Background Chimeric antigen receptor T (CART) cell therapy has resulted in impressive overall response rates in the treatment of cancer.¹ However, the durable response to therapy is limited by the development of exhaustion which impairs CART cell expansion and persistence.² Previously, our group identified IL-4 as a regulator of CART cell exhaustion through an evaluation of epigenetic changes seen with an in vitro model for CART cell exhaustion. We then showed that IL-4 supplementation of CART cells drives an exhausted phenotype as seen by an increase in the co-expression of multiple inhibitory receptors, a decrease in effector cytokine production, and a decrease in proliferative ability. Additionally, we showed that IL-4 neutralization with a monoclonal antibody can improve CART cell antitumor activity and in vivo expansion in a high tumor burden mantle cell lymphoma xenograft model.

Methods Building on our previous findings, we investigated gene editing strategies to interrupt the IL-4 pathway in CART cells. Gene editing during CART cell production is an attractive strategy due to increasing evidence that the epigenetic profile of pre-infusion CART cell products influences overall response to therapy.³ In particular, we used CRISPR Cas9 to create IL-4 and IL4R knockdown CART cells. These CART cells were used to both further investigate the mechanism of IL-4 induced CART cell exhaustion and to evaluate overall changes in CART cell activity.

Results In our in vitro model for CART cell exhaustion, IL-4 and IL4R knockdown CART cells display reduced phenotypical and functional signs of exhaustion as compared with control gRNA CART cells following chronic stimulation. This is seen by reduced co-expression of multiple inhibitory receptors, increased production of effector cytokines such as IL-2 (p = 0.006, p = 0.003), and an increase in proliferative ability (p = 0.0004, p = 0.009). This indicates that IL-4 drives CART cell exhaustion through an autocrine mechanism where both IL-4 production and reception by CART cells is important. Next, we evaluated the efficacy of IL-4 pathway edited CART cells in a high tumor burden JeKo-1 xenograft model. In this CART-stress model, both the IL-4 and IL4R knockdown CART cells improved tumor control (p = 0.001, p = 0.017) and expanded more than the control gRNA CART cells (p = 0.07, p = 0.009).

Conclusions Together, our data supports the further development and clinical testing of IL-4-edited CART cells to reduce the incidence of exhaustion and improve therapeutic response.

References

1. Neelapu SS, et al. 5-year follow-up supports curative potential of axicabtagene ciloleucel in refractory large b-cell lymphoma (zuma-1). Blood. 2023;141(19):2307–2315.

2. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) t cell therapy of chronic lymphocytic leukemia. Nature Medicine. 2018;24:563–571.

3. Deng Q, et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large b cell lymphomas. Nature Medicine. 2020;26:1878–1887.