Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors

Project Summary The goal of this proposal is to optimize the efficacy of CAR T cells against pancreatic tumors using engineered, synthetic cytokine receptors (velocity receptors, VRs) that enhance CAR T migration speed and tumor infiltration. We will test the motility and cell-killing capability of these new CAR T cells in vitro and select the ten most promising VR constructs for testing and validation in mouse models of pancreatic cancer. In 2024, an estimated 66,440 Americans will be diagnosed PDAC and 51,750 will die of the disease – the highest mortality rate of any major cancer. The five-year survival rate for pancreatic cancer patients (90% of whom have pancreatic ductal adenocarcinoma, PDAC) is just ~12%, yet the standard of care has not changed in years. CAR T cells express engineered chimeric antigen receptors (CARs) that bind specific antigens on cancer cells to trigger anti-tumor effects. They have shown exceptional clinical success for patients with hematologic malignan- cies; however, conventional CAR T cells show low specificity and poor infiltration for patients with solid tumors, especially pancreatic tumors. We hypothesize that a key reason for the lackluster performance of CAR T therapy (as well as molecular therapies) for patients with pancreatic tumors is the stromal matrix, which constitutes a major barrier to the infiltration of CAR T cells and hinders their interaction with target tumor cells. Our therapeutic approach focuses on the largely unaddressed challenge of crossing the stromal matrix. We recently engineered and expressed synthetic cytokine receptors (velocity receptors, VRs) on mesothelin CAR T cells (CAR TV cells) that enhance the migration speed of control CAR T cells 3- to 6-fold in vitro and prompt these cells to infiltrate solid tumors 10 to 50 times more than control CAR T cells in preclinical mouse models of PDAC. This enhanced infiltration correlates with tumor growth inhibition and prolonged survival, at doses for which control CAR T cells show little infiltration and no effect on tumor growth. This activation results from interactions between the engineered VR receptors and inflammatory cytokines secreted by the CAR T cells, including interleukin-5 (IL-5), IL-8, interferon γ (IFNγ), and tumor necrosis factor α (TNFα). As a proof of principle, we tested five different VR receptors that induced enhanced migration in vitro in PDAC mouse models. Our overall hypothesis is that hyper-migratory CAR TV cells lead to greater inhibition of solid tumor growth due to increased tumor infiltration. We propose to optimize the tumor-infiltration efficacy of CAR TV cells by enlarging the repertoire of VRs via a modular design based on the hinge, transmembrane, and signaling domains of the receptors for cytokines IL-5, IL-8, IFNγ, and TNFα. Our approach is “CAR-agnostic”; to compare with previous work, we will keep the (mesothelin) CAR construct unchanged. We will test the motility and cell-killing capability of these new CAR TV cells in vitro and select the ten most promising VR constructs for testing and validation in several mouse models of PDAC.