IL-18 variant polypeptides

Abstract

Interleukin-18 (IL-18) variant polypeptides are provided that include mutation(s) at one or more of G3, E6, D54, and N91 (amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). IL-18 variant polypeptides are also provided that include (e.g., further include) at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127 (amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). IL-18 variant polypeptides are also provided that bind to IL-18Rα and have substantially reduced binding to IL-18BP compared to wild-type IL-18. IL-18 variant polypeptides are also provided that have increased binding to IL-18Rα compared to wild-type IL-18 and substantially reduced binding to IL-18BP compared to wild-type IL-18.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] Electronic Sequence Listing Reference

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

[0005] The present application relates to interleukin-18 (IL-18) variant polypeptides that include at least one amino acid substitution compared to wild-type IL-18 (e.g., wild-type human IL-18), methods for producing such variant polypeptides, and methods for using such variant polypeptides in therapeutic applications. [Background technology]

[0006] IL-18 has been shown to stimulate innate lymphoid cells, myeloid cells, antigen-experienced non-naive T cells (see, e.g., Guo et al. (2012) Trends Immunol. 33, 598-606), and antigen-experienced natural killer (NK) cells. Therapeutically, recombinant IL-18 has been reported to synergize with immune checkpoint inhibitors (ICIs) (Ma et al. (2016) Clin Cancer Res 22:2969-2980) and chimeric antigen receptor T (CAR-T) cells in preclinical models (Hu et al. (2017) Cell Rep 20, 3025-3033). 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, clinical development of IL-18 has been curtailed due to its limited efficacy. Thus, there is a need in the art for compositions and methods for activating IL-18 receptor-mediated signaling in individuals and / or stimulating antigen-experienced T cells or NK cells in individuals to treat cancer and other IL-18-mediated diseases and disorders. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Guo et al.(2012)Trends Immunol.33,598-606 [Non-patent document 2] Ma et al. (2016) Clin Cancer Res 22:2969-2980 [Non-patent document 3] Hu et al.(2017)Cell Rep 20,3025-3033 [Non-patent document 4] Robertson et al. (2006) Clin Cancer Res 12,4265-4273 Summary of the Invention [Means for solving the problem]

[0008] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided that comprises at least one mutation at a residue selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149 (amino acid positions relative to wild-type human IL-18 set forth in SEQ ID NO: 1). In some embodiments, the IL-18 variant polypeptide comprises at least one mutation at a residue selected from the group consisting of G3, E6, D54, and N91 (amino acid positions relative to wild-type human IL-18 set forth in SEQ ID NO: 1). In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113, and N155 (amino acid positions relative to wild-type human IL-18 set forth in SEQ ID NO: 1).

[0009] In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127 (amino acid positions relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and a substitution at S117C. In some embodiments, the at least one mutation is selected from the group consisting of 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 the group consisting of: (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, interleukin-18 (IL-18) variant polypeptides are provided that comprise at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127 (amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and a substitution at S117C. In some embodiments, the at least one mutation is selected from the group consisting of 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 the group consisting of: (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.

[0011] In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and has reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide has increased binding to IL-18Rα compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide has an IL-18 binding activity of about 5×10 -5 K less than M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -5 M ~ approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at 5×10 -9 K over M DIn some embodiments, the IL-18 variant polypeptide exhibits no binding to IL-18BP (e.g., no detectable binding as measured by surface plasmon resonance).

[0012] In some embodiments, the variant polypeptide comprises a mutation at residue G3, where the mutation is selected from the group consisting of 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, where the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y. In some embodiments, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from the group consisting of E6R and E6K.

[0013] In some embodiments, the variant polypeptide comprises a mutation at residue D54, where the mutation is selected from the group consisting of D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T. In some embodiments, the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0014] In some embodiments, the variant polypeptide comprises a mutation at residue N91, where the mutation is selected from the group consisting of N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W. In some embodiments, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0015] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, where the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from the group consisting of R104S, R104Y, and R104T.

[0016] In some embodiments, the variant polypeptide does not include a mutation at residue R104.

[0017] In some embodiments, the variant polypeptide comprises a mutation at residue Q56, where the mutation is selected from the group consisting of 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 the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0018] In some embodiments, the variant polypeptide does not include a mutation at residue Q56.

[0019] In some embodiments, the variant polypeptide comprises a mutation at residue P57, where the mutation is selected from the group consisting of 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 the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0020] In some embodiments, the variant polypeptide does not comprise a mutation at residue P57.

[0021] In some embodiments, the variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the variant polypeptide comprises mutations at (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.

[0022] In some embodiments, the variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-299 and 307-318.

[0023] In some embodiments, a fusion polypeptide is provided comprising an IL-18 variant polypeptide described herein and a human IgG Fc domain or variant thereof. In some embodiments, the fusion polypeptide comprises a human IgG1 Fc domain variant comprising 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 variant thereof. In some embodiments, the C-terminus of the human IgG Fc domain or 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.

[0024] In some embodiments, a dimer is provided that comprises two fusion polypeptides described herein.In some embodiments, the dimer is a homodimer.In some embodiments, the dimer is a heterodimer.

[0025] In some embodiments, a nucleic acid encoding an IL-18 variant polypeptide or fusion polypeptide described herein is provided. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 301-306 and 333-335. In some embodiments, a vector comprising a nucleic acid described herein is provided. In some embodiments, a host cell comprising a nucleic acid or vector described herein is provided. In some embodiments, a method for producing an IL-18 variant polypeptide or fusion polypeptide is provided, comprising: (a) culturing a host cell described herein under conditions in which the IL-18 variant polypeptide or fusion polypeptide is expressed; and (b) recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell. 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 fusion polypeptide.

[0026] In some embodiments, a pharmaceutical composition is provided comprising an IL-18 variant polypeptide described herein, a fusion polypeptide described herein, a nucleic acid described herein, or a vector described herein.

[0027] In some embodiments, there is provided a method for treating a disease in an individual, comprising administering to the individual an effective amount of any one of the above pharmaceutical compositions. In some embodiments, the disease is cancer. In some embodiments, there is provided a method for activating hIL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of any one of the above pharmaceutical compositions. In some embodiments, there is provided a method for stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of any one of the above pharmaceutical compositions. In some embodiments, the stimulation comprises increasing the activity and / or number of antigen-experienced immune cells.

[0028] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0029] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0030] [Figure 1A] 1 illustrates a screening process for identifying IL-18 variant polypeptides described herein using methods based on magnetic cell separation (MACS) or fluorescence activated cell sorting (FACS).

[0031] [Figure 1B] The ability of output library pools from rounds 1, 2, 3, 4, and 5 to bind to either 1 μM IL-18BP or 50 nM IL-18Rα is shown.

[0032] [Figure 1C] Figure 1 shows the ability of representative single IL-18 variant clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43) to bind to IL-18Rα. SEQ ID NO: 1 is wild-type IL-18.

[0033] [Figure 2A] 1 shows SDS-PAGE and SEC-HPLC analysis of purified wild-type human IL-18 (SEQ ID NO: 1).

[0034] [Figure 2B] 1 shows SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant M12 (SEQ ID NO: 39).

[0035] [Figure 2C]1 shows SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant MM5 (SEQ ID NO: 133).

[0036] [Figure 2D] 1 shows SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant WM6 (SEQ ID NO: 145).

[0037] [Figure 2E] 1 shows SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant M17 (SEQ ID NO: 303).

[0038] [Figure 2F] 1 shows SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant M24 (SEQ ID NO: 105).

[0039] [Figure 3-1] 1 shows activation of signaling through the hIL-18 receptor by exemplary IL-18 variant polypeptides of the present application.

[0040] [Figure 3-2] Same as above

[0041] [Figure 3-3] Same as above

[0042] [Figure 4] 1 shows hIFNγ release induced by exemplary IL-18 variant polypeptides of the present application.

[0043] [Figure 5A] FIG. 1 shows activation of signaling through the hIL-18 receptor 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 an IL-18 binding protein ("BP").

[0044] [Figure 5B] 1 shows activation of signaling through the hIL-18 receptor 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 an IL-18 binding protein ("BP").

[0045] [Figure 5C] 1 shows activation of signaling through the hIL-18 receptor 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 an IL-18 binding protein ("BP").

[0046] [Figure 6A] Activation of signaling through the hIL-18 receptor by the fusion polypeptides WT IL-18, WT IL-18 in the presence of an IL-18 binding protein ("BP"), the fusion polypeptide M12-DB6-Fc_N297A, and M12-DB6-Fc_N297A in the presence of an IL-18 binding protein ("BP").

[0047] [Figure 6B] Activation of signaling through the hIL-18 receptor 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").

[0048] [Figure 6C] Activation of signaling through the hIL-18 receptor by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB9-Fc_N297A, and M12-DB9-Fc_N297A in the presence of an IL-18 binding protein ("BP").

[0049] [Figure 6D]1 shows activation of signal transduction via the hIL-18 receptor by the fusion polypeptide WT IL-18 and the fusion polypeptide M12-DB10-Fc_N297A.

[0050] [Figure 7A] Activation of signaling through the hIL-18 receptor by 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").

[0051] [Figure 7B] Activation of signaling through the hIL-18 receptor by 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").

[0052] [Figure 7C] 1 shows activation of signaling via the hIL-18 receptor by wild-type IL-18 and the fusion polypeptide Fc_N297A-MM5-DB6.

[0053] [Figure 7D] 1 shows activation of signal transduction via the hIL-18 receptor by wild-type IL-18 and the fusion polypeptide Fc_N297A-M12-DB6. DETAILED DESCRIPTION OF THE INVENTION

[0054] overview

[0055] Components of the interleukin-18 (IL-18) pathway have been found to be upregulated on tumor-infiltrating lymphocytes (TILs) (see, e.g., Zhou et al. (2020) Nature 583:609-614), suggesting that IL-18 therapy may enhance antitumor immunity. However, IL-18 has not shown significant efficacy in clinical trials. IL-18BP (IL-18BP), a high-affinity inhibitor of IL-18, has not been shown to be effective in tumor-infiltrating lymphocytes (TILs). D <1 nM (see, e.g., Dinarello et al. (2013) Front Immunol 4:289, doi:10.3389 / fimmu.2013.00289) is frequently upregulated in a variety of human and mouse tumors and is thought to limit the antitumor activity of IL-18 in mice and in clinical trials. This application is based, in part, on the applicant's identification of IL-18 variant polypeptides that induce signaling through the IL-18 receptor even in the presence of IL-18BP. By substituting one or a series of amino acids (e.g., G3, e.g., E6), the IL-18 variant polypeptides were found to exhibit resistance to IL-18BP while still being able to bind to and activate the IL-18 receptor. See, e.g., Example 6, Figures 3-4, and Tables 3 and 4. It has also been found that substitution of one or more amino acids at C38, C68, C76, S117, and / or C127 results in higher yields, more purified, and / or more potent IL-18 variant polypeptides (see, e.g., Example 7, Figures 5A-7D, Tables 5-9).

[0056] definition

[0057] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a cytokine and its receptor, that 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 is one that binds to that receptor with higher affinity, stronger avidity, more readily, and / or for longer than its binding to other targets. In some embodiments, the extent of binding of a cytokine to an unrelated target is less than about 10% of the binding of the cytokine to its receptor, as measured, for example, by radioimmunoassay (RIA). In some embodiments, a cytokine that specifically binds to a receptor has a dissociation constant (K D ) is ≦10 -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 includes, but is not required to be, 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, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ test, and peptide scan.

[0058] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from association with all components associated with the production environment. In some embodiments, an isolated nucleic acid molecule encoding a polypeptide or cytokine described herein exists in a form or setting that is different from that in which it is found in nature.

[0059] 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 as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0060] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0061] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0062] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For 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 attributable to a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of the present application contemplate any one or more of these aspects of treatment.

[0063] With respect to cancer, the term "treating" includes any or all of inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0064] The term "inhibition" or "inhibiting" refers to the reduction or elimination of any phenotypic characteristic, or the reduction or elimination of the occurrence, degree, or likelihood of that characteristic. "Reduce" or "inhibiting" refers to the decrease, lowering, or prevention of an activity, function, and / or amount compared to a reference. In certain embodiments, "reduce" or "inhibiting" refers to the ability to cause an overall reduction of 20% or more. In other embodiments, "reduce" or "inhibiting" refers to the ability to cause an overall reduction of 50% or more. In yet other embodiments, "reduce" or "inhibiting" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0065] The terms "subject," "individual," and "patient" are used interchangeably herein to 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.

[0066] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0067] Reference herein to "about" a value or parameter includes (and describes) a variation about that value or parameter itself. For example, a reference to "about X" includes a reference to "X."

[0068] As used herein, reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0069] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0070] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0071] Interleukin-18 (IL-18) variant polypeptides

[0072] Interleukin-18 (IL-18, also known as interferon-γ-inducing factor) is a protein encoded by the IL-18 gene in humans. It is a proinflammatory cytokine that can be produced by hematopoietic and non-hematopoietic cells. IL-18 can regulate both innate and adaptive immunity, and its dysregulation can lead to autoimmune or inflammatory diseases.

[0073] In one aspect, provided herein is an interleukin-18 ("IL-18") variant polypeptide comprising mutation(s) at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149 (amino acid positions are relative to wild-type (WT) human IL-18 as set forth 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. YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 1)

[0074] In some embodiments, the IL-18 variant polypeptide comprises mutations in two or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the two or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in three or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the three or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in four or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, for example in any combination. In some embodiments, the four or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in five or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the five or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in six or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination.In some embodiments, the six or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. 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 1149, for example in any combination. In some embodiments, the seven or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at eight or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the eight or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at nine or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the nine or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at ten or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, for example in any combination. In some embodiments, the ten or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149.In some embodiments, the IL-18 variant polypeptide comprises mutations at 11 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 11 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at 12 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 12 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at 13 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 13 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. 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 1149. 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 1149.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 two, at least three, or at least four mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0075] In some embodiments, the one or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations in two or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, the two or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations in three or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, the three or more mutations are selected from the group consisting 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, 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) 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 two, at least three, or at least four mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0076] In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113, and N155 (amino acid positions relative to wild-type human IL-18 as set forth in SEQ ID NO: 1).

[0077] In some embodiments, the IL-18 variant polypeptide comprises at least one (or two, or three) mutations in a residue selected from the group consisting of G3, E6, and M60 (amino acid positions relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). In some embodiments, the IL-18 variant polypeptide comprises at least one of: 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 in a residue selected from the group consisting of V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155. In some embodiments, the IL-18 variant polypeptide is selected from the group consisting of a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, and D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, and Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, P57Q, P57G, P57R; 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.

[0078] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6K, D54H, Q56D, P57R, N91V, and R104V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 combination set of substitutions comprising G3S, E6K, D54W, Q56P, P57D, N91G, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 set of substitutions comprising G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0081] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6K, D54W, Q56G, P57V, N91V, and R104F, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0082] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0083] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54W, Q56T, N91V, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0084] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54H, Q56T, P57A, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 combination set of substitutions including G3P, E6R, D54W, Q56P, P57A, N91A, and R104L, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 set of substitutions including G3P, E6R, D54W, Q56R, P57A, N91S, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54Q, Q56L, P57W, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0088] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54S, Q56R, P57N, N91G, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0089] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6K, D54G, Q56G, P57A, and N91T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0090] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6K, D54Q, Q56I, and P57W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0091] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3N, E6K, D54P, Q56S, P57S, N91R, and R104A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 combination set of substitutions comprising G3P, E6R, D54S, Q56Y, P57T, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0093] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54S, Q56R, P57N, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 combination set of substitutions comprising G3P, E6R, D54L, Q56T, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54S, Q56R, P57R, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0096] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6K, D54H, Q56E, P57Q, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54S, Q56R, P57S, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54S, Q56S, P57T, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0099] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0100] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54Y, Q56T, and P57R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54Y, Q56T, and P57S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 set of substitutions including G3P, E6R, D54L, Q56T, P57T, and N91R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0103] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0104] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0105] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6M, D54F, Q56D, P57R, and N91P, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0107] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6L, D54H, Q56T, P57V, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0108] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0109] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6G, D54S, Q56S, and P57R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0110] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3E, E6H, D54R, Q56T, and P57H, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0111] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54H, Q56R, P57N, N91V, and R104E, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0112] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54G, Q56G, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0113] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6S, D54A, Q56D, P57Q, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 set of substitutions comprising G3P, E6G, D54Q, Q56V, and P57W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 set of substitutions including G3P, E6S, D54W, Q56G, P57A, N91V, and R104I, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0116] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6R, D54W, Q56P, P57G, N91V, and R104L, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0117] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3D, E6K, D54P, Q56S, P57W, and N91W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0118] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution), which is G3P, at the amino acid position (position) relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0119] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6K, D54G, Q56G, and P57A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0120] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54L, Q56G, P57S, and N91V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0121] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, V11I, D54G, Q56G, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0122] In some embodiments, an IL-18 variant polypeptide comprises a combination set of substitutions including G3P, E6K, D54H, Q56Y, and P57S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0123] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising E6R, D54W, Q56S, and P57Q, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0124] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6K, D54L, Q56T, P57Q, and N91V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0125] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3A, E6Y, D54R, Q56S, P57L, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0126] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3P, E6R, D54L, Q56T, P57I, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0127] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising E6G, D54L, Q56T, P57E, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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.

[0128] In some embodiments, the IL-18 variant polypeptide comprises a combination set of substitutions comprising G3A, E6Y, D54R, Q56S, P57L, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, 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 a combination of a set of substitutions comprising M60K, and K96D, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0130] In some embodiments, the IL-18 variant polypeptide comprises a combination of a set of substitutions including G3P, E6R, and K96E, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0131] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) including M60K, where the amino acid position (position) is relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0132] In some embodiments, the IL-18 variant polypeptide comprises a combined set of substitutions involving G3P and E6R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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 a set of substitutions comprising G3P and E6K, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0134] In some embodiments, an IL-18 variant polypeptide comprises a combination of a set of substitutions comprising G3D, E6K, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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 a combination of a set of substitutions comprising G3S, and I149M, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0136] In some embodiments, an IL-18 variant polypeptide comprises a combination of a set of substitutions including G3P, E6R, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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) involving E6R, where the amino acid position (position) is relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0138] In some embodiments, the IL-18 variant polypeptide comprises a combination of a set of substitutions including E6R and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0139] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) including V11I, where the amino acid position (position) is relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0140] In some embodiments, the IL-18 variant polypeptide comprises a combination of a set of substitutions including G3S and K140R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equivalent or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra) and / or b) equivalent or lower binding affinity to an IL-18 binding protein compared to a corresponding IL-18 variant polypeptide that does not comprise the 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 the group consisting of C38I, C38V, C38L, and C38M, and 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 the group consisting of C68I, C68V, C68S, and C68D, and 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, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and 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.

[0141] In some embodiments, the IL-18 variant polypeptide comprises an 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.

[0142] In some embodiments, the IL-18 variant polypeptide comprises an 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.

[0143] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150.

[0144] In some embodiments, an 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 the group consisting of C38, C68, C76, D98, S117, and C127 (amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). In some embodiments, an IL-18 variant polypeptide comprises (or further comprises) a substitution at C38 (i.e., the wild-type cysteine ​​at position 38 is substituted with any amino acid), a substitution at C68 (i.e., the wild-type cysteine ​​at position 68 is substituted with any amino acid), and an S117C substitution. In some embodiments, an 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) a 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 the group consisting of C38S, C68S, C76S, and C127S (amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1). In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) the C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) the 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.

[0145] In some embodiments, the IL-18 variant polypeptide further comprises a mutation(s) at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises mutations at two 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 a mutation at a position other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant comprises (or further comprises) a mutation at a position 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 two, at least three, at least four, at least five, at least six, or at least seven mutations are selected from the group consisting of G3, E6, D54, Q56, P57, N91, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise substitutions at Q56 and P57 (amino acid positions relative to 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.

[0146] In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha ("IL-18Rα"), e.g., human IL-18Rα or "hIL-18α," and has substantially reduced binding to an IL-18 binding protein ("IL-18BP"), e.g., human IL-18 or "hIL-18BP." In some embodiments, the IL-18 variant polypeptide has substantially reduced binding to IL-18BP compared to wild-type human IL-18 set forth 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 wild-type human IL-18 set forth 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 WT human IL-18 set forth in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0147] In some embodiments, the IL-18 variant polypeptide is about 5×10 -5 Less than M, approximately 5 x 10 -6 Less than M, approximately 5 x 10 -7 Less than M, approximately 5 x 10 -8 Less than M, approximately 5 x 10 -9 Less than M, approximately 5 x 10 -10 Less than M, or about 5 x 10 -11 K less than M (including any range between these values) D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -5 M ~ approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 M ~ approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 M ~ approx. 5×10 -10 K of MD In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 M ~ approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 M ~ approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -9 M ~ approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 M ~ approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 M ~ approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -9 M ~ approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 M ~ approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 ~Approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant binds to IL-18Rα at about 5×10 -5 ~5×10 -11 M, for example, approximately 5 x 10 -5 , 6×10 -5 , 7×10 -5 , 8×10 -5 , 9×10 -5 , 1×10 -6 , 2 × 10 -6, 3×10 -6 , 4×10 -6 , 5×10 -6 , 6×10 -6 , 7×10 -6 , 8×10 -6 , 9×10 -6 , 1×10 -7 , 2 × 10 -7 , 3×10 -7 , 4×10 -7 , 5×10 -7 , 6×10 -7 , 7×10 -7 , 8×10 -7 , 9×10 -7 , 1×10 -8 , 2 × 10 -8 , 3×10 -8 , 4×10 -8 , 5×10 -8 , 6×10 -8 , 7×10 -8 , 8×10 -8 , 9×10 -8 , 1×10 -9 , 2 × 10 -9 , 3×10 -9 , 4×10 -9 , 5×10 -9 , 6×10 -9 , 7×10 -9 , 8×10 -9 , 9×10 -9 , 1×10 -10 , 2 × 10 -10 , 3×10 -10 , 4×10 -10 , 5×10 -10 , 6×10 -10 , 7×10 -10 , 8×10 -10 , 9×10 -10 , 1×10 -11 , 2 × 10 -11 , 3×10 -11 , 4×10 -11 , or 5 × 10 -11 Any one of K and M (including any range between these values) DIn some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is greater than the affinity of wild-type human IL-18 set forth 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 comparable (e.g., about the same) as the affinity of wild-type human IL-18 set forth in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0148] In some embodiments, the IL-18 variant polypeptide has substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide is at least 5×10 -9 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -8 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -7 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -6 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -5 K over M D In some embodiments, an IL-18 variant polypeptide exhibits no binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP). In some embodiments, an IL-18 variant polypeptide exhibits no binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP) at 10 -3 K over M D and binds to IL-18BP (e.g., hIL-18BP).

[0149] The affinity of IL-18 for IL-18Rα and / or IL-18BP as described herein can be experimentally determined by methods known in the art, including, but not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (EIA), surface plasmon resonance (SPR), peptide scanning, and fluorescence-activated cell sorting (FACS)-based kinetic competition screening.

[0150] In some embodiments, the IL-18 variant polypeptide comprises a mutation at residue G3, where the mutation is selected from the group consisting of G3P, G3D, G3E, G3N, and G3S. As used herein, "G3X" means that the wild-type amino acid G at position 3 of SEQ ID NO: 1 is replaced with the amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 of SEQ ID NO: 1 is replaced with the amino acid P.

[0151] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue E6, where the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from the group consisting of E6R and E6K.

[0152] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue D54, where the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, D54Y, D54P, D54A, D54F, D54G, and D54T. In some embodiments, the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0153] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue N91, where the mutation is selected from the group consisting of N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0154] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, where the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104V, R104A, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from the group consisting of R104S, R104Y, and R104T. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at residue R104.

[0155] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue Q56, where the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0156] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue P57, where the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57K, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0157] 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 a mutation at two or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at three or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at G3, E6, D54, and N91. In some embodiments, 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 two, at least three, or at least four mutations are selected from the group consisting of 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, hi 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 two, at least three, at least four mutations, at least five mutations, or at least six mutations are selected from the group consisting of G3, E6, D54, N91, Q56, and P57.

[0158] In some embodiments, an 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 any one of SEQ ID NOs: 2-299, 307-318. In some embodiments, an IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-299, 307-318. The amino acid sequences of SEQ ID NOs: 2-299, 307-318 are set forth in the sequence listing below in the Examples section.

[0159] Fusion Polypeptides

[0160] In some embodiments, the activatable IL-18 polypeptides of the present disclosure comprise a fusion polypeptide comprising wild-type IL-18 or an IL-18 variant polypeptide described herein. In some embodiments, the fusion polypeptide comprises (1) a wild-type IL-18 or IL-18 variant polypeptide, and (2) a dimerization domain. In some embodiments, the fusion polypeptide comprises (1) two or more wild-type IL-18, two or more IL-18 variant polypeptides, or any combination of two or more 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 have generally been identified as a stretch of approximately 35 amino acids containing four to five leucine residues separated from each other by six amino acids (Maniatis and Abel (1989) Nature 341:24-25). Exemplary leucine zippers are present in various 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 includes a helix-loop-helix domain (Murre, C. et al. (1989) Cell 58:537-544). Dimerization domains 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). Dimerization domains are described in further detail in Eisenman, U.S. Patent No. 5,624,818. In some embodiments, the dimerization domain is an antibody Fc domain.

[0161] In some embodiments, the fusion polypeptide comprises wild-type IL-18 (e.g., wild-type IL-18 whose amino acid sequence is 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 described herein) and an antibody Fc domain. In some embodiments, the C-terminus of wild-type IL-18 or the C-terminus of an 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 an IL-18 variant polypeptide. In some embodiments, the Fc domain is a human Fc domain or a variant thereof comprising one or more amino acid substitutions. In some embodiments, the Fc domain is a human IgG Fc domain or a variant thereof, e.g., a human IgG1, IgG2, or IgG4 Fc domain, or a variant of any of the foregoing. In some embodiments, a fusion polypeptide comprises, from N- to C-terminus, an IL-18 variant polypeptide described herein and a human IgG1 Fc variant comprising the N297A mutation, where amino acid numbering is according to the EU numbering system (also referred to 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, a fusion polypeptide comprises, from N- to C-terminus, wild-type IL-18 and a human IgG1 Fc variant comprising the N297A mutation. In some embodiments, a fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0162] Nucleic acids, vectors, host cells, and methods for producing IL-18 variant polypeptides

[0163] 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 the group consisting of SEQ ID NOs: 301-306 and 333-335. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 301-306 and 333-335. The nucleic acid sequences of SEQ ID NOs: 301-306 and 333-335 are set forth in the sequence listing below in the Examples section.

[0164] Also provided are vectors into which the nucleic acids described herein are inserted.

[0165] Briefly summarized, expression of an IL-18 variant polypeptide or fusion polypeptide described herein by 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, such as, for example, a promoter (e.g., a constitutive, regulatable, or tissue-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0166] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0167] Furthermore, the expression vector can be provided to cells 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) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (see, for example, WO 01 / 96584, WO 01 / 29058, and U.S. Patent No. 6,326,193).

[0168] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several 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 stable integration of the transgene over time and its propagation in daughter cells. Lentiviral vectors have an advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic.

[0169] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30–110 base pairs (bp) upstream of the start site, although several promoters have recently been shown to contain similarly functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or interlocked with one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0170] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of conferring high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), the long terminal repeat (LTR) promoter of human immunodeficiency virus (HIV), the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, and promoters of human genes, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0171] In some embodiments, expression of the nucleic acid(s) encoding the IL-18 variant polypeptide or fusion polypeptide is inducible. In some embodiments, the nucleic acid(s) encoding the IL-18 variant polypeptide or fusion polypeptide are operably linked to an inducible promoter, including inducible promoters known in the art. In some embodiments, the nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides described herein are designed to encode an epitope tag, for example, to facilitate purification or detection of the polypeptide. Exemplary epitope tags include, but are not limited to, 6xHis (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, e.g., 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).

[0172] The IL-18 variant polypeptides or fusion polypeptides of the present disclosure may be produced by any means known in the art. Exemplary techniques for polypeptide production are described below; however, these exemplary techniques are provided for illustrative purposes only and are not intended to be limiting.

[0173] The IL-18 variant polypeptides or fusion polypeptides described herein can be produced using recombinant methods. For recombinant production of IL-18 variant polypeptides or fusion polypeptides, nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or for expression. DNA encoding IL-18 variant polypeptides or fusion polypeptides can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the IL-18 variant polypeptide or fusion polypeptide). Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0174] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells, e.g., enables the vector to replicate independently of the host chromosomal DNA. This sequence may include an origin of replication or autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeast, and viruses. Generally, the origin of replication component is not needed for mammalian expression vectors (although the SV40 origin may be used because it contains the early promoter).

[0175] Expression and cloning vectors can contain a selection gene or selectable marker. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that complement auxotrophic deficiencies; or (c) proteins that supply critical nutrients unavailable from complex media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Other examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding an IL-18 variant polypeptide or fusion polypeptide, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. For example, Chinese hamster ovary (CHO) cell lines transformed with the DHFR gene and deficient in endogenous DHFR activity are identified by culturing the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR.

[0176] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an IL-18 variant polypeptide or fusion polypeptide of interest, a wild-type DHFR gene, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH) (specifically, wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, for example, an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418.

[0177] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide. Suitable promoters for use in prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, 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 can include inducible promoters / enhancers that are regulated by growth conditions. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription begins. Examples include, but are not limited to, 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 fusion polypeptide from a vector in a mammalian host cell can be controlled, for example, by a promoter obtained from a viral genome. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate-early promoter of the human cytomegalovirus is conveniently obtained as a HindIII restriction fragment. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0178] Transcription of a DNA encoding an IL-18 variant polypeptide or fusion polypeptide described herein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, commonly, enhancers from eukaryotic cell viruses are used.

[0179] Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing the mRNA.

[0180] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeast are suitable as 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 lower eukaryotic host microorganism. Certain fungal and yeast strains may be selected in which the glycosylation pathway has been "humanized" and which produce IL-18 variant polypeptides or fusion polypeptides with partially or fully human glycosylation patterns. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006).

[0181] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco may also be utilized as hosts.

[0182] Suitable host cells 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. Numerous baculovirus strains and mutants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori.

[0183] Vertebrate cells may be used as hosts, and growing vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian cell lines include, for example, SV40-transformed monkey kidney-derived cell line CV1 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 cells, or 293 cells subcloned in suspension culture for growth; 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-derived cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney-derived cells (MDCK, ATCC CCL 34), and buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and the human hepatoma line (Hep G2). Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including 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 specific mammalian host cell lines suitable for production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0184] 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. Further, see Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in U.S. Pat. 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. No. Re. 30,985 may be used as the host cell culture medium. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, The culture medium may be supplemented with nutrients (such as sodium, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be added at appropriate concentrations. Culture conditions, such as temperature, pH, and the like, will be those conventionally used with the host cell selected for expression and will be apparent to those of skill in the art.

[0185] When using recombinant technology, IL-18 variant polypeptide or fusion polypeptide can be produced intracellularly, produced in the periplasmic space, or directly secreted into the culture medium.When producing polypeptide intracellularly, the first step is to remove particulate debris, host cells, or lysed fragments, for example, by centrifugation or ultrafiltration.Carter et al., Bio / Technology 10:163-167(1992) describes a method for isolating antibodies secreted into the periplasmic space of E. coli.

[0186] Polypeptide compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being one of the preferred purification steps. In some embodiments, the IL-18 variant polypeptides or fusion polypeptides described herein comprise an epitope tag (e.g., a tag attached to the IL-18 variant polypeptide or fusion polypeptide via a cleavable linker) to facilitate purification. Exemplary epitope tags include, but are not limited to, e.g., 6xHis (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, e.g., 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).

[0187] Methods for treating diseases, activating signaling via the hIL-18 receptor, and stimulating antigen-experienced T cells or NK cells

[0188] Also provided herein are methods for treating a disease or condition in an individual. The methods comprise administering an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein to an individual having the disease or condition. In some embodiments, the disease or condition is a proliferative disorder. In some embodiments, the proliferative disorder is cancer.

[0189] In some embodiments, provided herein are methods for activating hIL-18 receptor-mediated signals in an individual, the methods comprising administering to the individual an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein. In some embodiments, provided herein are methods for stimulating antigen-experienced T cells or NK cells in an individual, the methods comprising administering to the individual an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein. In some embodiments of any of the methods herein, the individual is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, a laboratory animal, etc.

[0190] Compositions, kits and articles of manufacture

[0191] Also provided herein are compositions (eg, formulations) comprising an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell described herein.

[0192] Suitable compositions can be obtained by mixing an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0193] 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 may be useful for any of the therapeutic methods described herein.

[0194] The kits of the present application are in suitable packaging, including, but not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). The kits may optionally include additional components, such as buffers and instructional information.

[0195] Accordingly, the present application also provides an article of manufacture. The article of manufacture may comprise 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 packaging, and the like. Generally, the container holds the composition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle).

[0196] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0197] Illustrative Embodiments

[0198] Embodiment 1. An interleukin-18 (IL-18) variant polypeptide comprising at least one mutation at a residue selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein said amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO:1.

[0199] Embodiment 2. The IL-18 variant polypeptide of embodiment 1, comprising at least one mutation at a residue selected from the group consisting of G3, E6, D54, and N91, wherein said amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO:1.

[0200] Embodiment 3. The IL-18 variant polypeptide of embodiment 1 or 2, further comprising at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113, and N155, wherein said amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO:1.

[0201] Embodiment 4. The IL-18 variant polypeptide of any one of embodiments 1 to 3, further comprising at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, wherein said amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1.

[0202] Embodiment 5. The IL-18 variant polypeptide of embodiment 4, wherein the at least one mutation comprises a substitution at C38, a substitution at C68, and a S117C substitution.

[0203] Embodiment 6. The IL-18 variant polypeptide of embodiment 4, wherein the at least one mutation is selected from the group consisting of C38S, C68S, C76S, and C127S.

[0204] Embodiment 7. The IL-18 variant polypeptide of embodiment 4, wherein the variant polypeptide further comprises C38S, C68S, and C76S mutations.

[0205] Embodiment 8. The IL-18 variant polypeptide of embodiment 4, wherein the variant polypeptide further comprises a set of mutations selected from the group consisting of: (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.

[0206] Embodiment 9. An interleukin-18 (IL-18) variant polypeptide comprising at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, wherein said amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO:1.

[0207] Embodiment 10. The IL-18 variant polypeptide of embodiment 9, wherein the at least one mutation comprises a substitution at C38, a substitution at C68, and a S117C substitution.

[0208] Embodiment 11. The IL-18 variant polypeptide of embodiment 9, wherein the at least one mutation is selected from the group consisting of C38S, C68S, C76S, and C127S.

[0209] Embodiment 12. The IL-18 variant polypeptide of embodiment 10, wherein the variant polypeptide comprises C38S, C68S, and C76S mutations.

[0210] Embodiment 13. The IL-18 variant polypeptide of embodiment 9, wherein the variant polypeptide comprises a set of mutations selected from the group consisting of: (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.

[0211] Embodiment 14. The IL-18 variant polypeptide of any one of Embodiments 1 to 13, wherein the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO:1.

[0212] Embodiment 15. The IL-18 variant polypeptide of any one of Embodiments 1 to 14, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and has substantially reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18.

[0213] Embodiment 16. The IL-18 variant polypeptide of any one of Embodiments 1 to 15, wherein the IL-18 variant polypeptide has increased binding to IL-18Rα compared to wild-type IL-18.

[0214] Embodiment 17. The IL-18 variant polypeptide of any one of Embodiments 1 to 16, wherein the IL-18 variant polypeptide binds to IL-18Rα with a KD of less than about 5×10 −5 M.

[0215] Embodiment 18. The IL-18 variant polypeptide of any one of Embodiments 1 to 17, wherein the IL-18 variant polypeptide binds to IL-18Rα with a KD of about 5×10 −5 M to about 5×10 −11 M.

[0216] Embodiment 19. The IL-18 variant polypeptide of any one of Embodiments 1 to 18, wherein said IL-18 variant polypeptide binds to IL-18BP with a KD of greater than 5×10 −9 M.

[0217] Embodiment 20. The IL-18 variant polypeptide of any one of Embodiments 1 to 19, wherein said IL-18 variant polypeptide does not exhibit binding to IL-18BP.

[0218] Embodiment 21. The IL-18 variant polypeptide of any one of Embodiments 1 to 20, wherein the variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from the group consisting of G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N, and G3S.

[0219] Embodiment 22 The IL-18 variant polypeptide of embodiment 21, wherein the mutation is G3P.

[0220] Embodiment 23. The IL-18 variant polypeptide of any one of embodiments 1 to 22, wherein the variant polypeptide comprises a mutation at residue E6, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y.

[0221] Embodiment 24. The IL-18 variant polypeptide of embodiment 23, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S.

[0222] Embodiment 25. The IL-18 variant polypeptide of embodiment 24, wherein the mutation is selected from the group consisting of E6R and E6K.

[0223] Embodiment 26. The IL-18 variant polypeptide of any one of embodiments 1 to 25, wherein the variant polypeptide comprises a mutation at residue D54, wherein the mutation is selected from the group consisting of D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T.

[0224] Embodiment 27. The IL-18 variant polypeptide of embodiment 26, wherein the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0225] Embodiment 28. The IL-18 variant polypeptide of any one of embodiments 1 to 27, wherein the variant polypeptide comprises a mutation at residue N91, wherein the mutation is selected from the group consisting of N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W.

[0226] Embodiment 29. The IL-18 variant polypeptide of embodiment 28, wherein the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0227] Embodiment 30. The IL-18 variant polypeptide of any one of embodiments 1 to 29, wherein the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N.

[0228] Embodiment 31. The IL-18 variant polypeptide of embodiment 30, wherein the mutation is selected from the group consisting of R104S, R104Y, and R104T.

[0229] Embodiment 32. The IL-18 variant polypeptide of any one of embodiments 1 to 29, wherein the variant polypeptide does not comprise a mutation at residue R104.

[0230] Embodiment 33. The IL-18 variant polypeptide of any one of embodiments 1 to 32, wherein the variant polypeptide comprises a mutation at residue Q56, wherein the mutation is selected from the group consisting of Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N, and Q56V.

[0231] Embodiment 34. The IL-18 variant polypeptide of embodiment 33, wherein the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0232] Embodiment 35. The IL-18 variant polypeptide of any one of Embodiments 1 to 32, wherein the variant polypeptide does not comprise a mutation at residue Q56.

[0233] Embodiment 36. The IL-18 variant polypeptide of any one of embodiments 1 to 35, wherein the variant polypeptide comprises a mutation at residue P57, wherein the mutation is selected from the group consisting of P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D.

[0234] Embodiment 37. The IL-18 variant polypeptide of embodiment 36, wherein the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0235] Embodiment 38. The IL-18 variant polypeptide of any one of Embodiments 1 to 35, wherein the variant polypeptide does not comprise a mutation at residue P57.

[0236] Embodiment 39. The IL-18 variant polypeptide of any one of Embodiments 1 to 38, wherein the variant polypeptide comprises mutations at G3, E6, D54, and N91.

[0237] Embodiment 40. The IL-18 variant polypeptide of embodiment 39, wherein the variant polypeptide comprises the following mutations: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S.

[0238] Embodiment 41. The IL-18 variant polypeptide of any one of embodiments 1 to 34, 36 to 37, and 39 to 40, wherein the variant polypeptide further comprises mutations at Q56 and P57.

[0239] Embodiment 42. The IL-18 variant polypeptide of any one of embodiments 1 to 41, wherein the variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 299 and 307 to 318.

[0240] Embodiment 43. A fusion polypeptide comprising the IL-18 variant polypeptide of any one of Embodiments 1 to 42 and a human IgG Fc domain or a variant thereof.

[0241] Embodiment 44. The fusion polypeptide of embodiment 43, comprising a human IgG1 Fc domain variant comprising an N297A mutation (EU numbering).

[0242] Embodiment 45. The fusion polypeptide of embodiment 43 or 44, wherein the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of a human IgG Fc domain or a variant thereof.

[0243] Embodiment 46 The fusion polypeptide of embodiment 43 or 44, wherein the C-terminus of the human IgG Fc domain or variant thereof is fused to the N-terminus of the IL-18 variant polypeptide.

[0244] Embodiment 47. The fusion polypeptide of any one of embodiments 43 to 46, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 319 to 332 and 336 to 347.

[0245] Embodiment 48. A dimer comprising two fusion polypeptides according to any one of embodiments 43 to 47.

[0246] Embodiment 49 The dimer of embodiment 48, wherein the dimer is a homodimer.

[0247] Embodiment 50 The dimer of embodiment 48, wherein the dimer is a heterodimer.

[0248] Embodiment 51. A nucleic acid encoding an IL-18 variant polypeptide according to any one of embodiments 1 to 42 or a fusion polypeptide according to any one of embodiments 43 to 47.

[0249] Embodiment 52. The nucleic acid of embodiment 51, comprising a polynucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 301-306 and 333-335.

[0250] Embodiment 53. A vector comprising the nucleic acid of embodiment 51 or 52.

[0251] Embodiment 54. A host cell comprising the nucleic acid of embodiment 51 or 52 or the vector of embodiment 53.

[0252] Embodiment 55. A method for producing an IL-18 variant polypeptide or fusion polypeptide, comprising: (a) culturing the host cell of embodiment 54 under conditions in which the IL-18 variant polypeptide or fusion polypeptide is expressed; and (b) recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell.

[0253] Embodiment 56 The method of embodiment 55, further comprising purifying the IL-18 variant polypeptide or fusion polypeptide.

[0254] Embodiment 57. A pharmaceutical composition comprising an IL-18 variant polypeptide described in any one of Embodiments 1 to 42, a fusion polypeptide described in any one of Embodiments 43 to 47, a nucleic acid described in Embodiment 51 or 52, or a vector described in Embodiment 53.

[0255] Embodiment 58. A method of treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 57.

[0256] Embodiment 59 The method of embodiment 58, wherein the disease is cancer.

[0257] Embodiment 60 A method for activating a signal mediated by an hIL-18 receptor in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of Embodiment 57.

[0258] Embodiment 61 A method of stimulating antigen-experienced immune cells in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 57.

[0259] Embodiment 62 The method of embodiment 61, wherein the stimulating comprises increasing the activity and / or number of the antigen-experienced immune cells. [Example]

[0260] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and 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 accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. The following examples are intended to be purely illustrative of the present application and therefore should not be construed as limiting the present application in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0261] Example 1: Construction of a yeast expression library of IL-18 variant polypeptides that do not bind to IL-18BP

[0262] Interleukin-18 (IL-18) stimulates T cells and NK cells by binding to a heterodimeric receptor complex consisting of the IL-18Rα and IL-18Rβ subunits. IL-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 binding of IL-18BP to IL-18 inhibits the interaction between IL-18 and IL-18Rα. To develop variants of human IL-18 ("hIL-18") that do not bind to IL-18BP or have reduced binding affinity for IL-18BP, we used artificial intelligence-assisted molecular design methods to identify amino acid positions within wild-type hIL-18 for mutation. Next, a yeast library displaying hIL-18 variant polypeptides containing the identified mutation(s) compared to wild-type IL-18 (SEQ ID NO: 1) was constructed using NNK mutagenesis, resulting in a final library diversity of approximately 2.72 × 10 8 For expression studies, each of the IL-18 variants was fused to a c-myc tag.

[0263] Example 2: Screening for IL-18 variant polypeptides that bind to human IL-18Rα

[0264] Using a positive / negative selection strategy, the library constructed in Example 1 was screened 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 for hIL-18BP compared to wild-type IL-18. Briefly, recombinant hIL-18Rα extracellular domain ("hIL-18Rα ECD") was labeled with biotin and immobilized on a first set of streptavidin (SA) or anti-biotin magnetic microbeads for magnetic cell sorting (MACS)-based screening. Recombinant hIL-18BP was immobilized on a second set of SA or anti-biotin magnetic microbeads. Three rounds of selection were performed on the library constructed in Example 1 using hIL-18Rα ECD-bound beads and hIL-18BP-bound beads, as shown in Figure 1A.

[0265] Next, two more rounds of FACS-based kinetic competition screening (i.e., rounds 4 and 5 shown in Figure 1A) were performed to identify antibodies that (a) had higher or similar affinity for hIL-18Rα compared to WT hIL-18, and (b) had no or reduced binding to hIL-18BP (e.g., K for hIL-18BP). D >10 -3 M) IL-18 variant polypeptides were further enriched. Single yeast cells were then isolated from the output pools of rounds 4 and 5. Plasmids encoding the isolated IL-18 variant polypeptides were extracted from the yeast and sequenced by next-generation sequencing (NGS).

[0266] After each round of screening, the size of the output library was determined by FACS. The binding ability of the output library to 50 nM hIL-18Rα ECD or 1 μM hIL-18BP was also confirmed by FACS. As shown in Figure 1B, after the above screening, most of the displayed IL-18 variants did not bind to hIL-18BP or had reduced binding to hIL-18BP, while their binding ability to hIL-18Rα ECD was maintained or increased.

[0267] Example 3: Single clone fluorescence activated cell sorting (FACS) of yeast cells displaying IL-18 variant polypeptides.

[0268] Following the screening described in Example 2, single-clone FACS analysis was performed to examine the binding of yeast cell surface-expressed IL-18 variant polypeptides selected from rounds 4 and 5 to 10 nM hIL-18Rα-ECD. Briefly, the same density of each yeast clone was incubated with 10 nM IL-18Rα-ECD labeled with a fluorescent probe, followed by a washing step. The proportion of fluorescent-positive IL-18Rα ECD-binding yeast in each clonal population was then analyzed.

[0269] As shown illustratively in Table 1 and FIG. 1C (showing WT IL-18 (SEQ ID NO: 1), and clones M12 (SEQ ID NO: 39), and M17 (SEQ ID NO: 43) as examples), 117 of the 192 IL-18 variant clones tested (i.e., SEQ ID NOs: 2-118) were identified as positive clones (>0.1% fluorescent cells in the clonal population). SEQ ID NO: 1 is wild-type human IL-18. Table 1:

[0270] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0271] Example 4: Expression and purification of recombinant hIL-18

[0272] WT hIL-18 and the selected IL-18 variant polypeptides were expressed in the E. coli system. Briefly, the PET30a plasmid containing the codon-optimized sequence encoding WT hIL-18 or the hIL-18 variant polypeptide was transfected into E. coli BL21(DE3) cells. The transfected E. coli cells were then grown in culture, and the expressed polypeptides were collected and purified.

[0273] 2A-2F show WT hIL-18 and five exemplary variants (M12, MM5, WM6, M17, and M24) with purity greater than 95% as measured by SDS-PAGE and SEC-HPLC.

[0274] Example 5: Affinity of IL-18 variant polypeptides for hIL-18Rα

[0275] The affinity of IL-18 variant polypeptides for hIL-18Rα was assessed by biolayer interferometry (BLI) on an Octet RED96 using a Protein A sensor coated with hIL-18Rα ECD-Fc fusion protein, with WT hIL-18 used as a control. The binding affinities of WT hIL-18 and IL-18 variant polypeptides for hIL-18Rα ECD-Fc are shown in Table 2A.

[0276] [Table 2A-1] [Table 2A-2]

[0277] These IL-18 variant polypeptides (i.e., M1 to M41, LM1 to LM5, MM1 to MM2, MM4 to MM6, WM1 to WM6, WM8, WM10 to WM14, corresponding to SEQ ID NOs: 1 to 4, 8 to 9, 11 to 12, 17, 21 to 22, 26 to 28, 35, 38 to 39, 41, 43 to 44, 49 to 50, 54 to 56, 58 to 59, 65 to 66, 72 to 74, 81, 91, 94, 99, 104 to 105, 114, 116 to 117, 121 to 125, 131 to 134, 139 to 145, 147, 149 to 153) are shown in Table 2B.

[0278] [Table 2B-1] [Table 2B-2]

[0279] Example 6: Functional properties of IL-18 variant polypeptides

[0280] 6.1 Activation of hIL-18 Receptor-Mediated Signaling by IL-18 Variant Polypeptides

[0281] The NFκB-luc / hIL-18RαRβ HEK293 cell line, stably expressing hIL-18Rα, hIL-18Rβ, and an NFκB luciferase reporter gene, was used as reporter cells to determine the ability of IL-18 variant polypeptides to activate signaling through the hIL-18 receptor.

[0282] Briefly, reporter cells were first plated into wells of a white 96-well plate at 3.0 × 10 cells per well. 4 The cells were seeded at a density of 1 μg / mL. WT hIL-18 or IL-18 variant polypeptides were serially diluted in medium in the presence or absence of 1 μg / mL hIL-18 BP-hFc (a). After 16 hours of incubation at 37°C, luminescence intensity was measured.

[0283] As shown in Figure 3 and Table 3, all IL-18 variant polypeptides tested demonstrated the ability to activate signaling through the IL-18 receptor in a dose-dependent manner. Unlike WT hIL-18, whose activity was significantly (approximately 80-fold) reduced in the presence of 1 μg / mL hIL-18BP-hFc, IL-18 variant polypeptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM11, corresponding to 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, 149-150) exhibited EC 50 The Hil-18 BP-resistant activity was observed with a 3-fold or less change in IL-18 activity, indicating that the IL-18 variant polypeptides were significantly less susceptible to Hil-18 BP in activating Hil-18 receptor-mediated signaling. The only exception was WM6, whose EC 50 shows an approximately 12-fold reduction in potency in the presence of 1 μg / Ml HIL-18BP-hFc, but is still far superior to WT hIL-18.

[0284] [Table 3-1] [Table 3-2]

[0285] 6.2 Human PBMC-based hIFNγ release assay

[0286] A PBMC-based assay was performed to test the ability of IL-18 variant polypeptides to activate T / NK cells and induce the release of hIFNγ. Briefly, human PBMC cells were plated in wells of a 96-well plate at 5.0 × 10 per well. 5Cells were seeded at a density of 1000 x g and cultured for 5 hours before treatment. WT hIL-18 or IL-18 variant polypeptides were serially diluted in 1 ng / mL human IL-12 in the presence of 1 μg / mL hIL-18 BP-hFc or in the absence of 1 μg / mL hIL-18 BP-hFc and then added to the wells. After 16 hours of incubation at 37°C, the culture medium was collected from each well and hIFNγ concentrations were measured by FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0287] As shown in Figure 4, the tested IL-18 variant polypeptides demonstrated the ability to induce hIFNγ release from PBMCs in a dose-dependent manner. Furthermore, as shown in Table 4, the activity of WT hIL-18 was significantly attenuated in the presence of 1 μg / mL hIL-18BP-hFc, resulting in a significant increase in EC 50 In contrast, IL-18 variant polypeptides (i.e., M11 to M13, M15, M17, M21, M24, M29, M32 to M33, M35 to M36, MM5, WM4 to WM6, WM8, WM10 to WM11, corresponding to SEQ ID NOs: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116 to 117, 133, 143 to 150) inhibited EC 50 The change in hIFNγ induction was less than two-fold, indicating hIL-18 BP resistance activity, indicating that the IL-18 variant polypeptides were significantly less affected by hIL-18BP.

[0288] [Table 4]

[0289] 6.3 Mouse splenocyte-based mIFNγ release assay

[0290] To assess the in vivo efficacy of the IL-18 variant polypeptides of the present application, a mouse splenocyte-based mouse IFNγ (“mIFNγ”) release assay is performed as follows.

[0291] Briefly, freshly isolated mouse splenocytes were plated into wells of a 96-well plate at 6.5 x 10 cells per well. 5 Cells were seeded at a density of 1000 x g. WT hIL-18 or IL-18 variant polypeptides were serially diluted in 1 ng / mL mouse IL-12 (a) in the presence of 1 μg / mL mouse IL-18BP-Fc, or (b) in the absence of 1 μg / mL mouse IL-18BP-Fc, and then added to the wells. After 16 hours of incubation at 37°C, the culture medium was collected from each well and the mIFNγ concentration in the medium was measured by FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0292] Example 7: Generation and characterization of IL-18 variant polypeptides containing serine to cystine and / or cystine to serine substitutions

[0293] 7.1 Wild-type IL-18 or IL-18 polypeptide variants containing a cystine to serine substitution

[0294] Disulfide bond engineering can improve polypeptide stability and yield. The following experiments were performed to evaluate the effect of a Cys to Ser ("C→S") mutation on the efficacy of IL-18 variants and their expression in mammalian cells, such as CHO cells and 293F cells. Wild-type IL-18 and the IL-18 variant polypeptides M12 and MM5 were modified (or further modified) to contain C38S, C68S, and C76S substitutions, generating novel variants with the following amino acid sequences:

[0295] IL-18 variant containing C38S, C68S, and C76S substitutions in a WT hIL-18 background ("IL-18-SSS"): YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKDSQPRGMAVTISVKSEKISTLSSENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 307)

[0296] IL-18 variant containing C38S, C68S, and C76S substitutions in an M12 background ("M12-SSS"): YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 308)

[0297] IL-18 variant containing C38S, C68S, and C76S substitutions in an MM5 background (“MM5-SSS”): YFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKLSTERGMAVTISVKSEKISTLSSENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 309)

[0298] 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 an 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, with a purity of 65%. In contrast, the yield of WT IL-18-Fc_N297A obtained after one-step Protein A purification was approximately 16 mg / L, with a purity of 11%. The yield of M12-SSS-Fc_N297A obtained after one-step Protein A purification was 5.6 mg / ml, with a purity of 72%. In contrast, M12-Fc_N297A was not expressed at detectable levels in 100 mL CHO host cells. The yield of MM5-SSS-Fc_N297A obtained after one-step Protein A chromatography was 24.5 mg / L, with a purity of 46%. After a further purification step by gel filtration, the yield of MM5-SSS-Fc_N297 was 3.4 mg / L, with a purity of 99%. In contrast, MM5-Fc_N297A was not expressed at detectable levels in 100 mL CHO host cells.

[0299] M12-SSS was further modified to include a C127S substitution to generate M12-SSSS: YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLASEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 310)

[0300] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU numbering) to generate M12-SSSS-Fc_N297A (SEQ ID NO: 322). M12-SSSS-Fc_N297A was expressed in CHO cells, harvested, and purified by Protein A chromatography. The introduction of the C127S substitution (i.e., the fourth C→S substitution) reduced the yield and purity of M12-SSSS-Fc_N297A compared with that of M12-SSS-Fc_N297A (data not shown).

[0301] Next, the potency of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A to activate signaling through the IL-18 receptor was characterized using the in vitro assay described in Section 6.1 of Example 6. The potency of IL-18-SSS-Fc_N297A was shown to be equivalent to that of wild-type IL-18 (see Figure 5A), whereas M12-SSS-Fc_N297A showed approximately 7-fold reduced 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-18 BP-hFc ("BP"), while M12-SSS-Fc_N297A exhibited hIL-18 BP-resistant activity in the presence of 1 μg / ml hIL-18BP-hFc. See Figures 5A and 5B. As shown in Figure 5C, MM5-SSS-Fc_N297A showed approximately 220-fold reduced potency compared to MM5, but still exhibited hIL-18BP resistance activity in the presence of 1 μg / ml hIL-18BP-hFc. These results indicate that the three cystine-to-serine substitutions do not affect IL-18BP resistance. These data suggest that C38, C68, and C76 are important for improving the yield during production of IL-18 and IL-18 polypeptide-Fc fusion proteins, as well as the yield and purity after purification. MM5-SSS-Fc_N297A may exhibit a longer half-life than MM5.

[0302] 7.2 M12-Derived IL-18 Polypeptide Variants Comprising Amino Acid Substitutions That Remove and / or Introduce Cysteine ​​Residues

[0303] To evaluate the effects of substitutions that delete unique cysteines (i.e., naturally occurring cysteines in the polypeptide sequence) and / or that introduce disulfide bonds on the efficacy, expression, and purification of IL-18 variant polypeptides, the following experiments were performed. First, in silico screening based on molecular dynamics simulations was performed, and after AI-based stability assessment, mutation hotspots were identified at each of the four cysteines (C38, C68, C76, and C127) of M12, as described (see Table 5). Furthermore, structure-guided design was used to identify amino acids at spatial positions that, when substituted with Cys residue(s), would enable the formation of novel non-native disulfide bond(s). The mutation strategies shown in Table 5 (i.e., DB6, DB7, DB8, DB9, and DB10) are presented.

[0304] [Table 5]

[0305] The mutation strategies in Table 5 were introduced into the IL-18 polypeptide variant M12 to generate novel variants M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10.

[0306] M12-DB6 YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDIRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSCENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFECSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 311)

[0307] M12-DB7 YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKGSGARGMAVTISVKIEKISTLSCENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFECSSYEGYFLAAEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 312)

[0308] M12-DB8 YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKIEKISTLSCENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFECSSYEGYFLAIEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 313)

[0309] M12-DB9 YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDIRDNAPRTIFIISMYKGSGARGMAVTISVKIEKISTLSVENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAIEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 314)

[0310] M12-DB10 YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDIRDNAPRTIFIISMYKGSGARGMAVTISVKLEKISTLSYENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 315)

[0311] 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 an N297A substitution (EU numbering). The resulting fusion polypeptides, 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.

[0312] [Table 6]

[0313] M12-DB8-Fc_N297A was not expressed at detectable levels. M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A showed significantly higher expression yields or purity than 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 showed the highest expression yield (313 mg / L) after one-step Protein A purification, with a purity of 89.8% (measured by SEC-HPLC). The expression yield of M12-DB7-Fc_N297A was approximately 50% of that of M12-DB6-Fc_N297A. Although M12-DB7-Fc_N297A showed similar purity to M12-DB6-Fc_N297A after Protein A chromatography, such results indicate that the C127X substitution does not further improve polypeptide production in mammalian cells.

[0314] Next, 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 was characterized using the in vitro assay described in Section 6.1 of Example 6. As shown in Figures 6A-6D and Table 7 below, 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) exhibited significantly greater potency than WT IL-18 in activating IL-18 receptor-mediated signaling. The signaling activity of tested M12-DB6-Fc_N297A (FIG. 6A), M12-DB7-Fc_N297A (FIG. 6B), and M12-DB9-Fc_N297A (FIG. 6C) was not inhibited in the presence of 1 μg / ml hIL-18BP-hFc (see Table 7).

[0315] [Table 7]

[0316] Next, the "DB6" substitutions, namely C38I, C68S, and S117C, were introduced into variant MM5 to generate MM5-DB6.

[0317] MM5-DB6 YFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDIRDNAPRTIFIISMYKLSTERGMAVTISVKSEKISTLSCENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFECSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 316)

[0318] The "DB6" substitution was also introduced into wild-type IL-18 to generate WT IL18-DB6.

[0319] WT IL18-DB6 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDIRDNAPRTIFIISMYKDSQPRGMAVTISVKSEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFECSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 317)

[0320] An additional variant, WT-DBo, was designed by introducing an S117C substitution into wild-type IL-18.

[0321] WT IL-18-DBo YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFECSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 318)

[0322] The C-terminus 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 was not expressed at detectable levels. The expression yield of WT IL18-DB6-Fc was 157 mg / L, and WT IL18-DB6-Fc_N297A had a purity of 91.6% (measured by SEC-HPLC) after one-step Protein A chromatography (see Table 8). These data indicate that modifying the disulfide bond between C76 and C117 alone is not sufficient to improve expression of fusion polypeptides in mammalian cells. Improved expression in mammalian cells is achieved by fusion polypeptides containing IL-18 variants including (i) a modified C76-C117 disulfide bond, (ii) a C38 substitution (see Table 5), and (iii) a C68 substitution (see Table 5). MM5-DB6-Fc_N297A exhibits a much higher expression yield than WT IL18-DB6-Fc_N297A with similar purity (see Table 8). Such data also suggest that the "DB6" substitution is suitable for improving the yield of WT IL-18 and IL-18 variant polypeptides during production in mammalian cells and after purification.

[0323] Next, we designed fusion polypeptides by fusing the C-terminus of human IgG1 Fc containing the N297A substitution (EU numbering) to the N-terminus of each of the variants M12-DB6 and MM5-DB6. The resulting fusion polypeptides, 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, both Fc_N297A-MM5-DB6 and Fc_N297A-M12-DB6 showed high expression yields in CHO cells. After one-step Protein A chromatography purification, Fc_N297A-MM5-DB6 was approximately 97% pure (measured by SEC-HPLC), and Fc_N297A-M12-DB6 was 89% pure (measured by SEC-HPLC).

[0324] [Table 8]

[0325] Next, 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 was characterized using the in vitro assay described in Section 6.1 of Example 6. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A exhibited stronger IL-18 receptor cell activation signals than WT IL-18 (see Figures 7A and 7B, and Table 9). While Fc_N297A-MM5-DB6 exhibited similar potency to WT IL-18, Fc_N297A-M12-DB6 produced a much stronger activation signal on IL-18 reporter cells (see Figures 7C and 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, whereas the activity of MM5-DB6-Fc_N297A was resistant to inhibition by IL-18BP (Figures 7A and 7B; Table 9). These results indicate that introducing the "DB6" mutation set (i.e., C38I, C68S, and S117C; Table 5) into WT IL-18-Fc fusion polypeptides or IL-18 variant-Fc fusion polypeptides improves the expression yield of the fusion polypeptide during production in mammalian cells and the yield of purified fusion polypeptides after chromatography. Furthermore, WT IL-18 and IL-18 variants engineered to contain the "DB6" mutation set are more potent at activating signaling through the IL-18 receptor. Such results were observed in fusion polypeptides in which the C-terminus of the Fc region was fused to the N-terminus of an IL-18 variant, and in which the C-terminus of an IL-18 variant was fused to the N-terminus of an Fc domain.

[0326] [Table 9]

[0327] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover the modifications and variations of the present disclosure provided they come within the scope of the appended claims and their equivalents.

[0328] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0329] The present invention has been described in terms of specific embodiments known to the inventors or suggested by them to comprise preferred modes for carrying out the invention. In light of this disclosure, those skilled in the art will recognize that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, changes can be made in the protein structure without affecting the type or amount of biological action. All such modifications are intended to be within the scope of the appended claims.

[0330] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

Table 10-23

Table 10-24

Table 10-25

Table 10-26

Table 10-27

Table 10-28

Table 10-29

Table 10-30

Table 10-31

Table 10-32

Table 10-33

Table 10-34

Table 10-35

Table 10-36

Table 10-37

Table 10-38

Table 10-39

Table 10-40

Table 10-41

Table 10-42

Table 10-43

Table 10-44

Table 10-45

Table 10-46

Table 10-47

Table 10-48

Table 10-49

Table 10-50

Table 10-51

Claims

1. An interleukin 18 (IL-18) variant polypeptide comprising at least one mutation in a residue selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, Q56, P57, M60, A61, N91, K96, R104, R107, K140, N155, and I149, wherein the amino acid position is based on wild-type human IL-18 described in Sequence ID No.

1.

2. An IL-18 variant polypeptide according to claim 1, comprising at least one mutation in a residue selected from the group consisting of G3, E6, and M60, wherein the amino acid position is relative to wild-type human IL-18 as described in SEQ ID NO: 1; Preferably, the IL-18 variant polypeptide is a) G3P, G3S, G3E, G3N, G3D, or G3A, b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y, c) M60K Including at least one of the following: More preferably, the IL-18 variant polypeptide further comprises at least one or more mutations in residues selected from the group consisting of V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155; More preferably, the IL-18 variant polypeptide is 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 It further includes at least one of the following: More preferably, the IL-18 variant polypeptide is 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 Includes one permutation or set of permutation combinations selected from the group consisting of IL-18 variant polypeptide.

3. IL-18 variant polypeptide according to claim 1, comprising an amino acid sequence described in any 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, or the IL-18 variant polypeptide further comprising an S117C substitution, wherein the amino acid position is based on wild-type human IL-18 described in SEQ ID NO: 1, and optionally the S117C substitution promotes a C76-C117 disulfide bond; Preferably, the IL-18 variant polypeptide further comprises a substitution at C38, the amino acid position being relative to wild-type human IL-18 as described in SEQ ID NO: 1, and optionally, the substitution is selected from the group consisting of C38I, C38V, C38L, and C38M, and optionally, the substitution is C38I or C38V; More preferably, the IL-18 variant polypeptide further comprises a substitution at C68, wherein the amino acid position is relative to wild-type human IL-18 described in SEQ ID NO: 1, and optionally, the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and optionally, the substitution is C68I, C68L, or C38S; More preferably, the IL-18 variant polypeptide further comprises substitutions at C76 and / or C127, wherein the amino acid positions are based on wild-type human IL-18 as described in SEQ ID NO: 1, optionally the C76 substitution is C76Y or C76V, optionally the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and optionally the C127 substitution is C127A or C127I; More preferably, the IL-18 variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution; Most preferably, the IL-18 variant polypeptide is (a) C38I, C68S, and S117C, (b) C38V, C68I, S117C, and C127A, (c) C38S, C68I, S117C, and C127I, (d) C38I, C68I, C76V, and C127I, (e) C38I, C68L, and C76Y Further including a series of mutations selected from a group consisting of, IL-18 variant polypeptide.

4. An interleukin 18 (IL-18) variant polypeptide comprising at least one mutation in a residue selected from the group consisting of C38, C68, C76, S117, and C127, wherein the amino acid position is relative to wild-type human IL-18 as described in Sequence ID No. 1; Preferably, the at least one mutation includes a substitution at C38, a substitution at C68, and an S117C substitution; More preferably, the at least one mutation is selected from the group consisting of C38S, C68S, C76S, and C127S; More preferably, the variant polypeptide comprises C38S, C68S, and C76S mutations; More preferably, the variant polypeptide is (a) C38I, C68S, and S117C, (b) C38V, C68I, S117C, and C127A, (c) C38S, C68I, S117C, and C127I, (d) C38I, C68I, C76V, and C127I, (e) C38I, C68L, and C76Y An IL-18 variant polypeptide comprising a series of mutations selected from the group consisting of the following.

5. The IL-18 variant polypeptide specifically binds to IL-18 receptor α (IL-18Rα), and its binding to IL-18 binding protein (IL-18BP) is substantially reduced compared to the wild-type IL-18; Preferably, the IL-18 variant polypeptide has increased binding to IL-18Rα compared to the wild-type IL-18, and preferably, KD is less than approximately 5 × 10⁻⁵ M; Preferably, the variant polypeptide contains a mutation at residue G3, the mutation being selected from the group consisting of G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N, and G3S, and optionally the mutation being G3P; More preferably, the variant polypeptide comprises a mutation at residue E6, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y, and optionally, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S, and optionally, the mutation is selected from the group consisting of E6R and E6K; More preferably, the variant polypeptide comprises a mutation at residue D54, wherein the mutation is selected from the group consisting of D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T, and optionally, the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y; More preferably, the variant polypeptide contains a mutation at residue N91, and the mutation is selected from the group consisting of N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W, and optionally, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S. The IL-18 variant polypeptide according to any one of claims 1 to 4.

6. The variant polypeptide further comprises a mutation in residue R104, wherein the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N, and optionally the mutation is selected from the group consisting of R104S, R104Y, and R104T, or the variant polypeptide does not contain a mutation in residue R104; Preferably, the variant polypeptide contains a mutation at residue Q56, and the mutation is selected from the group consisting of Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N, and Q56V, and optionally the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y, or the variant polypeptide does not contain a mutation at residue Q56; More preferably, the variant polypeptide contains a mutation in residue P57, wherein the mutation is selected from the group consisting of P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D, and optionally the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V, or the variant polypeptide does not contain a mutation in residue P57; More preferably, the variant polypeptide includes mutations in G3, E6, D54, and N91, and optionally, the variant polypeptide is (i) G3P, (ii) E6R or E6K, (iii) D54W, D54H, D54S, or D54Q, and (iv) N91V, N91A, N91G, or N91S This includes the following mutation: The IL-18 variant polypeptide according to claim 1.

7. The IL-18 variant polypeptide according to claim 1, wherein the variant polypeptide further comprises mutations in Q56 and P57, or the variant polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 2-299 and 307-318, optionally comprising an amino acid sequence described in any one of SEQ ID NOs: 307-318, optionally comprising an amino acid sequence described in any one of SEQ ID NOs: 307-312 and 314-317.

8. A fusion polypeptide comprising the IL-18 variant polypeptide described in claim 1 and a human IgG Fc domain or a variant thereof; Preferably, the fusion polypeptide comprises a human IgG1 Fc domain variant containing the N297A mutation (EU numbering), and optionally, the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the human IgG Fc domain or its variant, or the N-terminus of the IL-18 variant polypeptide is fused to the C-terminus of the human IgG Fc domain or its variant, and optionally, the fusion polypeptide comprises one amino acid sequence from SEQ ID NOs. 319-332 and 336-347.

9. A dimer comprising two fusion polypeptides as described in claim 8.

10. A nucleic acid encoding the IL-18 variant polypeptide described in claim 1 or the fusion polypeptide described in claim 8.

11. A vector comprising the nucleic acid described in claim 10.

12. A host cell comprising the nucleic acid according to claim 10, or a vector comprising the nucleic acid according to claim 10.

13. A method for producing an IL-18 variant polypeptide or fusion polypeptide, (a) The host cells according to claim 12 are cultured under conditions in which the IL-18 variant polypeptide or fusion polypeptide is expressed. (b) The production method comprising recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell.

14. A pharmaceutical composition comprising an IL-18 variant polypeptide according to claim 1, a fusion polypeptide according to claim 8, a nucleic acid encoding the IL-18 variant polypeptide or the fusion polypeptide, or a vector containing the nucleic acid.

15. The pharmaceutical composition according to claim 14, for treating a disease in an individual, for activating signaling via the hIL-18 receptor in an individual, or for stimulating antigen-experienced immune cells in an individual where stimulation of antigen-experienced immune cells is required.

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