| Adoptive T-cell therapy, particularly chimeric antigen receptor (CAR) therapy, is a revolutionary and quickly-evolving means of treating cancer patients who can no longer be helped by standard therapies. In multiple clinical trials, including our own at Seattle Children's Hospital, CD19 CAR therapy for B-cell leukemia and lymphoma has achieved a complete remission rate of >90%. Unfortunately, in its present form, CAR therapy has had limited success against solid tumors. It is also not currently an option for patients who lack sufficient numbers of their own T-cells due to their disease or prior treatments. Thus, genome engineering strategies to overcome these limitations could be of great benefit to patients. We chose a two-pronged approach to achieve this goal: knock-out of the endogenous TCR and multiplex knock-out of the T-cell inhibitory checkpoints PD-1, Tim3, Lag3, and TIGIT. Knocking out these inhibitory checkpoint proteins specifically in the CAR T-cells will maintain the synergistic effects recently seen in combination monoclonal antibody therapy without the serious, sometimes fatal, immune-mediated side effects seen with systemic antibody therapy.;To this end, we first developed a linear mRNA expression vector with a long, encoded poly(A) tail to allow transient delivery of nucleases such as TALENs or CRISPR to primary human cells in a consistent, clinically applicable, and scalable fashion. We then used IVT mRNA made from this vector to deliver a TALEN pair targeting the TCR locus to CD19 CAR T-cells, and demonstrated that removal of the endogenous TCR does not hinder CAR T-cell function in vitro or in vivo in a murine xenograft tumor model. Knockout of the endogenous TCR will facilitate production of an allogeneic CAR T-cell product to be used as a bridge to HSCT in patients who cannot receive autologous CAR therapy. Removal of the endogenous TCR will also add a measure of safety when creating CAR T-cells lacking inhibitory checkpoint proteins by preventing GvHD while retaining anti-tumor effects. These technologies and methods may allow a wider variety of patients to benefit from the recent advances in CAR T-cell therapy. |
