Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling

Abstract

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Extended Data Figure 1:. Zip2R colocalization analysis by confocal microscopy.

(a) Pearson correlation analysis of mRuby and mClover in HEK293T cells transfected with indicated constructs (N=8-37, ****p<0.0001, one-way ANOVA). (b) Representative images.

Extended Data Figure 2:. Zip2R(2x) subcellular localization and trafficking determined by confocal microscopy.

(a) Schematic of SNAP/CLIP tag system. (b) Representative images of Zip2R(2x) with endosomal markers. Scale bar = 10μm. (c-f) Representative images of Zip2R(2x) colocalization with (c) cell membrane, (d) Lamp1, (e) Rab11, and (f) Rab5. (g) Comparison of Zip2R(2x) and Zip2R(1x) colocalization with indicated subcellular markers (N=4-14, *p<0.05, t test).

Extended Data Figure 3:. ZipR signaling is inhibited by ruxolitinib.

(a) pSTAT5 expression in Zip2R(2x) transduced T cells treated with increasing concentrations of ruxolitinib. IC50 is indicated with a dashed line (N=3, mean±SD). (b) pSTAT5 expression in Zip2R(2x) transduced T cells at baseline or following 24-hour incubation with 5 μM ruxolitinib (mean±SD, ***p<0.001, paired t-test). (c) pSTAT5 expression in Zip7R(2x) transduced T cells treated with increasing concentrations of ruxolitinib. IC₅₀ is indicated with a dashed line (N=3, mean±SD). (d) pSTAT5 expression in Zip7R(2x) transduced T cells at baseline or following 24-hour incubation with 5 μM ruxolitinib (mean±SD, ***p<0.001, paired t-test). (e) Viability of untreated or 5 μM ruxolitinib-treated T cells after 7 days of cytokine starvation (Dead : Viability dye⁺ Annexin V⁺; Apoptotic : Viability dye⁻ Annexin V⁺; Necrotic : Viability dye⁺ Annexin V⁻; Live: Viability dye⁻ Annexin V⁻) (N=3, mean±SD, ****p<0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test for live T cells).

Extended Data Figure 4:. ZipRs augment CAR T cell antitumor activity against LM7 osteosarcoma without altering antigen specificity.

(a,b) MTS assay after 24-hour co-culture of LM7 WT (left) or LM7 B7-H3 KO (right) cells with CAR T cells at indicated effector:target cell (E:T) ratios (N=3-4 biological replicates, mean±SD, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test). (c) Cytokine production after 24-hour co-culture of LM7 WT (left) or LM7 B7-H3 KO (right) cells with CAR or CAR.Zip2R T cells at a 2:1 E:T measured by multiplex analysis (N=2-3 biological replicates, mean±SD, two-way ANOVA with Tukey’s multiple comparisons test). (d) Cytokine production after 24-hour co-culture of A549 WT (left) or LM7 WT (right) cells with CAR or CAR.Zip7R T cells at a 2:1 E:T measured by multiplex analysis (N=2 biological replicates, mean±SD). (e) Number of stimulations in 7 day repeat stimulation assay with LM7 WT cells and CAR T cells at 2:1 E:T (mean±SD, *p<0.05, paired t-test). (f) Fold expansion of three representative donors used in repeat stimulation assays with LM7 WT cells. Data represented in (a). (g) Repeat stimulation assay with LM7 B7-H3 KO cells and CAR T cells at 2:1 E:T (N=4, mean±SD). (h) Stimulations of tumor cell killing in 7-day repeat stimulation assay with LM7 WT cells and CAR T cells at 2:1 E:T (mean±SD, *p<0.05, paired t-test). (i) Fold expansion of three representative donors used in repeat stimulation assays with LM7 WT cells. Data represented in (d). (j) Repeat stimulation assay with LM7 B7-H3 KO cells and CAR T cells at 2:1 E:T (N=3 biological replicates, mean±SD).

Extended Data Figure 5:. Phenotypic analysis of B7-H3-CAR T cells following stimulation with A549 WT.

B7-H3-CAR⁺, CAR⁺Zip2R⁺, or CAR⁺Zip7R⁺ T cells were stimulated twice with A549 WT. CAR positive or CAR and ZipR positive T cells were quantified by flow cytometry pre and post 2^nd stimulation (stim) and CD4 and CD8 positive T cells post 2^nd stim (N=2 donors). (a) Percent CAR⁺ or CAR⁺ZipR⁺ cells pre and post 2^nd stim (ns: nonsignificant, ***p<0.001, paired t-test). (b) Percent CD4⁺ (left panel) or CD8⁺ (right panel) cells post 2^nd stimulation (**p<0.01, ***p<0.001, paired t-test).

Extended Data Figure 6:. Zip2R and Zip7R augment EphA2-CAR T cell antitumor activity in vitro.

(a) Transduction efficiency of EphA2-CAR, EphA2-CAR.Zip2R, and EphA2-CAR.Zip7R T cells (N=3, mean±SD). (b) Frequency of CD4⁺ and CD8⁺ T cells transduced with indicated constructs (N=3, mean±SD). (c) Immunophenotype of CD4⁺ (left) or CD8⁺ (right) T cells with indicated constructs (T_N-Like: CCR7⁺ CD45RA⁺, T_EM: CCR7⁻ CD45RA⁻, T_CM: CCR7⁺ CD45RA⁻, T_EMRA: CCR7⁻ CD45RA⁺, N=3, mean±SD). (d) Fold expansion of ΔCAR (left) or CAR (right) T cells stimulated with A673 cells every 7 days (N=3, mean±SD). (e) Rounds of stimulation (left) and relative expansion compared to CAR (right) in repeat stimulation assays with A673 cells (N=3, mean±SD, **p<0.01, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test).

Extended Data Figure 7:. Zip2R augments the antitumor activity of EphA2-CAR in the A673 model.

(a) Experimental scheme of s.c. A673 model; mice received a single i.v. dose of 1×10⁶ CAR T cells on day 7 post tumor cell injection. (b) Tumor volume of mice treated with indicated constructs (N=4; donor 1). (c) Kaplan-Meier survival curve (*p<0.05, log-rank test). (d) Tumor volume of mice treated with indicated constructs (N=5; donor 2). (e) Tumor volume following rechallenge (dashed line) with A673 WT cells on the contralateral flank (N=4-5; tumor rejection: CAR: 0/5; CAR.Zip2R: 2/4). (f) Kaplan Meier survival following rechallenge.

Extended Data Figure 8:. Analysis of B7-H3-CAR.Zip7R T cell toxicity in vivo.

(a-d) Mice received a single i.v. dose of 3×10⁵ B7-H3.CAR.Zip7R.ffLuc T cells on day 7 post A549 cell injection; non-tumor bearing mice served as a control (N=5 per group). (a) Serial bioluminescence images. (b) Quantification of bioluminescence. (c) Kaplan-Meier survival (N=5). (d) Representative IHC of lung in non-tumor bearing mice treated with CAR.Zip7R T cells at day 48 post T cell injection. (e) Kaplan-Meier survival of A549-tumor bearing mice post i.v. injection of 3×10⁵ B7-H3-CAR.Zip7R control (AAVS1ko) or T cell receptor (TRACko) KO T cells (N=5). (f) A549 bearing mice received CAR.Zip7R.ffLuc T cells and on day 7 ruxolitinib was started (shaded area) in ½ of the mice (untreated: n=5; treated: n=5). Quantification of CAR.Zip7R.ffLuc T cell bioluminescence in untreated or ruxolitinib chow-treated mice. (g) Fold expansion on day 7 post start of ruxolitinb treatment (N=4-5, *p<0.05, Mann-Whitney U test). (h) Kaplan-Meier survival (N=4-5, **p<0.001, log rank test).

Extended Data Figure 9:. Immunophenotype and antigen specificity of CAR.Zip21R or CAR.Zip12R T cells.

(a) Frequency of CD4⁺ and CD8⁺ CAR and CAR.Zip21R T cells (N=4, mean±SD). (b) Immunophenotype of CD4⁺ (left) or CD8⁺ (right) T cells ((T_N-Like : CCR7⁺ CD45RA⁺, T_EM : CCR7⁻ CD45RA⁻, T^CM : CCR7⁺ CD45RA⁻, T_EMRA : CCR7⁻ CD45RA⁺, N=4, mean±SD). (c) Frequency of CD4⁺ and CD8⁺ CAR and CAR.Zip12R T cells (N=2-3, mean±SD). (d) Representative flow cytometry plots. (e) Immunophenotype of CD4⁺ (left) and CD8⁺ (right) T cells (N=2-3, mean±SD). (f) Transduction efficiency of ΔCAR and ΔCAR.Zip12R T cells (N=2, mean±SD). (g) Fold expansion of ΔCAR and ΔCAR.Zip12R T cells stimulated with A549 WT cells every seven days (N=2, mean ± SD).

Extended Data Figure 10:. Transcriptomic analysis of CD4⁺ CAR.ZipR T cells by scRNAseq.

(a) GSEA of unstimulated or stimulated CD4⁺ CAR and CAR.ZipR T cell populations. (b) Expression of memory, effector/cytotoxicity, inhibition/exhaustion, and activation markers in unstimulated or stimulated CD4⁺ CAR and CAR.ZipR T cell populations. (c) Expression of selected genes in unstimulated or stimulated CD4⁺ CAR and CAR.ZipR T cell populations (Wilcoxon rank sum test with Bonferroni correction; ****adjusted p value < 0.0001 and log₂ FC > 0.5 or < −0.5).

Figure 1:. Two leucine zipper motifs endow ZipRs with constitutive signal transduction.

(a) Schematic of IL-2 receptor-based leucine zipper receptors (Zip2R). (b) Zip2R(1x) and Zip2R(2x) constructs. (c) Transduction efficiency of human T cells as measured by mRuby [IL-2Rβ(1x), IL-2Rβ(2x)], mClover [IL-2Rγ(1x), IL-2Rγ(2x), Zip2R(2x)], or mRuby and mClover [Zip2R(1x)] expression (left). Phosphorylated STAT5 expression in transduced human T cells (right) (N=3-6 biological replicates, mean±SD, ****p<0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (d) Viability of transduced T cells after 7-day cytokine starvation as determined by flow cytometry (Dead : Viability dye⁺ Annexin V⁺; Apoptotic : Viability dye⁻ Annexin V⁺; Necrotic : Viability dye⁺ Annexin V⁻; Live: Viability dye⁻ Annexin V⁻) (N=3 biological replicates, mean±SD, **p<0.01, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test for live T cells). (e) Representative confocal microscopy images of transfected HEK293T cells (left). Colocalization area of mRuby and mClover (right) (N=80(L), N=89(R), *p<0.05, Kolmogorov-Smirnov test). (f) AlphaFold structure prediction of Zip2R(1x) and Zip2R(2x) as seen from the front and rotated 90 degrees. The starting residue, methionine, is shown as a green sphere. (g) Transduction efficiency of human T cells as measured by mClover expression (left). Phosphorylated STAT5 expression in transduced human T cells (right) (N=3-6 biological replicates, mean±SD, *p<0.05, t-test). (h) Viability of transduced T cells after 7-day cytokine starvation as determined by flow cytometry (Viability dye/Annexin V) (N=3 biological replicates, mean±SD, ****p<0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test for live T cells). (i) Viability of transduced T cells after 14- or 21-day cytokine starvation as determined by flow cytometry (Viability dye/Annexin V) (N=3 biological replicates, mean±SD, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test for live T cells). (j) Autonomous cell outgrowth assay: 1, 10, or 100 Jurkat cells or 1.5×10⁷ Zip2R(2x) or Zip7R(2x) transduced T cells were seeded in a G-REX cell culture plate and quantified weekly (N=3, *p<0.05, **p<0.01, two-way ANOVA of log transformed data with Dunnett’s multiple comparisons test). NT = non-transduced.

Figure 2:. Zip2R and Zip7R activate distinct signaling pathways as judged by multiplexed phosphoproteomics.

(a) Samples and experimental workflow of phosphoproteomic analysis. (b) Hierarchical clustering of top 700 differentially expressed proteins (p<0.05). (c) IKAP prediction of kinase activity in CD4⁺ (left) and CD8⁺ (right) cells. (d) Top upregulated proteins compared to NT cells in CD4⁺ (top) and CD8⁺ (bottom) cells. (e) Differentially expressed proteins in Zip7R versus Zip2R transduced CD4⁺ (top) and CD8⁺ (bottom) cells

Figure 3:. ZipRs improve CAR T cell effector function during chronic antigen exposure in vitro.

(a-b) Transduction efficiency of B7-H3-CAR and Zip2R (a) or Zip7R (b) in human T cells as measured by flow analysis for the ZipR (mClover) and CAR (anti-human F(ab’)₂) (N=3-6, mean±SD). (c-d) MTS assay after 24-hour co-culture of A549 WT (left) or A549 B7-H3 KO (right) cells with CAR T cells at indicated effector:target cell (E:T) ratios (N=3-4 biological replicates, mean±SD, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test). (e) Cytokine production after 24-hour co-culture of A549 WT (left) or A549 B7-H3 KO (right) cells with CAR or CAR.Zip2R T cells at a 2:1 E:T measured by multiplex analysis (N=2-3 biological replicates, mean±SD). (f) Stimulations of tumor cell killing in 7-day repeat simulation assay with A549 WT cells and CAR T cells at 2:1 E:T ratio (N=6 biological replicates, mean±SD, ***p<0.001, paired t-test). (g) Three representative donors used for repeat simulation assay with A549 WT cells represented in (f). (h) Repeat stimulation assay with A549 B7-H3 KO cells and CAR T cells at 2:1 E:T (N=4, mean±SD). (i) Stimulations of tumor cell killing in 7 day repeat stimulation assay with A549 WT cells and CAR T cells at 2:1 E:T (N=3 biological replicates, mean±SD, *p<0.05, paired t-test). (j) Three representative donors used for repeat stimulation assay with A549 WT cells represented in (i). (k) Repeat stimulation assay with A549 B7-H3 KO cells and CAR T cells at 2:1 E:T (N=3, mean±SD).

Figure 4:. ZipRs augment CAR T cell antitumor activity in vivo.

(a) Experimental scheme of i.v. A549.GFP.ffLuc model; mice received a single i.v. dose of 3×10⁵ CAR T cells on day 7 post tumor cell injection. (b) Tumor burden in the lungs as determined by serial bioluminescence imaging (N=5 [tumor and CAR.Zip7R] N=10 [CAR and CAR.Zip2R] from two donors). (c) Quantification of tumor flux in the lungs of treated mice (mean±SD, *p<0.05, ****p<0.0001, two-way ANOVA of log transformed BLI data). (d) scRNAseq experimental scheme. (e) UMAP projection of CAR or CAR.Zip2R T cells. (f) Quantification of cell population frequencies. (g) Expression of cytokines, chemokines, and surface effector markers. Red text indicates factors that are expressed log₂FC > 0.5 with an adjusted p value < 0.05 for CD4⁺ and CD8⁺ CAR.Zip2R compared to CAR T cells (Wilcoxon rank sum test with Bonferroni correction). (h) Ingenuity Pathway Analysis for CD8 and CD4 CAR.Zip2R T cells. (i) DEGs with log2FC > 0.5 or < −0.5 and adjusted p value < 0.05 in CD8⁺ and CD4⁺ T cells.

Figure 5:. Leucine zipper ectodomains enable the generation of ZipRs from diverse cytokine receptor families.

(a-c) Transduction efficiency (left) and pSTAT3 (a,b) or pSTAT4 (c) expression (right) in Zip21R (a), Zip10R or Zip22R (b), or Zip12R (c) T cells (mean±SD, **p<0.01, ***p<0.001, paired t-test (a,c), one-way ANOVA (b)). (d) Transduction efficiency of B7-H3 CAR and Zip21R in human T cells as measured by flow cytometry for ZipR (mClover) and CAR (anti-human F(ab’)₂) (N=4, mean±SD). (e) Fold expansion of CAR or CAR.Zip21R T cells in repeat stimulation assay with A549 WT cells (N=5). (f) Stimulations of tumor cell killing by CAR- or CAR.Zip21R-expressing cells in repeat stimulation assay with A549 WT cells (mean±SD, *p<0.05, paired t-test). (g) Transduction efficiency of B7-H3-CAR and Zip12R in human T cells as measured by flow analysis for the ZipR (mClover) and CAR (anti-human F(ab’)₂) (N=3, mean±SD). (h) Fold expansion of CAR or CAR.Zip12R T cells in repeat stimulation assay with A549 WT cells (N=3). (i) Stimulations of tumor cell killing by CAR- or CAR.Zip21R-expressing cells in repeat stimulation assay with A549 WT cells (N=3). (j) Experimental scheme of i.v. A549.GFP.ffLuc model; mice received a single i.v. dose of 3×10⁵ CAR T cells on day 7 post tumor cell injection. (k) Tumor burden in the lungs of treated mice as determined by serial bioluminescence imaging (N=4-5). (l) Kaplan-Meier survival curve (N=4-5, *p<0.05, log rank test). (m) Tumor burden in the lungs of treated mice as determined by serial bioluminescence imaging (N=5). (n) Kaplan-Meier survival curve (N=5, **p<0.001, log rank test).

Figure 6:. CAR.ZipR T cells sustain effector function through memory/survival or hybrid memory/effector transcriptional programs.

(a) scRNAseq experimental scheme. (b) Number of cells analyzed in each sample. (c) UMAP projection of unstimulated or stimulated CAR or CAR.ZipR T cell populations from two donors. (d) GSEA of unstimulated or stimulated CD8⁺ CAR.ZipR T cell populations in comparison to CAR T cells. (e) Expression of memory, effector/cytotoxicity, inhibition/exhaustion, and activation markers in unstimulated or stimulated CD8⁺ CAR and CAR.ZipR T cell populations. (f) Expression of selected genes in unstimulated or stimulated CD8⁺ CAR and CAR.ZipR T cell populations (Wilcoxon rank sum test with Bonferroni correction; ****adjusted p value < 0.0001 and log₂FC > 0.5 or < −0.5).