High-throughput antibody discovery directly from B cells using nanovial technology

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ABSTRACT Six out of ten top-grossing drugs in 2019 were monoclonal antibodies (mAbs), up from zero in 2010. By enabling highly-targeted therapies with enhanced efficacy and reduced side effects, mAbs have provided extraordinary benefits to patients. However, the discovery of mAb therapies remain slow and laborious owing to limited ability to quickly find antibodies with optimal functional properties (e.g., affinity). Most widely used methods still rely on immortalization of fragile B cells into hybridomas, followed by clonal expansion, ELISA screening, sequencing, and transient expression for downstream analysis of function. This entire process can take months and suffers from significant loss in the diversity of antibody sequences due to the immortalization process and the limited throughput of the standard well-plate formats. Emerging automated microfluidic workflows enable direct screening of B cells, improving diversity, but are still limited in the number of B cells screened. These specialized instruments are also not widely available, partly due to high capital equipment and consumable costs. Thus there is a critical need for a high-throughput single-cell screening workflow that does not rely on new specialized capital equipment and still maintains a linkage between antibody function and antibody sequence. To address these limitations, we will develop a lab on a particle-enabled workflow that enables customers to (i) select B cells secreting high affinity antibodies using standard fluorescence activated cell sorters (FACS) and (ii) link antibody function directly with paired heavy and light chain sequences in individual B cells. Our technology is based on microscale crescent-shaped hydrogel nanovials which capture cells, are functionalized to capture secretions, and enable formation of millions of uniform nanoliter droplets to prevent the loss and cross-talk of secretions. Like for standard ELISAs in microwells, nanovials can be exchanged between multiple solutions enabling functional assays of captured secreted antibodies, while maintaining the linkage of this information with the attached cells. In this Phase I SBIR, we will develop workflows to measure affinity of antibodies secreted by individual cells to a target antigen using an on-nanovial sandwich assay. We will use this assay to screen individual B cells from mice immunized against tetanus toxoid based on affinity using FACS and perform downstream single cell sequencing to identify matched heavy and light chain sequences. We will validate this process by transiently expressing identified VH and VL pairs with different measured affinities to compare with bulk ELISA. At the completion of the proposed work we will have demonstrated the ability to screen over 2 million B cells based on secreted antibody affinity in a single run, and identify potential antibody leads in <1 week. Following the successful completion of our aims we will have laid a strong foundation for an easily adopted reagent product that would provide significantly more antibody repertoire depth and the ability to select based on functional properties at early stages of discovery, ultimately reducing time and costs for antibody developers.