Project Summary. The human immune system participates in complex interactions with virtually all other systems in the body. In particular, the B cell component of the adaptive immune response plays a role in various disease settings, including infectious disease, cancer, autoimmunity, cardiovascular, hematologic, neurologic diseases, and others. In addition, antibodies (a product of B cells) are effectively used in diagnostics, therapy, and prevention. Yet, despite decades of antibody discovery efforts, there is still very limited data linking human antibody sequence to antigen specificity (the preferential recognition of target antigens by a given antibody). One of the major reasons for such limited data is the fact that even high- throughput antibody sequence identification methods such as next-generation sequencing (NGS) of B cell receptor (BCR) sequences are generally decoupled from the process of antibody functional characterization. As a result, even though there are typically thousands to millions of antibody sequences within a single NGS dataset, functional information is obtained only for a handful of antibodies against not more than 2-3 target antigens at a time. To address these significant challenges for current technologies for B cell characterization and antibody discovery, our group has been focusing on the development of a single-cell technology that, for a given sample, enables the mapping of antibody sequence to antigen specificity from a single high-throughput experiment for a large number of antigens and B cells at a time. The technology, LIBRA-seq (LInking B-cell Receptor to Antigen specificity through sequencing), involves physically mixing a B cell sample with a (theoretically unlimited) pool of DNA-barcoded antigens, thus transforming B cell-antigen binding into a “sequenceable event”. In essence, LIBRA-seq offers all of the following features: (a) Characterization of thousands to tens of thousands of B cells at a time, at the single-cell level; (b) Screening against a large number of antigens at a time; (c) For each B cell, determination of the paired heavy-light chain BCR sequence; (d) For each B cell, generation of a high-resolution antigen specificity map. We initially validated LIBRA-seq in proof-of-concept studies in the context of HIV-1, and subsequently coronavirus, infection. These initial studies lay the foundation for generalizing the LIBRA-seq technology for application toward diverse antigen targets, and highlight areas for technology optimization, which will be the focus of this technology development proposal. In particular, here we propose to optimize LIBRA-seq for generalized application toward a broad diversity of antigen targets. Ultimately, the LIBRA-seq technology will have a long-lasting impact on both basic and applied immunology, helping revolutionize our understanding of antibody-antigen interactions and leading to the discovery of novel antibody therapeutics targeting a large variety of disease areas of biomedical significance.

Project Narrative This project aims at generalizing the LIBRA-seq technology for high-throughput mapping of antibody sequence to antigen specificity to make it universally applicable to a broad diversity of antigen targets, in the context of a wide range of antibody discovery problems. Ultimately, the LIBRA-seq technology will have a long- lasting impact on both basic and applied immunology, helping revolutionize our understanding of antibody- antigen interactions and leading to the discovery of novel antibody therapeutics targeting a large variety of disease areas of biomedical significance.