Inducible MyD88/CD40 (iMC) Costimulation Provides Ligand-Dependent Tumor Eradication By CD123-Specific Chimeric Antigen Receptor T Cells
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AbstractIntroduction: Promising clinical results with CD19-specific chimeric antigen receptor (CAR)-directed T cells for the treatment of B cell leukemia and lymphoma suggest that CARs may be effective in other hematological malignancies, such as acute myeloid leukemia (AML). CD123/IL-3Rα is an attractive CAR-T cell target due to its high expression on both AML blasts and leukemic stem cells (AML-LSCs). However, the antigen is also expressed at lower levels on normal stem cell progenitors presenting a major toxicity concern should CD123-specific CAR-T cells show long-term persistence. Here, we describe a CAR platform, "GoCAR-T", which uses a proliferation-deficient, first generation, CD123-specific CAR together with a ligand (rimiducid (Rim))-dependent costimulatory switch (inducible MyD88/CD40 (iMC)) to provide physician-controlled eradication of CD123+ tumor cells and regulate long-term CAR-T cell engraftment.Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and first generation CAR targeting CD123 (SFG-iMC-CD123.ζ). The effects ofiMC costimulation on CD123-targeted CARs were assessed in coculture assays with CD123+, EGFPluciferase (EGFPluc)-modified leukemic cell lines (KG1, THP-1 and MOLM-13) with and without Rim using the IncuCyte live cell imaging system. IL-2 production was examined by ELISA from coculture supernatants. In vivo efficacy of iMC-CD123.ζ-modified T cells was assessed using an immune-deficient NSG tumor xenograft model. One million EGFPluc-expressing CD123+ THP-1 tumor cells were injected i.v. into the animals, followed by a single i.v. injection on day 7 with 2.5x106 non-transduced or iMC-CD123.ζ-modified T cells. Groups receiving CAR-T cells subsequently received i.p. injections of Rim (1 mg/kg) or vehicle only on days 0 and 15 post-T cell injection. Animals were evaluated for THP-1-EGFPluc tumor burden and weight change on a weekly basis using IVIS bioluminescent imaging (BLI) and for T cell persistence by flow cytometry and qPCR at day 30 post-T cell injection.Results: SFG-iMC-CD123.ζ efficiently transduced activated T cells (66±8%) and showed antigen-specific cytolytic function against CD123+ leukemic cell lines. However, in coculture assays both CAR antigen recognition and Rim-dependent iMC costimulation were required for IL-2 production (285±41 versus 2,541±255 pg/ml for control and 1 nM Rim, respectively), robust CAR-T cell proliferation (87-fold increase with Rim stimulation) and enhanced tumor cell killing. In NSG mice engrafted with CD123+ THP-1-EGFPluc tumor cells, only animals were treated with iMC-CD123.ζ-modified T cells and 1 mg/kg Rim on days 0 and 15 post-T cell injection controlled tumor growth, showing a 2-log (average radiance: NT = 3.0x108; CAR + Vehicle = 2.4x108; CAR + Rim=8.4x105 p/sec/cm2/sr) reduction in tumor burden compared to mice receiving CAR-T cells and vehicle only (Figure 1). Approximately two weeks after the second Rim injection (day 30), CAR-T cells were infrequent (<1.0%) in the spleen and bone marrow of both CAR groups suggesting that active costimulation is required for CAR-T persistence.Summary: GoCAR-T, a platform comprising a ligand-dependent activation switch and a proliferation-deficient first generation CAR, efficiently eradicates CD123+ leukemic cells when costimulation is provided by systemic rimiducid administration. Deprivation of iMC costimulation results in reduction of CAR-T levels, providing a user-controlled system for managing persistence and safety of CD123-specific CAR-T cells.DisclosuresFoster: Bellicum Pharmaceuticals: Employment. Duong:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.
