IL-18 variant polypeptides

Abstract

An interleukin 18 (IL-18) variant polypeptide is provided having at least one mutation at a residue selected from the group consisting of C38, C68, C76, S117 and C127 wherein the amino acid position is relative to the wild-type human IL-18 set forth in SEQ ID NO: 1. Also provided are IL-18 variant polypeptides that bind to IL-18R [alpha] and exhibit substantially reduced binding to IL-18BP relative to wild-type IL-18. Also provided are IL-18 variant polypeptides that exhibit increased binding to IL-18R [alpha] relative to wild-type IL-18, and substantially reduced binding to IL-18BP relative to wild-type IL-18. Related nucleic acids, vectors, host cells, methods of producing the IL-18 variant polypeptides, pharmaceutical compositions comprising the IL-18 variant polypeptides and uses of the pharmaceutical compositions are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Application No. PCT / CN2023 / 072886 filed on January 18, 2023, and International Application No. PCT / CN2023 / 117197 filed on September 6, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] References to electronic sequence listings

[0004] The contents of the electronic sequence listing (233002001543SEQLIST.xml; size: 362,677 bytes; creation date: January 16, 2024) are hereby incorporated by reference in their entirety. Technical Field

[0005] The present application relates to interleukin 18 (IL-18) variant polypeptides, methods of making such variant polypeptides, and methods of using such variant polypeptides in therapeutic applications, wherein the variant polypeptides comprise at least one amino acid substitution relative to wild-type IL-18 (e.g., wild-type human IL-18). Background Art

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

[0007] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. Summary of the Invention

[0008] In some embodiments, an interleukin 18 (IL-18) variant polypeptide is provided, 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 the 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 at least one mutation at a residue selected from the group consisting of G3, E6, D54, and N91, wherein the 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 further comprises at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. Exemplary IL-18 polypeptides can be found in, e.g., WO2020069398A1, WO2021097376A1, WO2023161853A1, WO2022038417A2, which are herein incorporated by reference in their entireties.

[0009] In some embodiments, the IL-18 polypeptide comprises at least one mutation at a residue selected from the group consisting of: G3, Q24, L29, Q56, P57, M60, A61, N91, K96, R104, R107, K140, N155, and I149, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1, optionally wherein the IL-18 polypeptide comprises a mutation at G3, E6, D54, and N91, further optionally wherein the variant polypeptide comprises a mutation 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 IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0010] In some embodiments, the IL-18 polypeptide comprises a substitution or a combination of substitutions selected from the group consisting of: 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, Q56 S, 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, Q 56T, 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, Q56 G, 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; 4 3) 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; 5 0) 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. ;

[0011] 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, wherein the 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 an S117C substitution. 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) a C38S, C68S, and C76S mutation. 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. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0012] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided, comprising at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, wherein the 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 an S117C substitution. 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 a C38S, C68S, and C76S mutation. 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. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0013] 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 exhibits reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide is expressed at a concentration of less than about 5×10 -5 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at a concentration of about 5×10 -5 to about 5×10 -11 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at a concentration greater than 5×10 -9 M's K D Binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide does not exhibit binding to IL-18BP (eg, no detectable binding as measured by surface plasmon resonance).

[0014] In some embodiments, 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. In some embodiments, the mutation is G3P. In some embodiments, the variant polypeptide comprises a mutation at residue E6, wherein the mutation is selected from 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.

[0015] In some embodiments, 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. In some embodiments, the mutation is selected from the group consisting of: D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0016] In some embodiments, 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. In some embodiments, the mutation is selected from the group consisting of: N91V, N91A, N91G, and N91S.

[0017] In some embodiments, 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. In some embodiments, the mutation is selected from the group consisting of: R104S, R104Y, and R104T.

[0018] In some embodiments, the variant polypeptide does not comprise a mutation at residue R104.

[0019] In some embodiments, 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. In some embodiments, the mutation is selected from the group consisting of: Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0020] In some embodiments, the variant polypeptide does not comprise a mutation at residue Q56.

[0021] In some embodiments, 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. In some embodiments, the mutation is selected from the group consisting of: P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0022] In some embodiments, the variant polypeptide does not comprise a mutation at residue P57.

[0023] 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.

[0024] 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.

[0025] In some embodiments, a fusion polypeptide is provided, which comprises an IL-18 variant polypeptide as described herein and a human IgG Fc domain or a 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 a human IgG Fc domain or a variant thereof. In some embodiments, the C-terminus of the human IgG Fc domain or a variant thereof is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0026] In some embodiments, a dimer is provided, comprising two fusion polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0027] 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 that express the IL-18 variant polypeptide or fusion polypeptide; 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 HEK293 cell). In some embodiments, the method comprises (e.g., further comprises) purifying the IL-18 variant polypeptide or fusion polypeptide.

[0028] In another aspect, the present application provides a method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising introducing cysteine ​​at position 117 and / or position 76 of the IL-18 polypeptide, thereby promoting a disulfide bond between C117 and C76, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. The engineering method is disclosed in the Examples of the present application, and in, for example, Cytokine. 2006 Apr; 34(1-2): 114-24, WO2022038417A2, Nat Commun 14, 2411(2023), the contents of which are incorporated herein in their entireties.

[0029] In another aspect, the present application provides a method for increasing the yield and / or purity of an IL-18 variant polypeptide, comprising engineering an IL-18 polypeptide by introducing cysteine ​​residues at positions 117 and / or 76 of the IL-18 polypeptide, thereby promoting disulfide bond formation between C117 and C76 of the variant polypeptide, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0030] In some embodiments according to any of the methods above, the method comprises a) introducing a cysteine ​​at position 117, and b) retaining the cysteine ​​at position 76 when the IL-18 polypeptide comprises a cysteine ​​at position 76, or introducing a cysteine ​​at position 76 when the IL-18 polypeptide comprises a non-cysteine ​​at position 76.

[0031] In some embodiments according to any of the above methods, the method comprises a) introducing a cysteine ​​at position 76, and b) retaining the cysteine ​​at position 117 when the IL-18 polypeptide comprises a cysteine ​​at position 117, or introducing a cysteine ​​at position 117 when the IL-18 polypeptide comprises a non-cysteine ​​at position 117.

[0032] In some embodiments according to any of the methods above, introducing cysteine ​​at position 117 or 76 comprises substituting cysteine ​​for the amino acid at position 117 or 76. In some embodiments, the substitution of the amino acid at position 117 comprises an S117C substitution.

[0033] In some embodiments according to any of the above methods, the method further comprises, when the IL-18 polypeptide comprises cysteine ​​at one or both of positions 38 and 68, removing the cysteine ​​at one or both of positions 38 and / or 68. In some embodiments, removing the cysteine ​​at one or both of positions 38 and / or 68 comprises replacing the cysteine ​​at positions 38 and / or 68 with a different amino acid. In some embodiments, the substitution of cysteine ​​at position 38 comprises C38S, C38I, C38L, C38V, or C38M, optionally wherein the substitution of cysteine ​​at position 38 comprises C38S, C38I, and C38V. In some embodiments, the substitution of cysteine ​​at position 68 comprises C68S, C68I, C68V, C68L, or C68D, optionally wherein the substitution of cysteine ​​at position 38 comprises C68S, C68I, and C68L.

[0034] In some embodiments according to any of the above methods, the method further comprises retaining the cysteine ​​at position 127 when the IL-18 polypeptide comprises a cysteine ​​at position 127, or introducing a cysteine ​​at position 127 when the IL-18 polypeptide comprises a non-cysteine ​​at position 127.

[0035] In some embodiments according to any of the above methods, the method further comprises introducing an alanine or an amino acid without a hydrophobic side chain at position 127, optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, further optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine, and threonine. In some embodiments, introducing an alanine or an amino acid without a hydrophobic side chain at position 127 comprises substituting the amino acid at position 127 of the IL-18 polypeptide with an alanine or an amino acid without a hydrophobic side chain.

[0036] In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises: a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0037] In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 311-312, 314-315, and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 311-312, 314-315, and 316-317.

[0038] In another aspect, the present application provides a method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising a) replacing the cysteine ​​at position 38 with another amino acid, b) replacing the cysteine ​​at position 68, and / or c) replacing the cysteine ​​at position 76, wherein the IL-18 variant polypeptide does not contain a free cysteine ​​at each of positions 38, 68, and 76. In some embodiments, the substituted amino acid in a), b), or c) is an amino acid having a hydrophobic side chain. In some embodiments, the amino acid with a hydrophobic side chain in a), b) or c) is selected from alanine, valine, isoleucine, leucine, methionine, aniline, tyrosine and tryptophan, optionally wherein the method comprises a) introducing any one of valine, isoleucine, leucine and methionine at position 38, b) introducing any one of valine, isoleucine and leucine at position 68, and / or c) introducing any one of valine and tyrosine at position 76, further optionally wherein introducing an amino acid with a hydrophobic side chain at position 38, 68 or 76 comprises substituting the amino acid at position 38, 68 or 76 with an amino acid with a hydrophobic side chain. In some embodiments, the method comprises a) introducing isoleucine at position 38, b) introducing isoleucine or leucine at position 68, and / or c) introducing any one of valine and tyrosine at position 76. In some embodiments, the IL-18 variant polypeptide comprises 38S, 38I, 38V, 38L, or 38M, optionally wherein the IL-18 variant polypeptide comprises 38S, 38I, or 38V, further optionally wherein the IL-18 variant polypeptide comprises 38I. In some embodiments, the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L, or 68D, optionally wherein the IL-18 variant polypeptide comprises 68S, 68I, or 68L, further optionally wherein the IL-18 variant polypeptide comprises 68S or 68L. In some embodiments, the IL-18 variant polypeptide comprises 76V or 76Y. In some embodiments, the IL-18 variant polypeptide comprises: a) 38I, b) 68I or 68L, and c) 76V or 76Y. In some embodiments, the IL-18 variant polypeptide does not comprise 117C. In some embodiments, the IL-18 variant polypeptide further comprises a cysteine, an alanine, or an amino acid without a hydrophobic side chain at position 127. In some embodiments, the IL-18 variant polypeptide comprises 38I, 68I, and 76V, optionally wherein the IL-18 variant polypeptide comprises 117S. In some embodiments, the IL-18 variant polypeptide further comprises 127I. In some embodiments, the IL-18 variant polypeptide comprises 38I, 68L, and 76Y, optionally wherein the IL-18 variant polypeptide comprises S117.In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 314-315, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 314-315.

[0039] In some embodiments according to any of the above methods, the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-299, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-299, optionally wherein the IL-18 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-299, NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133 and 143-150 having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereof, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, The amino acid sequence shown in any one of NO:1, 39 and 133, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO:1, 39 and 133.

[0040] In another aspect, the present application provides an IL-18 variant polypeptide produced by any one of the above methods.

[0041] In another aspect, the present application provides an IL-18 variant polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 311-312, 314-315 and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 311-312, 314-315 and 316-317.

[0042] In another aspect, the present application provides an IL-18 variant polypeptide comprising a cysteine ​​at position 117 and a cysteine ​​at position 76, wherein the 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 does not comprise a cysteine ​​at position 38, or the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68. In some embodiments, the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38, and the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68. In some embodiments, the IL-18 variant polypeptide comprises 38S, 38I, 38V, 38L, or 38M, optionally wherein the IL-18 variant polypeptide comprises 38S, 38I, or 38V. In some embodiments, the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L, or 68D, optionally wherein the IL-18 variant polypeptide comprises 68S, 68I, or 68L. In some embodiments, the IL-18 variant polypeptide comprises 127C. In some embodiments, the IL-18 variant polypeptide comprises an alanine at position 127 or an amino acid without a hydrophobic side chain at position 127, optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, further optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine, and threonine. In some embodiments, the IL-18 variant polypeptide comprises: a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127.

[0043] In another aspect, the present application provides a fusion polypeptide comprising a) any one of the above-described IL-18 variant polypeptides, and b) a second portion. In some embodiments, the second portion comprises a half-life extending moiety, optionally wherein the half-life extending moiety is an albumin binding moiety or an Fc domain. In some embodiments, the half-life extending moiety comprises an Fc domain, optionally wherein the Fc domain is a human IgG Fc domain, further optionally wherein the human IgG Fc domain is a human IgG1 domain. In some embodiments, the Fc domain is a modified Fc domain with reduced effector function, optionally wherein the Fc domain comprises a human IgG1 Fc domain comprising an N297A mutation (EU numbering). In some embodiments, the second portion is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the second portion is fused to the C-terminus of the IL-18 variant polypeptide. In some embodiments, the fusion polypeptide further comprises a linker between the IL-18 variant polypeptide and the second portion, optionally wherein the linker is a peptide linker (e.g., a GS linker). In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347.

[0044] In another aspect, the present application provides a dimer comprising two fusion polypeptides, wherein the fusion polypeptide is selected from any one or two of the fusion polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0045] In another aspect, the present application provides a nucleic acid encoding any one of the above-mentioned IL-18 variant polypeptides or any one of the fusion polypeptides.

[0046] In another aspect, the present application provides a nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NOs: 333-335.

[0047] In another aspect, the present application provides a vector comprising any one of the above-mentioned nucleic acids.

[0048] In another aspect, the present application provides a host cell comprising any one of the above nucleic acids or any one of the vectors. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a CHO cell or a HEK293 cell.

[0049] In another aspect, the present application provides a method for producing an IL-18 variant polypeptide or fusion polypeptide, comprising: (a) culturing any one of the above-mentioned host cells under conditions for expressing the IL-18 variant polypeptide or fusion polypeptide; and (b) recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell.

[0050] In another aspect, the present application provides a pharmaceutical composition comprising any one of the aforementioned IL-18 variant polypeptides, any one of the fusion polypeptides, any one of the dimers, any one of the nucleic acids, or any one of the vectors.

[0051] In another aspect, the present application provides a method for treating a disease in an individual, comprising administering to the individual an effective amount of any one of the above-mentioned pharmaceutical compositions.

[0052] In another aspect, the present application provides 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-mentioned pharmaceutical compositions.

[0053] In another aspect, the present application provides a method of stimulating immune cells that have experienced an antigen in an individual in need thereof, comprising administering to the individual an effective amount of any one of the above-described pharmaceutical compositions.

[0054] It should be understood that one, some or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description.

[0055] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1A Shown is a screening process in which methods based on magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) are used to identify the IL-18 variant polypeptides described herein.

[0057] Figure 1B The ability of output library pools from rounds 1, 2, 3, 4, and 5 to bind 1 μM IL-18BP or 50 nM IL-18Rα is shown.

[0058] Figure 1C The ability of representative individual IL-18 variant clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43) to bind to IL-18Rα is shown. SEQ ID NO: 1 is wild-type IL-18.

[0059] Figure 2 Figure A provides SDS-PAGE analysis and SEC-HPLC analysis of purified wild-type human IL-18 (SEQ ID NO: 1).

[0060] Figure 2 B provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M12 (SEQ ID NO: 39).

[0061] Figure 2 Panel C provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant MM5 (SEQ ID NO: 133).

[0062] Figure 2 Figure D provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant WM6 (SEQ ID NO: 145).

[0063] Figure 2 Figure E provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M17 (SEQ ID NO: 43).

[0064] Figure 2 Figure F provides SDS-PAGE analysis and SEC-HPLC analysis of purified IL-18 variant M24 (SEQ ID NO: 105).

[0065] Figure 3 Activation of hIL-18 receptor-mediated signaling by exemplary IL-18 variant polypeptides of the present application is shown.

[0066] Figure 4 Exemplary IL-18 variant polypeptides of the present application are shown to induce hIFNγ release.

[0067] Figure 5AActivation of hIL-18 receptor-mediated signaling by wild-type IL-18, fusion polypeptide IL-18-SSS-Fc_N297A, and fusion polypeptide IL-18-SSS-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0068] Figure 5B Activation of hIL-18 receptor-mediated signaling by IL-18 polypeptide variant M12, fusion polypeptide M12-SSS-Fc_N297A, and fusion polypeptide M12-SSS-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0069] Figure 5C Activation of hIL-18 receptor-mediated signaling by the IL-18 polypeptide variant MM5, the fusion polypeptide MM5-SSS-Fc_N297A, and the fusion polypeptide MM5-SSS-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0070] Figure 6A Activation of hIL-18 receptor-mediated signaling by the fusion polypeptide WT IL-18, WT IL-18 in the presence of IL-18 binding protein ("BP"), the fusion polypeptide M12-DB6-Fc_N297A, and M12-DB6-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0071] Figure 6B Activation of hIL-18 receptor-mediated signaling by fusion polypeptide WT IL-18, fusion polypeptide M12-DB7-Fc_N297A, and M12-DB7-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0072] Figure 6C Activation of hIL-18 receptor-mediated signaling by fusion polypeptide WT IL-18, fusion polypeptide M12-DB9-Fc_N297A, and M12-DB9-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0073] Figure 6D The results show that the fusion polypeptide WT IL-18 and the fusion polypeptide M12-DB10-Fc_N297A activate the hIL-18 receptor-mediated signaling.

[0074] Figure 7AActivation of hIL-18 receptor-mediated signaling by wild-type IL-18, wild-type IL-18 in the presence of IL-18 binding protein ("BP"), fusion polypeptide WT IL-18-DB6-Fc_N297A, and WT IL-18-DB6-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0075] Figure 7B Activation of hIL-18 receptor-mediated signaling by wild-type IL-18, wild-type IL-18 in the presence of IL-18 binding protein ("BP"), fusion polypeptide MM5-DB6-Fc_N297A, and MM5-DB6-Fc_N297A in the presence of IL-18 binding protein ("BP") is shown.

[0076] Figure 7C Activation of hIL-18 receptor-mediated signaling by wild-type IL-18 and the fusion polypeptide Fc_N297A-MM5-DB6 is shown.

[0077] Figure 7D Activation of hIL-18 receptor-mediated signaling by wild-type IL-18 and the fusion polypeptide Fc_N297A-M12-DB6 is shown. DETAILED DESCRIPTION

[0078] Overview

[0079] Components of the interleukin-18 (IL-18) pathway have been found to be upregulated in tumor-infiltrating lymphocytes (TILs) (see, e.g., Zhou et al. (2020) Nature 583:609–614), suggesting that IL-18 therapy could enhance anti-tumor immunity. However, IL-18 has not shown significant efficacy in clinical trials. IL-18BP is a high-affinity inhibitor of IL-18 (K D <1 nM, see, e.g., Dinarello et al. (2013) Front Immunol 4:289, doi:10.3389 / fimmu.2013.00289), which is often upregulated in a variety of human and murine tumors and is believed to limit the anti-tumor activity of IL-18 in mice and clinical trials. In addition, due to low yield and / or low purity, the efficient production of IL-18 is often challenging. Applicants have identified IL-18 variant polypeptides that can induce IL-18 receptor-mediated signaling even in the presence of IL-18BP. It has been found that by replacing one or a group of amino acids (e.g., G3, e.g., E6), IL-18 variant polypeptides exhibit resistance to IL-18BP while still being able to bind to and activate the IL-18 receptor. See, e.g., Example 6, Figure 3-Figure 4 , Table 3 and Table 4. Applicants have further discovered that by engineering one or more hotspots (e.g., by replacing C38, C68, C76, S117 and / or C127), higher yields, more purified and / or more potent IL-18 variant polypeptides can be obtained. See, for example, Example 7, Figures 5A-7D , Tables 5-9. Unexpectedly, the applicants discovered that by engineering the disulfide bond between C76 and C117 while removing the cysteines at C38 and C68, the yield and purity of the engineered variant IL-18 were significantly increased. See, for example, the DB6 and DB7 versions of the IL-18 polypeptide in Table 10.

[0080] definition

[0081] As used herein, the terms "specific binding," "specific recognition," and "specifically for" refer to a measurable and reproducible interaction, such as the binding between a cytokine and its receptor, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a cytokine that specifically recognizes a receptor is a cytokine that binds to that receptor with greater affinity, avidity, ease, and / or longer duration than it binds 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 its receptor has a specific binding affinity of ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The dissociation constant (K D In some embodiments, the specific binding may include but does not require exclusive binding. The binding specificity of the cytokine can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE TM -Testing and peptide scanning.

[0082] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine as described herein is one that has been identified and separated from at least one contaminant nucleic acid molecule that is normally associated with the environment in which it was produced. Preferably, the isolated nucleic acid is free from all components associated with the production environment. In some embodiments, the isolated nucleic acid molecule encoding a polypeptide or cytokine as described herein is in a form that is different from the form or environment in which it is found in nature.

[0083] 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 that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0084] As used herein, the terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell refers to a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0085] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid composition of the progeny may not be completely identical to that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0086] As used herein, " treatment " or " processing " is the method for obtaining the result (including clinical outcome) of benefit or expectation.For the purpose of the application, the clinical outcome of benefit or expectation includes but is not limited to following one or more: alleviate one or more symptoms caused by disease, alleviate the degree of disease, stabilize disease (for example, prevent or delay the deterioration of disease), prevent or delay the spread of disease (for example, metastasis), prevent or delay the recurrence of disease, delay or slow down the progress of disease, improve disease state, provide the alleviation (partial or complete) of disease, reduce the dosage of one or more other drugs needed for treating disease, delay the progress of disease, improve or improve quality of life, increase weight gain and / or prolong survival. " treatment " also encompasses the pathological consequences (for example, tumor volume) that reduce cancer. The method of the application contemplates any one or more of these aspects for the treatment of.

[0087] In the context of cancer, the term "treating" includes any or all of the following: 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.

[0088] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to a decrease, reduction, or prevention of an activity, function, and / or amount as compared to a reference value. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0089] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0090] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0091] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0092] As used herein, the expression "not for" a certain value or parameter generally means and describes "except" a certain value or parameter. For example, the method is not used to treat type X cancer, which means that the method is used to treat other types of cancer other than type X.

[0093] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0094] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0095] Interleukin-18 (IL-18) variant polypeptide

[0096] Interleukin-18 (IL-18, also known as interferon-gamma-inducing factor) is a human protein encoded by the IL-18 gene. It is a proinflammatory cytokine produced by both hematopoietic and non-hematopoietic cells. IL-18 regulates both innate and adaptive immunity, and its dysregulation can lead to autoimmune or inflammatory diseases.

[0097] In one aspect, provided herein are interleukin 18 ("IL-18") variant polypeptides comprising mutations at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, wherein the amino acid positions are relative to wild-type (WT) human IL-18 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.

[0098] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:1)

[0099] In some embodiments, the IL-18 variant polypeptide comprises a mutation at 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 a mutation at 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 at four 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 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 a mutation at 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 a mutation at 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, 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, e.g., in any combination. In some embodiments, 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 a mutation 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 a mutation 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, 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, e.g., in any combination. In some embodiments, 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 a mutation at eleven 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 eleven 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 a mutation at twelve 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, twelve 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 thirteen 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, thirteen 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 a mutation 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 a mutation at a position 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) a mutation at a position 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.

[0100] In some embodiments, 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 a mutation at two or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, 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 a mutation at three or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, 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 a mutation at G3, E6, D54, and N91. 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 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 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.

[0101] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) at least one mutation at a residue selected from Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1.

[0102] In some embodiments, the IL-18 variant polypeptide comprises at least one (or two, or three) mutation at a residue selected from G3, E6, and M60, wherein the 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 at least one of the following: a) G3P, G3S, G3E, G3N, G3D, or G3A; b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y; or c) M60K. In some embodiments, the IL-18 variant polypeptide further comprises at least one or more (e.g., two, three, four, or five) mutations at a residue selected from V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155. In some embodiments, the IL-18 variant polypeptide further comprises at least one of the following: a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, or D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, or Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, P57V, or P57V; h) K96D or K96E; i) I149M; or j) K140R.

[0103] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56D, P57R, N91V, and R104V, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0104] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S, E6K, D54W, Q56P, P57D, N91G, and R104T, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0105] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0106] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54W, Q56G, P57V, N91V, and R104F, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0107] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0108] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56T, N91V, and R104T, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0109] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56T, P57A, and N91A, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0110] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56P, P57A, N91A, and R104L, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0111] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56R, P57A, N91S, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0112] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Q, Q56L, P57W, and N91S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0113] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57N, N91G, and R104T, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0114] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54G, Q56G, P57A, and N91T, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0115] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54Q, Q56I, and P57W, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0116] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3N, E6K, D54P, Q56S, P57S, N91R, and R104A, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0117] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56Y, P57T, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0118] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57N, N91G, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57A, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0120] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57R, N91G, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0121] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56E, P57Q, and N91A, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0122] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57S, N91G, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0123] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56S, P57T, N91G, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0125] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Y, Q56T, and P57R, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0126] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Y, Q56T, and P57S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0127] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57T, and N91R, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0128] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, optionally wherein the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0129] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 a set of substitution combinations comprising G3P, E6M, D54F, Q56D, P57R, and N91P, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6L, D54H, Q56T, P57V, and N91S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6G, D54S, Q56S, and P57R, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3E, E6H, D54R, Q56T, and P57H, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56R, P57N, N91V, and R104E, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 a set of substitution combinations comprising G3P, E6R, D54G, Q56G, P57A, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6S, D54A, Q56D, P57Q, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 a set of substitution combinations comprising G3P, E6G, D54Q, Q56V, and P57W, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 set of substitution combinations comprising G3P, E6S, D54W, Q56G, P57A, N91V, and R104I, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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 a set of substitution combinations comprising G3P, E6R, D54W, Q56P, P57G, N91V, and R104L, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0142] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3D, E6K, D54P, Q56S, P57W, and N91W, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0143] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) that is G3P, wherein the amino acid position is relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0144] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54G, Q56G, and P57A, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0145] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56G, P57S, and N91V, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0146] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, V11I, D54G, Q56G, P57A, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0147] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56Y, and P57S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0148] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6R, D54W, Q56S, and P57Q, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0149] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54L, Q56T, P57Q, and N91V, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0150] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3A, E6Y, D54R, Q56S, P57L, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0151] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57I, and N91G, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0152] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6G, D54L, Q56T, P57E, N91G, and R104S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0153] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3A, E6Y, D54R, Q56S, P57L, and N91A, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises a C38S, C68S, and C76S substitution, 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.

[0154] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising M60K and K96D, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0155] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, and K96E, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0156] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) comprising M60K, wherein the amino acid position is relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0157] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P and E6R, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0158] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P and E6K, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0159] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3D, E6K, and N91S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0160] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S and I149M, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0161] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, and N91S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0162] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) comprising E6R, wherein the amino acid position is relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to the IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to the IL-18 binding protein than the corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0163] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6R and N91S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0164] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) comprising V11I, wherein the amino acid position is relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0165] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S and K140R, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions relative to wild-type human IL-18 as shown in SEQ ID NO: 1, optionally wherein the IL-18 variant polypeptide has a) comparable or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) comparable or lower binding affinity to an IL-18 binding protein than a corresponding IL-18 variant polypeptide without 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 C38I, C38V, C38L, and C38M, further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from C68I, C68V, C68S, and C68D, further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein C76 is substituted with C76Y or C76V, and optionally wherein C127 is substituted with C127A, C127I, C127Y, C127F, and C127L, further optionally wherein C127 is substituted with C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S and C76S substitutions, optionally wherein the variant polypeptide further comprises 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.

[0166] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-153.

[0167] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-150.

[0168] In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence set forth in any one of 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150.

[0169] In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, at least four, at least five, or six mutations at residues selected from C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a substitution at C38 (i.e., wherein the wild-type cysteine ​​at position 38 is substituted with any amino acid), a substitution at C68 (i.e., wherein the wild-type cysteine ​​at position 68 is substituted with any amino acid), and an S117C substitution. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C38I, C38V, C38L, C38M, or C38S mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C68S, C68I, C68D, C68V, or C68L mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C76S, C76V, or C76Y mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) an S117C mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C127A, C127Y, C127F, C127L, or C127I mutation. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, or four mutations selected from C38S, C68S, C76S, and C127S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68S, and S117C mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38V, C68I, S117C, and C127A mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38S, C68I, S117C, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68I, C76V, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68L, and C76Y mutations.

[0170] In some embodiments, the IL-18 variant polypeptide further comprises a mutation at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at two or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises a mutation 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 G3, E6, D54, Q56, P57, N91, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise a substitution at Q56 and P57, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0171] 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 exhibits significantly reduced binding to IL-18 binding protein ("IL-18BP"), e.g., human IL-18 or "hIL-18BP". In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18BP relative to WT human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide has an affinity for IL-18Rα (e.g., hIL-18Rα) that is comparable to the affinity of WT 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 (eg, hIL-18Rα) is increased compared to the affinity of WT human IL-18 shown in SEQ ID NO:1 for IL-18Rα (eg, hIL-18Rα).

[0172] In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D Less than approximately 5x 10 -5 M, less than about 5x 10-6 M, less than about 5x 10 -7 M, less than about 5x 10 -8 M, less than about 5x 10 -9 M, less than about 5x 10 -10 M or less than about 5x10 -11 M, including any range between these values. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -5 About 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 About 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 About 5 x 10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5x10 -7 About 5 x 10 -9 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 About 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -9 to about 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 to about 5x10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 About 5 x 10 - 10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -9 About 5 x 10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7About 5 x 10 -9 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 About 5 x 10 -9 M. In some embodiments, the IL-18 variant binds to IL-18Rα (e.g., hIL-18Rα), wherein K D About 5 x 10 -5 Up to 5x 10 -11 M, for example, about 5x 10 -5 , 6x 10 -5 , 7x 10 -5 , 8x 10 -5 , 9x 10 -5 , 1x 10 -6 , 2x 10 -6 , 3x 10 -6 , 4x 10 -6 , 5x 10 -6 , 6x10 -6 , 7x 10 -6 , 8x 10 -6 , 9x 10 -6 , 1x 10 -7 , 2x 10 -7 , 3x 10 -7 , 4x 10 -7 , 5x 10 -7 , 6x 10 -7 , 7x 10 -7 , 8x 10 -7 , 9x 10 -7 , 1x 10 -8 , 2x 10 -8 , 3x 10 -8 , 4x 10 -8 , 5x 10 -8 , 6x 10 -8 , 7x 10 -8 , 8x 10 -8 , 9x 10 -8 , 1x 10 -9 , 2x 10 -9 , 3x 10 -9 , 4x 10 -9 , 5x 10 -9 , 6x 10 -9 , 7x 10 -9 , 8x 10 -9 , 9x 10 -9 , 1x10 -10, 2x 10 -10 , 3x 10 -10 , 4x 10 -10 , 5x 10 -10 , 6x 10 -10 , 7x 10 -10 , 8x10 -10 , 9x 10 -10 , 1x 10 -11 , 2x 10 -11 , 3x 10 -11 , 4x 10 -11 or 5x 10 -11 M, including any range between these values. In 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 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is comparable to (e.g., approximately the same as) the affinity of wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0173] In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. -9 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -8 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -7 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -6 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5x10 -5 M's K D In some embodiments, the IL-18 variant polypeptide does not exhibit binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP). In some embodiments, the IL-18 variant polypeptide that does not exhibit binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP) exhibits a binding affinity greater than 10-3 M's K D Binds to IL-18BP (e.g., hIL-18BP).

[0174] The affinity of IL-18 described herein for IL-18Rα and / or IL-18BP can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL) assay, immunoradiometric (IRMA) assay, enzyme immunoassay (EIA), surface plasmon resonance (SPR), peptide scanning, and kinetic competitive screening based on fluorescence activated cell sorting (FACS).

[0175] In some embodiments, the IL-18 variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from G3P, G3D, G3E, G3N, and G3S. As used herein, "G3X" means that G, the wild-type amino acid at position 3 in SEQ ID NO: 1, has been substituted with amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 in SEQ ID NO: 1 has been substituted with amino acid P.

[0176] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue E6, wherein the mutation is selected from E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from E6R and E6K.

[0177] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue D54, wherein 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.

[0178] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue N91, wherein the mutation is selected from N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from N91V, N91A, N91G, and N91S.

[0179] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, wherein 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.

[0180] In some embodiments, wherein the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue Q56 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.

[0181] In some embodiments, wherein the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue P57 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.

[0182] 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, wherein the IL-18 variant polypeptide comprises a G3P mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an E6R or E6K mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises a D54W, D54H, D54S, or D54Q mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an N91V, N91A, N91G, or N91S mutation. In some embodiments, the IL-18 variant polypeptide does not contain mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant further contains mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide contains 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 G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide further contains mutations at Q56 and / or P57. In some embodiments, the IL-18 variant polypeptide does not contain mutations at positions other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at a position other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, at least four, at least five, or at least six mutations are selected from G3, E6, D54, N91, Q56, and P57.

[0183] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence that is 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% identical to the sequence of any one of SEQ ID NOs: 2-299, 307-318. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence 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 provided in the Sequence Summary Table following the Examples.

[0184] Fusion peptide

[0185] In some embodiments, the activatable IL-18 polypeptides of the present disclosure comprise a fusion polypeptide comprising a wild-type IL-18 or 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 a leucine zipper (LZ) element, or comprises a leucine zipper element. Leucine zippers are generally identified as approximately 35 amino acid segments containing 4 to 5 leucine residues separated by six amino acids (Maniatis and Abel (1989) Nature 341:24-25). Exemplary leucine zippers are present in a variety of eukaryotic DNA binding proteins, such as GCN4, C / EBP, c-Fos, c-Jun, c-Myc, and c-Max. In some embodiments, the dimerization domain is a helix-loop-helix domain, or comprises a helix-loop-helix domain (Murre, C. et al. (1989) Cell 58:537-544). The dimerization domain can also be selected from other proteins, such as retinoic acid receptors, thyroid hormone receptors, or other nuclear hormone receptors (Kurokawa et al. (1993) Genes Dev. 7:1423-1435), or 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 further described by Eisenman in U.S. Patent No. 5,624,818. In some embodiments, the dimerization domain is an antibody Fc domain.

[0186] In some embodiments, the fusion polypeptide comprises wild-type IL-18 (e.g., a wild-type IL-18 having an amino acid sequence 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 an antibody Fc domain. In some embodiments, the C-terminus of an 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, such as a human IgG1, IgG2, or IgG4 Fc domain or a variant of any of the foregoing. In some embodiments, the fusion polypeptide comprises (from N-terminus to C-terminus) an IL-18 variant polypeptide described herein and a human IgG1 Fc variant comprising an N297A mutation, wherein amino acid numbering is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the fusion polypeptide comprises (from N-terminus to C-terminus) wild-type IL-18 and a human IgG1 Fc variant comprising an N297A mutation. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

[0187] Nucleic acids, vectors, host cells, and methods for producing IL-18 variant polypeptides

[0188] Also contemplated are nucleic acid molecules encoding the IL-18 variant polypeptides described herein or the fusion polypeptides described herein. 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 that is 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% identical to a sequence selected from SEQ ID NOs: 301-306 and 333-335. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from SEQ ID NOs: 301-306 and 333-355. The nucleic acid sequences of SEQ ID NOs: 301-306 and 333-335 are provided in the sequence summary table following the Examples.

[0189] Also provided are vectors into which the nucleic acids described herein are inserted.

[0190] Briefly, expression of the IL-18 variant polypeptides or fusion polypeptides described herein can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a constitutive, regulatable, tissue-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0191] Nucleic acids can be cloned into various 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.

[0192] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art, such as described in Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally speaking, suitable vectors contain a functional origin of replication, promoter sequence, convenient restriction endonuclease site and one or more selectable markers in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0193] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncorretroviruses (e.g., murine leukemia viruses) because they can transduce non-proliferating cells (e.g., hepatocytes). They also have the additional advantage of low immunogenicity.

[0194] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, they are located in the region 30-110 base pairs (bp) upstream of the start site, although recently many promoters have been found to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or moved relative to each other, promoter function can still be retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp, and then activity will begin to decline.

[0195] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive the high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be 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 expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0196] In some embodiments, expression of the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide is inducible. In some embodiments, the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide is operably linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide described herein has been engineered to encode an epitope tag, for example to facilitate purification or detection of the polypeptide. Exemplary epitope tags include, but are not limited to, 6x His (also known as a His tag or a hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0197] The IL-18 variant polypeptides or fusion polypeptides of the present disclosure can be produced by any means known in the art. Exemplary techniques for polypeptide production are described below; however, these exemplary techniques are for illustrative purposes only and are not intended to be limiting.

[0198] Recombinant methods can be used to produce the IL-18 variant polypeptides or fusion polypeptides described herein. For recombinant production of IL-18 variant polypeptides or fusion polypeptides, nucleic acids encoding IL-18 variant polypeptides or fusion polypeptides are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or expression. DNA encoding IL-18 variant polypeptides or fusion polypeptides can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding IL-18 variant polypeptides or fusion polypeptides). A wide variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0199] Expression vectors and cloning vectors all contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, for example to allow the vector to replicate independently of the host chromosomal DNA. Such sequences may include an origin of replication or an autonomously replicating sequence. Such sequences are well known for various bacteria, yeasts, and viruses. Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin can be used because it contains an early promoter).

[0200] Expression and cloning vectors may contain a selection gene or selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement nutritional deficiencies, or (c) provide key nutrients not available in complex culture media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Another example of a suitable selectable marker for mammalian cells is one that can identify cells that can take up nucleic acids encoding IL-18 variant polypeptides or fusion polypeptides, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like. For example, a Chinese hamster ovary (CHO) cell line transformed with a DHFR gene that lacks endogenous DHFR activity can be identified by culturing transformants in culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR.

[0201] Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) 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), can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418.

[0202] Expression and cloning vectors typically contain a promoter recognized by the host organism, which is operably linked to a nucleic acid encoding an IL-18 variant polypeptide or fusion polypeptide. Promoters suitable for prokaryotic hosts include the pho A promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters, such as the tac promoter. However, other known bacterial promoters are also suitable. Promoter sequences are known for eukaryotic organisms. Yeast promoters are well known in the art and may include inducible promoters / enhancers that are regulated by growth conditions. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Examples include, but are not limited to, promoters for 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 the IL-18 variant polypeptide or fusion polypeptide from a vector in a mammalian host cell can be controlled by, for example, promoters obtained from viral genomes. The early and late promoters of the SV40 virus can be conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus can be conveniently obtained as a HindIII restriction fragment. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0203] Transcription of a DNA encoding an IL-18 variant polypeptide or fusion polypeptide described herein by higher eukaryotes is typically enhanced by inserting an enhancer sequence into the vector. Numerous enhancer sequences are known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic cells and viruses are commonly used.

[0204] Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insect, plant, animal, human or other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA.

[0205] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for IL-18 polypeptide variant encoding vectors or fusion polypeptide encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. Certain fungal and yeast strains can be selected in which the glycosylation pathways have been "humanized," thereby generating IL-18 variant polypeptides or fusion polypeptides having partially or fully human glycosylation patterns. See, for example, Li et al., Nat. Biotech. 24:210-215 (2006).

[0206] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts.

[0207] 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. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells, from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori.

[0208] Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0209] Host cells of the present disclosure can be cultured in a variety of culture media. Commercially available culture 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 host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds typically present at final micromolar concentrations), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be added at appropriate concentrations. Culture conditions (e.g., temperature, pH, etc.) are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0210] When recombinant techniques are used, the IL-18 variant polypeptide or fusion polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the polypeptide is produced intracellularly, the first step is to remove the particulate debris (host cell or lysed debris), for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of Escherichia coli.

[0211] The polypeptide composition prepared by the cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. In some embodiments, the IL-18 variant polypeptide or fusion polypeptide described herein comprises 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, 6x His (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0212] Methods for treating disease, activating hIL-18 receptor-mediated signaling, and stimulating T cells or NK cells that have experienced antigen

[0213] Also provided herein are methods of treating a disease or condition in an individual. The methods comprise administering to an individual suffering from the disease or condition 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, the disease or condition is a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0214] In some embodiments, a method of activating hIL-18 receptor-mediated signaling in an individual is provided, 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 is a method of stimulating T cells or NK cells that have experienced an antigen in an individual, 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, an experimental animal, etc.

[0215] Compositions, kits and articles of manufacture

[0216] Also provided herein are compositions (eg, formulations) comprising an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell described herein.

[0217] Suitable compositions are obtained by mixing IL-18 variant polypeptides, fusion polypeptides, nucleic acids, vectors or host cells having the desired purity, and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0218] Also provided are kits comprising an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell comprising a nucleic acid or vector as described herein. The kits can be used in any of the therapeutic methods described herein.

[0219] The kit of the present application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide other components, such as buffers and descriptive information.

[0220] Therefore, 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 (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Typically, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle).

[0221] Those skilled in the art will recognize that, within the scope and spirit of the present invention, multiple embodiments can be arranged. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but certainly should not be construed as limiting its scope in any way.

[0222] Exemplary embodiments

[0223] 1. An interleukin 18 (IL-18) variant polypeptide comprising at least one mutation at 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 shown in SEQ ID NO: 1.

[0224] 2. The IL-18 variant polypeptide of embodiment 1, wherein at least one mutation comprises a substitution at C38 and a substitution at C68.

[0225] 3. The IL-18 variant polypeptide of embodiment 2, wherein at least one mutation comprises an S117C substitution.

[0226] 4. The IL-18 variant polypeptide of embodiment 2 or embodiment 3, wherein the substitution at C38 comprises C38I, C38S, C38V, C38L or C38M, optionally wherein the substitution at C38 comprises C38I, C38S or C38V.

[0227] 5. The IL-18 variant polypeptide of any one of embodiments 2-4, wherein the substitution at C68 comprises C68I, C68V, C68S or C68D, optionally wherein the substitution at C68 comprises C68S, C68I or C68L.

[0228] 6. The IL-18 variant polypeptide according to any one of embodiments 2-5, wherein the variant polypeptide does not comprise a mutation at C76.

[0229] 7. The IL-18 variant polypeptide of any one of embodiments 2-6, wherein the variant polypeptide comprises a) a substitution at C38, b) a substitution at C68, c) C76, and d) an S117C substitution.

[0230] 8. The IL-18 variant polypeptide of embodiment 7, wherein the variant polypeptide comprises a) I38, S68, C76 and C117, or b) V38, I68, C76 and C117.

[0231] 9. The IL-18 variant polypeptide according to any one of embodiments 1 to 8, wherein the variant polypeptide does not comprise a mutation at C127.

[0232] 10. The IL-18 variant polypeptide of any one of embodiments 1-8, wherein the variant polypeptide comprises a mutation at C127, optionally wherein the variant polypeptide comprises C127A, C127Y, C127F or C127L, further optionally wherein the variant polypeptide comprises C127A.

[0233] 11. The IL-18 variant polypeptide of any one of embodiments 1-2, 4-5, and 9-10, wherein at least one mutation comprises a substitution at C76, optionally wherein the substitution at C76 comprises C76V or C76Y.

[0234] 12. The IL-18 variant polypeptide of embodiments 1-5 and 9-11, wherein at least one mutation comprises a substitution at C38, a substitution at C68, and a substitution at C76, optionally wherein the substitution at C38 comprises C38I, C38V, C38L, C38S, or C38M, optionally wherein the substitution at C68 comprises C68I, C68V, C68S, C68L, or C68D, and optionally wherein the substitution at C76 comprises C76Y, C76S, or C76V.

[0235] 13. The IL-18 variant polypeptide of embodiment 11, wherein the variant polypeptide comprises C38I, C68I and C76V.

[0236] 14. The IL-18 variant polypeptide of embodiment 11, wherein the variant polypeptide comprises C38I, C68L and C76Y.

[0237] 15. The IL-18 variant polypeptide of any one of embodiments 11-14, wherein the variant polypeptide further comprises a mutation at C127, optionally wherein the mutation at C127 is C127A, C127I, C127Y, S127S, C127F or C127L.

[0238] 16. The IL-18 variant polypeptide according to any one of embodiments 11-14, wherein the variant polypeptide does not comprise C127S.

[0239] 17. The IL-18 variant polypeptide of any one of embodiments 1 to 16, wherein the variant polypeptide comprises: a) S38, S68, S76, S117 and C127, b) S38, S68, S76, S117 and S127, c) I38, S68, C76, C117 and C127, d) V38, I68, C76, C117 and A127, e) I38, I68, V76, S117 and I127, or f) I38, L68, Y76, S117 and C127.

[0240] 18. The IL-18 variant polypeptide of any one of embodiments 1-17, wherein the variant polypeptide comprises I38, S68, C76, C117, and C127.

[0241] 19. The IL-18 variant polypeptide of any one of embodiments 1-18, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18.

[0242] 20. The IL-18 variant polypeptide according to any one of embodiments 1-19, wherein the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18.

[0243] 21. The IL-18 variant polypeptide of any one of embodiments 1-20, wherein the IL-18 variant polypeptide is expressed in an amount of less than about 5×10 -5 M's K D Binds to IL-18Rα.

[0244] 22. The IL-18 variant polypeptide according to any one of embodiments 1-21, wherein the IL-18 variant polypeptide is present at a concentration of about 5×10 -5 to about 5×10 -11 M's K D Binds to IL-18Rα.

[0245] 23. The IL-18 variant polypeptide according to any one of embodiments 1-22, wherein the IL-18 variant polypeptide is expressed in an amount greater than 5×10 -9 M's K D Binds to IL-18BP.

[0246] 24. The IL-18 variant polypeptide according to any one of embodiments 1-23, wherein said IL-18 variant polypeptide does not show binding to IL-18BP.

[0247] 25. The IL-18 variant polypeptide of any one of embodiments 1-24, 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, optionally wherein the mutation is G3P.

[0248] 26. The IL-18 variant polypeptide of any one of embodiments 1-25, 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, optionally wherein the mutation is selected from the group consisting of: E6R, E6K, E6G, E6T, E6A and E6S, further optionally wherein the mutation is selected from the group consisting of: E6R and E6K.

[0249] 27. The IL-18 variant polypeptide of any one of embodiments 1-26, 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, optionally wherein the mutation is selected from the group consisting of: D54W, D54H, D54S, D54Q, D54L and D54Y.

[0250] 28. The IL-18 variant polypeptide of any one of embodiments 1-27, 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, optionally wherein the mutation is selected from the group consisting of: N91V, N91A, N91G and N91S.

[0251] 29. The IL-18 variant polypeptide of any one of embodiments 1-28, 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, optionally wherein the mutation is selected from the group consisting of: R104S, R104Y and R104T.

[0252] 30. The IL-18 variant polypeptide of any one of embodiments 1-28, wherein the variant polypeptide does not comprise a mutation at residue R104.

[0253] 31. The IL-18 variant polypeptide of any one of embodiments 1-30, 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, optionally wherein the mutation is selected from the group consisting of: Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I and Q56Y.

[0254] 32. The IL-18 variant polypeptide of any one of embodiments 1-30, wherein the variant polypeptide does not comprise a mutation at residue Q56.

[0255] 33. The IL-18 variant polypeptide of any one of embodiments 1-32, 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, optionally wherein the mutation is selected from the group consisting of: P57A, P57G, P57R, P57W, P57S, P57T and P57V.

[0256] 34. The IL-18 variant polypeptide of any one of embodiments 1-32, wherein the variant polypeptide does not comprise a mutation at residue P57.

[0257] 35. The IL-18 variant polypeptide of any one of embodiments 1-34, wherein the variant polypeptide comprises mutations at G3, E6, D54, and N91, optionally wherein the variant polypeptide comprises mutations in: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S.

[0258] 36. The IL-18 variant polypeptide of any one of embodiments 1-31, 33 and 35, wherein the variant polypeptide further comprises mutations at Q56 and P57.

[0259] 37. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising S38, S68, S76, S117, and C127.

[0260] 38. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising S38, S68, S76, S117, and S127.

[0261] 39. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising I38, S68, C76, C117, and C127.

[0262] 40. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising V38, I68, C76, C117, and A127.

[0263] 41. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising I38, I68, V76, S117, and I127.

[0264] 42. The IL-18 variant polypeptide of any one of embodiments 17-36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1-299, except for comprising I38, L68, Y76, S117, and C127.

[0265] 43. The IL-18 variant polypeptide of any one of embodiments 1-42, wherein the variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 307-312 and 314-317.

[0266] 44. A fusion polypeptide comprising the IL-18 variant polypeptide of any one of embodiments 1-43 and a half-life extending moiety, optionally wherein the half-life extending moiety comprises an Fc domain, further optionally wherein the Fc domain comprises a human IgG Fc domain, further optionally wherein the Fc domain is a modified Fc domain with reduced effector function, further optionally wherein the Fc domain comprises a human IgG1 Fc domain comprising an N297A mutation (EU numbering).

[0267] 45. The fusion polypeptide of embodiment 44, wherein the half-life extending moiety is fused to the N-terminus of the IL-18 variant polypeptide.

[0268] 46. ​​The fusion polypeptide of embodiment 44, wherein the half-life extending moiety is fused to the C-terminus of the IL-18 variant polypeptide.

[0269] 47. A dimer comprising two fusion polypeptides according to any one of embodiments 44-46, optionally wherein the dimer is a) a homodimer or b) a heterodimer.

[0270] 48. A nucleic acid encoding the IL-18 variant polypeptide of any one of embodiments 1-43 or the fusion polypeptide of any one of embodiments 44-46.

[0271] 49. A vector comprising the nucleic acid of embodiment 48.

[0272] 50. A host cell comprising the nucleic acid of embodiment 48 or the vector of embodiment 49.

[0273] 51. A method for producing an IL-18 variant polypeptide or fusion polypeptide, comprising: (a) culturing the host cell of embodiment 50 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.

[0274] 52. A pharmaceutical composition comprising the IL-18 variant polypeptide of any one of embodiments 1-43, the fusion polypeptide of any one of embodiments 44-46, the nucleic acid of embodiment 48, or the vector of embodiment 49.

[0275] 53. A method of treating a disease in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 52.

[0276] 54. A method of activating hIL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 52.

[0277] 55. A method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 52.

[0278] 56. A method for increasing the yield and / or purity of an IL-18 variant polypeptide produced by a host cell, comprising removing one or more cysteines at positions 38, 68, 76 and / or 127 of the IL-18 polypeptide, wherein the amino acid position of the IL-18 polypeptide is relative to wild-type human IL-18 as shown in SEQ ID NO: 1.

[0279] 57. The method of embodiment 56, comprising removing at least two cysteines at positions 38 and 68, optionally wherein the removal of the cysteine ​​at position 38 comprises substitution of C38 with a cysteine ​​selected from the group consisting of C38I, C38S, C38V, C38L, and C38M, optionally wherein the removal of the cysteine ​​at position 68 comprises substitution of C68 with a cysteine ​​selected from the group consisting of C68I, C68S, C68V, C68S, and C68D.

[0280] 58. The method of embodiment 56 or embodiment 57, comprising introducing the S117C mutation and retaining the cysteine ​​at position 76.

[0281] 59. The method of any one of embodiments 56-58, comprising removing the cysteine ​​at position 127, optionally wherein the removal of the cysteine ​​at position 127 comprises substitution of C127 selected from the group consisting of C127S, C127A, C127I, C127Y, C127F and C127L.

[0282] 60. The method of any one of embodiments 56-57 and 59, comprising removing the cysteine ​​at position 76, optionally wherein removing the cysteine ​​at position 76 comprises substitution of C76 selected from the group consisting of C76S, C76V and C76Y.

[0283] 61. The method of any one of embodiments 56-60, comprising: a) introducing C38S, C68S, and C76S into the IL-18 polypeptide; b) introducing C38S, C68S, C76S, and C127S into the IL-18 polypeptide; c) introducing C38I, C68S, S117C into the IL-18 polypeptide; d) introducing C38V, C68I, S117C, C127A into the IL-18 polypeptide; e) introducing C38I, C68I, C76V, and C127I into the IL-18 polypeptide; or f) introducing C38I, C68L, and C76Y into the IL-18 polypeptide.

[0284] 62. The method of any one of embodiments 56-61, wherein the host cell is a mammalian cell, optionally wherein the host cell is a CHO cell or a HEK293 cell.

[0285] 63. The method of any one of embodiments 56-62, wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-299.

[0286] Example

[0287] The following examples are proposed to provide complete disclosure and description about how to make and use the present invention to those of ordinary skill in the art, which are not intended to limit the scope of its invention that the inventor thinks, nor are they intended to represent that the following experiments are all or unique experiments carried out. Efforts have been made to ensure the accuracy of the numerals (such as amounts, temperature, etc.) used, but some experimental errors and deviations should be considered. The following examples are only intended to serve as examples of the application and are therefore not to be considered as limiting the application in any way. The following examples and detailed description are only intended to illustrate, not to limit.

[0288] Example 1: Construction of a Yeast Expression Library of IL-18 Variant Polypeptides That Do Not Bind to IL-18BP

[0289] Interleukin 18 ("IL-18") stimulates T cells and NK cells by binding to its heterodimeric receptor complex, which is composed of 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 the binding of IL-18BP to IL-18 will block the interaction of IL-18 with IL-18Rα. In order to develop human IL-18 ("hIL-18") variants that do not bind to IL-18BP or have reduced binding affinity for IL-18BP, an artificial intelligence-guided molecular design approach was used to identify amino acid positions for mutation in wild-type hIL-18. Next, NNK mutagenesis technology was used to construct a yeast library displaying hIL-18 variant polypeptides containing the identified mutations relative to wild-type IL-18 (SEQ ID NO: 1), with a final library diversity of ∼2.72 × 10 8 A c-myc tag was fused to each IL-18 variant tested for expression.

[0290] Example 2: Screening for IL-18 variant polypeptides that bind to human IL-18Ra

[0291] The library constructed in Example 1 was screened using a positive / negative selection strategy to identify IL-18 variant polypeptides that bind to hIL-18Rα but not to hIL-18BP, as well as IL-18 variant polypeptides that have reduced binding affinity to hIL-18BP compared to wild-type IL-18. Briefly, the recombinant hIL-18Rα extracellular domain ("hIL-18RαECD") was labeled with biotin and immobilized on a first set of streptavidin (SA) magnetic microbeads or anti-biotin magnetic microbeads for magnetic activated cell sorting (MACS)-based screening. The recombinant hIL-18BP was immobilized on a second set of SA magnetic microbeads or anti-biotin magnetic microbeads. The library constructed in Example 1 was subjected to three rounds of screening using hIL-18RαECD-bound beads and hIL-18BP-bound beads, as shown in FIG. Figure 1A shown.

[0292] Then, two additional rounds of FACS-based kinetic competition screening were performed (i.e., Figure 1A ) to further enrich for IL-18 variant polypeptides that exhibit: (a) higher or comparable affinity for hIL-18Rα compared to WT hIL-18, and (b) undetectable or reduced binding to hIL-18BP (e.g., K for hIL-18BP). D >10 -3M). Then, single yeast cells from the output pools of rounds 4 and 5 were isolated. Plasmids encoding the isolated IL-18 variant polypeptides were extracted from the yeast and sequenced via next generation sequencing (NGS).

[0293] After each round of screening, the output library size was determined by FACS. The binding capacity of the output library to 50 nM hIL-18Rα ECD or 1 μM hIL-18BP was also confirmed by FACS. Figure 1B As shown, after the above screening, most of the displayed IL-18 variants did not bind to hIL-18BP or showed reduced binding to hIL-18BP, while the ability to bind to hIL-18RαECD was maintained or improved.

[0294] Example 3: Monoclonal Fluorescence Activated Cell Sorting (FACS) of Yeast Cells Displaying IL-18 Variant Polypeptides.

[0295] Following the screening described in Example 2, single-clone FACS analysis was performed to verify binding of IL-18 variant polypeptides expressed on the surface of yeast cells selected in rounds 4 and 5 to 10 nM hIL-18Rα-ECD. Briefly, equal densities of each yeast clone were incubated with 10 nM IL-18Rα-ECD labeled with a fluorescent probe, followed by a wash step. The proportion of fluorescently positive IL-18Rα-ECD-binding yeast in each clonal population was then analyzed.

[0296] Table 1 and Figure 1C As shown in the figure (using 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% of cells in the clone population were fluorescent). SEQ ID NO: 1 is wild-type human IL-18. Table 1:

[0297] Table 1. Monoclonal FACS results of 117 clones

[0298]

[0299]

[0300]

[0301]

[0302] Example 4: Expression and purification of recombinant hIL-18

[0303] WT hIL-18 and the selected IL-18 variant polypeptides described above were expressed in an Escherichia coli system. Briefly, a pET30a plasmid containing a codon-optimized sequence encoding WT hIL-18 or hIL-18 variant polypeptide was transfected into Escherichia coli BL21(DE3) cells. The transfected E. coli cells were then expanded in culture, and the expressed polypeptides were harvested and purified.

[0304] Figure 2 Figures AF show WT hIL-18 and five exemplary variants (M12, MM5, WM6, M17, and M24) with purity exceeding 95% as determined by SDS-PAGE and SEC-HPLC.

[0305] Example 5: Affinity of IL-18 variant polypeptides for hIL-18Ra

[0306] 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-18RαECD-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-18RαECD-Fc are shown in Table 2A.

[0307] Table 2A. Affinity of IL-18 variant polypeptides for hIL-18Rα (determined by BLI)

[0308]

[0309]

[0310]

[0311] Mutations in these IL-18 variant polypeptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM14, corresponding to 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, 149-153) are shown in Table 2B.

[0312] Table 2B

[0313]

[0314]

[0315] Example 6: Functional Characterization of IL-18 Variant Polypeptides

[0316] 6.1 Activation of hIL-18 Receptor-Mediated Signaling by IL-18 Variant Peptides

[0317] The ability of IL-18 variant polypeptides to activate hIL-18 receptor-mediated signaling was determined using the NFκB-luc / hIL-18RαRβ HEK293 cell line stably expressing hIL-18Rα, hIL-18Rβ, and the NFκB luciferase reporter gene as reporter cells.

[0318] Briefly, reporter cells were first seeded in the wells of a white 96-well plate at a density of 3.0 × 10 4 WT hIL-18 or IL-18 variant polypeptides were serially diluted in culture medium in the presence of (a) 1 μg / mL hIL-18BP-hFc and (b) the absence of 1 μg / mL hIL-18BP-hFc. After incubation at 37°C for 16 hours, luminescence intensity was measured.

[0319] like Figure 3 As shown in Table 3, all tested IL-18 variant polypeptides showed potency in activating IL-18 receptor-mediated signaling in a dose-dependent manner. Unlike WT hIL-18, whose activity was greatly reduced (approximately 80-fold) in the presence of 1 μg / mL hIL-18BP-hFc, the IL-18 variant polypeptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-11, corresponding to SEQ ID NO: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, 149-150) showed Hil-18 BP resistance activity, EC 50 The changes were no more than 3-fold, indicating that the IL-18 variant polypeptides were significantly less affected by Hil-18BP in terms of Hil-18 receptor-mediated signaling activation. An exception was WM6, whose EC 50 It showed about a 12-fold loss in potency, but was still much better than WThIL-18.

[0320] Table 3. EC of IL-18 variant polypeptides in the hIL-18 reporter gene assay in the presence or absence of 1 μg / mL hIL-18BP-hFc 50

[0321]

[0322]

[0323] 6.2 Human PBMC-based hIFNγ release assay

[0324] A PBMC-based assay was performed to test the ability of IL-18 variant polypeptides to activate T / NK cells and induce hIFNγ release. Briefly, human PBMCs were plated at 5.0 × 10 5 Cells were seeded at a density of 10 cells per well in a 96-well plate and cultured for 5 hours before treatment. WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL human IL-12 in the presence of (a) 1 μg / mL hIL-18BP-hFc or (b) the absence of 1 μg / mL hIL-18BP-hFc and then added to the wells. After incubation at 37°C for 16 hours, culture medium was collected from each well and hIFNγ concentration was measured via FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0325] like Figure 4 As shown in Table 4, the tested IL-18 variant polypeptides showed a dose-dependent effect in inducing the release of hIFNγ from PBMCs. In addition, as shown in Table 4, the activity of WT hIL-18 was greatly attenuated in the presence of 1 μg / mL hIL-18BP-hFc. 50 In contrast, IL-18 variant polypeptides (i.e., M11-M13, M15, M17, M21, M24, M29, M32-M33, M35-M36, MM5, WM4-WM6, WM8, WM10-11, corresponding to SEQ ID NOs: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, 143-150) exhibited hIL-18BP-resistant activity, EC 50 The change was no more than 2-fold, indicating that the IL-18 variant polypeptide was significantly less affected by hIL-18BP in terms of hIFNγ induction.

[0326] Table 4. EC of IL-18 variant polypeptides in PBMC assay in the presence or absence of 1 μg / mL hIL-18BP 50

[0327]

[0328] 6.3 mIFNγ Release Assay Based on Mouse Splenocytes

[0329] To evaluate the in vivo efficacy of the IL-18 variant polypeptides of the present application, a mouse splenocyte-based murine IFNγ ("mIFNγ") release assay was performed as follows.

[0330] Briefly, freshly isolated mouse splenocytes were seeded in wells of a 96-well plate at a density of 6.5 × 10 5 WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL mouse IL-12 in the presence or absence of (a) 1 μg / mL mouse IL-18BP-Fc or (b) 1 μg / mL mouse IL-18BP-Fc and then added to the wells. After incubation at 37°C for 16 hours, culture medium was collected from each well and the concentration of mIFNγ in the culture medium was measured via FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0331] Example 7: Generation and Characterization of IL-18 Variant Polypeptides Comprising Serine→Cysteine ​​and / or Cysteine→Serine Substitutions

[0332] 7.1 Wild-type IL-18 or IL-18 polypeptide variants containing a cysteine→serine substitution

[0333] Although the potent immunostimulatory activity of IL-18 has been reported, recombinant IL-18 is mainly expressed in the Escherichia coli system, which is not suitable for the production standards compatible with the good manufacturing practices (GMP) of mammalian host cells. Human IL-18 has four unpaired cysteines and does not contain disulfide bonds. Among the four cysteines, C38, C68 and C76 are highly exposed to the solvent, while C127 is partially exposed. During expression, due to the exposed free cysteine, IL-18 easily forms undesirable aggregates, resulting in reduced yield and / or purity. In order to improve the correct folding and stability of IL-18, that is, with higher yield and qualified purity as the goal, we designed IL-18 variants by using cysteine ​​mutation strategy and disulfide bond introduction strategy. The following experiments were conducted to evaluate the drugability of IL-18 variants and the effect of IL-18 variant expression in mammalian cells (such as CHO cells and 293F cells) by Cys to Ser ("C→S") mutation. The wild-type IL-18 and IL-18 variant polypeptides M12 and MM5 were modified (or further modified) to include C38S, C68S, and C76S substitutions to generate novel variants comprising the following amino acid sequence:

[0334] IL-18 variant containing C38S, C68S, and C76S substitutions in a WT hIL-18 background ("IL-18-SSS"):

[0335] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKDSQPRGMAVTISVKSEKISTLSSENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:307)

[0336] IL-18 variant containing C38S, C68S, and C76S substitutions in the M12 background ("M12-SSS"):

[0337] YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:308)

[0338] IL-18 variant containing C38S, C68S, and C76S substitutions in the MM5 background ("MM5-SSS"):

[0339] YFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKLSTERGMAVTISVKSEKISTLSSENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:309)

[0340] Fc fusion cytokines can improve pharmacokinetics and are suitable for widely used production processes. Next, the C-terminus of each of IL-18-SSS, M12-SSS, and M5-SSS was fused to the N-terminus of human IgG1 Fc containing the N297A substitution (EU numbering) to generate IL-18-SSS-Fc_N297A (SEQ ID NO: 319), M12-SSS_N297A (SEQ ID NO: 320), and MM5-SSS_N-297A (SEQ ID NO: 321). The fusion polypeptides were expressed in CHO cells, harvested, and purified via 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 comparison, the yield of WT IL-18-Fc_N297A obtained after one-step Protein A purification was ~16 mg / L with a purity of 11%. After a one-step Protein A purification, M12-SSS-Fc_N297A was obtained at a yield of 5.6 mg / mL and a purity of 72%. In contrast, M12-Fc_N297A was not expressed at detectable levels in 100 mL of CHO host cells. After a one-step Protein A chromatography step, MM5-SSS-Fc_N297A was obtained at a yield of 24.5 mg / L and a purity of 46%. After an additional gel filtration purification step, MM5-SSS-Fc_N297 was obtained at a yield of 3.4 mg / L and a purity of 99%. In contrast, MM5-Fc_N297A was not expressed at detectable levels in 100 mL of CHO host cells.

[0341] M12-SSS was further modified to include a C127S substitution to generate M12-SSSS:

[0342] YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLASEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:310)

[0343] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant comprising 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 via Protein A chromatography. The introduction of an additional C127S substitution (i.e., the fourth C→S substitution) resulted in better expression yield and purity than M12-Fc_N297A, however, it reduced the yield and purity of M12-SSS-Fc_N297A compared to those of M12-SSS-Fc_N297A (data not shown), suggesting that the C127 mutation may impair the conformational benefits of the first three cysteine ​​mutations.

[0344] Next, the in vitro assay described in Section 6.1 of Example 6 was used to characterize the potency of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A in activating IL-18 receptor-mediated signaling. The potency of IL-18-SSS-Fc_N297A was shown to be comparable to that of wild-type IL-18 (see Figure 5A ), while M12-SSS-Fc_N297A showed approximately 7-fold loss of potency compared to M12 (see Figure 5B ). The signaling activity of IL-18-SSS-Fc_N297A was reduced in the presence of 1 μg / ml hIL-18BP-hFc ("BP"), while M12-SSS-Fc_N297A exhibited hIL-18BP-resistant activity in the presence of 1 μg / ml hIL-18BP-hFc. Figure 5A and Figure 5B .like Figure 5C As shown, MM5-SSS-Fc_N297A showed a decrease in potency by approximately 220-fold compared to MM5, but exhibited hIL-18BP resistance activity in the presence of 1 μg / ml hIL-18BP-hFc. These results indicate that the three cysteine ​​→ serine substitutions do not affect the IL-18BP resistance characteristics. These data indicate that mutations at C38, C68, and C76 prevent the three exposed cysteines from forming undesirable intramolecular and intermolecular disulfide bonds, which is crucial for improving the yield and purity of IL-18 and IL-18 polypeptide-Fc fusion proteins. In addition, Fc-fused IL-18 and IL-18 variants are expected to exhibit longer half-lives.

[0345] 7.2 IL-18 polypeptides or IL-18 polypeptide variants comprising amino acid substitutions that remove and / or introduce cysteine ​​residues

[0346] The following experiments were performed to evaluate the effects of removing intrinsic cysteine ​​(i.e., naturally occurring cysteine ​​in the polypeptide sequence) and / or introducing disulfide bond substitutions on the drugability, expression, and purification of IL-18 variant polypeptides. M12 was used as a representative variant selected for the engineering strategy. First, computer screening was performed based on molecular dynamics simulations, followed by an AI-based stability assessment to determine the mutation hotspots on the four cysteines (C38, C68, C76, and C127) of M12, as listed below. See Table 5. In addition, structure-guided design was used to identify amino acids in spatial positions that, when substituted with Cys residues, would allow the formation of new non-natural disulfide bonds. The mutation strategies shown in Table 5 (i.e., DB6, DB7, DB8, DB9, and DB10) were proposed. It was assumed that introducing the S117C mutation while retaining C76 would promote the disulfide bond between C117 and C76.

[0347] Table 5. M12-derived IL-18 polypeptide variants

[0348]

[0349] The mutation strategy outlined in Table 5 was applied to the IL-18 polypeptide variant M12 to generate the new variants M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10. Mutation hotspots at individual sites were identified through in silico screening. Molecular dynamics simulation-based in silico screening, followed by AI-based stability assessment, identified mutation hotspots at each of the four cysteines (C38, C68, C76, and C127) of M12, as shown in the table.

[0350] M12-DB6

[0351]

[0352] M12-DB7

[0353]

[0354] M12-DB8

[0355]

[0356] M12-DB9

[0357]

[0358]

[0359] M12-DB10

[0360]

[0361] The C-terminal end of each variant of M12-DB6, M12-DB7, M12-DB8, M12-DB9 and M12-DB10 was fused to a human IgG1 Fc variant comprising 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 via a one-step Protein A chromatography. Purified preparations were analyzed via SEC and SDS PAGE (data not shown). See Table 6 below.

[0362] Table 6. Expression and purification of M12-DB6, M12-DB7, M12-DB8, M12-DB9 and M12-DB10 Fc_N297A fusion proteins

[0363]

[0364] “ / ” indicates that no purified protein was detected.

[0365] M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A showed greatly improved expression yield and / or purity compared to M12-Fc_N297A or M12-SSS-Fc_N297A (see Table 6). Among M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A, M12-DB6-Fc_N297A showed the highest expression yield (313 mg / L) after one-step Protein A purification, with a purity of 89.8% (determined by SEC-HPLC). M12-DB8-Fc_N297A was not expressed at detectable levels. The expression yield of M12-DB7-Fc_N297A was approximately 50% of that of M12-DB6-Fc_N297A. M12-DB7-Fc_N297A showed similar purity to M12-DB6-Fc_N297A after Protein A chromatography.

[0366] The high yield and purity of DB6 and DB7 are striking, especially the high yield. The M12-DB6 form is approximately 60 times purer than the SSS form. These data suggest that the strategy of removing cysteines at positions 38 and 68 and introducing C117 to promote a disulfide bond between C117 and C76 is highly advantageous.

[0367] The low yield of DB7 and the undetectability of DB8 suggest that the C127X substitution may not further improve peptide production in mammalian cells. Mutations at the partially exposed position C127 require careful design. Based on the C38X+C68X+C76+S117C strategy, position 127 favors C over A, and the hydrophobic 127I significantly impacts the proper folding of the IL-18 peptide. We further tested the corresponding variant IL-18-DB6 peptides with C127N, C127T, and C127S, all of which exhibited expression levels comparable to DB6. Given the partially exposed nature of position 127 and the results obtained, introducing amino acids with hydrophobic side chains at position 127 using this strategy should be avoided.

[0368] Next, the in vitro assay described in Section 6.1 of Example 6 was used to characterize the potency of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in activating IL-18 receptor-mediated signaling. 6A to 6D As shown in 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 ) showed much higher potency than WT IL-18 in activating IL-18 receptor-mediated signaling. In the presence of 1 μg / ml hIL-18BP-hFc, the tested M12-DB6-Fc_N297A ( Figure 6A )、M12-DB7-Fc_N297A( Figure 6B ) and M12-DB9-Fc_N297A ( Figure 6C )'s signal transduction activity was not inhibited. See Table 7.

[0369] Table 7. EC50 values ​​of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in in vitro IL-18 receptor-mediated signaling assays

[0370]

[0371] Next, the "DB6" substitutions (i.e., C38I, C68S, and S117C) were introduced into variant MM5 to generate MM5-DB6:

[0372] MM5-DB6

[0373]

[0374] A "DB6" substitution was also introduced into wild-type IL-18 to generate WT IL18-DB6:

[0375] WT IL18-DB6

[0376]

[0377] Another variant, WT-DBo, was designed by introducing the S117C substitution in wild-type IL-18:

[0378] WT IL-18-DBo

[0379]

[0380] The C-terminus of each of WT IL-18-DB6, WT IL-18-DBo and MM5-DB6 was fused to the N-terminus of a human IgG1 Fc variant comprising 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, after identical expression and purification procedures, no protein of WT IL18-DBo-Fc_N297A was detected. The expression yield of WT IL18-DB6-Fc was 157 mg / L, and the purity of WT IL18-DB6-Fc_N297A after one-step Protein A chromatography purification was 91.6% (determined by SEC-HPLC) (see Table 8). This data confirms that the strategy of promoting the disulfide bond between C76 and C117 and removing the cysteines at positions 38 and 68 is very advantageous. These data also indicate that introducing a disulfide bond between C76 and C117 alone may not be sufficient to improve fusion polypeptide expression in mammalian cells. Improved expression in mammalian cells was achieved using fusion polypeptides comprising IL-18 variants comprising (i) an engineered C76-C117 disulfide bond, (ii) a C38 substitution (see Table 5), and (iii) a C68 substitution (see Table 5). The expression yield and purity of MM5-DB6-Fc_N297A were consistently much higher than those of MM5-SSS-Fc_N297A (see Table 8). These data also suggest that the C38X+C68X+C76+S117C mutation strategy (e.g., "DB6" or "DB7" substitutions) is suitable for increasing the yield of WT IL-18 and IL-18 variant polypeptides during production and subsequent purification in mammalian cells.

[0381] Next, fusion polypeptides were designed in which the C-terminus of human IgG1 Fc containing the N297A substitution (EU numbering) was fused 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 via Protein A chromatography. As shown in Table 8, both Fc_N297A-MM5-DB6 and Fc_N297A-M12-DB6 exhibited comparable expression yield and purity in CHO cells compared to M12-DB6-Fc_N297A or MM5-DB6-Fc_N297A, respectively. After one-step Protein A chromatography purification, the purity of Fc_N297A-MM5-DB6 was approximately 97% (determined by SEC-HPLC), and the purity of Fc_N297A-M12-DB6 was 89% (determined by SEC-HPLC). These data suggest that the cystine mutation plus disulfide bond introduction strategy is applicable to Fc fusions of any fusion order.

[0382] Table 8. WT IL18-DB6-Fc_N297A, WT IL18-DBo-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6 and Fc_N297A-M12-DB6 were expressed in CHO cells and treated with Protein A.

[0383] Yield and purity after chromatography

[0384]

[0385] Next, the in vitro assay described in Section 6.1 of Example 6 was used to characterize the potency of WT IL18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 in activating IL-18 receptor-mediated signaling. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A exhibited potent IL-18 receptor cell activation signals that were stronger than those of WT IL-18 (see Figure 7A 、 Figure 7B and Table 9). Fc_N297A-MM5-DB6 showed similar potency to WT IL-18, whereas Fc_N297A-M12-DB6 gave a significantly stronger activation signal to IL-18 reporter cells (see Figure 7C 、 Figure 7Dand Table 9). In the presence of 1 μg / mL IL-18BP-hFc, the activity of WT IL18-DB6-Fc_N297A was significantly reduced, while the activity of MM5-DB6-Fc_N297A was resistant to inhibition by IL-18BP (see Figure 7A 、 Figure 7B and Table 9). Both M12-DB6-Fc_N297A and MM5-DB6-Fc_N297A maintained IL-18BP-resistant activity, whereas the activity of WT IL18-DB6-Fc_N297A was reduced by IL-18BP, indicating that the "DB6" mutant group (i.e., C38I, C68S, and S117C, see Table 5) improved the expression profiles of IL-18 and IL-18 variants without affecting IL-18BP dependency.

[0386] Taken together, these results demonstrate that the strategy of introducing C38X+C68X+C76+S117C mutations into WT IL-18-Fc fusion polypeptides or IL-18 variant-Fc fusion polypeptides significantly improves the expression yield of the fusion polypeptides during production in mammalian cells and improves the yield of the fusion polypeptides purified after chromatography, without affecting IL-18BP-resistant activity. Furthermore, WT IL-18 and IL-18 variants engineered to include the "DB6" mutation set exhibited increased potency in activating IL-18 receptor-mediated signaling. This was observed for both fusion polypeptides in which the C-terminus of the Fc region was fused to the N-terminus of the IL-18 variant and in which the C-terminus of the IL-18 variant was fused to the N-terminus of the Fc domain (see Table 10).

[0387] Table 9. EC50 values ​​of WT IL-18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and -Fc_N297A-M12-DB6 in in vitro IL-18 receptor-mediated signaling assays

[0388]

[0389]

[0390] See Table 10 below, which summarizes the above data.

[0391] Table 10.

[0392]

[0393] The scope of the present disclosure is not limited by the specific embodiments, which are intended to serve as independent illustrations of various aspects of the present disclosure, and any functionally equivalent compositions or methods are within the scope of the present disclosure. It will be understood by 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 present disclosure. Therefore, the present disclosure is intended to encompass modifications and variations of the present disclosure as long as they are within the scope of the appended claims and their equivalents.

[0394] 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.

[0395] The present invention has been described with reference to specific embodiments discovered or proposed by the inventors, which include preferred modes for carrying out the present invention. Those skilled in the art will recognize that, in light of this disclosure, various modifications and improvements may be made to the illustrated specific embodiments without departing from the intended scope of the present invention. For example, due to codon redundancy, the following DNA sequence may be altered without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, the protein structure may be altered without affecting the type or quantity of the biological effect. All such modifications are intended to be included within the scope of the appended claims.

[0396] Sequence Summary Table

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

Claims

1. A method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising introducing cysteine ​​at position 117 and / or position 76 of the IL-18 polypeptide, thereby promoting a disulfide bond between C117 and C76, wherein the amino acid position of the IL-18 polypeptide is relative to wild-type human IL-18 as shown in SEQ ID NO:

1.

2. The method of claim 1 , wherein the method comprises a) introducing a cysteine ​​at position 117, and b) retaining the cysteine ​​at position 76 when the IL-18 polypeptide comprises a cysteine ​​at position 76, or introducing a cysteine ​​at position 76 when the IL-18 polypeptide comprises a non-cysteine ​​at position 76.

3. The method of claim 1 or claim 2, wherein the method comprises a) introducing a cysteine ​​at position 76, and b) retaining the cysteine ​​at position 117 when the IL-18 polypeptide comprises a cysteine ​​at position 117, or introducing a cysteine ​​at position 117 when the IL-18 polypeptide comprises a non-cysteine ​​at position 117. 4 . The method according to claim 1 , wherein introducing cysteine ​​at position 117 or position 76 comprises substituting the amino acid at position 117 or position 76 with cysteine.

5. The method of claim 4, wherein the substitution of the amino acid at position 117 comprises an S117C substitution.

6. The method according to any one of claims 1 to 5, further comprising removing one or both cysteines at positions 38 and / or 68 when the IL-18 polypeptide comprises cysteines at one or both of positions 38 and 68.

7. The method of claim 6, wherein removing one or both cysteines at position 38 and / or position 68 comprises replacing the cysteines at position 38 and / or position 68 with different amino acids.

8. The method of claim 7, wherein the substitution of cysteine ​​at position 38 comprises C38S, C38I, C38L, C38V, or C38M, optionally wherein the substitution of cysteine ​​at position 38 comprises C38S, C38I, and C38V.

9. The method of claim 7 or claim 8, wherein the substitution of cysteine ​​at position 68 comprises C68S, C68I, C68V, C68L or C68D, optionally wherein the substitution of cysteine ​​at position 38 comprises C68S, C68I and C68L.

10. The method of any one of claims 1 to 9, wherein the method further comprises retaining the cysteine ​​at position 127 when the IL-18 polypeptide comprises a cysteine ​​at position 127, or introducing a cysteine ​​at position 127 when the IL-18 polypeptide comprises a non-cysteine ​​at position 127.

11. The method according to any one of claims 1 to 9, wherein the method further comprises introducing alanine or an amino acid without a hydrophobic side chain at position 127, optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine and proline, further optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine and threonine.

12. The method according to claim 11, wherein introducing alanine or an amino acid without a hydrophobic side chain at position 127 comprises substituting the amino acid at position 127 of the IL-18 polypeptide with alanine or an amino acid without a hydrophobic side chain.

13. The method of any one of claims 1 to 12, wherein the IL-18 variant polypeptide comprises: a) I38, V38 or S38, b) I68, S68 or L68, c) C76, and d)C117.

14. The method of claim 13, wherein the IL-18 variant polypeptide comprises I38, S68, C76, and C117.

15. The method of claim 13, wherein the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127.

16. The method according to any one of claims 1 to 15, wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-299, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1-299, optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133 and 143-150, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1-299, NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133 and 143-150 having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereof, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, The amino acid sequence shown in any one of NO:1, 39 and 133, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO:1, 39 and 133.

17. The method according to any one of claims 1 to 16, wherein the IL-18 variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 311-312, 314-315 and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 311-312, 314-315 and 316-317.

18. An IL-18 variant polypeptide produced by the method of any one of claims 1 to 17.

19. An IL-18 variant polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 311-312, 314-315 and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 311-312, 314-315 and 316-317.

20. An IL-18 variant polypeptide comprising a cysteine ​​at position 117 and a cysteine ​​at position 76, wherein the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO:

1.

21. The IL-18 variant polypeptide of claim 20, wherein the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38, or the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68.

22. The IL-18 variant polypeptide of claim 21, wherein the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38, and the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68.

23. The IL-18 variant polypeptide of any one of claims 20 to 22, wherein the IL-18 variant polypeptide comprises 38S, 38I, 38V, 38L or 38M, optionally wherein the IL-18 variant polypeptide comprises 38S, 38I or 38V.

24. The IL-18 variant polypeptide of any one of claims 20 to 23, wherein the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L or 68D, optionally wherein the IL-18 variant polypeptide comprises 68S, 68I or 68L.

25. The IL-18 variant polypeptide of any one of claims 20 to 24, wherein the IL-18 variant polypeptide comprises 127C.

26. The IL-18 variant polypeptide of any one of claims 20 to 24, wherein the IL-18 variant polypeptide comprises an alanine at position 127 or an amino acid without a hydrophobic side chain at position 127, optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine and proline, further optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine and threonine.

27. The IL-18 variant polypeptide according to any one of claims 20 to 25, wherein the IL-18 variant polypeptide comprises: a) I38, V38 or S38, b) I68, S68 or L68, c) C76, and d)C117.

28. The IL-18 variant polypeptide of claim 27, wherein the IL-18 variant polypeptide comprises I38, S68, C76, and C117.

29. The IL-18 variant polypeptide of claim 27, wherein the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127.

30. A fusion polypeptide comprising a) an IL-18 variant polypeptide according to any one of claims 18 to 29, and b) a second moiety.

31. The fusion polypeptide of claim 30, wherein the second portion comprises a half-life extending moiety, optionally wherein the half-life extending moiety is an albumin binding moiety or an Fc domain.

32. The fusion polypeptide of claim 31 , wherein the half-life extending moiety comprises an Fc domain, optionally wherein the Fc domain is a human IgG Fc domain, further optionally wherein the human IgG Fc domain is a human IgG1 domain.

33. The fusion polypeptide of claim 31 or claim 32, wherein the Fc domain is a modified Fc domain with reduced effector function, optionally wherein the Fc domain comprises a human IgG1 Fc domain comprising an N297A mutation (EU numbering).

34. The fusion polypeptide of any one of claims 30 to 33, wherein the second portion is fused to the N-terminus of the IL-18 variant polypeptide.

35. The fusion polypeptide of any one of claims 30 to 33, wherein the second portion is fused to the C-terminus of the IL-18 variant polypeptide.

36. The fusion polypeptide of any one of claims 30 to 35, wherein the fusion polypeptide further comprises a linker between the IL-18 variant polypeptide and the second moiety, optionally wherein the linker is a peptide linker.

37. The fusion polypeptide of any one of claims 30 to 36, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347.

38. A dimer comprising two fusion polypeptides according to any one of claims 30 to 37.

39. The dimer of claim 38, wherein the dimer is a homodimer.

40. The dimer of claim 38, wherein the dimer is a heterodimer.

41. A nucleic acid encoding the IL-18 variant polypeptide of any one of claims 18 to 29 or the fusion polypeptide of any one of claims 30 to 37.

42. A nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NOs: 333-335.

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

44. A host cell comprising the nucleic acid of claim 41 or claim 42 or the vector of claim 43, optionally wherein the host cell is a eukaryotic cell, further optionally wherein the host cell is a mammalian cell, further optionally wherein the host cell is a CHO cell or a HEK293 cell.

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

46. ​​A pharmaceutical composition comprising the IL-18 variant polypeptide according to any one of claims 18 to 29, the fusion polypeptide according to any one of claims 30 to 37, the dimer according to any one of claims 38 to 40, the nucleic acid according to claim 41 or claim 42, or the vector according to claim 43.

47. A method of treating a disease in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 46.

48. A method of activating hIL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 46.

49. A method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 46.

50. A method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising a) introducing an amino acid having a hydrophobic side chain at position 38, b) introducing an amino acid having a hydrophobic side chain at position 68, and / or c) introducing an amino acid having a hydrophobic side chain at position 76, wherein the IL-18 variant polypeptide comprises an amino acid having a hydrophobic side chain at each of positions 38, 68, and 76, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 as shown in SEQ ID NO:

1.

51. An IL-18 variant polypeptide comprising a) an amino acid with a hydrophobic side chain at position 38, b) an amino acid with a hydrophobic side chain at position 68, and / or c) an amino acid with a hydrophobic side chain at position 76, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 shown in SEQ ID NO: 1.

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