Thesis

TCR engineering of Natural Killer Cells for melanoma treatment

Melanoma is the most dangerous form of skin cancer because it is resistant to most common cancer treatments, such as chemotherapy and radiation. In 2014, a novel immunotherapy based on the blockage of the PD-1/PDL-1 signaling pathway was approved for malignant melanoma. However, this treatment is only effective with patients with high-level expression of PD-1/PDL-1. Therefore, developing an alternative melanoma treatment is an urgent mission. The goal of this proof of principle study was to augment the ability of natural killer (NK) cells to effectively target and kill melanoma cells by engineering them to express a T cell receptor (TCR) specific for melanoma antigen Mart-1. TCRs are not normally expressed on NK cells, although the protein components necessary for TCR signaling exist in these cells. By using this approach, the NK and T cell anti-melanoma properties would be consolidated within one cell type. Furthermore, as melanoma cells generally use different strategies to suppress T and NK cells, the TCR expressing NK cells could compensate for the lack of T cell activity when these cells are absent or viii immunosuppressed. In this study, human embryonic stem cells (hESC) were used as the source of genetically modified NKs, as they are relatively easy to genetically manipulate, expand to clinically relevant quantities, and to differentiate into NK cells that are phenotypically and functionally indistinguishable from their blood derived counterparts. TCRs require the CD3 complex of proteins for transport to the cell surface and proper signaling. To provide all the elements needed to support normal TCR function, the coding sequences for the CD3 γ, ζ, δ and ε chains, along with the Mart-1-specific TCR genes, were introduced into H1 hESC. Individual transgenic hESC lines were selected, the expressions of both Mart-1 TCR and CD3 on their surfaces were confirmed, and then they were differentiated into TCR/CD3 NK cells. In the future, Mart-1-specific TCR and CD3 expressing cells will be used in in vitro and in vivo experiments to establish that the presence of the Mart-1 TCR should endow these transgenic NKs with a higher anti- melanoma cytotoxic potential. Importantly, this technology could be adapted and expanded for treatment of other tumors and malignancies.

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