Poster Presentation Eradicate Cancer 2018

TCR silencing with shRNA as a foundation for a bank of allogeneic CAR19 T-cells generated by single-step genetic modification using the piggyBac transposase (#102)

David C Bishop 1 2 3 , David J Gottlieb 1 2 3 4 , Kenneth P Micklethwaite 1 2 3 4
  1. Westmead Institute for Medical Research, Westmead, NSW, Australia
  2. Department of Haematology, Westmead Hospital, Westmead, NSW, Australia
  3. Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
  4. Sydney Cellular Therapies Laboratory, Pathology West, Westmead, NSW, Australia

Aim: Autologous CD19-specific CAR (CAR19) T-cells have shown remarkable efficacy against B-cell malignancies, but manufacture of individualised products is expensive, delays patient treatment and is unfeasible in patients with insufficient healthy T-cells. We sought to evaluate TCR suppression by short hairpin RNA (shRNA) in low-cost piggyBac-generated CAR T-cells, in order to generate a ready-made allogeneic product devoid of alloreactivity.

Methods: PiggyBac transposon plasmids were generated for expression of CAR19-28z alone, or additionally with shRNA against either TCRα-chain or TCRβ-chain (shRNA designed by Takara Bio Inc). T-cells isolated by immunomagnetic separation were electroporated with piggyBac transposon and transposase plasmids (n=3 donors per construct), and expanded over 22 days via CD19 stimulation with IL-15 support. TCRneg cells were enriched by immunomagnetic CD3 depletion in CAR T-cell cultures that had been transfected with shRNA, to create a final product. Final products were further cultured to ensure stability of TCR suppression.

Results: CAR T-cell final products had expanded over 100-fold. Overall, the proportion of T cells with the desired TCRnegCAR+ phenotype varied significantly (5% for CAR19-28z alone, 41% with additional TCRα-chain shRNA and 70% with additional TCRβ-chain shRNA). CAR expression on T-cells was robust with CAR19-28z alone or with additional TCRβ-chain shRNA (96% and 98%, respectively), but was significantly lower with additional TCRα-chain shRNA (56%). The proportion of CAR T-cells that were TCRneg was significantly greater with additional TCRα-chain and TCRβ-chain shRNA (74% and 71%, respectively) compared to CAR19-28z alone (5%). TCR silencing was stable over a further 42 days with TCRβ-chain shRNA, but not TCRα-chain shRNA (CAR T-cells lacking TCR: 71% vs 1%, respectively).

Conclusion: CAR19 T-cells with stably suppressed TCR were successfully generated by introducing genes for both CAR and shRNA against TCRβ-chain into T-cells via single-step genetic modification with the piggyBac transposase system. This platform could form the foundation for a bank of inexpensive allogeneic TCRneg CAR T-cells with low GVHD potential, suitable for rapid treatment of many partially HLA-matched recipients.