Selective Support of CAR-T Cell Therapies By Cis-Targeted IL-2 or IL-21 Cytokines Results in Enhanced Anti-Tumor Activity

INTRODUCTION Approximately 50% of patients with acute lymphoid leukemia (ALL) or with aggressive lymphoma who receive anti-CD19 CART cells (CART-19) remain relapse-free at 1 year. In vivo behavior of CART-19 correlates with improved outcomes, spurring interest in the development of approaches to selectively control the in vivo expansion of infused CART cells . Administration of exogenous cytokines belonging to the IL-2 family is one such approach. However, the clinical potential of combining IL-2 family cytokines with CART cells is hampered by the pleiotropic nature of the current molecules, which leads to severe toxicities and expansion of multiple endogenous cells (e.g. Tregs) in addition to CAR-T cells. While mutations have been introduced into existing engineered IL-2 variants to alter potency, these molecules are not entirely selective since endogenous cells are still stimulated. Other approaches using orthogonal cytokine/cytokine receptors are interesting but require further genetic modification of the T cells. To address these challenges, we developed CAR-T specific IL-2 or IL-21 fusion molecules that selectively activate CART cells by recognizing an extracellular tag. METHODS Cis-targeted cytokine fusions are comprised of (1) a targeting antibody directed against a tag expressed on the CAR-T surface (truncated non-signaling epidermal growth factor receptor [EGFRt] that is co-expressed with the CAR in at least one clinical-stage CART19 product) and (2) a cytokine mutein with attenuated binding to its cognate cytokine receptor. Specifically, IL-2 muteins exhibit diminished binding to IL2R⍺ and IL2Rβ while the IL-21 mutein has diminished binding to the IL-21R subunit. The targeting arm of the fusion molecule provides avidity to the attenuated cytokines, resulting in selective activation of the associated cytokine receptors on CARTs. We engineered one cis-targeted EGFRt-IL2 fusion molecule and one EGFRt-IL21 fusion molecule. Both molecules were characterized in vitro using primary human CART cells. In vivo activity was tested in a "stress test” model of B-cell ALL by engrafting 1x106 NALM6 cells that expressed luciferase into NSG mice for six days, prior to IV injection with a low dose of human CART19 (0.1x106). Cis-targeted cytokine fusions were administered intraperitoneally once, one day after CAR-T infusion. Anti-tumor activity was measured weekly by bioluminescence imaging (BLI) and analysis of peripheral blood was performed to examine the phenotype of the CAR-Ts. RESULTS The specificity of the EGFRt-IL2 molecule was demonstrated by its ability to selectively induce pSTAT5 signaling and EGFRt-IL21 molecule by pSTAT3, resulting in >100-fold preferential STAT activity in CAR-expressing cells compared to CAR-negative cells. In vivo BLI revealed that when sub-optimal doses of CAR-Ts were injected into leukemia-bearing mice, both EGFRt-IL2 and EGFRt-IL21 induced substantial tumor regression (Figure 1). EGFRt-IL21 induced greater tumor clearance (CR 5/5) with continued tumor regression without relapse through day 35, post CAR-T infusion. Interestingly, while EGFRt-IL2 induced a stronger initial expansion of CART19 in vivo than EGFR2t-IL21 (median 19068 CAR19T cells/ul blood vs 56 CAR19T cells/ul blood, p< 0.0001 on day 16), the EGFRt-IL21 group retained the 1:1 ratio of CD4:CD8 T cells in the infusion product while there was a much greater level of CD4 T cells present in the EGFRt-IL2 group. Both therapies were superior to CART19 control (without exogenous cytokine) and were equally well tolerated in mice. These observations suggest that the IL-2 and IL-21 cytokine fusions mediate their effects through different mechanisms. CONCLUSIONS Cis-targeted IL-2 or IL-21 cytokine fusion molecules selectively augment CAR-Ts in vitro and enhance in vivo anti-tumor activity and survival. Temporal control of the cis-targeted cytokines directed by anti-tag antibodies represent a promising approach to enhance CART cell therapies. DISCLOSURES NDM, KDM, WC, PB, CK, DP, TP, AY, and ID are employees of Asher Biotherapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal