(590b) Glycan-Specific Antibody Discovery through Designer Glycopeptide Immunization

Sugar groups, or glycans, specific to or enriched on particular cell types and strains, such as tumor-associated carbohydrate antigens and immunogenic lipopolysaccharide on pathogenic bacteria, serve as identification markers that can be leveraged for targeting with monoclonal antibodies. In addition to being a powerful laboratory tool for purification, screening, and imaging, monoclonal antibodies have in recent years become increasingly important in treatment of cancer, autoimmune disorders, and an expanding list of other indications. However, to date, therapeutic monoclonal antibodies have almost exclusively targeted proteins, and there continues to be a lack of anti-glycan monoclonal antibodies for use in the laboratory and clinic. The poor immunogenicity of glycans contributes to this limited availability. Unlike protein antigens, carbohydrates often elicit an immune response independent of T cells, characterized by a lack of class-switched high affinity antibodies relevant for discovering therapeutic and diagnostic monoclonal antibodies.

Historically, monoclonal antibodies have been produced using hybridoma and display technologies. However, the emergence of next-generation sequencing approaches offers a rapid, refined antibody discovery alternative compared to the labor-intensive, time-consuming cell screening or panning steps required with current methods. High-throughput sequencing of entire cell populations are now widely accessible methods for obtaining sequences of target-binding immunoglobulin genes. This has previously been used to identify high-affinity anti-protein antibodies, and we propose that this approach can be expanded to glycan targets through utilization of glycosylated peptide antigen that elicits a robust T cell-dependent immune response while mitigating off-target antibody production.

Here we demonstrate that Escherichia coli engineered to synthesize designer glycans serves as a tractable platform for the production of rationally designed glycopeptides. These peptides contain motifs permitting in vivo glycosylation linked to amino acid epitopes known to bind with cell surface MHC II molecules necessary for a T cell-dependent humoral immune response. Using the O-antigen polysaccharide (O-PS) from Francisella tularensis as a model pathogenic bacterial glycan, we show immunization of mice with purified E. coli-expressed glycopeptides results in class-switched high affinity antibodies specific to the O-PS antigen. Analysis of the repertoire of immunoglobulin genes, obtained via high-throughput sequencing of the antibody-producing splenocytes isolated from immunized mice, can then be used to elucidate and recapitulate monoclonal antibodies with high affinity towards O-PS. Due to the modularity of the glycopeptide platform, whereby new glycans can be conjugated, immunized, and sequenced, this rapid monoclonal antibody pipeline can be exploited for use beyond our model O-PS antigen to other carbohydrate targets of interest.