Modeling Retreatment of Acute Lymphoblastic Leukemia with Anti-CD19 Chimeric Antigen Receptor T Cell Therapy

Tremendous progress has been achieved employing immunotherapy for B cell acute lymphoblastic leukemia (ALL), a leading cause of death in children from cancer. Recent trials using chimeric antigen receptor T cells (CART) targeting the B cell restricted antigen, CD19, that utilize the autologous transfer of patients' T cells, have demonstrated remarkable remission rates of 80% against relapsed or refractory ALL. Despite initial clearance of tumor, relapse with CD19 antigen loss ALL and with CD19 expressing ALL can occur. Attempts at retreatment of patients who have received CD19 CAR T cell therapy suggests that most patients will not respond to a second infusion of CD19 CAR T cells. It has been proposed that failure to respond to a second infusion of CAR T cells may be due to immunogenicity of the foreign CAR protein and elimination of CAR T cells due to immunological targeting. To evaluate the mechanism of retreatment failure in the setting of persistent antigen, we utilized a murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR transduced into mouse CD8 and CD4 polyclonal cells and tested against murine pre-B ALL in a syngeneic system. To investigate the issue of immunogenicity against CAR constructs, we immunized the mice with irradiated CAR T cells prior to CAR treatment to allow for anti-CAR T cell immunity. Following immunization, we inoculated the mice with leukemia on day 0 and treated the mice with 1 x 10⁶ CAR T cells on day 4. CAR treatment was able to clear leukemia and CAR T cell-reactive antibodies were not detected in the serum of the mice, suggesting that a mechanism other than classic host mediated immune rejection of CAR T cells may underlie CAR T cell retreatment failure. To further model the failure of CAR T cell retreatment, we evaluated the ability of a second CAR T cell infusion to eliminate a second leukemic challenge. Leukemia bearing mice were treated with a curative dose of CD19 CAR T cells post lymphodepleting regimen. 30 days after clearance of the primary leukemic challenge, the mice were rechallenged with leukemia and subsequently treated with mock T cells or CD19 CAR T cells. Mice treated with CAR T cells followed by retreatment with mock T cells demonstrated persistence of CAR T cells from the first treatment, which were able to expand and clear the second leukemia challenge. In mice treated with a second dose of CAR T cells, CAR T cells from the second infusion briefly expanded 10 days post infusion, but could not be detected at day 20 post infusion. In contrast, CAR T cells from the initial infusion were still detectable at both time points. These results demonstrate that CAR T cells are able to persist, and, in a model of leukemic relapse, are able to expand and clear leukemia. However, CAR T cells infused into mice with CAR T cells persisting after a prior infusion fail to persist and quickly contract without evidence of host immune rejection of CAR T cells. Our data suggests that the inability to successfully retreat CD19+ relapsed leukemia with subsequent doses of CAR T cells may also involve mechanisms beyond immune recognition and clearance of CAR T cells.