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Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao's base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation. [ABSTRACT FROM AUTHOR]

Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao’s base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation.

Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVβ3, αVβ5, αVβ6, αVβ8, and α5β1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 β-subunit partners: β6 = β8 > β3 > β1 = β5.

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens. [ABSTRACT FROM AUTHOR]

In a tour de force, Hart and colleagues recently used a technique known as BASEHIT (bacterial selection to elucidate host–microbe interactions in high throughput) to screen a yeast display library containing 3324 curated human exoproteins with 82 pathogen samples, focusing on vector-borne pathogens, to identify 1303 putative interactions. [ABSTRACT FROM AUTHOR]

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules. [ABSTRACT FROM AUTHOR]

Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from Streptococcus pneumoniae (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox. [ABSTRACT FROM AUTHOR]

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.

Leech hyaluronidase (LHyal) is a hyperactive hyaluronic acid (HA) hydrolase that belongs to the hyaluronoglucuronidase family. Traditionally, LHyal is extracted from the heads of leeches, but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides. However, at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps. Moreover, the enzyme is deactivated and removed after single use. To solve this problem, we developed a recyclable LHyal biocatalyst using a yeast surface display (YSD) system. After screening and characterization, we found that the cell wall protein Sed1p displayed stronger anchoring to the P. pastoris cell wall than other cell wall proteins. By optimizing the type and length of the linkers between LHyal and Sed1p, we increased the activity of enzymes displayed on the P. pastoris cell wall by 50.34% in flask cultures. LHyal-(GGGS)6-Sed1p activity further increased to 3.58 × 105 U mL−1 in fed-batch cultivation in a 5 L bioreactor. Enzymatic property analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme. Moreover, displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times. YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification process for HA oligosaccharides. Thus, this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

Abstract Molecular imaging plays important roles in many fields, including disease diagnosis, therapeutic efficacy evaluation, intraoperative imaging guidance, drug metabolism monitoring, and patient selection for appropriate treatment. As a key component, the targeting ligand determines the specificity, affinity, and in vivo performance of molecular imaging probes. In this review, high‐throughput screening and biological display platforms for the discovery of ligands applicable to molecular imaging are briefly reviewed. Basic information on ligand development for molecular imaging is first introduced, followed by a presentation of various selection platforms and typical or iterative cases. The features, advantages, limitations, and application scope of screening and display platforms are compared and discussed. Last, a basic selection strategy and a perspective for protein‐based ligands are provided.

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. [ABSTRACT FROM AUTHOR]

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

Synthetic cell–cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody–antigen pairs to a yeast–bacteria coincubation format and use flow cytometry to evaluate cell–cell interactions mediated by binding between surface‐displayed molecules. We further use fluorescence‐activated cell sorting to enrich a specific yeast‐displayed nanobody within a mixed yeast‐display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody–antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast–bacteria format supports efficient evaluation of ligand–target interactions. With further development, this format may facilitate systematic characterization and high‐throughput discovery of bacterial surface‐binding molecules. [ABSTRACT FROM AUTHOR]

Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from Streptococcus pneumoniae (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox.

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket. [ABSTRACT FROM AUTHOR]

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into crossreactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses. [ABSTRACT FROM AUTHOR]

Combinatorial biology methods such as phage and yeast display, suitable for the generation and screening of huge numbers of protein fragments and mutated variants, have been useful when dissecting the molecular details of the interactions between antibodies and their target antigens (mainly those of protein nature). The relevance of these studies goes far beyond the mere description of binding interfaces, as the information obtained has implications for the understanding of the chemistry of antibody–antigen binding reactions and the biological effects of antibodies. Further modification of the interactions through combinatorial methods to manipulate the key properties of antibodies (affinity and fine specificity) can result in the emergence of novel research tools and optimized therapeutics. [ABSTRACT FROM AUTHOR]

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into cross-reactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns. [ABSTRACT FROM AUTHOR]

We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN‐λ1, type 3 interferon (also known as IL‐29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL‐28 receptor 1 (IL‐28R1) and IL‐10 receptor 2 (IL‐10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN‐ λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine‐related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN‐λ. [ABSTRACT FROM AUTHOR]

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two‐in‐One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death‐ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties. [ABSTRACT FROM AUTHOR]

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two‐in‐One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death‐ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

Yeast has been established as a versatile platform for expressing functional molecules, owing to its well-characterized biology and extensive genetic modification tools. Compared to prokaryotic systems, yeast possesses advanced cellular mechanisms that ensure accurate protein folding and post-translational modifications. These capabilities are particularly advantageous for the expression of human-derived functional proteins. However, designing yeast strains as an expression platform for proteins requires the integration of molecular and cellular functions. By delving into the complexities of yeast-based expression systems, this review aims to empower researchers with the knowledge to fully exploit yeast as a functional platform to produce a diverse range of proteins. This review includes an exploration of the host strains, gene cassette structures, as well as considerations for maximizing the efficiency of the expression system. Through this in-depth analysis, the review anticipates stimulating further innovation in the field of yeast biotechnology and protein engineering.

Antiviral monoclonal antibody (mAb) discovery enables the development of antibody‐based antiviral therapeutics. Traditional antiviral mAb discovery relies on affinity between antibody and a viral antigen to discover potent neutralizing antibodies, but these approaches are inefficient because many high affinity mAbs have no neutralizing activity. We sought to determine whether screening for anti‐SARS‐CoV‐2 mAbs at reduced pH could provide more efficient neutralizing antibody discovery. We mined the antibody response of a convalescent COVID‐19 patient at both physiological pH (7.4) and reduced pH (4.5), revealing that SARS‐CoV‐2 neutralizing antibodies were preferentially enriched in pH 4.5 yeast display sorts. Structural analysis revealed that a potent new antibody called LP5 targets the SARS‐CoV‐2 N‐terminal domain supersite via a unique binding recognition mode. Our data combine with evidence from prior studies to support antibody screening at pH 4.5 to accelerate antiviral neutralizing antibody discovery. [ABSTRACT FROM AUTHOR]

Summary: Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

Abstract The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full‐length CD81 and expressed in EVs produced by HeLa cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin‐secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings.

Anaphylaxis represents one of the most severe and fatal forms of allergic reactions. Like most other allergies, it is caused by activation of basophils and mast cells by allergen-mediated cross-linking of IgE bound to its high-affinity receptor, FcεRI, on the cell surface. The systemic release of soluble mediators induces an inflammatory cascade, rapidly causing symptoms with peak severity in minutes to hours after allergen exposure. Primary treatment for anaphylaxis consists of immediate intramuscular administration of adrenaline. While adrenaline alleviates life-threatening symptoms of an anaphylactic reaction, there are currently no disease-modifying interventions available. We sought to develop potent and fast-acting IgE inhibitors with the potential to rapidly terminate acute allergic reactions. Using affinity maturation by yeast display and structure-guided molecular engineering, we generated 3 optimized disruptive IgE inhibitors based on designed ankyrin repeat proteins and assessed their ability to actively remove IgE from allergic effector cells in vitro as well as in vivo in mice. The engineered IgE inhibitors rapidly dissociate preformed IgE:FcεRI complexes, terminate IgE-mediated signaling in preactivated human blood basophils in vitro, and shut down preinitiated allergic reactions and anaphylaxis in mice in vivo. Fast-acting disruptive IgE inhibitors demonstrate the feasibility of developing kinetically optimized inhibitors for the treatment of anaphylaxis and the rapid desensitization of allergic individuals. [Display omitted] [ABSTRACT FROM AUTHOR]

The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full‐length CD81 and expressed in EVs produced by HeLa cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin‐secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings. [ABSTRACT FROM AUTHOR]

Yeast surface display is widely used in antibody discovery and optimization. However, traditional yeast display system could only display the antibody on the cell surface without the secretion of antibody to the cell culture supernatant for direct affinity measurement. To solve this problem, we generated the dual functional yeast display/ secretion system that displayed antibody crystallizable fragments (Fc) with Knob modified as "bait" to capture half molecular immunoglobulin (IgG) with Hole mutation, and secreted fully IgG assembled by Hole-Hole homodimer to the culture supernatant. Results showed that the heavy chain constant region 3 of immunoglobulin with Knob mutation was the best "bait" for antibody display. The dual functional yeast display/secretion system could not only efficiently identify the strains displayed high affinity antibodies from spiking library but also secrete the antibodies to the supernatant for direct affinity measurement. This study proves that the dual functional yeast display/ secretion system was successfully used for antibody affinity maturation, resulting in marked acceleration of the process. : [ABSTRACT FROM AUTHOR]

Abstract Background The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface. Results Fluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L. Conclusions FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti-HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP. [ABSTRACT FROM AUTHOR]

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies must have “drug-like” properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as “developability properties”: long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFβ-R2: transforming growth factor β-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda;

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8–3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl2 exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings. [ABSTRACT FROM AUTHOR]

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy. [ABSTRACT FROM AUTHOR]

• Developed and optimized a covalent SNAP tag quantification system for yeast protein display. • Demonstrated advantages of the SNAP tag at non-physiological pH, and to avoid cross-reactive labeling of antibody Fc genes. • Evaluated a synthetic SNAP-Fc gene library as a case study in Fc surface expression and binding to FcRn. • Quantitative analysis of sorted SNAP-Fc gene libraries using Next Generation Sequencing. Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.

Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein–protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.

Antibody discovery using invitro display technologies such as phage and/or yeast display has become acornerstone in many research and development projects, including the creation of new drugs for clinical use. Traditionally, after the selection phase, random clones are isolated for binding validation and Sanger sequencing. More recently, next-generation sequencing (NGS) technology has allowed deeper insight into the antibody population after aselection campaign, enabling the identification of many more specific binders. However, this approach only provides the DNA sequences of potential binders, the properties of which need to be fully elucidated by obtaining corresponding clones and expressing them for further validation. Here we present arapid novel method to harvest potential clones identified by NGS that uses asimple PCR and yeast recombination approach. The protocol was tested in selections against three different targets and was able to recover clones at an abundance level that would be impractical to identify using traditional methods.

Shedding of membrane-bound cell surface proteins, where the extracellular domain is released and found in the circulation is a common phenomenon. A prominent example is CEACAM5 (CEA, CD66e), where the shed domain plays a pivotal role in tumor progression and metastasis. For treatment of solid tumors, the presence of the tumor-specific antigen in the plasma can be problematic since tumor-specific antibodies might be intercepted by the soluble antigen before invading their desired tumor target area. To overcome this problem, we developed a generic procedure to generate bispecific antibodies, where one arm binds the antigen in a pH-dependent manner thereby enhancing antigen clearance upon endosomal uptake, while the other arm is able to target tumor cells pH-independently. This was achieved by incorporating pH-sensitive binding modalities in the common light chain IGKV3-15*01 of a CEACAM5 binding heavy chain only antibody. Screening of a histidine-doped light chain library using yeast surface display enabled the isolation of pH-dependent binders. When such a light chain was utilized as a common light chain in a bispecific antibody format, only the respective heavy/light chain combination, identified during selections, displayed pH-responsive binding. In addition, we found that the altered common light chain does not negatively impact the affinity of other heavy chain only binders toward their respective antigen. Our strategy may open new avenues for the generation of bispecifics, where one arm efficiently removes a shed antigen from the circulation while the other arm targets a tumor marker in a pH-independent manner.