Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research

Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time

Chromobodies have recently drawn great attention as bioimaging nanotools. They offer high antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Chromobodies can be used in live cell imaging for specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we develop multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies, and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we can control the release of His(6)-tagged chromobodies from MSNs in acidified endosomes and observe successful chromobody-antigen binding in the cytosol. Hence, by combining the two nanotools, chromobodies and MSNs, we establish a new powerful approach for chromobody applications in living cells

Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time

Chromobodies have recently drawn great attention as bioimaging nanotools. They offer high antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Chromobodies can be used in live cell imaging for specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we develop multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies, and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we can control the release of His(6)-tagged chromobodies from MSNs in acidified endosomes and observe successful chromobody-antigen binding in the cytosol. Hence, by combining the two nanotools, chromobodies and MSNs, we establish a new powerful approach for chromobody applications in living cells

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. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2-deoxyuridine (EdU) and 4,6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs

Bis‐ethynylphosphonamidates as an Modular Conjugation Platform to Generate Multi‐Functional Protein‐ and Antibody‐Drug‐Conjugates

Bis-ethynylphosphonamidates allow for a simple chemoselective addition of two thiol-containing modules in a row. We describe four such bis-electrophiles that carry different functional O-substituents with tunable hydrophilicity and enable further subsequent conjugations, thus facilitating a simple protocol for constructing protein-protein conjugates. An increased spacer length between the two ethynylphosphonamidates simplifies the formation of a conjugate from two bulky proteins. We apply these reagents to obtain homogeneous Antibody-Drug-Conjugates (ADCs) from DM1 and trastuzumab with excellent cytotoxicity and selectivity for the targeted cell line. Moreover, a bis-ethynylphosphonamidate, carrying an additional alkyne for a chemoselective triple conjugation, has been subjected to fluorescent labeling of an ADC specifically at the drug site give an Antibody-Drug-Fluorophore-Conjugate (ADFC), allowing for the observation of intracellular trafficking after ADC uptake into the targeted cell.LMU centerDeutsche Forschungsgemeinschaft (DFG)Einstein Foundation Berlin http://dx.doi.org/10.13039/501100006188Boehringer-Ingelheim FoundationFonds der Chemischen IndustrieLeibniz Association http://dx.doi.org/10.13039/501100001664German Federal Ministry for Economic Affairs and EnergyEuropean Social Fund http://dx.doi.org/10.13039/501100004895Bavarian Ministry of Economic Affairs, Regional Development and Energy http://dx.doi.org/10.13039/501100020639Peer Reviewe

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods

Stereoselective peptide catalysis in complex environments - from river water to cell lysates

Many stereoselective peptide catalysts have been established. They consist, like nature's catalysts, of amino acids but have significantly lower molecular weights than enzymes. Whereas enzymes operate with exquisite chemoselectivity in complex biological environments, peptide catalysts are used in pure organic solvents and at higher concentrations. Can a peptide catalyst exhibit chemoselectivity reminiscent of enzymes? Here, we investigated the properties of tripeptide catalysts in complex mixtures in hydrophobic and aqueous solvents. We challenged the catalysts with biomolecules bearing functional groups that could interfere by coordination or reaction with the peptide, the substrates, or intermediates. H-DPro-alpha MePro-Glu-NHC12H15 emerged through tailoring of the trans/cis ratio of the tertiary amide as a conformationally well-defined tripeptide that catalyzes C-C bond formations with high reactivity and stereoselectivity - regardless of the solvent and compound composition. The chemoselectivity of the tripeptide is so high that it even catalyzes reactions in cell lysates. The findings provoke the question of the potential role of peptide catalysis in nature and during the evolution of enzymes.ISSN:2041-6520ISSN:2041-653

Current Status: Site-Specific Antibody Drug Conjugates

Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability

Nanobodies and recombinant binders in cell biology.

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes