Refractory pediatric cancer is an ongoing clinical problem with significant morbidity and mortality. Adoptive therapy with chimeric antigen receptor (CAR) T cells has the potential to address this issue. However, specific targets for pediatric cancers have not been identified to date. In this work, we hypothesized that LeY and GRP78 are potential targets for CAR-T therapy of pediatric cancers. There are also fundamental questions around GMP production of the optimal CAR-T cell that remain unanswered, including the best activation and expansion protocol to generate the most potent CAR-T cells.

We assessed alternate activation protocols using bead-bound versus soluble activators for generating CAR-T products. We analyzed by flow cytometry the transduction efficiency by CAR expression, and the subtypes of T cells generated. We also assessed cytokine secretion and the cytotoxic capacity of these cells. We generated novel retroviral vectors encoding CAR specific for LeY and GRP78 and incorporated an extracellular domain of human CD34 (HuCD34) in order to track the cells in patients.

We obtained high transduction efficiency with all activation protocols (range 50-88%) that persist until 12 days post-activation; we observed higher LeY-CAR expression (88%) with the soluble activator CD3/CD28/CD2 and a stronger MFI at early stage, day 7 post activation. We successfully incorporated the huCD34 into the retroviral vectors encoding CAR specific for LeY and GRP78.The experiments to test these new constructs are ongoing.

These new hu-CD34 CARs against LeY and GRP78 tumor antigens could offer new targeted therapies with acceptable short and long-term toxicity for pediatric patients. We are currently selecting the pediatric tumors that express the LeY and the GRP78 antigens in order to select future patients for clinical trials. Furthermore this innovative tracking system will help us to understand the questions around CAR-T cell differentiation, functional polarisation and trafficking of the CAR-T cells in vivo.