Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

Abstract

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells. With a 5-year overall survival of approx. 30%, the prognosis of patients with acute myeloid leukemia remains poor, in particular for those with relapse or refractory disease. The adoptive transfer of T-cells genetically modified to express a chimeric antigen receptor (CAR) have shown impressive remission rates in patients with B-cell lymphoid malignancies. In contrast, CAR T-cell therapy for AML has induced durable remission only in a minority of patients so far, highlighting the need for strategies to improve anti-tumor efficacy and persistence of AML-specific CAR T-cell products. Adapting an established high throughput screening platform, we aimed to identify compounds with the potential to enhance CAR T-cell mediated cytotoxicity through interaction with tumor and/or CAR T-cells. Methods: We established a high throughput platform to determine the cytotoxicity of four different AML-specific CAR T-cell products targeting CD33 (CD33.CD28.41BB.zeta), CD70 (CD27.zeta), CLL1 (CLL1.CD28.zeta) and EMR2 (also known as ADGRE2; EMR2.41BB.zeta) in the presence of 32 compounds at five different concentrations in a 10,000-fold concentration range. CAR T-cells were generated by transduction of activated T-cells derived from peripheral blood mononuclear cells (PBMCs) of three healthy donors with a gamma retroviral vector containing the different CAR constructs. On day 11 after activation, CAR T-cells and non-CAR-transduced control T-cells were incubated with luciferase-expressing target cells at an effector-target ratio of 1:4 in 384-well plates. Viability of target cells cultured with CAR T-cells, control T-cells or in the absence of T-cells was calculated after 24 and 48 hours using a luciferase signal intensity assay. Combinatorial toxicities were calculated with the Bliss Independence model for synergistic effect and the highest single agent model for additive combination effect. Results: In quality analysis, the intensity of luminescence signals in control wells (tumor only, non-CAR-transduced control T-cells) of different plates was similar and principal component analysis (PCA) of samples derived from different days (batches) did not show significant differences ruling out relevant edge or batch effects that could negatively impact the screening results. Of all tested compounds birinapant, a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of apoptosis protein (IAP) family proteins and the hypomethylating agent (HMA) decitabine showed the strongest improvement of CAR T cell cytotoxicity in an additive but also in a synergistic manner. These effects were evident in CAR T-cell products derived from all different T cell donors without significant differences. Depending on the structural composition of the CAR construct, the strongest drug-induced improvement of CAR T-cell cytotoxicity was seen either after 24 or 48 hours. The third generation CAR T- cell construct (CD33.CD28.41BB.zeta) showed enhanced cytotoxicity already after 24 hours, whereas significant anti-leukemic efficacy of CD27.zeta, CLL1.CD28.zeta and EMR2.41BB.zeta CAR T-cells became evident at the later 48 hours timepoint. Importantly, co-treatment with birinapant and decitabine particularly enhanced the cytotoxicity of EMR2-specific CAR T-cells which exhibited the lowest baseline anti-tumor efficacy compared to all other CAR constructs highlighting the potential of distinct chemical compounds to improve the functionality of AML-specific CAR T-cells. Conclusion: Our data suggest that high throughput drug interaction screens are a reliable approach to investigate the impact of chemical compounds on the functionality of AML-specific CAR T-cell products. HMA and SMAC mimetics are promising candidates that may potentially enhance the cytotoxicity of CAR T-cells. Ongoing studies are evaluating the effect of HMAs and SMAC mimetics on CAR T-cell proliferation and anti-tumor efficacy against AML cell lines and primary AML patient samples upon serial antigen stimulation. Furthermore, in-depth proteogenomic characterization to identify molecular signatures of enhanced functionality are currently being performed.