PEGylated IL-10 (Pegilodecakin) Induces Systemic Immune Activation, CD8+ T Cell Invigoration and Polyclonal T Cell Expansion in Cancer Patients

Abstract

Tumor-reactive T cell exhaustion prevents the success of immune therapies. Pegilodecakin activates intratumoral CD8⁺ T cells in mice and induces objective tumor responses in patients. Here we report that pegilodecakin induces hallmarks of CD8⁺ T cell immunity in cancer patients, including elevation of interferon-γ and GranzymeB, expansion and activation of intratumoral CD8⁺ T cells, and proliferation and expansion of LAG-3⁺ PD-1⁺ CD8⁺ T cells. On pegilodecakin, newly expanded T cell clones, undetectable at baseline, become 1%-10% of the total T cell repertoire in the blood. Elevation of interleukin-18, expansion of LAG-3⁺ PD-1⁺ T cells and novel T cell clones each correlated with objective tumor responses. Combined pegilodecakin with anti-PD-1 increased the expansion of LAG-3⁺ PD-1⁺ CD8⁺ T cells.

Keywords: AM0010; CD8(+) T cell; IL-10; PEGylated Interleukin 10; T cell invigoration; Th1; clinical trial; clonal expansion; clonality; pegilodecakin.

Figure 1.. Immune Activation in the Serum on Pegilodecakin Monotherapy Correlates with Objective Response

(A) Comparison of the average concentration of 83 cytokines and proteins in the serum of cancer patients prior to and on pegilodecakin (20 μg/kg for 28 days; n = 30). Green, increased more than 3-fold; black, not changed; red, reduced more than 3-fold; blue, Th17. (B) Waterfall plot for the best tumor response in RCC patients on pegilodecakin monotherapy (n = 15) (PD, progressive disease; SD, stable disease; PR, partial response). (C–E) Th1 cytokines (IFN-γ, IL-18, and TNF-α) (C), FasL and lymphotoxin b (D), and TGF-β, and Th17 cytokines (IL-23, IL-12p40, and IL-17) (E) in the serum of RCC patients (predose day 1 and day 29, n = 16). (F) Thirteen-day time course of pegilodecakin in normal healthy volunteers (NHV) (n = 12), after single dose (day 1) and multiple dose (day 5–10) of pegilodecakin at indicated doses; serum concentration of pegilodecakin and serum cytokines (IFN-γ [pg/mL], IL-18, and FasL [fold induction over baseline]). (G) Three-month time course of daily pegilodecakin (20 μg/kg) in RCC patients (n = 6) (serum cytokines [IL-18, IFN-γ, and FasL]). (H) IL-18 induction in RCC (n = 16), non-squamous cell lung cancer (NSCLC) (n = 2), CRC (n = 6), and pancreatic cancer (PDAC) (n = 7) patients. (I) Correlation between best tumor response and IL-18 induction (fold over baseline; Pearson correlation, two-tailed p). (J) IL-18 (fold induction over baseline) after 28 days of treatment in RCC patients with progressive disease (n = 5); stable disease (n = 6) or partial response (n = 4). Data are represented as means ± SEM, p values represent results of t test, unless indicated. See also Figure S1 and Table S1.

Figure 2.. Sustained Expansion of LAG-3⁺ CD8⁺ T Cells in the Blood of RCC Patients Treated with Pegilodecakin

(A) Relative change in tumor burden (irRC) of an RCC patient over time on treatment with pegilodecakin (maximal tumor reduction −79%). (B) LAG-3 on CD8⁺ T cells in blood of the RCC patient in (A) by fluorescence-activated cell sorting (FACS). (C) LAG-3⁺ CD8⁺ T cells in all patients for pretreatment, after 1 or 2 months of pegilodecakin (n = 22). (D) LAG-3⁺ PD-1⁺ CD8⁺ T cells in the RCC patient in (A). (E) LAG-3⁺ PD-1⁺ CD8⁺ T cells in all patients (n = 22). (F) LAG-3⁺ PD-1⁺ Ki-67⁺ CD8⁺ T cells in patient (A). (G) LAG-3⁺ PD-1⁺ Ki-67⁺ CD8⁺ T cells in all patients (n = 22). (H) Ki-67⁺ TIM-3⁺ PD-1⁺ CD8⁺ T cells in the patient in (A). (I) Ki-67⁺ TIM-3⁺ PD-1⁺ CD8⁺ T cells in all patients (n = 22). (J and K) IL-10Ra and PD-1 expression on CD8⁺ T cells from pretreatment PBMCs; FACS analysis of one patient (J) and all patients (K) (n = 22, PD-1 is stratified to negative, low mean fluorescence intensity [MFI] < 1,000 and high MFI > 1,000). Data are represented as means ± SEM, p values represent results of t test (unless indicated). See also Figures S2 and S3.

Figure 3.. Correlation of CD8⁺ T Cell Phenotype in the Blood on Pegilodecakin with Tumor Response

(A and B) Correlation of LAG-3⁺ CD8⁺ T cells on pegilodecakin (day 57) (A) or before treatment (B) with best tumor response (following irRC criteria, PD ≥ 25% increase; PR ≥ 50% decrease). (C) Correlation of LAG-3⁺ PD-1⁺ Ki67⁺ CD8⁺ T cells (day 57) per patient with tumor response (C). (D and E) Percentage of LAG-3⁺ PD-1⁺ CD8⁺ T cells (day 57) (D) and LAG-3⁺ PD-1⁺ Ki-67⁺ CD8⁺ T cells (day 57) (E) stratified by tumor response. (F) Correlation of TIM-3⁺ PD-1⁺ CD8⁺ T cells (day 57) with best tumor response. (G) Percentage of TIM-3⁺ PD-1⁺ CD8⁺ T cells (day 57) stratified by tumor response. (H) Correlation of the best tumor response with the ratio of LAG-3⁺ PD-1⁺ Ki-67⁺ CD8⁺/TIM-3⁺ PD-1⁺ CD8⁺ T cells (day 57). Data are represented as best tumor response in each individual patient (A–C, F, and H) or means ± SEM (D, E, and G) (n = 22 patients). Spearman correlation was performed for (C, F, and H). A t test was used in (D, E, and G). See Figure S4.

Figure 4.. Increased Activation of Intratumoral CD8⁺ T cells in Patients Treated with Pegilodecakin

(A and B) Quantitation of intratumoral CD8⁺ T cells (A) and GranzymeB⁺ (GzmB) CD8⁺ T cells (B) based on analysis per mm² of tumor section at baseline and on treatment with pegilodecakin. (C and D) Immunofluorescence for CD8 (red) and GranzymeB (GzmB) (green, arrows point to GzmB-positive cells) in tumor sections prior to (C) and on treatment (D) with pegilodecakin. (E–G) Quantitation of phospho-STAT3⁺ CD8⁺ T cells in relation to all CD8⁺ T cells (E) and representative tumor section prior to (F) (arrow pointing to CD8⁺ T cell neighboring P-STAT3⁺ non-T cells) and on pegilodecakin (G) (arrows to P-STAT3⁺ CD8⁺ T cells). (H–J) Quantitation of LAG-3⁺ CD8⁺ T cells in relation to all CD8⁺ T cells (H) and representative tumor section prior to (I) (arrows to Lag3⁻ CD8⁺ T cell) and on pegilodecakin (J) (arrow to Lag3⁺ CD8⁺ T cell). (K) Quantitation of T-bet⁺ CD8⁺ T cells in relation to all intratumoral CD8⁺ T cells. (L–N) Quantitation of HLA-A⁺ tumor cells (L) and representative tumor section (lung cancer) prior to (M) and on pegilodecakin (N). At least 10 representative fields were quantified per section. Data are represented as means ± SEM (n = 4). Scale bar represents 50 μm (C, D, F, G, I, J, M, and N) in lower-magnification and 10 mm (C, D, F, G, I, and J) in high-magnification inserts. p Values represent the results of t tests.

Figure 5.. Pegilodecakin Sustains Activation and Proliferation of Human TCR-Activated CD8⁺ T Cell In Vitro

(A) Viability of PBMC-derived CD8⁺ T cells after activation with anti-CD3/CD28 antibodies (for 3 days) followed by 6 days of pegilodecakin or control treatment. (B) IL-10 receptor expression (MFI) in PBMC-derived CD8⁺ T cells (FACS analysis) after anti-CD3 or anti-CD3/CD28 stimulation. (C) STAT-3 phosphorylation in human CD8⁺ T cells after anti-CD3 or anti-CD3/anti-CD28 treatment and after pegilodecakin stimulation. (D) Percentage of PD-1- and LAG-3-expressing human CD8⁺ T cells after anti-CD3/CD28 stimulation. (E) Percentage of PD-1- and LAG-3-expressing CD8⁺ T cells stimulated for 3 days with anti-CD3/CD28 and then rested for 3 days or treated for 3 days with pegilodecakin in the presence or absence of anti-CD3-antibodies as indicated. (F) Percentage of PD-1- and Ki-67-expressing CD8⁺ T cells after anti-CD3/CD28 stimulation for 3 days followed by no treatment (control) or pegilodecakin for 3 days. (G) Percentage of PD-1- and GranzymeB-expressing CD8⁺ T cells in the presence or absence of pegilodecakin for 3 days, after anti-CD3/CD28 stimulation for 3 days. (H) GranzymeB in the supernatant of cells cultured as in (G) with increasing concentration of pegilodecakin. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, if not indicated in the panel. All experiments were performed at least three times. Data are represented as means ± SEM of one representative experiment (n = 5 for each experiment). Results were subjected to pairwise t tests. See also Figure S5.

Figure 6.. Polyclonal T Cell Expansion of Previously Undetectable T Cells in the Peripheral Blood of Patients

(A–D) Comparison of T cell clonal frequency between pretreatment (x axis) and on treatment (y axis) in the blood of a patient with melanoma (A), colorectal cancer (B), pancreatic cancer (C), and triple-negative breast cancer (D). Significantly expanding clones are colored in yellow or red, contracting clones are colored in blue and purple. Clones not detectable at baseline are shown as x = 0. Clones not detectable on treatment are y = 0. (E) Tumor burden at and after 21 weeks of pegilodecakin monotherapy in an RCC patient with a delayed tumor response. (F–H) The change in T cell clonality in the patient from (E) at week 5 (F) and at week 21 (G) of treatment. The quantification of expanded and contracted clones at weeks 5 and 21 (H). (I–K) Comparison of T cell clonal frequency between pretreatment and on treatment (at the indicated day of treatment) in patients with RCC: patient with progressive disease (+35% change [increase] in tumor burden) (I), patient with stable disease (+22%) (J), patient with partial response (−55%) (K). (L) Comparison of the absolute number of >10× expanding and >10× contracting T cell clones per patient (n = 12). (M) Total number of expanding T cell clones in the blood of patients based on the objective tumor response of the patient (n = 3 [PD]; n = 6 [SD]; n = 3 [PR]). (N) Time course of >10× expanding T cell clones on pegilodecakin as a percentage of the total T cell repertoire in the blood per patient (n = 12). (O) Correlation of the percentage of expanding T cell clones per patient (n = 12) with objective tumor response; “expanding clones” are defined as expanding at least 10-fold during the treatment period, the sum of the frequency of all expanding clones per time point per patient is plotted. Data in (L–N) are represented as means ± SEM, p values represent results of pairwise t test. Data in (O) represent Spearman correlation (r) and p.

Figure 7.. T Cell Responses in the Blood of Patients on Pegilodecakin and Pembrolizumab in Pretreated Cancer Patients

(A) Best response in pretreated cancer patients on pegilodecakin + pembrolizumab (indication as indicated; n = 19 evaluable patients). (B and C) Percentage of peripheral LAG-3⁺ PD-1⁺ CD8⁺ T cells (B) and LAG-3⁺ PD-1⁺ Ki-67⁺ CD8⁺ T cells (C) from patients on pegilodecakin and pembrolizumab (n = 23) after 21 and 57 days of treatment. (D) Comparison of T cell clonal frequency between pretreatment (x axis) and on treatment (y axis) in the blood of one RCC patient with a complete response (CR) on combination. (E) Comparison of >10× expanding clones in patients on pegilodecakin alone (n = 12) or on pegilodecakin and pembrolizumab (n = 13). (F) >10× expanding T cell clones in patients on pegilodecakin + pembrolizumab (n = 13) as a percentage of all T cell clones in the peripheral blood. (G) Comparison of the absolute number of expanded T cell clones with best response (n = 1 [PD]; n = 7 [SD]; n = 6 [PR]). All data are represented as means ± SEM, p values represent results of pairwise t test.