Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation

J Biochem. 2011 Mar;149(3):285-92. doi: 10.1093/jb/mvq135. Epub 2010 Nov 23.


Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / pharmacology
  • CD28 Antigens / immunology
  • CD3 Complex / immunology
  • Fibronectins / chemistry
  • Fibronectins / pharmacology*
  • Flow Cytometry
  • Genetic Vectors / genetics*
  • Humans
  • Immunotherapy, Adoptive
  • Lentivirus / genetics*
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / drug effects*
  • Leukocytes, Mononuclear / metabolism*
  • Lymphocyte Activation / drug effects
  • T-Lymphocytes / cytology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism
  • Transduction, Genetic / methods*


  • Antibodies, Monoclonal
  • CD28 Antigens
  • CD3 Complex
  • Fibronectins