Design of novel antitumor immunotherapies based on tumor-associated glycopeptide antigens

Division Of Basic Sciences - Nci

Investigators

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Abstract

Specific Aim 1. Optimization of both active (vaccine) and passive (antibodies) MUC4-TF-based agents for combined tumor immunotherapy: The commercialized antibody discussed in the last cycle was a first successful attempt at discovering a tumor-selective glycopeptide-based mAb. We have completed a biodistribution study that showed unequivocally that we can target MUC4 positive, but not MUC4 negative tumors in vivo with a mAb-Near-IR dye conjugate that we prepared. Cell-derived xenografts as well as patient-derived xenograft (PDX) models were selectively targeted in vivo. We also prepared both Fab and ScFv fragments, and the ScFv construct was successfully crystallized with our glycopeptide antigen. The ScFv fragment was used to prepare a CAR-T cell that we showed kills MUC4 positive tumor cells in vitro. Several Antibody-Drug conjugates were also prepared and showed selective toxicity when tested against MUC4-positive tumors. We have prepared several other glycopeptides and raised antibodies in mice to these new immunogens. Fluorescent probes will be prepared to explore both cell binding and in vivo targeting to tumors. Testing will be performed to find the optimum mix of glycan positioning on the peptide backbone and family of binding modules to identify the most therapeutically promising candidate. Our recent studies have shown that a specific glycopeptide generates antibodies that are completely glycopeptide specific (no binding to the unglycosylated peptide). Regarding our vaccine, the work on antibodies will also inform our design of the optimum vaccine immunogen to be used in several tumor models in vivo. The goal is to find the correct vaccine constructions for the most potent in vivo antitumor immune response. Specific Aim 2. Development of TACA or TACA glycomimetic-coated nanoparticles to inhibit tumor cell adhesion and metastasis: We have shown that gold nanoparticles with different TF constructs selectively inhibit growth of tumor cells that express Galectin-3. In addition, these particles are selectively active in vivo as antimetastatic agents depending on whether the TF antigen is attached to a serine (Ser) or threonine (Thr). We now have prepared serum-stable particles with new linkers and both Ser or Thr-linked TF antigen and showed similar biding to Galectin-3 (Gal-3), as well as inhibition of Gal-3 binding to TF antigen. Our latest effort has expanded to a new TACA, the LacdiNAc (LDN) structure (GalNAc(beta)1-4GlcNAc-beta-) which can be a unit in both N- and O-linked glycan structure on mammalian protein. Its diagnostic relevance in prostate and other cancer has fostered recent interest in this disaccharide. Very relevant to our work is that it also binds Galectin-3. There is a paucity of reports on both the synthesis and the biological function of LDN, in particular on its carbohydrate binding proteins interactions. We have developed a synthesis of this disaccharide with a handle to attach various linkers at the reducing end for attachment to nanoparticles or glycan arrays. We will attempt to define the binding properties of this important structure and expand this to biological work on how inhibition of LDN-Lectin binding may also be used in antitumor therapeutic design. This work, combined with our previous TF-based nanoparticle synthesis will guide us in future design of tools that selectively inhibit tumor-associated lectins in a highly selective manner