Interleukin 21 Enhances Survival and Expansion of CAR T Cells Via Inhibition of Their Terminal Differentiation during Interaction with Tumor Target Cells

Background: Efficiency of CAR T cell based therapies against cancer is often limited by a poor survival of CAR T following recognition of tumor target cells. Interaction of CAR T with target cells induces their rapid differentiation into late memory subtypes (Teff) which lack expression of CD27, CD28, CD62L and CCR7. Although these terminally differentiated T cells are highly cytotoxic, their in vivo engraftment capacity is lower which thus reduces their in vivo survival and enables only temporary antitumor effects. It is generally believed that CAR T cells with early memory phenotypes (Tscm + Tcm) would provide stronger antitumor effects due to better survival in vivo. Recently, we have developed a transposon-based protocol of clinical-grade CAR19 T manufacture (Otahal et al, Cytotherapy 2018) which uses a combination of cytokines IL-4, IL-7 and IL-21 which strongly enhance the generation of CAR T cells with Tscm/Tcm phenotypes.

Methods: We have thoroughly studied the effects of IL-21 on the survival, differentiation status and the expression of major immunoinhibitory receptors using CAR T cells specific to antigens CD19 and PSMA. After the co-culture of CAR T with their tumor target cells, the phenotypes were analyzed by multi-color flow cytometry, together with the assessment of effector functions and proliferation. We have compared the outcomes of signaling initiated by IL-21 on the fate of CAR T during this co-culture with the effects initiated by IL-2.

Results: We have found out that IL-21 is a strong regulator of CAR T memory differentiation initiated by recognition of tumor target cells. IL-21 supported expansion of CAR T with Tscm/Tcm phenotypes and inhibited their terminal differentiation into CD45RA+/- CD62L neg, CD27 neg, CD28 neg late memory subtypes (i.e. Teff and Tem). Additionally, IL-21 suppressed up-regulation of inhibitory receptors PD-1 and TIGIT by CAR T cells. Both IL-21 and IL-2 were indispensable to maintain proliferation of CAR T following their activation via the recognition of tumor target cells however, IL-2 induced a rapid differentiation of CAR T into late memory subtypes and resulted in significantly lower expansion than CAR T cells co-cultivated with tumor cells in the presence of IL-21.

Conclusions: Our data strongly suggest that the in vivo functions of CAR T cells can be significantly boosted by omitting the use of IL-2 during production because IL-2 drives CAR T towards their terminal effector differentiation state that reduces their ability to form long-lived memory cells. We are currently developing CAR T with transgenically expressed IL-21 and we are preparing a clinical testing of CAR T manufactured according to this protocol in patients diagnosed with relapsed-refractory B-ALL and B-NHL.

Supported by grants NV15-34498A and Primus/MED/34, MH CZ - DRO (Institute of hematology and blood transfusion, IN - 00023736) and by gifts from Heřmanský foundation.