455 Dual inactivation of SOCS1 and Regnase-1 in T cell therapies demonstrated enhanced anti-tumor activity by expanding discrete cell states in pre-clinical syngeneic mouse models

Background

The use of CRISPR/Cas9-gene-editing to enhance the anti-tumor activity of T cell therapies is a promising approach in the treatment of patients with solid tumors. We previously reported the utility of in vivo pair-wise CRISPR/Cas9 screens to identify the best target combinations in T cells able to enhance anti-tumor function, with SOCS1 and Regnase-1 identified as a top combination. In these studies, we further explore the mechanisms by which the dual-inactivation of SOCS1 and Regnase-1 in T cells enhances anti-tumor activity.

Methods

The efficacy of transferred SOCS1/Regnase-1 dual edited mouse OT1 TCR-Tg T cells were evaluated in syngeneic tumor models and compared to single edit, PD-1 and olfactory receptor (OLFR) controls. Anti-tumor activity and the phenotypic and transcriptional state of the transferred T cells were characterized by flow cytometry and scRNA-Seq.

Results

A single injection of SOCS1/Regnase-1 dual-edited OT1 T cells drove the complete clearance of large (>350mm³) established B16-OVA tumors in the absence of conditioning. Upon evaluation of OT1s present in tumor following transfer, we observed that dual editing of SOCS1 and Regnase-1 significantly enhanced OT1 accumulation in tumor when compared to single edit controls, with dual-edited OT1s comprising ~80% of intratumoral CD3⁺ T cells. Notably, our analysis detailed tumor-infiltrating dual-edited OT1 T cells to be enriched in discrete exhausted T cell (Tex) subsets displaying features of proliferative capacity and effector function in comparison to either single edit or PD-1 edit alone. Longitudinal evaluation of mice undergoing complete rejection of tumor demonstrated persistence of dual-edited OT1s and establishment of functional memory capable of fully rejecting secondary tumor re-challenge.

Conclusions

SOCS1 and Regnase-1 are a top dual-edit T cell target combination wherein CRISPR/Cas9-mediated inactivation robustly enhances the anti-tumor activity of adoptively transferred CD8⁺ T cells by driving the accumulation of intratumoral CD8⁺ T cells optimized for tumor control. These data demonstrate the potential of SOCS1/Regnase-1 dual edits to overcome functionality limitations associated with T cell therapies for the treatment of solid tumors.