55P - Engineering of chimeric cytokine receptors (CCR) to induce IL-7 signaling to CAR-T cells for solid tumor treatment

Background

Clinical efficacy of CAR-T cells in solid tumors has been limited due to poor expansion and survival. Most CAR constructs provide signal 1 and 2. Current strategies for providing signal 3 mainly rely on constitutive interleukin signaling, while achieving inducible signaling remains a challenging and highly pursued goal in the field. We hypothesized that adding the intracellular domain of the IL-7 cytokine receptor or its activation motifs to induce signal 3 upon antigen recognition would enhance CAR-T cell activation/persistence.

Methods

CCRs were designed by fusing a scFv to the IL-7Rα intracellular domain with the IL-7Rα or CD8 transmembrane domain. We also created second-generation ICOS or CD28-based CARs incorporating IL-7Rα motifs in different conformations. Construct expression and functionality were assessed in vitro and in vivo.

Results

CCRs were efficiently expressed on the T-cell membrane. In single transduction, CCRs failed to induce STAT5 signaling upon antigen recognition unlike a previously described constitutively active IL-7R (C7R). When the extracellular domain of the C7R was replaced by a scFv, STAT5 activation was not complete, suggesting either some steric concerns or that optimization of the CCR extracellular domain may be needed for correct homodimerization. In co-transduction with a CAR, CCRs did not provide a cytotoxic nor a proliferative advantage. Next, we explored the addition of IL-7Rα signaling motifs within a CAR construct. In vitro, we observed differences in terms of expression and cytotoxicity among the different constructs. Using a model of continuous antigen exposure, some IL7R-based constructs revealed enhanced cytotoxicity compared to the control. In vivo, IL7-based CARs behaved similarly to the control CAR, showing no clear advantage in terms of antitumor activity.

Conclusions

By generating and testing several conformations of IL-7R-based CARs and CCRs, we demonstrate that when IL-7Rα signaling domains are included in these receptors they lack signaling upon antigen recognition. Further analysis of CCR homo/heterodimerization with the IL-2Rɣ will be presented. We believe that these findings may shed light on dynamics of cytokine signaling and allow the design of an inducible CCR.

Legal entity responsible for the study

The authors.

Funding

Asociación Española Contra el Cáncer.

Disclosure

All authors have declared no conflicts of interest.