Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies

Cancer Res. 2015 Sep 15;75(18):3853-64. doi: 10.1158/0008-5472.CAN-14-3321. Epub 2015 Jul 16.


Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alemtuzumab
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal, Humanized / pharmacology
  • Antigens, CD / genetics
  • Antigens, CD19 / immunology
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology
  • Base Sequence
  • CD52 Antigen
  • Cytotoxicity, Immunologic
  • Drug Resistance
  • Gene Knockout Techniques*
  • Glycoproteins / deficiency
  • Glycoproteins / genetics
  • Graft vs Host Disease / prevention & control
  • Humans
  • Immunotherapy, Adoptive*
  • Lymphocyte Activation
  • Lymphoma / therapy
  • Mice
  • Mice, Mutant Strains
  • Molecular Sequence Data
  • RNA, Messenger
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Antigen, T-Cell, alpha-beta / deficiency
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / transplantation*
  • Transfection
  • Xenograft Model Antitumor Assays


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Antigens, CD
  • Antigens, CD19
  • Antigens, Neoplasm
  • CD52 Antigen
  • CD52 protein, human
  • Glycoproteins
  • RNA, Messenger
  • Receptors, Antigen, T-Cell
  • Receptors, Antigen, T-Cell, alpha-beta
  • Recombinant Fusion Proteins
  • Alemtuzumab