To overcome the complexity and cost of production of autologous CAR constructs, Frank et al. demonstrated that lentiviruses containing scFvs that simultaneously target and activate CD3 on T cells (LV-CD3) efficiently and specifically activate and transduce human CD4+ and CD8+ T cells in vitro, in whole blood, and in vivo (humanized mice). Off-target (CD45- or CD45+CD19+) transduction was minimal. In humanized mice, LV-CD3-CD19 constructs efficiently generated CD19-CARs that were durable and that depleted CD19+ B cells; both results were enhanced if mice were pretreated with IL-7, which preferentially stimulates naive and memory cells.
Contributed by Ed Fritsch
ABSTRACT: Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify resting or minimally stimulated cells, which is crucial for applications, such as efficient in vivo modification of T lymphocytes. Here, we introduce novel CD3-targeted LVs (CD3-LVs) capable of genetically modifying human T lymphocytes without prior activation. For CD3 attachment, agonistic CD3-specific single-chain variable fragments were chosen. Activation, proliferation, and expansion mediated by CD3-LVs were less rapid compared with conventional antibody-mediated activation owing to lack of T-cell receptor costimulation. CD3-LVs delivered genes not only selectively into T cells but also under nonactivating conditions, clearly outperforming the benchmark vector vesicular stomatitis-LV glycoproteins under these conditions. Remarkably, CD3-LVs were properly active in gene delivery even when added to whole human blood in absence of any further stimuli. Upon administration of CD3-LV into NSG mice transplanted with human peripheral blood mononuclear cells, efficient and exclusive transduction of CD3+ T cells in all analyzed organs was achieved. Finally, the most promising CD3-LV successfully delivered a CD19-specific chimeric antigen receptor (CAR) into T lymphocytes in vivo in humanized NSG mice. Generation of CAR T cells was accompanied by elimination of human CD19+ cells from blood. Taken together, the data strongly support implementation of T-cell-activating properties within T-cell-targeted vector particles. These particles may be ideally suited for T-cell-specific in vivo gene delivery.
Author Info: (1) Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany. Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany. (2) Molecular Biotechnolog
Author Info: (1) Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany. Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany. (2) Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany. (3) Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany. (4) Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany. (5) Irene Schneider Consulting, Hanau, Germany. (6) International Center for Infectiology, Research Team Enveloped Viruses, Vectors and Innate Responses, INSERM, Unit 1111, Centre National de la Recherche Scientifique, Unit Mixte de Recherche 5308, Ecole Normale Suprieure de Lyon, Universit Claude Bernard Lyon 1, University of Lyon, Lyon, France. (7) International Center for Infectiology, Research Team Enveloped Viruses, Vectors and Innate Responses, INSERM, Unit 1111, Centre National de la Recherche Scientifique, Unit Mixte de Recherche 5308, Ecole Normale Suprieure de Lyon, Universit Claude Bernard Lyon 1, University of Lyon, Lyon, France. (8) Translational Oncology at the University Hospital Mainz, Mainz, Germany; and. (9) Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany. (10) International Center for Infectiology, Research Team Enveloped Viruses, Vectors and Innate Responses, INSERM, Unit 1111, Centre National de la Recherche Scientifique, Unit Mixte de Recherche 5308, Ecole Normale Suprieure de Lyon, Universit Claude Bernard Lyon 1, University of Lyon, Lyon, France. Universit Cte d'Azur, INSERM, Centre Mditerranen de Mdecine Molculaire, Nice, France. (11) Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany. Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany. Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany.
