Abstract 1585: Primary and metastatic uveal melanoma cell lines express MelanA that can be used as a target antigen for transfection of mature dendritic cells
Authors: Britting Sabine , Schaft Niels , Dörrie Jan , Bösl Teresa , Bock Barbara , Mackensen Andreas , Bosch Jacobus J. 
Published: August 2012

Abstract
The most common intra-ocular malignancy is uveal melanoma (UM). Metastatic disease occurs in approximately 50% of UM patients and presents most frequently in the liver. Novel therapies that prevent the development of metastases are needed and immunotherapy is a potential option. Because of its origin in the immune-privileged eye, UM may be particularly responsive to T cell-based immunotherapy. MelanA is frequently over-expressed by human cutaneous melanomas. MelanA expression has also been detected in UM primary and metastatic tumor-tissue sections and could serve as a target antigen for immunotherapy of UM. Here we report that primary and metastatic UM cell lines express MelanA and that this protein can be used as a target antigen for transfection of mature dendritic cells (mDC). MelanA expression was confirmed in primary and metastatic UM cell lines using intracellular flow cytometry and quantitative PCR (qPCR). In addition, sequencing of UM-derived MelanA demonstrated that MelanA in primary and metastatic UM cells has an identical sequence as compared to the published MelanA consensus sequence. As an initial step towards activating UM-specific T cells by UM-derived mRNA transfected DC we are generating MelanA mRNA transfected mDC. To facilitate both MHC class I and class II presentation of MelanA epitopes after mRNA electroporation into mDC, we included the endosomal targeting sequence DCLamp linked to the MelanA protein. Monocytes were isolated from leukapheresis products of healthy donors by density centrifugation and plastic adherence. Monocytes were differentiated to monocyte-derived DC by culturing with IL-4 and GM-CSF for 6 days to obtain immature DC (iDC). iDCs were matured with a cytokine mixture containing IL-4, GM-CSF, IL-1α, IL-6, TNFα, and PGE2 for 48 hours. The resulting mDC expressed cell surface maturation markers CD25, CD83, and CD86 as confirmed by flow cytometry. mDC were transfected with either MelanA-mRNA of MelanA-DCLamp-mRNA, using an established electroporation protocol. To assess transfection efficiency and monitor MelanA expression in transfected mDC, MelanA-mRNA and MelanA-DCLamp-mRNA expression was demonstrated by qPCR and MelanA-protein expression was shown by intracellular flow cytometry at different time points post-transfection using variable mRNA concentrations. The detected amount of MelanA-mRNA and protein was dependent on the amount of transfected mRNA and presented transiently. Subsequently, total RNA was isolated from MelanA+ primary and metastatic UM cell lines, then reversely transcribed into cDNA, amplified by PCR and in vitro transcribed to mRNA. As confirmed by qPCR, MelanA-mRNA was present in the pool of UM-derived mRNA and can be used for transfection of mDC. Collectively, these data demonstrate that mDC can be transfected with MelanA-mRNA and suggest MelanA could serve as a target for immunotherapy of UM.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1585. doi:1538-7445.AM2012-1585