More than Toxins-Current Prospects in Designing the Next Generation of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) are rapidly becoming a cornerstone in targeted therapies, especially for the treatment of cancer. Currently, there are 12 FDA-approved ADCs, eight of which have been approved within the last five years, with numerous candidates in clinical trials. The promising clinical perspective of ADCs has led to the development of not only novel conjugation techniques, but also antibody formats, linkers, and payloads.

HP1β carries an acidic linker domain and requires H3K9me3 for phase separation.

Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β.

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody.

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads.

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates.

Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates.

Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties.

TuPPL: Tub-tag mediated C-terminal protein-protein-ligation using complementary click-chemistry handles.

We introduce a chemoenzymatic strategy for straightforward in vitro generation of C-terminally linked fusion proteins. Tubulin tyrosine ligase is used for the incorporation of complementary click chemistry handles facilitating subsequent formation of functional bispecific antibody-fragments. This simple strategy may serve as central conjugation hub for a modular protein ligation platform.

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies.

Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains.

A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation.

Actin and PIP3 waves in giant cells reveal the inherent length scale of an excited state.

The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion.

Membrane and actin reorganization in electropulse-induced cell fusion.

When cells of Dictyostelium discoideum are exposed to electric pulses they are induced to fuse, yielding motile polykaryotic cells. By combining electron microscopy and direct recording of fluorescent cells, we have studied the emergence of fusion pores in the membranes and the localization of actin to the cell cortex. In response to electric pulsing, the plasma membranes of two contiguous cells are turned into tangles of highly bent and interdigitated membranes.