Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy

Abstract

Purpose: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells.

Experimental design: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration.

Results: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both.

Conclusions: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

Fig. 1:. Stability and feasibility of engineering tumor-directed human T-cells with membrane-tethered IL-15/IL-21.

(A) Schema of expression plasmids. (B) Flow cytometry plots of co-transduced cells. The upper row shows the percentage of KKLC1-TCR⁺ T-cells. The bottom row depicts KKLC1-TCR⁺ T-cells with no (black), membrane-tethered IL-15 (green), IL-21 (purple), or IL15/IL-21 (red) cell surface expression. KKLC1-TCR⁺ T-cells served as flow controls. (C) Membrane-tethered IL-15/IL-21 expression in T-cells from 20 healthy donors after single-transduction. (D) Percentage of CD8⁺ and CD4⁺ T-cells with membrane-tethered IL-15/IL-21 expression after co- (grey bars) and single-transduction (white bars) from 29 donors. One-way ANOVA test. (E) Transduced T-cells or controls were added to respective tumors cells in cell kill assays at an effector-to-tumor (E:T) ratio of 1:1. After 24 hours, the supernatant of these cultures was sampled and tested for (E) IL-15 and (F) IL-21 (right), and other cytokines such as IFNγ (G), IL-2 (H), and TNFα (I). Expansion kinetics of cells after single-transduction (J) and co-transduction (K) after exogenous cytokine removal.

Fig. 2:. In vitro activity of tumor-directed T-cells with membrane-tethered cytokines.

(A) Assessment of cytotoxicity of KKLC1-TCR T-cell products against KKLC1^low (A375) and KKLC1^high tumors cells (JK-4156). E7-TCR and tNGFR serve as non-relevant controls. One-way ANOVA with multiple comparisons or Friedman test; **** <0.0001. (B) Experimental schema of the tumor rechallenge model. Tumor clearance with consecutive tumor rechallenges in a (C) GD2-CAR/neuroblastoma and (D) E7-TCR/epithelial tumor model. (E) Corresponding images of GFP⁺ CaSki tumor cells after 10 rechallenges. T-cell subset analysis of (F) untransduced (UT) mock and (G) E7-TCR T-cells after 10 tumor rechallenges. CM: central memory, EM: effector memory, TEMRA: effector memory cells re-expressing CD45RA.

Fig. 3:. Membrane-tethered IL-15/IL-21 limits the number of NK-like T-cells during continuous antigen exposure.

(A) Experimental design and T-cell expansion during continuous antigen exposure. (B) Distribution of hash-tagged samples. (C) Frequencies of large cellular subsets by cytokine group and day. (D) Uniform manifold approximation and projection (UMAP) with detailed and crude annotations of 25 clusters. (E) UMAP distinguishes the cytokine groups and demonstrates the evolution of the NK-like T-cell cluster (cluster 11) over time. Green, E7.mteth.IL15; red, E7.mteth.IL15.IL21. (F) Changes in the fraction of respective clusters over time. (G) Violin plots of marker genes to define NK-like T-cells.

Fig. 4:. Stability and biological effects of membrane-tethered IL-15/IL-21 in vivo.

(A) Experimental schema where tumor-free mice receive engineered T-cells. Their blood is subsequently analyzed for shed levels of cytokines. (B) Measured serum IL-15 (left) and IL-21 (right) levels from tumor-free mice after T-cell injection. (C) Experimental schema to measure shed cytokine levels in the serum of tumor-bearing mice receiving engineered T-cells and (D) corresponding measured serum IL-15 (left) and IL-21 (right) levels. (E) Absolute E7-TCR T-cells per gram of spleen (left) and tumor (right). One-way ANOVA test; ** <0.01, *** <0.001, **** <0.0001. (F) Percentages of E7-TCR T-cells in the spleen (left) and tumor (right). One-way ANOVA test; ** <0.01, *** <0.001, **** <0.0001. (G) Absolute numbers of KKLC1-TCR T-cells per gram of spleen (left) and tumor (right). Mann Whitney Test; * <0.05. (H) The tumor growth trajectory of mice used for Day 15 analysis of T-cell expansion.

Fig. 5:. Engineered tumor-directed T-cells with armored membrane-tethered IL-15/IL-21 outperform those with no or single cytokine expression in vivo.

(A) Schema of the heterotopic E7-TCR/epithelial tumor model. (B) Tumor growth curves of treated mice. The number of mice with a complete response (CR) is shown for each graph in the figure legend in parenthesis. (C) Survival of treated mice in a separate study cohort. Percentage (left) and absolute numbers (right) of E7-TCR T-cells in (D) the tumor or (E) spleens at the time the mice terminated the study. One-way ANOVA (tumor data) or Kruskal Wallis Test (spleen data), **<0.01. (F) Schema of the orthotopic K666-CAR/neuroblastoma tumor model. (G) Tumor and (H) spleen weights four weeks after CAR T-cell injection. Unpaired T-test, ** <0.01, **** <0.0001. Circles indicate mice completing the study, X symbols mark animals that died prior to the end of the study. (I) Splenic distribution of CD8 and CD4 ratio across therapy groups. (J) Residual CD8⁺ and CD4⁺ K666-CAR⁺ T-cells in the spleens. (K) Distribution of CD8 and CD4 ratio in the TME across therapy groups. (L) Residual CD8⁺ and CD4⁺ K666-CAR⁺ T-cells in the TME.