IL-18R supported CAR T cells targeting oncofetal tenascin C for the immunotherapy of pediatric sarcoma and brain tumors

Abstract

Background: Oncofetal splice variants of extracellular matrix (ECM) proteins present a unique group of target antigens for the immunotherapy of pediatric cancers. However, limited data is available if these splice variants can be targeted with T cells expressing chimeric antigen receptors (CARs).

Methods: To determine the expression of the oncofetal version of tenascin C (TNC) encoding the C domain (C.TNC) in pediatric brain and solid tumors, we used quantitative reverse transcription PCR and immunohistochemistry. Genetically modified T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo.

Results: We demonstrate that C.TNC is expressed on a protein level in pediatric tumors, including diffuse intrinsic pontine glioma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma. We generate C.TNC-CAR T cells and establish that these recognize and kill C.TNC-positive tumor cells. However, their antitumor activity in vivo is limited. To improve the effector function of C.TNC-CAR T cells, we design a leucine zipper-based chimeric cytokine receptor that activates interleukin-18 signaling pathways (Zip18R). Expression of Zip18R in C.TNC-CAR T cells improves their ability to secrete cytokines and expand in repeat stimulation assays. C.TNC-CAR.Zip18R T cells also have significantly greater antitumor activity in vivo compared with unmodified C.TNC-CAR T cells.

Conclusions: Our study identifies the C domain of the ECM protein TNC as a promising CAR T-cell therapy for pediatric solid tumors and brain tumors. While we focus here on pediatric cancer, our work has relevance to a broad range of adult cancers that express C.TNC.

Keywords: Chimeric antigen receptor - CAR; Immunotherapy; T cell.

Figure 1. C.TNC is expressed in solid and brain tumors. (A,B) Reverse transcription quantitative PCR of (A) pediatric cell lines (n=3 bioreplicates, mean+SEM) and (B) patient-derived xenografts for C.TNC. ΔC_T is relative to GAPDH. Acute lymphoblastic leukemia cell line CCRF-CEM had no C.TNC detected. (C) H-scores of primary human H3K27M+DIPG, ZFTA fusion-positive ependymoma (EPN), osteosarcoma (OS), rhabdomyosarcoma (RMS), and Ewing sarcoma (EWS) tumors. (D) Representative immunohistochemistry staining of primary FFPE human tumor samples and tonsils (negative control (neg Co)) with a C.TNC-specific antibody. 20× magnifications, 100 µM scale bar. Images were taken with an Olympus BX46 microscope and a Nikon DS-Fi3 camera at a 20× magnification and edited with Photoshop 25.5.1. C.TNC, tenascin C encoding the C domain; DIPG, diffuse intrinsic pontine glioma.

Figure 2. C.TNC-CAR T cells have antitumor activity in vitro and in vivo. (A) Schematic of C.TNC-CAR design. (B) Transduction efficiency of healthy donor T cells determined via flow cytometry on day 7 (n=5, mean+SEM). Graph shows the percentage of positive cells stained for each respective antibody for CAR transgene detection. (C,D) IFN-γ production measured by ELISA after 48 hours of co-culture with (C) sarcoma or (D) brain tumor cell lines at 2:1 effector to target (E:T) ratio. Negative values were plotted as zero (mean+SEM, n=3–4 for NT and C.TNC), two-way ANOVA, ***p<0.001, ****p<0.0001. (E,F) Cytotoxicity after 72 hours at a 4:1 E:T ratio determined by a luciferase-based assay (mean+SEM, n=3), two-way ANOVA, ****p<0.0001. (G) Cytotoxicity of C.TNC-negative cell line CCRF-CEM determined by a luciferase-based assay (n=4–5, mean+SEM). (H) Schematic of experiment in 5–6-week-old female NSG mice. 1×10⁶ LM7.green fluorescent protein.firefly luciferase cells were injected intraperitoneally (i.p.), followed by 1×10⁶ T cells injected i.p. 7 days later. (I) Flux values from weekly IVIS images (n=5 per cohort). (J) Overall survival of the mice, Mantel-Cox test, **p<0.01. (K) Schematic of experiment in 10–12-week-old male NSG mice. 1×10⁶ DIPG007.YFP.ffLuc cells were injected intracranially (i.c.), followed by 2×10⁶ T cells injected i.c. 7 days later. (L) Flux values from weekly IVIS images (n=5 per cohort). (M) Overall survival of the mice, Mantel-Cox test. ANOVA, analysis of variance; CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; DIPG, diffuse intrinsic pontine glioma; IFN, interferon; NT, non-transduced; scFv, single chain variable fragment.

Figure 3. Zip18R improves CAR T-cell effector function. (A) Schematic of 1X and 2X Zip18Rs, the leucine zippers (Zip) are connected with a (G₄S)₃ linker. The 1X and 2X Zip18Rs retroviral vectors also encode a P2A skip sequence and mClover. TM/IC: transmembrane and intracellular. (B) Representative transduction efficiency of Ramos-Blue and Ramos-Blue KD-MyD NF-κB/AP-1 reporter cells determined via flow cytometry. (C) Absorbance values of transduced Ramos-Blue NF-κB/AP-1 reporter cell supernatant mixed with QUANTI-Blue reagent (n=3 from two separate transductions). (D) Transduction of T cells with Zip18R (n=3, mean+SEM). (E,F) Populations after (E) 7 days or (F) 14 days of cytokine starvation as determined by flow cytometry (n=3, mean+SEM). Two-way analysis of variance, *p<0.05, **p<0.01, ***p<0.001. (G) Schematic of A673 model. T cells were sorted prior to injection. 7–9-week-old male NSG mice were used. (H) Tumor caliper measurements (n=4 for tumor and CAR only, n=5 for CAR.Zip18Rs). (I) Kaplan-Meier survival curve, Mantel-Cox test, **p<0.01. (J) Tumor caliper measurements. (n=3 for tumor and CAR.2XZip18R, n=4 for CAR and CAR.1XZip18R). (K) Kaplan-Meier survival curve, Mantel-Cox test, *p<0.05. CAR, chimeric antigen receptor; NT, non-transduced; Zip18R, interleukin-18 receptor-based leucine zipper receptor.

Figure 4. Zip18R bolsters C.TNC-CAR T-cell effector function in vitro. (A) Transduction efficiency of primary T cells was determined via flow cytometry on day 7 post-transduction (n=4, mean+SEM). Determined via F(ab’)₂ staining and mClover expression. (B) Repeat stimulation assay schematic. T cells were stimulated with LM7.green fluorescent protein.firefly luciferase at an effector to target ratio of 2:1 in the presence of IL-15 every 4 days. (C) Expansion of T cells in repeat stimulation assay. Each graph represents one donor. (D) Fold change of C.TNC-CAR and C.TNC.CAR.Zip18R T cells after stimulations 1 through 10, paired t-test, ****p<0.0001. (E–G) Quantification of (E) type 1 and (F) type 2/type 17 cytokines, and (G) chemokines 48 hours post first stimulation of the repeat stimulation assay (n=3, mean+SEM), data was log-transformed before statistical analysis, two-way analysis of variance, *p<0.05, **p<0.01, ***:p<0.001, ****p<0.0001. (H–J) Comparison of the sum of (H) type 1 cytokines, (I) type 2/type 17 cytokines, and (J) chemokines produced post first stimulation and fourth stimulation by C.TNC-CAR T cells and C.TNC-CAR.Zip18R T cells from repeat stimulation assay (n=3), unpaired t-test, *p<0.05, **p<0.01. CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; NT, non-transduced; TNF, tumor necrosis factor; Zip18R, interleukin-18 receptor-based leucine zipper receptor.

Figure 5. Zip18R signaling alters the transcriptome of C.TNC-CAR T cells. (A) Schematic of experiment. C.TNC-CAR.Zip18R T cells were collected from culture in IL-7/IL-15 and frozen for analysis or freshly collected from a co-culture assay with LM7.GFP.ffLuc (LM7.GL) tumor cells in the presence of IL-15 at 12, 24, and 48 hours post tumor cell stimulation. All four populations are present in the same culture. (B) Percentages of C.TNC-CAR+Zip18R+, C.TNC-CAR+, Zip18R+, and NT T cells at 12, 24, and 48 hours post co-culture. (C,D) Gene Set Enrichment Analysis comparing C.TNC-CAR.Zip18R T cells to C.TNC-CAR T cells at (C) baseline or (D) 48 hours post stimulation with LM7.GFP.ffLuc cells. Top 10 activated or suppressed significant (p.adjust<0.1) KEGG pathways are shown. (E–I) 1×10⁶ NT, Zip18R, C.TNC-CAR, and C.TNC-CAR.Zip18R T cells were cultured in media or against LM7 cells at a 2:1 effector to target ratio for 48 hours and then collected for flow cytometric analysis. (E) Transduction (TDX) percentage of NT, Zip18R, C.TNC-CAR, and C.TNC-CAR.Zip18R T cells alone and after 48 hours of stimulation. CD3+ expression is shown for NT samples. (n=3, mean+SEM), two-way ANOVA, ****p<0.0001. (F–I) Expression within pure isolated populations of T cells gated on CD3+ (NT), mClover+ (Zip18R+), (G₄S)₃+ (C.TNC-CAR+), and (G₄S)₃+ mClover+ (C.TNC-CAR+Zip18R+) for (F) CD69, (G) CD28, (H) CD39, and (I) TIM3 (n=3, mean+SEM), two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Only showing significance values for each cohort comparing Alone versus +LM7 and C.TNC-CAR versus C.TNC-CAR.Zip18R for both conditions. All p values are reported in online supplemental SFigure 16. ANOVA, analysis of variance; CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; ffLuc, firefly luciferase; GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; KEGG, Kyoto Encylopedia of Genes and Genomes; NT, non-transduced; TNF, tumor necrosis factor; Zip18R, interleukin-18 receptor-based leucine zipper receptor.

Figure 6. C.TNC-CAR.Zip18R T cells have superior antitumor activity in vivo. 1×10⁶ LM7.GFP.ffLuc cells were injected into 5–6-week-old female (Donor 1) or male (Donor 2) NSG mice. Seven days later, mice received an i.p. injection of 1×10⁶ sorted T cells. (A,B) Tumor flux values measured by IVIS imaging. (A) Donor 1, (B) Donor 2 (n=5 per cohort per donor). (C) Combined tumor flux values from both donors at weeks 2 and 5 (n=10, mean+SEM), one-way analysis of variance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D) Kaplan-Meier survival curve, Mantel-Cox test. P values listed in the table. (E) Schematic of rechallenge experiment. 5–6-week-old male mice were used for tumor only cohort. 1×10⁶ LM7.GFP.ffLuc cells were injected i.p. into naïve mice or C.TNC-CAR.Zip18R T-cell pretreated mice. (F) Tumor flux values measured by IVIS imaging (n=3–5). CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; ffLuc, firefly luciferase; GFP, green fluorescent protein; i.p., intraperitoneal; NT, non-transduced; Zip18R, interleukin-18 receptor-based leucine zipper receptor.