Novel Monoclonal Antibody Tools for Exploring the Altered Glycocalyx in Tumor Cells - Abstract The surface glycocalyx of tumor cells is comprised of key glycoproteins and glycolipids whose glycosylation is critical to tumor development and metastasis. Dysregulated glycan expression is a hallmark of cancer and contributes to altered cellular and matrix interactions, and immune evasion. Changes in glycosylation and generation of tumor-associated carbohydrate antigens (TACAs) arise through mutations in genes encoding glycosylation enzymes, altered localization of enzymes, altered glycoprotein expression, and many other factors that are poorly understood. We propose to develop, optimize, and validate novel monoclonal targeted antibody probes to aid in understanding these glycan changes in cancer and promote a multidisciplinary understanding of the cancer cell glycocalyx. We will achieve our goals with a convergent approach using 12 diverse types of cancer cells lines, that incorporates our novel glycan technology to aid in development of novel TACA-specific monoclonal antibodies (mAbs) that recognize tumor glycans. These mAbs are generated by immunizing the sea lamprey with tumor cells to induce lampreys to generate anti-glycan variable lymphocyte receptors (VLRs). Yeast surface display (YSD) libraries of such VLRs will be screened and enriched against tumor glycomes to identify TACA-specific VLRs, which are then expressed as recombinant VLR/Ig chimeras. These tools will be transformative in identification and targeting of signature TACAs (STACAs) and provide information to accelerate research of cancer diagnostics and treatment. The project has 3 specific aims. Aim 1 – We will use a panel of 12 diverse types of cancer cell lines to: a) exploit CORA (cellular O-glycome reporter/amplification) technology to orthogonally generate total cellular O-glycans including known and yet to be discovered TACAs; b) prepare total N-glycans and glycosphingolipids that may also contain TACAs by direct isolation and shotgun glycomics methods; and c) prepare printed tumor-cell glycan microarrays, and validate glycan recognition with well-characterized reagents. Aim 2 – We will: a) immunize lamprey with cells from Aim 1 and generate Yeast surface display (YSD) libraries; b) screen the YSD libraries on glycan arrays to identify specific VLRs for TACAs; c) generate recombinant VLR/Ig chimeric mAbs to TACAs; and d) characterize the specificity of mAbs for tumor glycans. Aim 3- We will use mAbs from Aim 2 for: a) immunohistochemical studies on TACA expression in human tumor cell lines, commercial tumor arrays, and for multiplexed analyses and spatial glycomics using a variety of human cancers to identify signature TACAs (STACAs); and b) glycolipid and glycoproteomic analyses of the human tumor cell lines to identify glycoproteins and their glycans carrying TACAs. Optimization of these innovative, convergent technologies will provide new tools and insights into the fundamental glycobiology of human cancers and facilitate research of cancer mechanisms.
