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Clinical scale zinc finger nuclease (ZFN)-driven gene-editing of PD-1 in tumor infiltrating lymphocytes (TIL) for the potential treatment of metastatic melanoma

  • Joal D Beane1,
  • Gary K Lee2,
  • Zhili Zheng1,
  • Nimisha Gandhi2,
  • Daniel Abate-Daga1,
  • Mini Bharathan1,
  • Mary Black1,
  • Matthew Mendel2,
  • Zhiya Yu1,
  • Sadik H Kassim1,
  • Smita Chandran1,
  • Martin Giedlin2,
  • Dale Ando2,
  • Ed Rebar2,
  • Andreas Reik2,
  • Michael Holmes2,
  • Philip D Gregory2,
  • Nicholas P Restifo3,
  • Steven A Rosenberg4,
  • Richard A Morgan5 and
  • Steven A Feldman1
Journal for ImmunoTherapy of Cancer20142(Suppl 3):P2

Published: 6 November 2014


MelanomaEffector MemoryMetastatic MelanomaHost Immune ResponseTumor Infiltrate Lymphocyte

Multiple inhibitory pathways exist to block the immune response to cancer potentially limiting 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. 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. To this end, our aim was to eliminate PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PD-1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 ZFNs, 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. Importantly, the PD-1 gene-edited TIL product displayed an effector memory phenotype and expanded approximately 500 - 2000 fold during a rapid cell expansion in vitro while retaining T cell effector function. Thus further study to determine the safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

Authors’ Affiliations

Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, USA
Sangamo Biosciences, Richmond, USA
National Cancer Institute, Bethesda, USA
US National Institutes of Health (NIH), Bethesda, USA
bluebird bio, Cambridge, USA


© Beane et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.