A conformation-selective protein binder for a KRAS mutant inhibits the interaction between RAS and RAF.

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.

Construction and Functionalization of a Clathrin Assembly for a Targeted Protein Delivery.

Protein assemblies have drawn much attention as platforms for biomedical applications, including gene/drug delivery and vaccine, due to biocompatibility and functional diversity. Here, the construction and functionalization of a protein assembly composed of human clathrin heavy chain and light chain for a targeted protein delivery, is presented. The clathrin heavy and light chains are redesigned and associated with each other, and the resulting triskelion unit further self-assembled into a clathrin assembly with the size of about 28 nm in diameter. The clathrin assembly is dual-functionalized with a protein cargo and a targeting moiety using two different orthogonal protein-ligand pairs through one-pot reaction. The functionalized clathrin assembly exhibits about a 900-fold decreased KD value for a cell-surface target due to avidity compared to a native targeting moiety. The utility of the clathrin assembly is demonstrated by an efficient delivery of a protein cargo into tumor cells in a target-specific manner, resulting in a strong cytotoxic effect. The present approach can be used in the creation of protein assemblies with multimodality.

Cysteamine-Gold Coated Carboxylated Fluorescent Nanoparticle Mediated Point-of-Care Dual-Modality Detection of the H5N1 Pathogenic Virus.

Globally, point-of-care testing (POCT) is the most preferable on-site technique for disease detection and includes a rapid diagnostic test (RDT) and fluorescent immunochromatographic strip test (FICT). The testing kits are generally insufficient in terms of signal enhancement, which is a major drawback of this approach. Sensitive and timely on-site POCT methods with high signal enhancement are therefore essential for the accurate diagnosis of infectious diseases. Herein, we prepare cysteamine-gold coated carboxylated europium chelated nanoparticle (Cys Au-EuNPs)-mediated POCT for the detection of the H5N1 avian influenza virus (AIV). Commercial nanoparticles were used for comparison. The spectral characteristics, surface morphologies, functional groups, surface charge and stability of the Cys AuNPs, EuNPs, and Cys Au-EuNPs were confirmed by UV-visible spectrophotometry, fluorescence spectrometry, transmission electron microscope with Selected area electron diffraction (TEM-SAED), Fourier-transform infrared spectroscopy (FTIR) and zeta potential analysis. The particle size distribution revealed an average size of ~130 ± 0.66 nm for the Cys Au-EuNPs. The Cys Au-EuNP-mediated RDT (colorimetric analysis) and FICT kit revealed a limit of detection (LOD) of 10 HAU/mL and 2.5 HAU/mL, respectively, for H5N1 under different titer conditions. The obtained LOD is eight-fold that of commercial nanoparticle conjugates. The photo luminance (PL) stability of ~3% the Cys Au-EuNPs conjugates that was obtained under UV light irradiation differs considerably from that of the commercial nanoparticle conjugates. Overall, the developed Cys Au-EuNPs-mediated dual-mode POCT kit can be used as an effective nanocomposite for the development of on-site monitoring systems for infectious disease surveillance.

In Vitro Evaluation of Two Novel Antimalarial Derivatives of SKM13: SKM13-MeO and SKM13-F.

Antimalarial drugs play an important role in the control and treatment of malaria, a deadly disease caused by the protozoan parasite Plasmodium spp. The development of novel antimalarial agents effective against drug-resistant malarial parasites is urgently needed. The novel derivatives, SKM13-MeO and SKM13-F, were designed based on an SKM13 template by replacing the phenyl group with electron-donating (-OMe) or electron-withdrawing groups (-F), respectively, to reverse the electron density. A colorimetric assay was used to quantify cytotoxicity, and in vitro inhibition assays were performed on 3 different blood stages (ring, trophozoite, and schizonts) of P. falciparum 3D7 and the ring/mixed stage of D6 strain after synchronization. The in vitro cytotoxicity analysis showed that 2 new SKM13 derivatives reduced the cytotoxicity of the SKM13 template. SKM13 maintained the IC50 at the ring and trophozoite stages but not at the schizont stage. The IC50 values for both the trophozoite stage of P. falciparum 3D7 and ring/mixed stages of D6 demonstrated that 2 SKM13 derivatives had decreased antimalarial efficacy, particularly for the SKM13-F derivative. SKM13 may be comparably effective in ring and trophozoite, and electron-donating groups (-OMe) may be better maintain the antimalarial activity than electron-withdrawing groups (-F) in SKM13 modification.

Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. METHODS: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. RESULTS: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. CONCLUSIONS: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Computer-guided binding mode identification and affinity improvement of an LRR protein binder without structure determination.

Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG1 (hIgG1). We performed computationally-guided identification of the Rb:hIgG1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding.

Epitopes of Protein Binders Are Related to the Structural Flexibility of a Target Protein Surface.

Protein binders including antibodies are known not to bind to random sites of target proteins, and their functional effectiveness mainly depends on the binding region, called the epitope. For the development of protein binders with desired functions, it is thus critical to understand which surface region protein binders prefer (or do not prefer) to bind. The current methods for epitope prediction focus on static indicators such as structural geometry or amino acid propensity, whereas protein binding events are in fact a consequence of dynamic interactions. Here, we demonstrate that the preference for a binding site by protein binders is strongly related to the structural flexibility of a target protein surface. Molecular dynamics simulations on unbound forms of antigen structures revealed that the antigen surface in direct contact with antibodies is less flexible than the rest of the surface. This tendency was shown to be similar in other non-antibody protein binders such as affibody, DARPin, monobody, and repebody. We also found that the relatedness of epitopes to the structural flexibility of a target protein surface is dependent on the secondary structure elements of paratopes. Monobody and repebody, whose binding sites are composed of ß-strands, distinctively prefer to bind to a relatively more rigid region of a target protein. These observations enabled us to develop a simple epitope prediction method which shows a comparable performance to the commonly used ones.

Synergistic anticancer effect of docosahexaenoic acid and isoliquiritigenin on human colorectal cancer cells through ROS-mediated regulation of the JNK and cytochrome c release.

A large body of research has demonstrated a synergistic anticancer effect between docosahexaenoic acid (DHA) and standard chemotherapy regimens against colorectal cancer (CRC). In this study, we investigated the chemotherapeutic potential of cotreatment with DHA and isoliquiritigenin (ISL) against CRC HCT-116 cells. Apoptosis was confirmed by Annexin V/PI staining and expression of apoptosis-associated proteins. The synergistic effect of DHA and ISL combination on apoptosis was detected using combination index approaches. Flow cytometry was carried out using fluorescent probes to measure the production of reactive oxygen species (ROS). DHA and ISL in combination synergistically enhanced the decrease in cell viability versus the compounds used alone. Moreover, we demonstrated that the synergistic anti-CRC activity of cotreatment with these two compounds was achieved by inducing the apoptosis caspase-dependently mediated through augmented ROS generation followed by increased Fas ligand mRNA expression and cytochrome c release. Our data also demonstrated that cotreating with DHA and ISL strongly upregulated the phosphorylation of ERK and JNK, which are functionally associated with ROS induced by the two compounds in combination. Interestingly, further study revealed that inhibiting ERK phosphorylation strongly enhanced Fas ligand mRNA expression and the combination of the two compounds induced stronger cytotoxicity, whereas inhibiting JNK phosphorylation significantly reduced the apoptotic signals mediated by cotreatment with these two compounds. Excessive ROS-induced JNK activation and cytochrome c release from mitochondria played a key role in the synergistic anticancer activity of CRC cells by cotreating with DHA and ISL.

Targeted delivery of a human Bcl-2-specific protein binder effectively induces apoptosis of cancer cells.

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.

A regulatory SH2 domain-targeting protein binder effectively inhibits the activity of Bruton's tyrosine kinase and its drug-resistant variants.

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.

Effective inhibition of C3a-mediated pro-inflammatory response by a human C3a-specific protein binder.

Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases.

GradDock: rapid simulation and tailored ranking functions for peptide-MHC Class I docking.

Motivation: The identification of T-cell epitopes has many profound translational applications in the areas of transplantation, disease diagnosis, vaccine/therapeutic protein development and personalized immunotherapy. While data-driven methods have been widely used for the prediction of peptide binders with notable successes, the structural modeling of peptide binding to MHC molecules is crucial for understanding the underlying molecular mechanism of the immunological processes. Results: We developed GradDock, a structure-based method for the rapid and accurate modeling of peptide binding to MHC Class I (pMHC-I). GradDock explicitly models diverse unbound peptides in vacuo and inserts them into the MHC-I groove through a steered gradient descent with a topological correction process. The simulation process yields diverse structural conformations including native-like peptides. We completely revised the Rosetta score terms and developed a new ranking function specifically for pMHC-I. Using the diverse peptides, a linear programming approach is applied to find the optimal weights for the individual Rosetta score terms. Our examination revealed that a refinement of the dihedral angles and a modification of the repulsion can dramatically improve the modeling quality. GradDock is five-times faster than a Rosetta-based docking approach for pMHC-I. We also demonstrate that the predictive capability of GradDock with the re-weighted Rosetta ranking function is consistently more accurate than the Rosetta-based method with the standard Rosetta score (approximately three-times better for a cross-docking set). Availability and implementation: GradDock is freely available for academic purposes. The program and the ranking score weights for Rosetta are available at http://bel.kaist.ac.kr/research/GradDock. Contact: hskim76@kaist.ac.kr. Supplementary information: Supplementary data are available at Bioinformatics online.

B-SIDER: Computational Algorithm for the Design of Complementary ß-Sheet Sequences.

The ß-sheet is an element of protein secondary structure, and intra-/intermolecular ß-sheet interactions play pivotal roles in biological regulatory processes including scaffolding, transporting, and oligomerization. In nature, a ß-sheet formation is tightly regulated because dysregulated ß-stacking often leads to severe diseases such as Alzheimer's, Parkinson's, systemic amyloidosis, or diabetes. Thus, the identification of intrinsic ß-sheet-forming propensities can provide valuable insight into protein designs for the development of novel therapeutics. However, structure-based design methods may not be generally applicable to such amyloidogenic peptides mainly owing to high structural plasticity and complexity. Therefore, an alternative design strategy based on complementary sequence information is of significant importance. Herein, we developed a database search method called ß-Stacking Interaction DEsign for Reciprocity (B-SIDER) for the design of complementary ß-strands. This method makes use of the structural database information and generates target-specific score matrices. The discriminatory power of the B-SIDER score function was tested on representative amyloidogenic peptide substructures against a sequence-based score matrix (PASTA 2.0) and two popular ab initio protein design score functions (Rosetta and FoldX). B-SIDER is able to distinguish wild-type amyloidogenic ß-strands as favored interactions in a more consistent manner than other methods. B-SIDER was prospectively applied to the design of complementary ß-strands for a splitGFP scaffold. Three variants were identified to have stronger interactions than the original sequence selected through a directed evolution, emitting higher fluorescence intensities. Our results indicate that B-SIDER can be applicable to the design of other ß-strands, assisting in the development of therapeutics against disease-related amyloidogenic peptides.

Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery.

With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.

Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway.

The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.

Repeat Module-Based Rational Design of a Photoswitchable Protein for Light-Driven Control of Biological Processes.

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 Cß 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.

Anti-malarial effect of novel chloroquine derivatives as agents for the treatment of malaria.

BACKGROUND: The widespread emergence of anti-malarial drug resistance has necessitated the discovery of novel anti-malarial drug candidates. In this study, chloroquine derivatives were evaluated for the improved anti-malarial activity. RESULTS: Novel two derivatives (SKM13 and SKM14) were synthesized based on the chloroquine (CQ) template containing modified side chains such as α,ß-unsaturated amides and phenylmethyl group. The selective index indicated that SKM13 was 1.28-fold more effective than CQ against the CQ-resistant strain Plasmodium falciparum. An in vivo mouse study demonstrated that SKM13 (20 mg/kg) could completely inhibit Plasmodium berghei growth in blood and increased the survival rate from 40 to 100% at 12 days after infection. Haematological parameters [red blood cell (RBC) count, haemoglobin level, and haematocrit level] were observed as an indication of clinical malarial anaemia during an evaluation of the efficacy of SKM13 in a 4-day suppression test. An in vivo study showed a decrease of greater than 70% in the number of RBC in P. berghei-infected mice over 12 days, but the SKM13 (20 mg/kg)-treated group showed no loss of RBC. CONCLUSIONS: CQ derivatives with substituents such as α,ß-unsaturated amides and phenylmethyl group have enhanced anti-malarial activity against the CQ-resistant strain P. falciparum, and SKM13 is an excellent anti-malarial drug candidate in mice model.

Radiolabeling and preliminary biodistribution study of 99mTc-labeled antibody-mimetic scaffold protein repebody for initial clearance properties.

Antibody-mimetic proteins are intensively being developed for biomedical applications including tumor imaging and therapy. Among them, repebody is a new class of protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His6)-tag bearing repebody (rEgH9) was labeled with [99mTc]-tricarbonyl, and biodistribution was performed following intravenous (I.V.) or intraperitoneal (I.P.) injection. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. Biodistribution data indicates radiotracer has a rapid clearance from blood and excreted through the kidneys for intravenous (I.V.) injection, but comparatively slow clearance for an intraperitoneal (I.P.) injection. SPECT-CT images were found to be in agreement with biodistribution data, high activity was found inside kidneys. The observed result for rapid blood clearance and renal excretion of repebody (rEgH9) provide useful information for the further development of therapeutic strategy.

Multi-pulse flash light sintering of bimodal Cu nanoparticle-ink for highly conductive printed Cu electrodes.

In this work, bimodal Cu nano-inks composed of two different sizes of Cu nanoparticles (NPs) (40 and 100 nm in diameter) were successfully sintered with a multi-pulse flashlight sintering technique. Bimodal Cu nano-inks were fabricated and printed with various mixing ratios and subsequently sintered by a flash light sintering method. The effects of the flashlight sintering conditions, including irradiation energy and pulse number, were investigated to optimize the sintering conditions. A detailed mechanism of the sintering of bimodal Cu nano-ink was also studied via real-time resistance measurement during the sintering process. The sintered Cu nano-ink films were characterized using x-ray photoelectron spectroscopy and scanning electron microscopy. From these results, it was found that the optimal ratio of 40-100 nm NPs was found to be 25:75 wt%, and the optimal multi-pulse flash light sintering condition (irradiation energy: 6 J cm-2, and pulse duration: 1 ms, off-time: 4 ms, and pulse number: 5) was found. The optimally sintered Cu nano-ink film exhibited the lowest resistivity of 5.68 µΩ cm and 5B adhesion level.

Protein Binders Specific for Immunoglobulin G from Different Species for Immunoassays and Multiplex Imaging.

An immunoassay is the most widely used method for analyzing a variety of analytes based on antigen-antibody interactions in the biological and medical sciences. However, the use of secondary antibodies has certain shortcomings, such as a high cost, cross-reactivity, and loss of binding affinity during labeling. Herein, we present the development of repebodies specifically binding to immunoglobulin G with a different origin, which is a small-sized nonantibody scaffold composed of leucine-rich repeat (LRR) modules, for use in immunoassays and imaging. Repebodies specific for IgG from different species (i.e., mouse, human, and rabbit) were selected through a phage display, and their affinities were matured using a modular engineering approach. The respective repebodies were labeled with various signal generators such as horseradish peroxidase (HRP), a fluorescent dye, and quantum dots, and the resulting repebodies were used as alternatives to conventional secondary antibodies in typical immunoassays and imaging. The labeled repebodies enabled the detection of diverse target analytes with high sensitivity and specificity, showing a negligible cross-reactivity. Moreover, the repebodies labeled with different color-emitting quantum dots allowed the imaging of cell-surface receptors and proteins in a multiplex manner. The developed repebodies can be effectively used for sensitive immunoassays and multiplex imaging.