Developing Novel Cell Therapy Strategies to Convert Macrophage Programming and Induce Synergistic, Multicellular Immunity Against AML

Acute myeloid leukemia (AML) is the most common leukemia among adults (80% of all cases) and the 2^nd most common leukemia in children, has one of the lowest 5-year survival rates among hematological cancers (32%). AML cells alter the programming of immune cells, supporting conversion of anti- to pro-tumorigenic cell types. Macrophages are effector myeloid cells of the innate immune system that can phagocytose tumor cells and secrete pro-inflammatory cytokines to recruit and induce anti-tumor immunity but are converted to pro-tumor programming by AML cells to enable AML tumor survival, inducing anti-cell death pathways and reducing drug sensitivity. Pro-tumor macrophages exhibit elevated CD206 (MRC1) expression; CD206 is significantly associated with negative outcomes in AML patients (**P<0.01) and is proposed as a prognostic factor for AML. Fortunately, macrophages exhibit plasticity and can be converted to anti-tumor programming with costimulatory signals. CD40 signaling has been shown to induce antitumor myeloid immunity and agonist CD40 antibodies have been tested in clinical trials. However, enthusiasm for CD40 agonist antibody therapies has been dampened by dose-limiting toxicities, inadequate pharmacokinetics and tissue penetration, and the non-tumor targeted delivery of the antibody modality. Another promising therapeutic approach, adoptive cell therapy (ACT), utilizes living, genetically-engineered T cells that actively localize to sites of tumor and specifically attack tumor cells. ACT is particularly attractive for AML therapy, as ACT is an established therapy for other hematological cancers and T cells naturally home to the sites of AML (blood, bone marrow). We innovate synthetic fusion proteins that combine a membrane-bound protein ectodomain with a costimulatory signaling endodomain (Oda et al, JEM, 2015; Oda et al, Blood, 2020) and fusion proteins that we developed are entering clinical trials in 2024 and 2025. We developed a combination therapy that employs both innate and adaptive immunity by using ACT to deliver CD40 signals to promote antitumor macrophages at the sites of AML. We developed fusion proteins that combine the ectodomain of CD40 ligand (CD40L) with a T cell costimulatory endodomain, expressed in the T cell membrane, and providing positive signals to both endogenous macrophages and tumor-targeted T cells. We developed several CD40L fusion proteins, termed Dual Costimulatory Receptors (DCR), with different intracellular costimulatory domains. In in vitro studies, CD40L DCRs showed high expression in T cells, enhanced accumulation/proliferation, and significantly enhanced T cell lysis of tumor cells in a Serial Killing Over-time (SKO) assay. We advanced the 3 leading CD40L DCRs to in vivo immunocompetent murine AML studies. In in vivo immunocompetent murine studies, we observed increased M1-like macrophages and increased overall survival following CD40L-HVEM DCR-T cell therapy, but not with control, CD40L-engineered T cell therapy. To determine if CD40L-HVEM DCR-T cell-reprogrammed macrophages would induce synergistic anti-tumor function, we developed a triple-cell SKO with T cells, macrophages, and tumor cells. While the combination of control T cells and macrophages showed similar lysis of tumor cells relative to T cells alone, CD40L-HVEM DCR-T cells and macrophages synergized to produce highly effective tumor eradiation. In studies with human T cells and tumor, CD40L-HVEM DCR-T cells exhibited increased activation and promoted conversion of macrophages to anti-tumor programming in vitro. Here, we show that engineered fusion proteins combined with a tumor-targeted cell therapy can redirect a pro-tumor immune cell subset associated with negative outcomes in AML patients, resulting in significantly enhanced outcomes.