Carbohydrate Antigen-bearing Nanoparticles for Antitumor Therapy

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. Over the years, we have prepared many optimized gold nanoparticles bearing the TF antigen attached to both threonine and serine amino acid residues with a capping acetate at the N-terminal amine group. Examination of these particles in various tumor cell lines for cytotoxicity has been performed and compared against each construct. These cells were chose to be either positive or negative for expression of the anti-apoptotic protein Galectin-3, an in vivo receptor for the TF antigen. Cells that are negative for Gal-3 do not respond to our particles, which suggest that cytotoxicity does go through a Gal-3 mediated pathway. Our latest iteration utilized a novel and more stable linker that imparts incredible stability to the particles in human serum and was published in this cycle. Our initial vaccine construct was previously described, and this 3-component vaccine gave a good immune response to glycopeptides bearing the TF antigen that were derived from a tandem repeat sequence from Mucin-4 (MUC4), a large protein which is overexpressed on Pancreatic Ductal Adenocarcinoma (PDAC) Cells and is a biomarker for PDAC-based tumors. This study identified a best construct, called MUC4-5TF, and this specific antigen was conjugated to KLH and polyclonal sera showed high specificity for the glycopeptide used for immunization. We subsequently collaborated with Rockland Immunochemicals to prepare a monoclonal antibody (mAb) which was found to be highly useful as a diagnostic and possibly a therapeutic agent against PDACS. We have shown this reagent to be 1) specific for tumor in tissue arrays, 2) Bind only to the sequence we vaccinated with, 3) Not cross react with a commercial antibody raised to an unglycosylated sequence and to not react at all with our linker technology. The reagent is now a commercial product with Rockland. In addition, we have re-tooled our vaccine construct and prepared gold nanoparticles with a coating of beta-1,3 glucans. These are glucose polysaccharides that bind to a specific C-type lectin on antigen presenting cells (APCs) such as macrophages and dendritic cells called Dectin-1. This facilitates entry into the APC and presentation of the attached antigen to T-cells. We showed after vaccination of mice that antibody titers to the antigen were very high, cytokine profiles were relevant to an antitumor response and CD4+ T-cells that react with the antigen are generated. We are in the process of optimizing this construct for therapeutic use. In addition, the mAb we produced with Rockland has been shown to distribute selectively to MUC4 positive tumors in vivo. An antibody drug conjugate *ADC) was also prepared and it shows high toxicity to MUC4 positive tumors in vitro. We have prepared a single chain fragment (ScFv) and are now developing a novel CAR-T cell is from this antibody.