David Fiebig

Ascendis Pharma

While yeast surface display (YSD) has gained traction for antibody hit discovery efforts with the first therapeutic YSD-isolated antibody sintilimab approved in 2018, a major drawback that remains is the time-consuming reformatting of monoclonal antibody (mAb) candidates. By using a Golden Gate cloning (GGC)-dependent workflow, the bulk transfer of...

The Tyro, Axl, and MerTK receptors (TAMRs) play a significant role in the clearance of apoptotic cells. In this work, the spotlight was set on MerTK, as it is one of the prominent TAMRs expressed on the surface of macrophages and dendritic cells. MerTK-specific antibodies were previously isolated from a transgenic rat-derived immune library with su...

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 o...

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these appr...

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 expres...

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...

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 rep...

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...

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here we report the isolation of common light chain (cLC)-based chicken monoclonal antibodies (mAbs) from an anti-epidermal growth factor receptor (EGFR) immune library utilizing yeast...

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Compl...

The Dispase autolysis-inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven-bladed β-propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes c...

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 five potential glutamines and ten lysines for MTG mediated cross-linking. Aim of the study was to determine the...

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....

Purification of the 39 kDa protein (β-lactamase Sml-1) by Fractogel EMD SO3¯ chromatography. Insert: protein pattern of the top fractions of peak 2 and peak 3. (TIF)

Separation of the 39 kDa protein (β-lactamase Sml-1) from SPI and SSTI by Fractogel EMD TMAE chromatography. Insert: protein pattern of the top fractions 1-3. (TIF)

Activity of the β-lactamase Sml-1 in surfaces colonies of S. mobaraensis. Overgrown agar plates were covered for 15 min (blue) and 30 min (red) using 0.15 mM nitrocefin. Increase in absorbance of nitrocefin supernatant aliquots was determined at 492 nm. (TIF)

Inhibition of the β-lactamase Sml-1 by penicillin G and ampicillin. The reaction mixtures contained besides ampicillin (blue) and penicillin G (red) 0.2 mM nitrocefin, 4 μM Sml-1, 50 mM NaCl and 50 mM Tris/HCl pH 7. (TIF)

Optimized gene sequence encoding the β-lactamase Sml-1 from S. mobaraensis for the production in E. coli. Modified codons are shown in red. (TIF)

Influence of pH (a) and temperature (b) on β-lactamase activity. The pH optimum was determined by monitoring continuously rSml-1 (1.6 μM) mediated hydrolysis of 0.1 mM nitrocefin in 50 mM buffer containing 50 mM NaCl up to 10 min at 492 nm (5.2 mU/ml = 100%). The used buffers were citrate (3–6.5, dark-blue), phosphate (6–8, green), Tris/HCl (7–9, p...

Transglutaminase mediated biotinylation of the recombinant 39 kDa protein (β-lactamase Sml-1) from S. mobaraensis. (a) Biotin blots showing streptavidin alkaline phosphatase stained rp39 linked to 1-N-biotinyl-6-N’-(carbobenzoxy-L-glutaminylglycyl)diamidohexane (labelling of lysines) and monobiotinylcadaverine (labelling of glutamines). Lanes 1/1’,...

Purification of the 39 kDa protein (β-lactamase Sml-1) from S. mobaraensis produced in E. coli BL21(DE3) RIL by IMAC. Insert: protein pattern of the top fractions of flowthrough (1) and the peaks 2/3. (TIF)

Hydrolysis of nitrocefin by the β-lactamase Sml-1 from S. mobaraensis. The reaction mediated by 3.5 μM Sml-1 in 50 mM Tris/HCl containing 50 mM NaCl were monitored at pH 7 for the indicated times and wave-length. The nitrocefin concentrations were 0 μM (pigeon blue), 60 μM (orange), 80 μM (light-blue), 100 μM (purple), 120 μM (green), 160 μM (Borde...