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The invention relates to methods of producing eukaryotic cell libraries encoding a repertoire of binding molecules (binders), wherein the methods use a site-specific nuclease for targeted cleavage of cellular DNA to enhance site-specific integration of binder genes through endogenous cellular repair mechanisms. Populations of eukaryotic cells are produced in which a repertoire of genes encoding binders are integrated into a desired locus in cellular DNA (e.g., a genomic locus) allowing expression of the encoded binding molecule, thereby creating a population of cells expressing different binders.


PubMed | Cancer Research UK Research Institute, Imperial College London, Roosevelt University and IONTAS Ltd
Type: Journal Article | Journal: The Biochemical journal | Year: 2015

Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes.


McCafferty J.,IONTAS Ltd.
Chemistry and Biology | Year: 2014

In this issue of Chemistry & Biology, Xie and colleagues describe a "phenotype directed" approach to identify antibodies that protect cells from death caused by rhinovirus infection. The cellular antibody library of 108 clones yielded two antibodies that prevented cell death via the same viral target: rhinovirus 3C protease. © 2014 Elsevier Ltd. All rights reserved.


Santamaria S.,University of Oxford | Santamaria S.,Imperial College London | Yamamoto K.,University of Oxford | Botkjaer K.,University of Cambridge | And 7 more authors.
Biochemical Journal | Year: 2015

Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes. © 2015 Authors; published by Portland Press Limited.


McCafferty J.,IONTAS Ltd | Schofield D.,MedImmune
Current Opinion in Chemical Biology | Year: 2015

The use of large genetically encoded binder libraries in co-operation with display technologies has matured over the past 25 years, and is now one of the primary methods used for selection of protein binders. Display technology has proven to be a robust and versatile method for generating binders to almost any antigen of interest. The evolution of this technology beyond antibody phage display has opened up new aspects for the concept of designer biologics. The ability to construct large populations of eukaryotic cells, including mammalian cells, where each cell expresses an individual antibody, peptide or engineered protein has added great value in identifying binders with desired properties. Here we review the evolution of display technology and highlight how it is being used today to generate binders with exquisite specificity, selectivity, affinity and developability characteristics. © 2015 .


The discovery and development of therapeutic antibodies to treat cancer or diseases of the immune system were the main subjects of the Biologics Congress 2015. Across the 2-day conference, approximately 150 participants, representing major European biopharmaceutical companies and visitors from the U.S., discussed the growth and development of the biologics market as more and more companies enter it. This report highlights presentations of interest from the 2 days. Copyright © 2015 Prous Science, S.A.U. or its licensors. All rights reserved.


Chapple S.D.,Iontas Ltd. | Dyson M.R.,Iontas Ltd.
Methods in Molecular Biology | Year: 2014

Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many variants including truncations, point mutations, investigation of orthologues, fusion to peptide or protein tags at the N-or C-terminus, the co-expression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multi-parallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multi-parallel method to express proteins in mammalian suspension cells by transient transfection in 24-well blocks. © 2014 Springer Science+Business Media, LLC.


PubMed | IONTAS Ltd.
Type: Comment | Journal: Chemistry & biology | Year: 2014

In this issue of Chemistry & Biology, Xie and colleagues describe a phenotype directed approach to identify antibodies that protect cells from death caused by rhinovirus infection. The cellular antibody library of 10(8) clones yielded two antibodies that prevented cell death via the same viral target: rhinovirus 3C protease.


Granata A.,University of Cambridge | Bernard W.G.,University of Cambridge | Zhao N.,Ohio State University | Mccafferty J.,Iontas Ltd. | And 2 more authors.
Stem Cells and Development | Year: 2015

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8-9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner. © Alessandra Granata et al. 2015; Published by Mary Ann Liebert, Inc.


PubMed | IONTAS Ltd
Type: | Journal: Advances in experimental medicine and biology | Year: 2016

Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions.

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