Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting

Abstract

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

Fig. 1. CD28 transmembrane in CARs containing a 4-1BB costimulatory domain enhances anti-tumor activity in vitro and in vivo.

a Illustration of a TAG72-CAR T-cell containing the humanized scFv targeting TAG72 with varying five extracellular spacer domains (EQ, dCH2, CD8h, HL, L), three transmembrane domains (CD4tm, CD8tm, CD28tm), and two intracellular costimulatory domains (4-1BB, CD28) followed by a cytolytic domain (CD3z). b Untransduced (UTD) and seven different TAG72-CAR T cells positively enriched for CD19t were evaluated by flow cytometry for Protein L to detect the scFv. c, d In vitro tumor cell killing activity relative to UTD (c) and IFNγ production by ELISA (d), of CAR T cells against tumor targets (TAG72- OVCAR8; TAG72+ OVCAR3, OV90, and OVCAR8-sTn) after 24 hr (for ELISA) or 72 hr of co-culture at an effector:target (E:T) ratio of 1:4. n = 9 from three independent experiments. Data are presented as mean values ±SEM. P values indicate differences between TAG72-dCH2(28tm)28z and TAG72-dCH2(28tm)BBz using a two-tailed Student’s t test. e Western blotting analysis of early downstream signaling mediators following CAR T-cell stimulation of indicated TAG72-CAR T cells. f Quantification of band density of phosphoprotein over their respective total protein levels. n = 2 per timepoint, representative of two independent experiments. Data are presented as mean values ±SD. g TAG72-CAR T-cell killing of OV90 cells measured by xCELLigence over 10 days (E:T = 1:20). h Schema of repetitive tumor cell challenge assay (top). TAG72-CAR T cells were co-cultured with OV90 cells (E:T = 1:2) and rechallenged with OV90 cells every two days. Remaining viable tumor cells and fold change in TAG72-CAR T cells were quantified as described in Methods prior to each tumor cell rechallenge. n = 6–9/group from at least two independent experiments. Data are presented as mean values ±SEM. P values indicate differences between 28tm28z and 28tmBBz using a two-tailed Student’s t test. i Representative bioluminescent flux imaging of intraperitoneal (i.p.) OVCAR3(eGFP/ffluc) tumor-bearing female NSG mice treated i.p. with UTD or indicated TAG72-CAR T cells. j Quantification of flux (individual mice in each group) from treated OVCAR3 tumor-bearing mice. UTD (n = 8/group); TAG72-CAR T cells (n = 10/group). Curative responses: UTD: 0/10, 4tmBBz: 0/10, 28tm28z: 0/10, 28tmBBz: 4/10. P < 0.005 comparing 28tm28z and 28tmBBz using a Multiple Mann–Whitney test.

Fig. 2. Membrane-bound IL-12 engineered TAG72-CAR T cells induce higher IFNγ, T-cell expansion, and anti-tumor activity in vitro.

a–c Tumor cell killing of OV90 cells by TAG72-CAR T cells (E:T = 1:20) with the addition of varying concentrations of anti-IFNγR1 blocking antibody, isotype control, and recombinant human IL-12 cytokine measured by xCELLigence over 10 days (a, b). n = 2/group at each timepoint. Data are presented as mean values ±SD. At day 10, IFNγ levels in supernatants were quantified by ELISA (c). n = 2/group, representative of two independent experiments. Data are presented as mean values ±SD. d Illustration of TAG72-CAR/mbIL12 T cell. e Flow cytometric analysis of surface or intracellular expression of mbIL12 in TAG72-CAR T cells stimulated with varying concentrations of plate-bound TAG72. n = 2/group, representative of two independent experiments. Data are presented as mean values ±SD. f Intracellular flow cytometric analysis of phosphorylated STAT3 (pSTAT3, pY705) (left) and pSTAT4 (right) in TAG72-CAR and TAG72-CAR/mbIL12 T cells stimulated with varying concentrations of plate-bound TAG72 or recombinant huIL12 (10 ng/mL). g Intracellular flow cytometric analysis of pSTAT4 in TAG72-CAR T cells co-cultured with HT1080 (TAG72−) cells transduced with mbIL12. Cells were stimulated with Immunocult CD3/CD28 per manufacturer’s recommendation. Cells were gated on CAR T cells and evaluated for pSTAT4. h TAG72-CAR/mbIL12 T cells were co-cultured with OV90 cells (E:T = 1:3) and rechallenged with OV90 cells every 2 days. The remaining viable tumor cells and TAG72-CAR T-cell proliferation were quantified as described in Methods prior to each tumor cell rechallenge. n = 3/group, representative of two independent experiments. Data are presented as mean values ±SD. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test.

Fig. 3. Locoregional intraperitoneal delivery of TAG72-CAR/mbIL12 T cells reduces tumor burden and increases regional and systemic CAR T-cell persistence in vivo.

a Representative bioluminescent flux imaging of i.p. OV90(eGFP/ffluc) tumor-bearing mice treated i.p. with CD19-CAR, CD19-CAR/mbIL12, TAG72-CAR or TAG72-CAR/mbIL12 T cells. b Quantification of flux (individual mice per group) from OV90(eGFP/ffluc) tumor-bearing mice treated i.p. with CD19-CAR T cells (n = 12/group), CD19-CAR/mbIL12 T cells (n = 12/group), TAG72-CAR T cells (n = 20/group) and TAG72-CAR/mbIL12 T cells (n = 17/group). Combined data are from two independent studies. P value indicates differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test. c Representative flow cytometric analysis of TAG72-CAR T cells per µL of peritoneal ascites. d Quantification of TAG72-CAR T cells per µL of peritoneal ascites (left) at weeks 2 and 4 post-treatment. n = 2/group per timepoint. Quantification of TAG72-CAR T cells per µL of peripheral blood (right) at weeks 1, 2, and 4 post-treatment. n = 5/group per timepoint. Data are presented as mean values ±SEM. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test. e Schematic for subcutaneous (s.c.) and i.p. OV90 dual-tumor model and treatment (top). Representative bioluminescent flux imaging of dual-tumor-bearing mice treated i.p. with TAG72-CAR or TAG72-CAR/mbIL12 T cells (bottom). f Quantification of flux (individual mice per group) from mice treated i.p. with TAG72-CAR T cells (n = 8/group) and TAG72-CAR/mbIL12 T cells (n = 8/group). g Quantification of subcutaneous tumor volume (individual mice per group) from mice treated i.p. with TAG72-CAR T cells. h Quantification of TAG72-CAR T cells per µL of peritoneal ascites at week 2 post-treatment. n = 2/group. i Quantification of TAG72-CAR T cells per µL of peripheral blood at days 7, 14, 21, and 28 post-treatment. n = 5/group per timepoint. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test. j Immunohistochemistry of CD3+ T cells in s.c. tumors at day 12 post-treatment. Data are representative of n = 3 mice/group. Scale bar = 100 µm.

Fig. 4. Locoregional intracerebroventricular delivery of HER2-CAR/mbIL12 T cells reduces tumor burden and increases regional and systemic CAR T-cell persistence in vivo.

a Schematic for intratibial (i.ti.) and intracranial (i.c.) BBM1 dual-tumor model and treatment. b Representative bioluminescent flux imaging of dual-tumor-bearing mice left untreated (no tx), or treated by intracerebroventricular (i.c.v.) injection of HER2-CAR or HER2-CAR/mbIL12 T cells. c Quantification of brain (top) or bone (bottom) flux from individual mice treated i.c.v. with HER2-CAR T cells (n = 11/group), HER2-CAR/mbIL12 T cells (n = 11/group), or no tx (n = 4/group). P < 0.05 for bone flux, and not significant (ns) for brain flux, comparing HER2-CAR and HER2-CAR/mbIL12 using a Multiple Mann–Whitney test. d, e Representative flow cytometric analysis (d) and quantification (e) of HER2-CAR T cells per µL of blood at weeks 1, 2, and 5 post-treatment. e n = 11–12/group. Data are presented as mean values ±SEM. P values indicate differences between HER2-CAR and HER2-CAR/mbIL12 using a two-tailed Student’s t test. f Immunohistochemistry of CD3+ T cells in i.ti. and i.c. tumors at day 7 post-treatment. Data are representative of n = 3 mice/group. Scale bar = 100 µm.

Fig. 5. Locoregional intraperitoneal delivery of TAG72-CAR/mbIL12 T cells safely and effectively target ovarian cancer peritoneal metastasis in an immune-competent syngeneic mouse model.

a Schematic for intraperitoneal (i.p.) ID8-mSTn tumor model and treatment. b Representative bioluminescence flux imaging of tumor-bearing mice, treated by intraperitoneal (i.p.) injection of indicated T cells. c Average tumor flux and d percent weight change in indicated T-cell treatments relative to pre-treatment weight (n = 5–7/group). P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 in tumor flux using a two-tailed Student’s t test, and between TAG72-CAR/mbIL12 and TAG72-CAR + sIL12 in percent weight change at both days 20 and 21 using a two-tailed Student’s t test. e Spleen photographs (n = 3/group) and f representative CD3 IHC in livers harvested at 7 days post treatment. Quantification of CD3+ counts per mm²: PSCA-CAR: 37.7 ± 1.7, TAG72-CAR: 54.5 ± 7.6, TAG72-CAR + sIL12: 233.5 ± 19.2, TAG72-CAR/mbIL12: 68.3 ± 5.1. Scale bar = 200 µm. g Quantification of serum levels of ALT (left) and AST (right) from mice at 6 days post treatment. n = 4–5/group. P values indicate differences between TAG72-CAR/mbIL12 and TAG72-CAR + sIL12 using a two-tailed Student’s t test. h Percent lymphocytes, monocytes, and neutrophils from complete blood count analysis collected at 7 days post treatment. n = 4/group. i, j ELISA quantification of IFNγ (i) and IL-12 (j) cytokines in mouse serum at day 18 and 20 (day 4 and 6 post treatment, respectively) post tumor injection (n = 6–7/group). All data are presented as mean values ±SEM.

Fig. 6. TAG72-CAR/mbIL12 T-cell therapy induces TME modifications in ovarian cancer peritoneal metastases in an immune-competent syngeneic mouse model.

a Representative H&E and CD3 IHC in solid tumor masses collected from the upper omental region of i.p. ID8-mSTn tumor-bearing mice treated with indicated T cells. Scale bar = 100 µm. b Representative flow cytometric analysis of tumor cells (CD3-CD45- double negative) in peritoneal ascites. c Quantification of tumor cells (CD3− CD45− double negative) and d immune subsets (CD45+, CD3+, CD11b+, and NK+) as cells/mL in peritoneal ascites. e Representative flow cytometric analysis of percent CAR T cells (CD3+ CD19t+) and f quantification counts of CD4+ and CD8+ CAR T cells/mL in peritoneal ascites. g Quantification of mean fluorescent intensity (MFI) of CD137+ in CAR T cells in peritoneal ascites. h Quantification of percent CD62L+ CD44+ (Tcm) in both CAR+ and CAR− T cells in peritoneal ascites. i Quantification of myeloid cell counts (Ly6G+, Ly6C+, Ly6G−/C− double negative tumor-associated macrophages (TAM) and CD11c+ CD103+ dendritic cells (DC) as cells/mL in peritoneal ascites gated from total CD11b+ cells. Representative flow cytometric analysis of percent (j) and quantification of MFI (k) on CD103+ MHC Class II+ double positive DC in peritoneal ascites. All analyses represent data collected from ascites of ID8-mSTn tumor-bearing mice at 7 days post treatment, n = 3/group. All data are presented as mean values ±SEM. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test.