63 research outputs found
Therapeutic antibody engineering by high efficiency cell screening
No full textAbstractIn recent years, several cell-based screening technologies for the isolation of antibodies with prescribed properties emerged. They rely on the multi-copy display of antibodies or antibody fragments on a cell surface in functional form followed by high through put screening and isolation of cell clones that carry an antibody variant with the desired affinity, specificity, and stability. Particularly yeast surface display in combination with high-throughput fluorescence-activated cell sorting has proven successful in the last fifteen years as a very powerful technology that has some advantages over classical generation of monoclonals using the hybridoma technology or bacteriophage-based antibody display and screening. Cell-based screening harbours the benefit of single-cell online and real-time analysis and characterisation of individual library candidates. Moreover, when using eukaryotic expression hosts, intrinsic quality control machineries for proper protein folding and stability exist that allow for co-selection of high-level expression and stability simultaneously to the binding functionality. Recently, promising technologies emerged that directly rely on antibody display on higher eukaryotic cell lines using lentiviral transfection or direct screening on B-cells. The combination of immunisation, B-cell screening and next generation sequencing may open new avenues for the isolation of therapeutic antibodies with prescribed physicochemical and functional characteristics
Design and testing of a thick-film dual-modality sensor for composition measurements in heterogeneous mixtures
No full textThe current paper focuses on design and laboratory evaluation of a dual-modality sensor, developed for the needs of oil and gas extraction industry to measure the composition of heterogeneous mixtures in harsh conditions. The sensor combines ultrasonic and electrical measurement techniques, which are non-destructive, rapid and can potentially provide an on-line industrial measurement. Such a ‘dual-modality’ measurement could potentially be reliable in a wider range of process conditions. A distinct feature of the sensors presented here is their construction, which makes use of the thick-film technology, enabling the construction of multi-layered structures of both conductive and non-conductive layers, some of which may exhibit piezoelectric properties for ultrasonic measurement purposes. These are later fired on a ceramic substrate to provide rugged sensors, capable of working in aggressive industrial environments. Laboratory experiments to investigate the feasibility of the dual-modality sensors were conducted and some comparisons with the theoretical predictions are presented
Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies
No full textRecent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies
Aptamers Binding to c-Met Inhibiting Tumor Cell Migration
No full textThe human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2’-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding
Investigation of Ruminococcus flavefaciens FD-1 cellulase
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Reason: ETDs are only available to UIUC Users without author permissionETDs are only available to UIUC Users without author permissionU of I OnlyThe cellulase system of the anaerobic bacterium Ruminococcus flavefaciens FD-1 was investigated. Through the use of ion-exchange chromatography and non-denaturing polyacrylamide gel electrophoresis, crude cellulase was found to contain no fewer than 18 endo--1,4-glucanase components and present in two cellulase complexes. No synergistic effect on \sp{14}C-cellulose degradation was observed when these two cellulase complexes were incubated together or when the cellulase complexes were incubated together or separately in the presence of an exo--1,4-glucanase produced by this organism (Exoglucanase A). Both cellulase complexes degraded xylan and the xylanase components migrated coincidently with all endo--1,4-glucanase components present in both cellulase complexes.A monoclonal antibody (MAb S1) generated against Exoglucanase A was used to investigate cellulolysis by R. flavefaciens. MAb S1 recognized few components in crude cellulase demonstrating its specificity for Exoglucanase A. MAb S1 was used to investigate the cellular location of the Exoglucanase A. Thin-sectioning of R. flavefaciens followed by immunolabelling, and examination using a transmission electron microscope showed a non-specific interaction of the monoclonal antibody for the embedding resin. Although, various treatments were tried to prevent this interaction, no conclusions about the cellular location of Exoglucanase A could be drawn. MAb S1 also inhibited the action of Exoglucanase A in vitro but failed to inhibit growth of R. flavefaciens when added to culture medium. This suggests the action of Exoglucanase A is not necessary for growth or the antibody concentration in the medium was too low to affect growth.Nutritional factors affecting cellulase expression in R. flavefaciens were also investigated. Cellobiose-grown cells produced more endo--1,4-glucanase and \sp{14}C-cellulase, but an equal amount of exo--1,4-glucanase compared to cellulose-grown cells. Growth on cellobiose or cellulose did not affect cellular location of either endo--1,4-glucanase or exo--1,4-glucanase, however cellulose-grown cells exhibited higher \sp{14}C-cellulase levels present in the supernatant as opposed to the cell pellet. Furthermore, addition of cellulose to a growing culture using cellobiose did not increase cellulase production compared to a control with no addition. Cellulase was constitutively produced in cellobiose- and cellulose-grown R. flavefaciens cells.Made available in DSpace on 2011-05-07T13:13:35Z (GMT). No. of bitstreams: 2
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Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.
No full textThe human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding
Analyses of the binding properties of CLN64.
No full text<p>(A–C) Representative dotblot analyses of the <sup>32</sup>P-ATP-labeled aptamer CLN64. The unselected rRfY aptamer pool R0, IgG1-Fc and EGFR-Fc served as negative control. Enlarged symbols represent the calculated K<sub>D</sub> value. (A) Specificity of CLN64 binding to c-Met in comparison to the unselected aptamer pool from 2’-fluoro pyrimidine composition (rRfY) analyzed with dotblot assays. (B) Cross-specificity of CLN64 binding to human c-Met and mouse c-Met by dotblot analyses. (C) Dotblot raw data; nitrocellulose readout for protein-bound CLN64 and PVDF readout for unbound CLN64. (D, E, F) Cellular binding analyses of truncated biotinylated aptamer CLN64 (CLN64-T). Previously described CLN3 was used as reference. CLN118 or CLN-F are unrelated aptamers with unmodified and 2’-fluoro modified compositions and served as negative controls. Biotinylated aptamers were detected with streptavidin phycoerythrin (SA-PE). Binding was tested on (D) EBC-1 cells with high c-Met surface levels, on (E) A549 cells with lower c-Met surface levels and on (F) MDA-MB-453 cells with no detectable c-Met surface levels. MDA-MB-453 cells express the related RON kinase on the cell surface. (G) For determination of the apparent cell surface binding constant, CLN64-T was titrated to EBC-1, A549 and Jurkat E6.1 cells (n = 2).</p
CLN3-T and CLN64-T influence on c-Met phosphorylation and signal transduction in A549 and EBC-1 cells.
No full text<p>(A, B) A549 cells or (C) EBC-1 cells were serum-starved and treated with indicated amounts of c-Met-specific CLN3-T, CLN64-T; non-c-Met binding aptamers CLN-X and CLNF. (A, B) A549 cells were additionally stimulated with HGF for 5 min. (A–C) Cell lysates were analyzed by immunoblotting for the availability of phosphorylated c-Met (Phospho-Met), c-Met, phosphorylated Akt (Phospho-Akt), Akt, phosphorylated MAPK (Phospho-MAPK), MAPK and the actin-binding protein Cofilin as loading control. (A) Both aptamers CLN3-T, CLN64-T inhibited HGF-mediated phosphorylation of c-Met and downstream transducers at 3 μM concentrations. No effect was observed for CLN-X and CLN-F. (B) CLN3-T, CLN64-T did not activate c-Met without HGF stimulation. (C) No inhibition of receptor phosphorylation was observed on EBC-1 cells expressing activated c-Met. Dotted lines indicate unconnected spots on the same membrane. The respective anti c-Met and anti Phospho-Met images were derived from two different gels.</p
Effects of CLN3-T and CLN64-T on HGF-induced migration on NCI-H441 cells and matrigel invasion of MDA-MB-231 cells.
No full text<p>NCI-H441 were grown until confluent and treated with CLN3-T, CLN64-T and CLN-X (negative control) at the indicated concentrations. A gap was introduced and cell migration was stimulated with 100 ng/ml HGF at maintained aptamer treatment. 1 μM of CLN3-T, CLN64-T inhibited HGF-mediated migration of NCI-H441 cells. Treatment of unrelated aptamer CLN-X did not show any effect on cell migration. Representative pictures of two independent experiments are shown.</p
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