Renewable antibodies to secreted proteins and single and multi-pass cell surface

University Of California, San Francisco, San Francisco CA

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Abstract

Antibodies for research and therapy Antibodies (Abs) are essential reagents for determining how proteins function under normal or pathophysiological conditions. Uses include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and identifying interacting partners. Such studies require Abs of high specificity that function in assays including Western blotting, immunoprecipitation, immunohistochemistry (IHC) and in vivo imaging. Over half the human proteome is not annotated, and functional Abs are not reliably available for these proteins. Where monoclonal or polyclonal Abs are commercially available, a high proportion show either poor specificity or fail to recognize their targets (1-5). For example, a recent editorial by Michel et al. highlighted the lack of target specificity for 49 Abs against 19 subtypes of GPCRs (6). An additional problem is lot-to-lot variability in Ab specificity, including monoclonal Abs (mAbs) made via hybridoma technology, resulting in inconsistent assay results (4). The purpose of TR&D Project One is to develop high throughput scalable technologies to generate widely available, renewable, validated and standardized sets of Ab reagents (rAbs) to a portion of the secretome consisting of plasma membrane and extracellular proteins. One key aspect of the technology that will be developed is that where possible, the expensive, time consuming and tedious task of antigen generation and purification will be bypassed by displaying the antigen at high levels on the surface of eukaryotic cells, including yeast, and mammalian cell lines. Antigens expressed on mammalian cells or yeast will be used for selection of phage Abs, as well as for validation and characterization. The use of phage display bypasses the low throughput, time consuming, and expensive immunization of animals to generate polyclonal Abs or the use of hybridoma technology to generate mAbs. Moreover, the Ab genes are cloned, the rAbs are forever renewable and can easily be formatted for expression as Ab fragments or traditional mAbs with any Fc. While we will primarily generate rAbs to a subset of the secretome in this Project (the extracellular portions of plasma membrane and extracellular proteins), this approach should be applicable to many or all of the secreted proteins, 20-40% of the proteome.