29 research outputs found
The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope.
No full textIn this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1
Wetlands and Water Framework Directive
No full textEnvironmental Science and Engineering; Water Policy/Water Governance/Water Management; Geoecology/Natural Processe
Wetlands and Water Framework Directive
No full textEnvironmental Science and Engineering; Water Policy/Water Governance/Water Management; Geoecology/Natural Processe
The shark strikes twice: Generation of Mono- and Bispecific High-Affinity vNAR Antibody Domains via Step-Wise Affinity Maturation
No full textThe aim of the work presented herein was the generation of high-affinity shark vNAR domains targeting different disease related antigens. For this, the natural IgNAR V domain repertoire of the bamboo shark (Chiloscyllium plagiosum) was analyzed and in analogy to these findings, a semi-synthetic complementarity determining region 3 (CDR3)-randomized Type IV vNAR library was constructed for yeast surface display. Through library screening against several disease-related antigens multiple different antigen-binding vNAR domains were isolated and characterized in terms of affinity, revealing moderate affinities in the triple-digit nano-molar to micro-molar range.
For optimization of antigen-binding vNAR domains, a new methodology for the affinity maturation was established that relies on the diversification of CDR1 of target-enriched binders. Sub-libraries were constructed in which five residues of the CDR1 loop were randomized and affinity-enhanced vNAR domains were identified by library screening using significantly decreased target concentrations. Affinities determined using yeast surface display revealed substantially affinity-optimized clones compared to parental molecules, obtained from initial library sorting.
Additionally, several vNAR domains were produced as soluble protein and characterized more meticulously in terms of affinity using bio-layer interferometry and in terms of stability via thermal shift assays. In this respect, affinities calculated by yeast surface display strongly correlated with affinities determined for soluble IgNAR V domains. Moreover, all produced vNAR variants exhibited high thermo-stability. Besides, an EpCAM-binding, affinity-matured vNAR domain was expressed as Fc-fusion protein in mammalian cells. Characterization of this formatted variant using bio-layer interferometry resulted in a moderately, but significantly enhanced affinity by the factor of three, presumably through avidity-effects.
Furthermore, also the generation of a new antigen-binding site into the IgNAR variable domain was in the scope of this work. This was achieved through the diversification of hypervariable loop 2 (HV2) of the IgNAR V domain and library screening. An EpCAM-specific vNAR was used as starting material and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM, resulting in bi-specific vNAR molecules. Essentially, it was demonstrated that a new paratope can be engineered into the vNAR scaffold that acts independently from the original antigen-binding site, composed of CDR3 and CDR1, as verified by yeast surface display
Spatial mapping of the leaf area index using remote sensing and ground measurements – the Biebrza National Park case study
No full textThe purpose of the described research was an attempt to estimate the leaf area index (LAI) parameter describing the structure of the vegetation based on the Landsat 5TM satellite imagery and field measurements made with the use of an optical plant canopy analyzer. The study was carried out in north-eastern Poland in the Biebrza river valley within the boundaries of the Biebrza National Park during the growing season of the year 2007. There were 13 spectral indices given in the literature known to be correlated with the LAI. The highest coefficient of determination and the highest correlation coefficient were obtained for the normalized difference vegetation index (NDVI) and the soil adjusted vegetation index (SAVI) indices for the wetland areas in the Biebrza river valley. The field measurements of the leaf area index and its spatial representation on satellite image show that the vegetation of natural river valleys is characterized by high spatial and seasonal variability. The study of the LAI on such large natural areas that are extensively used also requires knowledge of the methods of land use and the application of individual agrotechnical measures
Structural insights and biomedical potential of IgNAR scaffolds from sharks
No full textIn addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications
