Selinexor Reduces the Immunosuppressive Properties of Macrophages and Synergizes with CD19 CAR-T Cells Against B-Cell Lymphoma

Background: CD19 chimeric antigen receptor (CAR)-T cell therapy has achieved high response rates in patients with B-cell lymphoma (BCL). However, treatment failure and relapse can be attributable to CAR-T cell dysfunction and the immunosuppressive tumor microenvironment (TME). Selinexor (SEL), an exportin-1 (XPO-1) inhibitor, is clinically active in patients with multiple myeloma (MM) and BCL. Several studies reported that XPO-1 inhibitors rendered tumor cells more vulnerable to CAR-T cells. Patients with MM benefited from the combination of SEL and CAR-T cell therapy. In this study, we aimed to investigate the combination effects of SEL and CAR-T cells in BCL.

Methods: First, we isolated RNA of peripheral blood from patients enrolled in a clinical trial registered as NCT04008251 before CAR-T cell infusion. Subsequently, we analyzed the association of XPO-1 mRNA expression with clinical outcomes. BCL cell lines (Raji, CA46, and Ramos cells) were cocultured with CAR-T cells in supplementation with SEL (0nM, 50nM, 100nM). After 24 hours, specific cytotoxicity was evaluated by flow cytometry (FCM). We established two models by coculturing BCL cell lines with (the ex vivo TME model) or without (the control model) THP-1 cells in the administration of PMA and SEL, followed by a half-change medium and coculture with CAR-T cells. The phenotypes of CAR-T cells and the residual target tumor cells in the two models were assessed by FCM. We further evaluated the ratio of the phagocytized tumor cells and conducted transcriptome sequencing of CAR-T cells in the ex vivo TME model. Moreover, we established a syngeneic animal model by subcutaneous injection of A20 cells to decipher the role of SEL in the immune landscape. Finally, SEL was administered before CAR-T cells in mice engrafted with Raji-luciferase cells to identify the efficacy of the combination.

Results: Patients with lower expression of XPO-1 obtained longer overall survival (P = 0.0471) and progression-free survival (P = 0.0423). SEL impaired the specific cytotoxicity of CAR-T cells against target cells. SEL markedly decreased PD-1 and LAG-3 double expression on CAR-T cells in the ex vivo TME model, but only suppressed PD-1 expression in the control model. SEL administration resulted in more tumor death, which was more prominent in the ex vivo TME model than in the control model. Additionally, SEL did not affect the proportion of tumor cells phagocytized by macrophages. Bulk-RNA analysis of CAR-T cells in the ex vivo TME model showed that SEL upregulated T-cell activation-associated signaling pathways and cell cycle-related signaling pathways.

In the syngeneic animal model, SEL decreased M2 macrophages in tumor specimens. Next, we cocultured bone marrow-derived macrophages and A20 cells ex vivo, and FCM and RT-qPCR results indicated that SEL decreased M2 macrophages. Additionally, the IL-6 level in the coculture supernatants was slightly increased with SEL doses (P = 0.0534 at 100nM). In the xenograft animal model, the sequential use of SEL and CAR-T cells significantly suppressed tumor growth compared with SEL or CAR-T cell monotherapies. Meanwhile, SEL did not lead to obvious weight loss of the mice after CAR-T cell infusion. Furthermore, SEL significantly reduced the LAG-3 expression on CAR-T cells in tumor tissues (P = 0.0150).

Conclusions: Our study demonstrated that SEL could mitigate the immunosuppression of macrophages and improve CAR-T cell functionality, and the sequential administration of SEL and CAR-T cells was more effective in BCL.