647. Efficient Targeting of T Cell Malignancies In Vitro and In Vivo Using CD4-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells

Chimeric antigen receptor (CAR) immunotherapy has shown exceptional promise in targeting otherwise untreatable hematologic and solid tumor malignancies, providing new hope to both pediatric and adult patients. Although remarkable progress has been achieved in clinical trials for patients with relapsed/refractory B cell malignancies, CAR immunotherapy for patients with T cell leukemias and lymphomas has not yet been developed, despite a generally poorer prognosis. In light of this unmet clinical need, we engineered natural killer (NK) cells to express a third-generation CAR directed against CD4. Indeed, most aggressive peripheral T-cell lymphomas are CD4-positive with uniform expression of this surface molecule. Therefore, CD4 is potentially an ideal target for CAR. Furthermore, in contrast to donor T cells, CAR NK cells have the advantage of mediating anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). Also, their shorter lifespan relative to T cells may limit off-target events and thus eliminate the need for a “suicide switch” that would ablate the modified cells in the event of off-target effects. Other potential advantages of CAR NK cells over CAR T cells include the opportunity to be an off-the-shelf therapy, and simpler manufacturing. We generated a third generation CD4-specific CAR (CD4CAR) containing CD28, 4-1BB and CD3zeta signaling domains. This CAR was introduced into the NK-92 cell line, which has used in multiple clinical studies, resulting in CD4CAR NK cells. When assayed in co-culture, these CD4CAR NK cells had a profound ability to kill CD4 positive tumor cells in vitro using both CD4+ cell lines (Karpas 299, HL60, and CCRF-CEM) and two primary patient samples from pediatric and adult T cell leukemia and lymphomas (Figure 1Figure 1). To address any potential CD4CAR NK cell impact on the hematopoietic compartmentu0027s ability to repopulate, we also confirmed by CFU assay that CD34+ cells co-cultured with CD4CAR NK cells were able to differentiate into BFU-E and CFU-GM colonies at ratios statistically similar to CD34+ cells co-cultured with non-CAR NK cells. We then confirmed in vivo anti-CD4 positive tumor activity using xenogeneic mouse models. Together, our encouraging results of this preclinical study support the further development of anti-CD4 CAR-engineered NK cell immunotherapy for patients with T cell malignancies.Figure 1CD4CARNK cells kill both peripheral T cell lymphoma cell line and primary patient malignant cells at effector to target ratios of 2 to 1 and 5 to 1. Peripheral T cell lymphoma cell lines and primary patient T cell leukemia lymphoma samples were co-cultured for 24 hours with CD4CAR NK cells. The percent of malignant cell killing was determined by comparison to vector control transduced NK cells via flow cytometry analysis of cell survival.View Large Image | Download PowerPoint Slide