Enhanced Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV Specific Central Memory T Cells by CMV Vaccine.

Abstract 3014

Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of cloned and bulk T cell transfer as a therapy for patients. But potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. In contrast, the adoptive transfer of viral specific T cells has shown efficient efficacy for preventing progressive viral infections and exhibited long term persistence in patients, in part due to the viral specific T cells received optimal co-stimulation after engagement of their native receptors. Conceptually, engineering CMV specific T cells with CD19CAR to provide them with a second specificity for a tumor antigen may enable the transferred T cells (bi-specific T cells) to persist or numerically expand in vivo by stimulation of the endogenous TCR by virus antigen. Moreover, bi-specific T cell can be used in treatment for B cell malignancies in allo-settings without causing GVHD due to the pre-defined non-alloreactive TCR specificity. In this study, we explored the use of CMVxCD19CAR bi-specific T cells in CD19⁺tumor bearing NSG mice and evaluated their antitumor activity in response to CMVpp65 antigen stimulation as a consequence of CAR transduced T cell expansion.

CMV specific T cells derived from central memory T cells were selectively expanded by 2 rounds of stimulation with cGMP grade pp65 protein followed a rapid expansion containing OKT3 and feeder cells. The established CMV specific Tcm, in which majority of them are CMVpp65 tetramer positive, were then transduced with cGMP grade SIN lentivirus expressing CD19R:CD28:z/EGFRt. After stimulation with CD19 positive LCL, 40% of the resultant cells co-express pp65 tetramer and CAR as detected by EGFRt/Erbitux analysis. Functionally, the bi-specific T cells exhibit specific cytolytic activity and secret IFNg, IL2 and TNFα upon engagement with pp65 or CD19 antigen, indicating that the effector function of the bi-specific T cells can be induced through endogenous TCR or the introduced CAR. To evaluate the in vivo viral antigen driven anti-tumor efficacy of the adoptively transferred bi-specific T cells, CD19⁺LCL expressing GFPffluc were inoculated (i.v) into huIL-15 reconstituted NSG mice. Once the tumor engraftment was confirmed by in vivo imaging, bi-specific T cells were adoptively transferred (i.v) into the tumor bearing mice. Anti- tumor activity was observed 14 days post T cell infusion. As expected, this effect is transient and tumor re-progression occurred. In order to deliver CMV antigen for vaccine, we generated T-APC by loading CMVpp65 peptide into autologous T cells and injected the CMV T-APCs (I.v) into the bi-specific T cell treated mice, Influenza specific MP1 peptide pulsed autologous T cells were used as control T-APCs. CMV T-APC induced a second wave of antitumor activity 2 weeks post vaccine and mice survived for more than 2 months post adoptive transfer of T cells, while tumor grew vigorously when MP1-T-APCs were given as stimulators. The findings demonstrated that CD19CAR modified CMV specific T cells are capable of responding to viral antigen reactivation through their endogenous TCR, which could be used to magnify the antitumor activity of CAR transduced T cells in vivo.