Abstract 6714: Genetic screens identify T cell co-stimulation as a key modifier of the redirected cytotoxicity of bispecific T cell engager (BiTE®) molecules

BiTE® constructs redirect T cell cytotoxicity against cancer cells through simultaneous binding to CD3 on T cells and a tumor-associated antigen (TAA) on cancer cells. Blinatumomab, an anti-CD19 BiTE® molecule used to treat pre-B-ALL patients, leads to more than 40% complete remission rates. Only 30% of relapsed patients exhibit loss of CD19 on leukemia cells, indicating that the majority of patients acquire blinatumomab resistance independently of target loss. Understanding such mechanisms could lead to new predictive biomarkers and help improve current BiTE® immunotherapy. To gain insight into the mechanism of resistance to BiTE® cytotoxicity, we performed CRISPR screens to identify tumor-intrinsic genetic factors that modulate BiTE® molecule efficacy in vitro. Cas9 expressing human cancer cells transduced with a genome-wide sgRNA library were incubated with T cells and either a control BiTE® molecule that did not recognize the cancer cells, or an anti-TAA BiTE® molecule at a concentration that induced the lysis of ~90% of target cells. Next-gen sequencing was used to identify enriched or depleted sgRNA, indicative of genes whose loss confers resistance or susceptibility to BiTE®-mediated T cell killing. As expected, the most enriched candidates from the screen were involved in the expression, post-translational modification or surface presentation of the TAA targeted by the BiTE® molecules. In the case of a screen using an anti-MSLN BiTE® molecule, a panel of genes involved in GPI-anchor biosynthesis, required to anchor MSLN to the plasma membrane was uncovered. The screen also revealed genes known to promote immune evasion such as PD-L1 as well as novel genes whose roles in immunotherapy were not extensively studied before, including genes involved in apoptosis and the NF-κB pathway. One of the top enriched candidates was CD58, which encodes a co-stimulatory molecule that binds to CD2 on T cells. Genetic perturbation and pharmacological blockade of CD58-CD2 interaction significantly reduced BiTE®-mediated killing of target cells. Furthermore, re-establishment of CD58-CD2 interaction either by overexpressing CD58 in cancer cells or by using CD2-agonist large molecules improved T cell activation and target cell killing. Moreover, concurrent blocking of CD2 and CD28 co-stimulation inhibited BiTE®-mediated killing more profoundly than blocking either pathway alone, indicating redundancy between CD58- and CD80/86-mediated co-stimulation. Our study reveals a