Modular binder technology by NGS-aided, high-resolution selection in yeast of designed armadillo modules.

Establishing modular binders as diagnostic detection agents represents a cost- and time-efficient alternative to the commonly used binders that are generated one molecule at a time. In contrast to these conventional approaches, a modular binder can be designed in silico from individual modules to, in principle, recognize any desired linear epitope without going through a selection and hit-validation process, given a set of preexisting, amino acid-specific modules. Designed armadillo repeat proteins (dArmRP) have been developed as modular binder scaffolds, and we report here the generation of highly specific dArmRP modules by yeast surface display selection, performed on a rationally designed dArmRP library. A selection strategy was developed to distinguish the binding difference resulting from a single amino acid mutation in the target peptide. Our reverse-competitor strategy introduced here employs the designated target as a competitor to increase the sensitivity when separating specific from cross-reactive binders that show similar affinities for the target peptide. With this switch in selection focus from affinity to specificity, we found that the enrichment during this specificity sort is indicative of the desired phenotype, regardless of the binder abundance. Hence, deep sequencing of the selection pools allows retrieval of phenotypic hits with only 0.1% abundance in the selectivity sort pool from the next-generation sequencing data alone. In a proof-of-principle study, a binder was created by replacing all corresponding wild-type modules with a newly selected module, yielding a binder with very high affinity for the designated target that has been successfully validated as a detection agent in western blot analysis.

Prediction and design of protease enzyme specificity using a structure-aware graph convolutional network.

Site-specific proteolysis by the enzymatic cleavage of small linear sequence motifs is a key posttranslational modification involved in physiology and disease. The ability to robustly and rapidly predict protease-substrate specificity would also enable targeted proteolytic cleavage by designed proteases. Current methods for predicting protease specificity are limited to sequence pattern recognition in experimentally derived cleavage data obtained for libraries of potential substrates and generated separately for each protease variant. We reasoned that a more semantically rich and robust model of protease specificity could be developed by incorporating the energetics of molecular interactions between protease and substrates into machine learning workflows. We present Protein Graph Convolutional Network (PGCN), which develops a physically grounded, structure-based molecular interaction graph representation that describes molecular topology and interaction energetics to predict enzyme specificity. We show that PGCN accurately predicts the specificity landscapes of several variants of two model proteases. Node and edge ablation tests identified key graph elements for specificity prediction, some of which are consistent with known biochemical constraints for protease:substrate recognition. We used a pretrained PGCN model to guide the design of protease libraries for cleaving two noncanonical substrates, and found good agreement with experimental cleavage results. Importantly, the model can accurately assess designs featuring diversity at positions not present in the training data. The described methodology should enable the structure-based prediction of specificity landscapes of a wide variety of proteases and the construction of tailor-made protease editors for site-selectively and irreversibly modifying chosen target proteins.

A platform for the early selection of non-competitive antibody-fragments from yeast surface display libraries.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.

Leveraging yeast sequestration to study and engineer posttranslational modification enzymes.

Enzymes that catalyze posttranslational modifications (PTMs) of peptides and proteins (PTM-enzymes)-proteases, protein ligases, oxidoreductases, kinases, and other transferases-are foundational to our understanding of health and disease and empower applications in chemical biology, synthetic biology, and biomedicine. To fully harness the potential of PTM-enzymes, there is a critical need to decipher their enzymatic and biological mechanisms, develop molecules that can probe and modulate them, and endow them with improved and novel functions. These objectives are contingent upon implementation of high-throughput functional screens and selections that interrogate large sequence libraries to isolate desired PTM-enzyme properties. This review discusses the principles of Saccharomyces cerevisiae organelle sequestration to study and engineer PTM-enzymes. These include outer membrane sequestration, specifically methods that modify yeast surface display, and cytoplasmic sequestration based on enzyme-mediated transcription activation. Furthermore, we present a detailed discussion of yeast endoplasmic reticulum sequestration for the first time. Where appropriate, we highlight the major features and limitations of different systems, specifically how they can measure and control enzyme catalytic efficiencies. Taken together, yeast-based high-throughput sequestration approaches significantly lower the barrier to understanding how PTM-enzymes function and how to reprogram them.

An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.

Oral vaccination with recombinant Saccharomyces cerevisiae expressing Micropterus salmoides rhabdovirus G protein elicits protective immunity in largemouth bass.

Micropterus salmoides rhabdovirus (MSRV) is one of the main pathogens of largemouth bass, leading to serious economic losses. The G protein, as the only envelope protein present on the surface of MSRV virion, contains immune-related antigenic determinants, thereby becoming the primary target for the design of MSRV vaccines. Here, we displayed the G protein on the surface of yeast cells (named EBY100/pYD1-G) and conducted a preliminary assessment of the protective efficacy of the recombinant yeast vaccine. Upon oral vaccination, a robust immune response was observed in systemic and mucosal tissue. Remarkably, following the MSRV challenge, the relative percent survival of EBY100/pYD1-G treated largemouth bass significantly increased to 66.7 %. In addition, oral administration inhibited viral replication and alleviated the pathological symptoms of MSRV-infected largemouth bass. These results suggest that EBY100/pYD1-G could be used as a potential oral vaccine against MSRV infection.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1ß, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

Potent Apoptosis Induction by a Novel Trispecific B7-H3xCD16xTIGIT 2+1 Common Light Chain Natural Killer Cell Engager.

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity.

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10.

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. ß-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Construction of L-type lectin displaying Saccharomyces cerevisiae for Vibrio parahaemolyticus agglutination.

The utilization of Aga1P anchor protein in the display system for expressing heterologous proteins on the surface of Saccharomyces cerevisiae has been shown to be an ideal approach. This system has the ability to improve the expression of target proteins beyond the cell surface, resulting in increased activity and stability of the expression system. Recent studies have demonstrated that a new L-type lectin from Litopenaeus vannamei (LvLTLC1) has been found to possess the capability of agglutinating Vibrio parahaemolyticus, a pathogen responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, LvLTLC1 protein was designed to be expressed on the surface of S. cerevisiae via Aga1P anchor. The expression of LvLTLC1 protein on the surface of S. cerevisiae::pYIP-LvLTLC1-Aga1P was confirmed through the use of analytical techniques including SDS-PAGE, dot blot, and fluorescent immunoassay with LvLTC1-specific antibody. Subsequently, the newly generated yeast strain was evaluated for its ability to agglutinate V. parahaemolyticus and A. hydrophila. The obtained results indicated that S. cerevisiae expressing LvLTLC1 protein on its surface had the ability to agglutinate both AHPND-causing V. parahaemolyticus and A. hydrophila. This newly generated yeast strain could be served as a feed supplement for controlling bacteria in general and AHPND in particular.

Development of a TNF-α-mediated Trojan Horse for bacteria-based cancer therapy.

Tumor necrosis factor α (TNF-α) is upregulated in a chronic inflammatory environment, including tumors, and has been recognized as a pro-tumor factor in many cancers. Applying the traditional TNF-α antibodies that neutralize TNF-α activity, however, only exerts modest anti-tumor efficacy in clinical studies. Here, we develop an innovative approach to target TNF-α that is distinct from the neutralization mechanism. We employed phage display and yeast display to select non-neutralizing antibodies that can piggyback on TNF-α and co-internalize into cells through receptor ligation. When conjugating with toxins, the antibody exhibited cytotoxicity to cancer cells in a TNF-α-dependent manner. We further implemented the immunotoxin to an E. coli vehicle specially engineered for a high secretion level. In a syngeneic murine melanoma model, the bacteria stimulated TNF-α expression that synergized with the secreted immunotoxin and greatly inhibited tumor growth. The treatment also dramatically remodeled the tumor microenvironment in favor of several anti-tumor immune cells, including N1 neutrophils, M1 macrophages, and activated CD4+ and CD8+ lymphocytes. We anticipate that our new piggyback strategy is generalizable to targeting other soluble ligands and/or conjugates with different drugs for managing a diverse set of diseases.

Engineering a potent receptor superagonist or antagonist from a novel IL-6 family cytokine ligand.

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity.

Ubiquitination is the covalent attachment of ubiquitin (Ub) to substrate proteins and regulates several cellular processes, including protein degradation. Ub ligases (E3s) bring a Ub-conjugated enzyme E2 (E2-Ub) and the target protein closer to enable ubiquitination. In this study, we engineered a U-box domain of human U-box-type E3 E4B (E4BU) to enhance its function as a Ub ligase by accelerating the rate of Ub transfer directly from Ub-loaded human E2 UbcH5b (E2(UbcH5b)-Ub) to the proximal substrate. We developed a functional screening system for the E4BU library using a yeast surface display system combined with fluorescence-activated cell sorting (FACS) to isolate functionally improved variants. This phenotypic screening system yielded an E4BU variant, E4BU(#8), which exhibited an approximately 4-fold greater Ub ligase activity rate in the yeast displayed form than that of the E4BU wild-type. When E4BU(#8) was fused to a green fluorescent protein (GFP)-specific nanobody, the fusion protein polyubiquitinated GFP in proportion to the concentration and incubation time, with an approximately 3-fold faster Ub ligase activity rate than the previously isolated E4BU(NT) variant. Importantly, the engineered E4BU(#8) retained endogenous Lys48-linked polyubiquitination activity, which is essential for substrate degradation by the 26S proteasome. Our results indicated that E4BU(#8), which binds to and allosterically stimulates E2(UbcH5b)-Ub to enhance Ub transferase activity to a substrate, may be valuable in designing biological molecules for targeted protein degradation.

Safety profile of intravenous administration of live Pichia pastoris cells in mice.

Pichia pastoris has been widely used to produce antigenic proteins aimed to integrate subunit vaccines. Moreover, increasing interest in large-scale vaccine production at the lowest cost is rapidly focusing in the development of yeast surface display (YSD) systems for delivery of antigens. In this scenario, the safety of live yeast administration must be warranted, however, such information is very scarce. Here, we assess the intravenous administration (i.v.) of live P. pastoris cells in order to trace dissemination in BALB/c mice and to evaluate the immune response raised against the yeast compared to the well-defined pathogen Candida albicans. Our results demonstrate dissemination of P. pastoris to the heart, kidney, and spleen, but it is quickly eliminated during the first 48 h postinfection (hpi), with persistence in the liver along with mild mononuclear (MN) and polymorphonuclear (PMN) infiltrate, which was resolved at 144 hpi. In vivo delayed-type hypersensitivity test (DTH) or in vitro antigenic stimulation of mice splenocytes demonstrate that transient infection of P. pastoris did not induce a cell-mediated immune response nor increase the level of circulating IgG or IgM. These results demonstrate the innocuous profile of P. pastoris and support its use as a safe delivery system for vaccine development.

"Human and Mouse Cross-Reactive" Albumin-Binding Helix-Loop-Helix Peptide Tag for Prolonged Bioactivity of Therapeutic Proteins.

The effectiveness of protein and peptide pharmaceuticals depends essentially on their intrinsic pharmacokinetics. Small-sized pharmaceuticals in particular often suffer from short serum half-lives due to rapid renal clearance. To improve the pharmacokinetics by association with serum albumin (SA) in vivo, we generated an SA-binding tag of a helix-loop-helix (HLH) peptide to be linked with protein pharmaceuticals. For use in future preclinical studies, screening of yeast-displayed HLH peptide libraries against human SA (HSA) and mouse SA (MSA) was alternately repeated to give the SA-binding peptide AY-VE, which exhibited cross-binding activities to HSA and MSA with KD of 65 and 20 nM, respectively. As a proof of concept, we site-specifically conjugated peptide AY-VE with insulin to examine its bioactivity in vivo. In mouse bioassay monitoring the blood glucose level, the AY-VE conjugate was found to have a prolonged hypoglycemic effect for 12 h. The HLH peptide tag is a general platform for extending the bioactivity of therapeutic peptides or proteins.

Dietary intake of GDF11 delays the onset of several biomarkers of aging in male mice through anti-oxidant system via Smad2/3 pathway.

Current studies have generated controversy over the age-related change in concentration of growth differentiation factor 11 (GDF11) and its role in the genesis of rejuvenation conditions. In this study, we displayed rGDF11 on the surface of Yarrowic Lipolytica (Y. lipolytica), and proved the bioavailability of the yeast-displayed rGDF11 by oral delivery in aged male mice. On the basis of these findings, we started to explore the anti-aging activity and underlying mechanisms of displayed rGDF11. It was found that dietary intake of displayed rGDF11 had little influence on the body weight and biochemical parameters of aged male mice, but delayed the occurrence and development of age-related biomarkers such as lipofuscin (LF) and senescence-associated-ß-galactosidase, and to some extent, prolonged the lifespan of aged male mice. Moreover, we demonstrated once again that dietary intake of displayed rGDF11 enhanced the activity of anti-oxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX), reduced the reactive oxygen species (ROS) level, and slowed down the protein oxidation and lipid peroxidation. Importantly, we showed for the first time that rGDF11 enhanced the activity of CAT, SOD and GPX through activation of the Smad2/3 signaling pathway. Our study also provided a simple and safe route for delivery of recombinant GDF11, facilitating its therapeutic application in the future.