77. Clinical Scale Zinc Finger Nuclease (ZFN)-Driven Gene-Editing of PD-1 in Tumor Infiltrating Lymphocytes (TIL) for the Potential Treatment of Metastatic Melanoma

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Multiple inhibitory pathways are exploited by a number of cancers to block the body's immune response which may limit the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Importantly, its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor targeted T cells prior to ACT. Here we describe the elimination of PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PDCD1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 specific ZFNs, the clinical scale TIL production process yielded efficient modification of the PDCD1 gene locus (mean = 74.8% of alleles, n=3, range = 69.9 – 84.1%), which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 modified TIL products displayed an effector memory phenotype and expanded approximately 500 – 2000 fold during a rapid cell expansion. In addition, PD-1 modified and unmodified TILs showed equivalent levels of engraftment in a NSG-mouse model. PD-1 modified TILs demonstrated improved in vitro T cell effector functions relative to controls (statistically significant greater TNFα, GM-CSF and IFNγ cytokine secretion when co-cultured with MART-1+ tumor cells). Preliminary safety evaluations showed that PD-1 modified TILs maintained IL-2 dependent growth in vitro, and no tumors were observed in an NSG-mouse tumorigenicity study. These data support the further study of the efficacy and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma.