IL-18 variant polypeptides

Abstract

The present invention provides an interleukin 18 (IL-18) variant polypeptide comprising a mutation at one or more of G3, E6, D54 and N91 wherein the amino acid position is relative to the wild-type human IL-18 set forth in SEQ ID NO: 1. Also provided are IL-18 variant polypeptides comprising (e.g., further comprising) at least one mutation selected from the group consisting of C38, C68, C76, D98, S117 and C127 residues wherein the amino acid position is the amino acid position relative to the wild-type human IL-18 set forth in SEQ ID NO: 1. Also provided are IL-18 variant polypeptides that bind to IL-18R [alpha] and have significantly reduced binding to IL-18BP compared to wild-type IL-18. Also provided are IL-18 variant polypeptides that exhibit increased binding to IL-18R [alpha] relative to wild-type IL-18, and that exhibit significantly reduced binding to IL-18BP relative to wild-type IL-18. Related nucleic acids, vectors, host cells, methods of producing the IL-18 variant polypeptides, pharmaceutical compositions comprising the IL-18 variant polypeptides, and uses of such pharmaceutical compositions are also provided.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to International Application PCT / CN2022 / 117332, filed on September 6, 2022, and International Application PCT / CN2023 / 072886, filed on January 18, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence listing (233002001542SEQLIST.xml; size: 362,441 bytes; creation date: September 1, 2023) are incorporated herein by reference in their entirety. Technical field

[0005] This application relates to interleukin - 18 (IL - 18) variant polypeptides that contain at least one amino acid substitution relative to wild - type IL - 18 (e.g., wild - type human IL - 18), methods for preparing such variant polypeptides, and methods for using such variant polypeptides in therapeutic applications. Background art

[0006] It has been found that IL - 18 stimulates innate lymphocytes, myeloid cells, non - naive T cells that have experienced antigen (see, e.g., Guo et al. (2012) Trends Immunol. 33, 598–606), and natural killer (NK) cells that have experienced antigen. Therapeutically, recombinant IL - 18 has been reported to act synergistically with immune checkpoint inhibitors (ICI) (Ma et al. (2016) ClinCancer Res 22:2969–2980) and chimeric antigen receptor T (CAR - T) cells (Hu et al. (2017) Cell Rep 20, 3025–3033) in pre - clinical models. IL - 18 has been administered to patients in clinical trials and has been found to be safe and well - tolerated (Robertson et al. (2006) Clin Cancer Res 12, 4265–4273). However, the clinical development of IL - 18 has been limited by its limited efficacy. Thus, there is a need in the art for compositions and methods for activating IL - 18 receptor - mediated signaling and / or stimulating antigen - experienced T cells or NK cells in an individual to treat cancer and other IL - 18 - mediated diseases and disorders. Summary of the invention

[0007] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided that comprises at least one mutation at a residue selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises at least one mutation at a residue selected from: G3, E6, D54, and N91, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from: Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1.

[0008] In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from: C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution. In some embodiments, the at least one mutation is selected from: C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide further comprises a set of mutations selected from: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y.

[0009] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided that comprises at least one mutation at a residue selected from C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution. In some embodiments, the at least one mutation is selected from: C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 variant comprises C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises a set of mutations selected from: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y.

[0010] In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide specifically binds to interleukin-18 receptor alpha (IL-18Rα) and exhibits reduced binding to interleukin-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα with a K -5 d less than about 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα with a K -5 d between about 5 x 10 -11 M and about 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -9 d greater than 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide does not exhibit binding to IL-18BP (e.g., no detectable binding, as measured by surface plasmon resonance).

[0011] In some embodiments, the variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from: G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N, and G3S. In some embodiments, the mutation is G3P. In some embodiments, the variant polypeptide comprises a mutation at residue E6, wherein the mutation is selected from: E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y. In some embodiments, the mutation is selected from: E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from: E6R and E6K.

[0012] In some embodiments, the variant polypeptide comprises a mutation at residue D54, wherein the mutation is selected from: D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T. In some embodiments, the mutation is selected from: D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0013] In some embodiments, the variant polypeptide comprises a mutation at residue N91, wherein the mutation is selected from: N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W. In some embodiments, the mutation is selected from: N91V, N91A, N91G, and N91S.

[0014] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from: R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from: R104S, R104Y, and R104T.

[0015] In some embodiments, the variant polypeptide does not comprise a mutation at residue R104.

[0016] In some embodiments, the variant polypeptide comprises a mutation at residue Q56, wherein the mutation is selected from: Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from: Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0017] In some embodiments, the variant polypeptide does not comprise a mutation at residue Q56.

[0018] In some embodiments, the variant polypeptide comprises a mutation at residue P57, wherein the mutation is selected from: P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from: P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0019] In some embodiments, the variant polypeptide does not comprise a mutation at residue P57.

[0020] In some embodiments, the variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the variant polypeptide comprises the mutations: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S. In some embodiments, the variant polypeptide further comprises mutations at Q56 and P57.

[0021] In some embodiments, the variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-299 and 307-318.

[0022] In some embodiments, a fusion polypeptide is provided that comprises an IL-18 variant polypeptide as described herein and a human IgGFc domain or a variant thereof. In some embodiments, the fusion polypeptide comprises a variant of the human IgG1 Fc domain that contains an N297A mutation (EU numbering). In some embodiments, the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the human IgG Fc domain or a variant thereof. In some embodiments, the C-terminus of the human IgG Fc domain or a variant thereof is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0023] In some embodiments, dimers are provided that comprise two of the fusion polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0024] In some embodiments, nucleic acids are provided that encode an IL-18 variant polypeptide or a fusion polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 80% identity to any of SEQ ID NOs: 301-306 and 333-335. In some embodiments, vectors are provided that comprise the nucleic acids described herein. In some embodiments, host cells are provided that comprise the nucleic acids or vectors described herein. In some embodiments, a method is provided for producing an IL-18 variant polypeptide or a fusion polypeptide, the method comprising: (a) culturing the host cells described herein under conditions that express the IL-18 variant polypeptide or the fusion polypeptide; and (b) recovering the IL-18 variant polypeptide or the fusion polypeptide produced by the host cells. In some embodiments, the host cell is a mammalian host cell (e.g., a CHO cell or a HEK293 cell). In some embodiments, the method comprises (e.g., further comprises) purifying the IL-18 variant polypeptide or the fusion polypeptide.

[0025] In some embodiments, pharmaceutical compositions are provided that comprise an IL-18 variant polypeptide described herein, a fusion polypeptide described herein, a nucleic acid described herein, or a vector described herein.

[0026] In some embodiments, a method is provided for treating a disease in an individual, the method comprising administering to the individual an effective amount of the pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, a method is provided for activating hIL-18 receptor-mediated signaling in an individual, the method comprising administering to the individual an effective amount of the pharmaceutical composition described herein. In some embodiments, a method is provided for stimulating immune cells that have encountered an antigen in an individual in need thereof, the method comprising administering to the individual an effective amount of the pharmaceutical composition described herein. In some embodiments, the stimulation comprises increasing the activity and / or number of immune cells that have encountered an antigen.

[0027] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention will be further described by the following detailed description.

[0028] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. Brief Description of the Drawings

[0029] Figure 1A Shows the screening process, in which methods based on magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) are used to identify the IL-18 variant polypeptides described herein.

[0030] Figure 1B Shows the ability of the output library pools from rounds 1, 2, 3, 4, and 5 to bind 1 μM IL-18BP or 50 nM IL-18Rα.

[0031] Figure 1C Shows the ability of representative individual IL-18 variant clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43) to bind IL-18Rα. SEQ ID NO: 1 is wild-type IL-18.

[0032] Figure 2 A provides SDS-PAGE analysis and SEC-HPLC analysis of purified wild-type human IL-18 (SEQ ID NO: 1).

[0033] Figure 2 B provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M12 (SEQ ID NO: 39).

[0034] Figure 2 C provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant MM5 (SEQ ID NO: 133).

[0035] Figure 2 D provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant WM6 (SEQ ID NO: 145).

[0036] Figure 2 E provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M17 (SEQ ID NO: 303).

[0037] Figure 2 F provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M24 (SEQ ID NO: 105).

[0038] Figure 3 Shows the activation of hIL-18 receptor-mediated signal transduction by exemplary IL-18 variant polypeptides of the present application.

[0039] Figure 4 Shows the hIFNγ release induced by an exemplary IL-18 variant polypeptide of the present application.

[0040] Figure 5A Shows the activation of hIL-18 receptor-mediated signal transduction by wild-type IL-18, the fusion polypeptide IL-18-SSS-Fc_N297A, and the fusion polypeptide IL-18-SSS-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0041] Figure 5B Shows the activation of hIL-18 receptor-mediated signal transduction by the IL-18 polypeptide variant M12, the fusion polypeptide M12-SSS-Fc_N297A, and the fusion polypeptide M12-SSS-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0042] Figure 5C Shows the activation of hIL-18 receptor-mediated signal transduction by the IL-18 polypeptide variant MM5, the fusion polypeptide MM5-SSS-Fc_N297A, and the fusion polypeptide MM5-SSS-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0043] Figure 6A Shows the activation of hIL-18 receptor-mediated signal transduction by the fusion polypeptide WT IL-18, WT IL-18 in the presence of IL-18 binding protein (“BP”), the fusion polypeptide M12-DB6-Fc_N297A, and M12-DB6-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0044] Figure 6B Shows the activation of hIL-18 receptor-mediated signal transduction by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB7-Fc_N297A, and M12-DB7-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0045] Figure 6C Shows the activation of hIL-18 receptor-mediated signal transduction by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB9-Fc_N297A, and M12-DB9-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0046] Figure 6D Shows the activation of hIL-18 receptor-mediated signal transduction by the fusion polypeptide WT IL-18 and the fusion polypeptide M12-DB10-Fc_N297A.

[0047] Figure 7A Shows the activation of wild-type IL-18, wild-type IL-18 in the presence of IL-18 binding protein ("BP"), the fusion polypeptide WT IL-18-DB6-Fc_N297A, and WT IL-18-DB6-Fc_N297A in the presence of IL-18 binding protein ("BP") on hIL-18 receptor-mediated signal transduction.

[0048] Figure 7B Shows the activation of wild-type IL-18, wild-type IL-18 in the presence of IL-18 binding protein ("BP"), the fusion polypeptide MM5-DB6-Fc_N297A, and MM5-DB6-Fc_N297A in the presence of IL-18 binding protein ("BP") on hIL-18 receptor-mediated signal transduction.

[0049] Figure 7C Shows the activation of wild-type IL-18 and the fusion polypeptide Fc_N297A-MM5-DB6 on hIL-18 receptor-mediated signal transduction.

[0050] Figure 7D Shows the activation of wild-type IL-18 and the fusion polypeptide Fc_N297A-M12-DB6 on hIL-18 receptor-mediated signal transduction. Detailed Description

[0051] Overview

[0052] It has been found that components of the interleukin 18 (IL-18) pathway are upregulated in tumor infiltrating lymphocytes (TIL) (see, e.g., Zhou et al. (2020) Nature 583:609–614), suggesting that IL-18 therapy may enhance anti-tumor immunity. However, IL-18 has not shown significant efficacy in clinical trials. IL-18BP is a high-affinity inhibitor of IL-18 (K D <1 nM, see, e.g., Dinarello et al. (2013) Front Immunol 4:289, doi:10.3389 / fimmu.2013.00289), which is often upregulated in a variety of human and murine tumors and is thought to limit the anti-tumor activity of IL-18 in mice and clinical trials. This application is in part based on the applicant's identification of IL-18 variant polypeptides that can induce IL-18 receptor-mediated signal transduction even in the presence of IL-18BP. It was found that by substituting one or a group of amino acids (e.g., G3, e.g., E6), the IL-18 variant polypeptides exhibit resistance to IL-18BP while still being able to bind and activate the IL-18 receptor. See, e.g., Example 6, Figures 3 - 4, Tables 3 and 4. It was also found that higher yields, higher purities and / or more effective IL-18 variant polypeptides can be obtained by substituting one or more amino acids at C38, C68, C76, S117 and / or C127. For example, see Example 7, Figures 5A - 7D , Tables 5-9.

[0053] Definitions

[0054] As used herein, the terms "specifically bind", "specifically recognize", and "specific for" refer to a measurable and reproducible interaction, such as the binding between a cytokine and its receptor, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a cytokine that specifically recognizes a receptor refers to a cytokine that binds to that receptor with higher affinity, avidity, more readily, and / or for a longer duration compared to binding to other targets. In some embodiments, the binding of the cytokine to an irrelevant target is less than about 10% of the binding of the cytokine to its receptor, as measured by radioimmunoassay (RIA). In some embodiments, a cytokine that specifically binds to its receptor has a dissociation constant (K -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M). In some embodiments, the specific binding may include, but does not require, exclusive binding. The binding specificity of a cytokine can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, ELISA testing, RIA testing, ECL testing, IRMA testing, EIA testing, BIACORE D testing and peptide scanning. TM

[0055] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, the isolated nucleic acid is not bound to all of the components associated with the production environment. In some embodiments, the isolated nucleic acid molecule encoding a polypeptide or cytokine described herein is in a form different from its form or environment found in nature.

[0056] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of the host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are herein referred to as "expression vectors".

[0057] As used herein, the terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0058] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to that of the parental cell and may contain mutations. This application includes mutant progeny having the same function or biological activity as the function or biological activity selected or screened in the originally transformed cell.

[0059] "Treatment" or "treating" as used herein is a method of obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or enhancing the quality of life, increasing weight gain and / or extending survival. "Treatment" also includes reducing the pathological consequences of cancer (e.g., tumor volume). The methods of this application contemplate one or more of these aspects of treatment.

[0060] In the context of cancer, the term "treatment" includes any one or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden and improving one or more symptoms associated with the disease.

[0061] The terms "inhibit" or "inhibition" refer to a decrease or cessation of any phenotypic trait, or a decrease or cessation of the incidence, degree, or likelihood of that trait. "Decrease" or "inhibit" means a reduction, decrease, or prevention of activity, function, and / or amount compared to a reference value. In certain embodiments, "decrease" or "inhibit" means the ability to cause an overall decrease of 20% or more. In another embodiment, "decrease" or "inhibit" means the ability to cause an overall decrease of 50% or more. In yet another embodiment, "decrease" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more.

[0062] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0063] It should be understood that the embodiments of the present application described herein include embodiments of "consisting of" and / or "consisting essentially of".

[0064] References herein to "about" a value or parameter include (and describe) variations that are directed to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0065] As used herein, a reference to "not" a value or parameter generally means and describes "except" a value or parameter. For example, the method is not used to treat cancer of type X means that the method is used to treat other types of cancer other than type X.

[0066] The term "about X - Y" as used herein has the same meaning as "about X to about Y".

[0067] As used herein and in the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly dictates otherwise.

[0068] Interleukin-18 (IL-18) variant polypeptide

[0069] Interleukin-18 (IL-18, also known as interferon-gamma inducing factor) is a protein encoded by the human IL-18 gene. It is a pro-inflammatory cytokine that may be produced by hematopoietic and non-hematopoietic cells. IL-18 can regulate innate and adaptive immunity, and its dysregulation can lead to autoimmune or inflammatory diseases.

[0070] In one aspect, the present disclosure provides interleukin 18 (“IL-18”) variant polypeptides that comprise one or more mutations at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO:1. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO:1.

[0071] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMY KDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPG HDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:1)

[0072] In some embodiments, the IL-18 variant polypeptide comprises mutations at two or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, two or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at three or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, three or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at four or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, four or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at five or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, five or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at six or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, six or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at seven or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination.In some embodiments, seven or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises eight or more mutations among F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, eight or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises nine or more mutations among F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, nine or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises ten or more mutations among F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, ten or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises eleven or more mutations among F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, eleven or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises twelve or more mutations among F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, twelve or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149.In some embodiments, the IL-18 variant polypeptide comprises mutations at thirteen or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, for example, in any combination. In some embodiments, the thirteen or more mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant comprises (or further comprises) mutations at positions other than F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 2, at least 3, or at least 4 mutations are selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, wild-type IL-18 is human IL-18 comprising the amino acid sequence SEQ ID NO: 1.

[0073] In some embodiments, one or more mutations are selected from: G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises two or more mutations at two or more of G3, E6, D54, and N91, for example in any combination. In some embodiments, two or more mutations are selected from: G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises three or more mutations at three or more of G3, E6, D54, and N91, for example in any combination. In some embodiments, three or more mutations are selected from: G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant comprises (or further comprises) mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 2, at least 3, or at least 4 mutations are selected from: G3, E6, D54, and N91. In some embodiments, wild-type IL-18 is human IL-18 comprising the amino acid sequence SEQ ID NO: 1.

[0074] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) at least one mutation at a residue selected from: Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 shown in SEQ ID NO: 1.

[0075] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation (or two or three mutations) at a residue selected from G3, E6, and M60, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises at least one of the following: a) G3P, G3S, G3E, G3N, G3D, or G3A; b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y; or c) M60K. In some embodiments, the IL-18 variant polypeptide further comprises at least one or more (e.g., two, three, four, or five) mutations at a residue selected from V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155. In some embodiments, the IL-18 variant polypeptide further comprises at least one of the following: a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, or D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, or Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, P57Q, P57G, P57K, P57H, P57L, P57I, or P57E; d) N91V, N91G, N91A, N91S, N91T, N91R, N91K, N91P, N91I, or N91W; e) R104V, R104T, R104Y, R104F, R104T, R104L, R104S, R104A, R104E, R104I; f) N155K; g) V11I; h) K96D or K96E; i) I149M; or j) K140R.

[0076] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6K, D54H, Q56D, P57R, N91V, and R104V, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0077] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3S, E6K, D54W, Q56P, P57D, N91G, and R104T, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0078] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0079] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6K, D54W, Q56G, P57V, N91V, and R104F, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0080] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0081] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6R, D54W, Q56T, N91V, and R104T, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally where the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally where the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally where the substitution is selected from C38I, C38V, C38L, and C38M, further optionally where the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally where the substitution is selected from C68I, C68V, C68S, and C68D, further optionally where the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally where the C76 substitution is C76Y or C76V, and optionally where the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally where the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally where the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0082] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6R, D54H, Q56T, P57A, and N91A, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0083] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54W, Q56P, P57A, N91A, and R104L, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0084] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54W, Q56R, P57A, N91S, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0085] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54Q, Q56L, P57W, and N91S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0086] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54S, Q56R, P57N, N91G, and R104T, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0087] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6K, D54G, Q56G, P57A, and N91T, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0088] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6K, D54Q, Q56I, and P57W, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0089] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3N, E6K, D54P, Q56S, P57S, N91R, and R104A, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0090] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54S, Q56Y, P57T, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0091] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54S, Q56R, P57N, N91G, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0092] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54L, Q56T, P57A, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0093] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54S, Q56R, P57R, N91G, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0094] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6K, D54H, Q56E, P57Q, and N91A, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0095] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6R, D54S, Q56R, P57S, N91G, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0096] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54S, Q56S, P57T, N91G, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0097] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0098] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54Y, Q56T, and P57R, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0099] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54Y, Q56T, and P57S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0100] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54L, Q56T, P57T, and N91R, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0101] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0102] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0103] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations, said combination comprising G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide without one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0104] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6M, D54F, Q56D, P57R, and N91P, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0105] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6L, D54H, Q56T, P57V, and N91S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0106] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0107] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6G, D54S, Q56S, and P57R, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0108] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3E, E6H, D54R, Q56T, and P57H, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0109] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54H, Q56R, P57N, N91V, and R104E, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0110] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54G, Q56G, P57A, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0111] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6S, D54A, Q56D, P57Q, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have the one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0112] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6G, D54Q, Q56V, and P57W, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0113] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6S, D54W, Q56G, P57A, N91V, and R104I, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0114] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, D54W, Q56P, P57G, N91V, and R104L, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0115] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3D, E6K, D54P, Q56S, P57W, and N91W, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally where the IL-18 variant polypeptide has a) binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally where the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally where the substitution is selected from C38I, C38V, C38L, and C38M, further optionally where the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally where the substitution is selected from C68I, C68V, C68S, and C68D, further optionally where the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally where the C76 substitution is C76Y or C76V, and optionally where the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally where the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally where the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0116] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution), which is G3P, wherein the amino acid position is relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0117] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6K, D54G, Q56G, and P57A, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0118] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that includes G3P, E6R, D54L, Q56G, P57S, and N91V, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0119] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, V11I, D54G, Q56G, P57A, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have the one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0120] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6K, D54H, Q56Y, and P57S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0121] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes E6R, D54W, Q56S, and P57Q, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0122] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6K, D54L, Q56T, P57Q, and N91V, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0123] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3A, E6Y, D54R, Q56S, P57L, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0124] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include G3P, E6R, D54L, Q56T, P57I, and N91G, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0125] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations that include E6G, D54L, Q56T, P57E, N91G, and R104S, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally where the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally where the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally where the substitution is selected from C38I, C38V, C38L, and C38M, further optionally where the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally where the substitution is selected from C68I, C68V, C68S, and C68D, further optionally where the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally where the C76 substitution is C76Y or C76V, and optionally where the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally where the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally where the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0126] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3A, E6Y, D54R, Q56S, P57L, and N91A, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any additional mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally where the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is equivalent to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is equivalent to or lower than, that of the corresponding IL-18 variant polypeptide that does not have the one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally where the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally where the substitution is selected from C38I, C38V, C38L, and C38M, further optionally where the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally where the substitution is selected from C68I, C68V, C68S, and C68D, further optionally where the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally where the C76 substitution is C76Y or C76V, and optionally where the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally where the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally where the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0127] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes M60K and K96D, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0128] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, and K96E, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0129] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution), the substitution comprising M60K, wherein the amino acid position is relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide without one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0130] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P and E6R, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0131] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P and E6K, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0132] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3D, E6K, and N91S, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not contain any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0133] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3S and I149M, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0134] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3P, E6R, and N91S, where the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0135] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) that comprises E6R, wherein the amino acid position is relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0136] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes E6R and N91S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein, compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises an S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0137] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) that comprises V11I, wherein the amino acid position is relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity for the IL-18 receptor (e.g., IL-18Ra) that is comparable to or stronger than, and / or b) a binding affinity for the IL-18 binding protein that is comparable to or lower than, that of the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0138] In some embodiments, the IL-18 variant polypeptide comprises a combination of substitutions that includes G3S and K140R, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) a binding affinity equivalent to or stronger than that of the IL-18 receptor (e.g., IL-18Ra), and / or b) a binding affinity equivalent to or lower than that of the IL-18 binding protein, as compared to the corresponding IL-18 variant polypeptide that does not have one, two, or three additional mutations or substitutions. In some embodiments, the IL-18 variant polypeptide further comprises the S117C substitution, optionally wherein the S117C substitution promotes the C76-C117 disulfide bond. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C38, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises the C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises the C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0139] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-153.

[0140] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-150.

[0141] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150.

[0142] In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, at least four, at least five, or six mutations at residues selected from: C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a substitution at C38 (i.e., wherein the wild-type cysteine at position 38 is replaced with any amino acid), a substitution at C68 (i.e., wherein the wild-type cysteine at position 68 is replaced with any amino acid), and an S117C substitution. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C38I, C38V, C38L, C38M, or C38S mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C68S, C68I, C68D, C68V, or C68L mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C76S, C76V, or C76Y mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) an S117C mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C127A, C127Y, C127F, C127L, or C127I mutation. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, or four mutations selected from C38S, C68S, C76S, and C127S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68S, and S117C mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38V, C68I, S117C, and C127A mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38S, C68I, S117C, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68I, C76V, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68L, and C76Y mutations.

[0143] In some embodiments, the IL-18 variant polypeptide further comprises one or more mutations at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises two or more mutations at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises mutations at Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant comprises (or further comprises) mutations at positions other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 mutations are selected from: G3, E6, D54, Q56, P57, N91, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise substitutions at Q56 and P57, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence SEQ ID NO: 1.

[0144] In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor α (“IL-18Rα”), e.g., human IL-18Rα or “hIL-18α”, and exhibits a significantly reduced binding to IL-18 binding protein (“IL-18BP”, e.g., human IL-18 or “hIL-18BP”). In some embodiments, the IL-18 variant polypeptide exhibits a significantly reduced binding to IL-18BP relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is comparable to the affinity of the wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is increased compared to the affinity of the wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0145] In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα with a K D less than about 5x 10 -5 M, less than about 5x10-6 M, less than about 5 x 10 -7 M, less than about 5 x 10 -8 M, less than about 5 x 10 -9 M, less than about 5 x 10 -10 M or less than about 5 x 10 - 11 M, including any range between these values. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -5 to about 5 x 10 -11 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 D to about 5 x 10 -7 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -11 to about 5 x 10 D M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -7 to about 5 x 10 -10 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 D to about 5 x 10 -7 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -9 to about 5 x 10 D M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -8 to about 5 x 10 -11 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 D to about 5 x 10 -9 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -11 to about 5 x 10 D M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -8 to about 5 x 10 -10 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 D to about 5 x 10 -7 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -10 to about 5 x 10 D M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 -9 to about 5 x 10 -10 M binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide is at about 5 x 10 D to about 5 x 10 -7 to about 5 x 10 -9 M binds to IL-18Rα.D Binds to IL-18Rα. In some embodiments, the IL-18 variant polypeptide has a K -8 between about 5x 10 -9 and about 5x 10 D M for binding to IL-18Rα. In some embodiments, the IL-18 variant has a K -5 between about 5x 10 -11 and about 5x 10 D M for binding to IL-18Rα (e.g., hIL-18Rα), such as about 5x 10 -5 , 6x 10 -5 , 7x 10 -5 , 8x 10 -5 , 9x 10 -5 , 1x 10 -6 , 2x 10 -6 , 3x 10 -6 , 4x 10 -6 , 5x 10 -6 , 6x 10 -6 , 7x 10 -6 , 8x 10 -6 , 9x 10 -6 , 1x 10 -7 , 2x 10 -7 , 3x 10 -7 , 4x 10 -7 , 5x10 -7 , 6x 10 -7 , 7x 10 -7 , 8x 10 -7 , 9x 10 -7 , 1x 10 -8 , 2x 10 -8 , 3x 10 -8 , 4x 10 -8 , 5x 10 -8 , 6x10 -8 , 7x 10 -8 , 8x 10 -8 , 9x 10 -8 , 1x 10 -9 , 2x 10 -9 , 3x 10 -9 , 4x 10 -9 , 5x 10 -9 , 6x 10 -9 , 7x 10 -9 , 8x10 -9 , 9x 10 -9 , 1x 10-10 , 2 x 10 -10 , 3 x 10 -10 , 4 x 10 -10 , 5 x 10 -10 , 6 x 10 -10 , 7 x 10 -10 , 8 x 10 -10 , 9 x 10 -10 , 1 x 10 -11 , 2 x 10 -11 , 3 x 10 -11 , 4 x 10 -11 , or 5 x 10 -11 Any of those in M, including any range between these values. In some embodiments, the IL-18 variant polypeptide has a higher affinity for IL-18Rα (e.g., hIL-18Rα) than the wild-type human IL-18 shown in SEQ ID NO: 1 has for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the IL-18 variant polypeptide has an affinity for IL-18Rα (e.g., hIL-18Rα) that is comparable (e.g., approximately the same) to the affinity of the wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0146] In some embodiments, the IL-18 variant polypeptide exhibits significantly reduced binding to the IL-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -9 greater than 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -8 greater than 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -7 greater than 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -6 greater than 5 x 10 D M. In some embodiments, the IL-18 variant polypeptide binds to IL-18BP with a K -5 greater than 5 x 10 DBinds to IL-18BP. In some embodiments, the IL-18 variant polypeptide does not exhibit binding (e.g., undetectable binding) to IL-18BP (e.g., hIL-18BP). In some embodiments, the IL-18 variant polypeptide that does not exhibit binding (e.g., undetectable binding) to IL-18BP (e.g., hIL-18BP) has a K greater than 10 -3 M D Binds to IL-18BP (e.g., hIL-18BP).

[0147] The affinity of the IL-18 described herein for IL-18Rα and / or IL-18BP can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL) assay, immunoradiometric (IRMA) assay, enzyme immunoassay (EIA), surface plasmon resonance (SPR), peptide scanning, and fluorescence-activated cell sorting (FACS)-based kinetic competition screening.

[0148] In some embodiments, the IL-18 variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from G3P, G3D, G3E, G3N, and G3S. As used herein, "G3X" means that G, the wild-type amino acid at position 3 of SEQ ID NO: 1, has been replaced by amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 of SEQ ID NO: 1 has been replaced by amino acid P.

[0149] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue E6, wherein the mutation is selected from E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from E6R and E6K.

[0150] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue D54, wherein the mutation is selected from D54W, D54H, D54S, D54Q, D54L, D54Y, D54P, D54A, D54F, D54G, and D54T. In some embodiments, the mutation is selected from D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0151] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue N91, wherein the mutation is selected from N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from N91V, N91A, N91G, and N91S.

[0152] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from R104S, R104Y, R104T, R104L, R104V, R104A, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from R104S, R104Y, and R104T. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at residue R104.

[0153] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue Q56, the mutation being selected from Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0154] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue P57, the mutation being selected from P57A, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57K, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0155] In some embodiments, the IL-18 variant polypeptide comprises a mutation at one or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at two or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at three or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, wherein the IL-18 variant polypeptide comprises a G3P mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an E6R or E6K mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises a D54W, D54H, D54S, or D54Q mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an N91V, N91A, N91G, or N91S mutation. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant further comprises a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 2, at least 3, or at least 4 mutations are selected from: G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at Q56 and / or P57. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at a position other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 2, at least 3, at least 4 mutations, at least 5 mutations, or at least 6 mutations are selected from: G3, E6, D54, N91, Q56, and P57.

[0156] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of any one of SEQ ID NOs: 2-299, 307-318. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-299, 307-318. The amino acid sequences of SEQ ID NOs: 2-299, 307-318 are provided in the sequence summary table below the examples.

[0157] Fusion polypeptide

[0158] In some embodiments, the IL-18 polypeptides activatable by the present disclosure include fusion polypeptides that comprise wild-type IL-18 or an IL-18 variant polypeptide described herein. In some embodiments, the fusion polypeptide comprises (1) wild-type IL-18 or an IL-18 variant polypeptide and (2) a dimerization domain. In some embodiments, the fusion polypeptide comprises (1) two or more wild-type IL-18s, two or more IL-18 variant polypeptides, or two or more of any combination of wild-type IL-18 and IL-18 variant polypeptides, and (2) a dimerization domain. In some embodiments, the dimerization domain is or comprises a leucine zipper (LZ) element. Leucine zippers are generally identified as stretches of about 35 amino acids that contain 4-5 leucine residues separated from each other by 6 amino acids (Maniatis and Abel (1989) Nature 341:24-25). Exemplary leucine zippers occur in a variety of eukaryotic DNA-binding proteins such as GCN4, C / EBP, c-Fos, c-Jun, c-Myc, and c-Max. In some embodiments, the dimerization domain is or comprises a helix-loop-helix domain (Murre, C. et al. (1989) Cell 58:537-544). The dimerization domain can also be selected from other proteins such as retinoic acid receptors, thyroid hormone receptors, or other nuclear hormone receptors (Kurokawa et al. (1993) Genes Dev. 7:1423-1435), or from the yeast transcription factors GAL4 and HAP1 (Marmonstein et al. (1992) Nature 356:408-414; Zhang et al. (1993) Proc. Natl. Acad. Sci. USA 90:2851-2855). The dimerization domain is further described by Eisenman in U.S. Patent No. 5,624,818. In some embodiments, the dimerization domain is an antibody Fc domain.

[0159] In some embodiments, the fusion polypeptide comprises wild-type IL-18 (e.g., wild-type IL-18 having an amino acid sequence as set forth in SEQ ID NO: 1) and an antibody Fc domain. In some embodiments, the fusion polypeptide comprises an IL-18 variant polypeptide (e.g., an IL-18 variant polypeptide as described herein) and an antibody Fc domain. In some embodiments, the C-terminus of wild-type IL-18 or the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the antibody Fc domain. In some embodiments, the C-terminus of the antibody Fc domain is fused to the N-terminus of wild-type IL-18 or the N-terminus of the IL-18 variant polypeptide. In some embodiments, the Fc domain is a human Fc domain or a variant thereof, the variant comprising one or more amino acid substitutions. In some embodiments, the Fc domain is a human IgG Fc domain or a variant thereof, such as a human IgG1, IgG2, or IgG4 Fc domain or a variant of any of the foregoing. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, an IL-18 variant polypeptide as described herein and a human IgG1 Fc variant comprising an N297A mutation, wherein the amino acid numbering is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, wild-type IL-18 and a human IgG1 Fc variant comprising an N297A mutation. In some embodiments, the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0160] Nucleic acids, vectors, host cells, and methods for producing an IL-18 variant polypeptide

[0161] Nucleic acid molecules encoding the IL-18 variant polypeptides described herein or the fusion polypeptides described herein are also contemplated. In some embodiments, nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides described herein are provided. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence selected from SEQ ID NOs: 301-306 and 333-335. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from SEQ ID NOs: 301-306 and 333-355. The nucleic acid sequences of SEQ ID NOs: 301-306 and 333-335 are provided in the Sequence Listing table below the Examples.

[0162] Vectors incorporating the nucleic acids described herein are also provided.

[0163] Briefly, expression of the IL-18 variant polypeptides or fusion polypeptides described herein by a natural or synthetic nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements including, for example, a promoter (e.g., a constitutive, regulatable tissue-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in a eukaryotic host cell. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0164] The nucleic acid can be cloned into a variety of types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0165] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0166] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or ex vivo to the cells of a subject. A variety of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A variety of adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of the transgene and its transmission to daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of being less immunogenic.

[0167] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these elements are located in the region 30 - 110 base pairs (bp) upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so that promoter function is retained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0168] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that is capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include but are not limited to metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0169] In some embodiments, the expression of the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide is inducible. In some embodiments, the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide is operably linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide described herein has been engineered to encode an epitope tag, for example, to facilitate purification or detection of the polypeptide. Exemplary epitope tags include but are not limited to, for example, 6x His (also known as His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0170] The IL-18 variant polypeptides or fusion polypeptides of the present disclosure can be produced by any method known in the art. Exemplary techniques for polypeptide production are described below; however, these exemplary techniques are for illustrative purposes only and are not intended to be limiting.

[0171] The recombinant methods can be used to produce the IL-18 variant polypeptides or fusion polypeptides described herein. For the recombinant production of IL-18 variant polypeptides or fusion polypeptides, the nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides are isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the IL-18 variant polypeptides or fusion polypeptides can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the IL-18 variant polypeptide or fusion polypeptide). There are many vectors available. Vector elements generally include but are not limited to one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0172] Both expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, e.g., to replicate independently of the host chromosomal DNA. Such sequences can include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeasts, and viruses. Generally, mammalian expression vectors do not require an origin of replication component (the SV40 origin can be used as it contains an early promoter).

[0173] Expression vectors and cloning vectors can contain selectable genes or selectable markers. Typical selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement nutritional deficiencies, or (c) provide key nutrients not available in complex media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Another example of selectable markers applicable to mammalian cells are those that can identify cells that have taken up the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. For example, transformants are identified by culturing them in a medium containing methotrexate (Mtx) (a competitive antagonist of DHFR), for Chinese hamster ovary (CHO) cell lines lacking endogenous DHFR activity that have been transformed with the DHFR gene.

[0174] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding the IL-18 variant polypeptide or fusion polypeptide of interest, the wild-type DHFR gene, and another selectable marker (e.g., aminoglycoside 3'-phosphotransferase (APH)) can be selected by growing the cells in a medium containing a selection agent for the selectable marker (e.g., an aminoglycoside antibiotic such as kanamycin, neomycin, or G418).

[0175] Expression vectors and cloning vectors typically contain promoters recognized by the host organism and are operably linked to a nucleic acid encoding an IL-18 variant polypeptide or a fusion polypeptide. Promoters suitable for prokaryotic hosts include the phoA promoter, the beta-lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Eukaryotic promoter sequences are known. Yeast promoters are well known in the art and may include inducible promoters / enhancers regulated by growth conditions. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Examples include, but are not limited to, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Transcription of an IL-18 variant polypeptide or a fusion polypeptide from a vector in mammalian host cells can be under the control of, for example, a promoter obtained from a viral genome. The early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments, which also contain the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus can be conveniently obtained as a HindIII restriction fragment. Alternatively, the long terminal repeat of Rous sarcoma virus can be used as a promoter.

[0176] Transcription of DNA encoding an IL-18 variant polypeptide or a fusion polypeptide described herein in higher eukaryotes is usually increased by inserting enhancer sequences into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are currently known. However, enhancers from eukaryotic cell viruses are commonly used.

[0177] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing the mRNA.

[0178] Host cells suitable for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeasts or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as the Enterobacteriaceae, such as the genus Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, etc. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are also suitable cloning or expression hosts for vectors encoding IL-18 polypeptide variants or fusion polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized" to produce IL-18 variant polypeptides or fusion polypeptides with a partially or fully human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006).

[0179] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), Medicago truncatula, and tobacco can also be used as hosts.

[0180] Host cells suitable for expressing glycosylated IL-18 variant polypeptides or fusion polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified.

[0181] Vertebrate cells can serve as hosts, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or subclones of 293 cells adapted for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2 / 0. For a review of some mammalian host cell lines suitable for production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.

[0182] The host cells of the present disclosure can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described below can be used as the medium for the host cells: Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985. Any of these media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Appropriate concentrations of any other necessary supplements known to those skilled in the art can also be included. The culture conditions, such as temperature, pH, etc., are those previously selected for the host cells used for expression and will be apparent to those skilled in the art.

[0183] When recombinant techniques are utilized, the IL-18 variant polypeptide or fusion polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the polypeptide is produced intracellularly, the first step is to remove particulate debris (host cells or lysed fragments) by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of Escherichia coli.

[0184] Polypeptide compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, among which affinity chromatography is one of the commonly preferred purification steps. In some embodiments, the IL-18 variant polypeptides or fusion polypeptides described herein contain epitope tags (e.g., tags linked to the IL-18 variant polypeptides or fusion polypeptides via cleavable linkers) to facilitate purification. Exemplary epitope tags include, but are not limited to, for example, 6x His (also known as His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0185] Methods for treating diseases, activating hIL-18 receptor-mediated signal transduction, and stimulating T cells or NK cells that have encountered an antigen

[0186] Also provided herein are methods for treating a disease or disorder in an individual. The method includes administering to the individual having the disease or disorder an IL-18 variant polypeptide or fusion polypeptide, a nucleic acid, a vector, and / or a pharmaceutical composition described herein. In some embodiments, the disease or disorder is a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0187] In some embodiments, provided are methods for activating hIL-18 receptor-mediated signals in an individual, the method including administering to the individual an IL-18 variant polypeptide or fusion polypeptide, a nucleic acid, a vector, and / or a pharmaceutical composition described herein. In some embodiments, provided herein are methods for stimulating T cells or NK cells that have encountered an antigen in an individual, the method including administering to the individual an IL-18 variant polypeptide or fusion polypeptide, a nucleic acid, a vector, and / or a pharmaceutical composition described herein. In some embodiments of any method herein, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.

[0188] Compositions, kits, and articles of manufacture

[0189] Also provided herein are compositions (e.g., formulations) comprising an IL-18 variant polypeptide, a fusion polypeptide, a nucleic acid, a vector, or a host cell described herein.

[0190] A suitable composition can be obtained by mixing an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980)).

[0191] Also provided are kits comprising an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell comprising a nucleic acid or vector described herein. The kits can be used for any of the therapeutic methods described herein.

[0192] The kits of the present application employ suitable packaging. Suitable packaging includes, but is not limited to: vials, bottles, jars, flexible packages (such as sealed Mylar or plastic bags), etc. The kits can optionally provide additional components, such as buffers and interpretive information.

[0193] Accordingly, the present application also provides articles of manufacture. An article of manufacture can include a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packages, etc. Generally, the container contains the composition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0194] Those skilled in the art will recognize that there are a variety of possible embodiments within the scope and spirit of the present invention. The present invention will be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention, but should of course not be construed as limiting its scope in any way.

[0195] Exemplary Embodiments

[0196] Embodiment 1. An interleukin-18 (IL-18) variant polypeptide comprising at least one mutation at a residue selected from: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 as shown in SEQ ID NO: 1.

[0197] Embodiment 2. The IL-18 variant polypeptide of Embodiment 1, comprising at least one mutation at a residue selected from: G3, E6, D54, and N91, wherein the amino acid positions are relative to the amino acid positions of wild-type human IL-18 as shown in SEQ ID NO: 1.

[0198] Embodiment 3. The IL-18 variant polypeptide according to Embodiment 1 or 2, which further comprises at least one mutation at a residue selected from: Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO: 1.

[0199] Embodiment 4. The IL-18 variant polypeptide according to any one of Embodiments 1-3, which further comprises at least one mutation at a residue selected from: C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO: 1.

[0200] Embodiment 5. The IL-18 variant polypeptide according to Embodiment 4, wherein the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution.

[0201] Embodiment 6. The IL-18 variant polypeptide according to Embodiment 4, wherein the at least one mutation is selected from: C38S, C68S, C76S, and C127S.

[0202] Embodiment 7. The IL-18 variant polypeptide according to Embodiment 4, wherein the variant polypeptide further comprises C38S, C68S, and C76S mutations.

[0203] Embodiment 8. The IL-18 variant polypeptide according to Embodiment 4, wherein the variant polypeptide further comprises a set of mutations selected from: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y.

[0204] Embodiment 9. An interleukin-18 (IL-18) variant polypeptide, which comprises at least one mutation at a residue selected from: C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO: 1.

[0205] Embodiment 10. The IL-18 variant polypeptide according to Embodiment 9, wherein the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution.

[0206] Embodiment 11. The IL-18 variant polypeptide according to Embodiment 9, wherein at least one mutation is selected from: C38S, C68S, C76S, and C127S.

[0207] Embodiment 12. The IL-18 variant polypeptide according to Embodiment 10, wherein the variant polypeptide comprises the C38S, C68S, and C76S mutations.

[0208] Embodiment 13. The IL-18 variant polypeptide according to Embodiment 9, wherein the variant polypeptide comprises a set of mutations selected from: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y.

[0209] Embodiment 14. The IL-18 variant polypeptide according to any one of Embodiments 1-13, wherein the wild-type IL-18 is human IL-18 comprising the amino acid sequence SEQ ID NO: 1.

[0210] Embodiment 15. The IL-18 variant polypeptide according to any one of Embodiments 1-14, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor α (IL-18Rα) and exhibits significantly reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18.

[0211] Embodiment 16. The IL-18 variant polypeptide according to any one of Embodiments 1-15, wherein the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18.

[0212] Embodiment 17. The IL-18 variant polypeptide according to any one of Embodiments 1-16, wherein the IL-18 variant polypeptide binds to IL-18Rα with a KD of less than about 5x10 -5 M.

[0213] Embodiment 18. The IL-18 variant polypeptide according to any one of Embodiments 1-17, wherein the IL-18 variant polypeptide binds to IL-18Rα with a KD of about 5x10 -5 to about 5x10 -11 M.

[0214] Embodiment 19. The IL-18 variant polypeptide according to any one of Embodiments 1-18, wherein the IL-18 variant polypeptide binds to IL-18BP with a KD higher than 5x10 -9 M.

[0215] Embodiment 20. The IL-18 variant polypeptide according to any one of Embodiments 1-19, wherein the IL-18 variant polypeptide does not exhibit binding to IL-18BP.

[0216] Embodiment 21. The IL-18 variant polypeptide according to any one of Embodiments 1-20, wherein the variant polypeptide comprises a mutation at residue G3, and the mutation is selected from: G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N, and G3S.

[0217] Embodiment 22. The IL-18 variant polypeptide according to Embodiment 21, wherein the mutation is G3P.

[0218] Embodiment 23. The IL-18 variant polypeptide according to any one of Embodiments 1-22, wherein the variant polypeptide comprises a mutation at residue E6, and the mutation is selected from: E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y.

[0219] Embodiment 24. The IL-18 variant polypeptide according to Embodiment 23, wherein the mutation is selected from: E6R, E6K, E6G, E6T, E6A, and E6S.

[0220] Embodiment 25. The IL-18 variant polypeptide according to Embodiment 24, wherein the mutation is selected from: E6R and E6K.

[0221] Embodiment 26. The IL-18 variant polypeptide according to any one of Embodiments 1-25, wherein the variant polypeptide comprises a mutation at residue D54, and the mutation is selected from: D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T.

[0222] Embodiment 27. The IL-18 variant polypeptide according to Embodiment 26, wherein the mutation is selected from: D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0223] Embodiment 28. The IL-18 variant polypeptide according to any one of Embodiments 1-27, wherein the variant polypeptide comprises a mutation at residue N91, and the mutation is selected from: N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W.

[0224] Embodiment 29. The IL-18 variant polypeptide according to embodiment 28, wherein the mutation is selected from: N91V, N91A, N91G, and N91S.

[0225] Embodiment 30. The IL-18 variant polypeptide according to any one of embodiments 1-29, wherein the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from: R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N.

[0226] Embodiment 31. The IL-18 variant polypeptide according to embodiment 30, wherein the mutation is selected from: R104S, R104Y, and R104T.

[0227] Embodiment 32. The IL-18 variant polypeptide according to any one of embodiments 1-29, wherein the variant polypeptide does not comprise a mutation at residue R104.

[0228] Embodiment 33. The IL-18 variant polypeptide according to any one of embodiments 1-32, wherein the variant polypeptide comprises a mutation at residue Q56, wherein the mutation is selected from: Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N, and Q56V.

[0229] Embodiment 34. The IL-18 variant polypeptide according to embodiment 33, wherein the mutation is selected from: Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0230] Embodiment 35. The IL-18 variant polypeptide according to any one of embodiments 1-32, wherein the variant polypeptide does not comprise a mutation at residue Q56.

[0231] Embodiment 36. The IL-18 variant polypeptide according to any one of embodiments 1-35, wherein the variant polypeptide comprises a mutation at residue P57, wherein the mutation is selected from: P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D.

[0232] Embodiment 37. The IL-18 variant polypeptide according to Embodiment 36, wherein the mutation is selected from: P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0233] Embodiment 38. The IL-18 variant polypeptide according to any one of Embodiments 1-35, wherein the variant polypeptide does not contain a mutation at residue P57.

[0234] Embodiment 39. The IL-18 variant polypeptide according to any one of Embodiments 1-38, wherein the variant polypeptide contains mutations at G3, E6, D54, and N91.

[0235] Embodiment 40. The IL-18 variant polypeptide according to Embodiment 39, wherein the variant polypeptide contains the mutations: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S.

[0236] Embodiment 41. The IL-18 variant polypeptide according to any one of Embodiments 1-34, 36-37, and 39-40, wherein the variant polypeptide further contains mutations at Q56 and P57.

[0237] Embodiment 42. The IL-18 variant polypeptide according to any one of Embodiments 1-41, wherein the variant polypeptide contains the amino acid sequence shown in any one of SEQ ID NOs: 2-299 and 307-318.

[0238] Embodiment 43. A fusion polypeptide comprising the IL-18 variant polypeptide according to any one of Embodiments 1-42 and a human IgG Fc domain or a variant thereof.

[0239] Embodiment 44. The fusion polypeptide according to Embodiment 43, which comprises a variant of the human IgG1 Fc domain containing the N297A mutation (EU numbering).

[0240] Embodiment 45. The fusion polypeptide according to Embodiment 43 or 44, wherein the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the human IgG Fc domain or a variant thereof.

[0241] Embodiment 46. The fusion polypeptide according to Embodiment 43 or 44, wherein the C-terminus of the human IgG Fc domain or a variant thereof is fused to the N-terminus of the IL-18 variant polypeptide.

[0242] Embodiment 47. The fusion polypeptide according to any one of Embodiments 43-46, which comprises an amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0243] Embodiment 48. A dimer, which comprises two fusion polypeptides of any one of Embodiments 43-47.

[0244] Embodiment 49. The dimer according to Embodiment 48, wherein the dimer is a homodimer.

[0245] Embodiment 50. The dimer according to Embodiment 48, wherein the dimer is a heterodimer.

[0246] Embodiment 51. A nucleic acid, which encodes the IL-18 variant polypeptide according to any one of Embodiments 1-42 or the fusion polypeptide according to any one of Embodiments 43-47.

[0247] Embodiment 52. The nucleic acid according to Embodiment 51, which comprises a polynucleotide sequence having at least 80% identity with any one of SEQ ID NOs: 301-306 and 333-335.

[0248] Embodiment 53. A vector, which comprises the nucleic acid according to Embodiment 51 or 52.

[0249] Embodiment 54. A host cell, which comprises the nucleic acid according to Embodiment 51 or 52 or the vector according to Embodiment 53.

[0250] Embodiment 55. A method for producing an IL-18 variant polypeptide or a fusion polypeptide, comprising: (a) culturing the host cell of Embodiment 54 under conditions for expressing the IL-18 variant polypeptide or the fusion polypeptide; and (b) recovering the IL-18 variant polypeptide or the fusion polypeptide produced by the host cell.

[0251] Embodiment 56. The method according to Embodiment 55, further comprising purifying the IL-18 variant polypeptide or the fusion polypeptide.

[0252] Embodiment 57. A pharmaceutical composition, which comprises the IL-18 variant polypeptide according to any one of Embodiments 1-42, the fusion polypeptide according to any one of Embodiments 43-47, the nucleic acid according to Embodiment 51 or 52, or the vector according to Embodiment 53.

[0253] Embodiment 58. A method for treating an individual's disease, comprising administering to the individual an effective amount of the pharmaceutical composition according to Embodiment 57.

[0254] Embodiment 59. The method according to embodiment 58, wherein the disease is cancer.

[0255] Embodiment 60. A method of activating hIL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition according to embodiment 57.

[0256] Embodiment 61. A method of stimulating immune cells that have experienced an antigen in an individual in need thereof, the method comprising administering to the individual an effective amount of the pharmaceutical composition according to embodiment 57.

[0257] Embodiment 62. The method according to embodiment 61, wherein the stimulation comprises increasing the activity and / or number of immune cells that have experienced an antigen.

[0258] Examples

[0259] The following examples are presented in order to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present invention. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. The following examples are only used to illustrate the present application and should not be considered in any way to limit the present application. The following embodiments and detailed descriptions are for illustrative purposes only and not for limiting purposes.

[0260] Example 1: Construction of a yeast expression library of IL-18 variant polypeptides that do not bind IL-18BP

[0261] Interleukin 18 (“IL-18”) stimulates T cells and NK cells by binding to a heterodimeric receptor complex composed of the IL-18Rα and IL-18Rβ subunits. Interleukin 18 binding protein (“IL-18BP”) is a natural inhibitor of IL-18 activity. IL-18BP has a much higher affinity for IL-18 than IL-18Rα, and the binding of IL-18BP to IL-18 blocks the interaction of IL-18 with IL-18Rα. To develop human IL-18 (“hIL-18”) variants that do not bind IL-18BP or have reduced binding affinity for IL-18BP, we used an artificial intelligence-guided molecular design method to determine the amino acid positions of mutations in wild-type hIL-18. Next, a yeast library displaying hIL-18 variant polypeptides containing the identified mutations relative to wild-type IL-18 (SEQ ID NO: 1) was constructed using NNK mutagenesis, and the final library diversity was approximately 2.72×10 8The c-myc tag was fused to each IL-18 variant for expression testing.

[0262] Example 2: Screening of IL-18 variant polypeptides that bind to human IL-18Rα

[0263] The library constructed in Example 1 was screened using a positive / negative selection strategy to identify IL-18 variant polypeptides that bind to hIL-18Rα but not to hIL-18BP and IL-18 variant polypeptides with reduced binding affinity to hIL-18BP compared to wild-type IL-18. Briefly, the recombinant hIL-18Rα extracellular domain (“hIL-18RαECD”) was biotinylated and immobilized on the first group of streptavidin (SA) magnetic microbeads or anti-biotin magnetic microbeads for screening based on magnetic-activated cell sorting (MACS). Recombinant hIL-18BP was immobilized on the second group of SA magnetic microbeads or anti-biotin magnetic microbeads. The library constructed in Example 1 was screened three rounds using hIL-18RαECD-binding beads and hIL-18BP-binding beads, as Figure 1A shown.

[0264] Next, two additional rounds of FACS-based kinetic competition screening (i.e., Figure 1A the 4th and 5th rounds shown) were performed to further enrich IL-18 variant polypeptides that exhibited the following characteristics: (a) higher or comparable affinity for hIL-18Rα compared to WT hIL-18, and (b) undetectable binding to hIL-18BP or reduced binding to hIL-18BP (e.g., K D > 10 -3 M) for hIL-18BP. Then, single yeast cells were isolated from the output pools of the 4th and 5th rounds. Plasmids encoding the isolated IL-18 variant polypeptides were extracted from the yeast and sequenced by next-generation sequencing (NGS).

[0265] After each round of screening, the output library size was determined by FACS. The binding ability of the output library to 50 nM hIL-18RαECD or 1 μM hIL-18BP was also confirmed by FACS. As Figure 1B shown, after the above screening, most of the IL-18 variants displayed did not bind to hIL-18BP or had reduced binding to hIL-18BP, while maintaining or enhancing the binding ability to hIL-18RαECD.

[0266] Example 3: Monoclonal fluorescence-activated cell sorting (FACS) of yeast cells displaying IL-18 variant polypeptides.

[0267] According to the screening described in Example 2, monoclonal FACS analysis was performed to verify the binding of the IL-18 variant polypeptides expressed on the surface of yeast cells selected in the 4th and 5th rounds to 10 nM hIL-18Rα-ECD. Briefly, yeast clones of each identical density were incubated separately with 10 nM IL-18Rα-ECD labeled with a fluorescent probe, followed by a washing step. Then, the proportion of fluorescently positive IL-18Rα ECD-binding yeast in each clone population was analyzed.

[0268] As shown in Table 1 and Figure 1C as shown (which shows WT IL-18 (SEQ ID NO: 1), and taking clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43) as examples), among the 192 IL-18 variant clones tested, 117 (i.e., SEQ ID NOs: 2 - 118) were identified as positive clones (>0.1% of the cells in the clone population were fluorescent). SEQ ID NO: 1 is wild-type human IL-18. Table 1:

[0269] Table 1. Monoclonal FACS results of 117 clones

[0270]

[0271]

[0272]

[0273]

[0274] Example 4: Recombinant hIL-18 Expression and Purification

[0275] WT hIL-18 and the above-selected IL-18 variant polypeptides were expressed in an E. coli system. Briefly, the PET30a plasmid containing the codon-optimized sequence encoding WT hIL-18 or hIL-18 variant polypeptide was transfected into E. coli BL21(DE3) cells. Then, the transfected E. coli cells were amplified in the medium, and the expressed polypeptides were harvested and purified.

[0276] Figure 2 A- Figure 2 F shows that the purity of WT hIL-18 and five exemplary variants (M12, MM5, WM6, M17, and M24) exceeded 95% as determined by SDS-PAGE and SEC-HPLC.

[0277] Example 5: Affinity of IL-18 Variant Polypeptides for hIL-18Rα

[0278] Using a Protein A sensor coated with hIL-18RαECD-Fc fusion protein, the affinity of IL-18 variant polypeptides for hIL-18Rα was evaluated by Biolayer Interferometry (BLI) on an Octet RED96, and WT hIL-18 was used as a control. The binding affinities of WT hIL-18 and IL-18 variant polypeptides for hIL-18RαECD-Fc are shown in Table 2A.

[0279] Table 2A. Affinity of IL-18 variant polypeptides for hIL-18Rα (determined by BLI)

[0280]

[0281]

[0282]

[0283] The mutations of these IL-18 variant polypeptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM14, corresponding to SEQ ID NO: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, 149-153) are shown in Table 2B.

[0284] Table 2B.

[0285]

[0286]

[0287] Example 6: Functional Characterization of IL-18 Variant Polypeptides

[0288] 6.1 IL-18 Variant Polypeptides Activate hIL-18 Receptor-Mediated Signaling

[0289] The NFκB-luc / hIL-18RαRβ HEK293 cell line stably expressing hIL-18Rα, hIL-18Rβ, and NFκB luciferase reporter gene was used as a reporter cell to determine the ability of IL-18 variant polypeptides to activate hIL-18 receptor-mediated signaling.

[0290] Briefly, the reporter cells were first seeded at 3.0×10 per well 4Cells were seeded at a density of in the wells of a white 96-well plate. WT hIL-18 or IL-18 variant polypeptides were serially diluted in medium in the presence (a) and absence (b) of 1 μg / mL hIL-18BP-hFc. Luminescence intensity was measured after incubation at 37 °C for 16 h.

[0291] As Figure 3 shown in and Table 3, all tested IL-18 variant polypeptides exhibited potency to activate IL-18 receptor-mediated signaling in a dose-dependent manner. The activity of WT hIL-18 was greatly reduced (by approximately 80-fold) in the presence of 1 μg / mL hIL-18BP-hFc. In contrast, IL-18 variant polypeptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-11, corresponding to SEQ ID NO: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, 149-150) exhibited Hil-18 BP-resistant activity with an EC 50 change of no more than 3-fold, indicating that IL-18 variant polypeptides were significantly less affected by Hil-18BP in terms of activation of Hil-18 receptor-mediated signaling. One exception was WM6, whose EC 50 showed a potency loss of approximately 12-fold in the presence of 1 μg / Ml Hil-18BP-hFc but was still much better than WT hIL-18.

[0292] Table 3. EC of IL-18 variant polypeptides in the hIL-18 reporter gene assay in the presence or absence of 1 μg / mL hIL-18BP-hFc 50

[0293]

[0294]

[0295] 6.2 hIFNγ Release Assay Based on Human PBMC

[0296] A PBMC-based assay was performed to test the ability of IL-18 variant polypeptides to activate T / NK cells and induce hIFNγ release. Briefly, human PBMC cells were seeded at 5.0 × 10 per well 5Cells were seeded at a density of [number of cells] per well in 96-well plates and cultured for 5 hours before treatment. Serial dilutions of WT hIL-18 or IL-18 variant polypeptides with 1 ng / mL human IL-12 were then added to the wells in the presence (a) or absence (b) of 1 μg / mL hIL-18BP-hFc. After incubation at 37 °C for 16 hours, the culture medium was collected from each well, and the hIFNγ concentration was measured by FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0297] As Figure 4 shown, the tested IL-18 variant polypeptides exhibited the potency to induce the release of hIFNγ from PBMCs in a dose-dependent manner. In addition, as shown in Table 4, in the presence of 1 μg / mL hIL-18BP-hFc, the activity of WT hIL-18 was greatly attenuated, and the EC 50 was reduced by approximately 35-fold. In contrast, the IL-18 variant polypeptides (i.e., M11-M13, M15, M17, M21, M24, M29, M32-M33, M35-M36, MM5, WM4-WM6, WM8, WM10-11, corresponding to SEQ ID NO: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, 143-150) exhibited hIL-18BP-resistant activity, with an EC 50 change of no more than 2-fold, indicating that the IL-18 variant polypeptides were significantly less affected by hIL-18BP in terms of hIFNγ induction.

[0298] Table 4. EC 50

[0299]

[0300] 6.3 Mouse spleen cell-based mIFNγ release assay

[0301] To evaluate the in vivo efficacy of the IL-18 variant polypeptides of the present application, a mouse interferon-γ (“mIFNγ”) release assay based on mouse spleen cells was performed as follows.

[0302] Briefly, freshly isolated mouse spleen cells were seeded at 6.5×10 5Cells were seeded at a density of [number] cells per well in a 96-well plate. Serial dilutions of WThIL-18 or IL-18 variant polypeptides were made with 1 ng / mL murine IL-12 in the presence (a) or absence (b) of 1 μg / mL murine IL-18BP-Fc and then added to the wells. After incubation at 37 °C for 16 h, the culture medium was collected from each well and the concentration of mIFNγ in the culture medium was measured by FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0303] Example 7: Production and Characterization of IL-18 Variant Polypeptides Containing Serine→Cysteine and / or Cysteine→Serine Substitutions

[0304] 7.1 Wild-Type IL-18 or IL-18 Polypeptide Variants Containing Cysteine→Serine Substitutions

[0305] Disulfide engineering can improve polypeptide stability and yield. The following experiments were conducted to evaluate the impact of Cys to Ser ("C→S") mutations on the druggability of IL-18 variants and the expression of IL-18 variants in mammalian cells (e.g., CHO cells and 293F cells). Wild-type IL-18 and IL-18 variant polypeptides M12 and MM5 were modified (or further modified) to contain C38S, C68S, and C76S substitutions, generating new variants with the following amino acid sequences:

[0306] IL-18 variants containing C38S, C68S, and C76S substitutions in the WT hIL-18 background ("IL-18-SSS"):

[0307] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMY KDSQPRGMAVTISVKSEKISTLSSENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO: 307)

[0308] IL-18 variants containing C38S, C68S, and C76S substitutions in the M12 background ("M12-SSS"):

[0309] YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMY KGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO: 308)

[0310] IL-18 variants containing C38S, C68S, and C76S substitutions in the MM5 background ("MM5-SSS"):

[0311] YFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMY KLSTERGMAVTISVKSEKISTLSSENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO: 309)

[0312] Next, the C-terminus of each of IL-18-SSS, M12-SSS, and M5-SSS was fused to the N-terminus of human IgG1 Fc containing the N297A substitution (EU numbering) to generate IL-18-SSS-Fc_N297A (SEQ ID NO: 319), M12-SSS_N297A (SEQ ID NO: 320), and M5-SSS_N-297A (SEQ ID NO: 321). The fusion polypeptides were expressed in CHO cells, harvested, and purified by protein A chromatography. The yield of IL-18-SSS-Fc_N297A obtained after one-step protein A purification was 14.7 mg / L and the purity was 65%. In contrast, the yield of WT IL-18-Fc_N297A obtained after one-step protein A purification was approximately 16 mg / L and the purity was 11%. The yield of M12-SSS-Fc_N297A obtained after one-step protein A purification was 5.6 mg / ml and the purity was 72%. In contrast, the expression of M12-Fc_N297A did not reach a detectable level in 100 mL of CHO host cells. The yield of MM5-SSS-Fc_N297A obtained after one-step protein A chromatography was 24.5 mg / L and the purity was 46%. After further purification by gel filtration, the yield of MM5-SSS-Fc_N297 was 3.4 mg / L and the purity was 99%. In contrast, the expression of MM5-Fc_N297A did not reach a detectable level in 100 mL of CHO host cells.

[0313] M12-SSS was further modified to contain the C127S substitution to generate M12-SSSS:

[0314] YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLASEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO: 310)

[0315] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU numbering), resulting in M12-SSSS-Fc_N297A (SEQ ID NO: 322). M12-SSSS-Fc_N297A was expressed in CHO cells, harvested, and purified by Protein A chromatography. Introduction of the C127S substitution (i.e., the fourth C→S substitution) resulted in a decrease in the yield and purity of M12-SSSS-Fc_N297A compared to the yield and purity of M12-SSS-Fc_N297A (data not shown).

[0316] Next, the in vitro assay described in Section 6.1 of Example 6 was used to characterize the efficacy of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A in activating IL-18 receptor-mediated signaling. IL-18-SSS-Fc_N297A showed efficacy comparable to wild-type IL-18 (see Figure 5A ), while M12-SSS-Fc_N297A showed a ~7-fold loss of potency compared to M12 (see Figure 5B ). The signaling activity of IL-18-SSS-Fc_N297A was reduced in the presence of 1 μg / ml hIL-18BP-hFc (“BP”), while M12-SSS-Fc_N297A exhibited hIL-18BP-resistant activity in the presence of 1 μg / ml hIL-18BP-hFc. See Figure 5A and 5B . As Figure 5C shown, MM5-SSS-Fc_N297A showed a ~220-fold loss of potency compared to MM5, but exhibited hIL-18BP-resistant activity in the presence of 1 μg / ml hIL-18BP-hFc. These results indicate that the 3 cysteine→serine substitutions do not affect IL-18BP resistance. These data suggest that C38, C68, and C76 are critical for increasing the yield of IL-18 and IL-18 polypeptide-Fc fusion proteins during production and after purification, and can improve yield and purity. The half-life of MM5-SSS-Fc_N297A may be longer than that of MM5.

[0317] 7.2 IL-18 polypeptide variants derived from M12 with amino acid substitutions that remove and / or introduce cysteine residues

[0318] The following experiments were conducted to evaluate the effects of substitutions that remove native cysteines (i.e., cysteines that are naturally present in the polypeptide sequence) and / or introduce disulfide bonds on the druggability, expression, and purification of IL-18 variant polypeptides. First, in silico screening based on molecular dynamics simulations was performed, and mutation hotspots on each of the four cysteines (C38, C68, C76, and C127) of M12 were identified based on artificial intelligence-based stability assessments. See Table 5. In addition, structure-guided design was used to identify amino acids in spatial positions that, when substituted with Cys residues, would allow the formation of new non-native disulfide bonds. The mutation strategies shown in Table 5 (i.e., DB6, DB7, DB8, DB9, and DB10) were proposed.

[0319] Table 5. IL-18 polypeptide variants derived from M12

[0320]

[0321] The mutation strategies in Table 5 were introduced into the IL-18 polypeptide variant M12, resulting in new variants M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10.

[0322] M12-DB 6

[0323]

[0324] M12-DB7

[0325]

[0326] M12-DB8

[0327]

[0328] M12-DB9

[0329]

[0330] M12-DB10

[0331]

[0332] The C-terminus of each of the M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10 variants was fused to a human IgG1 Fc variant containing the N297A substitution (EU numbering). The resulting fusion polypeptides, namely M12-DB6-Fc_N297A (SEQ ID NO: 323), M12-DB7-Fc_N297A (SEQ ID NO: 324), M12-DB8-Fc_N297A (SEQ ID NO: 325), M12-DB9-Fc_N297A (SEQ ID NO: 326), and M12-DB10-Fc_N297A (SEQ ID NO: 327) were expressed in CHO cells, harvested, and purified by protein A chromatography. The purified preparations were analyzed by SEC and SDS PAGE (data not shown). See Table 6 below.

[0333] Table 6. M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10 Fc_N297A

[0334] Expression and purification of the fusion proteins

[0335]

[0336] M12-DB8-Fc_N297A did not reach detectable levels of expression. M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A showed greatly improved expression yields or purity compared to M12-Fc_N297A or M12-SSS-Fc_N297A (see Table 6). Among M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A, M12-DB6-Fc_N297A had the highest expression yield (313 mg / L) after one-step protein A purification and a purity of 89.8% (determined by SEC-HPLC). The expression yield of M12-DB7-Fc_N297A was approximately 50% of the yield of M12-DB6-Fc_N297A. M12-DB7-Fc_N297A showed a purity similar to that of M12-DB6-Fc_N297A after protein A chromatography, indicating that the C127X substitution did not further improve polypeptide production in mammalian cells.

[0337] Next, the in vitro assays described in Section 6.1 of Example 6 were used to characterize the potency of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in activating IL-18 receptor-mediated signaling. As Figures 6A - 6D and Table 7 below show, M12-DB6-Fc_N297A( Figure 6A ), M12-DB7-Fc_N297A( Figure 6B ), M12-DB9-Fc_N297A( Figure 6C ), and M12-DB10-Fc_N297A( Figure 6D ) were far more potent than WT IL-18 in activating IL-18 receptor-mediated signaling. The signaling activities of the tested M12-DB6-Fc_N297A( Figure 6A ), M12-DB7-Fc_N297A( Figure 6B ), and M12-DB9-Fc_N297A( Figure 6C ) were not inhibited in the presence of 1 μg / ml hIL-18BP-hFc. See Table 7.

[0338] Table 7. EC of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in in vitro IL-18 receptor-mediated signaling assays 50

[0339]

[0340] Next, the "DB6" substitutions (i.e., C38I, C68S, and S117C) were introduced into variant MM5 to generate MM5-DB6:

[0341] MM5-DB6

[0342]

[0343] The "DB6" substitutions were also introduced into wild-type IL-18 to generate WT IL18-DB6:

[0344] WT IL18-DB6

[0345]

[0346] Another variant, WT-DBo, was designed by introducing the S117C substitution into wild-type IL-18:

[0347] WT IL-18-DBo

[0348]

[0349] The C-terminus of each of WT IL-18-DB6, WT IL-18-DBo, and MM5-DB6 was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU numbering). The resulting fusion polypeptides WT IL18-DB6-Fc_N297A (SEQ ID NO: 328), WT IL-18-DBo-Fc_N297A (SEQ ID NO: 329), and MM5-DB6-Fc_N297A (SEQ ID NO: 330) were expressed in CHO cells, harvested, and purified by Protein A chromatography. The purified preparations were analyzed by SEC and SDS PAGE (data not shown). As shown in Table 8 below, WT IL18-DBo-Fc_N297A did not reach detectable levels of expression. The expression yield of WT IL18-DB6-Fc was 157 mg / L, and after one-step Protein A chromatography, the purity of WT IL18-DB6-Fc_N297A was 91.6% (determined by SEC-HPLC) (see Table 8). These data indicate that engineering a disulfide bond between C76 and C117 was not sufficient to improve the expression of the fusion polypeptide in mammalian cells. An increase in expression in mammalian cells was achieved using a fusion polypeptide containing an IL-18 variant, wherein the IL-18 variant contains (i) an engineered C76-C117 disulfide bond, (ii) a C38 substitution (see Table 5), and (iii) a C68 substitution (see Table 5). The expression yield of MM5-DB6-Fc_N297A was much higher than that of WT IL18-DB6-Fc_N297A, while the purity was similar (see Table 8). These data also indicate that the "DB6" substitution is suitable for increasing the yield of WT IL-18 and IL-18 variant polypeptides during production in mammalian cells and subsequent purification.

[0350] Next, fusion polypeptides were designed that contain the C-terminus of human IgG1 Fc with an N297A substitution (EU numbering) fused to the N-terminus of each of variants M12-DB6 and MM5-DB6. The resulting fusion polypeptides, namely Fc_N297A-MM5-DB6 (SEQ ID NO: 332) and Fc_N297A-M12-DB6 (SEQ ID NO: 331), were expressed in CHO cells, harvested, and purified by protein A chromatography. As shown in Table 8, Fc_N297A-MM5-DB6 and Fc_N297A-M12-DB6 showed high expression yields in CHO cells. After one-step protein A chromatography purification, the purity of Fc_N297A-MM5-DB6 was approximately 97% (determined by SEC-HPLC), and the purity of Fc_N297A-M12-DB6 was 89% (determined by SEC-HPLC).

[0351] Table 8. Yields and purities of WT IL18-DB6-Fc_N297A, WT IL18-DBo-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 after expression in CHO cells and protein A chromatography

[0352]

[0353] Next, the in vitro assay described in Section 6.1 of Example 6 was used to characterize the potency of WT IL18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 in activating IL-18 receptor-mediated signaling. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A showed strong IL-18 receptor cell activation signals, which were stronger than WT IL-18 (see Figure 7A 、 Figure 7B and Table 9). Fc_N297A-MM5-DB6 showed potency similar to WT IL-18, while Fc_N297A-M12-DB6 produced a stronger activation signal to IL-18 reporter cells (see Figure 7C 、 Figure 7D and Table 9). In the presence of 1 μg / mL IL-18BP-hFc, the activity of WT IL18-DB6-Fc_N297A was significantly reduced, while the activity of MM5-DB6-Fc_N297A was not inhibited by IL-18BP (see Figure 7A 、 Figure 7Band Table 9). These results demonstrate that introducing the "DB6" set of mutations (i.e., C38I, C68S, and S117C, see Table 5) into the WT IL-18-Fc fusion polypeptide or IL-18 variant-Fc fusion polypeptide increases the expression yield of the fusion polypeptide during production in mammalian cells and increases the yield of the fusion polypeptide purified after chromatography. In addition, WT IL-18 and IL-18 variants engineered to contain the "DB6" set of mutations exhibit higher potency in activating IL-18 receptor-mediated signaling. Such results were observed for both fusion polypeptides with the C-terminus of the Fc region fused to the N-terminus of the IL-18 variant and for fusion polypeptides with the C-terminus of the IL-18 variant fused to the N-terminus of the Fc domain.

[0354] Table 9. EC50 of WT IL-18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and -Fc_N297A-M12-DB6 in an in vitro IL-18 receptor-mediated signaling assay

[0355]

[0356] The present disclosure is not limited to the content of the specific embodiments described, which are intended to be illustrative of the various aspects of the present disclosure, and any combination or method that is functionally equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and combinations of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present invention is intended to cover modifications and variations of the present disclosure as long as they come within the scope of the appended claims and their equivalents.

[0357] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated herein by reference.

[0358] The present invention has been described in terms of specific embodiments found or proposed by the inventors to include the preferred modes of practicing the present invention. Those skilled in the art will appreciate that various modifications and changes can be made to the specific embodiments of the examples without departing from the intended scope of the present invention. For example, due to codon redundancy, the underlying DNA sequence can be altered without affecting the protein sequence. In addition, for considerations of biological functional equivalence, the protein structure can be altered without affecting the type or amount of biological action. All such modifications are intended to be included within the scope of the appended claims.

[0359] Sequence Summary Table

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

Claims

1. A variant polypeptide of interleukin-18 (IL-18), which comprises at least one mutation at a residue selected from: F2, G3, E6, V11, Q24, L29, D54, Q56, P57, M60, A61, N91, K96, R104, R107, K140, N155, and I149, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO:

1.

2. The IL-18 variant polypeptide according to claim 1, which comprises at least one mutation at a residue selected from: G3, E6, and M60, wherein the amino acid positions are relative to the amino acid positions of the wild-type human IL-18 shown in SEQ ID NO:

1.

3. The IL-18 variant polypeptide according to claim 2, which comprises at least one of the following: a) G3P, G3S, G3E, G3N, G3D, or G3A; b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y; or c) M60K.

4. The IL-18 variant polypeptide according to any one of claims 1-3, which further comprises at least one or more mutations at a residue selected from: V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155.

5. The IL-18 variant polypeptide according to claim 4, which comprises at least one of the following: a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, or D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, or Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, P57Q, P57G, P57K, P57H, P57L, P57I, or P57E; d) N91V, N91G, N91A, N91S, N91T, N91R, N91K, N91P, N91I, or N91W; e) R104V, R104T, R104Y, R104F, R104T, R104L, R104S, R104A, R104E, R104I; f) N155K; g) V11I; h) K96D or K96E; i) I149M; or j) K140R.

6. The IL-18 variant polypeptide according to any one of claims 1-5, which comprises one substitution or a combination of a set of substitutions selected from the following: 1) G3P, E6K, D54H, Q56D, P57R, N91V, and R104V; 2) G3S, E6K, D54W, Q56P, P57D, N91G, and R104T; 3) G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y; 4) G3P, E6K, D54W, Q56G, P57V, N91V, and R104F; 5) G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K; 6) G3P, E6R, D54W, Q56T, N91V, and R104T; 7) G3P, E6R, D54H, Q56T, P57A, and N91A; 8) G3P, E6R, D54W, Q56P, P57A, N91A, and R104L; 9) G3P, E6R, D54W, Q56R, P57A, N91S, and R104S; 10) G3P, E6R, D54Q, Q56L, P57W, and N91S; 11) G3P, E6R, D54S, Q56R, P57N, N91G, and R104T; 12) G3P, E6K, D54G, Q56G, P57A, and N91T; 13) G3P, E6K, D54Q, Q56I, and P57W; 14) G3N, E6K, D54P, Q56S, P57S, N91R, and R104A; 15) G3P, E6R, D54S, Q56Y, P57T, and N91G; 16) G3P, E6R, D54S, Q56R, P57N, N91G, and R104S; 17) G3P, E6R, D54L, Q56T, P57A, and N91G; 18) G3P, E6R, D54S, Q56R, P57R, N91G, and R104S; 19) G3P, E6K, D54H, Q56E, P57Q, and N91A; 20) G3P, E6R, D54S, Q56R, P57S, N91G, and R104S; 21) G3P, E6R, D54S, Q56S, P57T, N91G, and R104S; 22) G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S; 23) G3P, E6R, D54Y, Q56T, and P57R; 24) G3P, E6R, D54Y, Q56T, and P57S; 25) G3P, E6R, D54L, Q56T, P57T, and N91R; 26) G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y; 27) G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y; 28) G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y; 29) G3P, E6M, D54F, Q56D, P57R, and N91P; 30) G3P, E6L, D54H, Q56T, P57V, and N91S; 31) G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y; 32) G3P, E6G, D54S, Q56S, and P57R; 33) G3E, E6H, D54R, Q56T, and P57H; 34) G3P, E6R, D54H, Q56R, P57N, N91V, and R104E; 35) G3P, E6R, D54G, Q56G, P57A, and N91G; 36) G3P, E6S, D54A, Q56D, P57Q, and N91G; 37) G3P, E6G, D54Q, Q56V, and P57W; 38) G3P, E6S, D54W, Q56G, P57A, N91V, and R104I; 39) G3P, E6R, D54W, Q56P, P57G, N91V, and R104L; 40) G3D, E6K, D54P, Q56S, P57W, and N91W; 41) G3P; 42) G3P, E6K, D54G, Q56G, and P57A; 43) G3P, E6R, D54L, Q56G, P57S, and N91V; 44) G3P, E6R, V11I, D54G, Q56G, P57A, and N91G; 45) G3P, E6K, D54H, Q56Y, and P57S; 46) E6R, D54W, Q56S, and P57Q; 47) G3P, E6K, D54L, Q56T, P57Q, and N91V; 48) G3A, E6Y, D54R, Q56S, P57L, and N91G; 49) G3P, E6R, D54L, Q56T, P57I, and N91G; 50) E6G, D54L, Q56T, P57E, N91G, and R104S; 51) G3A, E6Y, D54R, Q56S, P57L, and N91A; 52) M60K and K96D; 53) G3P, E6R, and K96E; 54) M60K; 55) G3P and E6R; 56) G3P and E6K; 57) G3D, E6K, and N91S; 58) G3S and I149M; 59) G3P, E6R, and N91S; 60) E6R; 61) E6R and N91S; 62) V11I; and 63) G3S and K140R.

7. An IL-18 variant polypeptide according to any one of claims 1-6, which comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-153.

8. An IL-18 variant polypeptide according to any one of claims 1-6, which further comprises an S117C substitution, wherein the amino acid position is relative to the amino acid position of the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the S117C substitution promotes a C76-C117 disulfide bond.

9. The IL-18 variant polypeptide according to any one of claims 1-6 and 8, which further comprises a substitution at C38, wherein the amino acid position is relative to the amino acid position of the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the substitution is selected from C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V.

10. The IL-18 variant polypeptide according to any one of claims 1-6 and 8-9, which further comprises a substitution at C68, wherein the amino acid position is relative to the amino acid position of the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S.

11. The IL-18 variant polypeptide according to any one of claims 1-6 and 8-10, which further comprises a substitution at C76 and / or C127, wherein the amino acid position is relative to the amino acid position of the wild-type human IL-18 shown in SEQ ID NO: 1, optionally wherein the C76 substitution is C76Y or C76V, and optionally wherein the C127 substitution is selected from C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.

12. The IL-18 variant polypeptide according to any one of claims 1-6 and 8-11, wherein the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.

13. The IL-18 variant polypeptide according to any one of claims 1-6 and 8-12, wherein the variant polypeptide further comprises a set of mutations selected from: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y.

14. An interleukin-18 (IL-18) variant polypeptide, which comprises at least one mutation at a residue selected from C38, C68, C76, S117, and C127, wherein the amino acid position is relative to the amino acid position of the wild-type human IL-18 shown in SEQ ID NO:

1.

15. The IL-18 variant polypeptide according to claim 14, wherein at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution.

16. The IL-18 variant polypeptide according to claim 14, wherein at least one mutation is selected from: C38S, C68S, C76S, and C127S.

17. The IL-18 variant polypeptide according to claim 16, wherein the variant polypeptide comprises C38S, C68S and C76S mutations.

18. The IL-18 variant polypeptide according to claim 14, wherein the variant polypeptide comprises a set of mutations selected from: (a) C38I, C68S and S117C; (b) C38V, C68I, S117C and C127A; (c) C38S, C68I, S117C and C127I; (d) C38I, C68I, C76V and C127I; and (e) C38I, C68L and C76Y.

19. The IL-18 variant polypeptide according to any one of claims 1-18, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor α (IL-18Rα) and exhibits significantly reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18.

20. The IL-18 variant polypeptide according to any one of claims 1-19, wherein the IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to wild-type IL-18.

21. An IL-18 variant polypeptide according to any one of claims 1-20, wherein the IL-18 variant polypeptide binds to IL-18Rα with a K less than about 5 x 10 -5 M D .

22. The IL-18 variant polypeptide according to any one of claims 1-21, wherein the IL-18 variant polypeptide binds to IL-18Rα with a K of about 5x10 -5 to about 5x10 -11 M. D ​ 23. The IL-18 variant polypeptide according to any one of claims 1-22, wherein the IL-18 variant polypeptide binds to IL-18BP with a K -9 higher than 5x10 D M. -9 M D that binds to IL-18BP.

24. The IL-18 variant polypeptide according to any one of claims 1-23, wherein the IL-18 variant polypeptide does not exhibit binding to IL-18BP.

25. The IL-18 variant polypeptide according to any one of claims 1-24, wherein the variant polypeptide comprises a mutation at residue G3, and the mutation is selected from: G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N and G3S, optionally wherein the mutation is G3P.

26. The IL-18 variant polypeptide according to any one of claims 1-25, wherein the variant polypeptide comprises a mutation at residue E6, and the mutation is selected from: E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W and E6Y, optionally wherein the mutation is selected from: E6R, E6K, E6G, E6T, E6A and E6S, further optionally wherein the mutation is selected from: E6R and E6K.

27. The IL-18 variant polypeptide according to any one of claims 1-26, wherein the variant polypeptide comprises a mutation at residue D54, and the mutation is selected from: D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V and D54T, optionally wherein the mutation is selected from: D54W, D54H, D54S, D54Q, D54L and D54Y.

28. An IL-18 variant polypeptide according to any one of claims 1-27, wherein the variant polypeptide comprises a mutation at residue N91, wherein the mutation is selected from: N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y and N91W, optionally wherein the mutation is selected from: N91V, N91A, N91G and N91S.

29. An IL-18 variant polypeptide according to any one of claims 1-28, wherein the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from: R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I and R104N, optionally wherein the mutation is selected from: R104S, R104Y and R104T.

30. An IL-18 variant polypeptide according to any one of claims 1-28, wherein the variant polypeptide does not comprise a mutation at residue R104.

31. An IL-18 variant polypeptide according to any one of claims 1-30, wherein the variant polypeptide comprises a mutation at residue Q56, wherein the mutation is selected from: Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N and Q56V, optionally wherein the mutation is selected from: Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I and Q56Y.

32. An IL-18 variant polypeptide according to any one of claims 1-30, wherein the variant polypeptide does not comprise a mutation at residue Q56.

33. An IL-18 variant polypeptide according to any one of claims 1-32, wherein the variant polypeptide comprises a mutation at residue P57, wherein the mutation is selected from: P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y and P57D, optionally wherein the mutation is selected from: P57A, P57G, P57R, P57W, P57S, P57T and P57V.

34. An IL-18 variant polypeptide according to any one of claims 1-32, wherein the variant polypeptide does not comprise a mutation at residue P57.

35. An IL-18 variant polypeptide according to any one of claims 1-34, wherein the variant polypeptide comprises mutations at G3, E6, D54 and N91, optionally wherein the variant polypeptide comprises the mutations: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S or D54Q; and (iv) N91V, N91A, N91G or N91S.

36. An IL-18 variant polypeptide according to any one of claims 1-31, 33 and 35, wherein the variant polypeptide further comprises mutations at Q56 and P57.

37. An IL-18 variant polypeptide according to any one of claims 1-6 and 8-36, wherein the variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-299 and 307-318, optionally wherein the variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 307-318, optionally wherein the variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 307-312 and 314-317.

38. A fusion polypeptide comprising the IL-18 variant polypeptide according to any one of claims 1-37 and a human IgG Fc domain or a variant thereof.

39. The fusion polypeptide according to claim 38, which comprises a variant of the human IgG1 Fc domain containing an N297A mutation (EU numbering), optionally wherein the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the human IgG Fc domain or a variant thereof, or the N-terminus of the IL-18 variant polypeptide is fused to the C-terminus of the human IgG Fc domain or a variant thereof, optionally wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

40. A dimer comprising two fusion polypeptides according to claim 38 or 39.

41. A nucleic acid encoding the IL-18 variant polypeptide according to any one of claims 1-37 or the fusion polypeptide according to any one of claims 38-39.

42. A vector comprising the nucleic acid according to claim 41.

43. A host cell comprising the nucleic acid according to claim 41 or the vector according to claim 42.

44. A method for producing an IL-18 variant polypeptide or a fusion polypeptide, comprising: (a) culturing the host cell according to claim 43 under conditions for expressing the IL-18 variant polypeptide or the fusion polypeptide; and (b) recovering the IL-18 variant polypeptide or the fusion polypeptide produced by the host cell.

45. A pharmaceutical composition comprising the IL-18 variant polypeptide according to any one of claims 1-37, the fusion polypeptide according to any one of claims 38-39, the nucleic acid according to claim 41, or the vector according to claim 42.

46. A method for treating an individual's disease, comprising administering to the individual an effective amount of the pharmaceutical composition according to claim 45.

47. A method for activating hIL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition according to claim 45.

48. A method for stimulating immune cells experiencing an antigen in an individual in need thereof, the method comprising administering to the individual an effective amount of the pharmaceutical composition according to claim 45.

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