The Experts below are selected from a list of 1059 Experts worldwide ranked by ideXlab platform
Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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Abstract B27: A DARPin-based toolbox to understand and treat RAS-addicted cancers
Molecular Cancer Research, 2020Co-Authors: Jonas N. Kapp, Patrick Ernst, Jonas V. Schaefer, Wouter P. R. Verdurmen, Gabriela Nagy, Ralph Degen, Claudia Scholl, Andreas PlückthunAbstract:The repeating failure of small molecules as specific inhibitors of KRAS has drawn the attention to macromolecular structures, which can recognize their target with high affinity and specificity. Designed Ankyrin Repeat Proteins (DARPins) are multipurpose alternative affinity reagents that have proved to recognize targets with exceptional specificities and selectivity that often surpass those of antibodies. Due to their additional outstanding ability to act intracellularly within living cells and their recognition of structural rather than linear epitopes, DARPins have enabled a multitude of more advanced projects. In-house selections against GTP- or GDP-loaded KRAS were performed, and several hundred different DARPins could be identified. In subsequent validations, we not only analyzed their affinities, but also focused on the essential features of cross-reactivities, the recognition of various epitopes and their biologic functionalities. A number of candidates directly interfere with the RAS-Raf interaction and SOS-mediated nucleotide exchange by binding to the identical epitope on RAS as proved by crystal structures. One of these lead candidates with an affinity of 10 nM was used to elucidate whether anti-KRAS DARPins can mediate a biologic effect in model systems of human cancers. For this purpose, we chose cell lines categorized as KRAS dependent to generate stable cell lines expressing a highly active anti-KRAS DARPin under an inducible promotor, and it revealed potent antitumor activity, reducing the proliferation, colony formation and anchorage-independent growth. We could furthermore show that the observed effect resulted from reduced signaling of KRAS through its downstream MEK-ERK and PI3K-AKT pathways and the induction of apoptosis. Importantly, this anti-KRAS DARPin was shown to have no effects in the immortalized cell line HEK293T. In addition to traditional knockdown approaches, this model could be used to assess RAS dependency of human cancers. Combined with various suitable intracellular delivery techniques currently under development in our laboratory, the potential of this model will be further investigated for its potential in the treatment of solid tumors in a mouse model. In our broad effort many additional DARPins were identified that recognize different, nonoverlapping epitopes and most likely will interfere with other essential functions, such as the nanoclustering of RAS. This toolbox also allows the development of various biosensors in living cells. This large set of binders has the potential not just to help the community to gain detailed insights into the various functions and of RAS, but also might highlight novel vulnerabilities and innovative ways to finally make these key players druggable. Citation Format: Jonas N. Kapp, Jonas V. Schaefer, Wouter Verdurmen, Gabriela Nagy, Ralph Degen, Patrick Ernst, Claudia Scholl, Andreas Pluckthun. A DARPin-based toolbox to understand and treat RAS-addicted cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B27.
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Chaperone-assisted structure elucidation with DARPins
Current opinion in structural biology, 2020Co-Authors: Peer R. E. Mittl, Patrick Ernst, Andreas PlückthunAbstract:Designed ankyrin repeat proteins (DARPins) are artificial binding proteins that have found many uses in therapy, diagnostics and biochemical research. They substantially extend the scope of antibody-derived binders. Their high affinity and specificity, rigidity, extended paratope, and facile bacterial production make them attractive for structural biology. Complexes with simple DARPins have been crystallized for a long time, but particularly the rigid helix fusion strategy has opened new opportunities. Rigid DARPin fusions expand crystallization space, enable recruitment of targets in a host lattice and reduce the size limit for cryo-EM. Besides applications in structural biology, rigid DARPin fusions also serve as molecular probes in cells to investigate spatial restraints in targets.
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multispecific targeting with synthetic ankyrin repeat motif chimeric antigen receptors
Clinical Cancer Research, 2019Co-Authors: Ashwini Balakrishnan, Andreas Plückthun, Alexander I. Salter, Anusha Rajan, Paula L Kosasih, Jenna M Voutsinas, Michael C Jensen, Stanley R. RiddellAbstract:Purpose: The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. Experimental Design: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. Results: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen. Conclusions: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.
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rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts
Scientific Reports, 2019Co-Authors: Patrick Ernst, Annemarie Honegger, Peer R. E. Mittl, F Van Der Valk, Christina Ewald, Andreas PlückthunAbstract:Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.
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A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins
Nature Communications, 2019Co-Authors: Bunyarit Meksiriporn, Morgan B. Ludwicki, Erin A. Stephens, Allen Jiang, Lutz Kummer, Fabian Brandl, Hyeon-cheol Lee, Dujduan Waraho-zhmayev, Andreas Plückthun, Matthew P DelisaAbstract:There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.Protein phosphorylation helps to control many important cellular activities. Here the authors describe a genetic selection strategy to isolate designed ankyrin repeat proteins that bind specifically to phosphomodified targets.
G. M. Proshkina - One of the best experts on this subject based on the ideXlab platform.
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DARPin_9 29 targeted mini gold nanorods specifically eliminate her2 overexpressing cancer cells
ACS Applied Materials & Interfaces, 2019Co-Authors: G. M. Proshkina, A V Ryabova, S M Deyev, Francesco Tavanti, Maria Cristina Menziani, Roy Cohen, Liat Katrivas, Alexander KotlyarAbstract:We have demonstrated that designed ankyrin repeat protein (DARPin) _9-29, which specifically targets human epidermal growth factor receptor 2 (HER2), binds tightly to gold mini nanorods (GNRs). Molecular dynamic simulations showed that a single layer of DARPin_9-29 molecules is formed on the surface of the nanorod and that conjugation with the nanorod does not involve the protein’s domain responsible for specific binding to HER2. The nanorod-DARPin (DARPin-GNR) conjugate is specifically bound (in nanomolar concentrations) to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2. Illumination by near-infrared light (850 nm) led to almost complete eradication of the conjugate-treated SK-BR-3 cells; the viability of epithelial human breast cancer cells expressing normal amounts of the receptor was much less affected by the illumination. The results reported here pave the way toward application of DARPin-GNR conjugates in phototherapy of cancer.
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Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors.
Contrast media & molecular imaging, 2018Co-Authors: Anzhelika Vorobyeva, G. M. Proshkina, Mohamed Altai, Anna Orlova, Vladimir Tolmachev, Olga Bragina, Bogdan Mitran, Alexey Shulga, Vladimir Chernov, Sergey M. DeyevAbstract:High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9_29. DARPin 9_29 was labeled with iodine-125 by direct radioiodination and with [99mTc]Tc(CO)3 using the C-terminal hexahistidine tag. DARPin 9_29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [125I]I-DARPin 9_29 and [99mTc]Tc(CO)3-DARPin 9_29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 ± 0.7 %ID/g and 2.9 ± 0.7 %ID/g, respectively. This was significantly ( ) higher than the uptake of the same probes in HER2-negative Ramos lymphoma xenografts, 0.22 ± 0.09 %ID/g and 0.30 ± 0.05 %ID/g, respectively. Retention of [125I]I-DARPin 9_29 in the lung, liver, spleen, and kidneys was appreciably lower compared with [99mTc]Tc(CO)3-DARPin 9_29, which resulted in significantly ( ) higher tumor-to-organ ratios. The biodistribution data were confirmed by SPECT/CT imaging. In conclusion, radioiodine is a preferable label for DARPin 9_29.
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Synthesis, Characterization, and Selective Delivery of DARPin-Gold Nanoparticle Conjugates to Cancer Cells.
Bioconjugate chemistry, 2017Co-Authors: Sergey M. Deyev, G. M. Proshkina, Anastasiya V. Ryabova, Francesco Tavanti, Maria Cristina Menziani, Gennady Eidelshtein, Gavriel Avishai, Alexander KotlyarAbstract:We demonstrate that the designed ankyrin repeat protein (DARPin)_9–29, which specifically targets human epidermal growth factor receptor 2 (HER 2), binds tightly to gold nanoparticles (GNPs). Binding of the protein strongly increases the colloidal stability of the particles. The results of experimental analysis and molecular dynamics simulations show that approximately 35 DARPin_9–29 molecules are bound to the surface of a 5 nm GNP and that the binding does not involve the receptor-binding domain of the protein. The confocal fluorescent microscopy studies show that the DARPin-coated GNP conjugate specifically interacts with the surface of human cancer cells overexpressing epidermal growth factor receptor 2 (HER2) and enters the cells by endocytosis. The high stability under physiological conditions and high affinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer therapy.
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Cytotoxicity of targeted HER2-specific phototoxins based on flavoprotein miniSOG is determined by the rate of their internalization
Doklady Biochemistry and Biophysics, 2017Co-Authors: O. N. Shilova, G. M. Proshkina, A V Ryabova, S M Deyev, R. V. PetrovAbstract:The concept of targeted therapy implies the development of bifunctional agents complementing the therapeutic module with a targeting one. A promising target for the delivery of imaging and/or toxic modules is the HER2 (ErbB2) receptor. Earlier, we have functionally characterized the targeted photosensitizers 4D5scFv–miniSOG and DARPin–miniSOG, causing the death of HER2-overexpressing cells when irradiated with blue light. However, the cytotoxicity of targeted toxins 4D5scFv–miniSOG and DARPin–miniSOG (both having functionally active targeted and cytotoxic modules in recombinant proteins) against human breast adenocarcinoma cells differs 5 times. The study of the dynamics of internalization of 4D5scFv–miniSOG and DARPin–miniSOG proteins in the complex with HER2 in this work showed that the rate of internalization contributes most significantly to the toxicity of these photosensitizers, because it determines the duration of the presence of the phototoxin in the lipid bilayer of the cell membrane, where its damaging effect is maximum.
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Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo
Journal of controlled release : official journal of the Controlled Release Society, 2016Co-Authors: E. A. Sokolova, G. M. Proshkina, Olga M. Kutova, O. N. Shilova, Anastasiya V. Ryabova, Alexey A. Schulga, Oleg A. Stremovskiy, Tatiana A. Zdobnova, Irina V. Balalaeva, Sergey M. DeyevAbstract:DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin_9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth.
S M Deyev - One of the best experts on this subject based on the ideXlab platform.
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DARPin_9 29 targeted mini gold nanorods specifically eliminate her2 overexpressing cancer cells
ACS Applied Materials & Interfaces, 2019Co-Authors: G. M. Proshkina, A V Ryabova, S M Deyev, Francesco Tavanti, Maria Cristina Menziani, Roy Cohen, Liat Katrivas, Alexander KotlyarAbstract:We have demonstrated that designed ankyrin repeat protein (DARPin) _9-29, which specifically targets human epidermal growth factor receptor 2 (HER2), binds tightly to gold mini nanorods (GNRs). Molecular dynamic simulations showed that a single layer of DARPin_9-29 molecules is formed on the surface of the nanorod and that conjugation with the nanorod does not involve the protein’s domain responsible for specific binding to HER2. The nanorod-DARPin (DARPin-GNR) conjugate is specifically bound (in nanomolar concentrations) to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2. Illumination by near-infrared light (850 nm) led to almost complete eradication of the conjugate-treated SK-BR-3 cells; the viability of epithelial human breast cancer cells expressing normal amounts of the receptor was much less affected by the illumination. The results reported here pave the way toward application of DARPin-GNR conjugates in phototherapy of cancer.
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Cytotoxicity of targeted HER2-specific phototoxins based on flavoprotein miniSOG is determined by the rate of their internalization
Doklady Biochemistry and Biophysics, 2017Co-Authors: O. N. Shilova, G. M. Proshkina, A V Ryabova, S M Deyev, R. V. PetrovAbstract:The concept of targeted therapy implies the development of bifunctional agents complementing the therapeutic module with a targeting one. A promising target for the delivery of imaging and/or toxic modules is the HER2 (ErbB2) receptor. Earlier, we have functionally characterized the targeted photosensitizers 4D5scFv–miniSOG and DARPin–miniSOG, causing the death of HER2-overexpressing cells when irradiated with blue light. However, the cytotoxicity of targeted toxins 4D5scFv–miniSOG and DARPin–miniSOG (both having functionally active targeted and cytotoxic modules in recombinant proteins) against human breast adenocarcinoma cells differs 5 times. The study of the dynamics of internalization of 4D5scFv–miniSOG and DARPin–miniSOG proteins in the complex with HER2 in this work showed that the rate of internalization contributes most significantly to the toxicity of these photosensitizers, because it determines the duration of the presence of the phototoxin in the lipid bilayer of the cell membrane, where its damaging effect is maximum.
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Anti-HER2 phototoxin based on flavoprotein miniSOG causes the oxidative stress and necrosis of HER2-positive cancer cells
Moscow University Biological Sciences Bulletin, 2016Co-Authors: O. N. Shilova, G. M. Proshkina, A V Ryabova, S M DeyevAbstract:Development and functional characterization of novel, high-affinity protein compounds able to selectively kill human cancer cells is an urgent task of modern biomedical research. In this work, we studied the cytotoxicity of a recombinant phototoxic protein DARPin-miniSOG against the HER2-positive human breast adenocarcinoma cells. It was found that targeted phototoxin DARPin-miniSOG interacts specifically with HER2 receptor and causes the light-induced death of HER2-positive cells by the mechanism of necrosis. Irradiation of the cells in the presence of ascorbic acid eliminates the light-induced cytotoxicity of DARPin-miniSOG, which proves the prooxidant mechanism of phototoxin action.
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a new anticancer toxin based on her2 neu specific DARPin and photoactive flavoprotein minisog
Biochimie, 2015Co-Authors: G. M. Proshkina, O. N. Shilova, Oleg A. Stremovskiy, A V Ryabova, S M DeyevAbstract:Cytotoxic effects of a new targeted phototoxin DARPin-miniSOG and mechanism of its action were investigated in vitro. It was determined that DARPin-miniSOG causes light-induced death of HER2/neu-positive cancer cells (IC50 0.8 μM). Treatment of the cells with DARPin-miniSOG in the presence of ascorbic acid eliminated the light-induced cytotoxic action of the protein. This observation suggests the involvement of oxidative stress in the mechanism of the phototoxin action. DNA fragmentation analysis, caspase-3 activity assay and PI-staining of HER2/neu-positive cancer cells treated with DARPin-miniSOG indicated that phototoxin induces necrotic cell death under blue light illumination. Co-localization analysis showed that DARPin-miniSOG accumulates mostly in endosomes and lysosomes.
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highly specific hybrid protein DARPin mcherry for fluorescent visualization of cells overexpressing tumor marker her2 neu
Biochemistry, 2014Co-Authors: K. E. Mironova, G. M. Proshkina, Oleg A. Stremovskiy, O. N. Chernykh, A V Ryabova, S M DeyevAbstract:Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.
Sergey M. Deyev - One of the best experts on this subject based on the ideXlab platform.
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DARPins: Promising Scaffolds for Theranostics.
Acta naturae, 2019Co-Authors: O. N. Shilova, Sergey M. DeyevAbstract:Monoclonal antibodies are the classical basis for targeted therapy, but the development of alternative binding proteins has made it possible to use non-immunoglobulin proteins as targeting modules. The advantages of DARPins, scaffold proteins based on ankyrin repeats, over antibodies are as follows: small size, stability over a wide range of temperatures and pH values, low aggregation tendency, and ease of production in heterologous expression systems. The differences in the structure of the paratope of DARPin and antibodies broaden the spectrum of target molecules, while the ease of creating hybrid fusion proteins allows one to obtain bispecific and multivalent constructs. In this article, we summarize recent data on the development of therapeutic and imaging compounds based on DARPins.
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Indirect Radioiodination of DARPin G3 Using N-succinimidyl-Para-Iodobenzoate Improves the Contrast of HER2 Molecular Imaging.
International journal of molecular sciences, 2019Co-Authors: Anzhelika Vorobyeva, Alexey A. Schulga, Sergey M. Deyev, Sara S. Rinne, Tyran Günther, Anna Orlova, Vladimir TolmachevAbstract:Radionuclide molecular imaging of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal cancer might be used to stratify patients for HER2-targeted therapy as well as monitor treatment response and disease progression. Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins with favorable properties for molecular imaging. Herein we compared two methods for labeling the anti-HER2 DARPin (HE)3-G3, direct and indirect radioiodination. We hypothesized that the use of N-succinimidyl-para-iodobenzoate (SPIB) for radioiodination would facilitate the clearance of radiometabolites and improve the contrast of imaging. Both radiolabeled (HE)3-G3 variants preserved their binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was also demonstrated. A biodistribution comparison of [125I]I-(HE)3-G3 and [125I]I-PIB-(HE)3-G3, in mice bearing HER2 expressing SKOV3 xenografts, showed rapid clearance of [125I]I-PIB-(HE)3-G3 from normal organs and tissues and low accumulation of activity in organs with NaI-symporter expression. Both radiolabeled (HE)3-G3 variants had equal tumor uptake. Consequently, the indirect label provided higher tumor-to-blood and tumor-to-organ ratios compared with the direct label. Comparative Single Photon Emission Computed Tomography (SPECT)/CT imaging of HER2 expression in SKOV3 xenografts, using both radiolabeled DARPins, demonstrated the superior imaging contrast of the indirect label. Indirect radioiodination of (HE)3-G3 using SPIB could be further applied for SPECT and PET imaging with iodine-123 and iodine-124.
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Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors.
Contrast media & molecular imaging, 2018Co-Authors: Anzhelika Vorobyeva, G. M. Proshkina, Mohamed Altai, Anna Orlova, Vladimir Tolmachev, Olga Bragina, Bogdan Mitran, Alexey Shulga, Vladimir Chernov, Sergey M. DeyevAbstract:High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9_29. DARPin 9_29 was labeled with iodine-125 by direct radioiodination and with [99mTc]Tc(CO)3 using the C-terminal hexahistidine tag. DARPin 9_29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [125I]I-DARPin 9_29 and [99mTc]Tc(CO)3-DARPin 9_29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 ± 0.7 %ID/g and 2.9 ± 0.7 %ID/g, respectively. This was significantly ( ) higher than the uptake of the same probes in HER2-negative Ramos lymphoma xenografts, 0.22 ± 0.09 %ID/g and 0.30 ± 0.05 %ID/g, respectively. Retention of [125I]I-DARPin 9_29 in the lung, liver, spleen, and kidneys was appreciably lower compared with [99mTc]Tc(CO)3-DARPin 9_29, which resulted in significantly ( ) higher tumor-to-organ ratios. The biodistribution data were confirmed by SPECT/CT imaging. In conclusion, radioiodine is a preferable label for DARPin 9_29.
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Synthesis, Characterization, and Selective Delivery of DARPin-Gold Nanoparticle Conjugates to Cancer Cells.
Bioconjugate chemistry, 2017Co-Authors: Sergey M. Deyev, G. M. Proshkina, Anastasiya V. Ryabova, Francesco Tavanti, Maria Cristina Menziani, Gennady Eidelshtein, Gavriel Avishai, Alexander KotlyarAbstract:We demonstrate that the designed ankyrin repeat protein (DARPin)_9–29, which specifically targets human epidermal growth factor receptor 2 (HER 2), binds tightly to gold nanoparticles (GNPs). Binding of the protein strongly increases the colloidal stability of the particles. The results of experimental analysis and molecular dynamics simulations show that approximately 35 DARPin_9–29 molecules are bound to the surface of a 5 nm GNP and that the binding does not involve the receptor-binding domain of the protein. The confocal fluorescent microscopy studies show that the DARPin-coated GNP conjugate specifically interacts with the surface of human cancer cells overexpressing epidermal growth factor receptor 2 (HER2) and enters the cells by endocytosis. The high stability under physiological conditions and high affinity to the receptors overexpressed by cancer cells make conjugates of plasmonic gold nanostructures with DARPin molecules promising candidates for cancer therapy.
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Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo
Journal of controlled release : official journal of the Controlled Release Society, 2016Co-Authors: E. A. Sokolova, G. M. Proshkina, Olga M. Kutova, O. N. Shilova, Anastasiya V. Ryabova, Alexey A. Schulga, Oleg A. Stremovskiy, Tatiana A. Zdobnova, Irina V. Balalaeva, Sergey M. DeyevAbstract:DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin_9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth.
Ignacio Dolado - One of the best experts on this subject based on the ideXlab platform.
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design and characterization of mp0250 a tri specific anti hgf anti vegf DARPin drug candidate
mAbs, 2017Co-Authors: Kaspar H Binz, Savira Ekawardhani, Ulrike Fiedler, Talitha Bakker, Douglas Phillips, Andreas Cornelius, Christof Zitt, Thomas Göttler, Gabriel Sigrist, Ignacio DoladoAbstract:MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.
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Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate
mAbs, 2017Co-Authors: H. Kaspar Binz, Savira Ekawardhani, Ulrike Fiedler, Talitha Bakker, Douglas Phillips, Andreas Cornelius, Christof Zitt, Thomas Göttler, Gabriel Sigrist, Ignacio DoladoAbstract:MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.
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Abstract B25: First-in-human Phase I study to evaluate MP0250, a DARPin blocking HGF and VEGF, in patients with advanced solid tumors
Clinical Trials, 2015Co-Authors: Jordi Rodon, Ignacio Dolado, Christof Zitt, Aurelius Omlin, Karin H. Herbschleb, Javier Garcia-corbacho, Jan Steiner, Daniel Feurstein, Dascha Turner, Keith M. DawsonAbstract:Background: The VEGF/VEGFR and HGF/cMet pathways are implicated in tumor survival, growth, angiogenesis, invasion and metastasis. DARPins (designed ankyrin repeat proteins) are small genetically engineered proteins that bind to specific targets with high affinity. MP0250 is a first-in-class, tri-specific DARPin with the ability to simultaneously neutralize the activities of VEGF and HGF and also to bind to human serum albumin (HSA) to give an increased plasma half-life and potentially enhanced tumor penetration. MP0250 is currently being explored in a Phase I study. Methods: A phase I, open-label, repeated-dose, dose escalation, multi-center study to assess safety, tolerability and pharmacokinetics of MP0250 is in progress in patients with advanced solid tumors who have progressed on at least one prior standard therapy. Using a 3+3 design, eligible patients are being enrolled into dose escalation cohorts receiving MP0250 by intravenous infusion every other week until disease progression or unacceptable toxicity. Results: Twelve patients have been enrolled in the first three cohorts dosed with MP0250 at 0.5 (n = 3), 1.5 (n = 3) and 4 mg/kg (n = 6). MP0250 has been well tolerated and a maximum tolerated dose has not been reached. A single dose-limiting toxicity was observed at 4mg/kg (significant reduction in cardiac ejection fraction after 1st infusion in a patient with multiple cardiac risk factors). The most frequent adverse events (AEs, CTC version 4.03) were transient hypertension (42%), diarrhea (33%), fatigue (25%) and nausea (25%). With the exception of hypertension (grade 3 in 33% of all patients), all AEs were grade 1 or 2. Interim pharmacokinetic analyses indicated linear behavior between doses 1.5 and 4 mg/kg with a mean half-life of approximately 11 days (range 9-15 days). Sustained exposure was observed for all patients throughout the treatment periods analyzed, the longest to-date being 9 months. Stable disease for 10 months (treatment ongoing) has been observed in one patient with a head and neck tumor and for 8 months in a patient with a cervical adenocarcinoma. Conclusion: Preliminary data from the Phase I study of the first-in-class, tri-specific VEGF, HGF and HSA binding DARPin MP0250 shows it to be well tolerated, to have a mean half-life around 11 days and to have sustained exposure on repeated dosing. In addition, there was disease stabilization exceeding 8 months in two patients suggestive of anti-tumor activity. Further dose escalation is ongoing. Citation Format: Jordi Rodon, Aurelius Omlin, Karin H. Herbschleb, Javier Garcia-Corbacho, Jan Steiner, Ignacio Dolado, Christof Zitt, Daniel Feurstein, Dascha Turner, Keith M. Dawson, Michael T. Stumpp, Patrick Gilboy, Andreas Harstrick, Analia Azaro, Christoph J. Ackermann, Mark R. Middleton, Richard D. Baird. First-in-human Phase I study to evaluate MP0250, a DARPin blocking HGF and VEGF, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B25.
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Abstract P4-12-30: A bivalent Her2 targeting DARPin with high efficacy against Her2-low and Her2-positive tumors
Poster Session Abstracts, 2013Co-Authors: Ignacio Dolado, Michael T Stumpp, Ulrike Fiedler, H Strobel, Clara Metz, L RojkjaerAbstract:The approval of Herceptin, Perjeta and Kadcyla has dramatically improved the outcome of the 15% of breast cancer patients with high expression of Her2 (Her2-positive, IHC3+). In contrast, up to 50% of women with breast cancer express low levels of Her2 (Her2-low, IHC+2 or +1) at diagnosis, and these patients do not derive benefit from approved Her2 targeted therapies. It is estimated that 5-10% of Her2-positive patients will also develop Her2-low metastases refractory to treatment. Therefore, new therapeutic strategies are needed to alleviate the high tumor burden of Her2-low patients. We generated a bivalent Designed Ankyrin Repeat Protein (DARPin) containing two anti-Her2 moieties that target unique non-overlapping epitopes on Her2 (biparatopic). The biparatopic DARPin exerts a strong pro-apoptotic response on a panel of breast and gastric cancer cell lines, which cannot be fully recapitulated by treatment with Herceptin, Perjeta or both combined. DARPin treatment of Her2-positive breast cancer BT474 cells induces apoptosis (IC50 In summary, biparatopic targeting of Her2 enables inhibition of both Her2 and Her3, and blocks a tumor driver and its resistance mechanism. As a result, the DARPin interferes with both the proliferation and survival of tumor cells and triggers durable anti-tumor responses in vivo. DARPin treatment of Her2 positive tumors in mice show equivalent efficacy to Herceptin with faster kinetics; an effect that is magnified in Her2-low PDX tumors where the DARPin provides superior tumor control to Herceptin. Altogether, our data demonstrate the potential for the biparatopic DARPin to surpass some of the limitations of approved Her2 targeted agents, and warrants clinical investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-30.
