Ribosome Display Research Articles

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

BackgroundHuman interleukin-22 (IL-22) is known as a “dual function” cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis.MethodsWe used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis.ResultsWe demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A.ConclusionsWe developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

Development of uniform ribosome display technology enabling easy and efficient identification of full-length proteins that interact with bioactive small and large molecules.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

Non-invasive strategy: Developing a topical IL-4Rα-specific nanobody for the treatment of allergic airway diseases

Inhibiting IL-4 and IL-13 are critical cytokines that induce the pathogenic responses of allergic airway diseases. Currently, monoclonal antibodies targeting IL-4Rα are administered subcutaneously to treat eosinophilic rhinosinusitis and allergic asthma. However, these treatments have several drawbacks. To address these issues, we have developed a novel IL-4Rα-targeting nanobody designed for non-invasive delivery to local inflammatory sites in allergic airway diseases. H5, selected via the ribosomal display applied screening from synthetic nanobody library, underwent dimerization and in-silico affinity maturation using AlphaFold2 and GROMACS resulting in a substantial/dramatic enhancement of its binding affinity. H5 effectively controlled inflammatory markers such as MUC5AC, CCL26, and FOXJ1 in human nasal epithelial cells (HNECs) by inhibiting IL-4 and IL-13 signaling. The bivalent form of H5 showed efficacy in easily accessible cells, such as multi-ciliated cells, while the monovalent variant targeted hard-to-reach cells, such as basal cells of HNECs. In summary, we developed a nanobody that could effectively inhibit inflammatory signaling in HNECs via intranasal administration, showing promise as a non-invasive rhinitis treatment.

A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells

BackgroundInterleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target.MethodsAn albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking.ResultsWe demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells.ConclusionWe report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

Eukaryotic ribosome display for antibody discovery: A review.

Monoclonal antibody biologics have significantly transformed the therapeutic landscape within the biopharmaceutical industry, partly due to the utilisation of discovery technologies such as the hybridoma method and phage display. While these established platforms have streamlined the development process to date, their reliance on cell transformation for antibody identification faces limitations related to library diversification and the constraints of host cell physiology. Cell-free systems like ribosome display offer a complementary approach, enabling antibody selection in a completely in vitro setting while harnessing enriched cellular molecular machinery. This review aims to provide an overview of the fundamental principles underlying the ribosome display method and its potential for advancing antibody discovery and development.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders

Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of ∼104-fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.

Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies.

mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.

Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody–antigen interactions. The method, which we named ‘deep screening’, involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument’s flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

Single chain antibody fragment display systems: a review

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Antiviral Drugs and Vaccines for Omicron Variant: A Focused Review.

The Omicron variant of concern (VOC) replaced the delta variant rapidly and became the predominant strain due to more mutations in spike protein and receptor-binding domain (RBD) enhancing its infectivity and binding affinity. The severity of the illness is less than that of the delta variant. Omicron is nonsusceptible to REGEN-COV™ and bamlanivimab with etesevimab. Drugs that are effective against the Omicron variant are oral antiviral drugs such as Paxlovid (nirmatrelvir/ritonavir), remdesivir, sotrovimab, and molnupiravir. The potency of sotrovimab is reduced to 3-fold against Omicron, and 8-fold reduction in potency with sotrovimab is found in a particular variant of Omicron with a R346K substitution in spike protein. There are neither clinical trials comparing the efficacy of these 4 therapies with each other nor any data on a combination of two or more therapies. The current recommendation for mild-moderate, nonhospitalized patients who are at a high risk of disease progression is to use Paxlovid as the first-line option. If Paxlovid is not available or cannot be administered due to drug interactions, then the next best choice is sotrovimab. The third choice is remdesivir if sotrovimab is also not available and molnupiravir is to be given if the other three options are not available or cannot be administered. For prevention, 2130 (cilgavimab) in combination with COV2-2196 (tixagevimab) has been effective against BA.2 only. LY-CoV1404 (bebtelovimab) is recently authorized as it is effective against all sublineages of the Omicron variant. Regarding vaccine efficacy (VE), the 3-dose VE with mRNA vaccines at 14-60 days was found to be 71.6%, and after 60 days, it is 47.4%. There is a 34-38-fold reduction of neutralizing activity with prebooster sera and a 19-fold reduction with booster sera for the Omicron variant. This probably explains the reason for worldwide breakthrough infections with the Omicron variant with waning immunity. The neutralizing antibody response against Omicron elicited by the bivalent vaccine is superior to that of the ancestral Wuhan strain, without any safety concerns. For future advances, the ribosome display technology can be applied for the generation of human single-chain fragment variable (scFv) antibodies from B cells of recovered patients against Omicron and other Coronavirus variants as they are easier and faster to produce and have high affinity and high specificity.

Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10-8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

Exploring the ‘N-terminal arm’ & ‘Convex surface’ Binding Interfaces of the T3SS Chaperone-Translocator Complexes from P. Aeruginosa

One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial “toxins” via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator–chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH’s N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

Exploring the “N-Terminal Anchor” Binding Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa

The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bacterial cytosol through the host cell membrane and allows the direct injection of bacterial toxins. Effective pore formation is predicated by the translocator proteins binding to a small chaperone within the bacterial cytoplasm. Given the vital role of the chaperone-translocator interaction, we investigated the specificity of the "N-terminal anchor" binding interface present in both translocator-chaperone complexes from Pseudomonas aeruginosa. Isothermal calorimetry (ITC), alanine scanning, and the selection of a motif-based peptide library using ribosome display were used to characterize the major (PopB) and minor (PopD) translocator interactions with their chaperone PcrH. We show that 10 mer PopB51-60 and PopD47-56 peptides bind to PcrH with a KD of 148 ± 18 and 91 ± 9 μM, respectively. Moreover, mutation to alanine of each of the consensus residues (xxVxLxxPxx) of the PopB peptide severely affected or completely abrogated binding to PcrH. When the directed peptide library (X-X-hydrophobic-X-L-X-X-P-X-X) was panned against PcrH, there was no obvious convergence at the varied residues. The PopB/PopD wild-type (WT) sequences were also not prevalent. However, a consensus peptide was shown to bind to PcrH with micromolar affinity. Thus, selected sequences were binding with similar affinities to WT PopB/PopD peptides. These results demonstrate that only the conserved "xxLxxP" motif drives binding at this interface.

Combined invitro and cell-based selection display method producing specific binders against IL-9 receptor in high yields.

We combined cell-free ribosome display and cell-based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL-9Rα). The target, IL-9Rα, is the receptor involved in the signalling pathway of IL-9, a pro-inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths-the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target.

Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo.

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g-1 (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g-1; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.