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Bcl-2 family proteins are critical switches to control cell death and survival, and Bcl-2 is a key regulator in pro-survival signaling, causing various diseases including cancers. Bcl-2 has drawn a considerable attention as a potential target for developing a pro-apoptotic agent for cancers. We here present the development of a specific protein binder against human Bcl-2 and its cytosolic delivery to effectively induce apoptosis of cancer cells. The protein binder composed of leucine-rich repeat modules was selected for human Bcl-2, and its binding affinity was increased up to 60 nM through a modular evolution-based approach. The protein binder was efficiently delivered into cancer cells by an intracellular delivery system using a translocation domain from a bacterial exotoxin, resulting in a strong suppression of anti-apoptotic signaling in cancer cells. Our results demonstrate that the human Bcl-2-specific protein binder can act as a potent therapeutic agent for cancers. • We developed a protein binder which specifically binds to human Bcl-2. • The protein binder was selected against human Bcl-2 through phage display, and its binding affinity was matured by a modular approach. • Efficient intracellular delivery of the protein binder led to a significant suppression of anti-apoptotic signaling in cancer cells. • The protein binder showed a strong cytotoxicity against cancer cells in EGFR-specific manner, implying its great therapeutic potential. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors have drawn a consider attention as an effective cancer immunotherapy, and several monoclonal antibodies targeting the immune checkpoint receptors, such as human programmed cell death-1 (hPD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are clinically used for treatment of various cancers. Here we present the development of a small-sized protein binder which specifically binds to hPD‐1. The protein binder, which is composed of leucine-rich repeat (LRR) modules, was selected against hPD-1 through phage display, and its binding affinity was maturated up to 17 nM by modular evolution approach. The protein binder was shown to be highly specific for hPD-1, effectively inhibiting the interaction between hPD-1 and its ligand, hPD-L1. The protein binder restored T-cell function in vitro, and exhibited a strong anti-tumour activity in tumour mouse model, indicating that it acts as an effective checkpoint blockade. Based on the results, the developed protein binder specific for hPD-1 is likely to find a potential use in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Light‐driven control of biological processes using photoswitchable proteins allows high spatiotemporal interrogation or manipulation of such processes, assisting in understanding their functions. Despite considerable advances, however, the wide spread use of optical control has been hampered by a limited repertoire of photoswitchable proteins and a lack of generalized design strategy. Herein, we present a repeat module‐based rational design of a photoswitchable protein composed of LRR (Leucine‐rich repeat) modules using azobenzene as a photochromic ligand. Our design approach involves the rational selection of a Cβ pair between two nearby modules within a convex region and subsequent cross‐linking with a photochromic ligand. We demonstrate the general utility and potential of our strategy by showing the design of three target‐specific photoswitchable proteins and a light‐driven modulation of the cell signaling. With an abundance of LRR proteins in nature, our approach can expand the repertoire of photoswitchable proteins for light‐driven control of biological processes. [ABSTRACT FROM AUTHOR]

ABSTRACT A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Small GTPases are key signaling nodes that regulate the cellular processes and subcellular events, and their abnormal activities and dysregulations are closely linked with diverse cancers. Here, we report the development of conformation-selective protein binders for a KRAS mutant. The conformation-specific protein binders were selected from a repebody scaffold composed of LRR (Leucine-rich repeat) modules through phage display and modular engineering against constitute active conformation of KRAS. Epitope of the selected binders was mapped to be located close to switch I of KRAS. The conformation-selective protein binders were shown to effectively block the interaction between active KRAS and RAS-binding domain of BRAF, suppressing the KRAS-mediated downstream signaling. • We developed a conformation-selective protein binder against KRAS mutant. • The protein binder effectively inhibits the interaction of KRAS and BRAF. • The protein binder suppresses the KRAS-mediated downstream signaling. [ABSTRACT FROM AUTHOR]

The quest for highly sensitive and specific detection of disease biomarkers is high, despite many advances in analysis system. Here, we present a sensitive immunoassay platform using DNA-tethered gold nanoparticles and DNA-binding zinc fingers (ZFs). Monomeric alkaline phosphatase (mAP) and human TNF-α were employed as a signal generator and a disease biomarker, respectively. Gold nanoparticles (AuNPs) were first grafted with double-stranded DNAs having specific sequences for two different types of ZFs (QNK and zif268). The alkaline phosphatase and TNF-α-specific protein binder were genetically fused to each of two different types of ZFs, respectively, followed by conjugation with the DNA-tethered AuNPs in a sequence-specific manner. The use of the functionalized AuNPs as a signal generator in a colorimetric immunoassay of TNF-α led to LOD of 120 pg/ml, showing about 161-fold higher sensitivity than a protein binder-fused mAP. The present immunoassay platform could be applied to other analytes by simply replacing a targeting moiety, allowing a versatile and reproducible colorimetric immunoassay. Image 1 • AuNPs were functionalized with template DNA and zinc finger proteins (ZFs) in a sequence specific manner. • Functionalized AuNPs were used as a signal generator in colorimetric immunoassay. • The immunoassay platform showed a 161-fold lower limit of detection (LOD) for TNF-α than the previous system. • Developed immunoassay platform can be generally applied to other analytes by simply replacing a targeting moiety. • The immunoassay platform using functionalized AuNPs led to high sensitivity and reproducibility. [ABSTRACT FROM AUTHOR]

Human Bruton's tyrosine kinase (hBtk) plays a key role in growth and metabolism of B cells, but its dysfunctions cause various B-cell malignancies. Inhibitors targeting the ATP-binding pocket of hBtk have been developed, but they have several drawbacks such as adverse side effects and occurrence of drug-resistant mutations. Here, we present a protein binder which specifically binds to an allosteric regulatory SH2 domain of hBtk. The protein binder effectively inhibited the hBtk activity, indicating a critical role of the SH2 domain in allosteric regulation of the hBtk activity. Cytosolic delivery of the protein binder led to a significant inhibition on the BCR-mediated signaling and viability of B lymphoma cells. The utility of our approach was demonstrated by effective inhibition of drug-resistant hBtk variants by the protein binder. Based on the computationally predicted binding mode, the protein binder is likely to inhibit the hBtk activity by disrupting the interaction between the SH2 domain and kinase domain. The present approach can be used for developing therapeutic agents with improved efficacy for B-cell lymphoma. • We developed a protein binder which specifically binds to an allosteric regulatory SH2 domain of human Btk. • The protein binder effectively inhibits the hBtk activity of wild-type and even drug-resistant variants of hBtk. • Cytosolic delivery of the protein binder led to a significant suppression of BCR signaling pathway in B lymphoma cells. • We predicted binding interfaces of the SH2 domain and the kinase domain of hBtk based on a computational method. [ABSTRACT FROM AUTHOR]

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants. • Synthetic antibody A6 developed from repebody library blocks SARS-CoV-2 infections. • Bivalent A6 inhibit SARS-CoV-2 variants in vitro and in vivo with improved efficacy. • Linking to albumin binding domain enhances neutralization efficacy in mouse model. • Repebody scaffold-based synthetic antibody offer potential for COVID-19 treatment. [ABSTRACT FROM AUTHOR]

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas. [ABSTRACT FROM AUTHOR]

Small-sized non-antibody scaffolds have attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half-life is considered a drawback in the development of therapeutic agents. Here we demonstrate that a homo-dimeric form of a repebody enhances the anti-tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C-terminus of a respective human IL-6-specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo-dimeric form through interaction between spytag and spycatcher. The homo-dimeric repebody was detected as a single band in the SDS-PAGE analysis with an expected molecular size (78 kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL-6 molecules, and its binding affinity for hIL-6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half-life of small-sized protein binders, resulting in enhanced therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas. [ABSTRACT FROM AUTHOR]

The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli , respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli . The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment. [ABSTRACT FROM AUTHOR]

Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL −1 for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. [ABSTRACT FROM AUTHOR]

The strongest anaphylatoxin, C5a, plays a critical role in the proinflammatory responses, causing the pathogenesis of a number of inflammatory diseases including sepsis, asthma, and rheumatoid arthritis. Inhibitors of C5a thus have great potential as therapeutics for various inflammatory disorders. Herein, we present the development of a high-affinity repebody against human C5a (hC5a), which effectively suppresses the proinflammatory response. A repebody scaffold composed of leucine-rich repeat (LRR) modules was previously developed as an alternative protein scaffold. A repebody specifically binding to hC5a was selected through a phage display, and its affinity was increased up to 5 nM using modular engineering. The repebody was shown to effectively inhibit the production of C5a-induced proinflammatory cytokines by human monocytes. To obtain insight into a mode of action by the repebody, we determined its crystal structure in complex with hC5a. A structural analysis revealed that the repebody binds to the D1 and D3 regions of hC5a, overlapping several epitope residues with the hC5a receptor (hC5aR). It is thus likely that the repebody suppresses the hC5a-mediated immune response in monocytes by blocking the binding of hC5a to its receptor. The anti-hC5a repebody can be developed as a potential therapeutic for C5a-involved inflammatory diseases. [ABSTRACT FROM AUTHOR]