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Gaithersburg, MD, United States

Schwartz J.A.,Charles River Associates | Solomon J.A.,Charles River Associates | Henkelman K.,Charles River Associates | Leininger J.R.,MedImmune | And 2 more authors.
Toxicologic Pathology | Year: 2011

A single, solid, yellow-white thymic mass was found at necropsy of a two-year-old female cynomolgus macaque from a four-week, repeat-dose toxicity and immunogenicity study. Microscopically, the mass was multilobular and well encapsulated, surrounded by a thick connective tissue capsule, and composed of dense sheets of elongate or spindle-shaped cells and large cystic cavities separated by thick connective tissue stroma. Normal thymus was adjacent to the mass, but it was compressed. Within the mass were abundant interspersed Hassall's corpuscles; individual and small clusters of mature, small lymphocytes; scattered eosinophils; large areas of necrosis; focal mineralization; and cholesterol clefts. An interesting feature was the presence of large multinucleated giant cells, which varied widely in size and nuclear number. Immunohistochemical staining for two lymphocyte markers and two structural proteins confirmed the identity of the neoplastic spindle cells and other cellular components. There was no evidence of vascular invasion or metastasis. Features of the thymoma indicated it was a pre-existing condition and not treatment related. © 2011 by The Author(s).

Bannister D.,Medimmune Research | Popovic B.,Medimmune Research | Sridharan S.,Medimmune Research | Giannotta F.,ProGenosis S.A. | And 3 more authors.
Protein Engineering, Design and Selection | Year: 2011

Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure-activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes. © The Author 2011. Published by Oxford University Press. All rights reserved.

Buchanan A.,Medimmune Research | Ferraro F.,Medimmune Research | Rust S.,Medimmune Research | Sridharan S.,Medimmune Research | And 5 more authors.
Protein Engineering, Design and Selection | Year: 2012

Many natural human proteins have functional properties that make them useful as therapeutic drugs. However, not all these proteins are compatible with large-scale manufacturing processes or sufficiently stable to be stored for long periods prior to use. In this study, we focus on small four-helix bundle proteins and employ ribosome display in conjunction with three parallel selection pressures to favour the isolation of variant proteins with improved expression, solubility and stability. This in vitro evolution strategy was applied to two human proteins with known drug development issues, granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO). In the case of G-CSF, the soluble expression levels in Escherichia coli were improved 1000-fold, while for EPO the level of aggregation in an accelerated shelf-life study was reduced from over 80 to undetectable levels. These results exemplify the general utility of our in vitro evolution strategy for improving the drug-like properties of therapeutic proteins. © 2012 The Author.

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