An Engineered Viral Protein Exploits LCK Kinase to Rewire T Cell Signaling Pathways

Abstract

T cells are central mediators of human immunity that protect against infectious threats and eliminate malignant cells through a T cell response that is coordinated by specialized signaling pathways. For a T cell to attack a cancer cell, it must first become activated, proliferate, and acquire effector function. This process requires three critical signal inputs; Signal 1: Antigen; Signal 2: Co-stimulation; Signal 3: Cytokine. Once a T cell anti-tumor response is initiated, for cancer to persist, it must escape T cell mediated attack. Cancer can co-opt inhibitory T cell receptors to prevent T cell attack. Immunotherapies that block inhibitory receptors, such as PD1/L1 and CTLA4 antibodies, have revolutionized the treatment of metastatic cancer. However, many patients that fail to respond to immune checkpoint blockade do not possess an inflamed tumor and are hypothesized to require an activator that can promote a T cell response. Therefore, strategies to interface with T cell signaling pathways are necessary to effectively harness T cells as therapeutic entities for the treatment of cancer.

The TCR complex; coinhibitory and costimulatory receptors (e.g. CD28 and PD-1), as well as intracellular signaling proteins (e.g. ITK and ZAP-70) are phosphorylated by the SRC family kinase LCK. LCK kinase is basally active in resting T cells, which suggests that the recruitment of LCK activity to a protein, to induce its phosphorylation, could be a generalizable feature to be exploited for the targeted activation of T cell signaling pathways. Herpesvirus saimiri (HVS) infects T cells, expressing a tyrosine kinase interacting protein (TIP) to co-opt LCK by recruiting it to non-canonical substrates to phosphorylate and activate them. We therefore sought to determine whether HVS TIP protein could provide a blueprint to harness LCK kinase activity to promote the activation of intracellular signaling pathways in T cells.

The basal kinase activity of LCK is regulated by the inhibitory CSK kinase that phosphorylates the C-terminal tail of LCK to stabilize its closed conformation. It has been shown that the inhibition of CSK results in increased basal LCK activity that augments the activation of T cells in response to weak or partial TCR agonists. We isolated a minimal peptide derived from the wild-type TIP protein that binds to LCK kinase. Similar to CSK inhibition, the TIP-derived minimal peptide increased LCK basal kinase activity and enhanced TCR signaling; boosting T cell responsiveness to weak antigenic stimuli ex vivo, potentially offering a new avenue for improving the efficacy of T cell-based therapies that target weakly immunogenic tumors.

The efficacy of T cell therapies can be limited if cytokine-induced JAK-STAT signaling is dysregulated or insufficient to sustain functionality. We further explored the feasibility of recruiting LCK kinase activity to non-canonical protein substrates to directly activate targeted STAT proteins in T cells. STAT activation was accomplished by engineering the HVS TIP to provide a platform for the enforced recruitment of LCK to STAT proteins. We determined that the TIP Minimal peptide that binds to LCK could be combined with STAT binding sites derived from endogenous cytokine receptors. These constructs activated targeted STAT proteins in a cytokine-independent manner. A STAT5 activator enabled CD8+ T cells to retain their functionality under suppressive culture conditions ex vivo. These findings translated to reduced tumor outgrowth in vivo due to enhanced T cell persistence and functionality.