Biology subjects

Evers, A.

Publications and source records attributed to Evers, A..

6 recordsLinked to original sources

bioRxiv2026

Molecularly specific solubilization of therapeutic antibodies

Understanding the effects of formulation excipients on protein solubility is a key part of physical chemistry and pharmaceutical sciences. While excipients are routinely employed to reduce the self-association of biologic drugs, their mechanisms of action remain poorly understood and are often assumed to be broadly nonspecific. Using a high-throughput combinatorial droplet microfluidic platform, we systematically survey and quantify how common pharmaceutical excipients affect the solubility of a diverse panel of therapeutic monoclonal antibodies (mAbs). We show that, while excipients are generally solubilizing, their effects vary substantially across different mAbs, with excipient-specific solubilization scores spanning dynamic ranges of approximately 7-fold to >200-fold across the antibody panel. Histidine, arginine and sodium chloride, in particular, engage in interactions characterized by unique molecular specificity, whereas sucrose effects are largely governed by nonspecific, solvent-mediated interactions. Correlating excipient performance with dynamical mAb molecular features from solvated full-length homology models allows us to dissect and quantify the relative contributions of molecular features governing excipient-mediated solubilization. We envision this new physicochemical understanding lays the groundwork for rational excipient selection and bespoke formulation design, with direct implications for accelerating protein therapeutic development for preclinical scenarios.

biophysics↗

bioRxiv2023

PROxAb Shuttle: A non-covalent plug-and-play platform for the rapid generation of tumor-targeting antibody-PROTAC conjugates

Proteolysis-targeting chimeras (PROTACs) have evolved in recent years from an academic idea to a therapeutic modality with more than 25 active clinical programs. However, achieving oral bioavailability and cell-type specificity remains a challenge, especially for PROTACs recruiting the von Hippel-Lindau (VHL) E3 ligase. Herein, we present an unprecedented, plug- and-play platform for VHL-recruiting PROTACs to overcome these limitations. Our platform allows for the generation of non-covalent antibody-PROTAC complexes within minutes and obviates the need for prior PROTAC modification, antibody-drug linker chemistry optimization or bioconjugation. Our technology relies on camelid-derived antibody domains (VHHs) which can easily be engineered into existing therapeutic antibody scaffolds. The resulting targeted, bispecific fusion proteins can be complexed with PROTACs at defined PROTAC-to-antibody ratios and have been termed PROxAb Shuttles. PROxAb Shuttles can prolong the half-life of PROTACs from hours to days, demonstrate anti-tumor efficacy in vivo and have the potential for reloading in vivo to further boost efficacy.

biochemistry↗

bioRxiv2022

SUMO - In Silico Sequence Assessment Using Multiple Optimization Parameters

To select the most promising screening hits from antibody and VHH display campaigns for subsequent in-depth profiling and optimization, it is highly desirable to assess and select sequences on properties beyond only their binding signals from the sorting process. In addition, developability risk criteria, sequence diversity and the anticipated complexity for sequence optimization are relevant attributes for hit selection and optimization. Here, we describe an approach for the in silico developability assessment of antibody and VHH sequences. This method not only allows for ranking and filtering multiple sequences with regard to their predicted developability properties and diversity, but also visualizes relevant sequence and structural features of potentially problematic regions and thereby provides rationales and starting points for multi-parameter sequence optimization.

bioinformatics↗

bioRxiv2022

The SARS-CoV-2 accessory factor ORF7a downregulates MHC class I surface expression

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

microbiology↗

bioRxiv2020

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

This study examines how site-specific binding to the three identified neurosteroid binding sites in the 1{beta}3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3{beta}-epimer epi-allopregnanolone, binds to the canonical {beta}3(+)-1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the {beta}3 subunit, promoting receptor desensitization and the 1 subunit promoting ligand-specific effects. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for site-specific and general neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

biophysics↗

bioRxiv2019

Matrix-Assisted Laser Desorption-Ionization Time-of-Flight mass spectrometry using a custom-made database, biomarker assignment or mathematical classifiers does not differentiate Shigella spp. and Escherichia coli

PurposeShigella spp. and E. coli are closely related and cannot be distinguished using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish Shigella spp., E. coli, and its pathotype EIEC were explored. MethodsA custom-made database was developed, biomarkers were assigned, and classification models using machine learning were designed and evaluated using spectra of 456 Shigella spp., 42 E. coli, and 61 EIEC isolates, obtained by the direct smear method and the ethanol-formic acid extraction method. The isolates were identified using ipaH PCR and phenotypic and serological typing. ResultsIdentification with a custom-made database resulted in >94% Shigella identified at the genus level and >91% S. sonnei and S. flexneri at the species level, but distinction of S. dysenteriae, S. boydii, and E. coli was poor. Moreover, 10-15% of duplicates rendered discrepant results. With biomarker assignment, 98% S. sonnei isolates were correctly identified, although the S. sonnei biomarkers were not specific as other species, for instance S. boydii and E. coli were also identified as S. sonnei. Discriminating markers for S. dysenteriae, S. boydii, and E. coli were not assigned at all. Classifiers identified Shigella in 96% of isolates correctly, but most E. coli isolates were also assigned to Shigella. ConclusionNone of the proposed alternative approaches is suitable for use in clinical diagnostics for the identification of Shigella spp., E. coli, and EIEC, reflecting their relatedness and problematic taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the Shigella spp./E. coli complex, but other tests should be used for distinction.

microbiology↗