In a study published in Cell Reports Medicine on December 9, a research team led by Prof. WANG Haoyi from the Institute of Zoology (IOZ) of the Chinese Academy of Sciences reported the development of a chimeric antigen receptor T (CAR-T) cell exhaustion model and a functional screening platform for identifying compounds that can rejuvenate exhausted T cells. Using this innovative platform, the team identified the small-molecule compound miltefosine, which significantly enhances the tumor-killing activity of CAR-T cells.

T cell exhaustion is a differentiation state that arises when T cells are exposed to persistent antigen stimulation. This state is characterized by a progressive loss of effector functions, sustained expression of inhibitory receptors, impaired proliferation capacity, and compromised mitochondrial respiration and glycolytic function. Notably, T cell exhaustion has been identified as a critical obstacle to the efficacy of immune checkpoint blockade (ICB) and CAR-T cell immunotherapies. Manipulating the exhaustion process offers the potential to improve the therapeutic efficacy of T cell responses in cancer.

To address this challenge, Prof. WANG Haoyi's team generated hypofunction CAR-T cells through multiple rounds of tumor challenge and established a drug screening platform using hypofunctional CAR-T cells. By screening FDA-approved drugs, they discovered that miltefosine, a small molecule previously used as an antiparasitic drug for treating leishmaniasis, could restore the functionality of exhausted CAR-T cells. Remarkably, even in a terminally exhausted state where PD-1 antibody therapy proved ineffective, miltefosine was still capable of boosting CAR-T cell activity.

Single-cell RNA sequencing (scRNA-seq) analysis provided comprehensive evidence that miltefosine enhances effector function, reduces exhaustion, and improves metabolic activity in hypofunctional CAR-T cells. Further investigations revealed that miltefosine effectively rescues the glucose uptake deficit and enhances the impaired glycolytic and oxidative phosphorylation metabolism of hypofunctional CAR-T cells in a GLUT1-dependent manner, ultimately leading to improved efficacy in treating solid tumors.

In both allogeneic and syngeneic tumor models, miltefosine exhibited notable potential in enhancing the tumor-clearing capacity of CAR-T cells and T cells.

In summary, the team developed a drug screening platform utilizing a CAR-T cell exhaustion model to assess FDA-approved small molecules. Miltefosine emerged as a promising candidate, showing the capacity to improve CAR-T cell function and metabolic activity, suggesting its potential application in immunotherapy.

Diagram of the drug screening platform and the mechanism of miltefosine in CAR-T cells (Image by Prof. WANG's group)