Interleukin-18 variants and their uses

Abstract

The present invention provides compositions and methods comprising activators of IL-18 activity for therapeutic and non-therapeutic uses, which activators provide IL-18 signaling activity even in the presence of inhibitory molecules such as IL-18BP.

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 554,605, filed September 6, 2017, and U.S. Provisional Patent Application No. 62 / 652,279, filed April 3, 2018, the entire contents of each of which are incorporated herein by reference.

[0002] Interleukin-18 (IL-18) is a pro-inflammatory cytokine that stimulates T cells, NK cells, and myeloid cells. IL-18 has previously been proposed as an immunotherapeutic agent for treating cancer because of its ability to stimulate antitumor immune cells. However, the clinical efficacy of IL-18 has been limited. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, there is a need for compositions and methods that provide effective IL-18 signaling activity for the treatment and prevention of cancer and other diseases and disorders. The present invention addresses this unmet need. [Means for solving the problem]

[0004] In one aspect, the disclosure relates to compositions comprising IL-18 variant polypeptides. In some embodiments, the IL-18 variant polypeptides specifically bind to the IL-18 receptor (IL-18R) and exhibit substantially reduced binding to the IL-18 binding protein (IL-18BP).

[0005] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation relative to wild-type (WT) IL-18. In some embodiments, the WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

[0006] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X relative to SEQ ID NO: 30.

[0007] In some embodiments, the IL-18 variant polypeptide is selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60R, M60L, E77D, Q103 and at least one mutation selected from the group consisting of E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H.

[0008] In some embodiments, the IL-18 variant polypeptide comprises the following mutations relative to SEQ ID NO:30: M51X, M60X, S105X, D110X, and N111X.

[0009] In some embodiments, the IL-18 variant polypeptide comprises the following mutations relative to SEQ ID NO:30: M51X, K53X, Q56X, S105X, and N111X.

[0010] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 34-59, 73-91, 191-193, or a fragment thereof.

[0011] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X, and L151X relative to SEQ ID NO: 31.

[0012] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R104Q, R104S, N109D, and L151V relative to SEQ ID NO: 31.

[0013] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 60-72, or a fragment thereof.

[0014] In one embodiment, the present disclosure relates to a composition comprising a nucleic acid encoding an IL-18 variant polypeptide.

[0015] In some embodiments, the composition further comprises: (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-Ll, PDl, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, and CD40; (iii) a cancer cell opsonizing agent; or (iv) an agent that inhibits one or more of the following: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α v β3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAP1, and NRP1;

[0016] In one embodiment, the disclosure relates to a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising an IL-18 variant polypeptide or a nucleic acid encoding an IL-18 variant polypeptide.

[0017] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a cancer resistant to immune checkpoint inhibitors (ICIs). In some embodiments, the cancer is associated with tumors that have lost expression of MHC class I.

[0018] In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is an infectious disease.

[0019] In some embodiments, the method comprises administering to a subject an IL-18 variant polypeptide and at least one other agent. In some embodiments, the at least one other agent comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an agent that inhibits any one of PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, or CD40, or any combination thereof. In some embodiments, the at least one other agent comprises a cancer cell opsonizing agent. In some embodiments, the at least one other agent is CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSFIR, SLAMF7, integrin α vβ3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAP1, and NRP1. In some embodiments, the at least one other agent is conjugated to an IL-18 variant polypeptide. In some embodiments, the at least one other agent is an altered T cell or NK cell. In some embodiments, the at least one other agent is an oncolytic virus.

[0020] In one aspect, the present disclosure relates to a composition comprising an IL-18 binding protein (IL-18BP) inhibitor or IL-18BP antagonist, wherein the inhibitor or antagonist suppresses the ability of IL-18BP to neutralize endogenous IL-18. In some embodiments, the inhibitor or antagonist comprises at least one selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule compound, and an antisense nucleic acid molecule.

[0021] In some embodiments, the composition comprises an IL-18 variant polypeptide, wherein the IL-18 variant polypeptide specifically binds to IL-18BP and the IL-18 variant polypeptide exhibits substantially reduced binding to the IL-18 receptor (IL-18R).

[0022] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation relative to wild-type (WT) IL-18. In some embodiments, the WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

[0023] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X, and R131X relative to SEQ ID NO: 30.

[0024] In some embodiments, the IL-18 variant polypeptide is selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G, E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y, with respect to SEQ ID NO: 30. and containing at least one mutation selected from the group consisting of D40S, D40A, N41K, N41S, N41R, M51F, M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F, and R131S.

[0025] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 92-125, or a fragment thereof.

[0026] In some embodiments, the IL-18 variant polypeptide comprises the following mutations relative to SEQ ID NO:30: D17X, E30X, and Q103X.

[0027] In some embodiments, the IL-18 variant polypeptide comprises the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and Q103X.

[0028] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X, M111X, D129X, and D130X relative to SEQ ID NO: 31.

[0029] In some embodiments, the IL-18 variant polypeptide is selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G, D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, I35K, I35R, D36V, D36A, D36G, D36H, D36P, D36R, D36 and at least one mutation selected from the group consisting of L, M50F, M50L, Q102L, Q102I, R104E, R104A, R104P, R104G, R104Q, R104H, H108D, H108A, N109R, N109S, N109T, N109I, M111L, M111I, D129A, D129F, D129V, D129Y, D129S, D130E, D130T, D130G, D130N, D130R, D130S, D130Q, and D130H.

[0030] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 126-190, or a fragment thereof.

[0031] In one embodiment, the present disclosure relates to a composition comprising a nucleic acid encoding an IL-18 variant polypeptide.

[0032] In one embodiment, the composition further comprises: (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-Ll, PDl, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, and CD40; (iii) a cancer cell opsonizing agent; or (iv) an agent that inhibits one or more proteins selected from CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α v β3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAP1, and NRP1;

[0033] In one aspect, the present disclosure relates to a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising an IL-18 binding protein (IL-18BP) inhibitor or an IL-18BP antagonist.

[0034] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a cancer resistant to immune checkpoint inhibitors (ICIs). In some embodiments, the cancer is associated with tumors that have lost expression of MHC class I.

[0035] In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is an infectious disease.

[0036] In some embodiments, the method includes administering to the subject at least one other agent in addition to the IL-18BP inhibitor or IL-18BP antagonist. In some embodiments, the at least one other agent includes an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an agent that inhibits any one of PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, or CD40, or any combination thereof. In some embodiments, the at least one other agent includes a cancer cell opsonizing agent. In some embodiments, the at least one other agent is CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSFIR, SLAMF7, integrin α vThe inhibitor or antagonist targets one or more antigens selected from β3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAP1, and NRP1. In some embodiments, the inhibitor or antagonist is an IL-18 variant polypeptide, and the at least one other agent is conjugated to the IL-18 variant polypeptide. In some embodiments, the at least one other agent is an altered T cell or NK cell. In some embodiments, the at least one other agent is an oncolytic virus.

[0037] The following detailed description of the embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings, in which it is understood that the invention is not limited to the precise arrangements and equipment of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0038] [Figure 1A]Figures 1A and 1B show results from an example experiment demonstrating that the IL-18 pathway is a target for tumor immunotherapy. (Figure 1A) RNA-seq expression analysis of cytokines and receptors in CD8+ TILs demonstrates that the IL-18 pathway (including IL-18 and its receptor subunits) is upregulated in both activated and dysfunctional tumor T cell programs. Genes are assigned "activated" and "dysfunctional" scores relative to naive T cells. Highlighted in yellow are the IL-18 cytokine, IL-18R1 (Rα), and IL-18RAP (Rβ). Data modified from Singer et al. (Singer, M. et al., 2016, Cell 166:1500-1511, e1509). (Figure 1B) The IL-18 receptor subunits, IL-18Rα and IL-18Rβ, are part of the gene expression program that accompanies chronic antigen exposure, as seen after infection with LCMV (left; CD4) or VSV-OVA (right; CD8). Data are from the ImmGen database. [Figure 1B]Figures 1A and 1B show results from an example experiment demonstrating that the IL-18 pathway is a target for tumor immunotherapy. (Figure 1A) RNA-seq expression analysis of cytokines and receptors in CD8+ TILs demonstrates that the IL-18 pathway (including IL-18 and its receptor subunits) is upregulated in both activated and dysfunctional tumor T cell programs. Genes are assigned "activated" and "dysfunctional" scores relative to naive T cells. Highlighted in yellow are the IL-18 cytokine, IL-18R1 (Rα), and IL-18RAP (Rβ). Data modified from Singer et al. (Singer, M. et al., 2016, Cell 166:1500-1511, e1509). (Figure 1B) The IL-18 receptor subunits, IL-18Rα and IL-18Rβ, are part of the gene expression program that accompanies chronic antigen exposure, as seen after infection with LCMV (left; CD4) or VSV-OVA (right; CD8). Data are from the ImmGen database. [Figure 2] Figures 2A-2C show results from an example experiment demonstrating that IL-18BP possesses characteristics of a "soluble immunotherapy checkpoint." (Figure 2A) IL-18BP mediates interferon-γ (IFN-γ)-driven negative feedback of IL-18, reminiscent of the negative feedback of the immunotherapy checkpoint PD-L1. A schematic diagram of the IL-18 / IFN-γ / IL-18BP feedback loop is shown. Black arrows indicate stimulation, and red circuits indicate inhibition. (Figure 2B) IL-18BP is upregulated in gastric and breast cancer, as seen in data from the TCGA and Oncomine databases. (Figure 2C) PD-1 and IL-18BP expression are strongly correlated in a large number of breast and gastric cancer samples (from the TCGA database). R values ​​are 0.78 and 0.65, respectively. [Figure 3A]Figures 3A-3C show results from an example experiment demonstrating the use of yeast display to generate human IL-18 variants independent of IL-18BP. (Figure 3A) A structure-based library was designed to randomize residues in the IL-18:IL-18BP interface and introduced into the yeast display system. Yeast clones were selected for binding to IL-18Rα using magnetic and fluorescent cell sorting and counterselected for IL-18BP. (Figure 3B) Summary of directed evolution to generate IL-18BP-resistant IL-18 variants. Blue text indicates positive selection conditions, and red text indicates counterselection. (Figure 3C) Flow cytometry analysis of yeast-displayed WT IL-18 (left) or variants (right) after directed evolution. The y-axis indicates binding to IL-18BP, and the x-axis indicates binding to IL-18Rα. Clones remaining after five rounds of directed evolution showed a strong preference for IL-18Rα over IL-18BP. [Figure 3B-C] Figures 3A-3C show results from an example experiment demonstrating the use of yeast display to generate human IL-18 variants independent of IL-18BP. (Figure 3A) A structure-based library was designed to randomize residues in the IL-18:IL-18BP interface and introduced into the yeast display system. Yeast clones were selected for binding to IL-18Rα using magnetic and fluorescent cell sorting and counterselected for IL-18BP. (Figure 3B) Summary of directed evolution to generate IL-18BP-resistant IL-18 variants. Blue text indicates positive selection conditions, and red text indicates counterselection. (Figure 3C) Flow cytometry analysis of yeast-displayed WT IL-18 (left) or variants (right) after directed evolution. The y-axis indicates binding to IL-18BP, and the x-axis indicates binding to IL-18Rα. Clones remaining after five rounds of directed evolution showed a strong preference for IL-18Rα over IL-18BP. [Figure 4]Figure 4 shows results from an example experiment, summarizing the sequences of decoy-resistant human IL-18 (designated "DR-IL-18" and also referred to as "DR-18") variants. Each mutated position and its corresponding residue in the mature form of wild-type human IL-18 are indicated at the top of the table. hC4-hE12 represent sequences obtained after selection by directed evolution. hCS1-hCS4 are consensus sequences derived from the selected sequences. The crosshatched residues represent the five most conserved mutations observed. [Figure 5] Figures 5A and 5B show results from example experiments characterizing the biophysical properties of human DR-IL-18 variants. (Figure 5A) Yeast-displayed DR-IL-18 variants hCS1–hCS4 and A8 can bind to hIL-18Rα with binding isotherms comparable to those of wild-type human IL-18 (left). In contrast, little binding is observed between the same variants and hIL-18BP (right). (Figure 5B) Representative surface plasmon resonance sensorgrams between immobilized biotinylated human IL-18BP and DR-IL-18 variants. Recombinant hIL-18 (left) binds to IL-18BP with extremely high affinity, KD = 2.0 pM, whereas hCS1 (right) shows greatly reduced binding, with a much higher dissociation rate and KD = 15.2 nM. The data are summarized in Tables 6 and 7. [Figure 6]Figures 6A and 6B show results from an example experiment demonstrating that human DR-IL-18 variants are not inhibited by IL-18BP. (Figure 6A) Recombinant IL-18BP inhibits the binding of biotinylated IL-18Rα to yeast-displayed WT IL-18, but has no effect on DR-IL-18 variants hCS1-hCS4 and A8 (left). In contrast, IL-18BP effectively neutralizes the previously reported IL-18 E42A, K89A, and E42A / K89A variants (Kim et al., 2001, Proc. Natl. Acad. Sci., 98(6):3304-3309) (right). [E42 and K89 in Kim et al. correspond to E6 and K53 in SEQ ID NO: 30, respectively.] Biotinylated IL-18Rα was maintained at a fixed concentration of 100 nM in all samples (Figure 6B). WT IL-18, hCS1, hCS3, and hCS4 stimulate IL-18 HEK-Blue reporter cells with similar potency and efficacy (left). Wild-type IL-18 is highly sensitive to recombinant IL-18BP application in this assay (IC50 = 3 nM), whereas hCS1 and hCS3 are not inhibited by recombinant IL-18BP, even at 1 μM (right). hIL-18 was maintained at a fixed concentration of 5 nM, and hCS1 and hCS3 were maintained at a fixed concentration of 2.5 nM. [Figure 7]Figures 7A-7C show results from an example experiment demonstrating the use of yeast display to generate additional human IL-18 variants (version 2 variants) independent of IL-18BP. (Figure 7A) Summary of the positions in human IL-18 randomized in the version 2.0 library. For each position, the set of degenerate codons and encoded amino acids is shown. (Figure 7B) Summary of directed evolution to generate version 2.0 IL-18BP-resistant DR-IL-18 variants. Blue text indicates positive selection conditions, and red text indicates counterselection. (Figure 7C) Flow cytometric analysis of the progress of generation of version 2.0 DR-IL-18 variants. Yeast obtained after rounds 1, 4, and 6 were simultaneously stained with 250 nM IL-18BP streptavidin-PE tetramer or 100 nM IL-18Rα directly labeled with AlexaFluor 647. The y-axis shows binding to IL-18BP, and the x-axis shows binding to IL-18Rα. Clones remaining after six rounds of directed evolution strongly preferred IL-18Rα over IL-18BP. [Figure 8] Figure 8 shows the results from an example experiment, summarizing the sequences of version 2.0 decoy-resistant IL-18 (DR-IL-18) variants. Each mutated position and its corresponding residue in the mature form of wild-type human IL-18 are shown at the top of the table. The shaded rows indicate the recurrent sequence variants obtained in both rounds 5 and 6. [Figure 9]Figure 9 shows results from an example experiment characterizing the biophysical properties of the version 2.0 human DR-IL-18 variants. (Figure 9A) The yeast-displayed version 2.9 DR-IL-18 variants can bind to hIL-18Rα with a binding isotherm comparable to that of WT human IL-18. (Figure 9B) In contrast, little binding is observed between the same variants and hIL-18BP. (Figure 9C) The thermostability of the version 2.0 DR-IL-18 variants was assessed by heating the yeast-displayed variants through a temperature range over 15 minutes and then staining with hIL-18Rα. The version 2.0 DR-IL-18 variants were more thermostable than WT IL-18 (Tm = 47.6°C) and the first-generation consensus sequences (Tm = 50.9°C and 40.2°C for hCS1 and hCS2, respectively). (Figure 9D) Summary of receptor binding properties and thermostability of second-generation DR-IL-18 variants. NBD = no binding detected. ND = value not determined. [Figure 10A-B] Figures 10A-10C show results from an example experiment demonstrating the use of yeast display to generate IL-18BP-independent mouse IL-18 variants. (Figure 10A) Summary of directed evolution to generate IL-18BP-resistant mouse IL-18 variants. Blue text indicates positive selection conditions, and red text indicates counterselection. (Figure 10B) Flow cytometry analysis of yeast-displayed mouse IL-18 variants after five rounds of directed evolution. The y-axis indicates binding to IL-18BP, and the x-axis indicates binding to IL-18Rα. (Figure 10C) Summary of the sequences of decoy-resistant mouse IL-18 (DR-IL-18) variants. Each mutated position and its corresponding residue in the mature wild-type mouse IL-18 are indicated at the top of the table. mC1-mH3 represent the sequences obtained after selection in directed evolution. mCS1 and mCS2 are consensus sequences derived from the selected sequences. The crosshatched residues represent the five most conserved mutations observed. [Figure 10C]Figures 10A-10C show results from an example experiment demonstrating the use of yeast display to generate IL-18BP-independent mouse IL-18 variants. (Figure 10A) Summary of directed evolution to generate IL-18BP-resistant mouse IL-18 variants. Blue text indicates positive selection conditions, and red text indicates counterselection. (Figure 10B) Flow cytometry analysis of yeast-displayed mouse IL-18 variants after five rounds of directed evolution. The y-axis indicates binding to IL-18BP, and the x-axis indicates binding to IL-18Rα. (Figure 10C) Summary of the sequences of decoy-resistant mouse IL-18 (DR-IL-18) variants. Each mutated position and its corresponding residue in the mature wild-type mouse IL-18 are indicated at the top of the table. mC1-mH3 represent the sequences obtained after selection in directed evolution. mCS1 and mCS2 are consensus sequences derived from the selected sequences. The crosshatched residues represent the five most conserved mutations observed. [Figure 11] Figures 11A and 11B show results from example experiments characterizing the biophysical properties of murine DR-IL-18 variants. (Figure 11A) The yeast-displayed DR-IL-18 variants mA7, mB1, mC1, mE8, mCS1, and mCS2 can bind to hIL-18Rα with binding isotherms comparable to those of wild-type human IL-18 (left). In contrast, little binding is observed between the same variants and hIL-18BP (right). (Figure 11B) Representative surface plasmon resonance sensorgrams between immobilized mIL-18BP and murine DR-IL-18 variants. Recombinant mIL-18 (left) binds to mIL-18BP with an extremely high affinity, KD = 0.8 pM, whereas mCS2 (right) shows greatly reduced binding, with a KD value of >10 μM. The data are summarized in Tables 8 and 9. [Figure 12A-B]Figures 12A-12D show results from an example experiment investigating the pharmacodynamics of DR-IL-18 administered to mice. (Figure 12A) Schematic of the study design. Mice received seven daily doses of either vehicle (PBS), mIL-18 (1 mg / kg), or the DR-IL-18 variant mCS2 (1 mg / kg) (indicated by syringe). Blood samples were collected 2 days before the experiment and 5 hours after injection on days 0, 3, and 6. (Figure 12B) Peripheral blood CD4 cell, CD8 cell, NK cell, and monocyte counts on days 0, 3, and 6. Both IL-18 and mCS2 expanded NK cells and monocytes to similar extents by day 3. At each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents mCS2. (Figure 12C) Expression of CD69 on the surface of peripheral blood CD4, CD8, and NK cells. mCS2, but not IL-18, promoted CD69 expression on the surface of CD4 and CD8 cells. Both IL-18 and mCS2 increased CD69 on the surface of NK cells, but as evident at day 6, treatment with mCS2 maintained CD69 expression, whereas treatment with IL-18 restored CD69 levels to baseline. At each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents mCS2. (Figure 12D) Serum cytokine levels are shown for interferon-γ (IFN-γ), MIP-1b, and G-CSF. Treatment with mCS2 resulted in higher levels of IFN-γ, MIP-1b, and G-CSF compared with treatment with mIL-18. [Figure 12C-D]Figures 12A-12D show results from an example experiment investigating the pharmacodynamics of DR-IL-18 administered to mice. (Figure 12A) Schematic of the study design. Mice received seven daily doses of either vehicle (PBS), mIL-18 (1 mg / kg), or the DR-IL-18 variant mCS2 (1 mg / kg) (indicated by syringe). Blood samples were collected 2 days before the experiment and 5 hours after injection on days 0, 3, and 6. (Figure 12B) Peripheral blood CD4 cell, CD8 cell, NK cell, and monocyte counts on days 0, 3, and 6. Both IL-18 and mCS2 expanded NK cells and monocytes to similar extents by day 3. At each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents mCS2. (Figure 12C) Expression of CD69 on the surface of peripheral blood CD4, CD8, and NK cells. mCS2, but not IL-18, promoted CD69 expression on the surface of CD4 and CD8 cells. Both IL-18 and mCS2 increased CD69 on the surface of NK cells, but as evident at day 6, treatment with mCS2 maintained CD69 expression, whereas treatment with IL-18 restored CD69 levels to baseline. At each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents mCS2. (Figure 12D) Serum cytokine levels are shown for interferon-γ (IFN-γ), MIP-1b, and G-CSF. Treatment with mCS2 resulted in higher levels of IFN-γ, MIP-1b, and G-CSF compared with treatment with mIL-18. [Figure 13]Figure 13 shows results from an example experiment demonstrating that treatment with DR-IL-18 reduces body fat composition in mice. Body fat and lean body mass were measured in mice treated every three days with 0.01 mg / kg, 0.1 mg / kg, or 1 mg / kg of the DR-IL-18 variant mCS2, or 1 mg / kg of WT mIL-18. Treatment with mCS2 significantly reduced body fat as a percentage of total body weight (top panel). This manifested as a decrease or stable fat mass (left panel) and was consistent with an increase in lean body mass (right panel). Mice treated with vehicle and mIL-18 showed an increase in fat mass and stable lean body mass over the same treatment period. [Figure 14A] Figures 14A-14B show results from an example experiment demonstrating that DR-IL-18 is an effective immunotherapeutic agent in a melanoma model. (Figure 14A) Tumor growth spider plots in Yummer1.7 melanoma-bearing mice treated twice weekly with either saline (control), WT IL-18 (0.32 mg / kg), the DR-IL-18 variant mCS2 (0.32 mg / kg), anti-PD1 (8 mg / kg), IL-18 + anti-PD1, or mCS2 + anti-PD1. (Figure 14B) Survival curves from the same groups as in Figure 11A. mCS2 was effective as a monotherapy and synergistic when combined with anti-PD1 in this model. [Figure 14B] Figures 14A-14B show results from an example experiment demonstrating that DR-IL-18 is an effective immunotherapeutic agent in a melanoma model. (Figure 14A) Tumor growth spider plots in Yummer1.7 melanoma-bearing mice treated twice weekly with either saline (control), WT IL-18 (0.32 mg / kg), the DR-IL-18 variant mCS2 (0.32 mg / kg), anti-PD1 (8 mg / kg), IL-18 + anti-PD1, or mCS2 + anti-PD1. (Figure 14B) Survival curves from the same groups as in Figure 11A. mCS2 was effective as a monotherapy and synergistic when combined with anti-PD1 in this model. [Figure 15]Figures 15A and 15B show results from an example experiment demonstrating that the efficacy of DR-IL-18 in the melanoma model of Figure 14 is dependent on CD4 lymphocytes, CD8 lymphocytes, and interferon-gamma. (Figure 15A) Tumor growth spider plots in Yummer1.7 melanoma-bearing mice treated with saline (control), a DR-IL-18 variant (0.32 mg / kg) alone, or the DR-IL-18 variant mCS2 (0.32 mg / kg) in combination with depleting antibodies against either CD8, CD4, interferon-gamma, or NK1.1. (Figure 15B) Survival curves from the same groups as in Figure 15A. [Figure 16] Figure 16 shows results from an example experiment demonstrating that the effects of DR-IL-18 in the MC38 tumor model are dose-dependent. Spider plots of tumor growth from mice bearing MC38 colon cancer tumors treated every three days with saline (control), 1.0 mg / kg WT IL-18, 1.0 mg / kg mCS2, 0.1 mg / kg mCS, or 0.01 mg / kg mCS. WT IL-18 was ineffective at 1 mg / kg, whereas mCS was partially effective at 0.1 mg / kg and maximally effective at 1.0 mg / kg. [Figure 17] Figure 17 shows results from an example experiment demonstrating the effects of DR-IL-18 alone and in combination with the immune checkpoint inhibitor anti-PD1 in the MC38 tumor model. Spider plots of tumor growth from mice bearing MC38 colon cancer tumors treated with saline (control), 0.32 mg / kg WT IL-18, 0.32 mg / kg DR-IL-18 variant mCS2, 5 mg / kg anti-PD1, a combination of anti-PD1 and WT IL-18, or a combination of anti-PD1 and mCS2. All agents were administered intraperitoneally twice weekly for a total of six doses. [Figure 18A]Figures 18A and 18B show results from an example experiment examining antitumor mechanisms in MC38 tumor-bearing mice. (Figure 18A) Tumor immunophenotyping experiments in mice treated twice weekly with saline, WT IL-18, or the DR-IL-18 variant mCS2. Treatment with DR-IL-18 increased the number of CD8 and NK cells per mg of tumor (top two graphs on the left) and increased the expression of activation markers granzyme B and KLRG1 on the surface of CD8 and NK cells (top two graphs on the right). Treatment with DR-IL-18 did not improve the CD8:Treg ratio compared with saline treatment, whereas WT IL-18 worsened this ratio. However, treatment with DR-IL-18 increased the ratio of CD8 cells to suppressive myeloid populations, including tumor-associated macrophages (TAMs) and monocytic and granulocytic myeloid-derived suppressor cells (mMDSCs and gMDSCs). (Fig. 18B) Luminex measurements of serum cytokines collected from the same mice as in Fig. 18A 24 hours after the fourth dose. DR-IL-18 dramatically altered the secondary cytokine release profile relative to treatment with WT IL-18, resulting in a significant >100-fold increase in interferon-gamma, IL-7, and IL-15 levels. [Figure 18B]Figures 18A and 18B show results from an example experiment examining antitumor mechanisms in MC38 tumor-bearing mice. (Figure 18A) Tumor immunophenotyping experiments in mice treated twice weekly with saline, WT IL-18, or the DR-IL-18 variant mCS2. Treatment with DR-IL-18 increased the number of CD8 and NK cells per mg of tumor (top two graphs on the left) and increased the expression of activation markers granzyme B and KLRG1 on the surface of CD8 and NK cells (top two graphs on the right). Treatment with DR-IL-18 did not improve the CD8:Treg ratio compared with saline treatment, whereas WT IL-18 worsened this ratio. However, treatment with DR-IL-18 increased the ratio of CD8 cells to suppressive myeloid populations, including tumor-associated macrophages (TAMs) and monocytic and granulocytic myeloid-derived suppressor cells (mMDSCs and gMDSCs). (Fig. 18B) Luminex measurements of serum cytokines collected from the same mice as in Fig. 18A 24 hours after the fourth dose. DR-IL-18 dramatically altered the secondary cytokine release profile relative to treatment with WT IL-18, resulting in a significant >100-fold increase in interferon-gamma, IL-7, and IL-15 levels. [Figure 19]Figures 19A-19C show results from an example experiment demonstrating that DR-IL-18 can effectively treat tumors that lose MHC class I expression and thus become resistant to immune checkpoint inhibitors. (Figure 19A) Spider plots of tumor growth from mice bearing B2m-deficient Yummer1.7 tumors treated with saline, anti-PD1 + anti-CTLA4, DR-IL-18 variant mCS2, or mCS2 depleted of NK cells with anti-NK1.1 antibodies. DR-IL-18 had a robust effect on tumor growth and survival (Figure 19B), curing 60% of treated mice in this model, which was completely resistant even to combined treatment with anti-PD1 + anti-CTLA4. This effect was NK cell-dependent, as administration of anti-NK1.1 abolished the effect of mCS2 treatment. (Figure 19C) NK cells isolated from B2m-deficient Yummer1.7 tumors are nonfunctional, exhibiting reduced proliferation (Ki67 staining) and function (interferon-gamma secretion), but treatment with DR-IL-18 reverses this phenotype, allowing robust proliferation and cytokine secretion. [Figure 20]Figures 20A-20C show results from an example experiment demonstrating the use of yeast display to generate human IL-18 variants as IL-18BP antagonists (i.e., "decoy-to-decoy" D2D). These variants antagonize the effect of IL-18BP on endogenous IL-18 by binding to IL-18BP but not signaling. (Figure 20A) Summary of positions in human IL-18 randomized in the D2D library. For each position, the degenerate codon and encoded amino acid set are shown. (Figure 20B) Summary of directed evolution to generate D2D IL-18 variants that bind and neutralize IL-18BP but do not signal through IL-18R. Blue text indicates conditions for positive selection, and red text indicates counterselection. (Figure 20C) Flow cytometric analysis of the progress of D2D hIL-18 variant generation. Yeast obtained after rounds 1–4 were stained with 1 nM mouse IL-18BP (left panel), 1 nM human IL-18BP (center panel), or 1 μM IL-18Rα + 1 μM IL-18Rβ. Selected variants showed increased binding to IL-18BP but not to IL-18Rα or IL-18Rβ with each round of selection. [Figure 21] Figure 21 shows the results from an example experiment, summarizing the sequences of the D2D human IL-18 variants. Each mutated position and its corresponding residue in the mature form of wild-type human IL-18 are shown at the top of the table. [Figure 22]Figures 22A-C show results from example experiments characterizing the biophysical properties of human decoy-to-decoy (D2D) IL18 variants. (Figure 22A) Yeast-displayed D2D IL18 variants 5-B02, 5-E08, 5-F10, 5-F02, 5-F01, hD2D-CS1, hD2D-CS2, and hD2D-CS3 can bind hIL-18BP with isotherms comparable to those of WT human IL-18. (Figure 22B) In contrast, little binding is observed between the same variants and hIL-18Rα. (Figure 22C) Summary of receptor binding properties of D2D IL18 variants. NBD = no binding detected. [Figure 23] Figure 23 shows the results from an experiment performed in the Examples, summarizing the sequences of the D2D mouse IL-18 variants. Each mutated position and its corresponding residue in the mature form of wild-type mouse IL-18 are shown at the top of the table. [Figure 24] Figure 24 shows the results (sensorgrams) of the biophysical affinity of second-generation DR-IL-18 variants for binding to IL-18Rα and IL-18BP measured by surface plasmon resonance (SPR). Top row: Representative sensorgrams of the indicated IL-18 variants (soluble analyte) against hIL-18Rα (immobilized ligand). Bottom row: Representative sensorgrams of the indicated IL-18 variants against human hIL-18BP. The x-axis is time (in seconds) and the y-axis is response units (RU). The curves are time courses of data observed at different concentrations (starting at 1 nM and diluting 2-fold), with a best-fit curve superimposed assuming a 1:1 Langmuir binding model. [Figure 25]Figures 25A and 25B show data demonstrating the efficacy of DR-IL-18 against the CT26 colon tumor model. 250,000 CT26 cells were implanted subcutaneously, and treatment began on day 7, when tumors averaged approximately 60 mm3. Wild-type IL-18 and mCS2 were administered at a dose of 0.32 mg / kg twice weekly for a total of five doses. Anti-PD1 was administered at 10 mg / kg on the same schedule. (A) Overlay of multiple spider plots showing tumor growth: saline (PBS)-treated mice (black line), wild-type IL-18 (blue line), and DR-IL-18(mCS2) (pink line). Treatment with DR-IL-18, but not wild-type IL-18, alone resulted in tumor growth inhibition and tumor regression in a subset of mice. (B) Survival curves for mice treated with anti-PD-1, WT IL-18, and DR-IL-18 (mCS2). The number of complete responses is shown in parentheses. Use of DR-IL-18 rather than WT IL-18 led to prolonged survival and tumor clearance in 40% of mice, an improvement over the checkpoint inhibitor anti-PD-1. [Figure 26] Figures 26A and 26B show data demonstrating the efficacy of DR-IL-18 in the 4T1 breast cancer and B16-F10 melanoma models. (A) Tumor growth curves of BALB / C mice bearing 4T1 tumors after treatment with saline (PBS; black), WT IL-18 (blue), or the DR-IL-18 variant CS2 (pink). (B) Tumor growth curves of C57BL / 6 mice bearing B16-F10 tumors after treatment with saline (PBS; black), WT IL-18 (blue), or the DR-IL-18 variant CS2 (pink). In both models, only DR-IL-18, but not WT IL-18, resulted in tumor growth inhibition. Treatment was administered after the mean tumor volume exceeded 50 mm3, as indicated by the boxed "t." [Figure 27]Figures 27A and 27B expand on the data in Figures 19A–19C. Shown are data demonstrating the efficacy of DR-IL-18 in treating additional MHC class I-deficient tumor models resistant to immune checkpoint inhibitors. (A) B2m-deficient MC38 cells were generated using CRISPR / Cas9-mediated deletion as described for B2m-deficient YUMMER cells. Treatment began on day 7 after subcutaneous implantation of B2m- / - MC38 cells, when tumors averaged approximately 65 mm3. mCS2 was administered at a dose of 0.32 mg / kg twice weekly for five doses. Anti-PD1 and anti-CTLA4 were administered at 8 mg / kg on the same schedule. (B) RMA / S is a variant of an RNA lymphoma line containing a spontaneous mutation in tapasin, resulting in a defective antigen loading and therefore reduced surface expression of MHC class I. It is congenital to C57BL / 6 and is resistant to immune checkpoint inhibitors. Mice were implanted subcutaneously with 1,000,000 RMA / S cells, and treatment began on day 7. mCS2 was administered twice weekly at a dose of 0.32 mg / kg. Anti-PD1 was administered at 8 mg / kg on the same schedule. In both studies, only treatment with the DR-18 variant mCS2 demonstrated antitumor effects in the form of tumor growth inhibition (B2m- / - MC38) or tumor disappearance (RMA / S). [Figure 28]Figure 28 shows data demonstrating the effect of DR-IL-18 variants in enhancing antibody-dependent cellular cytotoxicity (ADCC). In vitro cytotoxicity studies were performed using CFSE-labeled Raji (B-cell lymphoma) cells and isolated human peripheral blood mononuclear cells (PBMCs). PBMCs and labeled Raji cells were incubated for 25 hours at an effector:target (E:T) ratio of 1:10. Human DR-IL-18 variant hCS-1 (1 μM), rituximab (10 μg / ml), or a combination of both agents was applied to the samples as indicated. Cytotoxicity was measured by flow cytometry and calculated as the percentage of CFSE-positive cells. DR-18 significantly enhanced tumor cell killing as a single agent and significantly enhanced killing by the therapeutic antibody rituximab. *p<0.05 by two-way ANOVA with Tukey's correction for multiple comparisons. [Figure 29A] Figures 29A and 29B show data demonstrating the antiviral efficacy of DR-18 variants in treating infectious diseases (e.g., here, systemic vaccinia virus infection). (A) Experimental design scheme. C57BL / 6 mice were infected intraperitoneally (IP) with 10 PFU of vaccinia virus (VACV) and then administered 1 mg / kg of WT mIL-18 or mCS2 IP. Three days after infection, mice were euthanized, and viral titers in the blood and ovaries were measured by RT-PCR. (B) Quantification of VACV viral copies in the blood and ovaries of treated mice at 3 days after infection. Treatment with CS2 significantly reduced viral titers, whereas WT IL-18 was ineffective. * p<0.05, ** p<0.01, *** p<0.001. [Figure 29B]Figures 29A and 29B show data demonstrating the antiviral efficacy of DR-18 variants in treating infectious diseases (e.g., here, systemic vaccinia virus infection). (A) Experimental design scheme. C57BL / 6 mice were infected intraperitoneally (IP) with 10 PFU of vaccinia virus (VACV) and then administered 1 mg / kg of WT mIL-18 or mCS2 IP. Three days after infection, mice were euthanized, and viral titers in the blood and ovaries were measured by RT-PCR. (B) Quantification of VACV viral copies in the blood and ovaries of treated mice at 3 days after infection. Treatment with CS2 significantly reduced viral titers, whereas WT IL-18 was ineffective. * p<0.05, ** p<0.01, *** p<0.001. [Figure 30] Figure 30A shows data demonstrating that the second-generation human DR-IL-18 variants are active. (Figure 30A) WT IL-18, as well as h6-12, h6-27, h6-29, and h6-31 stimulate IL-18 HEK-Blue reporter cells. h6-12, h6-27, and h6-29 show increased potency relative to WT IL-18, whereas h6-31 is equivalent in potency to WT IL-18. Thus, the data demonstrate that all second-generation human DR-IL-18 variants tested actively signal through the IL-18R. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention relates to IL-18 variants that induce or enhance IL-18 signaling. In one embodiment, the present invention relates to IL-18 variants that can bind to the IL-18 receptor (IL-18R) but not to the IL-18 binding protein (IL-18BP), thereby providing IL-18 signaling activity but not being inhibited by IL-18BP. In one embodiment, the present invention relates to IL-18 variants that bind to IL-18BP, thereby preventing the IL-18BP from binding to and inhibiting endogenous IL-18, thereby providing IL-18 signaling activity. Thus, the present invention provides compositions and methods that provide IL-18 signaling activity even in the presence of IL-18BP.

[0040] In various embodiments, the present invention relates to an IL-18 variant polypeptide or fragment thereof that specifically binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, an IL-18 variant polypeptide or fragment thereof that binds to IL-18R but does not substantially bind to IL-18BP is useful for providing IL-18 signaling activity that is not inhibited by the presence and activity of IL-18BP. In various embodiments, the present invention relates to an IL-18 variant polypeptide or fragment thereof that specifically binds to IL-18BP and thereby reduces or prevents the inhibition of endogenous IL-18 by that IL-18BP. In some embodiments, an IL-18 variant polypeptide or fragment thereof that binds to IL-18BP is useful for inhibiting IL-18BP activity, thereby inducing, enhancing, or promoting IL-18 signaling activity.

[0041] In some embodiments, IL-18 variant polypeptides or fragments thereof are useful for treating and preventing a disease or disorder. In various embodiments, the disease or disorder is cancer, an infectious disease (e.g., a poxvirus encoding an IL-18BP ortholog), a metabolic disease or disorder (including obesity and diabetes), or macular degeneration (e.g., wet macular degeneration, e.g., wet age-related macular degeneration, in which IL-18 variants can be used as anti-angiogenic agents; as a specific example, in some cases, IL-18 variant polypeptides of the invention can reduce choroidal neovascularization). Accordingly, in some embodiments, the invention is a composition comprising at least one IL-18 variant polypeptide or fragment thereof. In another embodiment, the invention is a method for treating or preventing a disease or disorder by administering at least one IL-18 variant polypeptide or fragment thereof, including, but not limited to, cancer, an infectious disease, a metabolic disease or disorder, or macular degeneration (e.g., wet macular degeneration, e.g., wet age-related macular degeneration).

[0042] In various embodiments, the IL-18 variant polypeptide comprises one mutation relative to the wild-type (WT) IL-18 polypeptide. In some embodiments, the WT IL-18 peptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 peptide comprises the amino acid sequence of SEQ ID NO: 31.

[0043] In some embodiments, the IL-18 variant polypeptides of the present invention exhibit decreased binding affinity for IL-18BP relative to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptides of the present invention exhibit increased binding affinity for IL-18BP relative to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptides of the present invention exhibit binding affinity for IL-18BP equivalent to that of WT IL-18 polypeptide.

[0044] In some embodiments, the IL-18 variant polypeptide exhibits increased binding affinity for IL-18R relative to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptide exhibits binding affinity for IL-18R equivalent to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptide exhibits decreased binding affinity for IL-18R relative to WT IL-18 polypeptide.

[0045] In some embodiments, the IL-18 variant polypeptide is a mammalian IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide is a human IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide is a mouse IL-18 variant polypeptide.

[0046] In various embodiments, the compositions and methods of the present invention include compositions and methods for the treatment and prevention of diseases and disorders, such as cancer, infectious diseases, and metabolic diseases and disorders. In some embodiments, a method comprises administering to a subject in need thereof a composition comprising at least one IL-18 variant polypeptide. In some embodiments, a method comprises administering to a subject in need thereof a composition comprising at least one IL-18 variant polypeptide and administering to the subject a composition comprising an additional agent.

[0047] In one such embodiment, the additional agent comprises an immunotherapeutic agent, the immunotherapeutic agent comprising at least one selected from the group including, by way of non-limiting example, altered T cells, chimeric antigen receptor T cells (CAR-T), armored CAR-T cells, viruses, antigens, vaccines, antibodies, immune checkpoint inhibitors, small molecules, chemotherapeutic agents, and stem cells. In some embodiments, a composition comprising at least one IL-18 variant polypeptide is used in a method for increasing immune system activity before, during, or after bacterial, viral, or other pathogen infection. In some embodiments, a composition comprising at least one IL-18 variant polypeptide is used in a method for increasing immune cell number and / or activity (e.g., T cell, and / or NK cell, and / or myeloid cell number and / or activity) in vitro, in vivo, or ex vivo.

[0048] definition

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology will be used.

[0050] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0051] The article "a" or "an" is used herein to refer to either one or to more than one (at least one) of the grammatical object of the article. For example, "an element" means one element or two or more elements.

[0052] "About," as used herein in reference to a measurable value (amount, time, etc.), is meant to include an open-ended variation of ±40%, or ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0053] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, means an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from an organism, tissue, cell, or component thereof that exhibits "normal" (expected) respective characteristics. A characteristic that is normal or expected for one cell or one type of tissue may be abnormal for a different cell or a different type of tissue.

[0054] The term "antibody," as used herein, refers to an immunoglobulin molecule capable of specifically binding to a particular epitope or antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and immunologically active portions of intact immunoglobulins. Antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, F(ab)2, as well as single chain antibodies (scFv), heavy chain antibodies (e.g., camelid antibodies), synthetic antibodies, chimeric antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0055] "Antibody heavy chain" as used herein means the larger of the two polypeptide chains present in all antibody molecules in their native conformation.

[0056] "Antibody light chain," as used herein, refers to the smaller of the two polypeptide chains present in all antibody molecules in their native conformation. Kappa and lambda light chains are the two major antibody light chain isotypes.

[0057] The term "synthetic antibody," as used herein, refers to an antibody produced using recombinant DNA techniques, an example of which is a bacteriophage-expressed antibody, as described herein. The term should also be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein, or by synthesizing the amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is obtained using well-known synthetic DNA or amino acid sequence techniques available in the art.

[0058] As used herein, "immunoassay" refers to any binding assay that utilizes an antibody capable of specifically binding to a target molecule to detect and quantitate that target molecule.

[0059] As used herein, the phrase "specifically binds" with respect to an IL-18 variant polypeptide refers to an IL-18 variant polypeptide that recognizes a particular receptor (e.g., IL-18R) or IL-18BP and binds to that receptor or IL-18BP. In some cases, the IL-18 variant polypeptide has substantially reduced binding to IL-18BP. For example, an IL-18 variant polypeptide that specifically binds to a receptor from one species may also bind to that receptor from one or more species. However, such species cross-reactivity does not in itself change the classification of the IL-18 variant polypeptide as specific. In another example, an IL-18 variant polypeptide that specifically binds to a receptor may also bind to different allelic forms of that receptor. However, such cross-reactivity does not in itself change the classification of the IL-18 variant polypeptide as specific. In some cases, the phrases "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the surface of that chemical species. For example, an IL-18 variant polypeptide recognizes and binds to one specific protein structure rather than proteins in general.

[0060] The term "applicator," as used herein, means any device for administering a composition of the present invention to a subject, non-limiting examples of which include hypodermic syringes, pipettes, iontophoresis devices, patches, and the like.

[0061] "Cancer," as used herein, refers to the abnormal growth or division of cells. Generally, the growth and / or survival of cancer cells exceeds and is uncoordinated with the growth and / or survival of surrounding normal cells or tissues. Cancers can be benign, precancerous, or malignant. Cancer can arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas), respiratory system (e.g., pharynx, lungs, bronchi), bones, joints, skin (e.g., basal cell, squamous, meningioma), breast, reproductive system (e.g., uterus, ovaries, prostate, testes), urinary system (e.g., bladder, kidneys, ureters), eyes, nervous system (e.g., brain), endocrine system (e.g., thyroid), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia).

[0062] The term "coding sequence" as used herein refers to a sequence of a nucleic acid, or its complement, or a portion thereof, that can be transcribed and / or translated to produce mRNA and / or a polypeptide or fragment thereof. Coding sequences include exons in genomic DNA or premature primary RNA transcripts, which are assembled by a cell's biochemical machinery to provide mature mRNA. The antisense strand is the complement of such a nucleic acid, from which a coding sequence can be derived. In contrast, the term "non-coding sequence" as used herein refers to a sequence of a nucleic acid or its complement, or a portion thereof, that is not translated into amino acids in vivo, i.e., with which tRNA does not interact or attempt to deliver amino acids. Non-coding sequences include both intron sequences in genomic DNA or premature primary RNA transcripts and gene-associated sequences (e.g., promoters, enhancers, silencers).

[0063] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," in which only a few of the nucleic acids' bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions, as well as detection reactions that depend on binding between nucleic acids.

[0064] A "disease" is a health condition in which an animal is unable to maintain homeostasis, and if the disease is not corrected, the animal's health will continue to deteriorate. In contrast, an animal "disorder" is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. A disorder, if left untreated, does not necessarily cause a further deterioration in the animal's health state.

[0065] By "effective amount" herein is meant an amount that provides a therapeutic, or prophylactic, or other desired benefit.

[0066] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide (e.g., gene, cDNA, mRNA) to serve as a template for the synthesis, in biological processes, of a defined sequence of nucleotides (i.e., rRNA, tRNA, mRNA) or other polymers or macromolecules having a defined sequence of amino acids and biological properties resulting therefrom. Thus, a gene encodes a protein when that protein is produced in a cell or other biological system by transcription and translation of its corresponding mRNA. Both the coding strand (whose nucleotide sequence is the same as the mRNA sequence and is usually given in a sequence listing) and the non-coding strand (used as a template to transcribe the gene or cDNA) can be said to encode the protein or other product of the gene or cDNA.

[0067] As used herein, the term "fragment," when applied to nucleic acids, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid is at least about 15 nucleotides in length; e.g., at least about 50 to about 100 nucleotides; or at least about 100 to about 500 nucleotides; or at least about 500 to about 1000 nucleotides; or at least about 1000 to about 1500 nucleotides; or at least about 1500 to about 2500 nucleotides; or about 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment," when applied to a protein, polypeptide, or peptide, refers to a subsequence of a larger protein, polypeptide, or peptide. A "fragment" of a protein, polypeptide, or peptide is at least about 5 amino acids in length; e.g., at least about 10 amino acids in length; or at least about 20 amino acids in length; or at least about 50 amino acids in length; or at least about 100 amino acids in length; or at least about 200 amino acids in length; or at least about 300 amino acids in length (and any integer value therebetween).

[0068] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that contains coding sequences necessary for the production of a polypeptide, or precursor, or RNA (e.g., mRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property of the full-length or fragment (e.g., enzymatic activity, receptor binding, signal transduction, immunogenicity, etc.) is retained. The term also encompasses the coding region of a structural gene and adjacent sequences located 5' and 3' of the coding region by at least about 2 kb, thereby corresponding to the length of the full-length mRNA and 5' regulatory sequences that influence the transcriptional characteristics of the gene. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' untranslated sequences. These 5' untranslated sequences usually contain regulatory sequences. Sequences located 3', or downstream, of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of genes. A genomic form of a gene or clone of a gene contains the coding region interrupted by non-coding regions called "introns" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed, or "spliced ​​out," from the nuclear or primary transcript; therefore, introns are absent in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in the polypeptide produced.

[0069] As used herein in the context of two or more nucleic acid or polypeptide sequences, "homologous," "matching," or "identical" means that the sequences share a specified percentage of the same residues across a specified region. The percentage can be calculated by aligning the two sequences, comparing the two sequences across a specified region, determining the number of positions with the same residue in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are different in length, i.e., alignment results in one or more offsets at the ends, and only one sequence is included in the specified region being compared, the residues of that single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Matching can be performed manually or using computer alignment algorithms (e.g., BLAST and BLAST 2.0).

[0070] As used herein, the term "instructional materials" includes publications, records, diagrams, or any other medium of expression that can be used to inform recipients that the nucleic acids, and / or peptides, and / or polypeptides, and / or compounds of the invention in the kit are useful in identifying, alleviating, or treating various diseases or disorders described herein. Optionally, or alternatively, the instructional materials can describe one or more methods for identifying or alleviating a disease or disorder in a cell or tissue of a subject. Kit instructional materials can be, for example, attached to a container containing the nucleic acids, and / or polypeptides, and / or compounds of the invention or shipped with a container containing the nucleic acids, and / or polypeptides, and / or compounds. Alternatively, the instructional materials can be shipped separately from the container, allowing the recipient to use the instructional materials in conjunction with the compounds.

[0071] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment (e.g., a host cell).

[0072] An "isolated nucleic acid" refers to a nucleic acid segment or fragment (e.g., a DNA fragment that has been removed from sequences that normally flank the fragment (e.g., sequences that flank the fragment in nature in a genome)) that is separated from sequences that normally flank the fragment. The term also applies to nucleic acids (e.g., RNA, DNA, proteins that naturally accompany the fragment in a cell) that have been substantially purified from other components that naturally accompany it. Thus, the term includes recombinant DNA that exists, for example, in a vector, an autonomously replicating plasmid or virus, or integrated into the genomic DNA of a prokaryote or eukaryote, or as a separate molecule independent of other sequences (e.g., cDNA, or genomic fragments, or cDNA fragments generated by PCR or restriction enzyme digestion). The term also includes recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0073] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to a probe to produce a "labeled" probe. The label can be one that is itself detectable (e.g., a radioisotope label or fluorescent label) or, in the case of an enzymatic label, one that is capable of catalyzing chemical alteration of a detectable substrate compound or composition (e.g., avidin-biotin). In some cases, primers can be labeled to allow detection of PCR products.

[0074] The term "modulate," as used herein, means mediating a detectable increase or decrease in mRNA, polypeptide activity and / or level, or mediating a response in a subject relative to the activity and / or level of the polypeptide, or mediating a response in a subject relative to the absence of treatment or compound and / or mRNA, polypeptide activity and / or level, or mediating a response in an otherwise equivalent subject without treatment. The term encompasses mediating a beneficial therapeutic, prophylactic, or other desired response in a subject (e.g., a human) by activating and / or suppressing and / or otherwise affecting the original signal or response.

[0075] "Mutation," "mutant," or "variant" as used herein refers to a change in a nucleic acid or polypeptide sequence compared to a reference sequence (which may be a naturally occurring, normal, or "wild-type" sequence), including a transition, deletion, insertion, or substitution / point mutation. A "mutant" or "variant," as used herein, refers to a nucleic acid or protein that contains a mutation.

[0076] "Nucleic acid" refers to polynucleotides, including poly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acids according to the present invention can contain any polymer or oligomer of pyrimidine bases (preferably cytosine, thymine, uracil) and purine bases (preferably adenine, guanine). (See Albert L. Lehninger, Principles of Biochemistry, pp. 793-800 (Worth Pub., 1982), which is incorporated herein in its entirety for all purposes. Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid building block, as well as any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from natural sources or artificially, i.e., synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0077] An "oligonucleotide" or "polynucleotide" is a nucleic acid, i.e., a compound, at least 2, preferably at least 8, 15, or 25 nucleotides in length, but which can range in length from 50, 100, 1,000, or 5,000 nucleotides, that specifically hybridizes to a polynucleotide. Polynucleotides include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mimetics thereof, isolated from natural sources, recombinantly produced, or artificially synthesized. Another example of a polynucleotide of the present invention can be a peptide nucleic acid (PNA). (See U.S. Patent No. 6,156,501, incorporated herein by reference in its entirety.) The present invention also encompasses situations where non-traditional base pairing is present, such as Hoosteen base pairing, which has been identified in some tRNA molecules and is postulated to exist in triple helices. "Polynucleotide" and "oligonucleotide" are used interchangeably in this disclosure. When a nucleotide sequence is represented herein in terms of a DNA sequence (e.g., A, T, G, C), it will be understood that the corresponding RNA sequence in which "T" is replaced by "U" (e.g., A, U, G, C) is also included.

[0078] The terms "patient," "subject," "individual," and the like are used interchangeably herein to refer to any animal, or cells thereof, to which the methods described herein can be applied, whether in vivo, in vitro, or in situ. In some non-limiting embodiments, the patient, subject, or individual is a human.

[0079] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a compound consisting of multiple amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a single protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains (commonly referred to in the art as peptides, oligopeptides, or oligomers, for example) and longer chains (commonly referred to in the art as proteins, of which there are many types). "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, polypeptide mutants, polypeptide variants, and combinations thereof.

[0080] As used herein, "polynucleotide" encompasses cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms, mixed polymers (both sense and antisense strands), and may be chemically or biochemically modified to contain non-natural, derivatized, synthetic, or semi-synthetic nucleotide bases. Modifications of wild-type or synthetic genes, non-limiting examples of which include deletion, insertion, or substitution of one or more nucleotides, or fusion to other polynucleotide sequences, are also contemplated.

[0081] The phrase "preventing" a disease or disorder, as used herein, means reducing the severity or frequency of at least one sign or symptom of the disease or disorder that a subject will experience.

[0082] "Sample" or "biological sample," as used herein, refers to biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting mRNA, polypeptides, or other markers of physiological or pathological processes in a subject, and may include bodily fluids, tissues, cellular material, and / or non-cellular material obtained from an individual.

[0083] As used herein, "substantially purified" means essentially free of other components, e.g., a substantially purified polypeptide is one that is separated from other components that normally accompany it in the natural state.

[0084] As used herein, the term "treatment" or "treatment regimen" refers to activities undertaken to prevent, treat, or alter a disease or disorder (e.g., a course of treatment using pharmacological, surgical, dietary, or other techniques to reduce or eliminate at least one sign or symptom of a disease or disorder). A treatment regimen may include prescribed doses of one or more compounds or surgery. While treatment is often beneficial and reduces or eliminates at least one sign or symptom of a disease or disorder, in some cases, the effects of treatment will result in unwanted effects or side effects. The effects of treatment will also be affected by the physiological state of the subject (e.g., age, sex, genetics, weight, other disease states, etc.).

[0085] The phrase "therapeutically effective amount" refers to that amount of a compound or composition of the invention that will induce the biological, physiological, clinical, or medical response desired by a researcher, veterinarian, physician, or other clinician in a cell, tissue, organ, system, or subject. The phrase "therapeutically effective amount" includes an amount of a compound or composition that, when administered, is sufficient to prevent the progression of, or treat to some extent, one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0086] The phrase "treating" a disease or disorder, as used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. The terms "treatment," "treating," "treat," and the like, as used herein, generally refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, meaning that the disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that the disease and / or its resulting side effects are partially or completely stabilized or cured. The term "treatment" includes the treatment of a disease in a mammal, particularly a human. "Treatment" includes (a) preventing a disease and / or condition from occurring in a subject who is predisposed to, but has not yet been diagnosed with, the disease or condition; or (b) inhibiting the disease and / or condition, e.g., slowing or halting its progression (e.g., halting tumor growth, reducing the rate of tumor growth, eliminating the rate of cancer cell growth, etc.); or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease and / or condition (e.g., causing a decrease in tumor size, reducing the number of cancer cells present, etc.). Subjects in need of treatment include those already suffering from the disease (e.g., subjects with cancer, subjects with an infection, subjects with a metabolic disorder, subjects with macular degeneration, etc.), as well as subjects in whom prevention is desirable (e.g., subjects susceptible to cancer, subjects susceptible to infection, subjects suspected of having cancer, subjects suspected of having an infection, subjects susceptible to a metabolic disorder, subjects susceptible to macular degeneration, etc.).

[0087] As used herein, "wild-type" refers to a gene or gene product isolated from a natural source. A wild-type gene is that which is most commonly observed in a population and is therefore referred to as the "normal" or "wild-type" form of that gene. In contrast, the terms "altered," "variant," and "mutant" refer to genes or gene products that have altered sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.

[0088] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as imposing an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to include all the possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to include all specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6. This applies regardless of the breadth of the range.

[0089] explanation

[0090] In some embodiments, the compositions and methods of the invention include an activator of IL-18 activity (e.g., signaling activity through IL-18R). In some embodiments, the activator is an IL-18 variant polypeptide. In some embodiments, the activator is a molecule that can bind to IL-18R and signal through the IL-18R. In some embodiments, the activator is a molecule that promotes IL-18 signaling by inhibiting IL-18BP.

[0091] In some embodiments, the present invention provides an IL-18 variant polypeptide, or fragment thereof, that specifically binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, an IL-18 variant polypeptide, or fragment thereof, that binds to IL-18R but does not substantially bind to IL-18BP is useful for providing IL-18 signaling activity that is not inhibited by the presence and activity of IL-18BP.

[0092] In some embodiments, the IL-18 variant polypeptide is resistant to negative regulation by an IL-18BP polypeptide or is independent of negative regulation by an IL-18BP polypeptide. In some embodiments, the IL-18BP polypeptide is substantially unable to bind to an IL-18 variant polypeptide. The IL-18 variant polypeptides of the present invention exhibit reduced binding affinity for IL-18BP relative to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptides exhibit increased binding affinity for IL-18R relative to WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptides exhibit similar binding affinity for IL-18R as WT IL-18 polypeptide. In some embodiments, the IL-18 variant polypeptides exhibit reduced binding affinity for IL-18R relative to WT IL-18 polypeptide.

[0093] In some embodiments, the present invention provides compositions comprising IL-18BP inhibitors that inhibit or reduce either or both of the expression and activity of IL-18BP. Non-limiting examples of IL-18BP inhibitors include chemical compounds, proteins, peptidomimetics, antibodies, ribozymes, and antisense nucleic acid molecules. In some embodiments, the IL-18BP inhibitor comprises an IL-18 variant that binds to IL-18BP and thereby reduces or prevents the IL-18BP from inhibiting IL-18 and IL-18 signaling.

[0094] In some embodiments, IL-18 variant polypeptides are useful in treating or preventing a disease or disorder. In various embodiments, the disease or disorder is cancer or a metabolic disease or disorder, including obesity and diabetes (e.g., methods of the invention can reduce body fat). Thus, in some embodiments, the invention is a composition comprising at least one IL-18 variant polypeptide, or a fragment thereof. In another embodiment, the invention is a method of treating or preventing a disease or disorder, non-limiting examples of which include cancer or a metabolic disease or disorder, by administering at least one IL-18 variant polypeptide, or a fragment thereof.

[0095] In some embodiments, an IL-18 variant polypeptide binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is between about 0.000000000001% and about 95% of the binding affinity of wild-type IL-18 for IL-18BP.

[0096] In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 95% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 90% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 85% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 80% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 75% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 70% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 65% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 60% of the binding affinity of wild-type IL-18 for IL-18BP.In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 55% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 50% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 45% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 40% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 35% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 30% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 25% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 20% of the binding affinity of wild-type IL-18 for IL-18BP.In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 15% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 10% of the binding affinity of wild-type IL-18 for IL-18BP.

[0097] In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 5% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 4% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 3% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 2% of the binding affinity of wild-type IL-18 to IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 1% of the binding affinity of wild-type IL-18 to IL-18BP.

[0098] In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.1% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.01% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.0001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.00001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.000001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.0000001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.00000001% of the binding affinity of wild-type IL-18 for IL-18BP.In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.000000001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.0000000001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.00000000001% of the binding affinity of wild-type IL-18 for IL-18BP. In some embodiments, an IL-18 variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is about 0.000000000001% of the binding affinity of wild-type IL-18 for IL-18BP.

[0099] In some embodiments, the IL-18 variant polypeptides of the present invention (DR-IL-18) that bind to IL-18R and exhibit substantially reduced binding to IL-18BP exhibit a K D is 10 nM or more (larger K D (meaning a lower binding affinity). In some embodiments, the DR-IL-18 variant polypeptides of the invention have a K D is 20 nM or more (e.g., 50 nM or more, or 100 nM or more, or 1 μM or more).

[0100] In some embodiments, an IL-18 variant polypeptide binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about two-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18 (note that an increased dissociation constant ratio means that binding to IL-18BP is reduced relative to binding to IL-18R). In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about 20-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 200-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 2,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 20,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, the variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about 200,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 2,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 20,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.

[0101] In some embodiments, an IL-18 variant polypeptide binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about three-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about 30-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 300-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 3,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 30,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, the variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about 300,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 3,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 30,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.

[0102] In some embodiments, an IL-18 variant polypeptide binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about 5-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about 50-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 500-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 5,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 50,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, the variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about 500,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 5,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 50,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.

[0103] In some embodiments, an IL-18 variant polypeptide binds to IL-18R and exhibits substantially reduced binding to IL-18BP. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about 10-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has a dissociation constant ratio of IL-18BP / IL-18R that is at least about 100-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 1,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 10,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 100,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, a variant polypeptide that binds to IL-18R and exhibits substantially reduced binding to IL-18BP has an IL-18BP / IL-18R dissociation constant ratio that is at least about 1,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 10,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18. In some embodiments, variant polypeptides that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a dissociation constant ratio of IL-18BP / IL-18R that is at least about 100,000,000-fold greater than the IL-18BP / IL-18R dissociation constant ratio of wild-type IL-18.

[0104] In some embodiments, IL-18 variant polypeptides of the invention (DR-IL-18) that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have an inhibition constant (Ki) of greater than 3 nM with respect to IL-18BP (e.g., 5 nM or greater, or 10 nM or greater, or 50 nM or greater, or 100 nM or greater, or 500 nM or greater, or 750 nM or greater, or 1 μM or greater). In some embodiments, DR-IL-18 variant polypeptides of the invention have a Ki of 500 nM or greater with respect to IL-18BP. In some embodiments, DR-IL-18 variant polypeptides of the invention have a Ki of 1 μM or greater with respect to IL-18BP.

[0105] In some embodiments, IL-18 variant polypeptides of the invention (DR-IL-18) that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have a K i of greater than 200 nM with respect to IL-18BP (e.g., 500 nM or greater, or 750 nM or greater, or 1 μM or greater). In some embodiments, DR-IL-18 variant polypeptides of the invention have a K i of 1 μM or greater with respect to IL-18BP.

[0106] In some embodiments, IL-18 variant polypeptides of the invention (DR-IL-18) that bind to IL-18R and exhibit substantially reduced binding to IL-18BP have an inhibition constant (Ki) with respect to IL-18BP that is at least two-fold greater than the Ki of wild-type IL-18 with respect to IL-18BP (i.e., the Ki of an IL-18 variant polypeptide of the invention with respect to IL-18BP is at least two-fold greater than the Ki of WT IL-18 with respect to IL-18BP). For example, in some cases, a DR-IL-18 variant polypeptide of the invention has a Ki with respect to IL-18BP that is at least five-fold greater (e.g., at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 200-fold, or at least 500-fold, or at least 1000-fold) than the Ki of wild-type IL-18 with respect to IL-18BP.

[0107] In some embodiments, the IL-18 variant polypeptides of the present invention (DR-IL-18) that bind to IL-18R and exhibit substantially reduced binding to IL-18BP exhibit reduced EC2 activity for IL-18BP. 50 However, the EC 50 is at least two-fold greater than the EC of the IL-18 variant polypeptide of the invention relative to IL-18BP. 50 is the EC of WT IL-18 with respect to IL-18BP 50 For example, in some cases, the DR-IL-18 variant polypeptides of the invention have an EC ratio that is at least 2-fold higher than that of IL-18BP. 50 However, the EC 50 at least 5 times greater (e.g., at least 10 times, or at least 50 times, or at least 100 times, or at least 200 times, or at least 500 times, or at least 1000 times).

[0108] In various embodiments, the IL-18 variant polypeptide comprises one mutation relative to the wild-type (WT) IL-18 polypeptide. In some embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 31. Unless otherwise specified, the symbol "X" is used below to represent any amino acid.

[0109] In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, where X represents any amino acid. In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least four mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least six mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X.In some embodiments, the human IL-18 variant polypeptide, or fragment thereof, is selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60R, M60L, E77D, Q103E, Q103K, Q103P, Q103A, Q103R, S10 and at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of: S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H.In some embodiments, the human IL-18 variant polypeptide is selected from the group consisting of hCS1 (SEQ ID NO: 34), hCS2 (SEQ ID NO: 35), hCS3 (SEQ ID NO: 36), hCS4 (SEQ ID NO: 37), hC4 (SEQ ID NO: 38), hA8 (SEQ ID NO: 39), hD6 (SEQ ID NO: 40), hH12 (SEQ ID NO: 41), hB11 (SEQ ID NO: 42), hC3 (SEQ ID NO: 43), hC2 (SEQ ID NO: 44), hG10 (SEQ ID NO: 45), hG11 (SEQ ID NO: 46), hG21 (SEQ ID NO: 47), hG12 (SEQ ID NO: 48), hG13 (SEQ ID NO: 49), hG14 (SEQ ID NO: 50), hG15 (SEQ ID NO: 51), hG16 (SEQ ID NO: 52), hG17 (SEQ ID NO: 53), hG18 (SEQ ID NO: 54), hG19 (SEQ ID NO: 55), hG20 (SEQ ID NO: 56), hG21 (SEQ ID NO: 57), hG22 (SEQ ID NO: 58), hG23 (SEQ ID NO: 59), hG24 (SEQ ID NO: 60), hG25 (SEQ ID NO: 61), hG26 (SEQ ID NO: 62), hG27 (SEQ ID NO: 63), hG28 (SEQ ID NO: 64), hG29 (SEQ ID NO: 65), hG30 (SEQ ID NO: 66), hG31 (SEQ ID NO: 67), hG32 (SEQ ID NO: 68), hG33 (SEQ ID NO: ID No. 45), hG1 (SEQ ID No. 46), hF1 (SEQ ID No. 47), hD2 (SEQ ID No. 48), hA1 (SEQ ID No. 49), hB3 (SEQ ID No. 50), hB4 (SEQ ID No. 51), hH3 (SEQ ID No. 52), hH5 (SEQ ID No. 53), hH4 (SEQ ID No. 54), hE1 (SEQ ID No. 55), hG2 (SEQ ID No. 56), hB9 (SEQ ID No. 57), hE12 (SEQ ID No. 58), hC5 (SEQ ID No. No. 59), 5-18 (SEQ ID NO: 73), 5-29 (SEQ ID NO: 74), 5-8 (SEQ ID NO: 75), 5-6 (SEQ ID NO: 76), 5-27 (SEQ ID NO: 77), 5-20 (SEQ ID NO: 78), 5-2 (SEQ ID NO: 79), 5-9 (SEQ ID NO: 80), 5-42 (SEQ ID NO: 81), 5-13 (SEQ ID NO: 82), 5-12 (SEQ ID NO: 83), 5-1 (SEQ ID NO: 84), 5-33 (SEQ ID NO: 85), 5-21 (SEQ ID NO: 86), 6-31 (SEQ ID NO: 87), 6-20 (SEQ ID NO: 88), 6-12 (SEQ ID NO: 89), 6-27 (SEQ ID NO: 90), 6-29 (SEQ ID NO: 91), 5-26 (SEQ ID NO: 191), 5-17 (SEQ ID NO: 192), 5-41 (SEQ ID NO: 193), or a fragment thereof.

[0110] In some cases, DR-IL-18 variants of the invention, or fragments thereof, comprise at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30. In some cases, DR-IL-18 variants of the invention, or fragments thereof, comprise at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30. In some cases, DR-IL-18 variants of the invention, or fragments thereof, comprise at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5 relative to SEQ ID NO: 30. X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant or fragment thereof of the invention comprises at least three mutations (e.g., at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30. X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant or fragment thereof of the invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30.wherein X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0111] In some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X, M60X, S105X, D110X, and N111X. For example, in some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T, K, D, E, R, or N; X2 is K, Q, L, or R; X3 is R, D, K, A, or N; X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. For example, in some instances, a DR-IL-18 variant or fragment thereof of the invention contains the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T or K; X2 is K or L; X3 is D, N, or A; X4 is K, N, S, or G; and X5 is H, Y, G, or R. In other words, in some cases, a DR-IL-18 variant or fragment thereof of the invention comprises the following mutations relative to SEQ ID NO: 30: {M51T or M51K}; {M60K or M60L}; {S105D, S105N, or S105A}; {D110K, D110N, D110S, or D110G}; or {N111H, N111Y, N111R, or N111G}.

[0112] In some instances, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. In some instances, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least three mutations selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. In some instances, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30. X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant or fragment thereof of the invention comprises at least three mutations selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30, wherein X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30.X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0113] In some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X, K53X, Q56X, S105X, and N111X. For example, in some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some instances, a DR-IL-18 variant or fragment thereof of the present invention comprises the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is K; X2 is G or S; X3 is G, R, or L; X4 is S, N, or G; and X5 is G or R. In other words, in some instances, a DR-IL-18 variant or fragment thereof of the present invention comprises the following mutations relative to SEQ ID NO: 30: {M51K}; {K53G or K53S}; {Q56G, Q56R, or Q56L}; {D110S, D110N, or D110G}; {N111R or N111G}.

[0114] In some cases, a DR-IL-18 variant or fragment thereof of the present invention comprises an amino acid sequence that is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30. Thus, in some cases, a DR-IL-18 variant or fragment thereof of the present invention comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0115] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the amino acid sequence set forth in any one of SEQ ID NOS: 34-59, 73-91, and 191-193. Thus, in some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the amino acid sequence set forth in any one of SEQ ID NOS: 34-59, 73-91, 191-193, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0116] In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) comprises an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least four mutations, relative to SEQ ID NO: 30, selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X.In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least six mutations, relative to SEQ ID NO: 30, selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) comprises an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, relative to SEQ ID NO: 30.

[0117] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant or fragment thereof of the present invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that includes at least three mutations selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO:30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30. wherein X1 is any of T, K, D, E, R, or N; X2 is any of K, Q, L, or R; X3 is any of R, D, K, A, or N; X4 is any of H, K, N, Q, E, N, S, or G; and X5 is any of H, D, Y, R, S, or G.In some cases, a DR-IL-18 variant or fragment thereof of the present invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that, relative to SEQ ID NO:30, includes at least three mutations selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5. wherein X1 is any of T, K, D, E, R, or N; X2 is any of K, Q, L, or R; X3 is any of R, D, K, A, or N; X4 is any of H, K, N, Q, E, N, S, or G; and X5 is any of H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30, wherein X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0118] In some cases, a DR-IL-18 variant or fragment thereof of the present invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X, M60X, S105X, D110X, and N111X. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T, K, D, E, R, or N; X2 is K, Q, L, or R; X3 is R, D, K, A, or N; X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0119] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO:30. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that includes at least three mutations selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO:30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30. X2 is either G, S, or T; X3 is either E, A, R, V, G, K, or L; X4 is either N, S, K, or G; and X5 is either R, S, G, or D.In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least three mutations selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30, wherein X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5, relative to SEQ ID NO: 30, wherein X1 is K; X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0120] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO:30: M51X, K53X, Q56X, S105X, or N111X. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is K; X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0121] In some embodiments, the murine IL-18 variant polypeptide comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X, and L151X, where X represents any amino acid. In some embodiments, the murine IL-18 variant polypeptide, or fragment thereof, is selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56L, V56M, V56R, V56A, V56R ... and at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of 6S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R104Q, R104S, N109D, and L151V. In some embodiments, the murine IL-18 variant polypeptide comprises at least one variant selected from the group consisting of mCS1 (SEQ ID NO: 60), mCS2 (SEQ ID NO: 61), mC1 (SEQ ID NO: 62), mA12 (SEQ ID NO: 63), mE8 (SEQ ID NO: 64), mC10 (SEQ ID NO: 65), mB7 (SEQ ID NO: 66), mB1 (SEQ ID NO: 67), mD1 (SEQ ID NO: 68), mH7 (SEQ ID NO: 69), mA7 (SEQ ID NO: 70), mE1 (SEQ ID NO: 71), mH3 (SEQ ID NO: 72), or a fragment thereof.

[0122] In some embodiments, the invention is a nucleic acid (eg, DNA, cDNA, mRNA, etc.) encoding at least one IL-18 variant polypeptide.

[0123] Suppressant compositions and methods for treating

[0124] In various embodiments, the present invention includes IL-18BP-inhibiting compositions and methods for treating or preventing diseases or disorders in which it is desirable to reduce IL-18B activity or levels. The efficacy of such agents is encompassed by the efficacy detailed above for DR-IL-18 variants. Non-limiting examples of diseases or disorders in which it is desirable to reduce IL-18B activity or levels that can be treated or prevented using the compositions and methods of the present invention include cancer, infectious diseases, metabolic diseases or disorders, and macular degeneration. In various embodiments, the IL-18BP-inhibiting compositions and methods of the present invention for treatment or prevention reduce the amount of IL-18BP polypeptide, the amount of IL-18BP mRNA, the amount of IL-18BP enzymatic activity, the amount of IL-18BP substrate binding activity, or a combination thereof.

[0125] Based on the disclosure provided herein, those skilled in the art will understand that a reduction in the level of IL-18BP encompasses a reduction in the expression of IL-18BP (including either or both of transcription and translation). Armed with the teachings of the present invention, those skilled in the art will also understand that a reduction in the level of IL-18BP includes a reduction in IL-18BP activity (e.g., enzymatic activity, substrate binding activity, etc.). Thus, non-limiting examples of a reduction in the level or activity of IL-18BP include a reduction in either or both of transcription and translation, a reduction in nucleic acid encoding IL-18BP, and a reduction in any activity of the IL-18BP polypeptide. The IL-18BP-inhibiting compositions and IL-18BP-inhibiting methods of the present invention can selectively inhibit IL-18BP or can inhibit both IL-18BP and another molecule.

[0126] Suppression of IL-18BP can be assessed using a variety of methods, including those disclosed herein as well as methods known in the art or developed in the future. That is, based on the disclosure provided herein, one of skill in the art will understand that a decrease in IL-18BP level or activity can be readily assessed using methods that assess the level of nucleic acid (e.g., mRNA) encoding IL-18BP, the level of IL-18BP polypeptide present in a biological sample, the level of IL-18BP activity (e.g., enzymatic activity, substrate binding activity, etc.), or a combination thereof.

[0127] Those skilled in the art will understand, based on the disclosure provided herein, that the present invention is useful for treating or preventing a disease or disorder in a subject in need thereof, regardless of whether the subject is currently being treated with another drug or therapy. Furthermore, those skilled in the art will understand, based on the teachings provided herein, that the diseases or disorders that can be treated by the compositions and methods described herein include any disease or disorder in which IL-18BP plays a role and in which reducing IL-18BP levels or activity promotes a favorable outcome of treatment.

[0128] Non-limiting examples of IL-18BP-inhibiting compositions and IL-18BP-inhibiting methods of the present invention that reduce the level or activity (e.g., enzymatic activity, ligand-binding activity, etc.) of IL-18BP include compounds, polypeptides, peptides, peptidomimetics, antibodies, ribozymes, small molecule compounds, antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.), or combinations thereof. Based on the disclosure provided herein, one of ordinary skill in the art would readily understand that IL-18BP-inhibiting compositions encompass compounds that reduce the level or activity of IL-18BP. In addition, it is well known to those skilled in the chemical arts that IL-18BP-inhibiting compositions encompass chemically modified compounds and derivatives.

[0129] The IL-18BP-inhibiting compositions and methods of the present invention that reduce the level or activity (e.g., enzymatic activity, ligand-binding activity, etc.) of IL-18BP include antibodies. The antibodies of the present invention include a variety of antibodies, examples of which include polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, F(ab)2, single-chain antibodies (scFv), heavy-chain antibodies (e.g., camel antibodies), synthetic antibodies, chimeric antibodies, and humanized antibodies. In some embodiments, the antibodies of the present invention are antibodies that specifically bind to IL-18BP.

[0130] In some embodiments, IL-18BP inhibitors comprise modified IL-18 variants designed to bind IL-18BP but not substantially bind to or interact with IL-18R (e.g., IL-18Rα and IL-18Rβ). This variant ("decoy-to-decoy" (D2D)) serves to bind IL-18BP, thereby preventing IL-18BP from suppressing endogenously produced IL-18 or IL-18 introduced for therapeutic purposes. It is contemplated that "decoy-to-decoy" variants can be generated using substantially the same yeast display selection strategy as described elsewhere herein. However, unlike the selection strategy, positive selection for IL-18BP binding is performed, followed by counterselection for substantial binding to IL-18Rα.

[0131] In various embodiments, an IL-18 variant polypeptide that binds to and inhibits IL-18BP comprises a single mutation relative to a wild-type (WT) IL-18 polypeptide. In some embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 31.

[0132] In various embodiments, the IL-18 variant polypeptide that binds to and inhibits IL-18BP comprises a human IL-18 variant polypeptide or fragment thereof comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X, and R131X, wherein X represents any amino acid. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G, E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y The human IL-18 variant polypeptide or fragment thereof comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D40S, D40A, N41K, N41S, N41R, M51F, M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F, and R131S.In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include hD2D-5F12 (SEQ ID NO: 92), hD2D-5F11 (SEQ ID NO: 93), hD2D-5F10 (SEQ ID NO: 94), hD2D-5F08 (SEQ ID NO: 95), hD2D-5F06 (SEQ ID NO: 96), hD2D-5F04 (SEQ ID NO: 97), hD2D-5F02 (SEQ ID NO: 98), hD2D-5F0 1 (SEQ ID NO: 99), hD2D-5E10 (SEQ ID NO: 100), hD2D-5E08 (SEQ ID NO: 101), hD2D-5E03 (SEQ ID NO: 102), hD2D-5E02 (SEQ ID NO: 103), hD2D-5D10 (SEQ ID NO: 104), hD2D-5D08 (SEQ ID NO: 105), hD2D-5D06 (SEQ ID NO: 106), hD2D-5D05 (SEQ ID NO: 107), hD2D-5D03 (SEQ ID NO: 108), hD2D -5D02 (SEQ ID NO: 109), hD2D-5C10 (SEQ ID NO: 110), hD2D-5C09 (SEQ ID NO: 111), hD2D-5C08 (SEQ ID NO: 112), hD2D-5C05 (SEQ ID NO: 113), hD2D-5C04 (SEQ ID NO: 114), hD2D-5C03 (SEQ ID NO: 115), hD2D-5B11 (SEQ ID NO: 116), hD2D-5B10 (SEQ ID NO: 117), hD2D-5B06 (SEQ ID NO: 118 ), hD2D-5B05 (SEQ ID NO: 119), hD2D-5B02 (SEQ ID NO: 120), hD2D-5A09 (SEQ ID NO: 121), hD2D-5A02 (SEQ ID NO: 122), hD2D-CS1 (SEQ ID NO: 123), hD2D-CS2 (SEQ ID NO: 124), hD2D-CS3 (SEQ ID NO: 125), or a fragment thereof.

[0133] In some cases, a D2D-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X, E30X, and Q103X relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X1, E30X2, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variant of the present invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17G, E30A, (Q103L or Q103I) relative to SEQ ID NO: 30.

[0134] In some instances, a D2D-IL-18 variant or fragment thereof of the present invention comprises a D17X, E30X, or Q103X mutation relative to SEQ ID NO: 30. For example, in some instances, a D2D-IL-18 variant or fragment thereof of the present invention comprises a D17X1, E30X2, or Q103X3 mutation relative to SEQ ID NO: 30, where X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some instances, a D2D-IL-18 variant or fragment thereof of the present invention comprises a D17G, E30A, or (Q103L or Q103I) mutation relative to SEQ ID NO: 30.

[0135] In some instances, a D2D-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. In some instances, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X1, E30X2, D35X3, M51X4, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some cases, the D2D-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17G, E30A, D35S, M51F, (Q103L or Q103I) relative to SEQ ID NO: 30.

[0136] In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and Q103X. In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, D35X3, M51X4, and Q103X3, where X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17G, E30A, D35S, M51F, and (Q103L or Q103I).

[0137] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30. Thus, in some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0138] In some cases, the D2D-IL-18 variants or fragments thereof of the present invention comprise an amino acid sequence that is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the amino acid sequence set forth in any one of SEQ ID NOS: 126-190. Thus, in some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the amino acid sequence set forth in any one of SEQ ID NOS: 126-190, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0139] In some cases, a D2D-IL-18 variant of the present invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X, and R131X.

[0140] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, and Q103X relative to SEQ ID NO: 30. In some cases, a D2D-IL-18 variant of the present invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X1, E30X2, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, and (Q103L or Q103I) relative to SEQ ID NO: 30.

[0141] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contain an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, Q103X. In some cases, the D2D-IL-18 variants or fragments thereof of the present invention (i) are 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, and Q103X3, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contain an amino acid sequence that includes a D17X, E30X, (Q103L or Q103I) mutation relative to SEQ ID NO: 30.

[0142] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contain at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X1, E30X2, D35X3, M51X4, and Q103X3 relative to SEQ ID NO: 30. wherein X1 is either G, H, R, or A; X2 is either A, T, G, K, or R; X3 is either S, A, or Y; X4 is either F, I, or L; and X5 is I or L.In some cases, the D2D-IL-18 variants or fragments thereof of the present invention (i) comprise an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and (Q103L or Q103I) relative to SEQ ID NO: 30.

[0143] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and Q103X. In some cases, a D2D-IL-18 variant of the present invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, D35X3, M51X4, and Q103X3, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and (Q103L or Q103I).

[0144] In some embodiments, an IL-18 variant polypeptide that binds to and inhibits IL-18BP comprises a murine IL-18 variant polypeptide comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X, M111X, D129X, and D130X, wherein X represents any amino acid. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G, D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, 135K, 135R, D36V, D36A, D36G, D36H, D36P, D 36R, D36L, M50F, M50L, Q102L, Q102I, R104E, R104A, R104P, R104G, R104Q, R104H, H108D, H108 and a murine IL-18 variant polypeptide or fragment thereof comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of: A, N109R, N109S, N109T, N109L M111L, M111I, D129A, D129F, D129V, D129Y, D129S, D130E, D130T, D130G, D130N, D130R, D130S, D130Q, and D130H. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include mD2D-A5 (SEQ ID NO: 126), mD2D-A6 (SEQ ID NO: 127), mD2D-A7 (SEQ ID NO: 128), mD2D-A8 (SEQ ID NO: 129), mD2D-A9 (SEQ ID NO: 130), mD2D-A11 (SEQ ID NO: 131),mD2D-A12 (Sequence ID No. 132), mD2D-B4 (Sequence ID No. 133), mD2D-B7 (Sequence ID No. 134), mD2D-B11 (Sequence ID No. 135), mD2D-B12 (Sequence ID No. 136), mD2D-C1 (Sequence ID No. 137), mD2D-C3 (Sequence ID No. 138), mD2D-C5 (Sequence ID No. 139), mD2D-C6 (Sequence ID No. 140), mD2D-C9 (Sequence ID No. 141), mD2D-C10 (Sequence ID No. 142), mD2D-C11 (Sequence ID No. 143), mD2D-D1 (Sequence ID No. No. 144), mD2D-D9 (SEQ ID NO. 145), mD2D-D12 (SEQ ID NO. 146), mD2D-E3 (SEQ ID NO. 147), mD2D-E4 (SEQ ID NO. 148), mD2D-E5 (SEQ ID NO. 149), mD2D-E7 (SEQ ID NO. 150), mD2D-E8 (SEQ ID NO. 151), mD2D-E9 (SEQ ID NO. 152), mD2D-E10 (SEQ ID NO. 153), mD2D-E11 (SEQ ID NO. 154), mD2D-E12 (SEQ ID NO. 155), mD2D-F3 (SEQ ID NO. 156), mD2D-F4 ( Sequence ID number 157), mD2D-F5 (Sequence ID number 158), mD2D-F7 (Sequence ID number 159), mD2D-F8 (Sequence ID number 160), mD2D-F9 (Sequence ID number 161), mD2D-G1 (Sequence ID number 162), mD2D-G7 (Sequence ID number 163), mD2D-G9 (Sequence ID number 164), mD2D-H7 (Sequence ID number 165), mD2D-E1 (Sequence ID number 166), mD2D-G8 (Sequence ID number 167), mD2D-H3 (Sequence ID number 168), mD2D-A10 (Sequence ID number 169), mD2D-H 1 (SEQ ID NO: 170), mD2D-F12 (SEQ ID NO: 171), mD2D-G10 (SEQ ID NO: 172), mD2D-G12 (SEQ ID NO: 173), mD2D-E2 (SEQ ID NO: 174), mD2D-G11 (SEQ ID NO: 175), mD2D-C4 (SEQ ID NO: 176), mD2D-F11 (SEQ ID NO: 177), mD2D-C2 (SEQ ID NO: 178), mD2D-F10 (SEQ ID NO: 179), mD2D-A2 (SEQ ID NO: 180), mD2D-F6 (SEQ ID NO: 181), mD2D-A1 (SEQ ID NO: 182),The present invention comprises a mouse IL-18 variant polypeptide or a fragment thereof selected from the group consisting of mD2D-E6 (SEQ ID NO: 183), mD2D-D4 (SEQ ID NO: 184), mD2D-D6 (SEQ ID NO: 185), mD2D-A3 (SEQ ID NO: 186), mD2D-A4 (SEQ ID NO: 187), mD2D-B10 (SEQ ID NO: 188), mD2D-B8 (SEQ ID NO: 189), and mD2D-B9 (SEQ ID NO: 190), or a fragment thereof.

[0145] In some embodiments, the invention is a nucleic acid (eg, DNA, cDNA, mRNA, etc.) encoding at least one IL-18 variant polypeptide.

[0146] In some embodiments, the IL-18 inhibitor is an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R. In some embodiments, the IL-18BP inhibitor that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is between about 0.000000000001% and about 95% of the binding affinity of wild-type IL-18 for IL-18R.

[0147] In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 95% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 90% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 85% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 80% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 75% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 70% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 65% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 60% of the binding affinity of wild-type IL-18 to IL-18R.In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 55% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 50% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 45% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 40% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 35% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 30% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 25% of the binding affinity of wild-type IL-18 to IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 20% of the binding affinity of wild-type IL-18 to IL-18R.In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 15% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 10% of the binding affinity of wild-type IL-18 for IL-18R.

[0148] In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 5% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 4% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 3% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 2% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 1% of the binding affinity of wild-type IL-18 for IL-18R.

[0149] In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.1% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.01% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.0001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.00001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.000001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.0000001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.00000001% of the binding affinity of wild-type IL-18 for IL-18R.In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.000000001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.0000000001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.0000000001% of the binding affinity of wild-type IL-18 for IL-18R. In some embodiments, an IL-18 variant polypeptide that binds to IL-18BP and exhibits substantially reduced binding to IL-18R binds to IL-18R with a binding affinity that is about 0.000000000001% of the binding affinity of wild-type IL-18 for IL-18R.

[0150] Furthermore, one of skill in the art, instructed by the present disclosure and the methods exemplified herein, will understand that IL-18BP-inhibitory compositions, as detailed herein and / or known in the art, include inhibitors discovered in the future and inhibitors identifiable by criteria well known in the art of pharmacology (e.g., physiological consequences of inhibiting IL-18BP). Accordingly, the present invention is in no way limited to any particular IL-18BP-inhibitory compositions exemplified or disclosed herein, but rather encompasses inhibitory compositions known in the art and those discovered in the future that one of skill in the art would understand to be useful.

[0151] Other methods for identifying and producing IL-18BP-inhibiting compositions are well known to those skilled in the art, and non-limiting examples include obtaining the inhibitors from natural sources (e.g., Streptomyces spp., Pseudomonas spp., Stylotella aurantium, etc.). Alternatively, IL-18BP inhibitors can be chemically synthesized. Furthermore, those skilled in the art will understand, based on the teachings provided herein, that IL-18BP-inhibiting compositions can be obtained from recombinant organisms. Compositions and methods for chemically synthesizing IL-18BP inhibitors and for obtaining IL-18BP inhibitors from natural sources are well known in the art and described in the prior art.

[0152] Those skilled in the art will understand that inhibitors can be administered as small molecule compounds, proteins, antibodies, nucleic acid constructs encoding proteins, antisense nucleic acids, nucleic acid constructs encoding antisense nucleic acids, or combinations thereof. Numerous vectors and other compositions and methods are known for administering proteins or nucleic acid constructs encoding proteins to cells or tissues. Thus, the present invention includes methods for administering proteins (IL-18BP inhibitors) or nucleic acids encoding proteins. (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0153] Those skilled in the art will understand that reducing the amount or activity of a molecule that itself increases the amount or activity of IL-18BP may be useful in reducing the amount or activity of IL-18BP in the compositions and methods of the present invention.

[0154] Antisense oligonucleotides are DNA or RNA molecules that are complementary to a portion of an RNA molecule. When present in a cell, antisense oligonucleotides hybridize to the existing RNA molecule and inhibit translation into a gene product. The use of antisense oligonucleotides to inhibit gene expression is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as is a method for expressing antisense oligonucleotides in cells (Inoue, U.S. Patent No. 5,190,931). The methods of the present invention include using antisense oligonucleotides to decrease the amount or activity of IL-18BP by reducing the amount of IL-18BP or by reducing the amount of a molecule that causes an increase in the amount or activity of IL-18BP.

[0155] The present invention contemplates antisense oligonucleotides that are synthesized and delivered to cells by methods well known to those of skill in the art. By way of example, antisense oligonucleotides can be synthesized with lengths of about 10 to about 100 nucleotides, more commonly about 15 to about 50 nucleotides. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides that have improved biological activity relative to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,244).

[0156] Similarly, gene expression can be inhibited by hybridizing an antisense molecule to the gene's promoter or other regulatory element, thereby affecting transcription of that gene. Methods for identifying promoters or other regulatory elements that interact with a gene of interest are well known in the art, and include methods such as the yeast two-hybrid system (Bartel and Fields, eds., The Yeast Two Hybrid System, Oxford University Press, Cary, NC).

[0157] Alternatively, the suppression of genes expressing IL-18BP or genes expressing proteins that increase the level or activity of IL-18BP can be achieved using ribozymes. The use of ribozymes to suppress gene expression is well known to those skilled in the art (see, for example, Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence-specific, and therefore can be engineered to recognize specific nucleotide sequences (Cech, 1988, J. Amer. Med. Assn. 260:3030), thereby enabling the selective cleavage of specific mRNA molecules. It is believed that one of ordinary skill in the art would be able to synthesize an antisense oligonucleotide or ribozyme, given the nucleotide sequence of the molecule, without undue experimentation, based on this disclosure and the references incorporated therein.

[0158] Those skilled in the art will understand that inhibitors of IL-18BP can be administered acutely (e.g., for a short period of time (e.g., one day, one week, one month, etc.)) or chronically (e.g., for a longer period of time (e.g., several months, one year or more, etc.)). Those skilled in the art will understand that inhibitors of IL-18BP can be administered alone or in any combination with other agents. Furthermore, those skilled in the art will understand that IL-18BP inhibitors can be administered alone or in any combination temporally (concurrently and / or before or after each other). Those skilled in the art will understand, based on the disclosure provided herein, that IL-18BP-inhibitory compositions can be used to treat or prevent diseases or disorders in a subject in need thereof, and that inhibitory compositions can be used alone or in any combination with other agents to affect the treatment outcome.

[0159] In various embodiments, any of the IL-18BP inhibitors of the invention described herein can be administered alone or in combination with other inhibitors of other molecules implicated in diseases or disorders disclosed herein or known in the art.

[0160] Armed with this disclosure, including the methods detailed herein, those skilled in the art will understand that the present invention is not limited to the treatment of diseases or disorders already identified. In particular, the disease or disorder need not be manifested in a subject at the site of injury. Indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant disease or disorder need not occur before the present invention provides a potential benefit. Accordingly, the present invention includes methods for preventing a disease or disorder in a subject, whereby administration of an IL-18BP-suppressing composition, as discussed elsewhere, to a subject prior to the onset of the disease or disorder can prevent the disease or disorder from progressing. The methods of prevention described herein also include treating a subject in remission to prevent the recurrence of the disease or disorder.

[0161] Those skilled in the art, armed with the disclosure herein, will understand that prevention of a disease or disorder includes administering an IL-18BP-inhibitory composition to a subject as a prophylactic measure against that disease or disorder. As discussed in more detail elsewhere herein, methods of reducing the level or activity of IL-18BP encompass a wide range of techniques that reduce not only the activity of IL-18BP, but also the expression (including either or both of transcription and translation) of nucleic acids encoding IL-18BP.

[0162] Additionally, as disclosed elsewhere herein, one of skill in the art, armed with the teachings provided herein, will understand that the present invention encompasses methods for preventing a variety of diseases, disorders, and conditions, which can be alleviated, treated, or prevented by reducing the expression and / or activity of IL-18BP. Methods for assessing whether a disease is associated with IL-18BP levels or activity are known in the art. Furthermore, the present invention encompasses future-discovered methods of treating or preventing such diseases.

[0163] The present invention encompasses the administration of an inhibitor of IL-18BP to practice the methods of the present invention. One of skill in the art would know, based on the disclosure provided herein, how to prepare and administer a suitable IL-18BP inhibitor to a subject. However, the present invention is not limited to any particular administration method or treatment regimen.

[0164] Cytokine inhibitors

[0165] In some embodiments, the compositions of the invention comprise an inhibitor of one or more cytokines. In some embodiments, the inhibitor of one or more cytokines comprises a compound, protein, peptide, peptidomimetic, antibody, ribozyme, small molecule compound, or antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.) that inhibits the expression and / or activity of one or more cytokines. In some embodiments, the inhibitor inhibits the expression and / or activity of IL-17, IL-5, or IL-3. In some embodiments, the cytokine inhibitor reduces toxicity. In some embodiments, the cytokine inhibitor enhances the effect of an administered IL-18 variant polypeptide or IL-18BP inhibitor.

[0166] Compositions and methods for treatment and prevention

[0167] In various embodiments, the present invention includes compositions comprising activators of IL-18 activity (e.g., signaling activity through at least one IL-18R) and methods for increasing IL-18 activity (e.g., signaling activity through at least one IL-18R) in cells, tissues, organs, systems, or subjects in need thereof. In various embodiments, the compositions and methods of the present invention for therapeutically activating IL-18 activity increase the amount of IL-18R signaling and / or immune cell activity. In various embodiments, non-limiting examples of diseases and disorders for which increasing IL-18R signaling may improve therapeutic outcomes include cancer, infectious diseases, macular degeneration, and metabolic diseases or disorders.

[0168] The following are non-limiting examples of cancers that can be treated or prevented by the methods and compositions of the present invention: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood visual pathway glioma, chordoma, chronic lymphocytic leukemia, chronic bronchial leukemia, and bronchial sarcoidosis. Myeloid leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, esophageal cancer, Ewing's tumor, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, mycosis fungoides, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (dystonia), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, glioma of the hypothalamus and visual pathway, hypothalamic tumor, intraocular (ocular) ) cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, pharyngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytosis and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycoses, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myeloid leukemia, myeloma, myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharynx Neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytosis of bone, osteosarcoma and malignant fibrous histiocytosis of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade malignant tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer,Cancer of the renal pelvis and ureter, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, glioma of the visual tract and hypothalamus, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0169] Thus, non-limiting examples of cancers that can be treated or prevented by the methods and compositions of the present disclosure include solid tumor cancers, liquid cancers, hematologic cancers, teratomas, sarcomas, and carcinomas.

[0170] In some embodiments, the methods of the present invention are useful for treating or preventing tumors or cancers that are resistant to immune checkpoint inhibitors (ICIs). Non-limiting examples of immune checkpoint inhibitors include anti-PD1 (e.g., nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-TIMS, anti-TIGIT, anti-LAG3, anti-B7H3, anti-B7H4, anti-VISTA, anti-ICOS, anti-GITR, anti-4-1BB, anti-OX40, and anti-CD40. Non-limiting examples of targets of immune checkpoint inhibitors include PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, and CD40. Thus, examples of immune checkpoint inhibitors include agents that inhibit proteins such as PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, or CD40. In some cases, an IL-18 variant polypeptide of the invention (e.g., a DR-IL-18 variant, a D2D-IL-18 variant) is administered with an immune checkpoint inhibitor (such as an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, or CD40, or any combination thereof).

[0171] Fusions / Conjugations

[0172] In some embodiments, the IL-18 variant polypeptides of the present disclosure are fused to another protein. That is, the IL-18 variant polypeptides or fragments thereof can be fused in-frame with a second polypeptide (fusion partner). In some embodiments, the second polypeptide (fusion partner) can increase the overall size of the fusion protein so that, for example, the fusion protein will not be rapidly cleared from circulation. In some cases, the IL-18 variant polypeptides or fragments thereof are not fused to a second polypeptide.

[0173] In some embodiments, the second polypeptide (the fusion partner of the IL-18 variant polypeptide or fragment thereof) is part or all of an immunoglobulin Fc region (i.e., an antibody Fc sequence). In other embodiments, the second polypeptide is any suitable polypeptide that substantially resembles Fc, e.g., to increase size and / or provide a multimerization domain and / or provide additional binding or interaction with Ig molecules. In some embodiments, the second polypeptide is part or all of human serum albumin (HSA). In some embodiments, the second polypeptide is part or all of an antibody, antibody fragment, camelid antibody or "nanobody," or other affinity reagent that binds to or interacts with HSA. These fusion proteins facilitate purification and multimerization and can exhibit extended half-life in vivo. Fusion proteins with disulfide-linked multimeric structures may also, in some cases, allow for more efficient binding to or neutralization of other molecules.

[0174] An IL-18 variant polypeptide, or a fragment thereof, when fused to a heterologous polypeptide can be referred to as an "IL-18 variant polypeptide portion" of an IL-18 variant polypeptide of the invention. In some cases, the "IL-18 variant polypeptide portion" can be 100 or more amino acids in length (e.g., 110 or more, or 125 or more, or 150 or more, or 90 or more, or 95 or more, or 100 or more, or 105 or more, or 110 or more, or 115 or more, or 120 or more, or 125 or more, or 130 or more, or 140 or more, or 150 or more), up to full-length IL-18, and can be fused to a heterologous polypeptide.

[0175] In some cases, the IL-18 variant polypeptide portion of the IL-18 variant polypeptide is in the range of 100 to 157 amino acids in length (e.g., 100 to 150 amino acids, 100 to 140 amino acids, 140 to 157 amino acids, 140 to 150 amino acids, 145 to 157 amino acids, 150 to 157 amino acids).

[0176] In some embodiments, the second polypeptide is a marker sequence (e.g., an affinity tag), such as a peptide that facilitates purification of the fused polypeptide. For example, the marker amino acid sequence can be, among other things, a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eaton Avenue, Chatsworth, CA 91311), many of which are commercially available. Hexa-histidine is useful for purifying fusion proteins, as described, for example, in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984. The addition of peptide moieties to polypeptides to facilitate handling is a well-known and routine technique in the art.

[0177] The IL-18 variant polypeptides of the present invention can be modified for a variety of purposes, e.g., conjugated / conjugated to a variety of other oligonucleotides, and / or proteins and / or nonproteinaceous moieties. For example, they can be post-translationally modified, e.g., by prenylation, acetylation, amidation, carboxylation, glycosylation, PEGylation (covalent attachment of polyethylene glycol (PEG) polymer chains), etc. Such modifications can also include glycosylation modifications, e.g., by altering the glycosylation pattern of the polypeptide by exposing the polypeptide to enzymes that affect glycosylation (e.g., mammalian glycosylation or deglycosylation enzymes) during polypeptide synthesis and processing or in further processing steps. In some embodiments, the IL-18 variant polypeptides of the present invention have one or more phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

[0178] In some other embodiments, the IL-18 variant polypeptides of the present disclosure include additional modifications to improve resistance to proteolysis, optimize solubility properties, or make them more suitable as therapeutic agents. For example, variants of the present disclosure further include analogs containing residues other than naturally occurring L-amino acids (e.g., D-amino acids or non-natural synthetic amino acids). Some or all of the amino acid residues can be substituted with D-amino acids.

[0179] Co-administration and multispecific IL-18 variant polypeptides

[0180] As noted elsewhere in this disclosure, in some cases, the IL-18 variant polypeptides of the present invention are administered with an additional agent. The terms "co-administration," "administered together," and "in combination with" include simultaneous administration of two or more therapeutic agents (e.g., a combination of an IL-18 variant polypeptide of the present invention (e.g., DR-IL-18 or D2D) and an additional agent), administration in conjunction with each other, or sequential administration without specific time limitations. In some embodiments, the agents are present in a cell or in a subject's body at the same time or exert their biological or therapeutic effect simultaneously. In some embodiments, the therapeutic agents are present in the same composition or unit dosage form. In other embodiments, the therapeutic agents are present in separate compositions or multiple unit dosage forms. In some embodiments, a first agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks before) administration of a second agent, simultaneously with administration of the second agent, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks after) administration of the second agent.

[0181] In some cases, an IL-18 variant (e.g., a DR-IL-18 variant or a D2D variant) of the present invention (e.g., formulated as a pharmaceutical composition) is administered with a cancer therapeutic, or a therapeutic for treating an infectious disease, or an antibody directed against cancer. With respect to administration of one or more agents of the present disclosure, such administration may include administration in conjunction with (i.e., simultaneously with) the agent / antibody, administration before the agent / antibody, or administration after the agent / antibody. One of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence, and dosage of administration of the individual agents and compositions of the present disclosure.

[0182] In some embodiments, treatment is achieved by administering a combination (co-administration) of an IL-18 variant of the invention (e.g., a DR-IL-18 variant or a D2D variant) with another agent (e.g., an immunostimulant, an agent for treating a chronic infection, a cytotoxic agent, an anti-cancer agent, etc.). One class of cytotoxic agents that can be used is chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, thiazolinone ... bicine, ifosfamide, interferon-alpha irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine, prochlorperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0183] While the IL-18 variants of the invention (e.g., DR-IL-18 variants or D2D variants) need not be formulated, they are optionally formulated with one or more agents that enhance their activity or therapeutic effect. In some embodiments, treatment is achieved by administering a combination (co-administration) of an IL-18 variant of the invention (e.g., DR-IL-18 variant) with an agent that opsonizes target cells. Accordingly, compositions comprising (and methods of using) (a) an IL-18 variant of the invention (e.g., DR-IL-18 variant) and (b) an agent that opsonizes target cells are also contemplated herein. In some instances, the agent that opsonizes target cells is rituximab. In some instances, the agent that opsonizes target cells is cetuximab.

[0184] An "agent that opsonizes a target cell" ("opsonizing agent") is any agent that can bind to a target cell (e.g., a cancer cell, a cell harboring an intracellular pathogen, etc.) and opsonize the target cell (e.g., mark the target cell for phagocytosis and / or antibody-dependent cellular cytotoxicity (ADCC)). For example, any antibody that has an Fc region and can bind to a target cell (e.g., a cancer cell, such as a tumor cell) is considered to be an agent that opsonizes a target cell. In some cases, an agent that opsonizes a target cell is an antibody that binds to a target cell (e.g., an anti-tumor antibody, an anti-cancer antibody, an anti-infection antibody, etc.).

[0185] For example, see Kwon et al., Proc. Natl. Acad. Sci USA 96:15074-15079 (1999) for antibodies selective for tumor cell markers, radiation, surgery, and / or hormone deprivation. Antiangiogenic agents can also be combined with the methods of the present invention. Numerous antibodies are currently in clinical use to treat cancer, and others are in various stages of clinical development. Numerous antigens and corresponding monoclonal antibodies exist for treating, for example, B-cell malignancies. One target antigen is CD20. Rituximab is a chimeric, unconjugated monoclonal antibody directed against the CD20 antigen. CD20 plays an important functional role in B-cell activation, proliferation, and differentiation. The CD52 antigen is the target of the monoclonal antibody alemtuzumab. Alemtuzumab is indicated for the treatment of chronic lymphocytic leukemia. CD22 is the target of numerous antibodies, which have recently demonstrated efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Two new monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes. Alemtuzumab (Campath) is used to treat chronic lymphocytic leukemia, gemtuzumab (Mylotarg) has found use in the treatment of acute myeloid leukemia, ibritumomab (Zevalin) has found use in the treatment of non-Hodgkin's lymphoma, and panitumumab (Vectibix) has found use in the treatment of colon cancer.

[0186] Non-limiting examples of monoclonal antibodies that have been used in solid tumors and are useful in the methods of the present disclosure include edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1A antigen found in colon and rectal cancer and is approved for use in Europe for these indications. Trastuzumab targets the HER-2 / neu antigen. Cetuximab (Erbitux) is also of interest for use in the methods of the present disclosure. This antibody binds to the EGF receptor (EGFR) and has been used to treat solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

[0187] The IL-18 variant polypeptides of the invention (e.g., DR-IL-18 variants or D2D-IL-18 variants) can be combined with any of the agents described above (e.g., agents such as antibodies that opsonize target cells). Thus, in some cases, the IL-18 variant polypeptides of the invention (e.g., DR-IL-18 variants or D2D-IL-18 variants) are used (co-administered) in combination therapy with one or more opsonizing agents selective for cancer cells (e.g., tumor cells). In some cases, an IL-18 variant polypeptide of the invention (e.g., a DR-IL-18 variant or a D2D-IL-18 variant) is used in combination therapy (co-administered) with one or more of cetuximab (which binds EGFR), panitumumab (which binds EGFR), rituximab (which binds CD20), trastuzumab (which binds HER2), pertuzumab (which binds HER2), alemtuzumab (which binds CD52), brentuximab (which binds CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, and edrecolomab (which binds 17-1A), or a combination thereof.

[0188] In some cases, an IL-18 variant polypeptide of the invention (e.g., a DR-IL-18 variant or a D2D-IL-18 variant) is co-administered with a cancer cell opsonizing agent (an opsonizing agent comprising an antigen-binding region that targets, for example, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSFIR, SLAMF7, integrin α v β3, TYRP1, GP MB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAPl, or NRP1, or any combination thereof).

[0189] In some cases, an IL-18 variant polypeptide of the invention (e.g., a DR-IL-18 variant or a D2D-IL-18 variant) is administered with an agent that targets one or more antigens selected from CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

[0190] In some cases, an IL-18 variant polypeptide of the invention (e.g., a DR-IL-18 variant or a D2D-IL-18 variant) may be used in combination therapy with any suitable immunomodulatory agent (e.g., an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, a TIGIT antibody, a TIM3 antibody, a LAG3 antibody, a VISTA antibody, a B7H3 antibody, a B7H4 antibody, a CD40 agonist, a 4-1BB modulator (e.g., a 4-1BB agonist), an OX-40 modulator (e.g., an OX-40 agonist), a GITR modulator (e.g., a GITR agonist), a CD47 binder (e.g., an anti-CD47 antibody or a high-affinity CD47 binder), a SIRPA binder (e.g., an anti-SIRPA antibody or a high-affinity SIRPA binder)), a TGFβ antagonist (e.g., an anti-TGFβ antibody), or a combination thereof. ), cytokines or cytokine variants (including IL-1, IL-2, IL-10, IL-12, IL-15, IL-18, IL-21, IL-33, interferon alpha, interferon beta, interferon gamma, TNF, TRAIL, lymphotoxin, LIGHT / TNSF14), agonists of Toll-like receptors (including TLR2, TLR4, TLR5, TLR7, TLR9), agonists of inflammasomes, agonists of the STING / cGAS pathway, agonists of the RIG-I pathway, antagonists of adenosine receptors A2aR / A2bR, antagonists of the aryl hydrocarbon receptor, antagonists of IDO and / or TDO, or oncolytic viruses.

[0191] In some cases, IL-18 variant polypeptides of the invention (e.g., DR-IL-18 variants or D2D-IL-18 variants) are used (co-administered) in combination therapy with inhibitors of BTLA and / or CD160. In some cases, IL-18 variant polypeptides of the invention (e.g., DR-IL-18 variants or D2D-IL-18 variants) are used (co-administered) in combination therapy with anti-CD47 / SIRPA agents (e.g., anti-CD47, anti-SIRPA, high-affinity CD47 binders, high-affinity SIRPA binders, etc.). In some cases, IL-18 variant polypeptides of the invention (e.g., DR-IL-18 variants or D2D-IL-18 variants) are used (co-administered) in combination therapy with inhibitors of TIM3 and / or CEACAM1.

[0192] As noted above, in some cases, an IL-18 variant polypeptide of the invention is fused to another protein (i.e., a "fusion partner," a "second polypeptide"). In some embodiments, the second polypeptide (the fusion partner for an IL-18 variant polypeptide of the invention) specifically binds to a target molecule that is different from the target molecule bound by the IL-18 variant polypeptide portion of the fusion protein (e.g., other than IL-18R for IL-18R-binding variants, or other than IL-18BP for IL-18BP-binding variants).

[0193] Thus, in some embodiments, the IL-18 variant polypeptides of the present invention are multispecific (e.g., bispecific). The terms "multispecific" or "bispecific" are generally used to refer to an agent that recognizes two or more different antigens by virtue of having at least one region specific for a first target (the ligand or Fab of a first antibody) and at least one region specific for a second target (the ligand or Fab of a second antibody). A bispecific agent is a type of multispecific agent because it specifically binds to two targets.

[0194] In some embodiments, the IL-18 variant polypeptides of the invention are multispecific (bispecific), such that a first region of the polypeptide comprises an IL-18 variant polypeptide sequence of the invention (i.e., the first region comprises an IL-18 variant polypeptide) and a second region that specifically binds to another target molecule (e.g., an antigen). For example, in some cases, the IL-18 variant polypeptide is fused to a second polypeptide that specifically binds to a target molecule different from the target molecule bound by the IL-18 variant polypeptide.

[0195] Any of the agents discussed above in the context of co-administration can be conjugated to an IL-18 variant polypeptide of the invention. As used herein, the term "co-administered" encompasses such conjugated compounds. For example, when agent 1 is co-administered with agent 2, the term encompasses embodiments in which agent 1 and agent 2 are not conjugated to each other, as well as embodiments in which agent 1 and agent 2 are conjugated to each other (e.g., when both agent 1 and agent 2 are proteins and agent 1 is fused to agent 2).

[0196] In some cases, the second region of the multispecific IL-18 variant polypeptide is a checkpoint inhibitor. In some cases, the second region of the multispecific IL-18 variant polypeptide inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 4-1BB, OX40, and CD40.

[0197] In some cases, the second region of the multispecific IL-18 variant polypeptide is a cancer cell opsonizing agent. In some cases, the second region of the multispecific IL-18 variant polypeptide targets one or more proteins selected from CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3). In some cases, the second region of the multispecific IL-18 variant polypeptide is an opsonizing agent that targets one or more proteins selected from CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

[0198] For example, in some cases, the second region of the multispecific IL-18 variant polypeptide comprises an ectodomain (e.g., an ectodomain from PD-1, PD-L1, CD47 (e.g., a high affinity CD47 variant / polypeptide), SIRPA (e.g., a high affinity SIRPA variant / polypeptide)). In some cases, the second region of the multispecific IL-18 variant polypeptide specifically binds to an antigen selected from CTLA-4, Lag-3, BTLA, Tim-3, CD244, CD40, CD40L, CD47, SIRPA, PD-1, and PD-L1.

[0199] In some embodiments, the IL-18 variant polypeptides of the present invention comprise a linker (e.g., a linker polypeptide). For example, in some embodiments, the IL-18 variant polypeptides of the present invention and the fusion partner are separated by a linker (e.g., a linker polypeptide). The linker polypeptide can have any one of a variety of amino acid sequences. Proteins can be joined by a flexible linker polypeptide (e.g., a flexible linker polypeptide), although other chemical linkages are not excluded. Suitable linkers include polypeptides from about 6 to about 40 amino acids in length, or from about 6 to about 25 amino acids in length. These linkers can be created by coupling proteins using synthetic oligonucleotides encoding the linker. Peptide linkers that are somewhat flexible can be used. The linking peptide can have virtually any amino acid sequence, although it should be understood that linker sequences can generally result in flexible peptides. The use of small amino acids (e.g., glycine, alanine, etc.) is useful for creating flexible peptides. Creating such sequences is routine for those skilled in the art. A variety of different linkers are commercially available and may be suitable for use.

[0200] In some embodiments, the IL-18 variant polypeptide is co-administered with engineered immune cells (e.g., CAR-T cells, CAR-NK cells, T cells, NK cells transduced with engineered T cell receptors). In another embodiment, the IL-18 variant polypeptide is co-administered with an oncolytic virus.

[0201] In some embodiments, nucleic acids encoding IL-18 variant polypeptides are comprised in engineered ("altered") immune cells (e.g., CAR-T cells, CAR-NK cells, T cells, NK cells transduced with an engineered T cell receptor). In this case, the engineered cells (altered T cells, altered NK cells) will secrete IL-18 variant polypeptides. The ability to secrete IL-18 variant polypeptides can be regulated in a context-dependent manner (e.g., turned on in the tumor microenvironment), for example, by a synthetic NOTCH receptor.

[0202] In some embodiments, the nucleic acid encoding the IL-18 variant polypeptide is contained within an oncolytic virus, in which case cells infected with the oncolytic virus will secrete the IL-18 variant polypeptide.

[0203] In some embodiments, the methods of the invention are useful for treating or preventing tumors or cancers that have lost surface MHC class I expression (e.g., tumors that have lost B2m (MHC locus) or tumors that have mutations in other members of the antigen-presenting and / or antigen-loading complex, such as tapasin).

[0204] Metabolic diseases and disorders include a variety of metabolic and endocrine-related diseases and disorders. Non-limiting examples of metabolic and endocrine-related diseases and disorders that can be treated or prevented by the methods and compositions of the present invention include obesity, diabetes, prediabetes, type 2 diabetes, maturity-onset diabetes of the young (MODY), hyperglycemia, metabolic syndrome, dyslipidemia, hypertriglyceridemia, and hypercholesterolemia.

[0205] Non-limiting examples of other diseases and disorders that can be treated or prevented using the compositions and methods of the present invention include viral infections, bacterial infections, parasitic infections, and low immune activity. In some embodiments, the viral infection is at least one of a poxvirus infection, a smallpox virus infection, a molluscum contagiosum infection, an HPV infection, or a viral wart. In some embodiments, the infection is a systemic infection. In some embodiments, the viral infection is a vaccinia virus infection. In some embodiments, the viral infection is a systemic vaccinia virus infection. In some embodiments, the bacterial infection is sepsis. In some embodiments, the low immune activity is neutropenia, which may be caused, for example, by chemotherapy.

[0206] Non-limiting examples of other diseases and disorders that can be treated or prevented using the compositions and methods of the present invention include macular degeneration. For example, in some cases, the disease or disorder is wet macular degeneration, and in some cases, the disease or disorder is wet age-related macular degeneration. In some such cases, IL-18 variants can be used as anti-angiogenic agents. For example, IL-18 variant polypeptides of the present invention can reduce choroidal neovascularization in some cases.

[0207] Thus, the present invention relates to the prevention and treatment of a disease or disorder, or its associated signs or symptoms, by administering a therapeutically effective amount of an IL-18 variant polypeptide, a recombinant IL-18 variant polypeptide, an active IL-18 variant polypeptide fragment (e.g., an IL-18 variant peptide), an activator of IL-18 variant expression or activity, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding at least one IL-18 variant polypeptide to a cell, tissue, organ, or subject in need thereof, for the purpose of treating or preventing the disease or disorder, or its associated signs or symptoms.

[0208] In some embodiments, compositions of the present invention are administered to cells, tissues, organs, systems, or subjects to treat or prevent a disease or disorder. In some embodiments, human IL-18 variant polypeptides are administered to cells, tissues, organs, systems, or subjects. In some embodiments, nucleic acids (e.g., DNA, cDNA, mRNA, etc.) encoding at least one human IL-18 variant polypeptide are administered to cells, tissues, organs, systems, or subjects.

[0209] In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least one mutation (e.g., at least two, at least three, at least four, at least five, at least six) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, N155X, where X represents any amino acid. In various embodiments, the human IL-18 variant polypeptide or fragment thereof comprises at least four mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, where X represents any amino acid. In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least six mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In various embodiments, the human IL-18 variant polypeptide, or fragment thereof, comprises at least one mutation (e.g., at least two, at least three, at least four, at least five, at least six mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X.

[0210] In some embodiments, the human IL-18 variant polypeptide or fragment thereof is selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60R, M60L, E77D, Q103E, Q103K, Q103P, Q103A, Q103R , S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, DUOS, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H.In some embodiments, the human IL-18 variant polypeptide or fragment thereof is selected from the group consisting of hCS1 (SEQ ID NO: 34), hCS2 (SEQ ID NO: 35), hCS3 (SEQ ID NO: 36), hCS4 (SEQ ID NO: 37), hC4 (SEQ ID NO: 38), hA8 (SEQ ID NO: 39), hD6 (SEQ ID NO: 40), hH12 (SEQ ID NO: 41), hB11 (SEQ ID NO: 42), hC3 (SEQ ID NO: 43), hC2 (SEQ ID NO: 44), h G10 (SEQ ID NO: 45), hG1 (SEQ ID NO: 46), hF1 (SEQ ID NO: 47), hD2 (SEQ ID NO: 48), hA1 (SEQ ID NO: 49), hB3 (SEQ ID NO: 50), hB4 (SEQ ID NO: 51), hH3 (SEQ ID NO: 52), hH5 (SEQ ID NO: 53), hH4 (SEQ ID NO: 54), hE1 (SEQ ID NO: 55), hG2 (SEQ ID NO: 56), hB9 (SEQ ID NO: 57), hE12 (SEQ ID NO: 58), hC5 (SEQ ID NO: 59), 5-18 (SEQ ID NO: 73), 5-29 (SEQ ID NO: 74), 5-8 (SEQ ID NO: 75), 5-6 (SEQ ID NO: 76), 5-27 (SEQ ID NO: 77), 5-20 (SEQ ID NO: 78), 5-2 (SEQ ID NO: 79), 5-9 (SEQ ID NO: 80), 5-42 (SEQ ID NO: 81), 5-13 (SEQ ID NO: 82), 5-12 (SEQ ID NO: 83), 5-1 (SEQ ID NO: 84), 5-33 (SEQ ID NO: 85) , 5-21 (SEQ ID NO: 86), 6-31 (SEQ ID NO: 87), 6-20 (SEQ ID NO: 88), 6-12 (SEQ ID NO: 89), 6-27 (SEQ ID NO: 90), 6-29 (SEQ ID NO: 91), 5-26 (SEQ ID NO: 191), 5-17 (SEQ ID NO: 192), 5-41 (SEQ ID NO: 193), or a fragment thereof.

[0211] In some instances, a DR-IL-18 variant or fragment thereof of the invention comprises at least one mutation (e.g., at least two, at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30. In some instances, a DR-IL-18 variant or fragment thereof of the invention comprises at least three mutations selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30. In some instances, a DR-IL-18 variant or fragment thereof of the invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5 relative to SEQ ID NO: 30. X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant or fragment thereof of the invention comprises at least three mutations selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30. X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant or fragment thereof of the invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30.wherein X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0212] In some instances, DR-IL-18 variants or fragments thereof of the invention comprise the following mutations relative to SEQ ID NO: 30: M51X, M60X, S105X, D110X, and N111X. For example, in some instances, DR-IL-18 variants or fragments thereof of the invention comprise the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T, K, D, E, R, or N; X2 is K, Q, L, or R; X3 is R, D, K, A, or N; X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. For example, in some instances, a DR-IL-18 variant or fragment thereof of the invention contains the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T or K; X2 is K or L; X3 is D, N, or A; X4 is K, N, S, or G; and X5 is H, Y, G, or R. In other words, in some cases, a DR-IL-18 variant or fragment thereof of the invention comprises the following mutations relative to SEQ ID NO: 30: {M51T or M51K}; {M60K or M60L}; {S105D, S105N, or S105A}; {D110K, D110N, D110S, or D110G}; {N111H, N111Y, N111R, or N111G}.

[0213] In some instances, DR-IL-18 variants of the invention, or fragments thereof, comprise at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. In some instances, DR-IL-18 variants of the invention, or fragments thereof, comprise at least three mutations selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. In some instances, DR-IL-18 variants of the invention, or fragments thereof, comprise at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30. X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least three mutations selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30, wherein X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30.X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0214] In some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X, K53X, Q56X, S105X, and N111X. For example, in some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations (e.g., at least two, or at least two, of SEQ ID NO: 30): M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some instances, DR-IL-18 variants of the invention, or fragments thereof, include the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. X4 is S, N, or G; and X5 is G or R. In other words, in some cases, DR-IL-18 variants of the invention, or fragments thereof, contain the following mutations relative to SEQ ID NO: 30: {M51K}; {K53G or K53S}; {Q56G, Q56R, or Q56L}; {D110S, D110N, or D110G}; {N111R or N111G}.

[0215] In some cases, the DR-IL-18 variants of the invention, or fragments thereof, comprise an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30. Thus, in some cases, a DR-IL-18 variant or fragment thereof of the present invention comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0216] In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) comprises an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least four mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, relative to SEQ ID NO: 30.In some cases, a DR-IL-18 variant or fragment thereof of the invention (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that, relative to SEQ ID NO: 30, includes at least six mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X, relative to SEQ ID NO: 30.

[0217] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least three mutations (e.g., at least three, or at least four mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30.wherein X1 is any of T, K, D, E, R, or N; X2 is any of K, Q, L, or R; X3 is any of R, D, K, A, or N; X4 is any of H, K, N, Q, E, N, S, or G; and X5 is any of H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that, relative to SEQ ID NO: 30, includes at least three mutations selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5. wherein X1 is any of T, K, D, E, R, or N; X2 is any of K, Q, L, or R; X3 is any of R, D, K, A, or N; X4 is any of H, K, N, Q, E, N, S, or G; and X5 is any of H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, M60X2, S105X3, D110X4, and N111X5, relative to SEQ ID NO: 30, wherein X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0218] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X, M60X, S105X, D110X, or N111X. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T, K, D, E, R, or N; X2 is K, Q, L, or R; X3 is R, D, K, A, or N; X4 is H, K, N, Q, E, N, S, or G; and X5 is H, D, Y, R, S, or G. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, M60X2, S105X3, D110X4, and N111X5, where X1 is T or K; X2 is K or L; X3 is either D, N, or A; X4 is either K, N, S, or G; and X5 is either H, Y, G, or R.

[0219] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO:30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that includes at least three mutations selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO:30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) contains at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO:30. X2 is either G, S, or T; X3 is either E, A, R, V, G, K, or L; X4 is either N, S, K, or G; and X5 is either R, S, G, or D.In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least three mutations selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5 relative to SEQ ID NO: 30, wherein X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of M51X1, K53X2, Q56X3, S105X4, and N111X5, relative to SEQ ID NO: 30, wherein X1 is K; X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0220] In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO:30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO:30: M51X, K53X, Q56X, S105X, and N111X. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is E, R, or K; X2 is G, S, or T; X3 is E, A, R, V, G, K, or L; X4 is N, S, K, or G; and X5 is R, S, G, or D. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: M51X1, K53X2, Q56X3, S105X4, and N111X5, where X1 is K; X2 is G or S; X3 is either G, R, or L; X4 is either S, N, or G; and X5 is G or R.

[0221] In various embodiments, the IL-18 variant polypeptide that binds to and inhibits IL-18BP comprises a human IL-18 variant polypeptide or fragment thereof comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X, and R131X, wherein X represents any amino acid. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G, E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y The human IL-18 variant polypeptide or fragment thereof comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D40S, D40A, N41K, N41S, N41R, M51F, M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F, and R131S.In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include hD2D-5F12 (SEQ ID NO: 92), hD2D-5F11 (SEQ ID NO: 93), hD2D-5F10 (SEQ ID NO: 94), hD2D-5F08 (SEQ ID NO: 95), hD2D-5F06 (SEQ ID NO: 96), hD2D-5F04 (SEQ ID NO: 97), hD2D-5F02 (SEQ ID NO: 98), hD2D-5F 01 (SEQ ID NO: 99), hD2D-5E10 (SEQ ID NO: 100), hD2D-5E08 (SEQ ID NO: 101), hD2D-5E03 (SEQ ID NO: 102), hD2D-5E02 (SEQ ID NO: 103), hD2D-5D10 (SEQ ID NO: 104), hD2D-5D08 (SEQ ID NO: 105), hD2D-5D06 (SEQ ID NO: 106), hD2D-5D05 (SEQ ID NO: 107), hD2D-5D03 (SEQ ID NO: 108), h hD2D-5D02 (SEQ ID NO: 109), hD2D-5C10 (SEQ ID NO: 110), hD2D-5C09 (SEQ ID NO: 111), hD2D-5C08 (SEQ ID NO: 112), hD2D-5C05 (SEQ ID NO: 113), hD2D-5C04 (SEQ ID NO: 114), hD2D-5C03 (SEQ ID NO: 115), hD2D-5B11 (SEQ ID NO: 116), hD2D-5B10 (SEQ ID NO: 117), hD2D-5B06 (SEQ ID NO: 118), No. 118), hD2D-5B05 (SEQ ID NO: 119), hD2D-5B02 (SEQ ID NO: 120), hD2D-5A09 (SEQ ID NO: 121), hD2D-5A02 (SEQ ID NO: 122), hD2D-CS1 (SEQ ID NO: 123), hD2D-CS2 (SEQ ID NO: 124), hD2D-CS3 (SEQ ID NO: 125), or a fragment thereof.

[0222] In some cases, a D2D-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X, E30X, and Q103X relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X1, E30X2, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variant or fragment thereof of the present invention comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17G, E30A, and (Q103L or Q103I).

[0223] In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X, E30X, and Q103X. For example, in some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, and Q103X3, where X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations: D17G, E30A, and (Q103L or Q103I).

[0224] In some cases, a D2D-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. In some cases, a DR-IL-18 variant of the invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17X1, E30X2, D35X3, M51X4, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some cases, the D2D-IL-18 variant of the present invention, or a fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four mutations) selected from the group consisting of D17G, E30A, D35S, M51F, and (Q103L or Q103I) relative to SEQ ID NO: 30.

[0225] In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and Q103X. In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, D35X3, M51X4, and Q103X3, where X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some instances, D2D-IL-18 variants of the invention, or fragments thereof, comprise the following mutations relative to SEQ ID NO: 30: D17G, E30A, D35S, M51F, and (Q103L or Q103I).

[0226] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30. Thus, in some cases, the D2D-IL-18 variants or fragments thereof of the present invention comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) relative to wild-type IL-18 (e.g., human IL-18).

[0227] In some cases, a D2D-IL-18 variant of the present invention, or a fragment thereof, comprises an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X, and R131X.

[0228] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, and Q103X relative to SEQ ID NO: 30. In some cases, a D2D-IL-18 variant of the present invention, or a fragment thereof, (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprises an amino acid sequence that includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X1, E30X2, and Q103X3 relative to SEQ ID NO: 30, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, and (Q103L or Q103I) relative to SEQ ID NO: 30.

[0229] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contain an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, Q103X. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, Q103X3, where X1 is G, H, R, or A; X2 is A, T, G, K, or R; and X3 is I or L. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, and (Q103L or Q103I).

[0230] In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) contain at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X1, E30X2, D35X3, M51X4, and Q103X3 relative to SEQ ID NO: 30. wherein X1 is either G, H, R, or A; X2 is either A, T, G, K, or R; X3 is either S, A, or Y; X4 is either F, I, or L; and X5 is I or L.In some cases, the D2D-IL-18 variants of the present invention, or fragments thereof, comprise an amino acid sequence that (i) is 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) includes at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of D17X, E30X, D35X, M51X, and (Q103L or Q103I) relative to SEQ ID NO: 30.

[0231] In some cases, the D2D-IL-18 variants or fragments thereof of the present invention (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, Q103X. In some cases, the D2D-IL-18 variants or fragments thereof of the present invention (i) are 85% or more identical (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X1, E30X2, D35X3, M51X4, and Q103X3, wherein X1 is G, H, R, or A; X2 is A, T, G, K, or R; X3 is S, A, or Y; X4 is F, I, or L; and X5 is I or L. In some cases, the D2D-IL-18 variants or fragments thereof of the present invention (i) are 85% or more (e.g., 90% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more) identical to the wild-type human IL-18 amino acid sequence set forth in SEQ ID NO: 30, and (ii) comprise an amino acid sequence that includes the following mutations relative to SEQ ID NO: 30: D17X, E30X, D35X, M51X, and (Q103L or Q103I).

[0232] In some embodiments, compositions of the invention are administered to a mouse cell, a mouse tissue, a mouse organ, a mouse system, or a mouse subject to treat or prevent a disease or disorder. In some embodiments, a mouse IL-18 variant polypeptide, or a fragment thereof, is administered to a cell, tissue, organ, system, or subject (e.g., a human cell, human tissue, human organ, human system, or human subject). In some embodiments, a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding at least one mouse IL-18 variant polypeptide is administered to a cell, tissue, organ, system, or subject.

[0233] In some embodiments, the murine IL-18 variant polypeptide, or fragment thereof, comprises at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X, and L151X, where X represents any amino acid. In some embodiments, the murine IL-18 variant polypeptide or fragment thereof is selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56L, V56M, V56R, V56A, V56R ... and at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of 6S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R104Q, R104S, N109D, and L151V. In some embodiments, the murine IL-18 variant polypeptide comprises at least one variant selected from the group consisting of mCS1 (SEQ ID NO: 60), mCS2 (SEQ ID NO: 61), mC1 (SEQ ID NO: 62), mA12 (SEQ ID NO: 63), mE8 (SEQ ID NO: 64), mC10 (SEQ ID NO: 65), mB7 (SEQ ID NO: 66), mB1 (SEQ ID NO: 67), mD1 (SEQ ID NO: 68), mH7 (SEQ ID NO: 69), mA7 (SEQ ID NO: 70), mE1 (SEQ ID NO: 71), mH3 (SEQ ID NO: 72), or a fragment thereof.

[0234] In some embodiments, an IL-18 variant polypeptide that binds to and inhibits IL-18BP comprises a murine IL-18 variant polypeptide comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X, M111X, D129X, and D130X, wherein X represents any amino acid. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G, D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, 135K, 135R, D36V, D36A, D36G, D36H, D36P, D 36R, D36L, M50F, M50L, Q102L, Q102I, R104E, R104A, R104P, R104G, R104Q, R104H, H108D, H108 and a murine IL-18 variant polypeptide, or fragment thereof, comprising at least one mutation (e.g., at least two, or at least three, or at least four, or at least five, or at least six mutations) selected from the group consisting of: A, N109R, N109S, N109T, N109L M111L, M111I, D129A, D129F, D129V, D129Y, D129S, D130E, D130T, D130G, D130N, D130R, D130S, D130Q, and D130H. In some embodiments, the IL-18 variant polypeptides that bind to and inhibit IL-18BP include mD2D-A5 (SEQ ID NO: 126), mD2D-A6 (SEQ ID NO: 127), mD2D-A7 (SEQ ID NO: 128), mD2D-A8 (SEQ ID NO: 129), mD2D-A9 (SEQ ID NO: 130), mD2D-A11 (SEQ ID NO: 131),mD2D-A12 (Sequence ID No. 132), mD2D-B4 (Sequence ID No. 133), mD2D-B7 (Sequence ID No. 134), mD2D-B11 (Sequence ID No. 135), mD2D-B12 (Sequence ID No. 136), mD2D-C1 (Sequence ID No. 137), mD2D-C3 (Sequence ID No. 138), mD2D-C5 (Sequence ID No. 139), mD2D-C6 (Sequence ID No. 140), mD2D-C9 (Sequence ID No. 141), mD2D-C10 (Sequence ID No. 142), mD2D-C11 (Sequence ID No. 143), mD2D-D1 (Sequence ID No. No. 144), mD2D-D9 (SEQ ID NO. 145), mD2D-D12 (SEQ ID NO. 146), mD2D-E3 (SEQ ID NO. 147), mD2D-E4 (SEQ ID NO. 148), mD2D-E5 (SEQ ID NO. 149), mD2D-E7 (SEQ ID NO. 150), mD2D-E8 (SEQ ID NO. 151), mD2D-E9 (SEQ ID NO. 152), mD2D-E10 (SEQ ID NO. 153), mD2D-E11 (SEQ ID NO. 154), mD2D-E12 (SEQ ID NO. 155), mD2D-F3 (SEQ ID NO. 156), mD2D-F4 ( Sequence ID number 157), mD2D-F5 (Sequence ID number 158), mD2D-F7 (Sequence ID number 159), mD2D-F8 (Sequence ID number 160), mD2D-F9 (Sequence ID number 161), mD2D-G1 (Sequence ID number 162), mD2D-G7 (Sequence ID number 163), mD2D-G9 (Sequence ID number 164), mD2D-H7 (Sequence ID number 165), mD2D-E1 (Sequence ID number 166), mD2D-G8 (Sequence ID number 167), mD2D-H3 (Sequence ID number 168), mD2D-A10 (Sequence ID number 169), mD2D-H 1 (SEQ ID NO: 170), mD2D-F12 (SEQ ID NO: 171), mD2D-G10 (SEQ ID NO: 172), mD2D-G12 (SEQ ID NO: 173), mD2D-E2 (SEQ ID NO: 174), mD2D-G11 (SEQ ID NO: 175), mD2D-C4 (SEQ ID NO: 176), mD2D-F11 (SEQ ID NO: 177), mD2D-C2 (SEQ ID NO: 178), mD2D-F10 (SEQ ID NO: 179), mD2D-A2 (SEQ ID NO: 180), mD2D-F6 (SEQ ID NO: 181), mD2D-A1 (SEQ ID NO: 182),The present invention relates to a method for producing a mouse IL-18 variant polypeptide, or a fragment thereof, selected from the group consisting of mD2D-E6 (SEQ ID NO: 183), mD2D-D4 (SEQ ID NO: 184), mD2D-D6 (SEQ ID NO: 185), mD2D-A3 (SEQ ID NO: 186), mD2D-A4 (SEQ ID NO: 187), mD2D-B10 (SEQ ID NO: 188), mD2D-B8 (SEQ ID NO: 189), and mD2D-B9 (SEQ ID NO: 190).

[0235] In some embodiments, the methods of the invention comprise administering to a subject, cell, or tissue an isolated nucleic acid molecule encoding an IL-18 variant polypeptide described herein.

[0236] Those skilled in the art will appreciate that increasing the level of IL-18 signaling through IL-18R encompasses increasing the amount of IL-18 or IL-18 variant polypeptide available to bind to and activate IL-18R. This can be achieved by increasing the level or activation of IL-18. Non-limiting examples of ways include direct or indirect administration of IL-18, direct or indirect administration of an IL-18 variant polypeptide, direct or indirect administration of an inhibitor of IL-18BP, as well as increasing the transcription and / or translation of nucleic acids encoding IL-18 or IL-18 variant polypeptides, including increasing any activity of IL-18 or IL-18 variant polypeptides.

[0237] Increased levels of IL-18 signaling (including increased levels due to the use of IL-18 variant polypeptides) can be assessed using a variety of methods, including those disclosed herein as well as methods known in the art and methods developed in the future. That is, based on the disclosure provided herein, one of skill in the art will understand that increased levels or activity of IL-18 signaling can be readily assessed using methods that assess the level of nucleic acid (e.g., mRNA) encoding IL-18, an IL-18 variant polypeptide, or a fragment thereof, and / or the level of IL-18, an IL-18 variant polypeptide, or a fragment polypeptide, and / or the level of activity of IL-18, an IL-18 variant polypeptide, or a fragment thereof in a biological sample obtained from a subject.

[0238] Based on the disclosure provided herein, one of skill in the art will understand that the present invention is useful for subjects currently being or will be treated for a disease or disorder in which increased IL-18 signaling activity would be beneficial, either in whole (e.g., systemic) or in part (e.g., local, cellular, tissue, organ) treatment. Based on the teachings provided herein, one of skill in the art will understand that the diseases and disorders treatable by the compositions and methods described herein include any disease or disorder in which increased IL-18 signaling promotes a positive biological, physiological, clinical, or therapeutic outcome.

[0239] Those skilled in the art will understand that in addition to directly increasing IL-18 signaling, decreasing the amount or activity of a molecule that itself reduces the amount or activity of IL-18 signaling can also serve to increase the activity of IL-18 signaling. Thus, activators of IL-18 activity can include, but should not be considered limited to, chemical compounds, proteins, peptidomimetics, antibodies, ribozymes, and antisense nucleic acid molecules. Based on the disclosure provided herein, those skilled in the art will readily understand that activators of IL-18 activity include compounds that increase the level of IL-18 signaling. In addition, activators of IL-18 activity include compounds that suppress the level or activity of molecules that themselves reduce the amount or activity of IL-18 signaling (i.e., IL-18BP). Contemplated herein are IL-18BP antagonists, non-limiting examples of which include monoclonal antibodies, small molecule therapeutic agents that neutralize IL-18BP, and modified IL-18 variants that bind to IL-18BP but do not substantially bind to or interact with IL-18R. Inhibition of IL-18BP in this manner increases the activity of endogenous IL-18 produced through disinhibition.

[0240] Armed with the teachings of the present invention, one of skill in the art will also understand that increasing the level of IL-18 signaling also includes increasing the level of IL-18 or an activity of IL-18 (e.g., receptor binding activity, receptor signaling activity, etc.). Accordingly, non-limiting examples of increasing the level or activity of IL-18 signaling include increasing the amount of available IL-18 polypeptide or IL-18 variant polypeptide, increasing the transcription and / or translation of a nucleic acid encoding an IL-18 polypeptide or IL-18 variant polypeptide, including increasing any activity of an IL-18 polypeptide or IL-18 variant polypeptide. The compositions and methods of the present invention for activating IL-18 activity can selectively activate IL-18 signaling or can activate both IL-18 signaling and another molecule or pathway. Thus, the present invention relates to the administration of an activator of IL-18 activity, or a recombinant activator of an IL-18 active polypeptide, or an active activator of an IL-18 active polypeptide fragment, or an activator of the expression or activity of an IL-18 signaling pathway component.

[0241] Furthermore, one of skill in the art, instructed by the present disclosure and the methods exemplified herein, will understand that activators of IL-18 activity include those activators that may be discovered in the future and those activators that may be identified by criteria well known in the art of pharmacology (e.g., physiological consequences of IL-18 signaling activation as detailed herein and / or known in the art). Thus, the present invention is in no way limited to any particular activators of IL-18 activity exemplified or disclosed herein, but rather, the present invention encompasses activators known in the art and those discovered in the future that one of skill in the art would understand to be useful.

[0242] Alternative methods for identifying and producing activators of IL-18 activity are well known to those of skill in the art, and non-limiting examples include obtaining the activator from a natural source (e.g., Streptomyces spp., Pseudomonas spp., Stylotella aurantium, etc.). Alternatively, activators of IL-18 activity can be chemically synthesized. Furthermore, those skilled in the art will understand, based on the teachings provided herein, that activators of IL-18 activity can be obtained from recombinant organisms. Compositions and methods for chemically synthesizing activators of IL-18 activity and for obtaining activators of IL-18 activity from natural sources are well known in the art and described in the prior art.

[0243] Those skilled in the art will appreciate that the activator can be administered as a small molecule compound, a protein, an antibody, a nucleic acid construct encoding a protein, an antisense nucleic acid, a nucleic acid construct encoding an antisense nucleic acid, or a combination thereof. Numerous vectors and other compositions and methods are known for administering proteins or nucleic acid constructs encoding proteins to cells or tissues. Thus, the present invention includes methods for administering a polypeptide (an activator of IL-18 signaling) or a nucleic acid encoding the polypeptide. (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0244] Those skilled in the art will understand that reducing the amount or activity of a molecule that itself reduces the amount or activity of IL-18 signaling can be useful for increasing the amount or activity of IL-18 signaling (e.g., reducing the amount or activity of IL-18BP). Antisense oligonucleotides are RNA molecules or RNA molecules complementary to a portion of an RNA molecule. Once present in a cell, antisense oligonucleotides hybridize to the existing RNA molecule and inhibit translation into a gene product. The use of antisense oligonucleotides to inhibit gene expression is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods for expressing antisense oligonucleotides in cells (Inoue, U.S. Patent No. 5,190,931). The methods of the present invention include increasing the amount or activity of IL-18 signaling by using antisense oligonucleotides to reduce the amount of a molecule that reduces the amount or activity of IL-18 signaling. The present invention contemplates antisense oligonucleotides that are synthesized and delivered to cells by methods well known to those skilled in the art. By way of example, antisense oligonucleotides can be synthesized to be about 10 to about 100 nucleotides in length, more typically about 15 to about 50 nucleotides in length. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity relative to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Patent No. 5,023,243).

[0245] Similarly, gene expression can be inhibited by hybridizing an antisense molecule to the gene's promoter or other regulatory element, thereby affecting transcription of that gene. Methods for identifying promoters or other regulatory elements that interact with a gene of interest are well known in the art, and include methods such as the yeast two-hybrid system (Bartel and Fields, eds., The Yeast Two Hybrid System, Oxford University Press, Cary, NC).

[0246] Alternatively, the suppression of genes expressing proteins that reduce the level or activity of IL-18 signaling can be achieved using ribozymes. The use of ribozymes to suppress gene expression is well known to those skilled in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA molecules capable of cleaving other single-stranded RNA molecules. Ribozymes are known to be sequence-specific and can be engineered to recognize specific nucleotide sequences (Cech, 1988, J. Amer. Med. Assn. 260:3030), thereby enabling the selective cleavage of specific mRNA molecules. It is believed that one of ordinary skill in the art would be able to synthesize an antisense oligonucleotide or ribozyme, given the nucleotide sequence of the molecule, without undue experimentation, based on the disclosure herein and the references incorporated therein.

[0247] Those skilled in the art will understand that activators of IL-18 activity (e.g., an IL-18 variant polypeptide or fragment thereof, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding an IL-18 variant polypeptide or fragment thereof) can be administered alone or in any combination thereof.

[0248] Those skilled in the art will also understand that administration can be acute (e.g., for a short period of time (e.g., one day, one week, one month, etc.)) or chronic (e.g., for an extended period of time (e.g., several months, one year or more, etc.)). Furthermore, it will be understood that activators of IL-18 activity (e.g., an IL-18 variant polypeptide or fragment thereof, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding an IL-18 variant polypeptide or fragment thereof) can be administered alone or in any combination, temporally, i.e., the activators can be administered simultaneously and / or before or after each other. Those skilled in the art will understand, based on the disclosure provided herein, that the therapeutic outcome can be affected by using an activator of IL-18 activity, or an activator of an IL-18 active polypeptide fragment, or a recombinant activator of an IL-18 active polypeptide fragment, or an active activator of an IL-18 active polypeptide fragment, alone or in any combination with another activator of IL-18 activity, or another activator of an IL-18 active polypeptide fragment, or another recombinant activator of an IL-18 active polypeptide fragment, or another active activator of an IL-18 active polypeptide fragment.

[0249] Those skilled in the art, armed with the present disclosure, including the methods detailed herein, will understand that the present invention is not limited to the treatment of diseases or disorders already identified. In particular, the disease or disorder need not be manifested in the subject at the site of injury. Indeed, the disease or disorder need not be detected in the subject before treatment is administered. That is, significant disease or disorder need not occur before the present invention provides a potential benefit. Accordingly, the present invention includes methods for preventing a disease or disorder in a subject, in which an activator of an IL-18 active molecule (e.g., a polypeptide, peptide, etc.) discussed elsewhere, or an activator of IL-18 activity, can be administered to the subject prior to the onset of the disease or disorder to prevent the disease or disorder from progressing.

[0250] The present invention encompasses practicing the methods of the invention by administering an activator of IL-18 activity, an activator of an IL-18 active polypeptide, a recombinant IL-18 signaling polypeptide, or an active IL-18 signaling polypeptide fragment. Based on the disclosure provided herein, one of skill in the art would know how to formulate and administer to a subject a suitable activator of IL-18 activity, or an activator of an IL-18 active polypeptide, a recombinant IL-18 signaling polypeptide, or an active IL-18 signaling polypeptide fragment. However, the present invention is not limited to any particular method of administration or treatment regimen. This is particularly true where one of skill in the art, instructed by the disclosure provided herein (including practicing the disclosure using art-recognized disease models), or skilled in the art of pharmacology, would understand that a method for administering an activator of IL-18 activity, an IL-18 signaling polypeptide, a recombinant IL-18 signaling polypeptide, or an active IL-18 signaling polypeptide fragment can be determined.

[0251] In some embodiments, a method comprises administering to a subject in need thereof a composition comprising at least one IL-18 variant polypeptide and administering to the subject a composition comprising an additional agent. In one such embodiment, the additional agent comprises an immunotherapeutic agent, including at least one selected from the group including, but not limited to, altered T cells, chimeric antigen receptor T cells (CAR-T), protected CAR-T cells, viruses, antigens, vaccines, antibodies, immune checkpoint inhibitors, small molecules, chemotherapeutic agents, and stem cells. In some embodiments, a composition comprising at least one IL-18 variant polypeptide is used in a method for increasing immune system activity before, during, or after bacterial, viral, or other pathogen infection. In some embodiments, a composition comprising at least one IL-18 variant polypeptide is used in a method for increasing immune cell number and / or activity (e.g., T cell, and / or NK cell, and / or myeloid cell number and / or activity) in vitro, in vivo, or ex vivo.

[0252] In some embodiments, the additional agent comprises an inhibitor of one or more cytokines. In some embodiments, the inhibitor of one or more cytokines comprises a compound, protein, peptide, peptidomimetic, antibody, ribozyme, small molecule compound, or antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.) that inhibits expression and / or activity of one or more cytokines. In some embodiments, the inhibitor inhibits expression and / or activity of IL-17, IL-5, or IL-3. In some embodiments, the cytokine inhibitor reduces toxicity. In some embodiments, the cytokine inhibitor enhances the effect of an administered IL-18 variant polypeptide or IL-18BP inhibitor.

[0253] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition that can be combined with a suitable IL-18 signaling modulator such that the suitable IL-18 signaling modulator can be administered to a subject using the combined chemical composition.

[0254] kit

[0255] The present invention also relates to kits useful in the methods of the invention. Such kits contain various combinations of components useful in any of the methods described elsewhere herein, including, for example, activators of IL-18 activity (such as IL-18 variant polypeptides and / or IL-18BP inhibitors), and / or materials for quantitatively analyzing IL-18 variant polypeptides or IL-18 variant nucleic acids, and / or instructional materials. For example, in some embodiments, the kits contain components useful for quantifying IL-18 variant nucleic acids in a biological sample. In another embodiment, the kits contain components useful for quantifying IL-18 variant polypeptides in a biological sample. In yet another embodiment, the kits contain components useful for assessing the activity (e.g., enzymatic activity, ligand binding activity, etc.) of IL-18 variant polypeptides in a biological sample.

[0256] In yet another embodiment, the kit contains assay components for monitoring the effectiveness of a treatment administered to a subject in need thereof, including instructional materials and components for determining whether the level of IL-18 signaling in a biological sample obtained from the subject has changed during or after administration of the treatment. In various embodiments, to determine whether the level of IL-18 signaling in the biological sample obtained from the subject has changed, the level of IL-18 signaling is compared to the level of at least one comparison control (e.g., a positive control, a negative control, a historical control, a historical standard, etc.) contained in the kit or the level of another reference molecule in the biological sample. In some embodiments, a ratio of the IL-18 signaling molecule to the reference molecule is determined to help monitor the treatment.

[0257] Pharmaceutical Compositions and Administration

[0258] As described below, compositions containing polypeptides, polypeptide fragments, activators of IL-18 signaling levels or activity, and inhibitors of IL-18BP levels or activity described elsewhere herein can be prepared and administered to a subject. As described below, compositions identified as activators of IL-18 activity (including, by way of non-limiting example, IL-18 variant polypeptides, recombinant IL-18 variant polypeptides, and active IL-18 variant polypeptide fragments) for treating and / or preventing a disease or disorder can be formulated and administered to a subject. As described below, compositions identified as useful IL-18BP inhibitors (including, by way of non-limiting example, compounds, proteins, peptides, peptidomimetics, antibodies, ribozymes, small molecule compounds, and antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.)) for treating and / or preventing a disease or disorder can be formulated and administered to a subject.

[0259] The present invention encompasses the preparation and use of pharmaceutical compositions, including those disclosed herein as active ingredients useful in the treatment or prevention of diseases or disorders. Such pharmaceutical compositions can consist solely of the active ingredient in a form suitable for administration to a subject, or can include the active ingredient together with one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combination thereof. The active ingredient can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt (e.g., in combination with a physiologically acceptable cation or anion), as is well known in the art. In various embodiments, the active ingredient is a polypeptide, a polypeptide fragment, an activator of IL-18 signaling levels or activity, an inhibitor of IL-18BP levels or activity, or a combination thereof, as described elsewhere herein.

[0260] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition that can be combined with a suitable IL-18 signaling modulator, such that the combined chemical composition can be used to administer the suitable modulator (e.g., activator, inhibitor, etc.) to a subject.

[0261] In some embodiments, pharmaceutical compositions can include large, slowly metabolized macromolecules, such as proteins, polysaccharides (such as chitosan), polylactic acids, polyglycolic acids, copolymers (such as latex-functionalized Sepharose™, agar, cellulose), polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets, liposomes), and the like.

[0262] Pharmaceutical compositions useful in practicing the present invention can be administered to deliver doses of about 0.1 ng / kg / day to 100 mg / kg / day or more.

[0263] In various embodiments, pharmaceutical compositions useful in the methods of the invention can be administered systemically, parenterally, or locally, for example, in the form of oral or inhaled formulations (including solid or aerosol), topical, or other similar formulations. In addition to the appropriate therapeutic composition, such pharmaceutical compositions can contain pharmaceutically acceptable carriers and other ingredients known to enhance or facilitate drug administration. Other possible formulations, such as nanoparticles containing the active ingredient, liposomes or other preparations, immunologically-based systems, and other suitable modulators thereof, can also be used to administer the methods of the invention.

[0264] Carriers can carry agents of the invention (e.g., IL-18 variant polypeptides) in a variety of ways (direct covalent bonding, covalent bonding via a linker group, or non-covalent bonding). Suitable covalently bonded carriers include proteins (e.g., albumin), peptides, and polysaccharides (e.g., aminodextran), each of which has multiple sites for attachment of various moieties. Carriers can also carry IL-18 variant polypeptides by non-covalent association (non-covalent bonding or encapsulation). The nature of the carrier can be soluble or insoluble for purposes of the present invention.

[0265] Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and examples include buffers (such as phosphates, citrates, and salts of other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, benzyl alcohol; alkylparabens (such as methylparaben and propylparaben); catechol; resorcino These include: alcohol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, and immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, and lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, and dextrin); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, and sorbitol); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as TWEEN™, PLURONICS™, and polyethylene glycol (PEG)). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0266] The active ingredient can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or interfacial polymerization, or in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. (ed.) (1980).

[0267] The compositions can be prepared as injectable solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The preparations can also be emulsified, as described above, or encapsulated in liposomes or microparticles (such as polylactides, polyglycolides, and copolymers for enhanced adjuvant effect). See Langer, Science 249:1527, 1990; Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of indwelling injectable or implantable preparations, which can be formulated to provide sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all regulations of the U.S. Food and Drug Administration's Good Manufacturing Practice (GMP).

[0268] As used herein, the expression "physiologically acceptable" ester or salt refers to an active ingredient in an ester or salt form that is compatible with any other ingredients of the pharmaceutical composition and is not harmful to the subject to whom the composition is to be administered.

[0269] The formulations of the pharmaceutical compositions described herein can be prepared by any method now known or hereafter developed in the art of pharmacology. In general, such methods include the step of combining the active ingredient with the carrier or one or more other accessory ingredients and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0270] While the description of pharmaceutical compositions presented herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to animals of all kinds. Methods for modifying pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and those skilled in the art of animal pharmacology can design and perform such modifications with no more than routine experimentation, if any.

[0271] Pharmaceutical compositions useful in the methods of the invention can be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, transdermal, intralesional, subcutaneous, intramuscular, ocular, intrathecal, or other known routes of administration. Other possible formulations include textured nanoparticles, liposomal preparations, other preparations containing the active ingredient, and immunologically-based formulations.

[0272] The pharmaceutical composition of the present invention can be prepared, packaged or sold as a single unit dose or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of the active ingredient that will be administered to a subject, or equal to a round fraction of such dosage (for example, half or one-third of such dosage).

[0273] The relative amounts of active ingredient, pharmaceutically acceptable carrier, and any additional ingredients contained in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, and will also depend on the route by which the composition is administered. The composition may contain, for example, 0.1% to 100% (w / w) active ingredient.

[0274] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active ingredients.

[0275] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

[0276] Formulations of pharmaceutical compositions of the invention suitable for oral administration may be prepared, packaged, or sold in the form of discrete solid unit doses, non-limiting examples of which include tablets, hard capsules, soft capsules, cachets, lozenges, and lozenges, each containing a predetermined amount of the active ingredient. Non-limiting examples of other formulations suitable for oral administration include powdered formulations, granular formulations, aqueous suspensions, oily suspensions, aqueous solutions, oily solutions, and emulsions.

[0277] Non-limiting examples of pharmaceutically acceptable excipients used in preparing pharmaceutical compositions include inert diluents, granulating agents, disintegrants, binders, and lubricants. Non-limiting examples of known dispersing agents include potato starch and sodium starch glycolate. Non-limiting examples of known surfactants include sodium lauryl sulfate. Non-limiting examples of known diluents include calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Non-limiting examples of known granulating and disintegrating agents include cornstarch and alginic acid. Non-limiting examples of known binders include gelatin, gum arabic, pregelatinized cornstarch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Non-limiting examples of known lubricants include magnesium stearate, stearic acid, silica, and talc.

[0278] Liquid formulations of pharmaceutical compositions of the invention may be prepared, packaged, or sold in liquid form or as a dry product for reconstitution with water or another suitable vehicle before use.

[0279] Suspensions can be prepared by conventional methods to suspend active ingredients in aqueous or oily vehicles.Aqueous vehicles include, for example, water and isotonic saline.Oil vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils (peanut oil, olive oil, sesame oil, coconut oil, fractionated vegetable oil, etc.), mineral oils (liquid paraffin, etc.).Suspensions can further include one or more additional ingredients, non-limiting examples of which include suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners.Oil suspensions can also include thickeners.

[0280] Non-limiting examples of known suspending agents include sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives (such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose). Non-limiting examples of known dispersing or wetting agents include natural phosphatides (such as lecithin), condensation products of alkylene oxides with fatty acids, condensation products of alkylene oxides with long-chain aliphatic alcohols, condensation products of alkylene oxides with partial esters derived from fatty acids and hexitols, and condensation products of alkylene oxides with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Non-limiting examples of known emulsifiers include lecithin and gum arabic. Non-limiting examples of known preservatives include methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, n-propyl para-hydroxybenzoate, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0281] The solution of active ingredient in aqueous or oily solvent can be prepared in substantially the same manner as suspension, but the main difference is that the active ingredient is dissolved in the solvent, not suspended.The solution of the pharmaceutical composition of the present invention can contain each of the components described for suspension, but it should be understood that the suspending agent does not necessarily help the active ingredient to dissolve in the solvent.Aqueous solvents include, for example, water and isotonic saline.Oil solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils (such as peanut oil, olive oil, sesame oil, coconut oil), fractionated vegetable oils, mineral oils (such as liquid paraffin).

[0282] Powder and granular formulations of the pharmaceutical compositions of the present invention can be prepared using known methods. Such formulations can be administered directly to a subject or can be used, for example, to form tablets, fill capsules, or add an aqueous or oily vehicle to prepare an aqueous or oily suspension or solution. Each of these formulations can further include one or more of the following: dispersing or wetting agents, suspending agents, and preservatives. Additional excipients (fillers, sweeteners, flavoring agents, coloring agents, etc.) can also be included in these formulations.

[0283] Pharmaceutical compositions of the invention can be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase can be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin), or a combination thereof. Such compositions can further include one or more emulsifying agents, such as natural gums (such as gum arabic or gum tragacanth), natural phosphatides (such as soybean or lecithin phosphatides), esters or partial esters derived from combinations of fatty acids and hexitol anhydrides (such as sorbitan monooleate), or condensation products of such partial esters with ethylene oxide (such as polyoxyethylenesorbitan monooleate). These emulsions can also contain additional ingredients, including, for example, sweeteners and flavoring agents.

[0284] Methods for impregnating or coating a material with a compound are known in the art, and non-limiting examples include depositing or binding a chemical composition to a surface, incorporating a chemical composition into the structure of a material during its synthesis (i.e., as in the case of physiologically degradable materials), and absorbing an aqueous or oily solution or suspension into an absorbent material, which may or may not be dried.

[0285] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically disrupting the tissue of a subject and administering the pharmaceutical composition through the disruption in the tissue. Non-limiting examples of parenteral administration therefore include administering the pharmaceutical composition by injection of the composition, by applying the composition through a surgical incision, or by applying the composition through a non-surgical wound that penetrates the tissue. Non-limiting examples of parenteral administration that are contemplated include cutaneous injection, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intracisternal injection, and kidney dialysis infusion techniques.

[0286] Pharmaceutical compositions suitable for parenteral administration comprise an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit-dose form, such as ampoules or multi-dose containers containing a preservative. Non-limiting examples of formulations for parenteral administration include suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, implantable sustained-release formulations, or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, non-limiting examples of which include suspending agents, stabilizers, and dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle, such as sterile, pyrogen-free water, and the reconstituted composition is administered parenterally.

[0287] Pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be prepared according to known techniques and may contain, in addition to the active ingredient, additional ingredients (such as dispersing agents, wetting agents, and suspending agents described herein). Such sterile injectable formulations can be prepared using non-toxic parenterally acceptable diluents or solvents (e.g., water, 1,3-butanediol). Non-limiting examples of other acceptable diluents and solvents include Ringer's solution, isotonic sodium chloride solution, and fixed oils (synthetic monoglycerides, synthetic diglycerides, etc.). Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can include pharmaceutically acceptable polymeric or hydrophobic materials (such as emulsions, ion exchange resins, sparingly soluble polymers, and sparingly soluble salts).

[0288] Non-limiting examples of formulations suitable for topical administration include liquid or semi-liquid preparations (liniments, lotions, oil-in-water emulsions, water-in-oil emulsions (creams, ointments, pastes, etc.), solutions, suspensions, etc.). Topically administrable formulations can contain, for example, about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient can be up to the solubility limit of the active ingredient in the solvent. Formulations for topical administration can further include one or more additional ingredients described herein.

[0289] Pharmaceutical compositions of the present invention can be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations can comprise dry particles containing the active ingredient having diameters ranging from about 0.5 to about 7 nanometers (preferably about 1 to about 6 nanometers). These compositions can be conveniently administered in dry powder form using a device with a dry powder reservoir (directing a stream of propellant into the dry powder reservoir to disperse the powder) or a self-propelling solvent / powder dispersion vessel (e.g., a device containing the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container). Such powders preferably contain particles where at least 98% by weight have a diameter greater than 0.5 nanometers and at least 95% by number have a diameter less than 7 nanometers. More preferably, at least 95% by weight have a diameter greater than 1 nanometer and at least 90% by number have a diameter less than 6 nanometers. Dry powder compositions preferably contain a solid fine powder diluent (e.g., sugar) and are conveniently provided in unit dosage form.

[0290] Low-boiling propellants generally comprise liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components such as a non-ionic liquid or an anionic solid surfactant, or a solid diluent (preferably having a particle size similar to that of the particles containing the active ingredient).

[0291] Pharmaceutical compositions of the present invention formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations can be prepared, packaged, or sold as an optionally sterile aqueous or dilute alcoholic solution or suspension containing the active ingredient, and can therefore be conveniently administered using any spray or atomizing device. Such formulations can further contain one or more additional ingredients, non-limiting examples of which include flavoring agents (e.g., saccharin sodium), volatile oils, buffers, surfactants, and preservatives (e.g., methyl hydroxybenzoate). The droplets provided by this route of administration preferably have an average diameter of about 0.1 to 200 nanometers. Formulations described herein as useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the present invention. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers.

[0292] Such formulations are administered by sniffing, i.e., by rapid inhalation through the nasal passages from a powder container held close to the nostril. Formulations suitable for nasal administration may, for example, contain as little as about 0.1% (w / w) or as much as 100% (w / w) active ingredient, and may further include one or more of the ingredients described herein.

[0293] Pharmaceutical compositions of the invention can be prepared, packaged, or sold in a formulation suitable for oral administration. Such formulations can be, for example, tablet or lozenge form, formulated using conventional techniques and contain, for example, 0.1 to 20% (w / w) of the active ingredient, with the remainder comprising a composition that dissolves or disintegrates in the mouth, and optionally one or more additional ingredients described herein. Alternatively, formulations suitable for oral administration can comprise a powder, or an aerosolized or atomized solution or suspension containing the active ingredient. Such powdered, aerosolized, or atomized formulations preferably have an average particle or droplet size in the range of about 0.1 to about 200 nanometers when dispersed and can further comprise one or more of the ingredients described herein.

[0294] The pharmaceutical compositions of the invention can be prepared, packaged, or sold in a formulation suitable for administration to the eye. Such formulations can be in the form of eye drops comprising, for example, a 0.1 to 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such eye drops can further comprise a buffer, salts, and one or more other additional ingredients described herein. Other useful formulations that can be administered to the eye include those comprising the active ingredient in microcrystalline form or in a liposomal preparation.

[0295] As used herein, non-limiting examples of "additional ingredients" include one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions (such as gelatin); aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, edited by Genaro, 1985, Mack Publishing Co., Easton, PA, the contents of which are incorporated herein by reference.

[0296] Typically, the dosage of a compound of the present invention that can be administered to an animal (preferably a human) ranges from about 0.001 mg to about 1000 mg per kg of the animal's body weight. The exact dosage will vary depending on numerous factors, non-limiting examples of which include the type of animal, the type of disease or disorder being treated, the age of the animal, and the route of administration. The dosage of the compound will vary from about 0.1 mg to about 10 mg per kg of the animal's body weight. The compound can be administered to an animal several times per day, or less frequently (such as once daily, once weekly, once every two weeks, or once monthly), or even less frequently (such as once every few months or even once a year or less). The frequency of administration will vary depending on numerous factors, as will be apparent to those skilled in the art. Non-limiting examples of such factors include the type and severity of the disease or disorder being treated, the type and age of the animal, etc. [Example]

[0297] Experimental Examples

[0298] The present invention is further described in detail by reference to the following experimental examples. These examples are presented for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples, but rather the present invention should be construed to encompass any variations that become evident as a result of the teachings provided herein.

[0299] Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the following specific examples, make and use the present invention and practice the claimed methods. Thus, the following working examples should not be construed as limiting in any way the remainder of the disclosure.

[0300] Example 1: IL-18 variant polypeptides

[0301] IL-18 is a pro-inflammatory cytokine capable of stimulating T cells, NK cells, and myeloid cells. Due to its ability to stimulate antitumor immune cells, IL-18 has previously been proposed as an immunotherapeutic agent for cancer. As demonstrated herein, the therapeutic efficacy of recombinant IL-18 therapy is severely limited by upregulation of the natural endogenous soluble inhibitor IL-18BP. The present invention is based, in part, on the development of both human and murine IL-18 variants that are almost completely independent of IL-18BP. These cytokine variants exhibit 100,000- to over 1,000,000-fold altered selectivity for the receptor (IL-18Rα) and IL-18BP. These variants have potent antitumor activity in preclinical tumor models, both as monotherapy and in combination with immune checkpoint inhibitors (e.g., anti-PD-1). As an additional application, since IL-18 also has a very clear anti-obesity role, we demonstrate herein that administration of these variants results in a greater reduction in body fat composition compared to WT IL-18 therapy. Thus, these novel variants have applications in endocrinology / metabolism / obesity in addition to tumor immunotherapy.

[0302] Also described herein are a group of additional IL-18 variants that act as IL-18BP antagonists by binding only to IL-18BP and exhibiting absent or greatly reduced binding to IL-18Rα. These proteins could be used to neutralize IL-18BP, thereby enhancing the activity of endogenous IL-18.

[0303] The materials and methods used in these experiments are described below.

[0304] Protein expression and purification

[0305] Human IL-18, mouse IL-18 (amino acids 1–157), and their variants were assembled as gBlocks (Integrated DNA Technologies, IDT) and cloned into the pET28a-smt vector. N-terminally SUMO-tagged and C-terminally hexahistidine-tagged proteins were expressed in Escherichia coli BL21(DE3) Rosetta strain. Protein expression was induced with 0.5 mM IPTG for 20 h at 16°C. These fusion proteins were first purified using Ni-chelating resin, followed by SUMO protease cleavage of the SUMO tag. Proteins were then separated from aggregates by sequential ammonium sulfate cleavage, followed by precipitation of aggregates with 20% ammonium sulfate and precipitation of the target protein with 70% ammonium sulfate. The protein pellet was resuspended and reapplied to Ni-chelating resin to remove the SUMO tag, and endotoxin was removed by washing with 0.1% Triton X-114. Finally, the eluted protein was buffer exchanged into PBS using a PD-10 column (GE Healthcare). The monodispersity of the protein samples was examined by size exclusion chromatography using an FPLC (Bio-Rad) and a SEC650 column (Bio-Rad).

[0306] Human IL-18Rα ectodomain (amino acids 19–329), IL-18Rβ ectodomain (amino acids 15–356), and IL-18BP (amino acids 31–194) were secreted and purified using a baculovirus expression system. Briefly, all construct sequences were cloned into the pAcBN-BH3 vector (BD Biosciences) containing an N-terminal gp67 signal peptide and C-terminal AviTag™ and hexahistidine tags. Plasmid constructs were transfected into Sf9 insect cells grown in SF900 II SFM medium (Invitrogen) at 27°C to establish high-titer recombinant viruses, which were then amplified. Recombinant protein was expressed by infection of High-Five insect cells (Invitrogen) grown in Insect Xpress medium (Lonza) at 27°C. Three days after infection, protein was extracted and concentrated by Ni-NTA (QIAGEN) affinity chromatography and purified to >98% homogeneity using a SEC650 sizing column (Bio-Rad) equilibrated in 10 mM HEPES (pH 7.5) and 150 mM NaCl.

[0307] The mouse IL-18Rα ectodomain (amino acids 19–329) and IL-18BP (amino acids 31–194) were produced as secreted proteins using the Expi293 expression system (Thermo Fisher). Briefly, all construct sequences were cloned into the BacMam expression vector pEZT_D_Lux, which contains an N-terminal H7 signal peptide and a C-terminal AviTag™ and hexahistidine tag. Plasmids were transfected into Expi293 cells cultured at 37°C in Expi293 expression medium (Thermo Fisher) using the ExpiFectamine 293 transfection kit (Thermo Fisher) according to the manufacturer's instructions. Cells were harvested 3–5 days postinfection. Protein purification procedures were the same as for the human proteins.

[0308] All IL-18 receptor constructs were fused with a C-terminal biotin acceptor peptide (AviTag)-GLNDIFEAQKIEWHE for protein biotinylation. Protein biotinylation was performed using soluble BirA ligase enzyme in 0.1 mM bisine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mM biotin (Sigma). Proteins were purified by size-exclusion chromatography on a SEC650 column as described above.

[0309] Yeast-based IL-18 presentation

[0310] Human and mouse IL-18 gene blocks (IDT) were synthesized, cloned into the pYAL vector, and displayed on the surface of Saccharomyces cerevisiae EBY100. Individual IL-18 colonies on the yeast surface were grown overnight in SDCAA liquid medium at 30°C and then plated in SGCAA liquid medium at 20°C for 1 day. IL-18 display levels on the yeast surface were confirmed by flow cytometry using an anti-cMyc tag antibody (anti-myc-PE; Cell Signaling Technologies). Receptor staining was performed with biotinylated IL-18Rα (with or without IL-18Rβ) or biotinylated IL-18BP in PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE) on ice. All analyses were performed on a Sony SA3800 flow cytometer.

[0311] Human IL-18 library construction and selection

[0312] For the first human decoy-resistant IL-18 library, 14 residues (Table 1) in hIL-18 that contact hIL-18Rα and hIL-18BP were identified from homologous positions by aligning the structure of the hIL-18 / hIL-18Rα / hIL-18Rβ complex (Protein Data Bank (PDB ID) code 3OW4) with the structure of IL-18 / IL-18BP (PDB ID 3F62). A library was constructed to randomize these residues using assembly PCR with degenerate primers listed in Table 2. The library had a theoretical diversity of unique protein sequences of approximately 1.96 × 10 11 The PCR product was further amplified using primers homologous to the pYAL vector and electroporated into EBY100 yeast along with the linearized pYAL. The resulting library contained 2.5 x 10 8 The transformants contained 10 transformants.

[0313] For the second V2.0 Star Decoy-resistant IL-18 library, 11 residues in hIL-18 that contact hIL-18Rα and hIL-18BP were selected and randomized, resulting in a theoretical diversity of variants of 3.44 × 10 9 The results were as shown in Figure 7A. A library randomizing these residues was constructed using assembly PCR with degenerate primers and electroporated into EBY100 yeast with pYAL. The resulting library had a transformant diversity of 6 × 10 8 It was a street.

[0314] [Table 1]

[0315] [Table 2]

[0316] For both libraries, transformed yeast were harvested, grown in liquid synthetic dextrose with casamino acids (SDCAA) medium at 30°C, and induced in liquid synthetic galactose with casamino acids (SGCAA) medium by diluting 1:10 and culturing for 24 hours at 20°C. By using an appropriate number of induced yeast in each round, at least 10-fold coverage of the expected library diversity was achieved at each step. 8The number of cells was kept below 1. All selection steps were performed at 4°C using PBE buffer (PBS containing 0.5% BSA and 2 mM EDTA). The selection reagents for each round of the first-generation library are listed in Table 5. In round 1, yeast were counterselected using anti-Cy5 / AlexaFluor 647 microbeads (Miltenyi) and an LS MACS column (Miltenyi) to remove nonspecifically bound beads. Yeast were labeled with 1 μM biotinylated hIL-18Rα for 1 hour at 4°C, followed by magnetic selection using SA / AlexaFluor 647 microbeads and an LS MACS column. In round 2, counterselection was performed using 1 μM biotinylated IL-18BP, and positive selection was the same as in round 1. In rounds 3–5, selection was performed by incubating yeast with 100 nM (rounds 3–4) or 10 nM (round 5) biotinylated IL-18Rα and 250 nM preformed biotin-capped hIL-18BP / SA-PE tetramer. After competitive binding, yeast were washed and labeled with SA / AlexaFluor 647 to detect IL-18Rα. Display levels were determined by staining with AlexaFluor 488-conjugated anti-cMyc (Cell Signaling Technologies), and the top 1% of IL-18Rα-binding yeast (among IL-18BP-nonbinding yeast) normalized for display were isolated by FACS using a Sony SA3800 cell sorter. After each round of selection, recovered yeast were grown overnight in SDCAA medium at 30°C and then induced for 24 hours at 20°C in SGCAA medium by diluting 1:10.

[0317] The V2.0 human DR-IL-18 library was similarly selected using a special selection process detailed in Figure 7B.

[0318] Mouse IL-18 library construction and selection

[0319] The construction and selection procedures were similar to those for human IL-18, with the following modifications: Library construction was guided by the in silico modeled mouse IL-18 / receptor complex structure (predicted by Phyer 2.0). 13 positions were selected for randomization (Table 3) using the primers listed in Table 4. Electroporation with pYAL resulted in 4 × 10 8 A library of transformants was obtained. The selection reagents used in each round are listed in Table 5.

[0320] [Table 3]

[0321] [Table 4]

[0322] [Table 5]

[0323] Surface plasmon resonance

[0324] Experiments were performed at 25°C using a Biacore T100. Biotinylated IL-18Rα or biotinylated IL-18BP was immobilized on the surface of a Biacore biotin capture chip (Series S CAP sensor chip, GE Healthcare), resulting in an Rmax of approximately 50 RU (IL-18Rα) or 10 RU (IL-18BP). Measurements were performed in HEPES-buffered saline (PBS). +Serial dilutions of IL-18 variants were performed in buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20). The surface was regenerated with three 60-second injections of regeneration buffer (3 / 4 (v / v) 8 M guanidine hydrochloride and 1 / 4 (v / v) 1 M sodium hydroxide). Experiments were performed simultaneously on multiple channels to look for incremental observations. All data were analyzed using Biacore T100 Evaluation Software version 2.0 using a 1:1 Langmuir binding model.

[0325] cell line

[0326] HEK-Blue IL-18 sensor cells (InvivoGen) were maintained in complete medium (DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 50 U / ml penicillin, and 50 μg / ml streptomycin) supplemented with 100 μg / ml normocin, 30 μg / ml blasticidin, 180 μg / ml zeocin, and 200 μg / ml hygromycin. YUMMER1.7 melanoma cells were cultured and prepared as previously described (Wang et al., 2017, Pigment Cell Melanoma Res. 30(4):428-435).

[0327] HEK-Blue cytokine activity assay

[0328] To measure cytokine activity, 50,000 HEK-Blue IL-18 sensor cells per well of a flat-bot...

Claims

1. 1. A composition comprising an interleukin-18 (IL-18) variant polypeptide comprising five or more mutations relative to wild-type (WT) interleukin-18 (IL-18) as set forth in SEQ ID NO: 30, wherein at least five of said five or more mutations are at any of positions M51, K53, P57, M60, S105, D110, and N111, wherein said mutations, if present, are T or K at M51, K, L, or Q at M60, and N111 at S105. is D, N, A, or R, D110 is K, S, G, H, Q, or N, and N111 is H, Y, G, R, or S, wherein the IL-18 variant polypeptide comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 30, and the IL-18 variant polypeptide specifically binds to the IL-18 receptor (IL-18R) and exhibits substantially reduced binding to the IL-18 binding protein (IL-18BP).

2. The composition of claim 1, wherein the IL-18 variant polypeptide binds to IL-18BP with a binding affinity that is about 10% or less of the binding affinity of wild-type IL-18 to IL-18BP.

3. The IL-18 variant polypeptide has a K of IL-18BP of 10 nM or more. D 2. The composition of claim 1, having:

4. A composition comprising an interleukin-18 (IL-18) variant polypeptide comprising five or more mutations relative to wild-type (WT) interleukin-18 (IL-18) as set forth in SEQ ID NO: 30, wherein at least five of said five or more mutations are at any of positions M51, K53, P57, M60, S105, D110, and N111, and wherein said mutations, if present, are T or K at M51, K, L, or Q at M60, and N111 at S105. is D, N, A or R, at D110 is K, S, G, H, Q or N, and at N111 is H, Y, G, R or S, wherein the IL-18 variant polypeptide comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 89, and the IL-18 variant polypeptide specifically binds to the IL-18 receptor (IL-18R) and exhibits substantially reduced binding to the IL-18 binding protein (IL-18BP).

5. 2. The composition of claim 1, wherein the IL-18 variant polypeptide further comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, G59X, E77X, Q103X, M113X, V153X, and N155X relative to SEQ ID NO: 30, wherein X represents any amino acid.

6. The IL-18 variant polypeptide is selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60L, E77D, Q103E, Q103K, Q10 6. The composition of claim 5, comprising a combination of mutations selected from the group consisting of 3P, Q103A, Q103R, S105R, S105D, S105N, S105A, D110H, D110K, D110N, D110Q, D110S, D110G, N111H, N111Y, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H.

7. 7. The composition of claim 6, wherein K at position 53 is mutated to R, G, S, or T; and P at position 57 is mutated to L, G, A, or K.

8. 8. The composition of claim 7, wherein K at position 53 is mutated to R, S, or G; P at position 57 is mutated to A; S at position 105 is mutated to D or N; D at position 110 is mutated to K, S, or G; and N at position 111 is mutated to H, Y, G, or R.

9. The composition of claim 1, wherein the IL-18 variant polypeptide further comprises a Q56X mutation relative to SEQ ID NO: 30, where X represents any amino acid.

10. 10. The composition of claim 9, wherein the Q at position 56 is mutated to E, A, R, V, G, K, or L.

11. 10. The composition of claim 9, wherein the Q at position 56 is mutated to L, G, or R.

12. The composition of claim 4, wherein the IL-18 variant polypeptide comprises an amino acid sequence having 95% or more sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, 39, 87, 89-91.

13. A composition comprising a nucleic acid encoding an IL-18 variant polypeptide of the composition of any one of claims 1 to 12.

14. (i) immune checkpoint inhibitors; (ii) an agent that inhibits one or more proteins selected from PD-Ll, PDl, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, and VISTA; (iii) CD40 agonist, 41BB agonist, OX40 agonist, or GITR agonist; (iv) cancer cell opsonizing agents; (v) CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α v β 3 an agent targeting one or more antigens selected from TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAP1, and NRP1; (vi) a cytokine selected from IL-1, IL-2, IL-10, IL-12, IL-15, IL-18, IL-21, IL-33, interferon alpha, interferon beta, interferon gamma, and TNF; (vii) an agonist of a Toll-like receptor selected from TLR2, TLR4, TLR5, TLR7, and TLR9; (viii) inflammasome agonists; (ix) STING / cGAS pathway agonists; and (x) RIG-I pathway agonists 14. The composition of any one of claims 1 to 13, further comprising one or more agents selected from:

15. 15. The composition of any one of claims 1 to 14 for use in a method for treating or preventing a disease or disorder in a subject in need thereof.

16. 16. The composition of claim 15, wherein the disease or disorder is cancer, and optionally the cancer is resistant to immune checkpoint inhibitors (ICIs) or is a cancer associated with tumors that have lost expression of MHC class I.

17. The IL-18 variant polypeptide is selected from the group consisting of: (a) an immune checkpoint inhibitor, optionally an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, or VISTA; (b) CD40 agonist, 41BB agonist, OX40 agonist, or GITR agonist; (c) cancer cell opsonizing agents; (d) CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HERl, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAMl, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSFIR, SLAMF7, integrin α v β 3 , an agent targeting one or more antigens selected from TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tenascin-3, STEAPl, and NRP1; (e) modified T cells or NK cells; (f) oncolytic viruses; (g) a cytokine optionally selected from IL-1, IL-2, IL-10, IL-12, IL-15, IL-18, IL-21, IL-33, interferon alpha, interferon beta, interferon gamma, and TNF; (h) a Toll-like receptor agonist optionally selected from TLR2, TLR4, TLR5, TLR7, and TLR9; (i) inflammasome agonists; (j) agonists of the STING / cGAS pathway; and (k) RIG-I pathway agonists; Suitable for administration with at least one other agent selected from 17. The composition of claim 15 or 16.

18. 18. The composition of claim 17, wherein the at least one other agent is conjugated to the IL-18 variant polypeptide.

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