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Original Investigation
Association of VA Surgeons
May 2014

MAGE-A3 With Cell-Penetrating Domain as an Efficient Therapeutic Cancer Vaccine

Author Affiliations
  • 1Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan
  • 2John D. Dingell VA Medical Center, Detroit, Michigan
  • 3Department of Biotechnology, Srinidhi Institute of Science and Technology, Hyderabad, India
  • 4Virocan Therapeutics Pvt Ltd, Guntur, India
JAMA Surg. 2014;149(5):451-457. doi:10.1001/jamasurg.2013.4113
Abstract

Importance  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.

Design  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.

Main Outcomes and Measures  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 and Relevance  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.

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