Large-Scale Transient Transfection of Suspension-Adapted Chinese Hamster Ovary Cells for the Production of the Trimeric SARS-CoV-2 Spike Protein.

The production and purification of the secreted ectodomain of SARS-CoV-2 spike protein (S protein) were performed by transiently transfecting suspension-adapted Chinese hamster ovary cells (ExpiCHO). The method involved the separate addition of plasmid DNA expressing the S protein and polyethyleneimine to a suspension culture at a density of 5 × 10 cells/mL; and the subsequent addition of dimethyl sulfoxide at 2% (v/v). The transfected ExpiCHO cells were cultivated at 31 °C with agitation by orbital shaking under 5% CO.

Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research.

Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV).

Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50 mL and 600 mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20 mL and 400 mL, respectively.

Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies.

Broadly potent anti-SARS-CoV-2 antibody shares 93% of epitope with ACE2 and provides full protection in monkeys.

Objectives: Due to the rapid evolution of SARS-CoV-2 to variants with reduced sensitivity to vaccine-induced humoral immunity and the near complete loss of protective efficacy of licensed therapeutic monoclonal antibodies, we isolated a potent, broad-spectrum neutralizing antibody that could potentially provide prophylactic protection to immunocompromised patient populations.

Methods: Spike-specific B-cell clones isolated from a vaccinated post-infected donor were profiled for those producing potent neutralizing antibodies against a panel of SARS-CoV-2 variants. The P4J15 antibody was further characterized to define the structural binding epitope, viral resistance, and in vivo efficacy.

Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology.

High-affinity peptides developed against calprotectin and their application as synthetic ligands in diagnostic assays.

Common inflammatory disorders such as ulcerative colitis and Crohn's disease are non-invasively diagnosed or monitored by the biomarker calprotectin. However, current quantitative tests for calprotectin are antibody-based and vary depending on the type of antibody and assay used. Additionally, the binding epitopes of applied antibodies are not characterized by structures and for most antibodies it is unclear if they detect calprotectin dimer, tetramer, or both.

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes.

CD4 T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4 T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry.

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range.

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period).

Patient-derived monoclonal antibody neutralizes SARS-CoV-2 Omicron variants and confers full protection in monkeys.

The SARS-CoV-2 Omicron variant has very high levels of transmission, is resistant to neutralization by authorized therapeutic human monoclonal antibodies (mAb) and is less sensitive to vaccine-mediated immunity. To provide additional therapies against Omicron, we isolated a mAb named P2G3 from a previously infected vaccinated donor and showed that it has picomolar-range neutralizing activity against Omicron BA.1, BA.

Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC values of 34 nM.

A highly potent antibody effective against SARS-CoV-2 variants of concern.

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies, and reduced sensitivity to vaccine-induced immunity. Here, we screen B cells from COVID-19 donors and identify P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOCs) identified to date. Structural characterization of P5C3 Fab in complex with the spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction.

A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies.

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains.

Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs).

Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays an essential role in immunity, neuronal function, and aging through catalysis of the rate-limiting step in the kynurenine pathway of tryptophan metabolism. Many IDO1 inhibitors with different chemotypes have been developed, mainly targeted for use in anti-cancer immunotherapy. Lead optimization of direct heme iron-binding inhibitors has proven difficult due to the remarkable selectivity and sensitivity of the heme-ligand interactions.

Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses to the spike (S) protein monomer, S protein native trimeric form, or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection ( = 93) and in individuals enrolled in a postinfection seroprevalence population study ( = 578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein, or within a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute-infection-phase samples. Interestingly, compared to anti-S antibody responses, those against the N protein appear to wane in the postinfection cohort.

High resolution CryoEM structure of the ring-shaped virulence factor EspB from .

The EspB protein of is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of EspB by cryo-electron microscopic examination of single particles have been thwarted by severe orientation bias of the particles. This was overcome by using detergent as a surfactant thereby allowing reconstruction of the EspB structure at 3.

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains.

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains.

De novo development of proteolytically resistant therapeutic peptides for oral administration.

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation.

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms.

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs).

Inhibition Mechanisms of Indoleamine 2,3-Dioxygenase 1 (IDO1).

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the rate-limiting step in the kynurenine pathway of tryptophan metabolism, which is involved in immunity, neuronal function, and aging. Its implication in pathologies such as cancer and neurodegenerative diseases has stimulated the development of IDO1 inhibitors. However, negative phase III clinical trial results of the IDO1 inhibitor epacadostat in cancer immunotherapy call for a better understanding of the role and the mechanisms of IDO1 inhibition.

Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase.

The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH-PH unit in solution.