CAR T Cells Releasing IL-18 Convert to T-Bethigh FoxO1low Effectors that Exhibit Augmented Activity against Advanced Solid Tumors  Markus Chmielewski, Hinrich Abken  Cell Reports  Volume 21, Issue 11, Pages 3205-3219 (December 2017) DOI: 10.1016/j.celrep.2017.11.063 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 CEA+ Pancreatic Adenocarcinoma Was Induced by Transplantation into the Pancreas of Immune-Competent, CEA Transgenic Mice Pancreas carcinoma was initiated by intra-pancreatic injection of CEA+ murine pancreatic carcinoma cells (Panc02, 2 × 105 cells/mouse), which express CBLuc, into CEA transgenic mice. (A and B) Tumor growth was monitored by recording (A) luciferase activity and (B) CEA serum levels and tumor weight. (C) Schematic depicting the modular composition of the CEA-specific CAR. Lκ, light chain kappa signal sequence; SCA431scFv, CEA-specific single chain (heavy and light) fragment variable; IgG1, CH2-CH3 IgG1 “spacer”-domain; CD4 TM, CD4 transmembrane domain; CD28-CD3ζ, combined intracellular CD28 and CD3ζ signaling domain. (D) Modified T cells express the CAR on the surface, as revealed by flow cytometry. (E) CEA is expressed on the surface of the modified Panc02 cells. (F) Mice with established tumors (day 10) or advanced tumors (day 17) were treated with one i.v. injection of 107 anti-CEA CAR T cells. The bioluminescence signals of CBLuc-marked Panc02 tumor cells were recorded on days 12, 17, and 26 and quantified as described in the Experimental Procedures. Data represent the mean ± SEM. Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 CAR T Cells with Inducible Release of IL-18 Increased T-Bet and Decreased FoxO1 Levels (A) A cytokine screen for inducing T-betlow FoxO1high CAR T cells. Human anti-CEA CAR T cells were activated by incubation with the immobilized anti-idiotypic antibody BW2064/36, which is specific for the anti-CEA CAR binding domain and used as a surrogate for CEA, in the presence of IL-1α, IL-1β, IL-2 IL-4, IL-6, IL-7, IL-10, IL-12, IL-17, IL-18, IL-21, TGF-β1, TNF-α, or IFN-γ (each 10 ng/mL) and for comparison without cytokine. T cells (5 × 104/well) were cultivated in triplicates for 36 hr and assayed for the expression of T-bet and FoxO1. The fold change in expression compared with cells without cytokine incubation is shown. Data represent the mean ± SEM; n = 3. (B) Schematic depicting the expression cassettes for the transgenic IL-18 and IL-12. Transgenic IL-12 and IL-18 expression, respectively, was controlled by the CAR-inducible NFAT6-IL-2 minimal promoter for CAR-inducible expression (iIL-12 and iIL-18) or the constitutive CMV promoter for constitutive expression (cIL-12 and cIL-18). (C) CAR T cells (5 × 104 CAR T cells/well) without transgenic cytokine or with iIL-12 or iIL-18 were co-incubated with CEA+ and CEA− Panc02 cells (2 × 104 cells/well), respectively. After 48 hr, IL-12 and IL-18 concentrations in the culture supernatants were monitored by ELISA. Data represent the mean ± SEM. (D) CAR T cells (8 × 102–105 CAR T cells per well) with and without transgenic IL-12 or IL-18 were co-incubated with CEA+ Panc02 cells (5 × 104 cells/well) for 48 hr. IFN-γ in the culture supernatant was determined by ELISA. Specific cytotoxicity was determined using a tetrazolium salt-based viability assay. Data represent the mean ± SEM. (E) Expansion kinetics of CAR T cells without transgenic cytokine or with iIL-12 or iIL-18. CAR T cells were cultivated for 10 days in plates pre-coated with the anti-idiotypic antibody BW2064/36 directed against the anti-CEA binding domain of the CAR. The number of viable cells was recorded daily. Data represent the mean ± SEM. (F) CAR T cells with and without transgenic IL-12 or IL-18 were cultivated for 24 hr in microtiter plates (105 cells/well) pre-coated with the anti-idiotypic antibody BW2064/36 or with an isotype matched antibody as a control. T-bet and FoxO1 expression levels were determined by flow cytometry (Student’s t test, ∗p < 0.05). (G) CAR T cells were separated into CD4+ and CD8+ T cell subsets and activated by the immobilized anti-idiotypic antibody BW2064/36 in the presence of IL-18 (100 ng/mL) or IL-12 (50 ng/mL) for comparison. T cells were cultivated for 24 hr and tested for T-bet and FoxO1 by flow cytometry (Student’s t test, ∗p < 0.05). Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Adoptive Therapy with Anti-CEA CAR T Cells with Inducible Release of IL-18 Prolongs the Survival of Mice with Advanced CEA+ Pancreatic Cancer (A) To establish pancreatic cancer, CEA+ and CEA− Panc02 cells, respectively, were inoculated into the pancreas of CEA transgenic mice (105 tumor cells/mouse, 7 mice/group). At around day 17 (advanced tumors, mean serum CEA level 30 ± 10 ng/mL of the mouse cohort), mice were treated by i.v. injection of one dose of CAR T cells with iIL-18 or iIL-12 or iIL-18 plus iIL-12 (107 CAR T cells/mouse). Mice treated with iIL-18 CAR T cells and the combination of iIL-12 and iIL-18 experienced an increased median survival compared with mice receiving iIL-12 CAR T cells or CAR T cells without transgenic cytokine (log rank test, ∗p < 0.05). The experiment was repeated twice. (B) Sera of mice 3 days before and 1 day after CAR T cell treatment (mice from A) were analyzed using a bead-based cytokine assay and analyzed in technical triplicates (6 mice/group). The cytokine detection limits were in the range of 0.1–1.0 pg/mL. Data represent the mean ± SEM. (C) Serum samples from mice were investigated by ELISA for the soluble form of CEA as a marker for tumor progression. Mice from (A) were used (IL-12, n = 6; IL-18, n = 5; IL-12 IL-18, n = 5; CAR, n = 5; w/o, n = 5 mice). The bold lines represent the mean CEA values. w/o, without. (D) Tumor tissue slides were analyzed for the presence of tumor-associated CD11c+ CD205+ DCs and expression of the activation marker CD80 and the suppression marker CD103. Scale bar, 20 μm. Statistical analysis was performed using Student’s t test; ∗p < 0.01. Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Adoptive Cell Therapy with iIL-18 CAR T Cells Alters the Inflammatory Cell Infiltrate in Targeted Pancreatic Tumors (A–E) CAR T cells with iIL-18 or iIL-12 or without transgenic cytokine were administered by i.v. injection (107 cells per mouse) into CEA transgenic mice with established CEA+ pancreatic adenocarcinoma. After 10 days, tumors were removed, and tissue slides were analyzed (A) for CEA expression and for the presence of activated (B) NKp46+ NKG2D+ NK cells, (C) F4/80+ CD86+ M1 and F4/80+ CD206+ M2 macrophages, (D) CAR-expressing CD4+ FoxP3+ Treg cells and CD4+ FoxP3− effector T cells, and (E) CD11b+ Ly6G− Ly6Chigh monocytic myeloid-derived suppressor cells (M-MDSCs) as well as CD11b+ Ly6G+ Ly6Clow granulocytic MDSCs (G-MDSCs). ImageJ software was used for quantitative analyses and the determination of fluorescence intensity (FI) per spot. Scale bars, 20 μm. Statistical analysis was performed using Student’s t test; ∗p < 0.01. Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Mice with Adoptively Transferred iIL-18 CAR T Cells Successfully Rejected Secondary Tumor Cell Challenge in the Long Term (A) Schedule of the challenge protocol. CEA transgenic mice were inoculated with irradiated CEA+ C15A3 tumor cells (106 cells/mouse) together with CEA-specific CAR iIL-18 T cells, CAR T cells without transgenic cytokine, and unmodified T cells as a control (107 cells/mouse, n = 6/group). All mice were challenged with non-irradiated CEA+ C15A3 tumor cells (106 cells/mouse) on day 20, and tumor growth was recorded until day 100. (B and C) Tumor volumes (B) and tumor-free survival after secondary tumor cell challenge (C) were recorded. Mean tumor tumor-free survival was analyzed by a Kaplan-Meier survival plot, and statistical significance was analyzed using a log rank test; ∗p < 0.05. (D) CAR T cells were recorded in peripheral blood samples by flow cytometry. (E) CAR+ iIL-18 and CAR+ T cells of total CD3+ T cells (percent) were plotted against the time after application. Data represent the mean ± SEM. (F) The number of CAR T cells with perforin (Prf1) and granzyme B (GzmB) in the peripheral blood of treated mice was recorded. (G) The expression of the inhibitory receptors PD-1, CTLA-4, and TIM-3 on engineered T cells was recorded by flow cytometry. Data represent the mean ± SEM. Statistical analysis was performed using Student’s t test; ∗p < 0.05. Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 iIL-18 CAR T Cells Eradicated Human Alveolar Cell Carcinoma in the Lungs of Immune-Compromised Mice Human lung carcinoma was induced in the Rag2−/−cγ−/− mouse by i.v. injection of CEA+ A549 carcinoma cells (106 cells/mouse) marked with CBLuc for bioluminescence imaging. (A) When tumors were established, human anti-CEA CAR T cells with or without iIL-18 were injected into the tail vein at day 0 (5 × 106 cells/mouse). Anti-CD30 CAR T cells of irrelevant specificity were injected for comparison. Up to six mice per cohort were treated. (B) Bioluminescence signals indicating tumor progression were quantified; tumor-free survival of the treated mice was determined. Statistical analysis was performed using a log rank test; ∗p < 0.05. (C) A549 carcinoma cells were engineered to express CEA on the cell membrane. Surface CEA and the CBLuc were recorded by flow cytometry. (D) Schematic depicting the modular composition of the human CEA-specific and CD30-specific CAR, respectively. hIgG1, human IgG1 CH2CH3; Lκ, leader of the Ig κ chain; tm, transmembrane. Cell Reports 2017 21, 3205-3219DOI: (10.1016/j.celrep.2017.11.063) Copyright © 2017 The Author(s) Terms and Conditions