IL-18 variant polypeptides

Abstract

Provided is an interleukin-18 (IL-18) variant polypeptide having at least one mutation at a residue selected from the group consisting of C38, C68, C76, S117, and C127, where the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. Also provided is an IL-18 variant polypeptide that binds to IL-18Rα and exhibits substantially reduced binding to IL-18BP compared to wild-type IL-18. Also provided is an IL-18 variant polypeptide that exhibits increased binding to IL-18Rα compared to wild-type IL-18 and exhibits substantially reduced binding to IL-18BP compared to wild-type IL-18.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to international applications PCT / CN2023 / 072886, filed January 18, 2023, and PCT / CN2023 / 117197, filed September 6, 2023, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (233002001543SEQLIST.xml, size: 362,677 bytes, and created on January 16, 2024) are incorporated herein by reference in their entirety.

[0003] Field of application The present application relates to interleukin-18 (IL-18) variant polypeptides that contain at least one amino acid substitution compared to wild-type IL-18 (e.g., wild-type human IL-18), methods of making such variant polypeptides, and methods of using such variant polypeptides for therapeutic applications. [Background technology]

[0004] Background of the application IL-18 has been found to stimulate innate lymphocytes, myeloid cells, antigen-experienced non-naive T cells (see, e.g., Guo et al. (2012) Trends Immunol. 33, 598-606), and antigen-experienced natural killer (NK) cells. Therapeutically, recombinant IL-18 has been reported to synergize with immune checkpoint inhibitors (ICIs) (Ma et al. (2016) Clin Cancer Res 22:2969-2980) and chimeric antigen receptor T (CAR-T) cells in preclinical models (Hu et al. (2017) Cell Rep 20, 3025-3033). IL-18 has been administered to patients in clinical trials and found to be safe and well-tolerated (Robertson et al. (2006) Clin Cancer Res 12, 4265-4273). However, clinical development of IL-18 has been hampered by its limited efficacy. Thus, there is a need in the art for compositions and methods for activating IL-18 receptor-mediated signaling in individuals and / or stimulating antigen-experienced T cells or NK cells in individuals to treat cancer and other IL-18-mediated diseases and disorders. The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Guo et al.(2012)Trends Immunol.33,598-606 [Non-patent document 2] Hu et al.(2017)Cell Rep 20,3025-3033 [Non-patent document 3] Ma et al. (2016) Clin Cancer Res 22:2969-2980 [Non-patent document 4] Robertson et al. (2006) Clin Cancer Res 12,4265-4273 Summary of the Invention

[0006] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided, comprising at least one mutation in 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 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises at least one mutation in 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 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, where the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. Exemplary IL-18 polypeptides can be found, for example, in WO2020069398A1, WO2021097376A1, WO2023161853A1, WO2022038417A2, which are incorporated by reference herein in their entireties.

[0007] 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 set forth in SEQ ID NO: 1; optionally, the IL-18 polypeptide comprises mutations at G3, E6, D54, and N91; and further optionally, 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 IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91 T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91 G, and 104S.

[0008] In some embodiments, the IL-18 polypeptide comprises one substitution or a set of combination 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 N 91T, 13) G3P, E6K, D54Q, Q56I, and P57W, 14) G3N, E6K, D54P, Q56S, P57S, N91R, and R104A, 15) G3P, E6R, D54S, Q56Y, P57T, and N91G, 16) G3P, E6R, D54S, Q56R, P57N, N91G, and R104S, 17) G3P, E6R, D54L, Q56T, P57A, and N91G, 18) G3P, E6R, D54S, Q56R, P57R, N91G, and R104S, 19) G3P, E6K, D54H, Q56E, P57Q, and N91A, 20 ) G3P, E6R, D54S, Q56R, P57S, N91G, and R104S, 21) G3P, E6R, D54S, Q56S, P57T, N91G, and R104S, 22) G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S, 23) G3P, E6R, D54Y, Q56T, and P57R, 24) G3P, E6R, D54Y, Q56T, and P57S, 25) G3P, E6R, D54L, Q56T, P57T, and N91R, 26) G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y, 27) G3P,E6R, D54H, Q56Y, P57T, N91V, and R104Y, 28) G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y, 29) G3P, E6M, D54F, Q56D, P57R, and N91P, 30) G3P, E6L, D54H, Q56T, P57V, and N91S, 31) G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y, 32) G3P, E6G, D54S, Q56S, and P57R, 33) G3E, E6H, D54R, Q56T, and P57H, 34) G3P, E6 R, D54H, Q56R, P57N, N91V, and R104E, 35) G3P, E6R, D54G, Q56G, P57A, and N91G, 36) G3P, E6S, D54A, Q56D, P57Q, and N91G, 37) G3P, E6G, D54Q, Q56V, and P57W, 38) G3P, E6S, D54W, Q56G, P57A, N91V, and R104I, 39) G3P, E6R, D54W, Q56P, P57G, N91V, and R104L, 40) G3D, E6K, D54P, Q56S, P57W, and N91W, 41) G3P, 42) G3P, E6K, D54G, Q56G, and P57A, 43) G3P, E6R, D54L, Q56G, P57S, and N91V, 44) G3P, E6R, V11I, D54G, Q56G, P57A, and N91G, 45) G3P, E6K, D54H, Q56Y, and P57S, 46) E6R, D54W, Q56S, and P57Q, 47) G3P, E6K, D54L, Q56T, P57Q, and N91V, 48) G3A, E6Y, D54R, Q56S, P57L, and N91G, 49) G3P, E6R, D54L, Q56T, P57I , and N91G, 50) E6G, D54L, Q56T, P57E, N91G, and R104S, 51) G3A, E6Y, D54R, Q56S, P57L, and N91A, 52) M60K and K96D, 53) G3P, E6R, and K96E, 54) M60K, 55) G3P and E6R, 56) G3P and E6K, 57) G3D, E6K, and N91S, 58) G3S and I149M, 59) G3P, E6R, and N91S, 60) E6R, 61) E6R and N91S, 62) V11I, and 63) G3S and K140R.

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

[0011] In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide is at about 5×10 -5 K less than M DIn some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -5 ~Approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at 5×10 -9 K exceeds M D In some embodiments, the IL-18 variant polypeptide does not exhibit binding to IL-18BP (e.g., does not exhibit detectable binding, such as measured by surface plasmon resonance).

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

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

[0017] In some embodiments, the variant polypeptide comprises a mutation at residue Q56, 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.

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

[0019] In some embodiments, the variant polypeptide comprises a mutation at residue P57, which mutation is selected from the group consisting of P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

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

[0021] In some embodiments, the variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the variant polypeptide comprises mutations: (i) G3P, (ii) E6R or E6K, (iii) D54W, D54H, D54S, or D54Q, and (iv) N91V, N91A, N91G, or N91S. In some embodiments, the variant polypeptide further comprises mutations at Q56 and P57.

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

[0023] In some embodiments, a fusion polypeptide is provided comprising an IL-18 variant polypeptide described herein and a human IgG Fc domain or variant thereof. In some embodiments, the fusion polypeptide comprises a human IgG1 Fc domain variant comprising an N297A mutation (EU numbering). In some embodiments, the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of a human IgG Fc domain or variant thereof. In some embodiments, the C-terminus of the human IgG Fc domain or variant thereof is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 336-347.

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

[0025] In some embodiments, nucleic acids encoding an IL-18 variant polypeptide or fusion polypeptide described herein are 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, vectors comprising the nucleic acids described herein are provided. In some embodiments, host cells comprising the nucleic acids or vectors described herein are provided. In some embodiments, methods for producing an IL-18 variant polypeptide or fusion polypeptide are provided, the methods comprising: (a) culturing a host cell described herein under conditions in which the IL-18 variant polypeptide or fusion polypeptide is expressed; and (b) recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell. In some embodiments, the host cell is a mammalian host cell (e.g., a CHO cell or a HEK293 cell). In some embodiments, the method comprises (e.g., further comprises) purifying the IL-18 variant polypeptide or fusion polypeptide.

[0026] In another aspect, the present application provides a method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising introducing cysteines at positions 117 and / or 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 set forth in SEQ ID NO: 1. Methods for engineering are disclosed in the examples of the present application, as well as 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.

[0027] In another aspect, the application provides a method for enhancing the yield and / or purity of an IL-18 variant polypeptide, comprising engineering an IL-18 polypeptide by introducing a cysteine ​​at positions 117 and / or 76 of the IL-18 polypeptide, thereby promoting a disulfide bond 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 set forth 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.

[0028] In some embodiments according to any one of the above methods, the method comprises: a) introducing a cysteine ​​at position 117; and b) retaining the cysteine ​​at position 76 if the IL-18 polypeptide comprises a cysteine ​​at position 76, or introducing a cysteine ​​at position 76 if the IL-18 polypeptide comprises a non-cysteine ​​at position 76.

[0029] In some embodiments according to any one of the above methods, the method comprises: a) introducing a cysteine ​​at position 76; and b) retaining the cysteine ​​at position 117 if the IL-18 polypeptide comprises a cysteine ​​at position 117, or introducing a cysteine ​​at position 117 if the IL-18 polypeptide comprises a non-cysteine ​​at position 117.

[0030] In some embodiments according to any one of the above methods, introducing a cysteine ​​at position 117 or 76 comprises substituting the amino acid at position 117 or 76 with cysteine. In some embodiments, the substitution of the amino acid at position 117 comprises an S117C substitution.

[0031] In some embodiments according to any one of the above methods, the method further comprises, when the IL-18 polypeptide comprises a cysteine ​​at one or both of positions 38 and 68, removing one or both cysteines at positions 38 and / or 68. In some embodiments, removing one or both cysteines at positions 38 and / or 68 comprises substituting the cysteine ​​with a different amino acid at position 38 and / or 68. In some embodiments, the substitution of cysteine ​​at position 38 comprises C38S, C38I, C38L, C38V, or C38M, optionally, 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, the substitution of cysteine ​​at position 38 comprises C68S, C68I, and C68L.

[0032] In some embodiments of any one of the above methods, the method further comprises retaining the cysteine ​​at position 127 if the IL-18 polypeptide comprises a cysteine ​​at position 127, or introducing a cysteine ​​at position 127 if the IL-18 polypeptide comprises a non-cysteine ​​at position 127.

[0033] In some embodiments according to any one of the above methods, the method further comprises introducing an alanine or amino acid without a hydrophobic side chain at position 127, wherein optionally the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, and further optionally the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, and threonine. In some embodiments, introducing an alanine or 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 amino acid without a hydrophobic side chain.

[0034] In some embodiments according to any one 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, and C117, and optionally, 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.

[0035] In some embodiments according to any one of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence set forth 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.

[0036] In another aspect, the application provides a method of engineering an IL-18 variant polypeptide from an IL-18 polypeptide, the method comprising: a) replacing a cysteine ​​at position 38 with another amino acid, b) replacing a cysteine ​​at position 68, and / or c) replacing a cysteine ​​at position 76, wherein the IL-18 variant polypeptide does not comprise 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 having a hydrophobic side chain in a), b), or c) is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylamine, tyrosine, and tryptophan; and optionally, 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, introducing an amino acid having a hydrophobic side chain at position 38, 68, or 76 comprises substituting an amino acid at position 38, 68, or 76 with an amino acid having 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, the IL-18 variant polypeptide comprises 38S, 38I, or 38V, and further optionally, the IL-18 variant polypeptide comprises 38I. In some embodiments, the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L, or 68D, optionally, the IL-18 variant polypeptide comprises 68S, 68I, or 68L, and further optionally, 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, 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, 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, the IL-18 variant polypeptide comprises S117. In some embodiments according to any one of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence set forth 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.

[0037] In some embodiments according to any one 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, and optionally 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, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150. and optionally, 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, 29, and 117, 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, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29, and 117, and further optionally, the IL-18 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 39, and 133, 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, 39, and 133.

[0038] In another aspect, the application provides an IL-18 variant polypeptide produced by any of the above methods.

[0039] In another aspect, the present application provides an IL-18 variant polypeptide comprising the amino acid sequence set forth 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.

[0040] In another aspect, the 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 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, or 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, and optionally 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, 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, the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, further optionally, the amino acid without a hydrophobic side chain is selected from the group consisting of 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 138, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, 168, C76, C117, and optionally, the variant polypeptide further comprises A127.

[0041] 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 moiety. In some embodiments, the second moiety comprises a half-life extending moiety, and optionally, 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, and optionally, the Fc domain is a human IgG Fc domain, and further optionally, 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, and optionally, the Fc domain comprises a human IgG1 Fc domain comprising an N297A mutation (EU numbering). In some embodiments, the second moiety is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the second moiety 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 moiety, which optionally is a peptide linker (such as a GS linker). In some embodiments, the fusion polypeptide comprises an 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.

[0042] In another aspect, the present application provides a dimer comprising two fusion polypeptides 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.

[0043] In another aspect, the application provides a nucleic acid encoding any of the IL-18 variant polypeptides or any of the fusion polypeptides described above.

[0044] In another aspect, the present application provides a nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NOs: 333-335.

[0045] In another aspect, the application provides a vector comprising any of the above nucleic acids.

[0046] In another aspect, the present application provides a host cell comprising any of the above nucleic acids or 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.

[0047] In another aspect, the application provides a method for producing an IL-18 variant polypeptide or fusion polypeptide, the method comprising: (a) culturing any of the host cells described above 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.

[0048] In another aspect, the application provides a pharmaceutical composition comprising any of the IL-18 variant polypeptides, any of the fusion polypeptides, any of the dimers, any of the nucleic acids, or any of the vectors described above.

[0049] In another aspect, the present application provides a method of treating a disease in an individual, comprising administering to the individual an effective amount of any of the above pharmaceutical compositions.

[0050] In another aspect, the present application provides a method of activating hIL-18 receptor-mediated signaling in an individual, the method comprising administering to the individual an effective amount of any of the above-described pharmaceutical compositions.

[0051] In another aspect, the present application provides a method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of any of the pharmaceutical compositions described above.

[0052] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0053] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0054] [Figure 1A] FIG. 1A illustrates the screening process using magnetic-activated cell sorting- (MACS-) or fluorescence-activated cell sorting- (FACS-) based methods to identify the IL-18 variant polypeptides described herein.

[0055] [Figure 1B] FIG. 1B shows the ability of output library pools from rounds 1, 2, 3, 4, and 5 to bind to either 1 μM IL-18BP or 50 nM IL-18Rα.

[0056] [Figure 1C] Figure 1C shows the ability of representative single IL-18 variant clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43) to bind to IL-18Rα. SEQ ID NO: 1 is wild-type IL-18.

[0057] [Figure 2-1] FIG. 2A provides SDS-PAGE and SEC-HPLC analysis of wild-type human IL-18 (SEQ ID NO: 1) after purification.

[0058] FIG. 2B provides SDS-PAGE and SEC-HPLC analysis of IL-18 variant M12 (SEQ ID NO: 39) after purification.

[0059] FIG. 2C provides SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant MM5 (SEQ ID NO: 133).

[0060] [Figure 2-2] FIG. 2D provides SDS-PAGE and SEC-HPLC analysis of IL-18 variant WM6 (SEQ ID NO: 145) after purification.

[0061] FIG. 2E provides SDS-PAGE and SEC-HPLC analysis of IL-18 variant M17 (SEQ ID NO: 43) after purification.

[0062] FIG. 2F provides SDS-PAGE and SEC-HPLC analysis of purified IL-18 variant M24 (SEQ ID NO: 105).

[0063] [Figure 3-1] FIG. 3 shows activation of hIL-18 receptor-mediated signaling by exemplary IL-18 variant polypeptides of the present application. [Figure 3-2] FIG. 3 shows activation of hIL-18 receptor-mediated signaling by exemplary IL-18 variant polypeptides of the present application. [Figure 3-3] FIG. 3 shows activation of hIL-18 receptor-mediated signaling by exemplary IL-18 variant polypeptides of the present application.

[0064] [Figure 4]FIG. 4 shows hIFNγ release induced by exemplary IL-18 variant polypeptides of the present application.

[0065] [Figure 5A] FIG. 5A shows activation of hIL-18 receptor-mediated signaling by wild-type IL-18, the fusion polypeptide IL-18-SSS-Fc_N297A, and the fusion polypeptide IL-18-SSS-Fc_N297A in the presence of an IL-18 binding protein ("BP").

[0066] [Figure 5B] FIG. 5B shows 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”).

[0067] [Figure 5C] Figure 5C shows 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 an IL-18 binding protein ("BP").

[0068] [Figure 6A] FIG. 6A shows 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”).

[0069] [Figure 6B]FIG. 6B shows activation of hIL-18 receptor-mediated signaling by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB7-Fc_N297A, and M12-DB7-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0070] [Figure 6C] FIG. 6C shows activation of hIL-18 receptor-mediated signaling by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB9-Fc_N297A, and M12-DB9-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0071] [Figure 6D] FIG. 6D shows activation of hIL-18 receptor-mediated signaling by the fusion polypeptide WT IL-18, the fusion polypeptide M12-DB10-Fc_N297A.

[0072] [Figure 7A] FIG. 7A shows 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”), the fusion polypeptide WT IL-18-DB6-Fc_N297A, and WT IL-18-DB6-Fc_N297A in the presence of IL-18 binding protein (“BP”).

[0073] [Figure 7B] Figure 7B shows 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").

[0074] [Figure 7C]FIG. 7C shows activation of hIL-18 receptor-mediated signaling by wild-type IL-18 and the fusion polypeptide Fc_N297A-MM5-DB6.

[0075] [Figure 7D] FIG. 7D shows activation of hIL-18 receptor-mediated signaling by wild-type IL-18 and the fusion polypeptide Fc_N297A-M12-DB6. DETAILED DESCRIPTION OF THE INVENTION

[0076] overview Components of the interleukin-18 (IL-18) pathway have been found to be upregulated on tumor-infiltrating lymphocytes (TILs) (see, e.g., Zhou et al. (2020) Nature 583:609-614), suggesting that IL-18 therapy may enhance antitumor immunity. However, IL-18 has not shown significant efficacy in clinical trials. IL-18BP, 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) is frequently upregulated in a variety of human and mouse tumors and is thought to limit the antitumor activity of IL-18 in mice and clinical trials. Furthermore, effective production of IL-18 is often difficult due to low yield and / or purity. Applicants have identified IL-18 variant polypeptides that induce IL-18 receptor-mediated signaling even in the presence of IL-18BP. By substituting one or a set of amino acids (e.g., G3, e.g., E6), the IL-18 variant polypeptides were found to exhibit resistance to IL-18BP while still being able to bind and activate the IL-18 receptor. See, e.g., Example 6, Figures 3-4, and Tables 3 and 4. Applicants have further found that by engineering one or more hot spots (e.g., with substitutions at C38, C68, C76, S117, and / or C127), higher yields, more purified, and / or more potent IL-18 variant polypeptides can be obtained. See, e.g., Example 7, Figures 5A-7D, and Tables 5-9. Surprisingly, Applicants have found 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 dramatically increases. See, e.g., the DB6 and DB7 versions of the IL-18 polypeptide in Table 10.

[0077] definition As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a cytokine and its receptor, 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 one that binds to this receptor with higher affinity, avidity, more readily, and / or with a 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 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 Dissociation constant of M (K D ) In some embodiments, the specific binding can include, but does not require, exclusive binding. The binding specificity of a cytokine can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-test, and peptide scan.

[0078] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, the isolated nucleic acid is free from association with all components associated with the production environment. In some embodiments, the isolated nucleic acid molecule encoding a polypeptide or cytokine described herein is in a form other than in the form or setting in which it is found in nature.

[0079] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked to them. Such vectors are referred to herein as "expression vectors."

[0080] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0081] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0082] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of the present application contemplate any one or more of these aspects of treatment.

[0083] In the context of cancer, the term "treating" includes any or all of inhibiting cancer cell growth, inhibiting cancer cell replication, reducing overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0084] The term "inhibition" or "inhibiting" refers to the reduction or elimination of any phenotypic characteristic, or the reduction or elimination of the occurrence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, lowering, or prevention of an activity, function, and / or amount compared to a reference. In certain embodiments, "reduce" or "inhibit" refers to the ability to produce an overall reduction of 20% or more. In other embodiments, "reduce" or "inhibit" refers to the ability to produce an overall reduction of 50% or more. "Reduce" or "inhibit" refers to the ability to produce an overall reduction of 75%, 85%, 90%, 95%, or more.

[0085] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0086] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0087] Reference herein to "about" a value or parameter includes (and describes) a variation about that value or parameter itself. For example, a reference to "about X" includes a reference to "X."

[0088] As used herein, reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0089] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0090] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Interleukin-18 (IL-18) variant polypeptides

[0091] Interleukin-18 (IL-18, also known as interferon-gamma inducer) is a protein encoded by the IL-18 gene in humans. It is a proinflammatory cytokine that can potentially be produced by hematopoietic and non-hematopoietic cells. IL-18 can regulate both innate and adaptive immunity, and its dysregulation can lead to autoimmune or inflammatory diseases.

[0092] In one aspect, provided herein is an interleukin-18 ("IL-18") variant polypeptide comprising mutation(s) at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, 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. YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 1)

[0093] In some embodiments, the IL-18 variant polypeptide comprises mutations in two or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the two or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in three or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the three or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in four or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, 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 mutations in five or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the five or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in six or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination.In some embodiments, the six or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations in 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, the seven or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at eight or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the eight or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at nine or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the nine or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at ten or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the ten or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149.In some embodiments, the IL-18 variant polypeptide comprises mutations at 11 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 11 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at 12 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 12 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at 13 or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the 13 or more mutations are selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant comprises (or further comprises) mutations at positions other than F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149.In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (such as 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.

[0094] In some embodiments, the one or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at two or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, the two or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at three or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, the three or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant comprises (or further comprises) a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0095] In some embodiments, the IL-18 variant polypeptide comprises (e.g., 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.

[0096] In some embodiments, the IL-18 variant polypeptide comprises at least one mutation (or two, or three) in a residue selected from the group consisting of G3, E6, and M60, where the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises at least one of: a) G3P, G3S, G3E, G3N, G3D, or G3A; b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y; or c) M60K. In some embodiments, the IL-18 variant polypeptide further comprises at least one or more (e.g., two, three, four, or five) mutations in residues selected from the group consisting of V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155. In some embodiments, the IL-18 variant polypeptide is selected from the group consisting of a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, and D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, and Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, P57Q, P57G, P d) N91V, N91G, N91A, N91S, N91T, N91R, N91K, N91P, N91I, or N91W; e) R104V, R104T, R104Y, R104F, R104T, R104L, R104S, R104A, R104E, R104I; f) N155K; g) V11I; h) K96D or K96E; i) I149M; or j) K140R.

[0097] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56D, P57R, N91V, and R104V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0098] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S, E6K, D54W, Q56P, P57D, N91G, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0099] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0100] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54W, Q56G, P57V, N91V, and R104F, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0101] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0102] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56T, N91V, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0103] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56T, P57A, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0104] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56P, P57A, N91A, and R104L, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0105] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56R, P57A, N91S, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0106] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Q, Q56L, P57W, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0107] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57N, N91G, and R104T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0108] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54G, Q56G, P57A, and N91T, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0109] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54Q, Q56I, and P57W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0110] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3N, E6K, D54P, Q56S, P57S, N91R, and R104A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0111] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56Y, P57T, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0112] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57N, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0113] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0114] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57R, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0115] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56E, P57Q, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0116] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56R, P57S, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0117] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54S, Q56S, P57T, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0118] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0119] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Y, Q56T, and P57R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0120] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54Y, Q56T, and P57S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0121] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57T, and N91R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0122] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0123] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0124] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0125] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6M, D54F, Q56D, P57R, and N91P, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0126] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6L, D54H, Q56T, P57V, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0127] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0128] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6G, D54S, Q56S, and P57R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0129] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3E, E6H, D54R, Q56T, and P57H, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0130] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54H, Q56R, P57N, N91V, and R104E, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0131] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54G, Q56G, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0132] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6S, D54A, Q56D, P57Q, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0133] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6G, D54Q, Q56V, and P57W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0134] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6S, D54W, Q56G, P57A, N91V, and R104I, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0135] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54W, Q56P, P57G, N91V, and R104L, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0136] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3D, E6K, D54P, Q56S, P57W, and N91W, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0137] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) that is G3P, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0138] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54G, Q56G, and P57A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0139] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56G, P57S, and N91V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0140] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, V11I, D54G, Q56G, P57A, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0141] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54H, Q56Y, and P57S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0142] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6R, D54W, Q56S, and P57Q, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0143] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6K, D54L, Q56T, P57Q, and N91V, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0144] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3A, E6Y, D54R, Q56S, P57L, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0145] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, D54L, Q56T, P57I, and N91G, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0146] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6G, D54L, Q56T, P57E, N91G, and R104S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0147] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3A, E6Y, D54R, Q56S, P57L, and N91A, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V; optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally wherein the C127 substitution is C127A or C127I.In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, and optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0148] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising M60K and K96D, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0149] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, and K96E, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0150] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) including M60K, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0151] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P and E6R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0152] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P and E6K, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0153] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3D, E6K, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0154] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S and I149M, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0155] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3P, E6R, and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0156] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) with E6R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0157] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising E6R and N91S, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0158] In some embodiments, the IL-18 variant polypeptide comprises one substitution (e.g., a single substitution) including V11I, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal to or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal to or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0159] In some embodiments, the IL-18 variant polypeptide comprises a set of substitution combinations comprising G3S and K140R, where the amino acid position(s) are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise any other mutations compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises one, two, or three mutations or substitutions compared to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide has a) equal or stronger binding affinity to an IL-18 receptor (e.g., IL-18Ra), and / or b) equal or lower binding affinity to an IL-18 binding protein, compared to 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 the group consisting of C38I, C38V, C38L, and C38M, and further optionally wherein the substitution is C38I or C38V. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C68, optionally wherein the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D, and further optionally wherein the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 variant polypeptide further comprises a substitution at C76 and / or C127, optionally wherein the C76 substitution is C76Y or C76V, optionally wherein the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L, and further optionally wherein the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions, optionally wherein the variant polypeptide further comprises a C127S substitution.In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant polypeptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant polypeptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant polypeptide further comprises C38I, C68L, and C76Y.

[0160] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-153.

[0161] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-150.

[0162] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence set forth in any one of 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150.

[0163] In some embodiments, an IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, at least four, at least five, or six mutations at residues selected from the group consisting of C38, C68, C76, D98, S117, and C127, where the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, an IL-18 variant polypeptide comprises (or further comprises) a substitution at C38 (i.e., the wild-type cysteine ​​at position 38 is substituted with any amino acid), a substitution at C68 (i.e., the wild-type cysteine ​​at position 68 is substituted with any amino acid), and an S117C substitution. In some embodiments, an IL-18 variant polypeptide comprises (or further comprises) a C38I, C38V, C38L, C38M, or C38S mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C68S, C68I, C68D, C68V, or C68L mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C76S, C76V, or C76Y mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a S117C mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C127A, C127Y, C127F, C127L, or C127I mutation. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, or four mutations selected from the group consisting of C38S, C68S, C76S, and C127S, where the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) the C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) the C38I, C68S, and S117C mutations.In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) C38V, C68I, S117C, and C127A mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) C38S, C68I, S117C, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) C38I, C68I, C76V, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises, or consists of) C38I, C68L, and C76Y mutations.

[0164] In some embodiments, the IL-18 variant polypeptide further comprises mutation(s) at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises mutations at two or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises mutations at Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise mutations at positions other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant comprises (or further comprises) mutations at positions other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 mutations are selected from the group consisting of G3, E6, D54, Q56, P57, N91, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise substitutions at Q56 and P57, where 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.

[0165] 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 substantially reduced binding to an IL-18 binding protein ("IL-18BP"), e.g., human IL-18 or "hIL-18BP." In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18BP compared to wild-type human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) corresponds to the affinity of wild-type human IL-18 set forth in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is increased compared to the affinity of WT human IL-18, as set forth in SEQ ID NO: 1, for IL-18Rα (e.g., hIL-18Rα).

[0166] In some embodiments, the IL-18 variant polypeptide is about 5×10 -5 Less than M, approximately 5 x 10 -6 Less than M, approximately 5 x 10 -7 Less than M, approximately 5 x 10 -8 Less than M, approximately 5 x 10 -9 Less than M, approximately 5 x 10 -10 Less than M, or about 5 x 10 -11 K less than M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -5 ~Approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 ~Approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 ~Approx. 5×10 -10 K of MD In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 ~Approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 ~Approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -9 ~Approx. 5×10 -11 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 ~Approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 ~Approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -9 ~Approx. 5×10 -10 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -7 ~Approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at about 5×10 -8 ~Approx. 5×10 -9 K of M D In some embodiments, the IL-18 variant binds to IL-18Rα at about 5×10 -5 ~Approx. 5×10 -11 M, e.g., about 5 x 10 -5 , 6×10 -5 , 7×10 -5 , 8×10 -5 , 9×10 -5 , 1×10 -6 , 2 × 10 -6 , 3×10-6 , 4×10 -6 , 5×10 -6 , 6×10 -6 , 7×10 -6 , 8×10 -6 , 9×10 -6 , 1×10 -7 , 2 × 10 -7 , 3×10 -7 , 4×10 -7 , 5×10 -7 , 6×10 -7 , 7×10 -7 , 8×10 -7 , 9×10 -7 , 1×10 -8 , 2 × 10 -8 , 3×10 -8 , 4×10 -8 , 5×10 -8 , 6×10 -8 , 7×10 -8 , 8×10 -8 , 9×10 -8 , 1×10 -9 , 2 × 10 -9 , 3×10 -9 , 4×10 -9 , 5×10 -9 , 6×10 -9 , 7×10 -9 , 8×10 -9 , 9×10 -9 , 1×10 -10 , 2 × 10 -10 , 3×10 -10 , 4×10 -10 , 5×10 -10 , 6×10 -10 , 7×10 -10 , 8×10 -10 , 9×10 -10 , 1×10 -11 , 2 × 10 -11 , 3×10 -11 , 4×10 -11 , or 5 × 10 -11 Any one of M's K Dand any range therebetween. 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, as set forth in SEQ ID NO: 1, for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is comparable to (e.g., about the same as) the affinity of wild-type human IL-18, as set forth in SEQ ID NO: 1, for IL-18Rα (e.g., hIL-18Rα).

[0167] In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18. -9 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -8 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -7 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -6 K over M D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at 5×10 -5 K over M D In some embodiments, an IL-18 variant polypeptide exhibits no binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP). In some embodiments, an IL-18 variant polypeptide exhibits no binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP) at 10 -3 K over M Dand binds to IL-18BP (e.g., hIL-18BP).

[0168] The affinity of IL-18 as described herein for IL-18Rα and / or IL-18BP can be determined experimentally by methods known in the art, including, but not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL) assay, immunoradiometric assay (IRMA) assay, enzyme-linked immunosorbent assay (EIA), surface plasmon resonance (SPR), peptide scanning, and fluorescence-activated cell sorting (FACS)-based kinetic competition screening.

[0169] In some embodiments, the IL-18 variant polypeptide comprises a mutation at residue G3, which mutation is selected from the group consisting of G3P, G3D, G3E, G3N, and G3S. As used herein, "G3X" means that G, i.e., the wild-type amino acid at position 3 of SEQ ID NO: 1, has been replaced with the amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 of SEQ ID NO: 1 has been replaced with the amino acid P.

[0170] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue E6, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from the group consisting of E6R and E6K.

[0171] 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.

[0172] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue N91, wherein the mutation is selected from the group consisting of N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0173] 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.

[0174] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue Q56, wherein the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0175] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue P57, wherein the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57K, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0176] In some embodiments, the IL-18 variant polypeptide comprises a mutation in one or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation in two or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation in three or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation in G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a G3P mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an E6R or E6K mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises a D54W, D54H, D54S, or D54Q mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an N91V, N91A, N91G, or N91S mutation. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant further comprises a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at Q56 and / or P57. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, N91, Q56, and / or P57. 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 (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least 1, at least 2, at least 3, at least 4 mutations, at least 5 mutations, or at least 6 mutations are selected from the group consisting of G3, E6, D54, N91, Q56, and P57.

[0177] In some embodiments, an IL-18 variant polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of any one of SEQ ID NOs: 2-299, 307-318. In some embodiments, an 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 below in the Examples.

[0178] Fusion Polypeptides 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 two or more of any combination of wild-type IL-18 and IL-18 variant polypeptides, and (2) a dimerization domain. In some embodiments, the dimerization domain is or comprises a leucine zipper (LZ) element. Leucine zippers have generally been identified as stretches of approximately 35 amino acids containing four to five leucine residues separated from each other by six amino acids (Maniatis and Abel (1989) Nature 341:24-25). Exemplary leucine zippers occur in various eukaryotic DNA-binding proteins, such as GCN4, C / EBP, c-Fos, c-Jun, c-Myc, and c-Max. In some embodiments, the dimerization domain is or includes a helix-loop-helix domain (Murre, C. et al. (1989) Cell 58:537-544). Dimerization domains can also be selected from other proteins, such as retinoic acid receptors, thyroid hormone receptors, or other nuclear hormone receptors (Kurokawa et al. (1993) Genes Dev. 7:1423-1435), or 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 in U.S. Patent No. 5,624,818 by Eisenman. In some embodiments, the dimerization domain is an antibody Fc domain.

[0179] In some embodiments, the fusion polypeptide comprises wild-type IL-18 (e.g., wild-type IL-18 whose amino acid sequence is set forth in SEQ ID NO: 1) and an antibody Fc domain. In some embodiments, the fusion polypeptide comprises an IL-18 variant polypeptide (e.g., an IL-18 variant polypeptide described herein) and an antibody Fc domain. In some embodiments, the C-terminus of wild-type IL-18 or the C-terminus of the IL-18 variant polypeptide is fused to the N-terminus of the antibody Fc domain. In some embodiments, the C-terminus of the antibody Fc domain is fused to the N-terminus of wild-type IL-18 or the N-terminus of the IL-18 variant polypeptide. In some embodiments, the Fc domain is a human Fc domain or a variant thereof comprising one or more amino acid substitutions. In some embodiments, the Fc domain is a human IgG Fc domain or a variant thereof, e.g., a human IgG1, IgG2, or IgG4 Fc domain, or a variant of any of the foregoing. In some embodiments, a fusion polypeptide comprises, from N-terminus to C-terminus, an IL-18 variant polypeptide described herein, and a human IgG1 Fc variant comprising an N297A mutation, where the amino acid numbering is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, a fusion polypeptide comprises, from N-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.

[0180] Nucleic acids, vectors, host cells, and methods for producing IL-18 variant polypeptides Nucleic acid molecules encoding the IL-18 variant polypeptides described herein or the fusion polypeptides described herein are also contemplated. In some embodiments, nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides described herein are provided. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 301-306 and 333-335. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 301-306 and 333-335. The nucleic acid sequences of SEQ ID NOs: 301-306 and 333-335 are provided in the sequence summary table below in the Examples.

[0181] Also provided are vectors into which the nucleic acids described herein can be inserted.

[0182] In summary, expression of an IL-18 variant polypeptide or fusion polypeptide described herein by a natural or synthetic nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a constitutive, regulatable, or tissue-specific promoter) and a 3' untranslated region (UTR). The vector may be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0183] Nucleic acids can be cloned into many types of 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.

[0184] Furthermore, the expression vector may be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0185] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic.

[0186] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 base pairs (bp) upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. Spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or interlocked with one another. In the thymidine kinase (tk) promoter, spacing between promoter elements can be increased to 50 bp before activity begins to decline.

[0187] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of conferring high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), 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, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0188] In some embodiments, expression of the nucleic acid(s) encoding the IL-18 variant polypeptide or fusion polypeptide is inducible. In some embodiments, the nucleic acid(s) encoding the IL-18 variant polypeptide or fusion polypeptide are operably linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid(s) encoding the IL-18 variant polypeptide or fusion polypeptide described herein are 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, 6xHis (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, e.g., enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0189] 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 provided for illustrative purposes only and are not intended to be limiting.

[0190] The IL-18 variant polypeptides or fusion polypeptides described herein can be produced using recombinant methods. For recombinant production of IL-18 variant polypeptides or fusion polypeptides, nucleic acids encoding the IL-18 variant polypeptides or fusion polypeptides are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or expression. DNA encoding IL-18 variant polypeptides or fusion polypeptides can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the IL-18 variant polypeptide or fusion polypeptide). Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0191] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells, e.g., enables the vector to replicate independently of the host chromosomal DNA. This sequence may include an origin of replication or autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeast, and viruses. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may be used because it contains the early promoter).

[0192] Expression and cloning vectors can contain a selection gene or selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) complement a nutritional deficiency; or (c) supply vital nutrients unavailable in complex media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Other examples of selectable markers suitable for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding an IL-18 variant polypeptide or fusion polypeptide, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. For example, Chinese hamster ovary (CHO) cell lines transformed with the DHFR gene and deficient in endogenous DHFR activity are identified by culturing the transformants in culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR.

[0193] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an IL-18 variant polypeptide or fusion polypeptide of interest, a wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR) 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.

[0194] Expression and cloning vectors generally contain a promoter recognized by the host organism and operably linked to the nucleic acid encoding the IL-18 variant polypeptide or fusion polypeptide. Suitable promoters for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter systems, and hybrid promoters, such as the tac promoter. However, other known bacterial promoters are also suitable. Eukaryotic promoter sequences are known. Yeast promoters are well known in the art and can contain inducible promoters / enhancers that are regulated by growth conditions. Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins. 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 an IL-18 variant polypeptide or fusion polypeptide from a vector in a mammalian host cell can be controlled, for example, by a promoter obtained from a viral genome. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate-early promoter of the human cytomegalovirus is conveniently obtained as a HindIII restriction fragment. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0195] Transcription of a DNA encoding an IL-18 variant polypeptide or fusion polypeptide described herein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers from eukaryotic cell viruses are used.

[0196] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA.

[0197] Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-mentioned prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes suitable for this purpose include fungi, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding IL-18 polypeptide variants or fusion polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism. Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized," resulting in the production of IL-18 variant polypeptides or fusion polypeptides with partial or fully human glycosylation patterns. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006).

[0198] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco may also be utilized as hosts.

[0199] Suitable host cells for expressing glycosylated IL-18 variant polypeptides or fusion polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori.

[0200] 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 include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney (CV1 ATCC CCL 70), African green monkey kidney (VERO-76, ATCC CRL-1587), human cervical carcinoma (HELA, ATCC CCL 2), canine kidney (MDCK, ATCC CCL 34), and buffalo rat liver (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and the human hepatoma line (Hep G2). Other useful mammalian host cell lines include DHFR - These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0201] The host cells of the present disclosure can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue No. 30,985 may be used as the culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (chloride, etc.). Other suitable supplements may be added, such as phosphate buffers (e.g., sodium, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug Gentamicin™), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0202] When using recombinant technology, IL-18 variant polypeptide or fusion polypeptide can be produced intracellularly in the periplasmic space or directly secreted into the culture medium.When the polypeptide is produced intracellularly, the first step is to remove particulate debris, host cells, or lysed fragments, for example, by centrifugation or ultrafiltration.Carter et al., Bio / Technology 10:163-167(1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli.

[0203] Polypeptide compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being one of the preferred purification steps. In some embodiments, the IL-18 variant polypeptides or fusion polypeptides described herein comprise an epitope tag (e.g., a tag attached to the IL-18 variant polypeptide or fusion polypeptide via a cleavable linker) to facilitate purification. Exemplary epitope tags include, but are not limited to, 6xHis (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, e.g., enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0204] Methods of treating disease, activating hIL-18 receptor-mediated signaling, and stimulating antigen-experienced T cells or NK cells Also provided herein are methods of treating a disease or condition in an individual. The methods comprise administering an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein to an individual having the disease or condition. In some embodiments, the disease or condition is a proliferative disorder. In some embodiments, the proliferative disorder is cancer.

[0205] In some embodiments, provided herein are methods of activating hIL-18 receptor-mediated signals in an individual, the methods comprising administering to the individual an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein. In some embodiments, provided herein are methods of stimulating antigen-experienced T cells or NK cells in an individual, the methods comprising administering to the individual an IL-18 variant polypeptide or fusion polypeptide described herein, a nucleic acid described herein, a vector described herein, and / or a pharmaceutical composition described herein. In some embodiments of any of the methods herein, the individual is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.

[0206] Compositions, kits and articles of manufacture Also provided herein are compositions (eg, formulations) comprising the IL-18 variant polypeptides, fusion polypeptides, nucleic acids, vectors, or host cells described herein.

[0207] Suitable compositions are obtained by mixing an IL-18 variant polypeptide, fusion polypeptide, nucleic acid, vector, or host cell having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0208] Also provided are kits comprising the IL-18 variant polypeptides, fusion polypeptides, nucleic acids, vectors, or host cells containing the nucleic acids or vectors described herein, which may be useful in any of the methods of treatment described herein.

[0209] The kits of the present application are in suitable packaging, including, but not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). The kits may optionally include additional components, such as buffers and instructional information.

[0210] Accordingly, the present application also provides an article of manufacture. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, plastic packaging, and the like. Generally, the container holds the composition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle).

[0211] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.

[0212] Illustrative Embodiments 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 positions are relative to wild-type human IL-18 set forth in SEQ ID NO:1.

[0213] 2. The IL-18 variant polypeptide of embodiment 1, wherein the at least one mutation comprises a substitution at C38 and a substitution at C68.

[0214] 3. The IL-18 variant polypeptide of embodiment 2, wherein said at least one mutation comprises a S117C substitution.

[0215] 4. The IL-18 variant polypeptide of embodiment 2 or embodiment 3, wherein said substitution at C38 comprises C38I, C38S, C38V, C38L, or C38M, optionally wherein said substitution at C38 comprises C38I, C38S, or C38V.

[0216] 5. The IL-18 variant polypeptide of any one of embodiments 2 to 4, wherein said substitution at C68 comprises C68I, C68V, C68S, or C68D, and optionally wherein said substitution at C68 comprises C68S, C68I, or C68L.

[0217] 6. The IL-18 variant polypeptide of any one of embodiments 2 to 5, wherein said variant polypeptide does not comprise a mutation at C76.

[0218] 7. The IL-18 variant polypeptide of any one of embodiments 2 to 6, wherein said variant polypeptide comprises a) a substitution at C38, b) a substitution at C68, c) C76, and d) an S117C substitution.

[0219] 8. The IL-18 variant polypeptide of embodiment 7, wherein said variant polypeptide comprises a) I38, S68, C76, and C117, or b) V38, I68, C76, and C117.

[0220] 9. The IL-18 variant polypeptide of any one of embodiments 1 to 8, wherein said variant polypeptide does not comprise a mutation at C127.

[0221] 10. The IL-18 variant polypeptide of any one of embodiments 1 to 8, wherein said variant polypeptide comprises a mutation at C127, optionally wherein said variant polypeptide comprises C127A, C127Y, C127F, or C127L, and further optionally wherein said variant polypeptide comprises C127A.

[0222] 11. The IL-18 variant polypeptide of any one of embodiments 1-2, 4-5, and 9-10, wherein said at least one mutation comprises a substitution at C76, and optionally said substitution at C76 comprises C76V or C76Y.

[0223] 12. The IL-18 variant polypeptide of embodiments 1 to 5 and 9 to 11, wherein said at least one mutation comprises a substitution at C38, a substitution at C68, and a substitution at C76, optionally wherein said substitution at C38 comprises C38I, C38V, C38L, C38S, or C38M, optionally wherein said substitution at C68 comprises C68I, C68V, C68S, C68L, or C68D, and optionally wherein said substitution at C76 comprises C76Y, C76S, or C76V.

[0224] 13. The IL-18 variant polypeptide of embodiment 11, wherein said variant polypeptide comprises C38I, C68I, and C76V.

[0225] 14. The IL-18 variant polypeptide of embodiment 11, wherein said variant polypeptide comprises C38I, C68L, and C76Y.

[0226] 15. The IL-18 variant polypeptide of any one of embodiments 11 to 14, wherein said variant polypeptide further comprises a mutation at C127, optionally wherein said mutation at C127 is C127A, C127I, C127Y, S127S, C127F, or C127L.

[0227] 16. The IL-18 variant polypeptide according to any one of embodiments 11 to 14, wherein said variant polypeptide does not comprise C127S.

[0228] 17. The IL-18 variant polypeptide of any one of embodiments 1 to 16, wherein said 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.

[0229] 18. The IL-18 variant polypeptide of any one of embodiments 1 to 17, wherein said variant polypeptide comprises I38, S68, C76, C117, and C127.

[0230] 19. The IL-18 variant polypeptide of any one of embodiments 1 to 18, wherein said 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) compared to wild-type IL-18.

[0231] 20. The IL-18 variant polypeptide according to any one of embodiments 1 to 19, wherein said IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to said wild-type IL-18.

[0232] 21. The IL-18 variant polypeptide is about 5 x 10 -5 K less than M D 21. An IL-18 variant polypeptide according to any one of embodiments 1 to 20, which binds to IL-18Rα at

[0233] 22. The IL-18 variant polypeptide is about 5 x 10 -5 ~Approx. 5×10 -11 K of M D 22. An IL-18 variant polypeptide according to any one of embodiments 1 to 21, which binds to IL-18Rα at

[0234] 23. The IL-18 variant polypeptide is 5 x 10 -9 K over M D 23. An IL-18 variant polypeptide according to any one of embodiments 1 to 22, which binds to IL-18BP at

[0235] 24. An IL-18 variant polypeptide according to any one of embodiments 1 to 23, wherein said IL-18 variant polypeptide does not exhibit binding to IL-18BP.

[0236] 25. The IL-18 variant polypeptide of any one of embodiments 1 to 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, and optionally the mutation is G3P.

[0237] 26. The IL-18 variant polypeptide of any one of embodiments 1 to 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.

[0238] 27. The IL-18 variant polypeptide of any one of embodiments 1 to 26, wherein said variant polypeptide comprises a mutation at residue D54, said mutation being selected from the group consisting of D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T, optionally wherein said mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0239] 28. The IL-18 variant polypeptide of any one of embodiments 1 to 27, wherein said variant polypeptide comprises a mutation at residue N91, said mutation being selected from the group consisting of N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W, and optionally said mutation being selected from the group consisting of N91V, N91A, N91G, and N91S.

[0240] 29. The IL-18 variant polypeptide of any one of embodiments 1 to 28, wherein said variant polypeptide further comprises a mutation at residue R104, said mutation being selected from the group consisting of R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N, and optionally said mutation being selected from the group consisting of R104S, R104Y, and R104T.

[0241] 30. The IL-18 variant polypeptide according to any one of embodiments 1 to 28, wherein said variant polypeptide does not comprise a mutation at residue R104.

[0242] 31. The IL-18 variant polypeptide of any one of embodiments 1 to 30, wherein said variant polypeptide comprises a mutation at residue Q56, said mutation being 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 said mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0243] 32. The IL-18 variant polypeptide according to any one of embodiments 1 to 30, wherein said variant polypeptide does not comprise a mutation at residue Q56.

[0244] 33. The IL-18 variant polypeptide of any one of embodiments 1 to 32, wherein said variant polypeptide comprises a mutation at residue P57, said mutation being selected from the group consisting of P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D, and optionally said mutation being selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0245] 34. The IL-18 variant polypeptide according to any one of embodiments 1 to 32, wherein said variant polypeptide does not comprise a mutation at residue P57.

[0246] 35. The IL-18 variant polypeptide of any one of embodiments 1 to 34, wherein the variant polypeptide comprises mutations at G3, E6, D54, and N91, and optionally wherein the variant polypeptide comprises the mutations: (i) G3P, (ii) E6R or E6K, (iii) D54W, D54H, D54S, or D54Q, and (iv) N91V, N91A, N91G, or N91S.

[0247] 36. The IL-18 variant polypeptide of any one of embodiments 1 to 31, 33, and 35, wherein the variant polypeptide further comprises mutations at Q56 and P57.

[0248] 37. The IL-18 variant polypeptide of any one of embodiments 17 to 36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1 to 299, except that the variant polypeptide comprises S38, S68, S76, S117, and C127.

[0249] 38. The IL-18 variant polypeptide of any one of embodiments 17 to 36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1 to 299, except that the variant polypeptide comprises S38, S68, S76, S117, and S127.

[0250] 39. The IL-18 variant polypeptide of any one of embodiments 17 to 36, wherein the variant polypeptide comprises a polypeptide identical to any one of SEQ ID NOs: 1 to 299, except that the variant polypeptide comprises I38, S68, C76, C117, and C127.

[0251] 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 that the variant polypeptide comprises V38, I68, C76, C117, and A127.

[0252] 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 that the variant polypeptide comprises I38, I68, V76, S117, and I127.

[0253] 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 that the variant polypeptide comprises I38, L68, Y76, S117, and C127.

[0254] 43. The IL-18 variant polypeptide of any one of embodiments 1 to 42, wherein the variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 307 to 312 and 314 to 317.

[0255] 44. A fusion polypeptide comprising an IL-18 variant polypeptide according to any one of embodiments 1 to 43 and a half-life extending moiety, optionally wherein said half-life extending moiety comprises an Fc domain, further optionally wherein said Fc domain comprises a human IgG Fc domain, further optionally wherein said Fc domain is a modified Fc domain with reduced effector function, further optionally wherein said Fc domain comprises a human IgG1 Fc domain comprising the N297A mutation (EU numbering).

[0256] 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.

[0257] 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.

[0258] 47. A dimer comprising two fusion polypeptides according to any one of embodiments 44 to 46, wherein the dimer is optionally a) a homodimer or b) a heterodimer.

[0259] 48. A nucleic acid encoding an IL-18 variant polypeptide according to any one of embodiments 1 to 43 or a fusion polypeptide according to any one of embodiments 44 to 46.

[0260] 49. A vector comprising the nucleic acid of embodiment 48.

[0261] 50. A host cell comprising a nucleic acid according to embodiment 48 or a vector according to embodiment 49.

[0262] 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.

[0263] 52. A pharmaceutical composition comprising an IL-18 variant polypeptide according to any one of embodiments 1 to 43, a fusion polypeptide according to any one of embodiments 44 to 46, a nucleic acid according to embodiment 48, or a vector according to embodiment 49.

[0264] 53. A method for treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 52.

[0265] 54. A method for activating a hIL-18 receptor-mediated signal in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 52.

[0266] 55. A method for 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.

[0267] 56. A method for increasing the yield and / or purity of an IL-18 variant polypeptide produced from a host cell, comprising deleting one or more cysteines at positions 38, 68, 76, and / or 127 of the IL-18 polypeptide, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 set forth in SEQ ID NO:1.

[0268] 57. The method of embodiment 56, comprising removing at least two cysteines at positions 38 and 68, optionally wherein removing said cysteine ​​at position 38 comprises a substitution of C38 selected from the group consisting of C38I, C38S, C38V, C38L, and C38M, and optionally wherein removing said cysteine ​​at position 68 comprises a substitution of C68 selected from the group consisting of C68I, C68S, C68V, C68S, and C68D.

[0269] 58. The method of embodiment 56 or embodiment 57, comprising introducing an S117C mutation and retaining a cysteine ​​at position 76.

[0270] 59. The method of any one of embodiments 56-58, comprising removing the cysteine ​​at position 127, optionally wherein removing the cysteine ​​at position 127 comprises substituting C127 selected from the group consisting of C127S, C127A, C127I, C127Y, C127F, and C127L.

[0271] 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 substituting C76 selected from the group consisting of C76S, C76V, and C76Y.

[0272] 61. The method of any one of embodiments 56 to 60, comprising: a) introducing into the IL-18 polypeptides C38S, C68S, and C76S; b) introducing into the IL-18 polypeptides C38S, C68S, C76S, and C127S; c) introducing into the IL-18 polypeptides C38I, C68S, S117C; d) introducing into the IL-18 polypeptides C38V, C68I, S117C, C127A; e) introducing into the IL-18 polypeptides C38I, C68I, C76V, and C127I; or f) introducing into the IL-18 polypeptides C38I, C68L, and C76Y.

[0273] 62. The method of any one of embodiments 56 to 61, wherein the host cell is a mammalian cell, optionally wherein the host cell is a CHO cell or a HEK293 cell.

[0274] 63. The method of any one of embodiments 56-62, wherein the IL-18 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-299. [Example]

[0275] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. The following examples are intended to be purely illustrative of the present application and therefore should not be construed as limiting the present application in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0276] Example 1: Construction of a yeast expression library of IL-18 variant polypeptides that do not bind to IL-18BP Interleukin-18 ("IL-18") stimulates T cells and NK cells by binding to its heterodimeric receptor complex 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 binding of IL-18BP to IL-18 interferes with IL-18 interaction with IL-18Rα. To develop variants of human IL-18 ("hIL-18") that do not bind to IL-18BP or have reduced binding affinity for IL-18BP, artificial intelligence-guided molecular design methods were used to identify amino acid positions within wild-type hIL-18 for mutation. A yeast library representing hIL-18 variant polypeptides containing the identified mutation(s) compared to wild-type IL-18 (SEQ ID NO: 1) was then constructed using NNK mutagenesis, resulting in a final library diversity of approximately 2.72 x 10 8 For expression studies, a c-myc tag was fused to each of the IL-18 variants.

[0277] Example 2: Screening for IL-18 variant polypeptides that bind to human IL-18Rα Using a positive / negative selection strategy, the library constructed in Example 1 was screened to identify IL-18 variant polypeptides that bind to hIL-18Rα, but not hIL-18BP or IL-18 variant polypeptides with reduced binding affinity for hIL-18BP compared to wild-type IL-18. Briefly, recombinant hIL-18Rα extracellular domain ("hIL-18Rα ECD") was labeled with biotin and immobilized on a first set of streptavidin (SA) or anti-biotin magnetic microbeads for magnetic-activated cell sorting (MACS)-based screening. Recombinant hIL-18BP was immobilized on a second set of SA or anti-biotin magnetic microbeads. Three rounds of selection were performed on the library constructed in Example 1 using hIL-18Rα ECD-bound beads and hIL-18BP-bound beads, as shown in Figure 1A.

[0278] Next, (a) higher or equal affinity for hIL-18Rα compared to WT hIL-18, and (b) (e.g., 10 -3 K over M D To further enrich IL-18 variant polypeptides that showed no detectable or reduced binding to hIL-18BP (see Figure 1A), two additional rounds of FACS-based kinetic competition screening (i.e., rounds 4 and 5, as shown in Figure 1A) were performed. Single yeast cells were then isolated from the output pools of rounds 4 and 5. Plasmids encoding the isolated IL-18 variant polypeptides were extracted from the yeast and sequenced via next-generation sequencing (NGS).

[0279] After each round of screening, the output library size was determined by FACS. The binding ability of the output library to 50 nM hIL-18Rα ECD or 1 μM hIL-18BP was also confirmed by FACS. As shown in Figure 1B, after the above screening, most of the IL-18 variants did not bind to hIL-18BP or showed reduced binding to hIL-18BP, while their binding ability to hIL-18Rα ECD was maintained or increased.

[0280] Example 3: Single clone fluorescence activated cell sorting (FACS) of yeast cells displaying IL-18 variant polypeptides. Following the screening described in Example 2, single-clone FACS analysis was performed to verify the binding of selected yeast cell surface-expressed IL-18 variant polypeptides from rounds 4 and 5 to 10 nM hIL-18Rα-ECD. Briefly, the same density of each yeast clone was incubated with 10 nM IL-18Rα-ECD labeled with a fluorescent probe, followed by a washing step. The proportion of fluorescently positive IL-18Rα ECD-binding yeast in each clonal population was then analyzed.

[0281] As exemplarily shown in Table 1 and FIG. 1C (which shows, as examples, WT IL-18 (SEQ ID NO: 1), and clones M12 (SEQ ID NO: 39) and M17 (SEQ ID NO: 43)), of the 192 IL-18 variant clones tested, 117 (i.e., SEQ ID NOs: 2-118) were identified as positive clones (more than 0.1% of cells in the population of clones have fluorescence). SEQ ID NO: 1 is wild-type human IL-18. Table 1: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0282] Example 4: Expression and purification of recombinant hIL-18 The above-mentioned WT hIL-18 and selected IL-18 variant polypeptides were expressed in the E. coli system. Briefly, the PET30a plasmid containing the codon-optimized sequence encoding WT hIL-18 or hIL-18 variant polypeptides was transfected into E. coli BL21(DE3) cells. The transfected E. coli cells were then grown in culture medium, and the expressed polypeptides were harvested and purified.

[0283] Figures 2A-2F show WT hIL-18 and five exemplary variants (M12, MM5, WM6, M17, and M24) with greater than 95% purity as determined via SDS-PAGE and SEC-HPLC.

[0284] Example 5: Affinity of IL-18 variant polypeptides for hIL-18Rα The affinity of IL-18 variant polypeptides for hIL-18Rα was assessed by Biolayer Interferometry (BLI) on an Octet RED96 using a Protein A sensor coated with hIL-18Rα ECD-Fc fusion protein, with WT hIL-18 used as a control. The binding affinities of WT hIL-18 and IL-18 variant polypeptides for hIL-18Rα ECD-Fc were shown in Table 2A. [Table 2A-1] [Table 2A-2]

[0285] The mutations of these IL-18 variant polypeptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM14, corresponding to SEQ ID 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) are shown in Table 2B. [Table 2B-1] [Table 2B-2]

[0286] Example 6: Functional characterization of IL-18 variant polypeptides 6.1 Activation of hIL-18 Receptor-Mediated Signaling by IL-18 Variant Polypeptides The NFκB-luc / hIL-18RαRβ HEK293 cell line, stably expressing hIL-18Rα, hIL-18Rβ, and an NFκB luciferase reporter gene, was used as reporter cells to determine the ability of IL-18 variant polypeptides in activating hIL-18 receptor-mediated signaling.

[0287] Briefly, reporter cells were initially plated at 3.0 × 10 per well. 4 Cells were seeded at a density of 1000x1000 cells / well into wells of a white 96-well plate. WT hIL-18 or IL-18 variant polypeptides were serially diluted in medium in the presence (a) and absence (b) of 1 μg / mL hIL-18BP-hFc. Luminescence intensity was measured after 16 hours of incubation at 37°C.

[0288] As shown in Figure 3 and Table 3, all IL-18 variant polypeptides tested demonstrated efficacy in activating IL-18 receptor-mediated signaling in a dose-dependent manner. Unlike hIL-18, whose activity was significantly reduced (approximately 80-fold) in the presence of 1 μg / mL hIL-18BP-hFc, IL-18 variant polypeptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, and WM10-WM11, corresponding to SEQ ID NOs: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-150) showed an EC 50 The IL-18 variant polypeptides showed significantly less effect of hIL-18BP in terms of activating hIL-18 receptor-mediated signaling. One exception was WM6, whose EC 50 showed approximately a 12-fold loss of titer in the presence of 1 μg / Ml hIL-18BP-hFc, which is still far superior to WT hIL-18. [Table 3-1] [Table 3-2]

[0289] 6.2 Human PBMC-based hIFNγ release assay A PBMC-based assay was performed to test the ability of IL-18 variant polypeptides to activate T / NK cells and induce hIFNγ release. Briefly, human PBMC cells were cultured at 5.0×10 per well. 5Cells were seeded into wells of a 96-well plate at a density of 1 / 3 cells and cultured for 5 hours before treatment. WT hIL-18 or IL-18 variant polypeptides were serially diluted in 1 ng / mL human IL-12 in the presence or absence of 1 μg / mL hIL-18BP-hFc (a) and then added to the wells. After 16 hours of incubation at 37°C, the culture medium was collected from each well, and hIFNγ concentrations were measured via FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0290] As shown in Figure 4, the tested IL-18 variant polypeptides demonstrated efficacy in inducing hIFNγ release from PBMCs in a dose-dependent manner. In addition, as shown in Table 4, the activity of WT hIL-18 was significantly attenuated in the presence of 1 μg / mL hIL-18BP-hFc, resulting in an EC 50 In contrast, IL-18 variant polypeptides (i.e., M11 to M13, M15, M17, M21, M24, M29, M32 to M33, M35 to M36, MM5, WM4 to WM6, WM8, and WM10 to WM11, corresponding to SEQ ID NOs: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116 to 117, 133, and 143 to 150) showed a 2-fold or less EC 50 The results showed that the IL-18 variant polypeptides were significantly less affected by hIL-18BP in terms of hIFNγ induction. [Table 4]

[0291] 6.3 Mouse splenocyte-based mIFNγ release assay To assess the in vivo efficacy of the IL-18 variant polypeptides of the present application, a mouse splenocyte-based mouse IFNγ (“mIFNγ”) release assay is performed as follows.

[0292] Briefly, freshly isolated mouse splenocytes were cultured at 6.5 × 10 cells per well. 5Cells were seeded into wells of a 96-well plate at a density of 1000x1000x1000 cells / well. WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL mouse IL-12 in the presence or absence of 1 μg / mL mouse IL-18BP-Fc (a) and then added to the wells. After 16 hours of incubation at 37°C, the culture medium was collected from each well and the mIFNγ concentration in the medium was measured via FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0293] Example 7: Generation and Characterization of IL-18 Variant Polypeptides Containing Serine to Cysteine ​​and / or Cysteine ​​to Serine Substitutions 7.1 Wild-type IL-18 or IL-18 polypeptide variants containing a cysteine ​​to serine substitution Although the potent immunostimulatory activity of IL-18 has been reported, recombinant IL-18 is mainly expressed in E. coli systems, which are less suitable for Good Manufacturing Practice (GMP)-compliant manufacturing standards that primarily use mammalian host cells. Human IL-18 has four unpaired cysteines and does not contain disulfide bonds. Of the four cysteines, C38, C68, and C76 are highly solvent-exposed, while C127 is partially exposed. When expressed, IL-18 tends to form undesirable aggregates due to the exposed free cysteines, resulting in reduced yield and / or purity. To improve the proper folding and stability of IL-18, i.e., to aim for a higher yield with acceptable purity, we engineered IL-18 variants by using cysteine ​​mutation and disulfide bond introduction strategies. The following experiments were performed to evaluate the effect of a Cys to Ser ("C→S") mutation on the druggability of IL-18 variants and on the expression of IL-18 variants in mammalian cells such as CHO cells and 293F cells. Wild-type IL-18 and IL-18 variant polypeptides M12 and MM5 were modified (or further modified) to contain C38S, C68S, and C76S substitutions to generate new variants containing the following amino acid sequences: IL-18 variant containing C38S, C68S, and C76S substitutions in a WT hIL-18 background ("IL-18-SSS"): YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKDSQPRGMAVTISVKSEKISTLSSENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 307) IL-18 variant containing C38S, C68S, and C76S substitutions in an M12 background ("M12-SSS"): YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 308) IL-18 variant containing C38S, C68S, and C76S substitutions in an MM5 background (“MM5-SSS”): YFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKLSTERGMAVTISVKSEKISTLSSENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 309)

[0294] Fc-fusion cytokines may improve pharmacokinetics and be suitable for widely applicable manufacturing 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, and the purity was 65%. In contrast, the yield of WT IL-18-Fc_N297A obtained after one-step Protein A purification was approximately 16 mg / L, with a purity of 11%. The yield of M12-SSS-Fc_N297A obtained after one-step Protein A purification was 5.6 mg / mL, with a purity of 72%. In contrast, M12-Fc_N297A was not expressed at detectable levels in 100 mL of CHO host cells. The yield of MM5-SSS-Fc_N297A obtained after one-step Protein A chromatography was 24.5 mg / L, with a purity of 46%. After a further purification step by gel filtration, the yield of MM5-SSS-Fc_N297 was 3.4 mg / L, with a purity of 99%. In contrast, MM5-Fc_N297A was not expressed at detectable levels in 100 mL of CHO host cells.

[0295] M12-SSS was further modified to include a C127S substitution to generate M12-SSSS: YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLASEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 310)

[0296] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU numbering) to generate M12-SSSS-Fc_N297A (SEQ ID NO: 322). M12-SSSS-Fc_N297A was expressed in CHO cells, harvested, and purified via protein A chromatography. Although the introduction of an extra C127S substitution (i.e., the fourth C→S substitution) resulted in better expression yield and purity than M12-Fc_N297A, the yield and purity of M12-SSSS-Fc_N297A were reduced compared to those of M12-SSS-Fc_N297A (data not shown), suggesting that the C127 mutation may compromise the conformational benefits of the first three cysteine ​​mutations.

[0297] Next, the potency of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A in activating IL-18 receptor-mediated signaling was characterized using the in vitro assay described in Section 6.1 of Example 6. The demonstrated potency of IL-18-SSS-Fc_N297A was comparable to that of wild-type IL-18 (see Figure 5A), whereas M12-SSS-Fc_N297A showed an approximately 7-fold loss in 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 of hIL-18BP-hFc ("BP"), whereas M12-SSS-Fc_N297A exhibited hIL-18BP-resistant activity in the presence of 1 μg / ml of hIL-18BP-hFc. See Figures 5A and 5B. As shown in Figure 5C, MM5-SSS-Fc_N297A showed a 220-fold loss in potency compared to MM5, but still exhibited hIL-18BP-resistant activity in the presence of 1 μg / ml of hIL-18BP-hFc. These results indicate that the three cysteine-to-serine substitutions do not affect the IL-18BP-resistant properties. These data suggest that mutations at C38, C68, and C76, which prevent undesired intramolecular and intermolecular disulfide bond formation at the three exposed cysteines, are important 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.

[0298] 7.2 IL-18 Polypeptides or IL-18 Polypeptide Variants Comprising Amino Acid Substitutions That Remove and / or Introduce Cysteine ​​Residues The following experiments were performed to evaluate the effects of substitutions that delete intrinsic cysteines (i.e., cysteines naturally occurring in the polypeptide sequence) and / or introduce disulfide bonds on the druggability, expression, and purification of IL-18 variant polypeptides. M12 is used as a representative variant for engineering strategy selection. First, in silico screening based on molecular dynamics simulations was performed, followed by AI-based stability assessment, to separately identify mutation hotspots for each of the four cysteines of M12 (C38, C68, C76, and C127), as listed. See Table 5. In addition, structure-guided design was used to identify amino acids located in spatial positions that, when substituted with Cys residue(s), could enable the formation of new non-native disulfide bond(s). The mutation strategies shown in Table 5 (i.e., DB6, DB7, DB8, DB9, and DB10) were proposed. It was hypothesized that introducing the S117C mutation while retaining C76 may promote a disulfide bond between C117 and C76. [Table 5]

[0299] The mutation strategies in Table 5 were introduced into the IL-18 polypeptide variant M12 to generate new variants M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10. Single-site mutation hotspots were identified from in silico screening. In silico screening was performed based on molecular dynamics simulations, and following AI-based stability assessment, mutation hotspots for each of the four cysteines (C38, C68, C76, and C127) of M12 were identified as listed. M12-DB6 [ka] M12-DB7 [ka] M12-DB8 [ka] M12-DB9 [ka] M12-DB10 [ka]

[0300] The C-terminus of each of the M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10 variants was fused to a human IgG1 Fc variant containing an N297A substitution (EU numbering). The resulting fusion polypeptides, M12-DB6-Fc_N297A (SEQ ID NO: 323), M12-DB7-Fc_N297A (SEQ ID NO: 324), M12-DB8-Fc_N297A (SEQ ID NO: 325), M12-DB9-Fc_N297A (SEQ ID NO: 326), and M12-DB10-Fc_N297A (SEQ ID NO: 327), were expressed in CHO cells, harvested, and purified by one-step protein A chromatography. The purified preparations were analyzed by SEC and SDS-PAGE (data not shown). See Table 6 below. [Table 6]

[0301] M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A showed significantly improved expression yield and / or purity (see Table 6) compared to either M12-Fc_N297A or M12-SSS-Fc_N297A. 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) with a purity of 89.8% (determined by SEC-HPLC) after one-step Protein A purification. 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.

[0302] The high yields and purities of DB6 and DB7, especially the high yields, are remarkable. The M12-DB6 format is approximately 60-fold higher than the SSS format. The data suggest that the strategy of removing cysteines at positions 38 and 68 while introducing C117 to promote a disulfide bond between C117 and C76 is highly advantageous. The low yield of DB7 and the undetectable yield of DB8 suggest that the C127X substitution may not further improve polypeptide production in mammalian cells. Mutations at the partially exposed C127 site require delicate design. Based on the C38X+C68X+C76+S117C strategy, the 127 site prefers C over A, but the hydrophobic 127I dramatically affects the proper folding of the IL-18 polypeptide. We further tested the corresponding variant IL-18-DB6 polypeptide with C127N, C127T, and C127S, and all three could be expressed at levels comparable to DB6. Considering the nature and consequences of the partially exposed 127 site, introducing amino acids with hydrophobic side chains at the 127 site should be avoided under this strategy.

[0303] Next, the potency of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in activating IL-18 receptor-mediated signaling was characterized using the in vitro assay described in Section 6.1 of Example 6. As shown in Figures 6A-6D and Table 7 below, M12-DB6-Fc_N297A (Figure 6A), M12-DB7-Fc_N297A (Figure 6B), M12-DB9-Fc_N297A (Figure 6C), and M12-DB10-Fc_N297A (Figure 6D) showed much higher potency than WT IL-18 in activating IL-18 receptor-mediated signaling. The signaling activity of tested M12-DB6-Fc_N297A (FIG. 6A), M12-DB7-Fc_N297A (FIG. 6B), and M12-DB9-Fc_N297A (FIG. 6C) was not inhibited in the presence of 1 μg / ml hIL-18BP-hFc (see Table 7). [Table 7]

[0304] Next, the "DB6" substitutions, i.e., C38I, C68S, and S117C, were introduced into variant MM5 to generate MM5-DB6: MM5-DB6 [ka]

[0305] The "DB6" substitution was also introduced into wild-type IL-18 to generate WT IL18-DB6: WT IL18-DB6 [ka]

[0306] An additional variant, WT-DBo, was designed by introducing a S117C substitution into wild-type IL-18: WT IL-18-DBo

change

[0307] The C-terminus of each of WT IL-18-DB6, WT IL-18-DBo, and MM5-DB6 was fused to the N-terminus of a human IgG1 Fc variant containing the 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 via Protein A chromatography. The purified preparations were analyzed by SEC and SDS PAGE (data not shown). As shown in Table 8 below, after the same expression and purification procedures, there was no detectable protein for WT IL18-DBo-Fc_N297A. The expression yield of WT IL18-DB6-Fc was 157 mg / L, and WT IL18-DB6-Fc_N297A was 91.6% pure (determined by SEC-HPLC) after one-step Protein A chromatography (see Table 8). This data confirmed the highly advantageous strategy of promoting a disulfide bond between C76 and C117 while removing cysteines at positions 38 and 68. Such data also indicate that simply introducing a disulfide bond between C76 and C117 may not be sufficient to improve fusion polypeptide expression in mammalian cells. Improved expression in mammalian cells is achieved by fusion polypeptides containing IL-18 variants, including (i) an engineered C76-C117 disulfide bond, (ii) a C38 substitution (see Table 5), and (iii) a C68 substitution (see Table 5). MM5-DB6-Fc_N297A consistently shows much higher expression yield and purity than MM5-SSS-Fc_N297A (see Table 8). Such data also suggest that a C38X+C68X+C76+S117C mutation strategy, such as the "DB6" or "DB7" substitution, is suitable for improving the yield of WT IL-18 and IL-18 variant polypeptides during production in mammalian cells and after purification.

[0308] 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 showed comparable expression yields and purity in CHO cells when compared to M12-DB6-Fc_N297A or MM5-DB6-Fc_N297A, respectively. After one-step protein A chromatography purification, Fc_N297A-MM5-DB6 was approximately 97% pure (determined by SEC-HPLC), and Fc_N297A-M12-DB6 was 89% pure (determined by SEC-HPLC). These data suggest that the cystine mutation + disulfide bond introduction strategy is suitable for Fc fusion in either fusion order. [Table 8]

[0309] Next, the potency of WT IL18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 in activating IL-18 receptor-mediated signaling was characterized using the in vitro assay described in section 6.1 of Example 6. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A exhibited potent IL-18 receptor cell activation signals that were stronger than that of WT IL-18 (see Figures 7A, 7B, and Table 9). Fc_N297A-MM5-DB6 showed similar potency to WT IL-18, whereas Fc_N297A-M12-DB6 produced a much stronger activation signal on IL-18 reporter cells (see Figures 7C, 7D, and Table 9). In the presence of 1 μg / mL IL-18BP-hFc, the activity of WT IL18-DB6-Fc_N297A was significantly reduced, whereas the activity of MM5-DB6-Fc_N297A was resistant to inhibition by IL-18BP (see Figures 7A, 7B, and Table 9). Both M12-DB6-Fc_N297A and MM5-DB6-Fc_N297A maintained IL-18BP-resistant activity, while the activity of WT IL18-DB6-Fc_N297A continued to be reduced by IL-18BP, indicating that the "DB6" set of mutations (i.e., C38I, C68S, and S117C, see Table 5) improves the expression profile of IL-18 and IL-18 variants without affecting IL-18BP dependence.

[0310] Notably, these results demonstrate that the strategy of introducing the C38X+C68X+C76+S117C mutations into a WT IL-18-Fc fusion polypeptide or an IL-18 variant Fc fusion polypeptide dramatically improves the expression yield of the fusion polypeptide during production in mammalian cells and improves the yield of purified fusion polypeptide after chromatography, without affecting IL-18BP resistance activity. Furthermore, WT IL-18 and IL-18 variants engineered to contain the "DB6" set of mutations exhibit higher potency in activating IL-18 receptor-mediated signaling. Such results were observed for fusion polypeptides in which the C-terminus of the Fc region was fused to the N-terminus of an IL-18 variant, as well as for fusion polypeptides in which the C-terminus of an IL-18 variant was fused to the N-terminus of the Fc domain (see Table 10). [Table 9]

[0311] For a summary of the above data, see Table 10 below.

[0312] [Table 10]

[0313] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It wil...

Claims

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

1.

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

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

4. 4. The method of claim 1, wherein introducing a cysteine ​​at position 117 or 76 comprises substituting the amino acid at position 117 or 76 with cysteine.

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

6. 6. The method of claim 1, further comprising removing the cysteines at one or both of positions 38 and 68 when the IL-18 polypeptide contains cysteines at one or both of positions 38 and 68.

7. 7. The method of claim 6, wherein removing one or both of the cysteines at positions 38 and / or 68 comprises substituting the cysteine ​​at positions 38 and / or 68 with a different amino acid.

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

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

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

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

12. 12. The method of claim 11, wherein 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.

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

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

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

16. 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, and optionally 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, 1 16-117, 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, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150, and optionally 16. The method of any one of claims 1 to 15, 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, 29, and 117, 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, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29, and 117, and further optionally, the IL-18 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 39, and 133, 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, 39, and 133.

17. 17. The method of any one of claims 1 to 16, wherein the IL-18 variant polypeptide comprises an amino acid sequence set forth 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. 1. An IL-18 variant polypeptide comprising the amino acid sequence set forth 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.

20. 1. 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. 21. The IL-18 variant polypeptide of claim 20, wherein the IL-18 variant polypeptide does not contain a cysteine ​​at position 38 or the IL-18 variant polypeptide does not contain a cysteine ​​at position 68.

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

26. 25. 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 the group consisting of cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, and further optionally wherein the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, and threonine.

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

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

29. 28. The IL-18 variant polypeptide of claim 27, wherein the IL-18 variant polypeptide comprises V38, I68, C76, C117, and optionally, 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. 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. 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, and further optionally wherein the human IgG Fc domain is a human IgG1 domain.

33. 33. The fusion polypeptide of claim 31 or claim 32, wherein the Fc domain is a modified Fc domain with reduced effector function, and optionally the Fc domain comprises a human IgGl 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. 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. 37. The fusion polypeptide of any one of claims 30-36, wherein the fusion polypeptide comprises an 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.

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

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

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

41. A nucleic acid encoding an IL-18 variant polypeptide according to any one of claims 18 to 29 or a fusion polypeptide according to 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 of claim 41 or claim 42.

44. 44. The 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. 1. A method for producing an IL-18 variant polypeptide or fusion polypeptide, comprising: (a) culturing the host cell of claim 44 under conditions in which the IL-18 variant polypeptide or fusion polypeptide is expressed; (b) recovering the IL-18 variant polypeptide or fusion polypeptide produced by the host cell.

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

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

48. 47. 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. 47. A method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to said individual an effective amount of the pharmaceutical composition of claim 46.

50. 1. A method of 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, and wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 as set forth in SEQ ID NO:

1.

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

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