Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

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 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.

Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.

Generation of high‐affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken‐derived antibodies. To this end, high‐affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high‐affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high‐affinity binders in the scFv and Fab formats. Next‐generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high‐throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained Km and kcat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (Ki of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn2+-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner.