A Dominant Negative SHP-2 Which Abrogates PD-1 Signalling Pathways and Restores Function of Cytotoxic CAR T Cells

Introduction

Recent successes in the clinic with monoclonal antibodies against cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed-death 1 (PD-1) have highlighted the importance of developing strategies to overcome T cell inhibition when investigating anti-tumour immunotherapies. Strategies to avoid inhibitory signals by the tumour microenvironment have recently been incorporated into CAR T cells with the most exploited strategy being the conversion of the inhibitory signal from one receptor into an activating one. Many of these inhibitory receptors signal through either the SHP-1/SHP-2 phosphatases. Here we present a method to block these phosphatases using a dominant negative protein. Unlike previous methods that focus on blocking one inhibitory receptor at a time, this approach enables the disarmament of multiple inhibitory receptors at once.

Methods and results

We generated dominant negative forms of SHP-2 (dnSHP2) that block the function of this endogenous inhibitory phosphatase. This was transduced into T cells coupled with an anti-CD19 CAR and CAR T function was assessed in the presence of PD-1/PD-L1 ligation. In the absence of dnSHP-2, CAR T cell killing, proliferation and cytokine release was significantly impaired when co-cultured with target cells that ligated both the CAR and PD-1. In contrast, CAR T cells that expressed dnSHP-2 observed no defect in killing response in the presence of PD1 ligation. Furthermore, dnSHP2 was shown to impair PD-1 mediated inhibition of cytokine release and proliferation in CAR T cells. This ability to block PD-1 mediated inhibition in CAR T cells was independent of the CAR generation, with similar results in first and second-generation CARs with either a 41BB or CD28 co-stimulatory domain.

Conclusions

We have successfully engineered a protein (dnSHP2) that is capable of abrogating the function of the inhibitory phosphatase SHP-2. The expression of dnSHP2 impaired PD-1 mediated inhibition of CAR T cells and could theoretically impair any inhibitory receptor that signals through SHP2, making this a universal approach to nullify inhibitory receptors on CAR T cells.