Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor

J Immunother. 2016 Jun;39(5):205-17. doi: 10.1097/CJI.0000000000000126.

Abstract

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

MeSH terms

  • Antigens, Neoplasm / immunology
  • CD8-Positive T-Lymphocytes / physiology*
  • Cancer Vaccines / immunology*
  • Cell Line, Tumor
  • Cell Proliferation
  • Coculture Techniques
  • ErbB Receptors / immunology
  • Genetic Engineering
  • Glioblastoma / immunology
  • Glioblastoma / therapy*
  • Humans
  • Immunologic Memory
  • Immunotherapy, Adoptive / methods*
  • Lymphocyte Activation
  • RNA, Messenger / genetics
  • Receptors, Antigen, T-Cell / genetics*
  • T-Cell Antigen Receptor Specificity
  • T-Lymphocyte Subsets / physiology*

Substances

  • Antigens, Neoplasm
  • Cancer Vaccines
  • RNA, Messenger
  • Receptors, Antigen, T-Cell
  • EGFR protein, human
  • ErbB Receptors