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.