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.
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Data availability
The RNA-seq data files are available through Synapse (https://www.synapse.org/#!Synapse:syn52457643). The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
Code availability
The code used to analyse the cluster size of colocalized Zip receptors can be accessed in the AutomatedImageAnalysis repository at https://github.com/Jorge-Ibanez-StJude/AutomatedImageAnalysis.
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Acknowledgements
We thank J. McCommon and A. George (St. Jude Animal Resource Center) and C. Coleman (St. Jude Center for In Vivo Imaging and Therapeutics) for assistance with in vivo mouse studies. We thank Q. Pan for assistance with bioinformatics and figure design. We also thank K. Nichols and S. Albeituni for helpful discussions on the use of ruxolitinib. Animal imaging was performed by the St. Jude Center for In Vivo Imaging and Therapeutics, which is supported by St. Jude Children’s Research Hospital and the National Cancer Institute (NCI; no. P30 CA021765). We thank the Computational Structural Biology Center in the Department of Structural Biology for support. Cellular images were acquired at the St. Jude Cell and Tissue Imaging Center, which is supported by St. Jude Children’s Research Hospital and NCI (no. P30 CA021765). The schematic shown in Fig. 1a was created with Biorender.com, for which we have a licence. This work was supported by NCI grant no. F31CA250401-01A1 to M.B., grant nos. R01NS121249 and R01NS122859 to G.K. and the American Lebanese Syrian Associated Charities to J.Y., J.P., M.M.B., G.K. and S.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.
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This study was conceptualized by M.B., G.K. and S.G. Experimentation and analysis were performed by M.B., S.L., B.I.S., H.Shi., X.S., X.M., J.I., P.N., M.S., J.W., A.K., D.L., S.L.P., H.T., R.V.P., Y.L., Z-F.Y., A.A.A., H.S., and P.V. M.H. provided the CT3 scFv. J.Y., J.P., H.C., M.M.B., G.K. and S.G. supervised the study. M.B., J.Y., J.P., H.C., M.M.B., G.K. and S.G provided funding and resources. M.B and S.G wrote the paper. All authors reviewed and edited the paper.
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M.B., B.I.S., A.A.A., M.M.B., G.K. and S.G. are coinventors on a patent application for the developed Zip receptor technology. S.L., A.A.A., G.K. and S.G. are coinventors on patent applications in the fields of cell or gene therapy for cancer. M.H. is an inventor on international patent application no. PCT/US2019/045338 assigned to NIH, ‘High affinity monoclonal antibodies targeting glypican-2 and uses thereof’. H.C. is a consultant of Kumquat Biosciences, Inc. S.G. is a consultant of TESSA Therapeutics, a member of the Data and Safety Monitoring Board of Immatics and has received honoraria from Tidal, Catamaran Bio, Sanofi and Novartis within the past 2 years. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Zip2R colocalization analysis by confocal microscopy.
a, Pearson correlation analysis of mRuby and mClover in HEK293T cells transfected with indicated constructs (IL-2Rβ(1x): N = 16, IL-2Rγ(1x): N = 8, Zip2R(1x): N = 37, IL-2Rβ(2x): N = 33, IL-2Rγ(2x): N = 24, Zip2R(2x): N = 28, mean ± SD, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). b, Representative images.
Extended Data Fig. 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 = 10 µm. c-f, Representative images and Zip2R(2x) colocalization analysis of indicated regions of interest (ROI) with (c) cell membrane, (d) Lamp1, (e) Rab11, and (f) Rab5. g, Comparison of Zip2R(2x) and Zip2R(1x) colocalization with indicated subcellular markers (Membrane: N = 14, Lamp1, Rab11, Rab5: N = 4, *p = 0.0224 (membrane), *p = 0.0479 (Lamp1), mean ± SD, two-tailed t-test).
Extended Data Fig. 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 = 2). b, pSTAT5 expression in Zip2R(2x) transduced T cells at baseline or following 24-hour incubation with 5 µM ruxolitinib (N = 4, mean ± SD, ***p = 0.0005, two-tailed 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 (N = 5, mean ± SD, ***p < 0.001, two-tailed t-test). e, Viability of untreated or 5 µM ruxolitinib-treated T cells after 7 days of cytokine starvation as determined by flow cytometry (Viability dye/Annexin V) (N = 3, mean ± SD, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test).
Extended Data Fig. 4 ZipRs augment CAR T cell antitumor activity against LM7 osteosarcoma without altering antigen specificity.
a,b, Cytotoxicity 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 = 4 (a), N = 3 (b) 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 (Left: CAR,CAR.Zip2R: N = 3, mean ± SD, ΔCAR, ΔCAR.Zip2R: N = 2; Right: N = 2, mean, 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). e, Number of stimulations in 7 day repeat stimulation assay with LM7 WT cells and CAR T cells at 2:1 E:T (N = 4, mean ± SD, *p = 0.0154, two-tailed t-test). f, Fold expansion of three representative donors used in repeat stimulation assays with LM7 WT cells. Data represented in (e). 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 (N = 3, mean ± SD, *p = 0.0202, two-tailed t-test). i, Fold expansion of three representative donors used in repeat stimulation assays with LM7 WT cells. Data represented in (h). 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 Fig. 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 2nd stimulation (stim) and CD4 and CD8 positive T cells post 2nd stim (N = 2 donors). a, Percent CAR+ or CAR+ZipR+ cells pre and post 2nd stim (***p = 0.0005, two-tailed t-test). b, Percent CD4+ (left panel) or CD8+ (right panel) cells post 2nd stimulation (**p = 0.001, ***p = 0.0003, two-tailed t-test).
Extended Data Fig. 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 (TN-Like: CCR7+ CD45RA+, TEM: CCR7- CD45RA−, TCM: CCR7+ CD45RA−, TEMRA: 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 = 0.0011, ****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test).
Extended Data Fig. 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 1x106 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 = 0.0438, 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 = 5 (CAR), N = 4 (CAR.Zip2R); tumor rejection: CAR: 0/5; CAR.Zip2R: 2/4). f, Kaplan Meier survival following rechallenge.
Extended Data Fig. 8 Analysis of B7−H3-CAR.Zip7R T cell toxicity in vivo.
a-d, Mice received a single i.v. dose of 3x105 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 3x105 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 (untreated), N = 5 (Ruxolitinib), mean ± SD, *p = 0.0159, two-tailed Mann-Whitney U test). h, Kaplan-Meier survival (N = 4 (untreated), N = 5 (Ruxolitinib), **p = 0.0027, log rank test).
Extended Data Fig. 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 ((TN-Like: CCR7+ CD45RA+, TEM: CCR7- CD45RA−, TCM: CCR7+ CD45RA−, TEMRA: CCR7− CD45RA+, N = 4, mean ± SD). c, Frequency of CD4+ and CD8+ CAR and CAR.Zip12R T cells (N = 3 (CAR), N = 2 (ΔCAR), mean ± SD). d, Representative flow cytometry plots. e, Immunophenotype of CD4+ (left) and CD8+ (right) T cells (N = 3 (CAR, CAR.Zip12R), N = 2 (ΔCAR, ΔCAR.Zip12R), mean ± SD, **p = 0.0031, *p = 0.0238 (TN-Like), *p = 0.0265 (TEMRA), two-way ANOVA with Tukey’s multiple comparisons test). f, Transduction efficiency of ΔCAR and ΔCAR.Zip12R T cells (N = 2, mean). g, Fold expansion of ΔCAR and ΔCAR.Zip12R T cells stimulated with A549 WT cells every seven days (N = 2, mean).
Extended Data Fig. 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).
Supplementary information
Supplementary Information
Supplementary Figs. 1–15.
Supplementary dataset 1
Phosphoproteomics dataset.
Supplementary dataset 2
Zip receptor sequences.
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Bell, M., Lange, S., Sejdiu, B.I. et al. Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01143-w
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DOI: https://doi.org/10.1038/s41551-023-01143-w
