Summary
This application addresses a major unmet need for technology that will facilitate the integration of glycomics and
glycan expression with genomics, transcriptomics, and metabolomics. The rapidly developing field of
glycoscience must have tools to identify and characterize the expression of human and animal glycans in the
context of normal development, homeostasis, and in disease processes. Traditional studies in the glycosciences
have relied on an odd assortment of plant-derived lectins and a few traditional mouse monoclonal antibodies,
but such reagents have multiple limitations. This application exploits our breakthrough technology that allows us
to develop suites of specific, rigorously characterized anti-carbohydrate antibodies (ACAs). We have developed
high-throughput immunization and screening technologies using variable-lymphocyte receptors (VLRBs) that are
generated upon immunization of ancient sea lampreys with cells, cell-conjugates, glycoproteins, or tissues.
Because of the evolutionary diversity of both their immune systems and glycomes as compared to mammals,
these ancient fish generate a diverse repertoire of anti-carbohydrate VLRBs with exquisite specificity, and are
able to discriminate between different linkages, motif presentation on N- and O-glycans, and the presence of
functional groups on individual monosaccharides. After immunization, the cloned cDNAs encoding the VLRBs
are expressed in a yeast surface display (YSD) library containing multiple individual specificities. This innovative
technology development allows us to both generate a ‘library of VLRBs’ in a single immunization protocol, as
well as sequentially screen these stable YSD libraries for ACAs using our numerous and diverse glycan and
glycopeptide microarrays and display technologies. Ultimately, after the VLRBs are screened for their binding to
glycan antigens, recombinant and reproducible VLRB-Ig chimeras are expressed, which can be used for all types
of immunological approaches to explore glycan expression and function. We propose 3 robust and innovative
Specific Aims to address the current technological deficiencies: Aim 1- Refine and expand our technology to
generate permanent VLRB YSD libraries against a diverse array of human glycan targets, using multiple
immunization and screening strategies. Aim 2- Enrich for and characterize monoclonal VLRBs that are specific
for human glycans, which will be critical in mapping the Human Glycome, by mining the created YSD libraries.
Aim 3- Generate novel VLRB specificities using targeted mutagenesis of characterized VLRBs, to create tailored
antibody specificities with potentially higher affinities. The success of our studies will climax with an arsenal of
rigorously vetted tools and robust reagents that will be made publicly available and accessible to the broader
research community. This will ultimately encourage widespread study of these incredibly important, complex
molecules that are vital in human health and disease, promote harmonization with other biological sciences, and
allow for the study of expression and functions of the Human Glycome.