in this study, we present a straightforward approach for functional cell-based screening by coencapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarosecontaining microdroplets. our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and fAcS. this indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins. The importance of biopharmaceuticals (biologics) in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 2025 1. Frequently applied biologics comprise substances such as cytokines, monoclonal antibodies, hormones, soluble receptors, recombinant DNAs, enzymes, and synthetic vaccines. While biologics in targeted therapies often demonstrate remarkable safety and specificity, especially in case of autoimmune diseases 2 and cancer 3 , the discovery of novel molecules and the necessary functional validation still represent a bottleneck in the development of novel biopharmaceuticals. To overcome these shortcomings, powerful display technologies such as phage display 4 and yeast surface display 5 have been developed which allow for the isolation of specific high-affinity molecules and respective genes from complex variant libraries. However, identified binders frequently show poor physiological activity in a biological context. Thus, extensive secondary functional screens are necessary for identification of hit molecules with the desired functional activity. Furthermore, those screens require elaborate subcloning of the surface-displayed hits into soluble expression formats and outcoming clones frequently demand further optimization of physicochemical and pharmacokinetic properties 6. Consequently, implementing functional assays and phenotypic screens in an earlier selection phase appears highly beneficial for the discovery of new potent biologic drugs or even first-in-class medicines with novel molecular mechanisms of action 7. In this context, a major limitation is represented by the relatively low throughput of classical phenotypic screens, falling far behind the performance of high-diversity library-based approaches resting on affinity-driven selection protocols 8. Complex p...

Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high‐throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence‐activated cell sorting (FACS) for the isolation of antigen‐specific chicken‐derived antibodies is demonstrated. To this end, yeast‐displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target‐specific scFv‐Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG‐binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns.

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)‐based chicken monoclonal antibodies from an anti‐epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence‐activated cell sorting (FACS). For the selection of high‐affinity antibodies, a yeast cell library presenting cLC‐comprising fragment antigen binding (Fab) fragments is panned against hEGFR‐overexpressing A431 cells. The resulting cell–cell‐complexes are sorted by FACS resulting in gradual enrichment of EGFR‐binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC‐based bispecific antibodies against native mammalian receptors.

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point‐of‐care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo‐triggered cycloaddition followed by site‐specific sortase A‐catalyzed transamidation. This site‐directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper‐based diagnostics.

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.

Triggering apoptosis of tumor cells has been in focus of cancer‐inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death‐inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis‐inducing conjugates (EC50=2–20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme‐catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5‐targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50=2–7 nm), providing an option for improved in vivo half‐life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

Antibody‐drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody‐drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug‐to‐antibody ratios, remarkable hydrophilicity, and high toxicity in vitro .

Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.