scientific, 4461146) and performed on a StepOne\u0026trade; Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). Samples were measured in triplicates and contained 3 \u0026micro;g of protein in 1x DPBS. A thermal profile from 25\u0026deg;C to 95\u0026deg;C with a ramp rate of 1% was acquired using StepOnePlus Software (Applied Biosystems, ThermoFisher Scientific) and analyzed using Protein Thermal Shift Software (Applied Biosystems, ThermoFisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsothermal titration calorimetry (ITC)\u003c/h2\u003e \u003cp\u003eProteins were dialyzed overnight at 4\u0026deg;C against 50 mM Tris (pH 7.0), 250 mM NaCl and 5% glycerol. The protein concentration was determined by measuring UV absorbance at 280 nm on a NanoDrop 2000c. ITC measurements were acquired on a MicroCal PEAQ-ITC instrument (Malvern Panalytical) and thermodynamic parameters were determined with the MicroCal PEAQ-ITC analysis software.\u003c/p\u003e \u003cp\u003eThe protein in the syringe (Abl SH2, 200 \u0026micro;M) was titrated to the monobody solution (AS25 or AS25\u003csub\u003eA57G\u003c/sub\u003e, 20 \u0026micro;M) in 19 steps with 0.5 \u0026micro;l for the first and 2 \u0026micro;l each for the other steps. The titration of Abl SH2 (300 \u0026micro;M) to AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e (30 \u0026micro;M) was done in 13 steps with 0.5 \u0026micro;l for the first and 3 \u0026micro;l for the subsequent steps. The duration of each injection was 4 s with 150 s spacing in between injections for all measurements. The reference power was set to 10 \u0026micro;cal/s, the stir speed to 750 rpm and feedback to high. All measurements were performed at 25\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003esecretion assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacteria day cultures were inoculated from stationary overnight cultures to an OD\u003csub\u003e600\u003c/sub\u003e of 0.15 in cultivation medium complemented with MgCl\u003csub\u003e2\u003c/sub\u003e (20 mM), glycerol (0.4% w/v), and EGTA (5 mM). The cultures were cultivated shaking for 90 min at 28\u0026deg;C and then shifted to a 37\u0026deg;C and incubated for 3 h. Protein expression from plasmids was induced with 0.2% L-arabinose (w/v), before shifting to 37\u0026deg;C. 2 ml of bacterial culture were collected by centrifugation (10 min at 16,000 x g), and proteins from 1800 \u0026micro;l supernatant were precipitated with 200 \u0026micro;l trichloroacetic acid (100% w/v) overnight at 4\u0026deg;C. The precipitated proteins were collected by centrifugation (15 min at 16,000 x g) and washed with ice-cold acetone. Samples were resuspended in SDS-PAGE loading buffer (SDS (2% w/v), Tris (0.1 M), glycerol (10% w/v), dithiothreitol (0.05 M), pH\u0026thinsp;=\u0026thinsp;6.8) and heated at 99\u0026deg;C for 5 min. Unless stated differently, proteins expressed by 1.2 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e bacteria or secreted by an equivalent of 2.4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e bacteria were loaded onto SDS-PAGE gels. The gels were run for 1.5 h (135 V, 500 mA), using BlueClassic Prestained Marker [Jena Biosciences (PS-107)] or Precision Plus Dual Color Protein Standard [Bio-Rad (1610374)] as size standards.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting of secretion assays\u003c/h2\u003e \u003cp\u003eSDS-PAGE gels were blotted on a Amersham\u0026trade; Protran\u0026reg; Western Blotting nitrocellulose membrane (0.2 \u0026micro;m) [Cytiva (10600001)] using a Trans-Blot Turbo Transfer System [Bio-Rad (1704150)] with the settings: 1.3 A, 25 V, 7 min. Immunoblots were carried out using primary rabbit antibodies against the FLAG peptide [Rockland (600-401-383), 1:5,000] in combination with a secondary goat anti-rabbit antibody conjugated to a peroxidase [Sigma (A8275) 1:10,000] and visualized with Immobilon Forte Western HRP substrate [Merck (WBLUF0500)] on a LAS-4000 Luminescence Image Analyzer. Unprocessed blots can be found in Supplementary Figs.\u0026nbsp;4 and 8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInfection of adherent eukaryotic cells (HEK293 and HeLa cells)\u003c/h2\u003e \u003cp\u003eA day prior to infections, HeLa and HEK293 were seeded at 30% confluency in cell culture medium (DMEM GlutaMAX with 10% FBS) without antibiotics. Bacteria day cultures were inoculated from stationary overnight cultures to an OD\u003csub\u003e600\u003c/sub\u003e of 0.12 in cultivation medium complemented with MgCl\u003csub\u003e2\u003c/sub\u003e (20 mM), glycerol (0.4% w/v), and 200 \u0026micro;g/ml ampicillin. CaCl\u003csub\u003e2\u003c/sub\u003e (5 mM) was added for non-secreting conditions. The cultures were cultivated shaking for 90 min at 28\u0026deg;C. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with 0.2% L-arabinose (w/v) and cultures were shifted to 37\u0026deg;C for 120 min to induce T3SS formation. After that, bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v). Medium of the eukaryotic cell culture was changed to colorless RPMI, supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v). For the infection, \u003cem\u003eYersinia\u003c/em\u003e were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C (non-shaking). After 2 h incubation, the bacteria were removed and the eukaryotic cells were further incubated for 1 h in cell culture medium (DMEM GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin) supplemented with 200 \u0026micro;g/ml gentamicin. Cells were washed twice with 1 x phosphate buffered saline (PBS) and maintained in normal cell culture medium (DMEM GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin) until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInfection of non-adherent eukaryotic cells (K562 and Jurkat cells)\u003c/h2\u003e \u003cp\u003eK562 and Jurkat cells were seeded at 3 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml in cell culture medium (RPMI 1640 GlutaMAX with 10% FBS) without antibiotic supplementation. Bacterial cells were prepared as described for the infection of adherent eukaryotic cells. For the infection, \u003cem\u003eYersinia\u003c/em\u003e were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated at 37\u0026deg;C (non-shaking) at 5% CO\u003csub\u003e2\u003c/sub\u003e. After 2 h incubation, Jurkat and K562 cells were diluted in cell culture medium (RPMI 1640 GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin) supplemented with 200 \u0026micro;g/ml gentamicin or 100 \u0026micro;g/ml gentamicin, respectively, and further incubated for 1 h. Cells were centrifuged (5 min at 500 x g) and washed twice with 1 x PBS. After the wash, the cells were further diluted to a confluency of 0.5 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml and maintained in normal cell culture medium (RPMI 1640 GlutaMAX with 10% FBS, 50 U/ml Penicillin and 50 \u0026micro;g/ml Streptomycin) until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of injection kinetics into HeLa LgBiT, K562 LgBiT and Jurkat LgBiT cells\u003c/h2\u003e \u003cp\u003eFor the kinetics measurements displayed in Fig.\u0026nbsp;2bc and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Hela LgBiT cells were seeded at 20,000 cells/well in RPMI medium without antibiotic supplementation into a black 96-well microtitration plate [BRAND (781668)] on the day prior to infections. Suspension cell lines, Jurkat LgBiT cells and K562 LgBiT cells, were seeded at 360,000 cells/well in Opti-MEM\u0026trade; (31985070, Gibco), supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v), without antibiotic supplementation into a black 96-well microtitration plate [BRAND (781668)] on the day of infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBacteria day cultures were inoculated from stationary overnight cultures to an OD\u003csub\u003e600\u003c/sub\u003e of 0.12 in cultivation medium complemented with MgCl\u003csub\u003e2\u003c/sub\u003e (20 mM), glycerol (0.4% w/v), and CaCl\u003csub\u003e2\u003c/sub\u003e (5 mM). The cultures were cultivated shaking for 90 min at 28\u0026deg;C and then shifted to a 37\u0026deg;C and incubated for 120 min to induce T3SS formation. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with 0.2% L-arabinose (w/v). Bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v). For the infection, \u003cem\u003eYersinia\u003c/em\u003e cells were added to the eukaryotic cells at a multiplicity of infection (MOI) of 20. The enzymatic Nano-Glo\u0026reg; Luciferase Assay System [Promega (N1110)] was used according to manufacturer instructions. 30 \u0026micro;l of Nano-Glo\u0026reg; Luciferase Assay Reagent (substrate:buffer 1:50) was added to each sample. Bioluminescence was detected every 3 min in a microplate reader [Tecan Infinite 200 PRO], for 2 h at 37\u0026deg;C with an integration and settle time of 200 ms, each. The background signal was subtracted from the obtained values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis of monobody levels after translocation\u003c/h2\u003e \u003cp\u003eMonobody levels after translocation were assessed by taking samples directly after (0 h) and 24 h after gentamicin treatment. Total protein extraction was done in lysis buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), 5 mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% NP-40) supplemented with 50 mM NaF, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 \u0026micro;l/ml Tosyl-L-phenylalaninchloromethylketon (TPCK) and protease inhibitors (cOmplete\u0026trade;, CO-RO, Roche) and cleared by centrifugation (20 min at 20,000 x g). Protein concentrations were determined using a Bradford assay (Bio-Rad). Equal amounts of proteins (50 \u0026micro;g cell lysate) were separated on a SDS-polyacrylamide electrophoresis (PAGE) gel and transferred to a nitrocellulose membrane (0.2 \u0026micro;m) by wet transfer. Membranes were blocked for 1 h at room temperature in blocking buffer (2.5% BSA and 2.5% milk powder in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20; pH7.6)). Subsequently, the membranes were probed with mouse anti-HiBiT (1:1000) diluted in blocking buffer at 4\u0026deg;C overnight. This was followed by incubation with secondary antibodies, IRDye\u0026reg;800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000 in TBS-T) and anti-beta tubulin-DyLight\u0026trade; 680 (1:500 in TBS-T). Fluorescent detection was performed using the LI-COR Imaging system. Protein levels were quantified using the Empiria Studio\u0026reg; software and normalized to tubulin and control protein. 24 h sample was normalized to the 0 h sample of the respective protein. All blots were performed in three independent experiments. Unprocessed blots can be found in Supplementary Figs.\u0026nbsp;5 and 7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative immunoblotting for intracellular concentration\u003c/h2\u003e \u003cp\u003eIntracellular monobody concentrations in HeLa and K562 cells were determined with quantitative immunoblotting. Samples were taken after gentamicin treatment and total protein extraction was done as described above. Equal amounts of proteins (30 or 50 \u0026micro;g cell lysate) were loaded on a SDS-PAGE gel along with various amounts of purified HiBiT-Monobody protein. After transfer to nitrocellulose membrane, the membrane was probed with anti-HiBiT and anti-beta tubulin antibodies, as described above. Fluorescent detection was performed using the LI-COR Imaging system. Protein levels were quantified using the Empiria Studio\u0026reg; software. The purified protein samples were used to obtain a standard curve. Then, the protein amount in the cell lysate samples was determined using the standard curve. The final concentration in HeLa and K562 cells was calculated with the cell number used for the blot and a single cell volume of 4.2 pL\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and 1.65 pL\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, respectively. All blots were performed in three independent experiments. Unprocessed blots can be found in Supplementary Fig.\u0026nbsp;6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular stability of the monobody\u003c/h2\u003e \u003cp\u003eEukaryotic cell culture was prepared as described above. Bacteria day cultures were inoculated from stationary overnight cultures to an OD\u003csub\u003e600\u003c/sub\u003e of 0.12 in cultivation medium complemented with MgCl\u003csub\u003e2\u003c/sub\u003e (20 mM), glycerol (0.4% w/v), and 200 \u0026micro;g/ml ampicillin. CaCl\u003csub\u003e2\u003c/sub\u003e (5 mM) was added for non-secreting conditions. The cultures were cultivated shaking for 90 min at 28\u0026deg;C and shifted to 37\u0026deg;C for 60 min to induce T3SS formation. Subsequently, expression of the monobody cargo protein from the pBAD plasmid was induced with L-arabinose (w/v) and cultures were incubated for another 60 min at 37\u0026deg;C. After that, bacterial cells were collected (2 min at 2,400 x g) and the pellet was washed with culture grade PBS, supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v). Medium of the eukaryotic cell culture was changed to colorless RPMI, supplemented with DAP (60 \u0026micro;g/ml) and 0.2% L-arabinose (w/v). For the infection, \u003cem\u003eYersinia\u003c/em\u003e cells were added to the eukaryotic cells at a multiplicity of infection (MOI) of 100 and incubated at 37\u0026deg;C (non-shaking). After 2 h incubation, 150 \u0026micro;g/ml gentamicin was added prior to a further incubation for 1 h. Finally, the eukaryotic cells were washed with cell culture medium (RPMI) supplemented with 150 \u0026micro;g/ml gentamicin, 10% FCS (v/v) and endurazine (1:100), as stated in the manufacturer\u0026rsquo;s instructions [Promega Nano-Glo\u0026reg; Endurazine\u0026trade; Live Cell Substrate (N2570)]. Bioluminescence was detected every 3 min in a microplate reader [Tecan Infinite 200 PRO], for 24 h and 37\u0026deg;C with an integration and settle time of 200 ms, each. The background signal was subtracted from the obtained values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNanoBiT Protein-Protein Interaction for determining monobody and target interaction\u003c/h2\u003e \u003cp\u003eA day prior to infection, HeLa cells stably expressing Abl-SH2-LgBit or Lck-SH2-LgBiT were seeded at 25,000 per well into a white 96 well LUMITRAC microplate (655074, Greiner Bio-One) in cell culture medium (DMEM GlutaMAX with 10% FBS) without antibiotics. Cells were infected and treated as described above. After gentamicin treatment, cells were washed and kept in 100 \u0026micro;l Opti-MEM (31985070, Gibco) supplemented with 10% FBS. Nano-Glo Live Cell Reagent (N2011, Promega) was prepared by combining 1 volume of Nano-Glo Live Cell Substrate with 19 volumes of Nano-Glo LCS Dilution buffer. 25 \u0026micro;l of Nano-Glo Live Cell Reagent was added to each well and the plate was gently mixed on an orbital shaker (30 seconds for 300 rpm). Luminescence was immediately measured on a SpectraMax M5 (Molecular Devices) with an exposure time of 500 ms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePhospho-STAT5 (pY694-STAT5)\u003c/h2\u003e \u003cp\u003eSTAT5 phosphorylation (pY694) in K562 cells was assessed upon monobody translocation. Cells were infected and treated as described above. K562 cells continuously treated with 1 \u0026micro;M imatinib or 10 \u0026micro;M imatinib served as positive controls. Samples were taken 5 h and 24 h after the start of the infection or imatinib treatment. 1 x 10\u003csup\u003e6\u003c/sup\u003e cells were spun down (5 min at 500 x g) and resuspended in 1 x PBS. Then, the cells were fixed in 3.2% paraformaldehyde (PFA, E15710, Science Services) for 10 min at room temperature. After fixation, the cells were spun down (5 min at 300 x g) and stored in 95% ice-cold methanol at -20\u0026deg;C overnight. On the next day, the cells were washed with 1 x PBS, spun down (5 min at 400 x g), resuspended in 1 x PBS with 4% FBS (FACS buffer) and incubated at 4\u0026deg;C for 2 h. Cells were spun down (5 min at 500 x g) and resuspended in Human SeroBlock (1:20 in FACS buffer; BUF070A, Bio-Rad). After blocking for 15 min, the cells were stained with BD Phosflow\u0026trade; Alexa Fluor\u0026reg; 647 Mouse Anti-Stat5 (pY694) (1:5 in FACS buffer; 612599, BD Biosciences) for 45 min on ice. Lastly, cells were spun down (5 min at 400 x g), resuspended in 1 x PBS and analyzed on a Guava easyCyte\u0026trade; (Luminex) using the 642 nm laser and a 661/15 nm bandpass filter. Data was analyzed using FlowJo (v10). Gating strategy is shown in Supplementary Fig.\u0026nbsp;10. Analysis of STAT5 phosphorylation was done in three independent experiments. In each experiment, the mean fluorescence intensity (MFI) of untreated cells was set to 1 and the relative MFI of all samples was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis Assay (Activated Caspase 3/7 and Dead Cell Stain)\u003c/h2\u003e \u003cp\u003eCellEvent\u0026trade; Caspase-3/7 Green Flow Cytometry Assay Kit (C10427, Molecular Probes) was used to study initiation of apoptosis in K562 upon monobody translocation. Cells were infected and treated as described above. K562 cells continuously treated with 1 \u0026micro;M imatinib or 10 \u0026micro;M imatinib served as positive controls. Samples were taken 24 h and 48 h after the start of the infection or imatinib treatment. 0.5 x 10\u003csup\u003e6\u003c/sup\u003e cells were centrifuged (5 min at 500 x g) and resuspended in 1 x PBS with 2% FBS. Then, the cells were stained with the CellEvent\u0026trade; Caspase-3/7 Green Detection Reagent (1:1000) and incubated for 30 min at 37\u0026deg;C. Afterwards, cells were stained with SYTOX\u0026trade; AADvanced\u0026trade; Dead Cell Stain (1:1000) for 5 min at 37\u0026deg;C. After staining, samples were directly analyzed on a Guava easyCyte\u0026trade; (Luminex) using the 488 nm laser and a 525/30 nm bandpass filter and 642 nm laser and a 695/50 nm bandpass filter. Single stained samples were used for compensation. Data was analyzed using FlowJo (v10). Gating strategy is shown in Supplementary Fig.\u0026nbsp;10. Induction of apoptosis was analyzed in three to five independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eQuantification and statistical analysis were performed using GraphPad Prism 10 and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, as specified in the figure legends. Statistical analyses were performed with an ordinary one-way ANOVA followed by Šid\u0026aacute;k multiple comparisons tests. \u003cem\u003eP\u003c/em\u003e values below 0.05 were considered statistically significant. Sample sizes (\u003cem\u003en\u003c/em\u003e) are provided in the respective figure legend. Asterisks represent statistical significance (ns denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, \u003csup\u003e*\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003e denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eEngineering of monobody variants with reduced stability\u003c/h2\u003e \u003cp\u003eWe wanted to exploit the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e for the direct cytosolic delivery of monobodies that bind intracellular oncoproteins. Besides an N-terminal T3SS secretion signal\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e for recognition by the system, the translocation through the needle requires unfolding of the cargo protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Less stable proteins are more efficiently translocated\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, whereas very stable proteins like GFP can block the needle\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Monobodies, including AS25, have high thermodynamic stability (Supplementary Fig.\u0026nbsp;1). Therefore, we attempted to engineer less stably folded monobody variants that would translocate more efficiently whilst retaining target binding. The D7K mutation in monobodies had been introduced to enhance its stability based on the identification of electrostatic repulsion involving Asp7 in the 10th FN3 domain (10FN3) of human fibronectin, the scaffolding domain for monobody engineering\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, and thus we reverted it. Sequence comparison of 10FN3 with the 3rd FN3 domain (3FN3), as well structural modeling pointed us towards another mutation, A57G, that might decrease monobody stability without affecting target binding. AS25 with the A57G mutation was selected for further characterization, as much higher soluble expression of this variant in \u003cem\u003eE. coli\u003c/em\u003e was observed than for AS25 with the K7D reversion (data not shown). Additionally, analysis of the co-crystal structure of AS25 with its target identified Y45 as a possible critical residue for Abl1 SH2 domain binding, and we therefore included a Y45A mutation as a negative control with possible decreased binding to Abl1 SH2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;2). We assessed thermodynamic stability of the AS25 variants with a thermal shift assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). While the wildtype AS25 monobody had a high melting temperature of ~\u0026thinsp;74\u0026deg;C, the inclusion of the A57G mutation decreased the melting temperature by more than 13\u0026deg;C. The addition of the Y45A mutation only led to a minor decrease of further 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Next, to monitor effects of the mutations on target binding, we determined thermodynamic binding parameters of these monobody mutants using isothermal titration calorimetry (ITC) measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f, Supplementary Fig.\u0026nbsp;3). All measurements suggested an Abl1 SH2:AS25 monobody binding stoichiometry of 1:1. The AS25\u003csub\u003eA57G\u003c/sub\u003e variant showed no decreased binding affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 156 nM; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) when compared to the wildtype AS25 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 180 nM; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In contrast, the AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e variant resulted in a\u0026thinsp;~\u0026thinsp;15-fold decreased binding affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 2690 nM, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Also, the binding enthalpy of AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e was half of the binding enthalpy of the other variants (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eIn summary, we engineered a destabilized variant of AS25 (AS25\u003csub\u003eA57G\u003c/sub\u003e) that retained binding affinity to its target, as well as a variant with a strongly reduced binding affinity (AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e), for testing secretion specificity and efficiency with the T3SS system. This low-affinity AS25 variant (AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e) will be used as negative control for all experiments and termed \u0026lsquo;non-binding\u0026rsquo; for simplicity from here onwards.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003esecretion of monobody variants using the\u003c/b\u003e \u003cb\u003eY. enterocolitica\u003c/b\u003e \u003cb\u003eT3SS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing the N-terminal secretion signal of a native T3SS effector, YopE\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;138\u003c/sub\u003e, all three variants of AS25 were expressed by the bacteria and efficiently secreted in an \u003cem\u003ein vitro\u003c/em\u003e secretion assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-h). Notably, the destabilized variants allowed for a stronger concurrent secretion of SctA, a protein that is essential for the formation of the translocon in the host cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, left), indicating that indeed, the destabilized variants of the monobody prevented the blocking of the needle. Importantly, we verified that a strain lacking SctQ (\u003cem\u003eΔsctQ\u003c/em\u003e; non-secreting strain) showed expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), but no detectable secretion of the monobody into the culture supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, right).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTranslocation of monobodies into eukaryotic cells\u003c/h2\u003e \u003cp\u003eIn order to monitor translocation of monobodies into eukaryotic cells in real-time, we employed a live cell split-NanoLuc luciferase system\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We used HeLa and Jurkat cell lines stably expressing the large domain of the NanoLuc luciferase (LgBiT) in the cytoplasm. Upon addition of bacteria expressing monobodies tagged with the HiBiT peptide, only successful cytoplasmic delivery would result in high affinity binding of HiBiT to LgBiT (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 0.7 nM), leading to a reconstitution of a functional NanoLuc enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Starting measurements immediately after the addition of the bacteria, we observed a strong luciferase signal upon translocation of AS25\u003csub\u003eA57G\u003c/sub\u003e and AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e into HeLa cells, which increased over 120 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). AS25, without destabilizing mutation, resulted in much weaker translocation. The secretion-deficient strain (Δ\u003cem\u003esctQ\u003c/em\u003e) expressing AS25\u003csub\u003eA57G\u003c/sub\u003e showed weak luminescence signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In Jurkat cells, also AS25\u003csub\u003eA57G\u003c/sub\u003e was translocated strongest, while AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e showed similar translocation kinetics as AS25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe next tested whether the monobodies are translocated into different human cell lines and analyzed monobody levels by immunoblotting after infection. Besides HeLa, we used human embryonic kidney (HEK293) as a second adherent cell line. In addition, we chose two hematopoietic, non-adherent cell lines: Jurkat, the most commonly used T lymphocyte cell line, and K562, the most commonly used cell line expressing BCR::ABL1. All cell lines were incubated with bacteria expressing AS25\u003csub\u003eA57G\u003c/sub\u003e or AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e for 2 h. A non-secreting bacterial strain (Δ\u003cem\u003esctQ\u003c/em\u003e), expressing AS25\u003csub\u003eA57G\u003c/sub\u003e, was used as a negative control. After incubation, the cells were treated with gentamicin to kill bacteria before preparing cellular extracts for immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). To obtain a high translocation efficiency with non-adherent cell lines, we used higher cell densities of the target cells while maintaining a MOI of 100.\u003c/p\u003e \u003cp\u003eTranslocation of monobodies was achieved in all four cell lines and robustly detected by immunoblotting of a 29 kDa band, which is in line with the expected molecular weight of the monobody with secretion signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-h). In the two adherent cell lines, HEK293 and HeLa, the non-binding AS25 variant (AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e) was detected at slightly higher concentrations than the binding counterpart (AS25\u003csub\u003eA57G\u003c/sub\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-h). In contrast, in the two suspension cell lines, Jurkat and K562, less of the non-binding AS25 was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f). Due to the higher abundance of BCR::ABL1 and Abl1 in K562 and Jurkat, respectively, it is possible that the binding of AS25 to its target leads to a longer half-life, whereas the non-binding AS25 could be less stable in these cells, leading to a lower abundance even though the efficiency of translocation is comparable. In the experiment using adherent cell lines, we observed some signal from cells incubated with a non-secreting strain (\u003cem\u003eΔsctQ)\u003c/em\u003e. As this strain was clearly deficient in cytosolic delivery of monobodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c), we suspected that this signal could stem from adherent bacteria that were not efficiently cleared by the gentamicin treatment. In contrast, the non-secreting strain only resulted in less than 10% of the signal in the suspension cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f).\u003c/p\u003e \u003cp\u003eCytosolic concentrations substantially higher than the binding affinity of the AS25 monobody (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 156 nM, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) are required for significant target inhibition. Therefore, we assessed the intracellular concentrations of monobodies that were translocated after monobody translocation by the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e in HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). For absolute quantifications, we used serial dilutions of known amounts of recombinant AS25 monobody (without secretion signal) as a reference in immunoblots next to cell lysates after translocation. Taking the cell numbers and cell volume into account, we estimate the intracellular concentrations of the binding and non-binding AS25 in HeLa cells to be ~\u0026thinsp;40 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). This indicated that sufficient amounts of monobodies were translocated to enable intracellular target binding and inhibition.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eIntracellular stability of monobodies after translocation\u003c/h2\u003e \u003cp\u003eTo assess the duration of the inhibitory effect on the target protein, we next analyzed monobody half-life after translocation in HeLa and Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To obtain first insights, we compared samples directly after gentamicin treatment (0 h) to samples taken 24 h later. We observed that initial monobody amounts decreased to ~\u0026thinsp;10% of the initial amounts for both binding and non-binding variants in HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Also, in Jurkat cells, a strong decrease was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) but less pronounced for the binding AS25 as compared to its non-binding counterpart, in line with the observations from above (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo study the degradation kinetics of monobodies after translocation in more detail, we translocated HiBiT-tagged monobodies into HeLa and Jurkat cells stably expressing LgBiT and then treated the cells with gentamicin, which allowed us to monitor the change of luminescence over time as a readout for monobody degradation. Luminescence intensity, corresponding to LgBit complementation by the HiBiT-tagged monobody, peaked after 2\u0026ndash;4 hours and then gradually decreased in HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Wildtype AS25 showed both the slowest increase and the weakest decrease over time (\u0026lt;\u0026thinsp;40% degradation after 24 h, compared to the peak intensity), whereas the non-binding mutant was degraded most strongly (\u0026gt;\u0026thinsp;60% degradation after 24 h, compared to the 2 h peak concentration). The Δ\u003cem\u003esctQ\u003c/em\u003e strain shows a very weak translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), supporting the interpretation that the signal obtained in the immunoblot analysis for this strain with the adherent cell lines (Fig.\u0026nbsp;2gh, 3a) is due to unspecific binding of non-lysed bacteria and not due to translocation. Translocation into Jurkat cells showed similar kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), albeit with a stronger signal for the wild-type AS25 monobody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eTarget binding after monobody translocation\u003c/h2\u003e \u003cp\u003eWe so far detected the cytosolic delivery of AS25 monobody variants in a split-NanoLuc assay and by immunoblotting. The translocated monobodies were detectable for several hours. Still, although monobodies and their parental 10FN3 are known to rapidly and reversibly refold\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e these experiments do not allow to conclude if the monobodies refold after translocation and are thus functional binders. Thus, we next set up an assay to measure the monobody-target interaction in the cytoplasm. For this purpose, we employed HeLa cell lines stably expressing LgBiT fused to the Abl1 SH2 domain (AS25 monobody target) or the Lck SH2 domain (negative control) and delivered monobodies tagged with SmBiT using T3SS. As the binding affinity of the LgBiT-SmBiT interaction is in the high micromolar range (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 190 \u0026micro;M), only the interaction of monobody and target will result in reconstituted, functional luciferase\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Therefore, this system can be used to assess translocation of functional refolded monobody and target engagement in living cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Similar to the untagged variants of the monobody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), AS25-SmBiT was secreted in an \u003cem\u003ein vitro\u003c/em\u003e secretion assay, with an increased secretion of monobody and translocator SctA for the destabilized AS25\u003csub\u003eA57G\u003c/sub\u003e variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, left). Again, we verified that all monobody variants are expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), while the strain lacking SctQ (\u003cem\u003eΔsctQ\u003c/em\u003e) showed no detectable secretion into the culture supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, right).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAS25\u003csub\u003eA57G\u003c/sub\u003e-SmBiT resulted in a very strong increase in luminescence showing interaction of AS25 with the Abl1 SH2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The high specificity of this interaction was underlined by the low signal of the non-binding AS25, as well as the non-secreting strain. The difference between the wildtype AS25 and the destabilized AS25\u003csub\u003eA57G\u003c/sub\u003e again shows the significantly enhanced translocation of the destabilized variant. Importantly, in the control cell line expressing LgBiT fused to the Lck-SH2 domain, we could only detect background signal for all AS25 variants/strains, underlining the high selectivity of AS25 and the highly specific readout of this assay system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In summary, these results show refolding and specific target interaction of AS25 after translocation into HeLa cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eMonobody translocation leads to inhibition of BCR::ABL1 signaling\u003c/h2\u003e \u003cp\u003eWe next assessed if the translocated monobodies exert an inhibitory effect on target signaling. Therefore, we chose the BCR::ABL1-expressing cell line K562, which is dependent on active BCR::ABL1 signaling for growth and survival. We first determined AS25 translocation and degradation kinetics, as well as the intracellular monobody concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). As for HeLa cells (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), both AS25\u003csub\u003eA57G\u003c/sub\u003e and AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e showed efficient translocation into K562 cells, which plateaued after ~\u0026thinsp;75 minutes, while the wildtype AS25 showed slower kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The intracellular monobody stability was similar to the other cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d for comparison). The intracellular concentration of the binding AS25\u003csub\u003eA57G\u003c/sub\u003e was ~\u0026thinsp;30 \u0026micro;M, which surpasses the binding affinity of AS25-Abl SH2 interaction by far (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), while the non-binding AS25\u003csub\u003eY45A\u0026thinsp;\u0026minus;\u0026thinsp;A57G\u003c/sub\u003e accumulated to around half the concentration (~\u0026thinsp;13 \u0026micro;M; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe then studied the functional consequences of AS25 monobody binding on BCR::ABL1 signaling in K562 cells after translocation into the cytoplasm. Inhibition of BCR::ABL1 kinase activity results in inhibition of STAT5 phosphorylation on Tyr-694 (pY-694), a critical BCR::ABL1 substrate and signaling mediator \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, which can be conveniently and reliably measured by intracellular FACS staining.\u003c/p\u003e \u003cp\u003eWe observed a strong reduction of STAT5 phosphorylation 5 h after translocation of AS25\u003csub\u003eA57G\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). A similar degree of reduction was obtained by treating the cells with the BCR::ABL1 tyrosine kinase inhibiting drug imatinib. In contrast, no reduction was observed with the non-binding variant or a non-secreting strain (\u003cem\u003eΔsctQ\u003c/em\u003e). Also, translocation of an unrelated destabilized monobody targeting the SHP1 tyrosine phosphatase (MbC\u003csub\u003eA57G\u003c/sub\u003e) showed no reduced STAT5 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These results show that the observed inhibition of BCR::ABL1 signaling by AS25\u003csub\u003eA57G\u003c/sub\u003e was dependent on functional translocation and monobody-target binding. In line with the monobody degradation kinetics, the inhibitory effect of AS25\u003csub\u003eA57G\u003c/sub\u003e after 24 h was weaker as compared to 5 h, but still significant in comparison to the negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eInduction of apoptosis after monobody translocation\u003c/h2\u003e \u003cp\u003eFinally, we wanted to monitor if the pronounced inhibition of STAT5 phosphorylation in K562 cells resulted in apoptosis induction and cell death as monitored by caspase 3/7 activation and staining for dead cells, respectively, using FACS. The translocation of AS25\u003csub\u003eA57G\u003c/sub\u003e led to a strong increase in apoptotic cells after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and c). After 48 h, the effect was still significant, albeit weaker, possibly due to degradation of the translocated monobody and continued cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c). As expected, imatinib treatment showed a more pronounced effect after 48 h than after 24 h. The non-binding AS25 variant and the unrelated monobody (MbC\u003csub\u003eA57G\u003c/sub\u003e) did not result in a significant increase of apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c), showing that induction of apoptosis was a direct consequence of target binding. Infection with the non-secreting strain (Δ\u003cem\u003esctQ\u003c/em\u003e) expressing AS25\u003csub\u003eA57G\u003c/sub\u003e led to a weaker, but detectable response at 24 h, but not at 48 h (Fig.\u0026nbsp;7a-c). As the strain is deficient in monobody delivery (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and did not affect BCR::ABL1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e), we suspect this to be an unspecific effect at the earlier timepoint of this particular strain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, delivery of binding AS25 into K562 cells resulted in selective inhibition of BCR::ABL1 signaling and led to induction of apoptosis in CML cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe demonstrated that the T3SS of an avirulent \u003cem\u003eYersinia enterocolitica\u003c/em\u003e strain can be re-engineered to serve as a versatile and highly efficient system for protein delivery of a functional BCR::ABL1-targeting monobody. The delivered monobodies are able to engage and inhibit their target in cells, which results in perturbation of BCR:ABL1 signaling. We further demonstrate that this selective inhibition after delivery leads to induction of apoptosis in BCR:ABL1-dependent cells.\u003c/p\u003e \u003cp\u003eTo improve translocation efficiency, we created a destabilized monobody variant by mutating Ala-57 that faces the hydrophobic core of the monobody. As this position is not used for making a combinatorial library and is located on the opposite side relative to the intended target binding interfaces\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, we believe to have established a general strategy that can be adopted for efficient delivery of any monobody. This view is supported by the efficient delivery of the A57G mutant SHP1 SH2-targeting monobody MbC (see Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003eUsing the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e for the delivery of monobody proteins has several positive features: Firstly, we observed that T3SS-mediated delivery of the destabilized cargo into different cell lines resulted in high cytosolic concentrations in the mid-micromolar concentration range already shortly after initiation of delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Such concentrations exceed the binding affinities of monobody-target interaction by \u0026gt;\u0026thinsp;10-fold and hence enable efficient target inhibition in cells. Additionally, high concentrations of translocated cargo are desirable as this lowers the dosage required to elicit a functional effect. So far, only few studies have determined the amount of translocated cargo. In HeLa cells, we were able to translocate around 10\u003csup\u003e8\u003c/sup\u003e molecules per cell, which amounts to a concentration of 40 \u0026micro;M. Previous studies using the T3SS of \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium SPI-1 or \u003cem\u003eEscherichia coli\u003c/em\u003e to translocate binding proteins into HeLa showed concentrations around 200 nM\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e or around 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e translocated molecules per cell\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Delivery of AS25 with the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e, therefore, appears to be at least 100-fold more efficient.\u003c/p\u003e \u003cp\u003eSecondly, delivery to the cytoplasm of target cells is ensured given the direct injection of the cargo through the plasma membrane without the need to cross other membranes or requirement for specific receptors. In contrast, other protein delivery approaches depend on specific receptors and endocytic uptake pathways\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. T3SS-mediated delivery circumvents the challenge to enable escape from the endo-/lysosomal compartments, which can result in entrapment of cargo and subsequent degradation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThirdly, the intrinsic ability of \u003cem\u003eYersinia\u003c/em\u003e to target tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e makes it an ideal candidate for monobody delivery and inhibition of oncogenic signalling. Furthermore, this system can be engineered to specifically target cancer cells\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e or to respond to external stimuli \u0026ndash; an engineered version of the \u003cem\u003eY. enterocolitica\u003c/em\u003e T3SS incorporating an optogenetic switch can be activated by illumination with high temporal and spatial resolution\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Controlled delivery combined with high intracellular concentrations of translocated monobody would thus further lower the toxicity of bacterial application and safer for future clinical application.\u003c/p\u003e \u003cp\u003eOn the other hand, T3SS-mediated monobody delivery has also limitations. One restraint is the limitation to genetically encoded cargos: Approaches to enhance stability and half-life using \u003cem\u003ein vitro\u003c/em\u003e synthesized mirror-image monobodies composed of D-amino acids (Hantschel lab, unpublished observations) cannot be combined with the T3SS. Similarly, cargo that is labeled with small-molecule fluorescent dyes cannot be easily translocated by the T3SS to follow translocation kinetics and intracellular fate. The use of self-labeling tags, e.g. Halo, SNAP or CLIP, may circumvent this problem\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, but requires additional experimental steps and may perturb translocation kinetics\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, whereas the split-NanoLuc luciferase employed by us only required minor modifications of the cargo.\u003c/p\u003e \u003cp\u003eOur kinetics experiments showed a fast injection of monobodies, with peak concentrations 1\u0026ndash;4 h after addition of the bacteria, followed by a decrease to 25\u0026ndash;50% 24 h after T3SS-mediated delivery (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is important to understand the pathways that led to this reduction in addition to dilution effects due to cell growth and division. Proteasomal degradation via K48-polyubiquitination is a possible candidate and removal of Lys residues in the monobody sequence and/or the N-terminal secretion signal could therefore result in increased intracellular half-life. Another important factor influencing cytosolic monobody stability appears to be target binding. We observed faster kinetics and higher levels of binding AS25 when compared to its non-binding counterpart in Jurkat and K562 cells that have high expression of the AS25 target proteins ABL1 and BCR::ABL1. This was despite very similar \u003cem\u003ein vitro\u003c/em\u003e secretion of the two AS25 variants. A possible explanation could be that target binding may stabilize monobody levels, whereas non-binding monobodies or cells that express only low levels of target result in faster turn-over and degradation. This is in line with a previous study showing that the expression of the target in the cells led to a higher accumulation of the translocated binder\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Notably, both translocation and degradation kinetics varied between cell lines, indicating potential variations in refolding and proteolysis kinetics or different amounts of stabilizing target proteins. A related question concerns the observation why the relatively mild (~\u0026thinsp;15-fold) reduction in target binding affinity \u003cem\u003ein vitro\u003c/em\u003e caused by the Y45A mutation in AS25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) was sufficient to abolish cytosolic target engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), to prevent inhibition of STAT5 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e) and to abolish induction of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). As the mechanism of inhibition of AS25 requires competition with the intramolecular SH2-kinase domain interface of BCR::ABL1\u003csup\u003e15\u003c/sup\u003e, a higher AS25 concentration may be required than what would be predicted from the binding affinity to the isolated ABL1 SH2 domain, as previously observed\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Hence, even a mild mutation, such as Y45A, can result in a loss-of-function in cells.\u003c/p\u003e \u003cp\u003eFuture applications of T3SS-mediated monobody delivery may include targeted protein degradation approaches, such as AdPROMs or bioPROTACs.\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e For these approaches, monobodies were fused to different E3 ubiquitin ligases to induce the degradation of monobody target protein after transfection or viral transduction in cancer cell lines.\u003c/p\u003e \u003cp\u003eWhile not unique to T3SS-mediated delivery, there is a high flexibility in terms of delivered cargo. For example, subcellular targeting moieties can be fused to delivered monobodies to enable e.g. nuclear or membrane localization\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Also, two monobodies targeting different domains in a target protein can be delivered as a tandem fusion to enhance target selectivity and efficacy of inhibition, as previously demonstrated\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Alternatively, one could also envision tandem fusion monobodies with specificity for two different targets, which can induce \u003cem\u003ede novo\u003c/em\u003e protein-protein interactions. Still, all these approaches may need further optimization as the larger size of the cargo might decrease translocation efficiency.\u003c/p\u003e \u003cp\u003eThe delivery of immunomodulating proteins to cancer cells and the tumour microenvironment by the \u003cem\u003eY. enterocolitica\u003c/em\u003e T3SS is currently evaluated in a clinical trial\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and hence indicates a clear path to clinical translation. While type I interferons and certain natural pro-apoptotic proteins have been delivered before\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, our work provides the groundwork for the delivery selective protein-based signaling inhibitors to cancer cells by the \u003cem\u003eY. enterocolitica\u003c/em\u003e T3SS, which resulted in inhibition of a central oncogene, its downstream pathways and induction of apoptosis. For the future \u003cem\u003ein vivo\u003c/em\u003e applications of T3SS-mediated delivery, enhancement of anti-tumor immunity by the immunogenic properties of most bacteria can be advantageous and is realized by different biotech start-up companies\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. On the other hand, a fine balance needs to be struck to prevent an overactivation of the immune system resulting in acute inflammation and cytokine storm. Some of these limitations may be addressed by bioengineering, which is increasingly used to improve the characteristics of bacteria as drug delivery vehicles, including improvements of their safety profiles, and modification of their immunogenicity and targeting specificity\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Recent advances for the specific use of the T3SS for drug delivery include the engineering of carrier bacteria for lower immunogenicity\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e, the modification of the T3SS for further increased translocation speed\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, transfer or even synthetic \u003cem\u003ede novo\u003c/em\u003e assembly of the T3SS in selected carrier bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. In addition, the development of a light-controlled T3SS using optogenetic switches allows for control of protein delivery with high temporal and spatial precision\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, we showed that the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e can serve as an efficient system for the delivery of monobody proteins to cancer cells, which resulted in oncogene-dependent perturbation of signaling and cell proliferation. This delivery approach holds great promise for future therapeutic use without the need to genetically manipulate target cells.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e10FN3:\u0026nbsp;\u003c/strong\u003e10\u003csup\u003eth\u003c/sup\u003e FN3 domain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3FN3:\u0026nbsp;\u003c/strong\u003e3\u003csup\u003erd\u003c/sup\u003e FN3 domain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbl:\u0026nbsp;\u003c/strong\u003eAbelson tyrosine kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBCR:\u0026nbsp;\u003c/strong\u003ebreakpoint cluster region\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCML:\u0026nbsp;\u003c/strong\u003echronic myeloid leukemia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCPP:\u0026nbsp;\u003c/strong\u003ecell-penetrating peptides\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFACS:\u0026nbsp;\u003c/strong\u003efluorescence activated cell sorting\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHEK293:\u0026nbsp;\u003c/strong\u003ehuman embryonic kidney\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eITC:\u0026nbsp;\u003c/strong\u003eisothermal titration calorimetry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLgBiT:\u0026nbsp;\u003c/strong\u003elarge domain of the NanoLuc luciferase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMb:\u0026nbsp;\u003c/strong\u003eMonobodies\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMOI:\u0026nbsp;\u003c/strong\u003emultiplicity of infection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSH2:\u0026nbsp;\u003c/strong\u003eSrc Homology 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTAT5:\u0026nbsp;\u003c/strong\u003esignal transducer and activator of transcription 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT3SS:\u0026nbsp;\u003c/strong\u003eType III secretion system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003csub\u003em\u003c/sub\u003e:\u0026nbsp;\u003c/strong\u003emelting temperature\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSA:\u0026nbsp;\u003c/strong\u003ethermal shift assay\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthors information\u003c/h2\u003e \u003cp\u003eChiara Lebon and Sebastian Grossmann contributed equally to this work.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e \u003cp\u003eInstitute of Physiological Chemistry, Faculty of Medicine, Philipps-University of Marburg, 35043 Marburg, Germany\u003c/p\u003e \u003cp\u003eChiara Lebon \u0026amp; Oliver Hantschel\u003c/p\u003e \u003cp\u003eDepartment of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany\u003c/p\u003e \u003cp\u003eSebastian Grossmann, Florian Lindner \u0026amp; Andreas Diepold\u003c/p\u003e \u003cp\u003eSwiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, \u0026Eacute;cole polytechnique f\u0026eacute;d\u0026eacute;rale de Lausanne, 1015 Lausanne, Switzerland\u003c/p\u003e \u003cp\u003eGreg Mann\u003c/p\u003e \u003cp\u003eDepartment of Medicine, New York University School of Medicine, Laura and Isaac Perlmutter Cancer Center, New York University Langone Health, Department of Biochemistry and Molecular Pharmacology, New York, NY 10016, USA\u003c/p\u003e \u003cp\u003eAkiko Koide \u0026amp; Shohei Koide\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eContributions\u003c/strong\u003e \u003cp\u003eCL and SG planned, conducted and analyzed most experiments. GM performed work on engineering and characterization of destabilized monobodies. FL conducted and analyzed initial experiments. AK and SK provided critical advice on the project and design of destabilized monobodies. AD and OH designed and coordinated the study, planned the experiments and analyzed data. AD, OH, CL and SG wrote the manuscript. All authors edited the manuscript.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCorresponding authors\u003c/strong\u003e \u003cp\u003eCorrepondence to Oliver Hantschel or Andreas Diepold\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eAK and SK are listed as inventors on issued and pending patents on the monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications). SK is a co-founder, receives consulting fees and hold equity in Aethon Therapeutics; is a co-founder and holds equity in Revalia Bio; has received research funding from Aethon Therapeutics, Argenx BVBA, Black Diamond Therapeutics, and Puretech Health, all outside of the current work. AD and FL are listed as inventors on an issued patent on the light-controlled T3SS filed by Max Planck Innovation (WO/2020/201115). The other authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e \u003ch2\u003eElectronic supplementary material\u003c/h2\u003e \u003cp\u003eSupplementary material includes ten figures and eight tables.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eWe acknowledge support by the European Research Council (Consolidator Grant; ERC-2016-CoG 682311) to O.H., C.L. and G.M., and by the Max Planck Society to A.D., S.G. and F.L.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCL and SG planned, conducted and analyzed most experiments. GM performed work on engineering and characterization of destabilized monobodies. FL conducted and analyzed initial experiments. AK and SK provided critical advice on the project and design of destabilized monobodies. AD and OH designed and coordinated the study, planned the experiments and analyzed data. AD, OH, CL and SG wrote the manuscript. All authors edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank all members of the Diepold and Hantschel labs for input and discussions. We additionally thank Katja Langenfeld (Diepold lab) for assistance with eukaryotic cell culture, Dominique Brandt (Worzfeld lab) for assistance with the generation of stable cell lines, and Prof. Samuel Wagner (University of T\u0026uuml;bingen) for the provision of the HeLa + LgBiT cell line. We acknowledge support by the European Research Council (Consolidator Grant; ERC-2016-CoG 682311) to O.H., C.L. and G.M., and by the Max Planck Society to A.D., S.G. and F.L.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhong L, et al. Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. 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Infect Immun. 2004;72:2879\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/iai.72.5.2879-2888.2004\u003c/span\u003e\u003cspan address=\"10.1128/iai.72.5.2879-2888.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4705983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4705983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe 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 \u003cem\u003eY. enterocolitica\u003c/em\u003e, 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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTo 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.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eHence, we establish the T3SS of \u003cem\u003eY. enterocolitica\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR::ABL1 signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 11:03:10","doi":"10.21203/rs.3.rs-4705983/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-15T18:26:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T09:19:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35151863594179406270034852130844514957","date":"2024-07-15T10:52:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79488795349637271038236193836945097319","date":"2024-07-14T10:48:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-13T16:08:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-10T23:08:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-10T23:07:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2024-07-08T13:39:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0bd7fa0b-307b-40e0-98ad-7bf6f54e86f5","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:08:18+00:00","versionOfRecord":{"articleIdentity":"rs-4705983","link":"https://doi.org/10.1186/s12964-024-01874-6","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2024-10-16 15:57:50","publishedOnDateReadable":"October 16th, 2024"},"versionCreatedAt":"2024-08-05 11:03:10","video":"","vorDoi":"10.1186/s12964-024-01874-6","vorDoiUrl":"https://doi.org/10.1186/s12964-024-01874-6","workflowStages":[]},"version":"v1","identity":"rs-4705983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4705983","identity":"rs-4705983","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}