Develop universal CAR macrophage cell therapy for pancreatic cancer

SUMMARY/ABSTRACT Macrophages (Mφs) are innate immune cells, phagocytes and professional antigen presenting cells, which are crucial for tissue homeostasis and adaptive immune response regulation. The high phenotypic and functional plasticity and the high penetration rate in several solid tumor types including pancreatic ductal adenocarcinoma (PDAC) make Mφ cell therapy ideal for solid tumor treatment. To overwrite their immune suppressive program once infiltrated in solid tumors, Mφs can be engineered with chimeric antigen receptors (CARs) to redirect their specificity and function. Phosphatidylserine (PS) is a negatively charged amino-phospholipid mostly confined to the inner leaflet of the plasma membrane bilayers, but flipped to the outer membrane under lethal stress. PS is a universal “eat-me” antigen of apoptotic cells and often abnormally externalized by cancer cells. We hypothesize that PS targeted CAR Mφs (α-PS CAR-Ms) may be a universal “third-party, off-the-shelf” adjuvant cell therapy of traditional therapies for solid tumor treatment, because cancer treatment modalities such as targeted therapies can induce transient but massive tumor cell apoptosis. To verify it, we employed KRAS-addicted PDAC models for pilot studies and have created an α-PS CAR that can provoke antigen specific phagocytotic and cytotoxic activities in Mφs against KRAS inhibitor (KRASi)-treated PDAC cells. Due to structure conserveness of PS in human and mouse, α-PS CAR-Ms reacted with both mouse and human PDAC cells post KRASi treatment. In the project, we propose to demonstrate the anti-tumor effect of α-PS CAR-Ms as an adjuvant therapy of KRASi in human PDAC xenograft models and mouse spontaneous PDAC models and dissect the working mechanisms of α-PS CAR-Ms. Aim 1. Demonstrate α-PS CAR activities in human PDAC models. Aim 1a will generate α- PS CAR-Ms using human primary Mφs and perform functional assays in vitro to determine antigen-specific phagocytosis and cell killing. In addition, we will explore whether combination with inhibitors targeting natural PS receptors on Mφs and blockage of the “don’t-eat-me” signal will further enhance tumoricidal activities of α-PS CAR-Ms. Aim 1b will determine the polarization status of α-PS CAR-Ms by flow cytometry analysis and dissect the molecular reprogramming by -omic analysis. Aim 1c will demonstrate the tumoricidal activity of α-PS CAR- Ms as an adjuvant therapy of KRASi in human PDAC xenograft models. Aim 2. Determine the tumoricidal activity of α-PS CAR-Ms in immunocompetent mouse models. We hypothesize that murine α-PS CAR-Ms can not only kill PDAC cells directly but may also rewire the tumor immune microenvironment to further provoke anti-tumor immune response. Aim 2a will produce murine α-PS CAR-Ms and determine their phagocytotic and killing activities against KRASi-treated PDAC cells. After validation, we will examine whether α-PS CAR-Ms are effective to impair tumor growth and prevent tumor recurrence as an adjuvant therapy of KRASi in spontaneous PDAC mouse models. Aim 2b will characterize the TME remodeling by α-PS CAR-Ms via immunophenotyping and delineate the potential intercellular crosstalk within immune cell types.