A Competition-Based Strategy for the Isolation of an Anti-Idiotypic Blocking Module and Fine-Tuning for Conditional Activation of a Therapeutic Antibody.

The masking of therapeutic antibodies by the installation of a blocking module that can be removed under certain physiological conditions, is becoming increasingly important to improve their safety and toxicity profile. To gain access to such masking units, we used chicken immunization in combination with yeast surface display and a competition-based FACS screening campaign to obtain anti-idiotypic single-chain Fv (scFv) fragments. This approach promotes the identification of functional masking units, since specificity and high affinity do not necessarily guarantee a paratope blocking effect.

Tailor-Made Bioactive Papers by Site-Specific and Orthogonal Covalent Immobilization of Proteins.

A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells.

Conditional Cell Penetration of Masked CPPs by an ADEPT-like Approach.

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished.

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity.

Self-decorating cells surface-initiated enzymatic controlled radical polymerization.

Through the innovative use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant change in yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering.

Artificial cell synthesis using biocatalytic polymerization-induced self-assembly.

Artificial cells are biomimetic microstructures that mimic functions of natural cells, can be applied as building blocks for molecular systems engineering, and host synthetic biology pathways. Here we report enzymatically synthesized polymer-based artificial cells with the ability to express proteins. Artificial cells were synthesized using biocatalytic atom transfer radical polymerization-induced self-assembly, in which myoglobin synthesizes amphiphilic block co-polymers that self-assemble into structures such as micelles, worm-like micelles, polymersomes and giant unilamellar vesicles (GUVs).

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides.

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.

The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG.