Cancer Germline Antigen Specific CTL As Immunotherapy for Neuroblastoma

Cancer germline antigens (CGA) are expressed on several solid tumors including neuroblastoma.  Their expression can be epigenetically upregulated upon treatment with decitabine (DAC), a demethylating chemotherapy drug.  We have previously shown that DAC treatment improves the recognition of neuroblastoma and sarcoma cells to CGA-specific cytotoxic T lymphocytes (CTL) by upregulating the expression of MAGE-A1, MAGE-A3 and NY-ESO-1. ^[1,2]   We have conducted a Phase I/II clinical trial in which low dose DAC is followed by administration of autologous dendritic cell (DC) vaccine pulsed with MAGE-A1, MAGE-A3 and NY-ESO-1 peptide antigens. ^[3] An alternative therapy that does not depend upon the ability of a patient to respond to a vaccine is the use of tumor antigen-specific T cells. Nevertheless it can be difficult to expand CTL with tumor antigen specific killing functions, and not just cytokine secretion.  Li and group reported that a combination of CD25 depletion followed by culture of monocyte depleted lymphocytes with tumor antigens and IL-21 could result in T cell responses to cancer antigens. ^[4,5] IL-21 exerts potent antitumor effects by expanding cytotoxic CD8+ T cells, and suppressing FOXp3 expression and expansion of regulatory T cells (Tregs).  The first objective of this study was to determine if CD25 depletion followed by IL-21 in culture could facilitate the generation of CGA-CTL.  We have successfully cultured MAGE-A1, MAGE-A3 and NY-ESO-1 CTL following 3-5 weekly stimulations of CD25 depleted peripheral blood lymphocytes (PBL) with autologous dendritic cells (DC) pulsed with overlapping peptides derived from each of these antigens from 4/4 healthy donors.  After the second antigen stimulation, T cells were supplemented with IL-2, IL-7 along with IL-21.  Antigen-specific cytotoxicity was detected against autologous B cell blasts (BB) pulsed with MAGE-A1, MAGE-A3 or NY-ESO-1 peptides as well as tumor cell lines that have been treated with DAC and/or IFN-γ.  Without performing CD25 depletion and culture in IL-21, we were unable to generate CGA-CTL from these donors, indicating that CD25 depletion and IL-21 in culture favors the generation of CGA-CTL.  The second objective was to study if the addition of IL-21 alone, without CD25 depletion, may be sufficient for the expansion of these CTL. In a translational setting, omitting CD25 depletion would greatly simplify the expansion of CGA-CTL from patients.  CTL cultured from PBL (without CD25 depletion) displayed equal or greater antigen-specific cytotoxicity when compared to CTL cultured from the CD25 depleted cell fraction in all donors, indicating that CD25 depletion is not necessary and the effects of IL-21 in culture seem sufficient to facilitate the expansion of CGA specific CTL.  These CGA-CTL preferentially lysed HLA partially matched neuroblastoma cell lines treated with DAC and/or IFN-γ, the later to upregulate MHC class I expression on neuroblastoma cells.  To understand potential mechanisms whereby DAC and IFN-γ could enhance tumor cell susceptibility to killing by CGA-specific CTL, we stained three neuroblastoma lines, BE2C, SKH-SH and IMR-32 for Fas, Fas ligand, TRAIL-R1, TRAIL-R2, mannose-6-phosphate receptor (M6PR) and intracellular-adhesion molecule (ICAM-1) that are associated with cytotoxicity. The increased percentages of Fas⁺, M6PR⁺ and ICAM-1⁺ on tumor lines post-treated with DAC and IFN-γ for five days indicates enhanced cytotoxicity could be Fas, M6PR or ICAM-1 mediated.  Further studies are underway to determine the mechanisms whereby DAC and IFN-γ treated tumor cells are killed by CGA CTL cultured in this manner.