Tumor-Associated Carbohydrate Antigens (TACAs) are aberrant glycan structures covalently linked to proteins and/or lipids on the surface of tumor cells. They are very different than the glycan repertoire presented on a normal cell phenotype, and hence can invoke an immune response that is protective against tumor growth and progression. TACAs are often presented on large glycoproteins called mucins, many that are overexpressed in different cancers. Mucin4 (MUC4) is aberrantly expressed in pancreatic ductal adenocarcinoma’s (PDACs). MUC4 peptides, glycosylated with a specific TACA, the Thomsen Friedenreich antigen (Galb1-3GalNAca-O-Ser/Thr) were previously used as immunogens in vaccine constructs which led to the development of a tumor specific monoclonal antibody (mAb) referred as F5. The present study investigated the tumor homing ability of F5 mAb in in-vivo PDAC mice models. F5 was fluorescently labeled with Frederick near infrared dye (F5-FNIR), and binding to the MUC4 glycopeptide was confirmed by SPR and ELISA. MUC4⁺ and MUC4^-PDAC cell-derived xenograft models in immunocompromised mice were developed to determine the in-vivo tumor specificity of F5-FNIR. Fluorescently labelled F5 accumulated in various MUC4⁺ xenografts much more than those that were MUC4^-. Excised organs and tumors showed significant localization of F5-FNIR in MUC4⁺ tumors. To assess F5 biodistribution in a more relevant model, F5-FNIR tumor homing in PDAC MUC4+ human patient derived xenografts (PDXs) was assessed. F5-FNIR specifically localized in MUC4+ PDXs compared to isotype control. These results led us to develop an F5 antibody-drug conjugate (ADC). We designed and synthesized an ADC with F5 conjugated to monomethyl auristatin E (MMAE) as a cytotoxic payload via a cathepsin B-sensitive valine-citrulline linker. Successful conjugation of MMAE to the F5 was confirmed by UV and mass spectrometry. The ADC was found to be significantly cytotoxic to MUC4⁺ PDAC cells compared to MUC4^-cells. In addition, the ADC also exhibited cytotoxicity in 3D tumor organoids of MUC4+ PDAC cells. We are currently exploring in-vivo efficacy of F5-ADC in relevant tumor mice model with a functioning humanized immune system. Our data shows that F5 conjugates may have significant potential as a therapy against pancreatic cancer.

Joseph Barchi

National Cancer Institute

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