MedicalResearch.com Interview with:
Scott A. Gruber, M.D., Ph.D., MBA, FACS, FCP, FACHE, CPE
Chief of Staff, John D. Dingell VA Medical Center
Associate Dean for Veterans Affairs & Professor of Surgery
Wayne State University School of Medicine
John D. Dingell VA Medical Center Chief of Staff
Detroit, MI 48201
MedicalResearch: What are the main findings of the study?
Dr. Gruber: We successfully addressed the problem of inadequate intracellular delivery of tumor- specific antigens (TSAs) to dendritic cells (DCs) by using synthetic cell-penetrating domains or peptides (CPPs) to create fusion tumor antigens (Ags) that readily penetrate through the plasma membrane. We demonstrated cloning and purification of the TSA melanoma-associated antigen 3 (MAGE-A3) in frame with CPP, producing enhanced cytosolic bioavailability in dendritic cells without altering cell functionality. Further, we showed that recombinant bacterial proteins can be easily engineered to purify large amounts of CPP-MAGE-A3. Use of full-length proteins circumvents the need to define HLA class I allele binding before vaccination and increases the number of epitopes recognized by CD8+ cytotoxic T lymphocytes (CTLs) when compared with peptide-pulsed dendritic cells. Finally, the use of proteins rather than plasmids or viral vectors for in vitro dendritic cell vaccine preparation avoids the practical and theoretical safety concerns regarding genomic modification.
MedicalResearch: Were any of the findings unexpected?
Dr. Gruber: We expected to get increased membrane penetration and dendritic cell entry of CPP-MAGE-A3 versus MAGE-A3 alone, but we were surprised by the high efficiency. We examined the ability of MAGE-A3 to access the cytoplasm of dendritic cells by pulsing with recombinant tumor Ag on day 6 of culture. We observed very little fluorescent staining in MAGE-A3-pulsed DCs even after 2 h. In contrast, CPP-MAGE-A3 penetrated the DCs within 5 min after pulsing. The rapidity of this increased uptake was unexpected, and clearly demonstrated a rapid way to introduce CPP-MAGE-A3 into the cytoplasm.
MedicalResearch: What should clinicians and patients take away from your report?
Dr. Gruber: Effective cancer immunotherapy relies on efficient intracellular tumor- specific antigen delivery to access HLA class I molecules in the cytoplasm in order to generate robust cytotoxic T lymphocyte responses and produce tumor cell lysis. Here we provide the rationale for use of cell-penetrating peptides to enhance introduction and intracellular expression of the highly tumorigenic Ag MAGE-A3 in ex vivo generated DCs, potentially leading towards development of a more potent therapeutic cancer vaccine when compared with existing MAGE-A3 protein and peptide vaccines.
MedicalResearch: What recommendations do you have for future research as a result of this study?
Dr. Gruber: Although we addressed the issue of inadequate cytoplasmic expression of tumor- specific antigen for efficient CTL generation, the production of CTLs is dampened by the tumor-induced immunosuppressive microenvironment that acts as a substantial barrier to uptake and processing of tumor Ag by DCs and further inhibits development of therapeutically-relevant CTL responses. Some progress has been made in the identification of factors responsible for tumor-induced immunosuppression countering the development of robust CTLs or tumor-infiltrating lymphocytes. Myeloid-derived suppressor cells, tumor cell expression of programmed death receptor-ligand1 and tumor bed infiltration by regulatory T cells all diminish anti-tumor immunity via different mechanisms. In order to improve outcomes with DC-based therapeutic cancer vaccines, it is necessary to block the immunosuppressive environment while at the same time providing the means for effective CTL generation.