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Abstract

Adoptive cell therapy using tumor antigen-specific CD8+ T cells is a promising approach to treat patients with advanced or metastasizing cancer including malignant melanoma. The general procedure is based on the isolation, in vitro stimulation and expansion of tumor-infiltrating lymphocytes (TILs). These activated and expanded TILs, which are specific for common tumor antigens are then transferred back into the patients to attack and neutralize cancer cells. However, current studies report disease relapse in treated patients because of T cell exhaustion after ongoing TCR stimulation resulting in a decreased effector function and a loss of their replicative capacity. The use of induced pluripotent stem cells (iPSCs) as a source for the generation of “off-the-shelf” CD8+ T cells could enable T cell-based immunotherapy on a large scale. Similar to embryonic stem cells, iPSCs have the potential to give rise to nearly unlimited amounts of any cell type, including CD8+ T cells. The aim of this project was to develop strategies for the in vitro generation of melanoma antigen-specific CD8+ T cells from human iPSCs. First, iPSCs were transfected with S/MAR DNA vectors coding for a T cell receptor (TCR) against MART-1 or a chimeric antigen receptor (CAR) directed against MCSP. S/MAR DNA vectors are effective gene delivery systems since they contain a scaffold/matrix attachment region, which binds to the nuclear matrix and ensures a persistent gene expression over hundreds of cell divisions. The transfection of iPSCs with the S/MAR DNA vector did not alter the gene expression of the pluripotency genes NANOG and OCT4 as well as other T cell-related differentiation genes. Next, a 2D co-culture system with OP9 murine stromal cells was used to facilitate the differentiation of CD34+ hematopoietic stem and progenitors cells (HSPCs) from iPSCs. Further differentiation of CD34+ HSPCs into CD4+CD8+ double-positive (DP) T cells was achieved by long-term coculture with bioengineered OP9 stromal cells expressing T cell-specific cytokines like FLT3-ligand, CXCL12 and SCF as well as the Notch ligand DLL4 and supplementation of the growth medium with recombinant IL7. Since clinical applications require clean populations of effector T cells without contamination with murine cells, the stromal cell based co-culture system was replaced with a commercially available differentiation kit. Following the manufacturer’s instructions, I was able to generate a limited amount of CD8+ single-positive (SP) T cells. Flow cytometric analysis of common T cell markers revealed a low CD3 expression in generated T cells but a persistent MART-1 TCR or MCSP CAR expression, respectively. In comparison to CD4+CD8+ DP T cells, CD8+ SP T cells demonstrated the capability to produce cytokines, showed degranulation capacity after stimulation and exhibited cytotoxic effects against melanoma cell lines. Hence, these findings revealed the possibilities as well as the challenges connected with the use of human iPSCs for the generation of CD8+ T cells. However, the use of S/MAR DNA vectors and novel differentiation strategies could enable further clinical applications in cancer treatment and help to improve common immunotherapies for melanoma patients.