In vivo persistence of genetically modified T cells generated ex vivo using the fibronectin CH296 stimulation method.
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.
Yu
S S
SS
Center for Cell and Gene Therapy, Takara Bio, Otsu, Shiga, Japan. seungshin@viromed.co.kr
Nukaya
I
I
Enoki
T
T
Chatani
E
E
Kato
A
A
Goto
Y
Y
Dan
K
K
Sasaki
M
M
Tomita
K
K
Tanabe
M
M
Chono
H
H
Mineno
J
J
Kato
I
I
D000818:Animals; D001483:Base Sequence; D017690:Cell Transplantation; D017931:DNA Primers; D005353:Fibronectins / Q000008:administration & dosage*; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D016130:Immunophenotyping; D008213:Lymphocyte Activation; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D010446:Peptide Fragments / Q000008:administration & dosage*; D012190:Retroviridae / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014161:Transduction, Genetic
D017931:DNA Primers; D005353:Fibronectins; D010446:Peptide Fragments