Superkine IL-2 and IL-33 Armored CAR T Cells Reshape the Tumor Microenvironment and Reduce Growth of Multiple Solid Tumors

Abstract

Chimeric-antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against hematologic tumors. Yet, CAR T-cell therapy has had little success against solid tumors due to obstacles presented by the tumor microenvironment (TME) of these cancers. Here, we show that CAR T cells armored with the engineered IL-2 superkine Super2 and IL-33 were able to promote tumor control as a single-agent therapy. IFNγ and perforin were dispensable for the effects of Super2- and IL-33-armored CAR T cells. Super2 and IL-33 synergized to shift leukocyte proportions in the TME and to recruit and activate a broad repertoire of endogenous innate and adaptive immune cells including tumor-specific T cells. However, depletion of CD8+ T cells or NK cells did not disrupt tumor control, suggesting that broad immune activation compensated for loss of individual cell subsets. Thus, we have shown that Super2 and IL-33 CAR T cells can promote antitumor immunity in multiple solid tumor models and can potentially overcome antigen loss, highlighting the potential of this universal CAR T-cell platform for the treatment of solid tumors.

Figure 1.. Effects of cytokine expressing CAR T cells on B16F10 tumors.

a) CAR design consisting of TA99 scFv, CD28 transmembrane and costimulatory domains, and CD3ζ signaling domain. Cytokine constructs were designed with a T2A (self-cleaving peptide) separating cytokine combinations to release independent interleukins and contained an IRES GFP to determine transduction efficiency. b) Experimental schematic showing C57BL/6 mice challenged with 2x10⁵ B16F10 tumors i.d. on day 0 and then treated with 7x10⁶ TA99 CAR T cells i.v. in the tail vein 6 days post tumor inoculation. c) Tumor growth and d) survival of B16F10 tumor–bearing mice treated with TA99 CAR T cells engineered to express Super2, IL33, or both in combination. e) B16F10 tumor growth in mice treated with 7x10⁶ Super2 + IL33 TA99 CAR T cells on day 6, 11 or 14 post tumor inoculation. f) Tumor growth and g) survival of B16F10 tumor–bearing mice treated with TA99 CAR T cells engineered to express Super2 in combination with either IL4, IL5, IL25, TSLP, or IL33. c, e, f) For tumor growth, Tukey’s two-way ANOVA was performed with n=5–10 mice per group and combining at least 2 independent experiments. Data are represented as mean ± stdev. P value: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. d, g) For survival, Kaplan Meier curves were analyzed using the Log-rank (Mantel-Cox) test with n=9–10 and combining at least 2 independent experiments.

Figure 2.. Role of CAR, IFNγ and Perforin on the effects of TA99 CAR T–cell therapy on B16F10 tumors.

a) Tumor growth and b) final tumor weights 19 days post tumor inoculation of B16F10 tumors following no treatment or treated with Super2 + IL33 TA99 CAR T cells engineered from wildtype (WT), Prf1 KO or IFNγ KO mice. c) B16F10 tumor growth in mice following no treatment or treatment with 7x10⁶ Super2 + IL33 T cells expressing either a full-length or tailless TA99 CAR. d) Tumor growth and e) final tumor weights of B16F10 tumors following no treatment or treatment with Super2 + IL33 TA99 CAR T cells or T cells expressing Super2 + IL33 without CAR expression. a, c, d) For tumor growth, Tukey’s two-way ANOVA was performed with n=6–10 mice per group and combining at least 2 independent experiments. b, e) For tumor weights, Tukey’s ordinary one-way ANOVA was performed with n=6–10 mice per group and combining at least 2 independent experiments. a – e) Data are represented as mean ± stdev. P value: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure 3.. TILs in mice treated with Super2 + IL33 TA99 CAR T cells.

a) TA99 CAR T cells and Super2 + IL33 TA99 CAR T cells were engineered from CD45.1 mice. B16F10 tumors from CD45.2 mice receiving no treatment or treatment with the CD45.1 cells were extracted and analyzed by flow cytometry for tumor-infiltrating CAR T cells, identified by CD45.1 expression and normalized per gram of tumor. b) Experimental schematic showing that C57BL/6 albino mice challenged i.d. with 2x10⁵ B16F10 tumors were treated with 7x10⁶ luciferase expressing TA99 CAR T cells or Super2 + IL33 TA99 CAR T cells 6 days post tumor inoculation. c) Total luminescent signal and d) representative images of CAR T cells in mice imaged on an IVIS imager following i.p. injection of luciferin substrate on the indicated days. e) Tumor-infiltrating endogenous CD8⁺ T cells, CD4⁺ T cells, NK cells were identified by CD45.2 expression and analyzed as in a). c) For luminescence analysis, mixed effects two-way ANOVA analysis was performed with n=5–10 mice per group and combining 2 independent experiments. e) For TIL analysis, Tukey’s ordinary one-way ANOVA was performed with n=7–10 mice per group and combining 2 independent experiments. All data are represented as mean ± stdev. P value: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure 4.. Effects of Super2 + IL33 CAR T cells in depleted recipient mice.

a) Schematic representing tumor depletion mouse models. NK cells and CD4⁺ cells were depleted using 250 ug/dose every 3–4 days of either anti-NK1.1 or anti-CD4 starting at Day −1, respectively. CD8-deficient mice (CD8 KO) were used to eliminate CD8⁺ cells. Mice were administered Super2 + IL33 TA99 CAR T cells six days after tumor inoculation. Tumor growth curves and final tumor weights in b, c) CD4-depleted recipients, d, e) NK1.1-depleted recipients, and f, g) CD8-deficient recipients. b, d, f) For tumor growth, Tukey’s two-way ANOVA was performed with n=8–10 mice per group and combining 2 independent experiments. c, e, g) For tumor weights, Tukey’s ordinary one-way ANOVA was performed with n=8–10 mice per group and combining 2 independent experiments. All data are represented as mean ± stdev. P value: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure 5.. scRNA-seq of tumor infiltrates.

C57BL/6 mice inoculated with 2x10⁵ B16F10 tumors received no treatment (NT) or were treated with 7x10⁶ TA99 CAR T cells (CAR) or Super2 + IL33 TA99 CAR T cells after 6 days. Fifteen days post tumor inoculation, tumors were harvested, subjected to a percoll gradient and submitted for scRNA-seq. Aggregated normalized scRNA-seq data were clustered using the Seurat implementation of shared nearest neighbor unsupervised clustering and visualized using UMAP method. Cluster cell identities were determined using the top upregulated genes in each cluster. a) Cell clusters and their cell identities, b) sample id, c) cell type frequencies in each sample, d) macrophage and B cell/DC clusters, e) cell type frequencies in each sample, f) dotplot of highly differentially expressed transcripts, g) NK and T cell clusters, h) cell type frequencies in each sample, i) dotplot of highly differentially expressed transcripts.

Figure 6.. Effects of Super2 + IL33 TA99 CAR T–cell therapy on TME compartments.

C57BL/6 mice inoculated with 2x10⁵ B16F10 tumors i.v. through the tail vein. Six days post tumor inoculation, mice were left not treated or treated with 7x10⁶ TA99 CAR T cells or Super2 + IL33 TA99 CAR T cells. Seventeen days post tumor inoculation, TILs were analyzed by flow cytometry to identify a, b) CD3⁺ T cells and NK cells, c, d) CD4⁺ and CD8⁺ T cells, e, f) PD-1 and Tim-3 expression on CD8⁺ T cells, g, h) Treg cells, I, j, k) TAMs and their expression of MHC class II. b, d, f, h, k) All data are represented as mean ± stdev. For % of CD45.2 frequencies, two-way ANOVA, Sidak’s multiple comparison test was performed. h) For CD8⁺ to Treg ratio, Kruskal-Wallis multiple comparison test was performed with n=4 mice per group, representative flow plots and data from 2 experiments. P value: ****p<0.0001, **p<0.01, *p<0.05.

Figure 7.. Effects of Super2 + IL33 CAR therapy on B16F10 metastatic tumors, MC38 colon tumors, and B16F10-B7H6 tumors.

a, b) C57BL/6 mice inoculated with 2x10⁵ B16F10 tumors i.v. through the tail vein. Six days post tumor inoculation, mice were left not treated or treated with 7x10⁶ TA99 CAR T cells or Super2 + IL33 TA99 CAR T cells. Fifteen days post tumor inoculation, mice lungs were harvested for quantification and to determine tumor burden. c, d) C57BL/6 mice were inoculated with 1x10⁶ MC38 tumors s.c. and left not treated or treated with 7x10⁶ NKG2D CAR T cells or Super2 + IL33 NKG2D CAR T cells 6 days later. c) Tumor growth and d) final tumor weights of MC38 tumors. e-g) C57BL/6 mice were inoculated i.d. with 2x10⁵ B16F10 tumors that express human antigen B7H6 and left not treated or treated with 4x10⁶ TZ47 CAR T cells or Super2 + IL33 TZ47 CAR T cells on day 6. e) Tumor growth and f) final tumor weights on Day 19. g) Number of TRP2 tetramer⁺ T cells identified by flow cytometry. c, e) For tumor growth, Tukey’s two-way ANOVA was performed with n=9–10 mice per group and combining 2 independent experiments. d, f) For tumor weights, Tukey’s ordinary one-way ANOVA was performed with n=9–10 mice per group and combining 2 independent experiments. g) For TRP2 cell analysis, Tukey’s ordinary one-way ANOVA was performed with n=9–10 mice per group and combining 2 independent experiments. All data are represented as mean ± stdev. P value: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.