title: Annexin-directed β-glucuronidase for the targeted treatment of solid tumors.
abstract: Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme β-glucuronidase (βG), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted βG to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting. To create fusion proteins, human annexin A1/A5 was genetically fused to the activity-enhancing 16a3 mutant of human βG, expressed in chemically defined, fed-batch suspension culture, and chromatographically purified. All fusion constructs achieved >95% purity with yields up to 740 μg/l. Fusion proteins displayed cancer selective cell-surface binding with cell line-dependent binding stability. One fusion protein in combination with the prodrug SN-38 glucuronide was as effective as the drug SN-38 on Panc-1 pancreatic cancer cells and HAAE-1 endothelial cells, and demonstrated efficacy against MCF-7 breast cancer cells. βG fusion proteins effectively enable localized combination therapy that can be tailored to each patient via prodrug selection, with promising clinical potential based on their near fully human design.
authors.affiliation: Biomedical Engineering Program and School of Chemical, Biological and Materials Engineering, University of Oklahoma, 100 E. Boyd St., Norman, OK 73019, USA.
authors.forename: Katrin P
authors.initials: KP
authors.lastname: Guillen
authors.affiliation: Protein Production Core, Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
authors.forename: Eliza A
authors.initials: EA
authors.lastname: Ruben
authors.affiliation: Biomedical Engineering Program and School of Chemical, Biological and Materials Engineering, University of Oklahoma, 100 E. Boyd St., Norman, OK 73019, USA.
authors.forename: Needa
authors.initials: N
authors.lastname: Virani
authors.affiliation: Biomedical Engineering Program and School of Chemical, Biological and Materials Engineering, University of Oklahoma, 100 E. Boyd St., Norman, OK 73019, USA rharrison@ou.edu.|Stephenson Cancer Center, Health Sciences Center, University of Oklahoma, 800 Northeast 10th St., Oklahoma City, OK 73104, USA.
authors.forename: Roger G
authors.initials: RG
authors.lastname: Harrison
mesh_terms: D017305:Annexin A1 / Q000235:genetics*; D017304:Annexin A5 / Q000235:genetics*; D045744:Cell Line, Tumor; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D058990:Molecular Targeted Therapy; D009154:Mutation; D011355:Prodrugs / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017305:Annexin A1; D017304:Annexin A5; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase
keywords: SN-38 glucuronide; enzyme prodrug therapy; phosphatidylserine; vascular targeted; β-glucuronidase