Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a ‘decoy-resistant’ IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.
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We thank all members of the Ring and Bosenberg labs for advice and technical assistance; E. Folta-Stogniew for assistance with SPR; and A. Iwasaki for mice and reagents. The cartoon in Fig. 1a was created using BioRender (https://biorender.com/). This work was supported by grants from the National Cancer Institute Immuno-Oncology Translation Network (U01CA233096; to A.M.R. and M.W.B.), Gabrielle’s Angel Foundation (to A.M.R.), and the Blavatnik Fund for Innovation at Yale (to A.M.R.). A.M.R. is additionally supported by an NIH Director’s Early Independence Award (DP5OD023088), the Pew-Stewart Scholars Program, and the Robert T. McCluskey Foundation. M.F.S. is supported by a Miguel Servet contract from Instituto de Salud Carlos III, Fondo de Investigacion Sanitaria (Spain). W.D. is supported by a Career Development Award from the Dermatology Foundation. O.-E.W. is supported by a NSF Graduate Research Fellowship (1752134) and by a NIH training grant (T32AI055403). The T100 Biacore instrumentation used was supported by NIH Award S10RR026992-0110.
A.M.R., T.Z., and S.F. are named inventors on a patent application that describes the DR-18 molecule. A.M.R. is the founder of Simcha Therapeutics, the commercial licensee of DR-18, and holds equity in the immuno-oncology companies Forty-Seven Inc., ALX Oncology, and Medicenna Therapeutics. W.D. and M.W.B. serve as consultants for Eli Lilly. W.D. has research funding from Pfizer for unrelated work.
Peer review information Nature thanks Jim Wells, Dietmar Zehn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Distribution of intratumoral CD8+ T cell transcripts by dysfunction and activation gene scores, adapted from Singer et al.43. Blue points mark a curated list of cytokine and cytokine receptors. Red points mark Il18, Il18r1, Il18rap. b–f, Wild-type C57BL/6 mice were subcutaneously engrafted with the indicated mouse syngeneic tumour. Tumour and spleen were removed and analysed by flow cytometry at day 7 after engraftment. b, c, Representative flow plots (b) and quantification of percentage of IL-18Rα expression (c) on splenic and intratumoral CD4+ T cells (NK1.1−, CD3+, TCRβ+, CD4+), CD8+ T cells (NK1.1−, CD3+, TCRβ+, CD8+), NK cells (CD3−, TCRβ−, NK1.1+, CD200R1−) from wild-type mice engrafted with MC38 cells. d, Percentage IL-18Rα expression on splenic and intratumoral CD4+ T cells, CD8+ T cells, and NK cells from wild-type mice engrafted with YUMMER1.7 cells. e, f, Representative flow plots (e) and quantification of surface CD44 and IL-18Rα expression (f) on CD8+ T cells in spleen and tumour from MC38 tumour-bearing mice. g, Normalized quantification of Il18bp by qPCR in blood and tumour lysate assessed at day 7 after s.c. engraftment of wild-type mice with MC38 tumours, treated with PBS, IL-18 or anti-IFN-γ + IL-18. h, Quantification of plasma IL-18BP protein level by ELISA at day 7 after s.c. engraftment of wild-type mice with MC38 tumours, treated with either PBS or IL-18. i, Representative immunohistochemistical staining for IL-18BP from l18bp−/− mouse spleen, wild-type spleen, MC38 tumour, or MC38 tumour treated with one dose of IL-18, and assessed on day 7. Scale bar, 25 μm. j, Quantification of IL-18BP+ cells per high power field (HPF) in representative sections from each group in i. b–f, i, Data are representative of two independent experiments with n = 5 mice per group. g, h, j, Data are pooled from two independent experiments with n = 5 mice per group. P values were calculated using two-way ANOVA (c, d, f) or two-tailed unpaired Student’s t-tests (g, h, j), and data are presented as mean ± s.e.m.
Extended Data Fig. 2 IL-18BP level is elevated in human cancers and correlated with T cell infiltration.
a, Expression of IL18BP transcripts in normal (blue) or cancer (red) tissues from the TCGA database. CHOL, cholangiocarcinoma; DLBC, diffuse large B cell lymphoma; GBM, glioblastoma multiforme; HSNC, head and neck squamous carcinoma; KIRC, kidney renal clear cell carcinoma; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma (*P < 0.01). b–d, Correlation of IL18BP expression with T cell markers CD3E (b), CD8A (c) and PDCD1 (d) from the TCGA database for SKCM (n = 558), BRCA (breast adenocarcinoma, n = 1,085), HNSC (n = 44), STAD (n = 221), and OV (ovarian cancer, n = 426). e, Frequency of IL-18BP immunohistochemistry staining levels in human tumour tissue microarrays. Each sample was scored as negative (0) or positive (1+, 2+, or 3+). Representative images are shown for each staining level. f, Quantification of plasma IL-18BP protein level by ELISA for healthy donors (n = 22) and patients with NSCLC (n = 52) at baseline before treatment and at the time of the following CT scan after receiving treatment with anti-PD-(L)1 (n = 52). g, Representative mean tumour growth in wild-type (left) and Il18bp−/− (right) mice engrafted s.c. with MC38 tumours and treated with PBS or IL-18. Data are representative of three independent experiments with n = 5 mice per group. P values were calculated using one-way ANOVA (a, f) or two-way ANOVA (g), and data are presented as mean ± s.e.m.
Related to Fig. 1. a, Structural alignment of hIL-18 (green)–hIL-18Rα/Rβ (cyan) complex (PDB ID 3WO4) with hIL-18–vIL-18BP (blue) complex (PDB ID 3F62). b, c, Representative surface plasmon resonance (SPR) sensorgrams of mouse WT IL-18 binding to IL-18Rα (b) or IL-18BP (c). IL-18Rα measurements were conducted using a conventional multiple cycle program, whereas IL-18BP measurements were conducted using a single-cycle program. d, Dose–response curves of IL-18BP protein antagonizing IL-18Rα in complex with indicated IL-18 and mutants (E42A, K89A and E42A/K89A). Experiments were performed in duplicate (n = 2). e, Randomized positions of murine IL-18 to create DR_18, with the corresponding degenerate codon and the potential amino acid at each position. f, Summary of the experimental design for directed evolution and yeast selection process to generate DR-18. Yeast libraries were selected for IL-18Rα binding and counter-selected against IL-18BP using magnetic-activated cell sorting (MACS; rounds 1 and 2) and subsequently FACS (rounds 3–5). Blue text (right) indicates positive selection reagent, and red text (left) shows the counter-selection reagent. g, Structural representation of DR-18 mutation positions in IL-18Rα and IL-1BP binding overlap region. Side chains from a minimized set of mutations up to six consensus residues (1N, 50M, 52K, 55E, 56V and 59L) are displayed as stick models. b–d, Data are representative of two independent experiments and are presented as mean ± s.e.m.
Related to Fig. 2. Wild-type mice were engrafted s.c. with 0.5 × 106 MC38 or YUMMER1.7 tumour cells. From day 7 after engraftment, when tumour size reached 50–100 mm3, mice were treated twice weekly with PBS, or 0.32 mg/kg IL-18 or DR-18. a, Representative spider plots depicting MC38 tumour growth in wild-type mice treated with PBS, IL-18, DR-18, anti-PD-1, IL-18 + anti-PD-1, or DR-18 + anti-PD-1. b, e, Representative mean tumour growth (a) and Kaplan–Meier survival curves (e) of wild-type mice engrafted s.c. with YUMMER1.7 cells and treated with PBS, IL-18, DR-18, anti-PD-1, IL-18 + anti-PD-1, or DR-18 + anti-PD-1. Arrows signify days of treatment. c, f, Mean tumour growth (c) and Kaplan–Meier survival curves (f) of wild-type Balb/c mice engrafted s.c. with 0.25 × 106 CT-26 colon carcinoma cells and treated with PBS, anti-PD-1, IL-18, or DR-18. d, g, Mean tumour growth (d) and and Kaplan–Meier survival curves (g) of wild-type mice engrafted s.c. with 0.25 × 106 B16-F10 melanoma cells and treated with PBS, IL-18, or DR-18 from day 7. h, Mean tumour growth of wild-type mice engrafted s.c. with MC38 cells and treated with PBS, IL-18, CS1 or CS2 (DR-18), with the indicated doses. a–d, h, Data are representative of three independent experiments with n = 5 mice per group. e–g, Data are summary of three (e) or two (f–h) independent experiments with n = 5 mice per group. Data are presented as mean ± s.e.m.
Related to Fig. 2. a, Kaplan–Meier survival curves of mice engrafted with YUMMER1.7 tumours and treated with PBS, DR-18, or DR-18 and anti-CD4, anti-CD8, anti-NK1.1, or anti-IFN-γ antibodies. Treatment parameters were the same as for MC38 tumours (Fig. 2a, see Methods). b, Kaplan–Meier survival curves of wild-type or Rag2−/− mice engrafted s.c. with MC38 tumours and treated with PBS or DR-18. c, Kaplan–Meier survival curves of mice re-challenged with 1.0 × 106 MC38 tumour cells after successful DR-18 treatment compared to naive mice engrafted with 1.0 × 106 MC38 cells for the first time. d–i, Thy1.1+ P14 CD8 T cells (2.0 × 106) were adoptively transferred into wild-type mice, which were then engrafted s.c. with 0.25 × 106 B16F10-gp33 tumour cells one day later. Mice were then treated twice weekly with PBS or DR-18 (0.32 mg/kg) once tumours reached 50–100 mm3 for a total of three treatments. d–f, Representative flow plots (d), quantification of frequency (e), and absolute number of antigen-specific tumour-infiltrating P14 CD8+ T cells (f), as defined by Thy1.1+ and Gp33-tetramer. g, Percentage of IFN-γ on intratumoral antigen-specific (Thy1.1+Tetramer+) P14 CD8+ cells after PBS (n = 4) or DR-18 (n = 4) treatment. h, i, Representative flow plots (h) and quantification (i) of CD44 and CD39 expression on endogenous (Thy1.1−Tetramer−) tumour-infiltrating CD8+ T cells. j, Quantification of CD44 and PD-1 expression on tumour-infiltrating CD8+ T cells from wild-type mice engrafted with MC38 cells and treated with PBS or DR-18 in combination with FTY720. k, Representative flow cytometry plots of IL-18Rα expression on CD4+ T cells (CD45+, NK1.1−, CD3+, TCRβ+, CD4+), CD8+ T cells (CD45+, NK1.1−, CD3+, TCRβ+, CD8+), NK cells (CD45+, CD3−, TCRβ−, NK1.1+), B cells (CD45+, CD3−, TCRβ−, NK1.1−, CD19+), monocytes (CD45+, CD64+, CD11b+, Ly6C+), macrophages (CD45+, CD64+, MHCII+, F4/80+), dendritic cells (CD45+, CD64−, CD11c+), and neutrophils (CD45+, CD64−, CD11b+, Ly6G+) from MC38 tumours (n = 5), matched spleens (n = 5), un-engrafted wild-type spleens (n = 3), or Il18r1−/− spleens (n = 3). l, Representative mean tumour growth of MC38 tumours implanted into Rag2−/− mice that were adoptively transferred with T cells isolated from wild-type (n = 5) or Il18r1−/− (n = 8) mice and treated with PBS or DR-18. Black arrows signify day of treatment. m, Representative mean tumour growth of MC38 tumours implanted into wild-type (n = 4) and XCR1DTR (n = 5) mice treated with PBS or DR-18 in combination with DT. Green arrows signify DT treatment regimen. d–j, Data are representative of two independent experiments and presented as mean ± s.e.m. P values were calculated using one-tailed (e, f) or two-tailed (g) unpaired Student’s t-tests or two-way ANOVA (i, j).
Related to Fig. 3. Wild-type mice were engrafted s.c. with 0.5 × 106 YUMMER1.7 cells. From day 7 after engraftment, mice were treated twice weekly with PBS, IL-18 or DR-18 (s.c.). On day 15, tumours were removed and sorted, and 5,000 cells per condition were used for scRNA-seq analysis. a, t-SNE projection showing all cells present in YUMMER1.7 and YUMMER1.7-B2m−/− tumours, coloured by cluster. Cells from the YUMMER1.7-B2m−/− experiments are shown in light grey and are discussed later. b, t-SNE projection showing distribution of cells coloured by treatment condition (dark grey, PBS; blue, IL-18; magenta, DR-18). c, Percentage of cells in each cluster by treatment. d, t-SNE projection of all cells analysed showing expression of genes supporting cell-type assignments. e, t-SNE projections of all cells analysed showing expression of IL-18 pathway-related genes. f, t-SNE projections showing expression of selected genes in computationally isolated Cd3e-positive T cells. g, t-SNE projections of Cd68+ cells, showing expression of Cx3cr1, Nos2, Arg1, and Mertk. h, t-SNE projection of Cd68+ cells, coloured by graph-based cluster. i, t-SNE projection of myeloid cell distribution as a function of treatment condition. j, Frequency of macrophages within each cluster identified in h. k, Heatmap showing differentially expressed genes in Cd68+ cells by clusters identified in h. l, m, Frequency (l) and absolute numbers (m) of Nos2+ macrophages from MC38 engrafted tumours following PBS, IL-18 or DR-18 treatment. Data are representative of three independent experiments with n = 5 mice per group and presented as mean ± s.e.m. P values calculated using one-way ANOVA.
Related to Fig. 3. Wild-type mice were engrafted s.c. with 0.5 × 106 MC38 or YUMMER1.7 cells. From day 7 after engraftment mice were treated twice weekly i.p. with PBS, IL-18, or DR-18. Tumours were harvested and analysed on day 15 after engraftment. a, Absolute number of tumour-infiltrating CD4+ T cells, CD8+ T cells and NK cells from YUMMER1.7 tumours in each treatment group. b, Quantification of intracellular Ki-67+ staining by percentage of indicated intratumoral lymphocyte population. c, Absolute number of antigen-experienced (CD44+) CD8+ T cells from MC38 tumours in each treatment group. d, Representative flow plots of PD-1 and CD39 staining on intratumoral CD44+ CD8+ T cells from MC38 tumours in indicated treatment groups. e, Absolute number of PD-1−CD39−, PD-1+CD39− and PD-1+CD39+ CD44+CD8+ T cells from d in each treatment group. f, Quantification by Luminex of IFN-γ protein level from tumour lysate of indicated treatment groups. g, Quantification of intracellular IFN-γ staining by percentage of indicated intratumoral lymphocyte populations from YUMMER1.7 tumours. h, i, Quantification of intracellular IFN-γ staining by percentage in tumour (h) or spleen (i) for each indicated intratumoral lymphocyte population from mice engrafted with MC38 cells. j, k, Quantification of intracellular granzyme B staining by MFI (j) and CD107 staining in intratumoral CD8+ T cells and NK cells (k) from MC38 tumours in the indicated treatment groups. l, m, Absolute number (l) and frequency (m) of polyfunctional NK cells measured by co-staining of intracellular IFN-γ and granzyme B following ex vivo stimulation with PMA and ionomycin. Data are representative of three independent experiments with five mice per group. P values calculated using two-tailed unpaired Student’s t-tests (a), one-way ANOVA (b, f, g, j, k) or two-way ANOVA (c, e, h, i, l) and data presented as mean ± s.e.m.
Related to Fig. 3. a, Frequency of surface PD-1 and TIM3 staining on MC38 intratumoral CD44+CD8+ T cells in indicated treatment groups. b, Absolute cell number of DN (PD-1−TIM3−), SP (PD-1+TIM3−) or DP (PD-1+TIM3+) CD8+ T cells from YUMMER1.7 tumours in each treatment group. c, Frequency of intracellular TCF1 in intratumoral CD44+CD8+ T cells in MC38 tumours in each treatment group. d, Absolute number of TCF1+CD44+CD8+ T cells from MC38 tumours in each treatment group. e, Frequency of intracellular TCF1 staining in MC38 intratumoral CD44+CD8+ T cells subsets based on PD-1 and TIM3 surface expression. f, Representative histograms of intracellular TCF1 staining in intratumoral SP PD1+TIM3−CD44+CD8+ T cells from YUMMER1.7 tumours. g, Absolute number of TCF1+ SP PD1+TIM3− CD44+CD8+ T cells from YUMMER1.7 tumours in each treatment group. h, i, Frequency of intracellular IFN-γ in DP (PD-1+TIM3+) CD44+CD8+ T cells from MC38 (h) and YUMMER1.7 (i) tumours in each treatment group. j, Absolute number of TCF1+ cells in SP (PD-1+TIM3−) CD44+CD8+ T cells in draining lymph nodes from MC38 tumours in each treatment group. Data are representative of three independent experiments with five mice per group. P values were calculated using one-way (c, d, g–j) or two-way ANOVA (a, b, e) and data are presented as mean ± s.e.m.
Related to Fig. 4. a, Representative spider plots showing tumour growth in wild-type mice engrafted s.c. with MC38-B2m−/− tumours and treated with PBS, or anti-PD-1 + anti-CTLA-4, IL-18 or DR-18. b, c, Kaplan–Meier survival curves (b) and representative spider plots (c) of wild-type mice engrafted s.c. with YUMMER1.7-B2m−/− tumours and treated twice weekly with PBS, anti-PD-1 + anti-CTLA-4, IL-18, DR-18, or DR-18 + anti-CD8 and anti-NK1.1 antibodies. Depleting antibodies were given on the same schedule as for MC38-B2m−/− (see Methods). d, e, Kaplan–Meier survival curves (d) and spider plots showing tumour growth (e) of wild-type mice engrafted s.c. with 1 × 106 RMA-S tumour cells and treated with PBS, anti-PD-1, IL-18, DR-18 or DR-18 + anti-NK1.1. Spider plot data are representative of two independent experiments and survival curve data are pooled from two independent experiments. f, Absolute number of intratumoral NK cells (NK1.1+CD3−EOMES+DX5+CD200R−), NKT cells (NK1.1+CD3+) and ILC1 cells (NK1.1+CD3−EOMES−DX5−CD200R+) in wild-type mice engrafted s.c. with MC38-B2m−/− tumours in indicated treatment groups. g, t-SNE projection of different clusters in YUMMER1.7-B2m−/− tumours as a function of treatment. YUMMER1.7 tumours are shown in light grey (discussed in Fig. 3, see Methods). h, Representative flow cytometry plots of IFNγ and Ki67 staining on intratumoral NK cells from YUMMER1.7-B2m−/− tumours treated with PBS or DR-18. i, j, Frequency of Ki67+IFNγ+ NK cells from YUMMER1.7-B2m−/− (i) and MC38-B2m−/− (j) tumours treated with PBS or DR-18. k, Representative mean tumour growth in wild-type mice engrafted s.c. with MC38-B2m−/− tumours and treated with PBS, DR-18, or DR-18 + anti-FasL or anti-IFN-γ. l, Representative mean tumour growth of Prf1−/− mice engrafted s.c. with MC38-B2m−/− cells and treated with PBS (n = 3) and DR-18 (n = 5) as indicated. h–l, Data are representative of two independent experiments with five mice per group. P values calculated using two-tailed unpaired Student’s t-test and data presented as mean ± s.e.m.
a, SPR sensorgrams of human DR-18 (hDR-18) binding to human or cynomolgus macaque (M. fascicularis) IL-18Rα and IL-18BP. b, Concentration–response curves of hIL-18 or hDR-18 on HEK Blue IL-18R reporter cells. c, Titration of hIL-18BP on a fixed concentration of hIL-18 (1 ng/ml) or hDR-18 (0.1 ng/ml) on HEK Blue IL-18R reporter cells. d, e, Concentration–response curves of IFN-γ stimulation by hIL-18 or hDR-18 on human PBMCs (d) or cynomolgus macaque PBMCs (e). IFN-γ concentration measured by ELISA. Data are representative of at least two independent experiments with n = 2 for b–e and presented as mean ± s.e.m.
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Zhou, T., Damsky, W., Weizman, OE. et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature 583, 609–614 (2020). https://doi.org/10.1038/s41586-020-2422-6
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