Overexpression of mutant EZH2 augments the efficacy of anti-PSMA CAR T therapy against prostate cancer
Introduction
Immunotherapy has transformed the field of cancer therapeutics, with CAR T therapy being one of its major breakthroughs in the past decade. CAR T therapy has seen incredible success in patients with hematological malignancies. However, the same is not seen in solid tumors. CAR T cells face multiple challenges in solid tumors, one of which is persistence. In this study, we developed a novel CAR T therapy against prostate cancer, and further engineered it to express a constitutively active form of EZH2, aiming to improve the persistence of the T cells by regulating their memory phenotype.
Methods
We used retroviral transduction to generate CAR T cells that recognize PSMA, a frequently overexpressed antigen in prostate cancer. EZH2 is a methyltransferase playing a crucial role in the maintenance of the memory phenotype of CAR T cells. This activity is regulated by Akt, as phosphorylation of EZH2 at serine-21 by Akt deactivates EZH2 methyltransferase activity on histone H3. Thus, in these PSMA CAR T cells, we further expressed an Akt-insensitive EZH2 mutant, with the serine-21 mutated to alanine (EZH2-S21A). This EZH2-S21A represents a constitutively active form of EZH2. We then evaluated the efficacy of the CAR T cells using in vitro T cell cytotoxicity assays and in vivo immunocompromised mouse model xenograft experiments.
Results
EZH2-S21A CAR T cells targeting PSMA have shown superior tumor-killing activity both in vitro and in vivo. Using T cell cytotoxicity assays, we found that EZH2-S21A CAR T cells were able to eliminate PSMA-positive prostate cancer cells at a lower E/T (effector to target cell) ratio compared to PSMA-CAR T. These results were also recapitulated in vivo, using NSG mice subcutaneously injected with prostate cancer tumor cells. Upon intravenous injection of CAR T cells, it was found that the EZH2-S21A CAR T eradicated the tumor faster than the control PSMA-CAR T cells.
Conclusion
Our study suggests that by engineering anti-PSMA CAR T cells to express EZH2-S21A, we can significantly enhance the tumor-killing activity of CAR T cells, both in vitro and in vivo. Further work will be performed to evaluate the persistence of these T cells, such as analyzing the memory markers upon repeated stimulation with tumor cells, and re-injecting tumors into mice to evaluate the in vivo efficacy of these T cells in a tumor rechallenge model.