A5026212691- Monoclonal and Polyclonal Antibodies Research
- Glycosylation and Glycoproteins Research
- Protein purification and stability
- Cell Adhesion Molecules Research
- Osteoarthritis Treatment and Mechanisms
- Transgenic Plants and Applications
- Extracellular vesicles in disease
- T-cell and B-cell Immunology
- RNA Interference and Gene Delivery
- Immunotherapy and Immune Responses
- Knee injuries and reconstruction techniques
- Toxin Mechanisms and Immunotoxins
- Advanced Biosensing Techniques and Applications
- SARS-CoV-2 and COVID-19 Research
- CAR-T cell therapy research
- Peptidase Inhibition and Analysis
- Protease and Inhibitor Mechanisms
- Galectins and Cancer Biology
- Immune Cell Function and Interaction
- HER2/EGFR in Cancer Research
- Allergic Rhinitis and Sensitization
- Viral Infectious Diseases and Gene Expression in Insects
- Food Allergy and Anaphylaxis Research
- Biosensors and Analytical Detection
- SARS-CoV-2 detection and testing
BOKU University
2015-2024
Institute of Molecular Biotechnology
2020-2024
Austrian Centre of Industrial Biotechnology (Austria)
2018-2024
Christian Doppler Laboratory for Thermoelectricity
2016
Blood Transfusion Centre of Slovenia
2001-2008
National Institute of Environmental Health Sciences
2004
Duke University Hospital
2004
Duke Medical Center
2004
University of Ljubljana
1997-2000
Educell
2000
Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip CH3 domain randomized. A high percentage these library members bound to soluble CD64 and Protein indicating that randomization step did not grossly interfere with overall structure displayed Fc. Sorting by FACS for binders against HER2/neu yielded antigen-specific (Fcab; antigen binding) one was affinity matured, resulting Fcab clone H10-03-6 showed >10-fold improvement...
The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode therapeutic action. As more therapeutics pass through the pipeline; however, it is clear that non-optimal biophysical can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems lead delay failure in traversing process. Aggregation propensity also correlated with increased...
Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody have primarily provided qualitative results, accurate seroprevalence studies and tracking of levels over time require highly specific, sensitive quantitative test setups.
We report the stabilization of human IgG1 Fc fragment by engineered intradomain disulfide bonds. One these bonds, which connects N-terminus CH3 domain with F-strand, led to an increase melting temperature this 10°C as compared in context wild-type region. Another bond, BC loop D-strand, resulted Tm 5°C. Combined one molecule, both bonds about 15°C. All mutations had no impact on thermal stability CH2 domain. Importantly, binding neonatal receptor was also not influenced mutations. Overall,...
Significance The broadly neutralizing anti–HIV-1 monoclonal antibody (mAb) PG9 requires multiple posttranslational modifications to exhibit its full biological activity, including proper N -glycosylation and tyrosine sulfation. We now describe a technology that permits the controlled synthesis of these in Nicotiana benthamiana . This allowed us show sulfated neutralizes HIV-1 with much higher potency than unsulfated antibody. also found glycooptimized mAb versions made plants are superior...
Abstract Introduction Allergen‐specific IgE‐blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal in serum are technically challenging we evaluated the applicability acidic disruption antibody‐allergen complexes by a modified ELISA protocol with monoclonal (mAbs) specific for relevant major allergens Betv1 and Mald1. Then, AIT‐induced blocking non‐blocking Mald1‐specific sera...
Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It highly glycosylated with five N-glycosylation sites that might impact it structurally and/or functionally. produced in mammalian cells exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression aflibercept plant-based system targeted profiles. Nicotiana benthamiana-based...
Abstract The research of extracellular vesicles (EVs) has boomed in the last decade, with promise them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties recipient cell that are vital for health host organism. To enhance ability their targeted delivery, we employed an intrinsically overrepresented protein, CD81, serve recognition desired target antigen. Yeast libraries displaying mutant variants large loop...
The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target virus-neutralizing antibodies present in convalescent sera COVID-19 patients and thus considered key antigen for immunosurveillance studies vaccine development. Although recombinant expression RBD has been achieved several eukaryotic systems, mammalian cells have proven particularly useful. authors aimed to optimize produced HEK293-6E towards stable homogeneous preparation addressed its O-glycosylation as...
Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage well-folded clones for efficient binder selection, information about correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination overall whole libraries IgG1-Fc on surface yeast flow cytometry. Libraries...
The application of recombinant antibodies for the analysis foods and food contaminants is now a major focus, given their capacity to be engineered tailor specificity, enhance stability, modify structural formats fit desired analytical platform. In this study, human scFv antibody fragments generated against aflatoxin B1 (AFB1) were selected as model explore effect on binding activity evaluate potential use immunoreagents contaminant analysis. Four (AFB1), previously isolated by chain...
Abstract Antigen‐binding Fc fragments (Fcabs) are a new unique class of immunotherapeutics. They small (50 kD) fully functional antibody alternatives that bind antigen and elicit effector functions such as antibody‐dependent cytotoxicity (ADCC) complement‐dependent cytotoxicity. Since Fcabs carry the natural FcRn binding site antibodies, they have very favorable pharmacokinetics. We showed recently Fcab H10‐03‐6 is high‐affinity binder Her‐2/neu (ErbB2/neu) mediating killing...
The binding site of ochratoxin A (OTA) within domain 2A human serum albumin (HSA) is examined by theoretical simulations and site-directed mutagenesis experiments. calculated constant, based on docking experiments affinity constants derived from the empirical free energy as implemented in AutoDock 3.0, for OTA dianion (3.7 × 106 M-1) good agreement with experimental value 5.2 M-1. carboxy terminus associates R218 R222 protein. Binding reduced over an order magnitude mutant R218A both...
Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically wide variety of target proteins. We describe the selection and affinity maturation Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate extent engineering permissible Fcabs we applied targeted mutagenesis all three C-terminal loop structures C-terminus CH3 domain isolate high-affinity binders by directed evolution yeast display. The matured clone, CT6,...
Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains attractive targets for engineering towards specific antigen recognition units. To enhance tolerance a tetraspanin fold to modification, we achieved significant thermal stabilization human CD81 large loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown exhibit positive shift melting temperature (Tm) up 25 °C. combination two most potent bonds...
We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for pair of CH3 efficient pairing heavy light variable domain is achieved using "Knobs-into-Holes" strategy. This construct, composed only naturally occurring immunoglobulin sequences without artificial linkers, expressed at high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed amino acid residues located interface was applied to improve biophysical properties...
Abstract Bispecific antibodies and antibody fragments are therapeutics of growing importance. They clinically applied for effector cell engagement, enhanced targeting selectivity, addressing multiple cellular pathways active transfer certain activities into difficult-to-reach compartments. These functionalities could profit from a third antigen specificity. In this work we have employed symmetrical bispecific parental mAb 2 format, which feature novel binding site in the C H 3 domains,...