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Consequences of sequence variants for the expression of a dual targeting novel format antibody construct
[Thesis]. Manchester, UK: The University of Manchester; 2014.
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Abstract
Antibody engineering is an innovative field of research that has generated a wide range of novel antibody-based formats that both exploit and improve natural antibody properties. Novel format antibodies have the potential to offer significant advantages over natural antibodies when used as biopharmaceuticals, however these non-natural structures often pose a great challenge to the host cell used for their manufacture. Protein expression is a highly regulated process, and quality control mechanisms at each stage can result in a block, or “bottleneck” in expression. This can impact product yield, cost of goods and entry into the clinical pipeline. The molecular determinants that govern novel-format expression in host cells are poorly defined, however there is growing evidence that limited variations in both nucleotide and amino acid sequence can have a severe impact on antibody expression.Therefore this Thesis aims to investigate the consequences of sequence variation on the expression of a novel antibody format (mAbdAb) in mammalian host cells in order to determine the molecular mechanisms that govern their expression.A diverse panel of mAbdAbs with sequence variations limited to the dAb domain were generated through phage display and cloning technologies. It was determined that amino acid variations located within the CDRs of the dAb results in a range of expression titres in both transient HEK and stable CHO expression platforms. In vitro translation of mAbdAb heavy chain proteins in rabbit reticulocyte lysates (RRL) showed no difference in expression between sequence variants, therefore cell-free translation was suggested as a potential expression platform. Examination of each stage of expression in stable CHO cells revealed that the amount of mRNA was not limiting to expression and distinct expression profiles were observed at the protein level. The majority of mAbdAb constructs showed little evidence of intracellular heavy chain polypeptide which was not altered through chemical inhibition of proteolytic degradation pathways, indicating that degradation was not responsible for poor expression. This led to the hypothesis that low titres were related to how the CHO cell utilises the heavy chain message.