De Novo Selection

De novo selection strategies involve testing a large number of combinations of novel heavy and light chains in a high-throughput manner. The design of such a strategy should provide confidence that inherent diversities are surveyed and that events with low frequency will be identified. As opposed to leveraging existing reagents to guide selection, de novo screening generally provides opportunities to identify antibodies against multiple epitopes with potentially different functions.

The reiterative nature of the ACTIVMAb^® discovery platform provides fast and efficient enrichment of even low frequency events making de novo screening a possibility. Furthermore, the modular nature of our libraries coupled with innovative selection strategies allow us to screen diversities that rival other platforms.

Figure 1. De novo screening can embody many different forms. For example, we may choose to conduct a true de novo screen using libraries of heavy and light chains that together produce large combinatorial diversities. Alternatively, we may choose to limit either the light or heavy chain diversity to a restricted population. Selection is achieved by expressing antibody on the surface of cells and introducing labeled antigen. Cells that express antibodies that bind antigen are separated by FACS. The virus within these cells is harvested and amplified and used in a subsequent round of enrichment until a suitably enriched positive population of antibodies is identified. The primary sequence of individual clones are then examined and unique clones are validated through a variety of in vitro assays.

Figure 2. Flow cytometry data from a de novo screen of novel heavy and light chain combinations demonstrates essentially background staining prior to sorting and almost 75 fold enrichment after a single round of FACS. The population in the upper right hand quadrant of the post-sort with-antigen histogram was subjected to a further round of enrichment followed by selection of individual clones.