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IL-2Rα-biased agonist enhances antitumor immunity by invigorating tumor-infiltrating CD25+CD8+ T cells

Abstract

To avoid regulatory T cell promotion and vascular toxicity, the interleukin-2 receptor-β/interleukin-2 receptor-γ (IL-2Rβγ)-biased approach is used by most IL-2 analogs in immuno-oncology. However, recent clinical disappointments in these IL-2 agonists have questioned this strategy. Here we show that both wild-type (IL-2wt) and IL-2Rβγ-attenuated (IL-2α-bias) agonists that preserve IL-2Rα (CD25) activities can effectively expand tumor-specific CD8+ T cells (TSTs) and exhibit better antitumor efficacy and safety than the ‘non-α’ counterpart (IL-2). Mechanistically, TSTs coexpress elevated CD25 and PD-1 and are more susceptible to stimulation by IL-2Rα-proficient agonists. Moreover, the antitumor efficacy of anti-PD-1 depends on activation of PD-1+CD25+ TSTs through autocrine IL-2–CD25 signaling. In individuals with cancer, a low IL-2 signature correlates with non-responsiveness to anti-PD-1 treatment. In mouse models, IL-2α-bias, but not IL-2, restores the IL-2 signature and synergizes with anti-PD-1 to eradicate large established tumors. These findings underscore the indispensable function of CD25 in IL-2-based immunotherapy and provide rationales for evaluating IL-2Rα-biased agonists in individuals with cancer.

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Fig. 1: IL-2wt exhibits stronger antitumor efficacy than IL-2 through selective expansion of TST cells.
Fig. 2: IL-2wt and IL-2 differentially reshape the TME.
Fig. 3: IL-2α-bias elevates the CD8+ Teff cell-to-Treg cell ratio in tumors, but not in the periphery, to promote antitumor efficacy.
Fig. 4: PD-1+CD25+CD8+ TILs are enriched for TSTs in both mouse and human tumors.
Fig. 5: Antitumor efficacy of anti-PD-1 is highly dependent on the activation of PD-1+CD25+CD8+ TILs through autocrine IL-2–CD25 signaling.
Fig. 6: The IL-2 pathway signature in tumors is associated with clinical responsiveness to anti-PD-1.
Fig. 7: An IL-2Rα-biased, but not IL-2Rβγ-biased, agonist reprograms the tumor immune landscape and synergizes with anti-PD-1 therapy.

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Data availability

In-house-generated mouse scRNA-seq and bulk RNA-seq data that support the findings of this study have been deposited in the ArrayExpress database under accession numbers E-MTAB-12373 and E-MTAB-12372. Human scRNA-seq and bulk RNA-seq data reanalyzed here are available under accession numbers GSE176021 (LUAD and SCC data from Caushi et al.25), GSE144945 (NSCLC data from Cho et al.26), GSE178341 (CRC data from Pelka et al.27), GSE162498 (NSCLC data from Gueguen et al.28), GSE123814 (BCC and SCC data from Yost et al.29) and GSE156728 (ESCA, MM, OV, PACA, RC, THCA and UCEC data from Zheng et al.30). The melanoma bulk RNA-seq data are available from the European Nucleotide Archive under accession number PRJEB23709. Human nsqNSCLC bulk RNA-seq data were collected from the ORIENT-11 study (ClinicalTrials.gov identifier NCT03607539) published previously35.

Requests for further information, resources and reagents should be directed to the lead contact K.H. under a fully executed material and transfer agreement. Source data are provided with this paper.

Code availability

The bioinformatics analyses were performed using open-source software, including CellRanger (version 6.1.2), STAR (version 2.5.3a), StringTie (version 1.3.1), gffcompare (version 0.9.9c), GSEA (version 4.2.3), Seurat (version 4.1.3) and GSEA (version 1.42.0) and the Immunedeconv R package (version 2.1.0).

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Acknowledgements

We thank D. Yu, Y.-J. Liu, L. L. Lanier, L. Fong and C. Garcia-Echeverria for helpful discussions and staff at the Innovent Flow Cytometry Core and Pathology Core for assistance in experiments. K.H. was supported by the Innovent Guoqing Academy discovery research fund.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization and supervision: K.H. In vitro cell experiments: J.G. Animal studies: W.W., J.L., Y.L., Y.F., Y.Y., Y.Z., J.X., M.W., B.C. and H.L. Bioinformatics: T.C., X.L., J.S. and J.Z. Protein sciences: F.F., S.Z., X.L., Z.W., F.W. and X.L. Immunohistochemistry: J.D. Writing, original draft: K.H. Writing, review and editing: K.H., W.W., T.C., X.L., J.G. and J.Z.

Corresponding author

Correspondence to Kaijie He.

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Competing interests

All authors are current employees of Innovent Biologics. K.H., F.F., W.W. and S.Z. are inventors of a related patent (WO2021185361A1).

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Extended data

Extended Data Fig. 1 In vitro and in vivo activities of IL-2wt or IL-2.

(a–c) Binding kinetics of anti-human B7H3 antibody (hz20G5) fused with either IL-2wt or IL-2 detected by bio-layer interferometry (BLI). IL-2wt (A) and IL-2 (B) binding kinetics to human IL-2Rα, human IL-2Rβγ, mouse IL-2Rα and mouse IL-2Rβγ. Table summarizing binding affinity of each molecule (C). (d) Binding kinetics of antibody hz20G5 to human B7H3 and mouse B7H3, showing high affinity of hz20G5 to human B7H3 but no binding activity to mouse B7H3 antigen. (e) Dose response curves of pSTAT5% in CD4+CD25-T cell and NK cells treated with serial diluted IL-2wt or IL-2 (experiment performed in duplicates). (f) Individual MC38 tumor growth curves in each treatment group (related to Fig. 1c). (g–i) Three repeated experiments to confirm in vivo efficacy and safety in MC38 tumor-bearing mice. Average tumor volume changes, body weight changes, and individual tumor growth curves were shown for each experiment. High dose (10 mg/kg) of IL-2 agonists was used in one experiment (G), and the remaining experiments used 3mg/kg. n = 7 (G) or n = 6 (H-I) mice per group. (j–k) Evaluation of wet lung weight in wild-type C57BL/6 mice (J) or Rag1-/- C57BL/6 mice (K) after a single administration of hIgG, IL-2wt or IL-2 at indicated dosage 5 days post treatment. n = 5 (J) or n = 8 (K) mice per group. Mean + s.e.m. is presented, and p-values are calculated using two-way ANOVA for tumor growth curves and one-way ANOVA for bar graphs.

Source data

Extended Data Fig. 2 PK and PD analyses in MC38 tumor-bearing mice after IL-2wt or IL-2 treatment.

(a) Pharmacokinetics of IL-2wt and IL-2 in C57BL/6 mice after single 3mg/kg intravenous administration. n = 9 mice per group. (b) Frequency of CD8+T and Treg in CD45+ cells and CD8/ Treg ratio in tumor, spleen and blood from MC38 tumor-bearing mice at the end of study after repeated dosing of indicated reagents (n = 4~5 mice, 3mg/kg, ip. injection, QWx3). (c) Representative FACS plots and bar graphs showing the frequencies of MuLV p15E-tetramer+ in different CD8+TILs subsets from IgG control treated tumors. (n = 4 mice). (d) CD39+% and CD39-PD-1-% in CD8+TILs after treatment. (n = 4 mice). (e) Representative contour plots of PD-1 versus CD39 in CD8+TILs (black) and dot plots showing p15E-tetramer+CD8+TILs (red) after treatment, and bar graphs showing p15E-tetramer+% in CD8+TILs on the right. (n = 4 mice). (f) CD25 mean fluorescent intensity (MFI) on total CD8+TILs (left panel); CD25 MFI on CD39- or CD39+ CD8+TILs after treatment (right panel). (n = 4 mice). (g) CD25 MFI on p15E-tetramer+CD8+TILs after treatment. (n = 4 mice). (h) Representative FACS plots and bar graphs showing Treg cells in each treatment group. (n = 4 mice). Mean + s.e.m. is presented, and p-values are calculated using two-way ANOVA for PK analysis and one-way ANOVA for bar graphs.

Source data

Extended Data Fig. 3 In vivo efficacy, safety and TIL analysis in B16F10-OVA and CT26 tumors.

(a–n) B16F10-OVA tumor study related to Fig. 1f–l. (n = 7 mice per group. But n = 6 (hIgG), n = 7 (IL-2wt) and n = 5 (IL-2) mice were analyzed for (b–n), as data was not available due to tumor ulceration (n = 1 in hIgG) and premature deaths (n = 2 in IL-2 treated group)). (a) Individual tumor growth curves. (n = 7 mice). (b, c) Tumor weight and wet lung weight measured at the end of the study. (d) Intratumoral CD8 to Treg ratio in each treatment group. (e) Representative contour plots of PD-1 versus CD39 in CD8+TILs (black) and dot plots showing OVA-tetramer+CD8+TILs (red) after treatment. (f) Frequency of CD25+% and CD25 expression levels (MFI) in CD39- or CD39+ CD8+TIL subsets. (g) Frequency (in CD45+ cells) and absolute cell numbers of OVA-tetramer+CD8+TILs in tumors. (h) Frequency (in CD45+ cells) and absolute cell numbers of Ki67+OVA-tetramer+ CD8+TILs in tumors. (i-j) Frequency (in CD45+ cells) and absolute cell numbers of CD8+ and Ki67+CD8+ TILs in tumors. (k-l) Frequency (in CD45+ cells) and absolute cell numbers of CD4+ and Ki67+CD4+ TILs in tumors. (m-n) Frequency (in CD45+ cells) and absolute cell numbers of intratumoral Treg and Ki67+Treg cells. (o–r) Efficacy and safety evaluation in CT26 tumor model. Average tumor volume changes (o), body weight changes (p), individual tumor growth curves (q) and wet lung weight (r) were shown. n = 7 mice per group. One mouse died prematurely in IL-2 group, so only n = 6 data points were shown in IL-2 group in (r). Mean + s.e.m. is presented, and p-values calculated using one-way ANOVA for bar graphs, two-way ANOVA for tumor growth curves and body weight changes, and two-sided paired t-test for symbols & lines plots.

Source data

Extended Data Fig. 4 scRNA-seq analysis of immune cell clusters in MC38 tumors.

Related to Fig. 2. C57BL/6 mice bearing MC38 tumors (n = 3 mice) were treated weekly with 3 mg/kg of IL-2wt, IL-2 or IgG for 3 times. Tumors were dissected five days after the last dose and CD45+ cells were isolated for scRNA-seq. (a) UMAP, (b) cell cluster composition and (c) heatmap of CD45+ cells. (d) Heatmap and (e) feature plots of CD3+ cell clusters. (f) Heatmap comparing the CD8+ naïve T cluster and the CD8+ (IFN-stimulated genes) cluster. Genes associated with interferon stimulation are highlighted. (g) UMAP, (h) cell cluster composition, (i) heat map and (j) PD-L1 violin plots (total n = 5865, IgG=3304, IL-2wt= 1637, IL-2 =924) of the mononuclear phagocytes cluster from (A). The center lines of boxplots within violin plots indicate median values, box limits indicate the 25th and 75th percentiles and whiskers extend 1.5x the interquartile range from 25th and 75th percentiles. (k) Feature plots showing PD-L1 expression (top) and the average expression levels of genes from “GOBP Response To Interferon Gamma’ signature (bottom).

Extended Data Fig. 5 In vitro and in vivo activities of IL-2α-bias or IL-2nα/N88D.

(a, b) Binding kinetics of IL-2α-bias (a) and IL-2nα/N88D (b) detected by bio-layer interferometry (BLI) to human IL-2Rα, human IL-2Rβγ, mouse IL-2Rα and mouse IL-2Rβγ. (c) Table summarizing binding affinity of each molecule to IL-2Rs. (d) Dose response curves of pSTAT5% in CD4+CD25-T cell and NK cells after stimulation. (experiment performed in duplicates). (e) Table summarizing in vitro activities (pSTAT5 stimulation) of four IL-2 agonists. (f) Dose response curves of pSTAT5 activation in CTLL-2 pSTAT5 reporter cells after IL-2 agonists stimulation. (experiment performed in triplicates). (g) Tumor weight dot plot and body weight change curves related to Fig. 3d. (n = 6 mice). (h) Average and individual tumor growth curves of MC38 tumors in Rag1-/- mice after transferring 5 million congenic CD8+T cells on day7 post implantation with or without 3mg/kg IL-2α-bias treatment (n = 6 mice). (i-j) In vivo efficacy of IL-2α-bias in OT-I cells transferred B16F10-OVA model. 0.2 million OT-I cells were transferred into Rag1-/- or NOG mice when B16F10-OVA tumors were palpable (4~7 days post implantation), then tumor-bearing mice were treated with IgG or IL-2α-bias @3mg/kg weekly for three times. (i) Individual tumor growth curves and survival plot of Rag1-/- mice (n = 8 mice). (j) Individual tumor growth curves and tumor volumes at the end of study in NOG mice (n = 8 mice). Mean + s.e.m. is presented, and p-values are calculated using one-way ANOVA for bar graphs and two-way ANOVA for tumor growth curves.

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Extended Data Fig. 6 In vivo efficacy of IL-2α-bias with or without depletion of CD4+T, CD8 +T and NK cells.

(a) Individual tumor growth curves in each treatment group (related to Fig. 3e). (b) Tumor weight measured at the end of study. (c) Body weight change curves. (d) Representative dot plots showing the cell depletion efficiency in tumors. (e) Frequency of CD8+T, CD4+T, Treg and NK cells in CD45+ cells in tumors (top) and blood (bottom). N = 7 mice per group. Mean + s.e.m. is presented, and p-values are calculated using one-way ANOVA for bar graphs in B and E (Dunnett’s multiple comparisons test).

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Extended Data Fig. 7 In vivo activity and safety profiles of IL-2α-bias in 5 different syngeneic tumors.

(a–d) Tumor growth curves and body weight changes in B16F10 (a), LLC1 (b), CT26 (c) and EMT6 (d) tumor models after indicated treatments (n = 7 mice). (e) Wet lung weight measured from treated mice at the end of study (n = 7 mice). The center lines indicate median values, box limits indicate the 25th and 75th percentiles and whiskers extend 1.5x the interquartile range from 25th and 75th percentiles. (F-H) In vivo activities of IL-2α-bias in MC38 tumors (n = 8 mice), related to Fig. 4e, f. Dot plots showing PD-1 and CD25 expressions on CD8+TILs in MC38 tumors (f), frequency and absolute number of IFN-γ+CD8+TILs (g), and enrichment fold of IFN-γ+% in PD-1-CD25-, PD-1+CD25- and PD-1+CD25+ subsets of CD8+TILs after treatment (H). (i) Tumor growth curves showing efficacy of IL-2α-bias in B16F10-OVA tumors (n = 6~7 mice). (J) Dot plots showing PD-1 and CD25 expressions on CD8+TILs in B16F10-OVA tumors. (k) OVA-tetramer+% in PD-1-CD25-, PD-1+CD25- and PD-1+CD25+ subsets of CD8+TILs after IgG or IL-2α-bias treatment (n = 6~7 mice). Mean + s.e.m. is presented, and p-values are calculated using two-way ANOVA for tumor growth curves (A-D and I, Tukey’s multiple comparisons test) and for H and K (Šídák’s multiple comparisons test), one-way ANOVA (Tukey’s multiple comparisons test) for box plots (E), and two-sided t-test for bar graphs (G).

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Extended Data Fig. 8 Dynamics of immune cell subsets in tumors and spleens derived from five syngeneic tumor models.

MC38, B16F10, CT26, EMT6 and LLC were collected and digested 1 week, 2 weeks and 3 weeks post implantations. Cell suspensions of tumors and spleens were stained and analyzed by flow cytometry (n = 6~8 mice). (a) Stacked bar graphs showing the proportion and absolute number of each immune cell subpopulation in different tumor models. (n = 6~8 mice). (b) Quantitation of CD25 expressions (MFI, measured by flow cytometry) in PD-1- versus PD-1+ CD8+TILs from different timepoints. Two-side paired t-test was used to calculate p-values. (c) Representative histograms showing mouse CD25 expression levels in CD8+TILs among five different tumor types (left). Correlational analysis between CD25+CD8+ frequency in intratumor CD8 and tumor growth inhibition of IL-2α-bias in five different syngeneic tumor models (right). (d) Correlational analysis of intratumor CD8+T, Treg or CD8/Treg ratio versus tumor growth inhibition (TGI%) of IL-2α-bias in five different syngeneic tumor models. (e) Correlational analysis of other intratumor immune subpopulations versus TGI% of IL-2α-bias in five different syngeneic tumor models (n = 6~8 mice). (f) Cell number/area and the percentage of CD8+T cell in tumor and adjacent tissue from CRC tissue microarray (n = 50 tumor samples) in Fig. 4g. Mean ± s.e.m. is presented, and p-values for linear regressions (C-E) were calculated by default using Graphpad Prism8 software, and p-values for dot plots were calculated by two-sided t-test (F).

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Extended Data Fig. 9 αPD-1 efficacy is dependent on the activation of PD-1+CD25+CD8+TILs through autocrine IL-2/CD25 signaling.

(a) Quantification of intracellular IL-2% in CD8+T cells after 24hrs activation by aAPC in the lower chamber. Dash line indicates data from PBMC only control. Statistical analysis by two-sided student’s t-test (Experiment done once, with 5 technical replicates). (b-c) Representative histograms (b) and bar graphs (c) showing intracellular IL-2% in different CD8+T cell subsets after 24hrs activation by aAPC (Experiment done once, with 5 technical replicates). (d-e) Ex vivo measurement of intracellular IFN-γ (D) and TNF-α (e) in CD8+TILs from MC38 tumors. Similar to Fig. 5h–j. (f) MC38 tumors treated with αPD-1 antibody (5mg/kg, QWx2 as indicated by arrows) with or without CD25 blocking antibody (n = 7 mice). Experiments were repeated three times. Tumor growth curves (left), representative FACS plots of GZMB expression (middle) and the corresponding MFI plots of the indicated cell subsets (right). Mean + s.e.m. is presented, and p-values are calculated using two-way ANOVA unless otherwise indicated.

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Extended Data Fig. 10 IL-2 pathway signature correlates with αPD-1 response.

(a-g) Data from ORIENT-11 and melanoma studies. (A) GSEA of combo group responders versus non-responders, supplementary to Fig. 6. (b) GSEA of “PID_IL2_1PATHWAY’ signature in nsqNSCLC patients responding to chemotherapy versus non-responders. (c) Waterfall plot in chemo group. (d) Bar plot representing statistical difference (two-sided student t-test) between responders and non-responders for each signaling pathway. Red dashed line: p = 0.05. X-axis represents –log10(p-value); Y-axis represents signaling pathways. (e) The relative percentage of distinct cell types (including CD45+ immune cells and CD45- non-immune cells) were deconvoluted from the RNA-seq data of each patient sample. (f) Box and whisker plots showing “CD8+ T cells ratio in CD45+ (% CD8)’ and “PID_IL2_1PATHWAY’ signature score among non-responders (NR) or responders (R) to different treatments. The center lines indicate median values, box limits indicate the 25th and 75th percentiles and whiskers extend 1.5x the interquartile range from 25th and 75th percentiles. P-values were calculated using two-sided Wilcoxon test. (g) Kaplan–Meier analysis of progression-free survival and Overall survival in Combo_High_%CD8, Combo_Low_%CD8, Chemo_high_%CD8 and Chemo_low_%CD8 groups. HR, hazard ratio; p-values were computed using log-rank (Mantel–Cox) test. (h-i) Small tumors (60–80 mm3) and large tumors (200–300 mm3) with or without the indicated IL-2 treatment were extracted for RNAseq analysis. Four mice in each group. S, small tumor; L, large tumor; UNT, untreated. (h) Heatmap of GSVA immune signature scores. Each lane represents the signature score from a single mouse. (i) GSEA of small tumor versus large tumor. (j-k) Individual tumor growth curves in mice bearing MC38 (j) or EMT6 (k) tumors from different treatment groups as in Fig. 7g, h respectively. Low dose: 3mg/kg; High dose: 10mg/kg. n = 7 mice per group.

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Tables 1 and 2.

Reporting Summary

Source data

Source Data Figs. 1, 3 and 5 and Extended Data Figs. 1–3 and 5–10

Raw assay readout.

Source Data Fig. 4

Raw assay readout and raw bioinformatics analysis output.

Source Data Fig. 6

Raw bioinformatics analysis output.

Source Data Fig. 7

Raw assay readout and raw RNA-seq output.

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Wu, W., Chia, T., Lu, J. et al. IL-2Rα-biased agonist enhances antitumor immunity by invigorating tumor-infiltrating CD25+CD8+ T cells. Nat Cancer 4, 1309–1325 (2023). https://doi.org/10.1038/s43018-023-00612-0

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