Coupling IL-2 with IL-10 to mitigate toxicity and enhance antitumor immunity

Abstract

Wild-type interleukin (IL)-2 induces anti-tumor immunity and toxicity, predominated by vascular leak syndrome (VLS) leading to edema, hypotension, organ toxicity, and regulatory T cell (Treg) expansion. Efforts to uncouple IL-2 toxicity from its potency have failed in the clinic. We hypothesize that IL-2 toxicity is driven by cytokine release syndrome (CRS) followed by VLS and that coupling IL-2 with IL-10 will ameliorate toxicity. Our data, generated using human primary cells, mouse models, and non-human primates, suggest that coupling of these cytokines prevents toxicity while retaining cytotoxic T cell activation and limiting Treg expansion. In syngeneic murine tumor models, DK2¹⁰ epidermal growth factor receptor (EGFR), an IL-2/IL-10 fusion molecule targeted to EGFR via an anti-EGFR single-chain variable fragment (scFV), potently activates T cells and natural killer (NK) cells and elicits interferon (IFN)γ-dependent anti-tumor function without peripheral inflammatory toxicity or Treg accumulation. Therefore, combining IL-2 with IL-10 uncouples toxicity from immune activation, leading to a balanced and pleiotropic anti-tumor immune response.

Keywords: EGFR; IFNγ; IL-10; IL-2; T cell exhaustion; cytokine; cytokine release syndrome; immunotherapy; targeted therapy; vascular leak syndrome.

Graphical abstract

Figure 1

IL-10 controls IL-2-mediated induction of inflammatory cytokines PBMCs were cultured with a titration of IL-2, IL-10, or IL-2+IL-10 (0–13 nM) for 24 h followed by measurement of supernatant analytes either before (A and B) or following anti-CD3 (C and D). Statistical analyses are depicted between IL-2 and IL-2+IL-10 groups at the highest concentration (13 nM) by donor-matched pairwise t test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001). Results are reported as the mean ± SEM.

Figure 2

Concomitant treatment of IL-2 with IL-10 prevents IL-2-induced systemic inflammation and edema without stunting immune activation (A–D) Mice were subcutaneously administered vehicle control, IL-2 (5 μg), IL-10 (2.4 μg), or IL-2+IL-10 (5 and 2.5 μg, respectively) daily for 9 days, as indicated. Statistical analyses comparing the control, IL-2, and IL-2+IL-10 groups are reported. (A–D) Serum analytes on day 9 after final administration were measured. Data are representative of 3 animals per group. Statistical analyses performed by two-way ANOVA with Tukey’s multiple comparisons test (∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). (E) After final administration, Evans blue dye extravasation into the lungs was measured. Data are representative of 3–9 animals per group. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). (F–J) The frequency of immune cell subsets in spleen, namely eosinophils (SSC^hi/CD11b⁺/SiglecF⁺), GzmB⁺/Perforin⁺ double-positive NK cells (CD3⁻/CD19⁻/NKp44⁺), CD8⁺ Tem (CD3⁺/CD8⁺/CD44⁺/CD62L⁻), and Tregs (CD3⁺/CD4⁺/CD25⁺/Foxp3⁺). Data are representative of 3 animals per group. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (p value; ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). Results are reported as the mean ± SEM.

Figure 3

IFNγ- and TNF-α-dependent IL-2 toxicity is ameliorated by IL-10 (A–D) PBMCs were untreated (control) or cultured with cytokines (1.3 nM) and receptor-blocking antibodies as follows: IL-2, IL-10, IL-2+anti-IFNγR1 (5 μg/mL), or IL-2+anti-IL-10RA (30 μg/mL) for up to 48 h, and supernatant analytes were measured. Data are representative of 5–9 independent donor experiments. Statistical analyses performed by donor pairwise t test on samples collected at 48 h. Statistically significant differences reported between IL-2, IL-2+anti-IFNγR1, and IL-2+anti-IL-10RA (p value; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). (E–G) PBMCs were cultured with IL-10 (1.3 nM), IFNγ (10 ng/mL), and IL-10+IFNγ or untreated (control), and supernatant analytes were measured. Data are representative of 4 independent donor experiments. Statistical analyses of the 48 h results were performed by pairwise t test (p value; ns, not significant; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). (H) Monocytes were stimulated for up to 16 h with IFNγ alone or with IL-10 (1.3 nM) and stained for IRF1 and IRF8 protein levels. Reported is the median fluorescence intensity (MFI) of IFNγ+IL-10-treated cells relative to the IFNγ-treated cells. Data are representative of 3 independent donor experiments. Statistical analyses performed by one-sample t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01). (I and J) Quantitation of Evans blue dye extravasation into the lungs after IL-2 treatment of wild-type, IFNγ-knockout, and TNF-α-knockout animals (I) and wild-type mice administered anti-TNF-α or an isotype control (J). Data are representative of 6–9 animals per group. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). Results are reported as the mean ± SEM.

Figure 4

Structural combination of wild-type IL-2 and an IL-10 mutein (A) Monocytes treated with LPS alone (controls) or in addition to a titration (0–13 nM) of wild-type or EBV IL-10. TNF-α reduction reported after 24 h compared to controls. Data are representative of 4 independent donor experiments. Statistical analyses performed by two-way ANOVA with Tukey’s multiple comparisons test and concentrations with statistical differences are indicated (∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001). (B) CTLs (CD8⁺) treated with a titration (0–13 nM) of wild-type or EBV IL-10 (0–13 nM) and IFNγ measured after 24 h. Data are representative of 14 independent donor experiments. Statistical analyses performed by two-way ANOVA with Tukey’s multiple comparisons test (∗∗p ≤ 0.01). (C) Monocytes treated with LPS alone (controls) or in addition to a titration (0–13 nM) of ScFV:EBV IL-10 wild type, ScFv:EBV IL-10 muteins (M1 or M2), or recombinant human IL-10 and TNF-α measured after 24 h. Data are reported as TNF-α reduction compared to controls. Data are representative of 2–4 independent donor experiments. (D) CTLs (CD8⁺) treated with a titration (0–13 nM) of ScFV:EBV IL-10 wild type, ScFv:EBV IL-10 muteins (M1 or M2), or recombinant human IL-10 and IFNγ measured after 24 h. Data are representative of 6 independent donor experiments. Statistical analyses performed by one-way ANOVA at the highest concentration (∗∗p ≤ 0.01). (E) Schematic of the full-length DK2¹⁰ (EGFR) construction depicting each domain. (F) A predicted computational model of DK2¹⁰ (EGFR) generated using chimera, with domains indicated. (G and H) The far- (G) and near-ultraviolet (H) spectra of DK2¹⁰ (EGFR) acquired via circular dichroism. (I–K) The binding kinetics of DK2¹⁰ (EGFR) measured by Octet using streptavidin (SA) immobilized biotinylated EGFR (I), IL-2Rα (J), and IL-10RA (K). Results are reported as the mean ± SEM.

Figure 5

Coupling IL-2 and IL-10 together retains immune activation and enhances tumor cytolysis (A) Monocytes were treated with LPS alone (controls) or also with a titration (0–1.3 nM) of IL-2, IL-10, IL-2+IL-10, or DK2¹⁰ (EGFR) and TNF-α measured after 24 h. Reported as TNF-α reduction compared to controls and representative of 8 independent donor experiments. Statistical analyses indicated between IL-2, IL-2+IL-10, and DK2¹⁰ (EGFR) performed by two-way ANOVA with Tukey’s multiple comparisons test (∗p ≤ 0.05). (B and C) CTLs (CD8⁺) (B) or CD4⁺ T cells (C) were treated with a titration (0–1.3 nM) of IL-2, IL-10, IL-2+IL-10, or DK2¹⁰ (EGFR) and IFNγ measured after 24 h. Data are representative of 8 independent donor experiments. Statistical analyses indicated between IL-2, IL-2+IL-10, and DK2¹⁰ (EGFR) performed by two-way ANOVA with Tukey’s multiple comparisons test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). (D and E) PBMCs were treated with a titration (0–1.3 nM) of DK2¹⁰ (EGFR) for 24 h (pre-anti-CD3) or stimulated an additional 24 h with anti-CD3 (post-anti-CD3). Anti-tumor factors (D) or CRS-associated factors (E) were measured in supernatant. Data are representative of 13–14 independent donor experiments. Statistical analyses were conducted by mix-effects analysis comparing analyte level at each dose of DK2¹⁰ (EGFR) to the respective pre/post-anti-CD3 untreated control (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). (F) IRF1/IRF8 protein measured in monocytes stimulated up to 16 h with IFNγ (10 ng/mL) alone (controls) or with DK2¹⁰ (EGFR) (1.3 nM), and median fluorescence intensity (MFI) relative to the controls is reported. Data are representative of 3 independent donor experiments. Statistical analyses performed by one-sample t test (ns, not significant; ∗p ≤ 0.05). (G–J) CTLs (CD8⁺) co-cultured with SK-BR3-GFP at a 10:1 E-to-T ratio. As indicated, cells were left untreated (controls) or treated with 1.3 nM of IL-2, IL-10, IL-2+IL-10, or DK2¹⁰ (EGFR) for up to 5 cycles of cytolysis. (H–I) During cycle 4 and 5 of cytolysis, DK2¹⁰ (EGFR) was additionally removed. (J) DK2¹⁰ (EGFR)-mediated cytolysis was measured after MHC I was knocked down in SK-BR-3-GFP cells by siRNA or MHC I antibody blocking. Data are representative of 3 independent donor experiments. Statistical analyses were performed by one-way ANOVA of the results at the final time point measured (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). Results are reported as the mean ± SEM.

Figure 6

Coupling and targeting IL-2 and IL-10 together retains broad immune activation and increases anti-tumor potency in mice (A–M) C57BL/6 mice (C57BL/6 and IFNγ-knockout mice in H) were implanted subcutaneously with a human EGFR-expressing B16F10 cell line (B16F10^hEGFR+). B16F10^hEGFR+ tumor-bearing mice were treated three times per week with vehicle control or the reagent(s) indicated. (A–F, H–L) Data are representative of the tumor volumes (mm³) from 4 to 15 animals per group. (A) Mice treated with vehicle, IL-2 (0.4 mg/kg), IL-10 (1.0 mg/kg), or both (n = 10). Statistical analyses were performed by Mann-Whitney test. (B) Mice treated with vehicle, IL-2 (scFv) (0.4 mg/kg), IL-10 (scFv) (1.0 mg/kg), or both (n = 10). Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. (C) Mice treated with vehicle, IL-2 (EGFR) (0.4 mg/kg), IL-10 (EGFR) (1.0 mg/kg), or both (n = 10). Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. (D) Mice treated with vehicle control or varying doses (0–6 mg/kg) of DK2¹⁰ (EGFR) (n = 7). Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. (E) Mice treated with vehicle, DK2¹⁰ (scFv) (2.0 mg/kg), or DK2¹⁰ (EGFR) (2.0 mg/kg) (n = 9–10). Statistical analyses were performed by Mann-Whitney test. (F) Mice treated with vehicle, DK2¹⁰ (EGFR) (2.0 mg/kg), or varying doses of IL-2 (0–4.0 mg/kg) (n = 4–7). Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparisons test. (G) Mice treated with vehicle, DK2¹⁰ (EGFR) (2.0 mg/kg), or IL-2 (2.0 mg/kg) (n = 4–7). Serum was collected, and cytokine concentrations are reported. Statistical analyses were performed by Student’s t test. Results are reported as the mean ± SD. (H) Wild-type or IFNγ-knockout mice were treated with vehicle or DK2¹⁰ (EGFR) (2.0 mg/kg) (n = 7–9). Statistical analyses were performed by Welch’s t test. (I–K) Mice treated with vehicle or DK2¹⁰ (EGFR) (2.0 mg/kg) with and without cell ablation of CD8⁺ (I), CD4⁺ (J), or NK cells (K) (n = 5–6). Statistical analyses were performed by Mann-Whitney test. (L) Mice treated with vehicle or DK2¹⁰ (EGFR) (2.0 mg/kg) with and without FTY720 (n = 11–15). Statistical analyses were performed by Mann-Whitney test. (M–P) Mice were treated with vehicle or DK2¹⁰ (EGFR) (2.0 mg/kg) for 6–8 days (n = 4). (M) The frequencies of CD4⁺ and CD8⁺ T cells among TILs. (N) Following treatment, CD8⁺ Tem (CD3⁺/CD8⁺/CD44⁺/CD62L⁻) TILs were sorted, and gene expression was performed by nCounter analysis. Genes with a fold change of 1.5 from control animals and p value ≤0.05 are reported. (O) The oxygen consumption rate of isolated CD8⁺ TILs (n = 9). (P) Isolated CD8⁺ TILs from treated mice were rested overnight before co-culturing with B16F10^hEGFR+ cells, and an Elispot was performed to detect IFNγ-producing tumor-reactive cells (n = 5–9). Statistical analyses were performed by Welch’s t test. (Q) Tumor volumes (mm³) from mice bearing LL2^hEGFR+ tumors treated with vehicle or DK2¹⁰ (EGFR) with or without anti-PD-1 (2.5 mg/dose) for 1 week (n = 9). Statistical analyses were performed by Welch’s t test. (R) A patient-derived xenograft (PDX) model (non-small cell lung cancer) was established by subcutaneous implant of patient tumor cells, and mice were treated with vehicle, anti-PD-1 (10 mg/kg; every 5 days intraperitoneally), or DK2¹⁰ (EGFR) (0.5 or 2.0 mg/kg, three times a week subcutaneously) (n = 2). Tumor volumes are reported (mm³). Statistical analyses were performed by Welch’s t test. (ns, not significant; p value; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). Results are reported as the mean ± SEM, unless otherwise noted.

Figure 7

Coupling and targeting of IL-2 and IL-10 exerts broad immune activation in non-human primates and shows a strong safety profile in a GLP study (A–L) Male, and female, cynomolgus monkeys were treated with DK2¹⁰ (EGFR) (0–2.5 mg/kg) dosed subcutaneously three times a week for 27 days. Each group had both males and females (n = 6–10). (A) DK2¹⁰ (EGFR) plasma concentration plotted up to 72 h after the first dose. (B–E) Plasma cytokines (pg/mL) from animals at the indicated study time points. Concentrations below the lower limit of quantification (LLOQ) are reported as 150 pg/mL. (F) Hematology analyses, represented as fold change, comparing week −1 (pre-dose) to day 28 (post-dose) cell concentrations. Reported are eosinophils, platelets, red blood cells (RBCs), and white blood cells (WBCs). (G) Clinical chemistry on day 28. Reported are alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), hemoglobin (HGB), red blood cell distribution width (RDW), and cholesterol. (H–L) Blood was collected at the indicated times (time from first dose and the most recent dose are indicated on the x axis) to determine immune cell subset frequencies. Subsets reported are total T cells among CD45⁺ lymphocytes (H), frequency of proliferating (Ki67⁺) Tregs (CD3⁺/CD4⁺/CD127^low/CD25⁺) (I), CD4⁺ T cells (J), CTLs (K), and the ratio of CTLs to Tregs (L). Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test on measurement at the final time point. Results are reported as the mean ± SD. LLOQ is indicated on several plots.