IL-38 blockade induces anti-tumor immunity by abrogating tumor-mediated suppression of early immune activation

Figures & data

Figure 1. IL-38 is targeted by B cells in cancer patients and is highly expressed in multiple tumor types. (a) Identification of IL-38 as the specific target of IMM20130 assessed by protein microarray. Binding signal and S-Scores shown for the top 200 proteins based on binding signal. (b) Assessment of IL-38 transcript expression in tumor and normal tissue using TCGA RNAseq data. Samples were normalized for IL-38 expression and subdivided based on high (brown) and low (green) expression using thresholds set according to a value of 10–15% of normal tissues. Numbers in each plot represent frequency of IL-38 positive samples in all tumor samples using this threshold. (c) Immunohistochemical analysis of IL-38 expression in three cancer types. Head and neck squamous carcinoma (HNSC), lung tumors comprising multiple subtypes including adenocarcinoma and squamous cell carcinoma, and cervical squamous carcinoma (CESC) were stained with IMM20324 or isotype control antibodies to detect IL-38 expression. Pathological scoring was performed according to staining density. Representative examples of specimens with a range of expression intensity within each indication are shown. (d) Average intensity of IL-38 expression across cancer indications tested. Intensity scoring analysis was performed using a 7-point scale, with 0 corresponding to no expression and 6 being strong staining. Scoring was performed in both cytoplasmic and nuclear compartments and averaged to calculate final scores.

(a) XY scatter plot depicting binding intensity versus s-score, with the IL-38 point in the top right quadrant and the remaining proteins clustered in the bottom left. (b) Four column scatter plots comparing IL-38 expression in tumor and normal from four tissues, showing positive clusters present in the tumor samples, but not the normal tissues. (c) Matrix of tissue sections depicting brown patches of expression for IL-38 stained sections, but no brown areas in isotype control stained sections. (d) Three violin plots comparing IHC staining in normal versus staged tumor sections for three cancers, showing no differences, except higher expression in stage IA CESC compared to normal cervical tissue.

Figure 2. IL-38 expression is associated with reduced immune infiltration and HPV status in head and neck squamous carcinoma. (a) Signature analysis of immune lineages in IL-38 high versus IL-38 low head and neck tumors. Negative values represent reduced expression of lineage-associated genes and lower infiltration. Positive values represent increased infiltration of indicated lineages. HNSC: head and neck squamous cell carcinoma; LUSC: lung squamous cell carcinoma; ESCA: esophageal carcinoma: LUAD: Lung adenocarcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma: SKCM: skin cutaneous melanoma. (b) IL-38 expression in HPV-negative and HPV-positive populations of head and neck cancers.

(a) A matrix of five zero-centered horizontal bar charts representing immune lineages, with all showing values below zero for six different tumor types tested. (b) Bar chart with 2 bars showing significantly decreased expression in HPV-positive versus HPV-negative tumors.

Table 1. IL-38 expression in selected cancer types using Tempus data.

Cancer type	Mean expression [Log2(TPM+1)]	Stdev	Sample size	% Positive
Gastroesophageal squamous carcinoma	0.774	0.885	88	86.4
Head and neck squamous carcinoma	0.646	0.940	467	63.8
Lung squamous carcinoma	0.260	0.497	753	47.7

Table 2. IL-38 expression is associated with immune cell infiltration in selected cancers.

Cancer type, cell population	Correlation	p Value
Gastroesophageal squamous carcinoma (n = 62)
CD4^+ T cells	−0.178	0.167
CD8^+ T cells	−0.176	0.172
Macrophage	−0.159	0.216
NK	−0.015	0.907
Head and neck squamous carcinoma (n = 396)
CD4^+ T cells	−0.189	0.078
CD8^+ T cells	−0.243	<0.001
Macrophage	−0.217	<0.001
NK	−0.147	0.003
Lung squamous carcinoma (n = 594)
CD4^+ T cells	−0.035	0.397
CD8^+ T cells	−0.149	<0.001
Macrophage	−0.186	<0.001
NK	0.002	0.961

Figure 3. IMM20324 is specific for IL-38, binds human and mouse protein with different affinities and neutralizes binding to putative human IL-38 receptors. (a) Identification of IL-38 as the specific target of IMM20324H assessed by protein microarray. Binding signal and S-scores shown for the top 200 proteins based on binding signal. (b) (Top) Binding of recombinantly produced IMM20324 mIgG2a antibody to recombinant human and mouse IL-38 using BLI. Two-fold titrations (1.52–0.095 µM) were performed to generate K_D values for binding to species-specific proteins. (Bottom) Values shown are average of two experiments. (c) ELISA-based binding analysis of IMM20324 to recombinant full-length human and mouse IL-38 using direct ELISA. (d) Flow cytometry-based binding to human and mouse IL-38 overexpressing CHO cells. Cells were fixed and permeabilized to assess binding of intracellular IL-38. Mock-transfected controls, in addition to isotype staining controls for human (gray) and mouse (black) cells also shown on graph. (e) Inhibition of IL-38 receptor binding to its receptors. Inhibition of binding to IL-36R is shown in the circles, IL1RAPL1 is shown in squares. Isotype control-treated samples are in blue, while samples treated with IMM20324 are shown in red.

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(a) XY scatter plot depicting binding intensity versus s-score, with the IL-38 point in the top right quadrant and the remaining proteins clustered in the bottom left. (b) A table and two kinetic plots both showing five lines of increasing height that all rise rapidly and then plateau, with the y-axis for mouse binding being slightly lower than that of the human plot. (c) Two binding curves which overlay each other. (d) A set of binding curves, most depicting no or low binding and two showing dose-dependent IMM20324 binding to mouse and human IL-38, with mouse binding comparably lower than human. (e) Four binding curves, two blue lines showing constant binding at increasing concentrations, and two red lines showing dose-dependent inhibition of binding that is complete at the highest concentration.

Figure 4. IMM20324H exhibits favorable PK profile and is well tolerated in non-tumor-bearing mice. (a) In vivo pharmacokinetic analysis of IMM20324H following administration of antibody by intravenous (IV) and intraperitoneal (IP) routes performed in C57BL/6 mice. Dose shown is 10 mg/kg. (b) Analysis of serum IMM20324H concentrations on D21 following administration of antibody by IP injection. Groups were dosed once weekly (QW) or twice weekly (Q2W) with the labeled dose in mg/kg. (c) Average body weight of animals from each treatment group was monitored throughout the duration of the study (d) Average spleen weights of animals comprising each treatment group were assessed upon study termination on D21. (e) Assessment of circulating cytokine levels in serum following administration of IMM20324H. Blood was sampled following study termination. Data presented as log2 transformation of the cytokine concentration+1. Dotted line indicates the lower limit of quantification (LLOQ) at 10 pg/mL.

(a) Kinetics plot showing two lines with an incomplete but gradual decrease over time. (b) Bar chart with individual points showing a dose-dependent increase of IMM20324H concentration with increasing frequency and dose. (c) Kinetics plot showing average animal weight over 20 d where seven lines show a similar pattern of a small gradual increase over time. (d) Bar chart showing no differences in spleen weight when comparing seven groups. (e) Scatter plot showing low concentrations of a series of 31 cytokines, where only 1 cytokine (IL-12p70) shows a noticeable increase in two of four replicates.

Figure 5. Targeting of IL-38 inhibits tumor growth and elicits immunological memory against EMT6 breast tumors in vivo. (a–e) Mice were randomized at D1 based on tumor volume and treated with three different doses of IMM20324H or a PBS vehicle control twice per week for 6 total doses. (a) Mean tumor volume up to D17 (b) Tumor volume on D17, the final day all mice were on study. (c) Individual growth curves for each mouse in the indicated group up to D45. (d–e) EMT6 cells were implanted into the opposite flank of IMM20324H-treated mice following full regression of the primary tumor. No additional antibody was administrated during secondary challenge. (d) Secondary EMT6 tumor growth curves compared to untreated age-matched mice. (e) Survival curves of rechallenged mice showing % survival per group. *p <0.05.

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(a) A line graph showing mean tumor volume growth over 18 d, where tumor growth in IMM20324H-treated mice is delayed compared to the isotype control treated group. (b) Scatter plot showing tumor volumes from four groups of 10 individual animals where the left-most group has low variability around the mean, while the three other groups show a subset of animals with markedly decreased tumor sizes. (c) Four tumor growth kinetic plots for groups in panels (a) and (b) showing rapid size increase in the vehicle-treated group and delayed growth or disappearance of tumors in a subset of animals in IMM20324H-treated groups. (d) Kinetic growth plot showing a black line representing untreated animals which starts low and increases over time and a blue line representing IMM20324H-treated mice that starts low and is reduced to zero over time. (e) A survival kinetics plot depicting a black line that progressively drops to approximately 20% over time and a blue line that remains at 100% for the entire study.

Table 3. Linear mixed effects modeling of IMM20324H treatment in the EMT6 syngeneic model.

		Volume
Predictors	Estimates	CI	p	df
(Intercept)	−91.65	−382.94 to 199.65	0.532	66.73
IMM20324H (10 mg/kg)	−31.68	−44.23 –to −19.13	<0.001	490.57
IMM20324H (15 mg/kg)	−55.79	−68.44 to −43.14	<0.001	491.87
IMM20324H (25 mg/kg)	−56.56	−68.43 to −44.69	<0.001	489.62

Figure 6. IMM20324 inhibits growth of B16.F10 tumors in vivo. (a–e) Mice were randomized at D0 based on tumor volume and treated with 10 or 50 mg/kg dose of IMM20324 or isotype control intraperitoneally every 3days for six total doses. Tumor volumes were measured every 3 days. Tumor growth curves were fitted with exponential model. (a) Mean tumor volume up to D 10. (b) Interim tumor volumes of individual animal on D10. (c) Growth curves of individual animal from the indicated group over the duration of the study. (d) Calculated average tumor growth rate over the duration of study. (e) Calculated days when tumor volume reaches 1500 mm³ based on fitting of tumor growth curve. * p < 0.05, ** p < 0.01, ns = not significant.

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(a) A line graph showing mean tumor volume growth over 10 d, where tumor growth in IMM20324H-treated mice at two doses is delayed compared to the isotype control-treated group. (b) Scatter plot showing tumor volumes from three groups of 15 individual animals where the left-most group shows a range of values whereas the right-most groups show lower values with a more uniform size. (c) Three tumor growth kinetic plots for isotype and two treatment groups where each animal is depicted by a line and some animals in the treatment groups show reduced growth over time. (d) Scatter plot showing tumor volumes from three groups of 15 individual animals where the left-most group shows more variability and a higher mean compared to more uniform groups on the left with significantly reduced mean tumor volumes. (e) Scatter plot showing tumor volumes from three groups of 15 individual animals where the left-most group shows a lower mean and the mean of the right-most groups are elevated in comparison.

Table 4. Linear mixed effects modeling of IMM20324 treatment in the B16.F10 syngeneic model.

		Volume
Predictors	Estimates	CI	p	df
(Intercept)	−418.67	−774.01 to −63.33	0.021	186.93
IMM20324 (10 mg/kg)	−48.24	−93.35 to −3.14	0.036	248.38
IMM20324 (50 mg/kg)	−12.90	−58.27 to 32.46	0.576	247.38

Figure 7. IMM20324 treatment decreases tumor volume and increases intra-tumoral chemokines in a dose-dependent manner. (a) B16.F10 tumor-bearing mice given a single dose of IMM20324 at 3, 10, or 30 mg/kg and mice were bled at indicated timepoints. IMM20324 exposure was measured by ELISA and plotted over time (n = 3 mice per timepoint). (b) Mice were randomized at D0 based on tumor volume and treated indicated dose of IMM20324 or isotype control intraperitonially every 3 days for four total doses. Mice were taken down on D11, 24 hours after the final dose. Terminal exposure of IMM20324 was plotted against tumor volume on D11. (c) Intra-tumoral chemokine concentrations detected by Luminex analysis. Mice in all IMM20324 treatment groups are shown. r and p values were calculated using Pearson correlation co-efficients.

(a) Kinetics plot showing three lines, all with a sharp initial increase followed by a gradual decrease over time, but with all three showing different overall concentrations related to the initial dose. (b) Single scatter plot depicting concentration versus tumor volume where mice from three treatment groups are color-coded and low tumor volume correlates with concentration and the highest dose group. (c) Five scatter plots of pooled data from IMM20324 treament groups showing that increased concentration of IMM20324 and five chemokines are correlated.