Huazhen Liu

HI-TOPK-032 decreases the expression of LAG-3 and TIM-3 and frequencies of CCR7+PD-1+ subset in CD8+ CAR-T cells of mice bearing subcutaneous Huh7-HCC.

Activation of mTOR diminishes the effects of HI-TOPK-032 on GPC3-CAR-T therapy and reduces CD8+ TCM frequency in tumor-bearing mice.

Gating strategy used for CD8+ CAR-T cells from the tumor-bearing mice

HI-TOPK-032 enhances the specific effector responses of CAR-T cells to GPC3.

The gene or protein expression of GPC3-CAR.

HI-TOPK-032 inhibits AKT/mTOR signaling in CAR-T cells.

Gating strategy used for CD8+ CAR-T cells in vitro.

Continuous treatment with HI-TOPK-032 increases the expression of effector molecules but reduces some of the exhaustion molecules in CAR-T cells in vitro.

Chimeric antigen receptor (CAR) T cells are emerging as an effective antitumoral therapy. However, their therapeutic effects on solid tumors are limited because of their short survival time and the immunosuppressive tumor microenvironment. Memory T cells respond more vigorously and persist longer than their naïve/effector counterparts. Therefore, promoting CAR T-cell development into memory T cells could further enhance their antitumoral effects. HI-TOPK-032 is a T-LAK cell–originated protein kinase (TOPK)-specific inhibitor that moderately represses some types of tumors. However, it is unknown whether HI-TOPK-032 works on hepatocellular carcinoma (HCC) and whether it impacts antitumoral immunity. Using both subcutaneous and orthotopic xenograft tumor models of two human HCC cell lines, Huh-7 and HepG2, we found that HI-TOPK-032 significantly improved proliferation/persistence of CD8+ CAR T cells, as evidenced by an increase in CAR T-cell counts or frequency of Ki-67+CD8+ cells and a decrease in PD-1+LAG-3+TIM-3+CD8+ CAR T cells in vivo. Although HI-TOPK-032 did not significantly suppress HCC growth, it enhanced the capacity of CAR T cells to inhibit tumor growth. Moreover, HI-TOPK-032 augmented central memory CD8+ T cell (TCM) frequency while increasing eomesodermin expression in CD8+ CAR T cells in tumor-bearing mice. Moreover, it augmented CD8+ CAR TCM cells in vitro and reduced their expression of immune checkpoint molecules. Finally, HI-TOPK-032 inhibited mTOR activation in CAR T cells in vitro and in tumors, whereas overactivation of mTOR reversed the effects of HI-TOPK-032 on CD8+ TCM cells and tumor growth. Thus, our studies have revealed mechanisms underlying the antitumoral effects of HI-TOPK-032 while advancing CAR T-cell immunotherapy.