Potentiate Immune Activation and Function By Targeting Inhibitor of Apoptosis Proteins (IAPs) in Relapse/Refractory DLBCL

Background: The prognosis of relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) that had progressed after or are ineligible for high dose chemotherapy and autologous stem cell support (HDC-ASCS) is extremely poor (median overall survival of 6.3 months). Cellular based immunotherapy with Chimeric Antigen Receptor T-cell against CD19 (CART-19) has proven high effective in r/r DLBCL with an overall response rate of 52-83% and an overall survival rate of 40%. While r/r DLBCL benefits from CART-19 therapy, a significant number of progress after initial response stresses the need to improve the efficacy of this novel approach. A significant amount of work is being done in improving the manufacturing or activation of CART cells. However, there is few data on lymphoma associated factors that could influence response to CART-19 therapy. Previously we reported an imbalance and dysfunction of pro- and anti-apoptotic proteins, including Bak/Bax, Mcl-1/BCLxL/Survivin and upregulation of inhibitor of apoptosis (IAP) family proteins in rituximab+chemotherapy (R-chemo) resistant DLBCL. Targeting IAPs by small inhibitor LCL161 in lymphoma cells significantly overcame chemotherapy resistance in vitro and in vivo. LCL161 potentiated CART-19 cell cytotoxicity mediated in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma pre-clinical models. Here, we investigate the mechanism of action to enhance immune activation by targeting IAPs in r/r DLBCL.

Methods: Primary T-cells were isolated and activated from peripheral blood apheresis samples from consenting adults (healthy human donors or patients with DLBCL) using Pan T-Cell Isolation Kit (MACS) and activated by T-cell Dynabeads human activator CD3/CD28 kit. Cells were expanded in RPMI media supplemented with IL2 for 2 weeks. At day 14, cells phenotype analysis was performed (CD3, CD4, CD8) by flow cytometry. T-cell number was calculated by trypan blue exclusion assay and proliferation was measured by presto blue assay after exposure to LCL161 or control for 48hrs. After exposure, CD4 and CD8 ratios were determined by cellular surface staining. T-cell function assay was determined by intracellular staining with perforin and granzyme B. Regulatory T-cells were determined as CD25+ and FOXP3+ staining cells. T-cell cytolytic activity was accessed by ⁵¹Cr release assay using ⁵¹Cr labeled rituximab-chemotherapy sensitive (Raji and RL cells) or resistant (Raji 4RH and RL 4RH) cell lines as target cells (effector/target ratio 10/1). To confirm the role of IAP proteins in the effects of LCL161 in T-cell activation, we conducted cytotoxicity assays using XIAP knock out lymphoma cell lines as target cells. Stable XIAP knock out cell lines were generated by CRISPR-Cas9 gene editing system. Lentiviral Cas9 nuclease particles were transduced into Raji and Raji 4RH cells followed by lentiviral XIAP sgRNA transduction. Lentiviral sgRNA non-targeting control was also used as negative control.

Result: In vitro exposure of T-cells to LCL161 did not affect cell viability. On the other hand, it led to an increase in CD8+ T-cells and in the CD8:CD4 ratio. In addition, the percentage of regulatory T cells (CD25+ and FOXP3+) was reduced following LCL161 exposure. Using the T-cell function assay, we found that perforin and granzyme B inside T-cell was significantly increased post LCL161 exposure. The presence of LCL161 significantly improved the T-cell killing activity towards rituximab-chemotherapy sensitive and more importantly resistant cell lines. XIAP knock out in target cells resulted in abrogation of the enhance cytotoxicity exhibited by T-cells exposed to LCL161.

Conclusion: Our data suggest that besides its previously documented direct anti-tumor activity, LCL161 has dual functions modulating immune cells function. LCL161 improve T-cell function by increasing the number of CD8+ T-cells and intracellular levels of granzyme B and perforin leading to an increase in T-cell cytotoxicity. In addition, LCL161 decreases the number of T-reg cells further enhancing immune activation. Taking together, targeting IAPs would be an attractive approach that potentiate current CART-19 immunotherapy in the clinical setting.