title: MAGE-A3 with cell-penetrating domain as an efficient therapeutic cancer vaccine.
abstract: OBJECTIVE
  In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane.
  
  
  OBJECTIVE
  To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3.
  
  
  METHODS
  MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD-MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel-nitrilotriacetic acid affinity purification of recombinant proteins.
  
  
  METHODS
  Measurement of DC membrane penetration of CPD-MAGE-A3 vs MAGE-A3 and determination of the effect of CPD-MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens.
  
  
  RESULTS
  Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD-MAGE-A3 proteins, while CPD antibodies recognized only CPD-MAGE-A3. Purified CPD-MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD-MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation.
  
  
  CONCLUSIONS
  We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.
authors.affiliation: Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan2John D. Dingell VA Medical Center, Detroit, Michigan.
authors.forename: Ramesh B
authors.initials: RB
authors.lastname: Batchu
authors.affiliation: Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan2John D. Dingell VA Medical Center, Detroit, Michigan.
authors.forename: Oksana
authors.initials: O
authors.lastname: Gruzdyn
authors.affiliation: Department of Biotechnology, Srinidhi Institute of Science and Technology, Hyderabad, India4Virocan Therapeutics Pvt Ltd, Guntur, India.
authors.forename: Ravindra B
authors.initials: RB
authors.lastname: Potti
authors.affiliation: Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan.
authors.forename: Donald W
authors.initials: DW
authors.lastname: Weaver
authors.affiliation: Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan2John D. Dingell VA Medical Center, Detroit, Michigan.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gruber
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology / Q000627:therapeutic use*; D001682:Biological Availability; D019496:Cancer Vaccines / Q000276:immunology / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000276:immunology; D057846:Cell-Penetrating Peptides; D003001:Cloning, Molecular; D003600:Cytosol / Q000187:drug effects* / Q000276:immunology*; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology*; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D009363:Neoplasm Proteins / Q000276:immunology / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D057846:Cell-Penetrating Peptides; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins