Pancreatic Cancer Cell Lysis by Cell-Penetrating Peptide-MAGE-A3–Induced Cytotoxic T Lymphocytes

In contrast to the therapeutic advances for other malignant neoplasms, the therapeutic advances for pancreatic cancer have been slow, with the 5-year survival rate currently at less than 8% for patients with pancreatic cancer.¹ Immunotherapy is a particularly appealing approach to pancreatic cancer owing to its potential for eliminating tumor cells that are often unreachable by conventional therapies and its negligible side effects. Along these lines, dendritic cells (DCs) are central to the generation of effector cytotoxic CD8⁺ T lymphocytes (CTLs) that recognize tumor-specific antigens (TSAs) expressed on the surface of cancer cells. MAGE-A3 (melanoma antigen family A, 3) is a TSA expressed in a significant fraction of pancreatic cancers,² thus providing an opportunity for introducing DC-based immunotherapy. However, clinically meaningful antitumor immune responses in DC vaccine trials have been sparse owing, in part, to suboptimal intracellular bioavailability of TSA to HLA class I molecules. Various cell-penetrating peptide (CPP) domains are known to ferry covalently linked heterologous TSAs across the plasma membrane into the cytosolic compartment to access HLA class I molecules.³ We and others have previously demonstrated that CPP effectively increased the intracellular entry of TSAs.^4,5 We extend this work by investigating whether DCs pulsed with MAGE-A3 linked to CPP could elicit more effective antitumor CTL responses.

PANC-1, an established HLA-A*0201 MAGE-A3⁺ human pancreatic cancer cell line, was obtained from the American Type Culture Collection and propagated at standard conditions in RPMI 1640 medium with 10% fetal bovine serum. The CPP-MAGE-A3 and MAGE-A3 (control) recombinant proteins expressed in Escherichia coli were purified as previously described using his-tag affinity chromatography.⁴ Dendritic cells were generated from the adherent fraction of peripheral blood mononuclear cells obtained from HLA-A*0201 donors and pulsed with 3 μmol/L of recombinant protein. Mixed lymphocyte reaction assays were conducted according to our previous method,⁶ to assess the effect of recombinant protein pulsing on the functional activity of DCs. MAGE-A3–specific CTLs were generated by coculturing autologous T lymphocytes with DCs pulsed with recombinant protein. We measured the resulting secretion of interleukin 4 (IL-4), IL-12p70, and IFN-γ, as well the degree of CTL-mediated killing of PANC-1 cells.⁶P < .05 was considered to be statistically significant.

MAGE-A3, CPP-MAGE-A3, and mock-treated DCs all demonstrated similar degrees of cell proliferation in mixed lymphocyte reaction assays, indicating retention of DC functionality after pulsing with recombinant protein (Figure 1A). Coculture of autologous T lymphocytes with DCs treated with CPP-MAGE-A3 demonstrated significantly higher IFN-γ secretion but a similar degree of IL-4 and IL-12p70 release when compared with MAGE-A3 alone (Figure 1B). In addition, overall levels of the cytokines stimulating CD8⁺ T cells and the HLA class I pathway (IFN-γ and IL-12p70) were significantly higher than levels of IL-4, a cytokine promoting the CD4⁺ HLA class II pathway. Finally, we found that a significantly higher level of PANC-1 target cell lysis was observed following treatment of CTL with CPP-MAGE-A3 compared with MAGE-A3 alone (81.5% vs 60%; P < .05) (Figure 2).

In this study, we demonstrate that the addition of a CPP to MAGE-A3 enhances IFN-γ secretion and pancreatic cancer cell killing without altering DC functionality, potentially improving the potency of existing MAGE-A3 protein and peptide vaccines. Although the addition of the CPP did not enhance the secretion levels of IL-12p70, we did observe a significant enhancement in the release of IFN-γ that is more directly responsible for tumor cell lysis. Our demonstration of more potent cell-mediated immune responses and enhanced tumor cell lysis of the PANC-1 cell line indicate that the CPP could be a crucial factor in enhancing the killing activity of MAGE-A3⁺ pancreatic tumors and could also be linked to TSAs targeting other malignant neoplasms. This form of DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for the management of pancreatic cancer.

Corresponding Author: Ramesh B. Batchu, PhD, John D. Dingell VA Medical Center, 4646 John R St, Detroit, MI 48201 (rbatchu@med.wayne.edu).

Published Online: August 17, 2016. doi:10.1001/jamasurg.2016.2346.

Author Contributions: Dr Batchu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Batchu, Qazi, Weaver.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Batchu, Qazi, Gruber.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Batchu, Qazi, Mahmud.

Obtained funding: Batchu, Weaver.

Administrative, technical, or material support: Gruzdyn, Mahmud, Mostafa, Weaver, Gruber.

Study supervision: Qazi, Gruber.

Conflict of Interest Disclosures: None reported.

Previous Presentation: This paper was presented at the 40th Annual Meeting of the Association of VA Surgeons; April 11, 2016; Virginia Beach, Virginia.

Additional Contributions: We thank Rajesh Dachepalli, MS, from Virocan Therapeutics, for support with the purification of recombinant proteins. No compensation was received from a funding sponsor.