Cancer Germline Antigen Specific CTL As Immunotherapy for Neuroblastoma
Authors: Li Dan , Bai Fanqi , Sundaram Kumaran , Krishnadas Deepa K. , Lucas Kenneth 
Published: October 2019

Abstract
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.

Disclosures
No relevant conflicts of interest to declare.