Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ('nanobody') library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model's performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that provides a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

Au Nanoparticle-Based Amplified DNA Detection on Poly-l-lysine Monolayer-Functionalized Electrodes

Affinity sensing of nucleic acids is among the most investigated areas in biosensing due to the growing importance of DNA diagnostics in healthcare research and clinical applications. Here, we report a simple electrochemical DNA detection layer, based on poly-l-lysine (PLL), in combination with gold nanoparticles (AuNPs) as a signal amplifier. The layer shows excellent reduction of non-specific binding and thereby high contrast between amplified and non-amplified signals with functionalized AuNPs; the relative change in current was 10-fold compared to the non-amplified signal. The present work may provide a general method for the detection of tumor markers based on electrochemical DNA sensing.

Electrochemical Detection of Waterborne Bacteria Using Bi-Functional Magnetic Nanoparticle Conjugates

Detection of microbial contamination in water is imperative to ensure water quality. We have developed an electrochemical method for the detection of E. coli using bi-functional magnetic nanoparticle (MNP) conjugates. The bi-functional MNP conjugates were prepared by terminal-specific conjugation of anti-E. coli IgG antibody and the electroactive marker ferrocene. The bi-functional MNP conjugate possesses both E. coli-specific binding and electroactive properties, which were studied in detail. The conjugation efficiency of ferrocene and IgG antibodies with amine-functionalized MNPs was investigated. Square-wave voltammetry enabled the detection of E. coli concentrations ranging from 101–107 cells/mL in a dose-dependent manner, as ferrocene-specific current signals were inversely dependent on E. coli concentrations, completely suppressed at concentrations higher than 107 cells/mL. The developed electrochemical method is highly sensitive (10 cells/mL) and, coupled to magnetic separation, provides specific signals within 1h. Overall, the bi-functional conjugates serve as ideal candidates for electrochemical detection of waterborne bacteria. This approach can be applied for the detection of other bacteria and viruses.

Radiosynthesis and Evaluation of Cyclohexyl (5-(2-[11C-Carbonyl]acetamidobenzo[d]thiazol-6-yl)-2-Methylpyridin-3-yl)Carbamate ([11C]PK68) As a New Radioligand For Imaging Receptor-Interacting Protein 1 Kinase

Background: Receptor-interacting protein 1 kinase (RIPK1) is a key enzyme in the regulation of cellular necroptosis. Recently, cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate (PK68, 5) has been developed as a potent inhibitor of RIPK1. Herein, we radiosynthesized [11C]PK68 as a new positron emission tomography (PET) ligand for imaging RIPK1 and evaluated its potential in vivo.Results: We synthesized [11C]PK68 by reacting amine precursor 14 with [11C]acetyl chloride. At the end of synthesis, we obtained [11C]PK68 of 1200–1790 MBq (n = 10) with >99% radiochemical purity and a molar activity of 37–99 GBq/μmol starting from 18–33 GBq of [11C]CO2. The fully automated synthesis took 30 min from the end of irradiation. In a small-animal PET study, [11C]PK68 was rapidly distributed in the liver and kidneys of healthy mice after injection, and was subsequently cleared from their bodies via hepatobiliary excretion and the intestinal reuptake pathway. Although there was no obvious specific binding of RIPK1 in the PET study, [11C]PK68 demonstrated relatively high stability in vivo, and may be used as a lead compound for further candidate development.Conclusions: In the present study, we successfully radiosynthesized [11C]PK68 and evaluated its potential in vivo. We are planning to optimize the chemical structure of [11C]PK68 and conduct further PET studies on it using pathological models.

Fine pore engineering in a series of isoreticular metal-organic frameworks for efficient C2H2/CO2 separation

The separation of C2H2/CO2 is not only industrially important for acetylene purification but also scientifically challenging owing to their high similarities in physical properties and molecular sizes. Ultramicroporous metal-organic frameworks (MOFs) can exhibit a pore confinement effect to differentiate gas molecules of similar size. Herein, we report the fine-tuning of pore sizes in sub-nanometer scale on a series of isoreticular MOFs that can realize highly efficient C2H2/CO2 separation. The subtle structural differences lead to remarkable adsorption performances enhancement. Among four MOF analogs, by integrating appropriate pore size and specific binding sites, [Cu(dps)2(SiF6)] (SIFSIX-dps-Cu, SIFSIX = SiF62-, dps = 4.4’-dipyridylsulfide, also termed as NCU-100) exhibits the highest C2H2 uptake capacity and C2H2/CO2 selectivity. At room temperature, the pore space of SIFSIX-dps-Cu significantly inhibits CO2 molecules but takes up a large amount of C2H2 (4.57 mmol g−1), resulting in a high IAST selectivity of 1787 for C2H2/CO2 separation. The multiple host-guest interactions for C2H2 in both inter- and intralayer cavities are further revealed by dispersion-corrected density functional theory and grand canonical Monte Carlo simulations. Dynamic breakthrough experiments show a clean C2H2/CO2 separation with a high C2H2 working capacity of 2.48 mmol g−1.

An Original Approach to Measure Ligand/receptor Binding Affinity in Non-purified Samples

Several biochemical and biophysical methods are available to determine dissociation constants between a biological target and its ligands. Most of them require purification, labelling or surface immobilisation. However, these measurements remain challenging concerning membrane proteins because purification requires their extraction from the native lipid environment using different approaches, a process that may impact receptor conformation and functionality. We have developed a novel experimental procedure to determine binding affinities of a ligand to a membrane protein, the dopamine D2 receptor (D2R), directly from cell membrane fragments, using microscale thermophoresis (MST). Two main challenges had to be overcome: to determine the concentration of dopamine D2R in the crude sample; to find ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to be about 36.8 ± 2.6 pmol/mg. Then titration curves allowed the determination of a KD about 5.3 ± 1.7 nM, that is very close to the reported value. Important details of the experimental procedure are detailed to allow the transposition of this novel method to various membrane proteins.

Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of ‘Almost Natural’ Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, which results in its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule bound M87 in the crystal, but in solution recruited additional LL-37 molecules, consistent with a protein trap neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain, and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

Passive transfer of Ad26.COV2.S-elicited IgG from humans attenuates SARS-CoV-2 disease in hamsters

SARS-CoV-2 Spike-specific binding and neutralizing antibodies, elicited either by natural infection or vaccination, have emerged as potential correlates of protection. An important question, however, is whether vaccine-elicited antibodies in humans provide direct, functional protection from SARS-CoV-2 infection and disease. In this study, we explored directly the protective efficacy of human antibodies elicited by Ad26.COV2.S vaccination by adoptive transfer studies. IgG from plasma of Ad26.COV2.S vaccinated individuals was purified and transferred into naïve golden Syrian hamster recipients, followed by intra-nasal challenge of the hamsters with SARS-CoV-2. IgG purified from Ad26.COV2.S-vaccinated individuals provided dose-dependent protection in the recipient hamsters from weight loss following challenge. In contrast, IgG purified from placebo recipients provided no protection in this adoptive transfer model. Attenuation of weight loss correlated with binding and neutralizing antibody titers of the passively transferred IgG. This study suggests that Ad26.COV2.S-elicited antibodies in humans are mechanistically involved in protection against SARS-CoV-2.