T cell engineering using V(D)J recombination allows tumor growth inhibition in mice
2Ichilov, Tel Aviv Sourasky Medical Center
3San Raffaele Telethon, Institute for Gene Therapy Safety of Gene therapy and Insertional Mutagenesis Milano - Italy
Introduction: T cell therapies have shown clinical success in hematological malignancies, but they rely on prolonged, expensive ex vivo manipulations and their efficacy is hampered by early T cell exhaustion, promoted by the prolonged culturing required for the scarcely retrieved cells. We propose “VDJ targeting”: a novel immunotherapy approach targeting AAV-delivered immune genes into the genome using V(D)J recombination in developing T cells.
Material and methods: We use AAV vectors coding for a transgenic TCR/CAR gene followed by a recognition signal sequence (RSS) for the recombination activating gene (RAG) complex, which is active only during T cell development. We targeted ex vivo differentiated T cells from human cord blood and bone marrow and from mouse bone marrow. Mouse bone marrow cells were grown on OP9DL1 feeder cells in IL-7 and FLT-3 containing medium for 5 days, and then transduced with 1e6 vector genomes per cell of an AAV-DJ vector coding for a CAR against CD19. Human cord blood or bone marrow derived HSPCs were grown on feeder cells with an SPEM-2 medium for 5 days and then transduced with 7e5 vector genomes per cell of an AAV6 vector coding for either a CAR anti-CD19 or a TCR targeting an NY-ESO1 peptide presented on HLA-A*02.
Results and discussion: Following transduction, mouse bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) CD3+ were found to express the CAR and were activated by CD19-expressing cancer cells. Similarly, human cord blood and bone marrow derived HSPCs CD3+ cells successfully expressed the TCR and were activated by NY-ESO-1 expressing cancer cells. Importantly, human T cells, engineered by VDJ recombination to express the anti-NY-ESO-1 TCR, inhibited tumor growth and prolonged survival in immunodeficient mice implanted with a human NY-ESO-1+ melanoma. Moreover, our VDJ-targeted T cells vastly outperformed conventionally engineered T cells, transduced with a lentiviral vector coding the anti-NY-ESO-1 TCR. Conclusion: VDJ targeting produces potent naïve T cells that may be transplanted in lower numbers and have increased persistence compared to T cells engineered with state-of-the-art technologies. VDJ targeting is thus an efficient T cell engineering technology applicable in the autologous, allogeneic and in vivo engineering settings.