In vivo persistence of genetically modified T cells generated ex vivo using the fibronectin CH296 stimulation method

Abstract

Recombinant human fibronectin fragment (FN-CH296, RetroNectin) has been widely used for retroviral gene therapy to enhance gene transfer efficiency. Based on the observation that immobilized FN-CH296 together with anti-CD3 monoclonal antibodies (anti-CD3) enhanced cell proliferation while conserving the naive phenotype of T cells, we used FN-CH296 costimulation to generate engineered T cells. For comparison, human peripheral blood mononuclear cells were stimulated under three kinds of conditions including anti-CD3 only, anti-CD3 and anti-CD28 monoclonal antibodies conjugated with beads (anti-CD3/anti-CD28) and immobilized FN-CH296 together with anti-CD3 (anti-CD3/FN-CH296); all three treatments were followed by retroviral gene transfer. Of all the stimulation methods, the one involving anti-CD3/FN-CH296 produced the most cell expansion with conservation of the naive phenotype. Engineered T cells were transplanted into NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice, and all the mice were killed 14 days later. Transplanted T cells were detected in all the mice; however, mice injected with anti-CD3/FN-CH296-stimulated T cells showed higher transgene expression in organs than mice injected with anti-CD3-stimulated cells. These results demonstrate that the anti-CD3/FN-CH296 stimulation can be an efficient way to generate large numbers of genetically modified T cells that can provide higher and longer lasting levels of transgene expression in vivo and that are suitable for adoptive T-cell transfer therapy.