Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

Mol Ther. 2015 Aug;23(8):1380-1390. doi: 10.1038/mt.2015.71. Epub 2015 May 5.


Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

MeSH terms

  • Alleles
  • Animals
  • Cell Separation
  • Cytokines / metabolism
  • Endoribonucleases / genetics*
  • Female
  • Flow Cytometry
  • Gene Expression Regulation, Neoplastic*
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunologic Memory
  • Immunotherapy, Adoptive
  • Interferon-gamma / metabolism
  • Lymphocyte Activation / immunology
  • Lymphocytes, Tumor-Infiltrating / immunology*
  • Melanoma / therapy*
  • Mice
  • Neoplasm Metastasis
  • Neoplasm Transplantation
  • Phenotype
  • Programmed Cell Death 1 Receptor / genetics*
  • Programmed Cell Death 1 Receptor / metabolism
  • Tumor Necrosis Factor-alpha / metabolism
  • Zinc Fingers*


  • Cytokines
  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor
  • Tumor Necrosis Factor-alpha
  • Green Fluorescent Proteins
  • Interferon-gamma
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Endoribonucleases