Activatable interleukin-18 polypeptide

Abstract

Activatable interleukin-18 (IL-18) polypeptides are provided, comprising: (a) an IL-18 polypeptide (e.g., a wild-type IL-18, an IL-18 variant polypeptide, or a fusion protein comprising a wild-type IL-18 or an IL-18 variant polypeptide); and (b) a masking moiety, wherein the wild-type IL-18, an IL-18 variant polypeptide, or a fusion protein comprising a wild-type IL-18 or an IL-18 variant polypeptide is linked to the masking moiety via a cleavable linker. Also provided are related nucleic acids, vectors, host cells, methods of producing the IL-18 variant polypeptides, pharmaceutical compositions comprising the activatable IL-18 polypeptides, and therapeutic uses of such pharmaceutical compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to international applications PCT / CN2023 / 072869, filed January 18, 2023, 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 (233002001641SEQLIST.xml, size: 445,962 bytes, and creation date: January 17, 2024) are incorporated herein by reference in their entirety.

[0003] Field of application The present application relates to activatable interleukin-18 (IL-18) polypeptides comprising: (a) an IL-18 polypeptide (e.g., wild-type IL18, a variant thereof comprising at least one amino acid substitution relative to wild-type IL-18 (e.g., wild-type human IL-18), or a fusion polypeptide comprising any of the foregoing); and (b) a masking moiety (e.g., a receptor polypeptide, receptor, or subdomain(s)). The present application also relates to methods of making such activatable IL-18 polypeptides and methods of using such activatable IL-18 polypeptides for therapeutic applications. [Background technology]

[0004] Background of the application Interleukin-18 (IL-18, also known as interferon-γ-inducing factor) is a proinflammatory cytokine that 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). 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. 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 hindered by its limited efficacy. 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 preclinical models (e.g., mice) and clinical trials. IL-18BP-resistant IL-18 variant polypeptides have been engineered that retain signaling potential but are not inhibited by IL-18BP (see, e.g., Zhou et al. (2020) Nature 583:609-614).

[0005] Furthermore, because IL-18 can regulate both innate and adaptive immunity, its dysregulation can lead to autoimmune or inflammatory diseases. For example, IL-18 plays a role in Th1-mediated immune responses by activating NK cells and Th1 cells, which are involved in host defense against intracellular pathogen infections through IFNγ production. IL-18 also induces the production of TNF and FasL, which are involved in growth, survival, and apoptosis. IL-18 is thought to play a role in driving Th2 responses by inducing the production of IL-4 and IL-13 in T cells, NK cells, mast cells, and basophils. Other mediators induced by IL-18 include inducible nitric oxide; cyclooxygenase (Cox-2); proinflammatory cytokines IL-1β and IL-6; chemokines IL-8, MCP-1, and MIP-1α; the intracellular adhesion molecule ICAM-1; and the growth factor GM-CSF. IL-18 also induces the production of cytokines such as IL-12 and IL-2. Given the pleiotropic functions of IL-18, regulation of IL-18 activity is essential to prevent aberrant immune responses.

[0006] There is a need in the art for compositions and methods for activating IL-18 receptor-mediated signaling in an individual in a regulated manner, e.g., by either wild-type IL-18 or IL-18BP-resistant IL-18 variant polypeptides, and / or stimulating antigen-experienced T cells or NK cells in an individual in a regulated manner (e.g., activation at the site of a tumor) 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]

[0007] [Non-Patent Document 1] Guo et al.(2012)Trends Immunol.33,598-606

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Summary of the Invention

[0008] Summary 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.

[0009] In some embodiments, the IL-18 polypeptide comprises at least one mutation at a residue selected from the group consisting of G3, Q24, L29, Q56, P57, M60, A61, N91, K96, R104, R107, K140, N155, and I149, wherein the amino acid positions are relative to wild-type human IL-18 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.

[0010] 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.

[0011] In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, 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.

[0012] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided, comprising at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, 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.

[0013] In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits reduced binding to IL-18 binding protein (IL-18BP) 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).

[0014] In some embodiments, the variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from the group consisting of G3P, G3D, G3E, G3F, G3K, G3T, G3W, G3N, and G3S. In some embodiments, the mutation is G3P. In some embodiments, the variant polypeptide comprises a mutation at residue E6, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6H, E6L, E6M, E6N, E6P, E6Q, E6V, E6W, and E6Y. In some embodiments, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from the group consisting of E6R and E6K.

[0015] In some embodiments, the variant polypeptide comprises a mutation at residue D54, wherein the mutation is selected from the group consisting of D54W, D54H, D54I, D54S, D54Q, D54L, D54M, D54Y, D54P, D54R, D54A, D54F, D54G, D54V, and D54T. In some embodiments, the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0016] In some embodiments, the variant polypeptide comprises a mutation at residue N91, wherein the mutation is selected from the group consisting of N91V, N91A, N91D, N91F, N91G, N91S, N91I, N91P, N91R, N91L, N91T, N91C, N91K, N91Y, and N91W. In some embodiments, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0017] In some embodiments, the variant polypeptide further comprises a mutation at residue R104, wherein the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104M, R104V, R104A, R104C, R104E, R104G, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from the group consisting of R104S, R104Y, and R104T.

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

[0019] In some embodiments, the variant polypeptide comprises a mutation at residue Q56, wherein the mutation is selected from the group consisting of Q56A, Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56I, Q56K, Q56W, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

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

[0021] 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.

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

[0023] In some embodiments, the variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the variant polypeptide comprises mutations: (i) G3P, (ii) E6R or E6K, (iii) D54W, D54H, D54S, or D54Q, and (iv) N91V, N91A, N91G, or N91S. In some embodiments, the variant polypeptide further comprises mutations at Q56 and P57.

[0024] In some embodiments, the variant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-299 and 307-318.

[0025] In another aspect, the application provides 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 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. 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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. A functional variant as described herein in the context of an IL-18 variant polypeptide refers to a variant that exhibits IL-18 function comparable to a reference IL-18 polypeptide (e.g., at least 50%, 60%, 70%, 75%, or 80%) of the function as measured, for example, by the method described in Example 1.

[0035] 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.

[0036] 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.

[0037] In another aspect, the application provides an IL-18 variant polypeptide produced by any of the above methods.

[0038] 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.

[0039] 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. 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In another aspect, the application provides a nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NOs: 333-335. In some embodiments, an activatable interleukin-18 (IL-18) polypeptide is provided, comprising (a) an IL-18 polypeptide and (b) a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker. In some embodiments, the masking moiety inhibits the IL-18 polypeptide from activating IL-18 receptor-mediated signaling when the masking moiety is linked to the IL-18 polypeptide via the cleavable linker. In some embodiments, the masking moiety comprises an IL-18 pro-peptide, an extracellular domain of IL-18Rα, an extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any one of the foregoing, or a variant of any one of the foregoing. In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 333, 336, and 353-356.

[0044] In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more proteases. In some embodiments, the one or more proteases are one or more tumor microenvironment (TME) proteases. In some embodiments, the one or more TME proteases are selected from the group consisting of urokinase-type plasminogen activator, matriptase, legumain, prostate-specific antigen, dipeptidyl peptidase, hepsin, matrix metalloproteinase, a disintegrin and metalloproteinase, human leukocyte elastase, proteinase 3, prourokinase, plasminogen, staphylokinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase. In some embodiments, the one or more TME proteases are matrix metalloproteases selected from the group consisting of matrix metalloprotease 1, matrix metalloprotease 2, matrix metalloprotease 3, matrix metalloprotease 8, matrix metalloprotease 9, matrix metalloprotease 10, matrix metalloprotease 12, and matrix metalloprotease 14. In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloprotease 9, matrix metalloprotease 10, and legumain. In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NOs: 372-377.

[0045] In some embodiments, the IL-18 polypeptide comprises wild-type IL-18. In some embodiments, the wild-type IL-18 comprises wild-type human IL-18. In some embodiments, the wild-type human IL-18 comprises the amino acid sequence of SEQ ID NO: 1.

[0046] In some embodiments, the IL-18 polypeptide comprises an IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18, or (ii) no binding to IL-18BP. 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 comprises any one of SEQ ID NOs: 2-299 and 307-318. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150.

[0047] In some embodiments, the IL-18 polypeptide comprises a fusion protein comprising (1) a wild-type IL-18 or IL-18 variant polypeptide and (2) an antibody Fc domain or variant thereof. In some embodiments, the IL-18 variant polypeptide of the fusion protein specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18, or (ii) no binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide of the fusion protein exhibits increased binding to IL-18Rα compared to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide of the fusion protein comprises any one of SEQ ID NOs: 2-299 and 307-318. In some embodiments, the human IgG1 Fc domain variant of the fusion protein comprises an N297A mutation (EU numbering). In some embodiments, the human IgG1 Fc domain variant comprises the amino acid sequence of SEQ ID NO: 371 or 390. In some embodiments, the C-terminus of the IL-18 variant polypeptide of the fusion protein is fused to the N-terminus of the human IgG Fc domain or variant thereof of the fusion protein. In some embodiments, the C-terminus of the human IgG Fc domain or variant thereof of the fusion protein is fused to the N-terminus of the IL-18 variant polypeptide of the fusion protein. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 378-389.

[0048] In some embodiments, the activatable IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 340-343, 345-352, 357-362, 364, 366-370, and 391-398.

[0049] In some embodiments, a dimer comprising two activatable IL-18 polypeptides described herein is provided. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0050] In some embodiments, nucleic acids encoding the activatable IL-18 polypeptides described herein are provided. In some embodiments, vectors comprising the nucleic acids described herein are provided. In some embodiments, host cells comprising the nucleic acids described herein or the vectors described herein are provided. In some embodiments, methods of producing an activatable IL-18 polypeptide are provided, the methods comprising: (a) culturing a host cell described herein under conditions in which the activatable IL-18 polypeptide is expressed; and (b) recovering the activatable IL-18 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 activatable IL-18 polypeptide.

[0051] In some embodiments, a pharmaceutical composition comprising an activatable IL-18 polypeptide described herein, a nucleic acid described herein, or a vector described herein is provided.

[0052] In some embodiments, methods are provided for treating a disease in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, methods are provided for activating IL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition described herein. In some embodiments, methods are provided for stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition described herein. In some embodiments, the stimulation comprises increasing the activity and / or number of antigen-experienced immune cells.

[0053] In some embodiments, the individual is a human.

[0054] 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.

[0055] 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]

[0056] [Figure 1A] 1 shows the results of biolayer interferometry experiments performed to assess the binding of WT hIL-18 and proIL-18 to hIL-18Rα.

[0057] [Figure 1B] Activation of hIL-18 receptor-mediated signaling by pro-IL-18, before and after treatment of pro-IL-18 with caspase-1, and by caspase-1-treated pro-IL-18 in the presence of hIL-18 binding protein.

[0058] [Figure 2A] 1 shows activation of hIL-18 receptor-mediated signaling by pro-IL-18-MMP9L before and after treatment of pro-IL-18-MMP9L with MMP9.

[0059] [Figure 2B] Figure 1 shows activation of hIL-18 receptor-mediated signaling by pro-IL-18-MMP10L.

[0060] [Figure 2C] 1 shows activation of hIL-18 receptor-mediated signaling by pro-IL-18-Legu L before and after treatment of pro-IL-18-Legu L with legumain.

[0061] [Figure 3A] Size-exclusion chromatography curves of proM12, proM12 cleaved with MMP9 ("proM12 cleaved"), and M12 are shown.

[0062] [Figure 3B] Size exclusion chromatography curves of proMM5, MMP9-cleaved proMM5 ("proMM5 cleaved"), and MM5 are shown.

[0063] [Figure 3C] Activation of hIL-18 receptor-mediated signaling by proM12, M12, proM12 cleavage, and proM12 cleavage plus hIL-18 binding peptide (BP) is shown.

[0064] [Figure 3D] Activation of hIL-18 receptor-mediated signaling by proMM5, MM5, proMM5 cleavage, and proMM5 cleavage plus hIL-18 binding peptide (BP) is shown.

[0065] [Figure 3E]Activation of hIL-18 receptor-mediated signaling by proM21, M21, and proM21 cleavage.

[0066] [Figure 3F] Activation of hIL-18 receptor-mediated signaling by proM13, M13, and proM13 cleavage.

[0067] [Figure 3G] Activation of hIL-18 receptor-mediated signaling by proM24, M24, and proM24 cleavage.

[0068] [Figure 3H] Activation of hIL-18 receptor-mediated signaling by proWM4, WM4, and proWM4 cleavage.

[0069] [Figure 3I] Activation of hIL-18 receptor-mediated signaling by proWM5, WM5, and proWM5 cleavage.

[0070] [Figure 3J] Activation of hIL-18 receptor-mediated signaling by proWM6, WM6, and proWM6 cleavage.

[0071] [Figure 3K] Activation of hIL-18 receptor-mediated signaling by proM12, M12, MMP9-digested proM12, and caspase-1-digested proM12 is shown.

[0072] [Figure 4A] 1 shows the results of a PBMC-based assay performed to assess the ability of M12, proM12, and proM12 cleavage in inducing hIFNγ release.

[0073] [Figure 4B]1 shows the results of a PBMC-based assay performed to assess the ability of MM5, proMM5, and proMM5 cleaved in inducing hIFNγ release.

[0074] [Figure 5A] Activation of hIL-18 receptor-mediated signaling by M12-DB6-Fc_N297A, Ra-M12-DB6-Fc_N297A, and Ra-M12-DB6-Fc_N297A cleavages.

[0075] [Figure 5B] Activation of hIL-18 receptor-mediated signaling by M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, and D12-M12-DB6-Fc_N297A truncations.

[0076] [Figure 5C] Activation of hIL-18 receptor-mediated signaling by M12-DB6-Fc_N297A, BPm-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297A cleavages.

[0077] [Figure 6A] 1 shows activation of hIL-18 receptor-mediated signaling by MM5-DB6-Fc_N297A, Ra-MM5-DB6-Fc_N297A, and Ra-MM5-DB6-Fc_N297A cleavages.

[0078] [Figure 6B] 1 shows activation of hIL-18 receptor-mediated signaling by MM5-DB6-Fc_N297A, D12-MM5-DB6-Fc_N297A, and D12-MM5-DB6-Fc_N297A cleavages.

[0079] [Figure 6C] Activation of hIL-18 receptor-mediated signaling by Fc_N297A-MM5-DB6, Fc_N297A-Ra-MM5-DB6, and Fc_N297A-Ra-MM5-DB6 cleavage.

[0080] [Figure 6D] Activation of hIL-18 receptor-mediated signaling by Fc_N297A-MM5-DB6, Fc_N297A-MM5-DB6-Ra, and Fc_N297A-MM5-DB6-Ra cleavage.

[0081] [Figure 7] Activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with linkers of variable lengths, and cleavage of Fc_N297A-M12-DB6-Ra by MMP9.

[0082] [Figure 8A] 1 shows activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with a multiprotease recognition linker, and cleavage of Fc_N297A-M12-DB6-Ra by MMP9. [Figure 8B] 1 shows activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with a multiprotease recognition linker, and cleavage of Fc_N297A-M12-DB6-Ra by MMP9.

[0083] [Figure 9A] 1 shows activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with the multiprotease recognition linker UM-2, and Fc_N297A-M12-DB6-Ra-UM2 cleaved by MMP9, MMP14, or uPA, respectively.

[0084] [Figure 9B]1 shows activation of hIL-18 receptor-mediated signaling by Fc_N297A-MM5-DB6, Fc_N297A-MM5-DB6-Ra with the multiprotease recognition linker UM-2, and Fc_N297A-MM5-DB6-Ra-UM2 cleaved by MMP9, MMP14, or uPA, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0085] overview The present application is based, in part, on Applicant's identification of an activatable IL-18 polypeptide comprising: (a) wild-type IL-18 (e.g., wild-type human IL-18), a variant thereof comprising at least one amino acid substitution relative to wild-type IL-18 (e.g., wild-type human IL-18), or a fusion polypeptide comprising any of the foregoing; and (b) a masking moiety. The masking moiety is linked to the wild-type IL-18, IL-18 variant polypeptide, or fusion polypeptide via a cleavable linker comprising, for example, a site recognized and cleaved by a tumor microenvironment (TME) protease. The activatable IL-18 polypeptide is unable to stimulate IL-18 receptor-mediated signaling or is unable to fully activate IL-18 receptor-mediated signaling until the masking moiety is separated from the wild-type IL-18, IL-18 variant polypeptide, or fusion polypeptide after cleavage by a protease, such as a TME protease.

[0086] definition

[0087] 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.

[0088] 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.

[0089] 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."

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0097] 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."

[0098] 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.

[0099] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0100] 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.

[0101] Activatable interleukin-18 (IL-18) polypeptide In some embodiments, an activatable interleukin-18 (IL-18) polypeptide is provided, comprising (a) an IL-18 polypeptide and (b) a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker. As used herein, "IL-18 polypeptide" refers to wild-type IL-18, an IL-18 variant polypeptide, or a fusion protein comprising a wild-type IL-18 or an IL-18 variant polypeptide. In some embodiments, the masking moiety prevents (e.g., inhibits or reduces) the IL-18 polypeptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling) when the masking moiety is linked to the IL-18 polypeptide via the cleavable linker.

[0102] In some embodiments, the masking moiety comprises or is derived from (i) an IL-18 pro-peptide (e.g., human IL-18 propeptide), an extracellular domain of IL-18Rα (e.g., the extracellular domain of human IL-18Rα), an extracellular domain of IL-18Rβ (e.g., the extracellular domain of human IL-18Rβ), an IL-18 binding protein (e.g., a human IL-18 binding protein), a fragment of any one of the foregoing, or a variant of any one of the foregoing. In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 333, 336, and 353-356. In some embodiments, the masking moiety comprises two or more sets of amino acid sequences set forth in SEQ ID NOs: 333, 336, and 353-356. In some embodiments, the cleavable linker comprises at least one amino acid sequence that is recognized and cleaved by a protease. In some embodiments, the cleavable linker comprises two or more sequences that are recognized and cleaved by a protease. In some embodiments, the two or more sequences are recognized and cleaved by different proteases. In some embodiments, the two or more sequences are recognized and cleaved by the same protease. In some embodiments, the protease is a tumor microenvironment (TME) protease.In some embodiments, the TME protease is urokinase-type plasminogen activator (uPA), matriptase (MTSP-1), legumain, prostate-specific antigen (PSA), dipeptidyl peptidase (e.g., DPP4), hepsin, matrix metalloproteinase (e.g., matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 1 (MMP1 ... These include, but are not limited to, matrix metalloproteinase 10 (MMP10), matrix metalloproteinase 12 (MMP12), or matrix metalloproteinase 14 (MMP14), a disintegrin and metalloproteinase (such as ADAM10, 17), human leukocyte elastase (HLE), proteinase 3 (PR3), pro-urokinase, plasminogen, staphylokinase, serine proteases, plasmin, cathepsins (B, L, S), tissue kallikrein, and / or kallikrein-related peptidases (KLK1, 2, 3, 6, 7). In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of VLK, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NOs: 372-377, the sequences described in Kridel et al. (2002) J Biol Chem. 277(26):23788-93, and the sequences described in Chen et al. (2002) J Biol Chem. 277(6):4485-4491.

[0103] In some embodiments, the IL-18 polypeptide is or comprises wild-type IL-18. In some embodiments, the wild-type IL-18 is wild-type human IL-18 (hIL-18). In some embodiments, the wild-type hIL18 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide is or comprises an IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18, or (ii) no binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to wild-type IL-18. Exemplary IL-18 variant polypeptides that may be included in an activatable IL-18 polypeptide are described in more detail elsewhere herein. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-299 and 307-318. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 polypeptide is or comprises a fusion polypeptide. In some embodiments, the fusion polypeptide comprises (1) a wild-type IL-18 or an IL-18 variant polypeptide (e.g., an IL-18 variant described herein) and (2) an antibody Fc domain. In some embodiments, the antibody Fc domain is a human Fc domain. In some embodiments, the human Fc domain is a human IgG Fc domain, such as an IgG1, IgG2, or IgG4 Fc domain. In some embodiments, the Fc domain is an Fc variant, eg, a variant of a human Fc domain that comprises one or more amino acid insertions, deletions, or substitutions relative to the wild-type human Fc domain.In some embodiments, the Fc domain variant is a variant of a human IgG1 Fc domain. In some embodiments, the human IgG1 Fc domain variant comprises an N297A substitution, 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, the human IgG1 Fc domain variant comprises an N297A substitution comprising the amino acid sequence set forth in SEQ ID NO: 371 or SEQ ID NO: 390. Exemplary fusion polypeptides that may be included are described in more detail elsewhere herein. In some embodiments, the human IgG1 Fc domain variant comprising an N297A substitution comprises the amino acid sequence of SEQ ID NO: 371 (see Sequence Summary Table).

[0104] In some embodiments, the activatable IL-18 polypeptide further comprises a spacer sequence (e.g., a non-cleavable linker sequence). In some embodiments, the spacer sequence comprises between 3 and 200 amino acids. Suitable spacer sequences are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues such as glycine and serine. In some embodiments, the spacer sequence is or comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 363) or GGGGSGGGSGSGGG (SEQ ID NO: 365).

[0105] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety, (ii) a cleavable linker, and (iii) a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, the masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any one of the foregoing, or a variant of any one of the foregoing. In some embodiments, the masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), optionally wherein the IL-18 pro-peptide comprises a pro-peptide of human IL-18 (hIL-18), optionally wherein the pro-peptide of hIL-18 comprises the amino acid sequence of SEQ ID NO: 333. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (ULM, LM9, etc., see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) adjacent to the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the variant polypeptide comprises a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117.In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316.

[0106] In some embodiments, an activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety comprising a truncated pro-peptide of pro-IL-18 comprising the amino acid sequence of SEQ ID NO: 333, (ii) a cleavable linker, and (iii) a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) adjacent to the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the variant polypeptide comprises a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, and optionally, the variant polypeptide further comprises A127.In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316.

[0107] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety comprising an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2), (ii) a cleavable linker, and (iii) a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, the masking moiety comprises an amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the variant polypeptide comprises a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, and optionally, the variant polypeptide further comprises A127.In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316.

[0108] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a wild-type IL-18 or IL-18 variant polypeptide, (ii) a cleavable linker, and (iii) a masking moiety. In some embodiments, the masking moiety comprises an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2). In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the 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, 168, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316.

[0109] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) an Fc domain (e.g., a human IgG1 Fc domain), (ii) a masking moiety, (iii) a cleavable linker, and (iv) a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, the masking moiety comprises an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2). In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the 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, 168, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316. In some embodiments, the activatable IL-18 polypeptide further comprises a spacer (e.g., a GS linker) between the Fc domain and its adjacent domain. In some embodiments, the spacer is a peptide linker having about 5, 10, 15, 20, 25, 30, or more amino acids.

[0110] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety, (ii) a cleavable linker, (iii) a wild-type IL-18 or IL-18 variant polypeptide, and (iv) an Fc domain (e.g., a human IgG1 Fc domain). In some embodiments, the masking moiety comprises an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2). In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the 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, 168, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316. In some embodiments, the activatable IL-18 polypeptide further comprises a spacer (e.g., a GS linker) between the Fc domain and its adjacent domain. In some embodiments, the spacer is a peptide linker having about 5, 10, 15, 20, 25, 30, or more amino acids.

[0111] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) an Fc domain (e.g., a human IgG1 Fc domain), (ii) a wild-type IL-18 or IL-18 variant polypeptide, (iii) a cleavable linker, and (iv) a masking moiety. In some embodiments, the masking moiety comprises an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2). In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the 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, 168, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316. In some embodiments, the activatable IL-18 polypeptide further comprises a spacer (e.g., a GS linker) between the Fc domain and its adjacent domain. In some embodiments, the spacer is a peptide linker having about 5, 10, 15, 20, 25, 30, or more amino acids.

[0112] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a wild-type IL-18 or IL-18 variant polypeptide, (ii) a cleavable linker, (iii) a masking moiety, and (iv) an Fc domain (e.g., a human IgG1 Fc domain). In some embodiments, the masking moiety comprises an extracellular domain of IL-18Rα (e.g., domains 1-3, e.g., domains 1-2). In some embodiments, the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence that is recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumain). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, etc.; see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) flanking the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker has a length of about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids. 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. In some embodiments, the variant IL-18 polypeptide further comprises C76 and C117, and the amino acids at positions 38 and 68 are not cysteines. In some embodiments, the 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, 168, C76, C117, and optionally, the variant polypeptide further comprises A127. In some embodiments, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316. In some embodiments, the activatable IL-18 polypeptide further comprises a spacer (e.g., a GS linker) between the Fc domain and its adjacent domain. In some embodiments, the spacer is a peptide linker having about 5, 10, 15, 20, 25, 30, or more amino acids.

[0113] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety, (ii) a cleavable linker, (iii) a wild-type IL-18 or IL-18 variant polypeptide, and (iv) a human IgG1 Fc domain or variant thereof. In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a wild-type IL-18 or IL-18 variant polypeptide, (ii) a cleavable linker, (iii) a masking moiety, and (iv) a human IgG1 Fc domain or variant thereof. In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a human IgG1 Fc domain or variant thereof, (ii) a spacer sequence, (iii) a masking moiety, (iv) a cleavable linker, and (v) a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a human IgG1 Fc domain or a variant thereof, (ii) a spacer sequence, (iii) a wild-type IL-18 or IL-18 variant polypeptide, (iv) a cleavable linker, and (v) a masking moiety. In some embodiments, the activatable IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 378-389. In some embodiments, the activatable IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 341-343, 345-352, 357-362, 364, 366-370, and 391-398.

[0114] In some embodiments, a dimer comprising two activatable IL-18 polypeptides described herein is provided. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0115] Interleukin-18 (IL-18) variant polypeptides In some embodiments, the activatable IL-18 polypeptide comprises an IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide comprises mutation(s) at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, where 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)

[0116] In some embodiments, the IL-18 variant polypeptide comprises mutations at two or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, where the amino acid positions are relative to wild-type (WT) human IL-18, e.g., in any combination, as set forth in SEQ ID NO: 1. 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 I149. In some embodiments, the IL-18 variant polypeptide comprises mutations at three or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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).

[0122] 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).

[0123] 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).

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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 amino acid sequence of any one of SEQ ID NOs: 2-299 and 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 and 307-318. The amino acid sequences of SEQ ID NOs: 2-299 and 307-318 are provided in the Sequence Summary Table below in the Examples.

[0134] Fusion Polypeptides In some embodiments, an activatable IL-18 polypeptide of the present disclosure comprises 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 fusion polypeptide comprises a mask that prevents (e.g., inhibits) the wild-type IL-18 and / or IL-18 variant polypeptides from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, one or more of the wild-type IL-18 and / or IL-18 variant polypeptides in the fusion polypeptide are masked to prevent (e.g., inhibit) the wild-type IL-18 and / or IL-18 variant polypeptides from activating (e.g., inhibit) IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, each of the wild-type IL-18 and / or IL-18 variant polypeptides in the fusion polypeptide is masked to prevent (e.g., inhibit) the wild-type IL-18 and / or IL-18 variant polypeptides from activating (e.g., inhibit) IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the dimerization domain is or includes a leucine zipper (LZ) element. Leucine zippers have generally been identified as stretches of about 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.

[0135] 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 378-389.

[0136] Nucleic acids, vectors, host cells, and methods for producing activatable IL-18 polypeptides Nucleic acid molecules encoding the activatable IL-18 polypeptides described herein are also contemplated. In some embodiments, nucleic acids encoding the activatable IL-18 polypeptides described herein are provided. Vectors into which the nucleic acids described herein can be inserted are also provided.

[0137] In summary, expression of an activatable IL-18 polypeptide described herein by a natural or synthetic nucleic acid encoding the activatable IL-18 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.

[0138] 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.

[0139] 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).

[0140] 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.

[0141] 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.

[0142] 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.

[0143] In some embodiments, expression of the nucleic acid(s) encoding the activatable IL-18 polypeptide is inducible. In some embodiments, the nucleic acid(s) encoding the activatable IL-18 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 activatable IL-18 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).

[0144] The activatable IL-18 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.

[0145] The activatable IL-18 polypeptides described herein can be produced using recombinant methods. For recombinant production of activatable IL-18 polypeptides, nucleic acids encoding the activatable IL-18 polypeptides are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or expression. DNA encoding activatable IL-18 polypeptides can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding activatable IL-18 polypeptides). 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.

[0146] 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).

[0147] 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 suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding an activatable IL-18 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.

[0148] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an activatable IL-18 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.

[0149] Expression and cloning vectors generally contain a promoter recognized by the host organism and operably linked to a nucleic acid encoding an activatable IL-18 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 activatable IL-18 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.

[0150] Transcription of a DNA encoding an activatable IL-18 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, an enhancer from a eukaryotic cell virus is used.

[0151] 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.

[0152] 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 variant polypeptides or fusion polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized," resulting in the production of activatable IL-18 polypeptides with partial or fully human glycosylation patterns. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006).

[0153] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco may also be utilized as hosts.

[0154] Suitable host cells for the expression of glycosylated activatable IL-18 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.

[0155] 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.

[0156] 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.

[0157] When using recombinant technology, activatable IL-18 polypeptides can be produced intracellularly in the periplasmic space or directly secreted into the culture medium.If the polypeptide is produced intracellularly, the first step is to remove particulate debris, either 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.

[0158] 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 activatable IL-18 polypeptides described herein include an epitope tag (e.g., a tag attached to the activatable IL-18 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).

[0159] 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 include administering an activatable IL-18 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.

[0160] In some embodiments, provided herein are methods of activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling) in an individual (e.g., a human individual), the methods comprising administering to the individual an activatable IL-18 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 activatable IL-18 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.

[0161] Compositions, kits and articles of manufacture Also provided herein are compositions (eg, formulations) comprising the activatable IL-18 polypeptides, nucleic acids, vectors, or host cells described herein.

[0162] Suitable compositions are obtained by mixing the activatable IL-18 polypeptide, nucleic acid, vector, or host cells having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0163] Also provided are kits comprising the activatable IL-18 polypeptides, nucleic acids, vectors, or host cells comprising the nucleic acids or vectors described herein, which may be useful in any of the methods of treatment described herein.

[0164] 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.

[0165] 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).

[0166] 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.

[0167] Illustrative Embodiments 1. An activatable interleukin-18 (IL-18) polypeptide comprising: (a) an IL-18 polypeptide; and (b) a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker, and optionally, 1) b) is fused to the N-terminus of a), or 2) b) is fused to the C-terminus of a).

[0168] 2. The activatable IL-18 polypeptide of embodiment 1, wherein said masking moiety, when attached to said IL-18 polypeptide via said cleavable linker, inhibits said IL-18 polypeptide from activating IL-18 receptor-mediated signaling.

[0169] 3. The activatable IL-18 polypeptide of embodiment 1 or 2, wherein said masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), an extracellular domain of IL-18Rα, an extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any one of the foregoing, or a variant of any one of the foregoing.

[0170] 4. The activatable IL-18 polypeptide of any one of embodiments 1 to 3, wherein said masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), optionally wherein said IL-18 pro-peptide comprises a pro-peptide of human IL-18 (hIL-18), optionally wherein said hIL-18 pro-peptide comprises the amino acid sequence of SEQ ID NO: 333.

[0171] 5. The activatable IL-18 polypeptide of embodiment 4, wherein said masking moiety comprises a truncated pro-peptide of pro-IL-18.

[0172] 6. The activatable IL-18 polypeptide of embodiment 5, wherein said truncated pro-peptide of pro-IL-18 comprises the amino acid sequence of SEQ ID NO: 336.

[0173] 7. The activatable IL-18 polypeptide of any one of embodiments 1 to 3, wherein said masking moiety comprises the extracellular domain of IL-18Ra or a fragment thereof, and optionally said fragment of the extracellular domain of IL-18Ra comprises a) the first and second domains of said extracellular domain, or b) the third domain of said extracellular domain.

[0174] 8. The activatable IL-18 polypeptide of embodiment 3 or embodiment 7, wherein the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356.

[0175] 9. The activatable IL-18 polypeptide of any one of embodiments 1 to 8, wherein said cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more proteases, and optionally said one or more proteases are one or more tumor microenvironment (TME) proteases.

[0176] 10. The activatable IL-18 polypeptide of embodiment 9, wherein said one or more proteases are one or more tumor microenvironment (TME) proteases, wherein said one or more TME proteases are selected from the group consisting of urokinase-type plasminogen activator (uPA), matriptase, legumain, prostate-specific antigen, dipeptidyl peptidase, hepsin, matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase, human leukocyte elastase, proteinase 3, prourokinase, plasminogen, staphylokinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase.

[0177] 11. The activatable IL-18 polypeptide of embodiment 10, wherein said one or more TME proteases is a matrix metalloprotease, said matrix metalloprotease being selected from the group consisting of matrix metalloprotease 1, matrix metalloprotease 2, matrix metalloprotease 3, matrix metalloprotease 8, matrix metalloprotease 9, matrix metalloprotease 10, matrix metalloprotease 12, and matrix metalloprotease 14.

[0178] 12. The activatable IL-18 polypeptide of any one of embodiments 1 to 11, wherein the cleavable linker comprises one or more amino acid sequences recognized and cleaved by one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 14, urokinase-type plasminogen activator, matriptase, and legumain.

[0179] 13. The activatable IL-18 polypeptide of any one of embodiments 1 to 12, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases, and optionally the one or more amino acid sequences are recognized and cleaved by a) both legumain and MMP9 / MMP2 / MMP14, b) both uPA and MMP9 / MMP2 / MMP14, or c) all of uPA, legumain, MMP2, MMP14, and MMP9.

[0180] 14. The activatable IL-18 polypeptide of any one of embodiments 1 to 13, wherein the cleavable linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NOs: 372 to 377.

[0181] 15. The activatable IL-18 polypeptide of any one of embodiments 1 to 14, wherein the cleavable linker further comprises a spacer sequence, and optionally, said cleavable linker comprises two spacer sequences flanking both the N-terminus and the C-terminus of said amino acid sequence that are recognized and cleaved by one or more proteases.

[0182] 16. The activatable IL-18 polypeptide of any one of embodiments 1 to 15, wherein the cleavable linker comprises two or more sets of amino acid sequences that are recognized and cleaved by one or more proteases.

[0183] 17. The activatable IL-18 polypeptide of embodiment 16, wherein at least one of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is flanked by two spacer sequences within the cleavable linker, and wherein each of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is flanked by two spacer sequences within the cleavable linker.

[0184] 18. The activatable IL-18 polypeptide of any one of embodiments 1 to 17, wherein the spacer sequence comprises a GS linker, and optionally, the GS linker has a length of no more than about 10, 9, 8, 7, 6, or 5 amino acids.

[0185] 19. The activatable IL-18 polypeptide of any one of embodiments 1-18, wherein the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids.

[0186] 20. The activatable IL-18 polypeptide of any one of embodiments 1-19, wherein the cleavable linker consists of about 10, 15, 20, or 25 or more amino acids.

[0187] 21. An activatable IL-18 polypeptide, wherein the cleavable linker consists of about 25 to about 30 amino acids, and wherein the masking moiety comprises the extracellular domain of IL-18Rα, and further optionally, wherein the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356.

[0188] 22. The activatable IL-18 polypeptide of any one of embodiments 1 to 21, wherein the cleavable linker comprises any one of the amino acid sequences of SEQ ID NOs: 399 to 407.

[0189] 23. The activatable IL-18 polypeptide of any one of embodiments 1 to 22, wherein said IL-18 polypeptide comprises wild-type IL-18, optionally wherein said wild-type IL-18 comprises wild-type human IL-18, optionally wherein said wild-type human IL-18 comprises the amino acid sequence of SEQ ID NO: 1.

[0190] 24. The activatable IL-18 polypeptide of any one of embodiments 1 to 23, wherein said IL-18 polypeptide comprises an IL-18 variant polypeptide.

[0191] 25. The activatable IL-18 polypeptide of embodiment 24, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18, or (ii) no binding to IL-18BP.

[0192] 26. The activatable IL-18 polypeptide of embodiment 24 or 25, wherein said IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to said wild-type IL-18.

[0193] 27. The activatable IL-18 polypeptide of any one of embodiments 24 to 26, wherein said IL-18 variant polypeptide comprises at least three, four, five, or six mutations at G3, E6, D54, Q56, P57, N91, and R104 of the IL-18 polypeptide, and the amino acid positions are relative to wild-type (WT) human IL-18 as set forth in SEQ ID NO: 1.

[0194] 28. The activatable IL-18 polypeptide of any one of embodiments 24 to 27, wherein said IL-18 variant polypeptide comprises a cysteine ​​at position 117 and a cysteine ​​at position 76, said amino acid positions being relative to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally wherein said IL-18 variant polypeptide does not comprise a cysteine ​​at position 38 and / or wherein said IL-18 variant polypeptide does not comprise a cysteine ​​at position 68.

[0195] 29. The activatable IL-18 polypeptide of any one of embodiments 24-28, wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally, the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150.

[0196] 30. The activatable IL-18 polypeptide of any one of embodiments 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking moiety, wherein the masking moiety comprises a truncated pro-peptide of pro-IL-18, and the masking moiety comprises the amino acid sequence of SEQ ID NO: 336, b) a cleavable linker, and c) an IL-18 variant polypeptide comprising any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150, and optionally wherein the cleavable linker has a length of about 50, 40, 30, 25, 20, 15, 12, or 10 or less amino acids.

[0197] 31. The activatable IL-18 polypeptide of any one of embodiments 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking moiety comprising the amino acid sequence of any one of SEQ ID NOs: 353-356, b) a cleavable linker, and c) an IL-18 variant polypeptide comprising any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150, and optionally wherein the cleavable linker has a length of about 10-40, 10-30, 20-30, or 25-30 amino acids.

[0198] 32. The activatable IL-18 polypeptide of any one of embodiments 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; b) a cleavable linker; and c) a masking moiety comprising the amino acid sequence of any one of SEQ ID NOs: 353-356, wherein optionally the cleavable linker has a length of about 10-40, 10-30, 20-30, or 25-30 amino acids.

[0199] 33. The activatable IL-18 polypeptide according to any one of embodiments 1 to 32, wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 340-343 and 345-352, or a) a functional variant thereof of an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 340-343 and 345-352.

[0200] 34. The activatable IL-18 polypeptide of any one of embodiments 1-33, wherein the IL-18 polypeptide comprises a fusion protein comprising (1) a wild-type IL-18 or an IL-18 variant polypeptide, and (2) a half-life prolonging domain, optionally wherein the half-life prolonging domain comprises an albumin binding moiety or an antibody Fc domain or a variant thereof, and optionally wherein the half-life prolonging domain is a human IgG Fc domain (such as hIgG1 Fc).

[0201] 35. The activatable IL-18 polypeptide of embodiment 34, wherein the IL-18 variant polypeptide of the fusion protein specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18, or (ii) no binding to IL-18BP.

[0202] 36. The activatable IL-18 polypeptide of embodiment 34 or 35, wherein the IL-18 variant polypeptide of the fusion protein exhibits increased binding to IL-18Rα compared to the wild-type IL-18.

[0203] 37. The activatable IL-18 polypeptide of any one of embodiments 34-36, wherein the human IgG1 Fc domain variant of the fusion protein comprises an N297A mutation (EU numbering), and optionally wherein the human IgG1 Fc domain variant comprises the amino acid sequence of SEQ ID NO: 371 or 390.

[0204] 38. The activatable IL-18 polypeptide of any one of embodiments 34 to 37, wherein the C-terminus of the IL-18 variant polypeptide of the fusion protein is fused to the N-terminus of the human IgG Fc domain or variant thereof of the fusion protein.

[0205] 39. The activatable IL-18 polypeptide of any one of embodiments 34 to 37, wherein the C-terminus of the human IgG Fc domain or variant thereof of the fusion protein is fused to the N-terminus of the IL-18 variant polypeptide of the fusion protein.

[0206] 40. The activatable IL-18 polypeptide of any one of embodiments 34-39, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332, 378-389, and 408-418, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 319-332, 378-389, and 408-418.

[0207] 41. The activatable IL-18 polypeptide of any one of embodiments 1 to 40, comprising the amino acid sequence of any one of SEQ ID NOs: 340-343, 345-352, 357-362, 364, 366-370, 391-398, and 408-418, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 319-332, 378-389, and 408-418.

[0208] 42. A dimer comprising two activatable IL-18 polypeptides according to any one of claims 1 to 41.

[0209] 43. The dimer according to embodiment 42, wherein the dimer is a homodimer.

[0210] 44. The dimer according to embodiment 42, wherein the dimer is a heterodimer.

[0211] 45. A nucleic acid encoding an activatable IL-18 polypeptide according to any one of embodiments 1 to 41.

[0212] 46. ​​A vector comprising the nucleic acid of embodiment 45.

[0213] 47. A host cell comprising a nucleic acid according to embodiment 45 or a vector according to embodiment 46.

[0214] 48. A method for producing an activatable IL-18 polypeptide, comprising: (a) culturing the host cell of embodiment 47 under conditions in which the activatable IL-18 polypeptide is expressed; and (b) recovering the activatable IL-18 polypeptide produced by the host cell.

[0215] 49. The method of embodiment 48, further comprising purifying the activatable IL-18 polypeptide.

[0216] 50. A pharmaceutical composition comprising an activatable IL-18 polypeptide according to any one of embodiments 1 to 41, a nucleic acid according to embodiment 45, or a vector according to embodiment 46.

[0217] 51. A method for treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 50.

[0218] 52. The method of embodiment 51, wherein the disease is cancer.

[0219] 53. A method for activating IL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 50.

[0220] 54. 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 50.

[0221] 55. The method of embodiment 54, wherein the stimulation comprises increasing the activity and / or number of immune cells that have experienced the antigen.

[0222] 56. The method of any one of embodiments 36 to 40, wherein the individual is a human. [Example]

[0223] 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.

[0224] Example 1: Materials and Methods for Examples 2-4 Expression and purification of recombinant WT hIL-18 and IL-18 variant polypeptides Wild-type ("WT") hIL-18, IL-18 variant polypeptides M12, MM5, M21, M13, M24, WM4, WM5, and WM6, activatable IL-18, activatable IL-18 variant polypeptides (described in the Examples below), and fusion polypeptides containing the foregoing were expressed in an E. coli system. Briefly, PET30a plasmids containing codon-optimized sequences encoding WT hIL-18, IL-18 variant polypeptides, and activatable hIL-18 polypeptides were each transfected into E. coli BL21(DE3) cells. The transfected E. coli cells were grown in culture medium, and the expressed polypeptides were harvested and purified. IL18 variant polypeptides M12, MM5, M21, M13, M24, WM4, WM5, and WM6 were previously described in PCT / CN2022 / 117332, filed September 6, 2022.

[0225] Recombinant hIL-18 expression and purification in CHO cells Fusion polypeptides containing wild-type ("WT") hIL-18, IL-18 variant polypeptides, or activatable IL-18 polypeptides (described in the Examples below) were expressed in mammalian cell lines (293F or CHO). Briefly, a plasmid containing a signal peptide with a codon-optimized sequence encoding the fusion polypeptides described in these Examples was transfected into CHO cells. The transfected CHO cells were grown in culture medium and cultured for 4 days. The cell culture medium containing the secreted fusion polypeptide was then collected and purified via protein A chromatography. Certain eluates were subjected to additional gel filtration or ion exchange steps.

[0226] IL-18 activity assessed by reporter assay The NFκB-luc / hIL18RαRβ 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 wild-type IL-18, IL-18 variant polypeptides, activatable IL-18 polypeptides (before treatment with tumor microenvironment proteases), and activatable IL-18 polypeptides (after treatment with tumor microenvironment proteases) in activating hIL-18 receptor-mediated signaling.

[0227] Briefly, reporter cells were initially plated at 3.0 × 10 per well. 4 Cells were seeded at a density of 1 / 4 of a 96-well white-bottom plate into wells. IL-18 was 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.

[0228] Human PBMC-based hIFNγ release assay A PBMC-based assay was performed to test the ability of wild-type IL-18, activatable IL-18 polypeptides (before treatment with tumor microenvironment proteases), and activatable IL-18 polypeptides (after treatment with tumor microenvironment proteases) to activate T / NK cells and induce hIFNγ release. Briefly, human PBMC cells were cultured at 5.0 x 10 per well. 5 Cells were seeded into wells of a 96-well plate at a density of 1 / 3 cells and cultured for 5 hours before treatment. hIL-18 was 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.

[0229] Mouse splenocyte-based mIFNγ release assay A mouse splenocyte-based mouse IFNγ ("mIFNγ") release assay is performed as follows: Briefly, freshly isolated mouse splenocytes were added at 6.5×10 per well. 5 Cells 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.

[0230] Digestion of activatable IL-18 polypeptide The activatable IL-18 polypeptides described in Examples 2 and 3 were cleaved by either caspase-1 or tumor microenvironment ("TME") proteases (i.e., matrix metalloproteases MMP2, MMP9, MMP10, MMP14) to assess the cleavage efficiency and activity of the resulting "cleaved" forms of the activatable IL-18 polypeptides. For the caspase-1 cleavage reaction, the activatable IL-18 polypeptides were incubated with pre-activated caspase-1 (Abcam catalog no. ab39901) at a ratio of 100-500 μg protein:1 unit caspase for 1-16 hours at room temperature. For MMP2, MMP9, MMP10, and MMP14 cleavage reactions, activatable IL-18 polypeptide was incubated with preactivated MMP2, MMP9, MMP10, or MMP14 at a weight ratio of 100:1 to 500:1 (µg polypeptide:µg protease) for 1 to 16 hours at room temperature or 37°C. MMP2, MMP9, MMP10, and MMP14 cleavage reactions were terminated by placing the samples on ice or storing them at -80°C.

[0231] Example 2: Activatable IL-18 polypeptides containing a masking moiety derived from the native pro-peptide of pro-IL-18 The IL-18 precursor (or "pro-IL18") remains inactive in the cytosol until a signal, such as inflammasome activation, induces its maturation. Inflammasomes are multiprotein assemblies composed of three proteins: nucleotide-binding oligomerization domain (NOD)-like receptors, apoptosis-associated speck-like protein-containing CARD (ASC), and caspase-1. They form upon detection of pathogens or other harmful substances, such as reactive oxygen species and uric acid crystals, in the cytosol. The inflammasome then autocatalyzes the activation of the cysteine ​​protease caspase-1, which removes the propeptide from the IL-18 precursor to produce mature IL-18. The native pro-peptide of human pro-IL-18 (i.e., MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESD (SEQ ID NO: 333)) was used as a starting point to develop a masking moiety that blocks binding of WT hIL-18 (i.e., mature IL-18), IL-18 variant polypeptides, or fusion polypeptides containing the foregoing to IL-18Rα and prevents activation of hIL-18 receptor-mediated signaling. The native caspase-1 recognition sequence of SEQ ID NO: 333 (i.e., AEDDENLESD (SEQ ID NO: 334), corresponding to amino acids 27-36 of SEQ ID NO: 333) was replaced with the protease recognition sequence of MMP-2, MMP-9, MMP-10, MMP-14, legumain, or another tumor microenvironment ("TME")-specific protease, or a combination of one or more of the foregoing recognition sequences.

[0232] 2.1 Proof of concept: The native pro-peptide of human pro-IL-18 blocks mature WT human IL-18 from binding to IL-18Ra and activating IL-18 receptor-mediated signaling. Full-length human pro-IL-18 (SEQ ID NO: 335) was produced in E. coli as described above for recombinant WT IL-18 (i.e., "mature" WT IL-18). The ability of pro-IL-18 to bind to hIL-18Rα was assessed via biolayer interferometry (BLI), and the ability of pro-IL-18 to activate IL-18 receptor-mediated signaling was assessed using the reporter assay described in Example 1. Signaling assay experiments were performed with caspase-1-treated pro-IL-18. (Caspase-1-treated pro-IL-18 is also referred to herein as "truncated pro-IL-18." Truncated pro-IL-18 has the same amino acid sequence as mature WT IL-18 (i.e., SEQ ID NO: 1).) As shown in Figure 1A, pro-IL-18 showed no detectable binding to hIL-18Rα under the experimental conditions, i.e., when pro-IL-18 was present at a concentration of 32 nM. Pro-IL-18 also did not activate hIL-18 receptor-mediated signaling. See Figure 1B. In contrast, truncated pro-IL-18 activates hIL-18 receptor-mediated signaling to the same extent as recombinant WT IL-18. See Figure 1B. The signaling activity of truncated pro-IL-18 is inhibited in the presence of 1 μg / ml of human IL-18 binding protein ("BP"). See Figure 1B.

[0233] 2.2 Design, Preparation, and Characterization of Exemplary Cleavable Linkers Activatable IL-18 polypeptides were designed that comprise, from N- to C-terminus, amino acids 1-26 of the native pro-peptide of proIL-18 (i.e., MAAEPVEDNCINFVAMKFIDNTLYFI, SEQ ID NO: 336), an amino acid linker sequence containing a protease recognition site for caspase-1 or a TME protease, and WT hIL-18, respectively. See Table 1. [Table 1]

[0234] Pro-IL18, pro-IL18-MMP9L, pro-IL18-MMP10L, pro-IL18-MMP2L, and pro-IL18-Legu L were expressed and purified. Pro-IL18-MMP2 / MMP14L could not be purified satisfactorily. Pro-IL18, pro-IL18-MMP9L, pro-IL18-MMP10L, and pro-IL18-Legu L were expressed satisfactorily in E. coli and purified via SEC to >95% purity, as determined by SDS-PAGE and SEC (data not shown).

[0235] The potency of proIL18-MMP9L, proIL18-MMP10L, and proIL18-Legu L to activate hIL-18 receptor-mediated signaling was assessed using the hIL-18 reporter cell assay described in Example 1. As shown in Figures 2A-2C, the potencies of proIL18-MMP9L, proIL18-MMP10L, and proIL18-Legu L were found to be approximately 1000-fold lower than WT hIL-18 (i.e., SEQ ID NO: 1), with Cmax reduced by up to 100 nM.

[0236] The potency of proIL18-MMP9L and proIL18-Legu L to activate hIL-18 receptor-mediated signaling was partially restored after treatment with MMP9 and legumain, respectively (see Figures 2A and 2C). The potency of "truncated" proIL18-MMP9L (i.e., after MMP9 treatment) was approximately 16-fold lower (EC50: 2.46 nM) than that of recombinant WT hIL-18 (EC50: 0.16 nM). See Figure 2A.

[0237] Example 3: Design and Characterization of Exemplary IL-18 Variant Polypeptides Comprising a Masking Moiety Derived from a Pro-IL-18 Pro-Peptide and an MMP9-Cleavable Linker Constructs containing the masking portion of SEQ ID NO: 344, including amino acids 1-26 of the native pro-peptide of pro-IL-18 (i.e., MAAEPVEDNCINFVAMKFIDNTLYFI, SEQ ID NO: 336) and an MMP9-cleavable linker sequence of SGGPGPAGMKGLPG (SEQ ID NO: 337), were fused to the N-terminus of the IL-18 variant polypeptides M12 and MM5 to generate proM12 (SEQ ID NO: 345) and proMM5 (SEQ ID NO: 346). Pro M12 MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPGSYFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKGSGARGMAVTISVKCEKISTLSCENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED Pro MM5 MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPGSYFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKLSTERGMAVTISVKCEKISTLSCENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0238] ProM12 and proMM5 were then expressed in E. coli, harvested, and purified to greater than 95% purity as determined by SDS-PAGE and SEC. Next, proM12 and proMM5 were subjected to MMP9 treatment (as described in Example 1). The efficiency of cleavage of proM12 and proMM5 by MMP9 was assessed via HPLC-SEC. Briefly, as shown in Figure 3A, proM12 had a shorter retention time than M12 without the masking moiety. ProM12 treated with MMP9 (i.e., "proM12 cleavage") had a retention time located between proM12 and M12. Similar results were observed with proMM5, MM5, and proMM5 treated with MMP9 (i.e., "proMM5 cleavage"). See Figure 3B. The results in Figures 3A and 3B demonstrate complete cleavage (i.e., removal of the masking moiety) of proM12 and proMM5 by MMP9. Without being bound by theory, the difference in molecular weight between, for example, M12 and "pro-M12 cleavage" and, for example, MM5 and "pro-MM5 cleavage" may be due to amino acid residues within the MMP9 linker that remain attached to the N-terminus of M12 or MM5 after MMP9 treatment.

[0239] The efficacy of activatable IL-18 variant polypeptides to activate hIL-18 receptor-mediated signaling was assessed using the hIL-18 reporter cell assay described in Example 1. The following sets of polypeptides were tested: (1) proM12 (SEQ ID NO: 345), M12, and proM12 cleavage (i.e., proM12 treated with MMP9), (2) proMM5 (SEQ ID NO: 346), MM5, and proMM5 cleavage (i.e., proMM5 treated with MMP9), (3) proM21 (SEQ ID NO: 347), M21, and proM21 cleavage (i.e., proM21 treated with MMP9), (4) proM13 (SEQ ID NO: 348), M13, and proM13 cleavage (i.e., proM21 treated with MMP9). (5) proM24 (SEQ ID NO: 349), M24, and proM24 cleavage (i.e., proM24 treated with MMP9), (6) proWM4 (SEQ ID NO: 350), WM4, and proWM4 cleavage (i.e., WM4 treated with MMP9), (7) proWM5 (SEQ ID NO: 351), WM5, and proWM5 cleavage (i.e., WM5 treated with MMP9), and (8) proWM6 (SEQ ID NO: 352), WM6, and proWM6 cleavage (i.e., WM6 treated with MMP9). As shown in Figures 3C-3J, all activatable IL-18 polypeptides tested were significantly reduced in potency compared to the corresponding "unmasked" IL-18 variant polypeptides, with Cmax reduced by up to 100 nM. Of the activatable IL-18 polypeptides shown in Figures 3C-3J, only minimal hIL-18 receptor-mediated signaling was observed for proWM4, proWM5, and proWM6 at 100 nM (see Figures 3H-3J). The proM12, proMM5, proM21, proM13, proM24, proWM4, proWM5, and proWM6 truncations demonstrated restored hIL-18 receptor-mediated signaling. The proM12, proM21, and proM13 truncations demonstrated nearly fully restored signaling (see Figures 3C, 3E, and 3F and Table 2). The proMM5, proM24, proWM4, proWM5, and proWM6 truncations demonstrated only partially restored hIL-18 receptor-mediated signaling.See Figures 3D, 3G, 3H, 3I, and 3J and Table 2. [Table 2]

[0240] The effect of BP on hIL-18 receptor-mediated signaling by proM12 cleavage and proMM5 cleavage was also evaluated. As shown in Figures 3C and 3D, proM12 cleavage and proMM5 cleavage retain BP resistance and exhibit similar signaling activity in the presence and absence of 1 μg / mL of human IL-18 binding protein ("BP").

[0241] To confirm that the MMP9-recognition linker-fused masking moiety MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPG (SEQ ID NO: 344) was specifically cleaved by MMP9 but not by caspase-1, proM12 was treated with either MMP9 or caspase-1 in vitro, and the MMP9- or caspase-1-treated proM12 was evaluated in the hIL-18 reporter cell assay described in Example 1. As shown in Figure 3K, proM12 cleaved after MMP9 treatment exhibited restored signaling activity, whereas proM12 cleaved after caspase-1 treatment exhibited signaling activity equivalent to that of untreated proM12. These results indicated that the MMP9-recognition linker-fused masking moiety (SEQ ID NO: 344) was no longer susceptible to caspase-1 cleavage.

[0242] Next, a PBMC-based assay was performed to test the ability of proM12 and proMM5 (i.e., before and after treatment with MMP9) to induce hIFNγ release. Figures 4A and 4B show that WT hIL-18 had an EC of approximately 0.1 nM. 50These results show that, whereas proM12 and proMM5 were able to induce human IFNγ release in the presence of IL-12 containing the cleavage fragment, neither proM12 nor proMM5 were able to induce human IFNγ release, even at up to 10 nM. After treatment with MMP9, both proM12 and proMM5 cleavage fragments exhibited hIFNγ-releasing activity. See Figures 4A and 4B. The proM12 cleavage fragment exhibited activity similar to that of WT hIL-18 or M12, while the unmasked proMM5 (proMM5 cleavage fragment) exhibited approximately 7-fold lower activity compared to WT hIL-18. See Table 3 below. [Table 3]

[0243] Example 4: Activatable IL-18 polypeptides comprising masking moieties derived from IL-18Rα, IL-18Rα subdomain, IL-18Rβ, IL-18BP, and IL-18BP variants. 4.1 Design and Characterization of Activatable IL-18 Polypeptides Containing Masking Moieties Derived from IL-18Rα, IL-18Rα Subdomains, IL-18Rβ, IL-18BP, and IL-18BP Variants Additional masking moieties were designed derived from IL-18Rα, IL-18Rα subdomains, IL-18Rβ, IL-18BP, and IL-18BP variants, see below. IL-18Ra (also referred to herein as "18Ra") - the extracellular domain of IL-18Ra (AA20-318) ESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKAD (SEQ ID NO: 353) IL-18Ra D12 (also referred to herein as "D12") - the first two domains of IL-18Ra (AA20-208) ESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIV (SEQ ID NO: 354) IL-18Ra D3 (also referred to herein as "D3") - the third domain of IL-18Ra (AA210-318) RSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKAD (SEQ ID NO: 355) IL-18BPm (also referred to herein as "BPm") - a modified form of IL-18BP that exhibits the ability to bind to and inhibit the activity of IL-18BP-resistant IL18 variant polypeptides. AAKQSPALEVTWPEVEVPLNGTLSLSSVASSIFPNFSILYWLGNGSFIEHLPGRLWEGSTSEERGSTGTQLSKALVLEQLTPALHSTNFSSVLVDPEQVVQRHVVL (SEQ ID NO: 356)

[0244] The masking moieties shown above were each linked to M12-DB6-Fc_N297A (SEQ ID NO: 323) via a linker sequence containing an MMP9 recognition site (i.e., SGGPGPAGMKGLPGS, SEQ ID NO: 337) to produce the activatable M12-DB6-Fc_N297A polypeptide, the sequence of which is shown below.

[0245] Ra-M12-DB6-Fc_N297A The IL-18Ra sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0246] D12-M12-DB6-Fc_N297A The IL-18Ra D12 sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0247] D3-M12-DB6-Fc_N297A The IL-18Ra D3 sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0248] BPm-M12-DB6-Fc_N297A The IL-18BPm sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0249] M12-DB6-D3-Fc_N297A The IL-18Ra D3 sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0250] Fc_N29A-D3-M12-DB6 The IL-18Ra D3 sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. An additional spacer sequence (GGGGSGGGGSGGGGS, SEQ ID NO: 363) is shown in double-underlined italic text. [ka]

[0251] Fc_N297A-M12-DB6-D3 The IL-18Ra D3 sequence is underlined, the cleavable linker is in italic text, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. An additional linker sequence (GGGGSGGGGSGSGGG, SEQ ID NO: 365) is shown in double-underlined italic text. [ka]

[0252] Ra-M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, D3-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297A were expressed in CHO cells and purified via protein A chromatography. These activatable IL-18 polypeptides exhibited 74%-96% purity (as measured via SEC-HPLC) and approximately 80%-95% purity (as measured via SDS-PAGE) after one-step protein A purification (data not shown).

[0253] The potency of Ra-M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297A to activate hIL-18 receptor-mediated signaling was assessed before and after MMP9 treatment using the hIL-18 reporter cell assay described in Example 1. The potency of WT IL-18 (SEQ ID NO: 1), M12-DB6-Fc_N297A (SEQ ID NO: 323), and Fc_N297A-M12-DB6 (SEQ ID NO: 331) was assessed in parallel.

[0254] As shown in Figures 5A-5C and Table 4 below, the IL-18Ra, IL-18RaD12, and IL-BPm masking moieties each significantly inhibited the signaling activity of Ra-M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297A (i.e., signaling was reduced 100- to 1790-fold compared to M12-DB6-Fc_N297A). The IL-18Ra masking moiety exhibited the highest masking capacity (see Figure 5A and Table 4). After MMP9 treatment, the Ra-M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297A cleavages exhibited signaling activity comparable to that of M12-DB6-Fc_N297A (see Figures 5A-5C and Table 4), with EC50 values ​​varying within 3-fold (see Table 4). [Table 4]

[0255] Next, the masking moieties Ra, D12, D3, and BPm (described above) were each linked to MM5-DB6-Fc_N297A (SEQ ID NO: 330) via a cleavable linker containing an MMP9 recognition site (i.e., SGGPGPAGMKGLPGS, SEQ ID NO: 337), producing the activatable MM5-DB6-Fc_N297A polypeptide, the sequence of which is shown below.

[0256] Ra-MM5-DB6-Fc_N297A The IL-18Ra sequence is underlined, the cleavable linker is in italic text, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0257] D12-MM5-DB6-Fc_N297A The IL-18Ra D12 sequence is underlined, the cleavable linker is in italic text, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0258] MM5-DB6-Ra-Fc_N297A The IL-18Ra sequence is underlined, the cleavable linker is in italic text, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. [ka] [ka]

[0259] Fc_N297A-Ra-MM5-DB6 The IL-18Ra sequence is underlined, the cleavable linker is in italic text, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. The additional linker sequence GGGGSGGGSGGGGS (SEQ ID NO: 363) is shown in double-underlined italic text. [ka]

[0260] Fc_N297A-MM5-DB6-Ra The IL-18Ra sequence is underlined, the cleavable linker is in italic text, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. An additional linker sequence (GGGGSGGGGSGSGGG, SEQ ID NO: 365) is shown in double-underlined italic text. [ka]

[0261] The potency of Ra-MM5-DB6-Fc_N297A, D12-MM5-DB6-Fc_N297A, Fc_N297A-Ra-MM5-DB6, and Fc_N297A-MM5-DB6-Ra to activate hIL-18 receptor-mediated signaling was assessed before and after MMP9 treatment using the hIL-18 reporter cell assay described in Example 1. The potency of WT IL-18 (SEQ ID NO: 1), MM5-DB6-Fc_N297A (SEQ ID NO: 330), and Fc_N297A-MM5-DB6 (SEQ ID NO: 332) was assessed in parallel.

[0262] As shown in Figures 6A-6D and Table 5 below, both IL-18Ra and IL-18RaD12 masking moieties significantly inhibited the signaling activity of Ra-MM5-DB6-Fc_N297A, D12-MM5-DB6-Fc_N297A, Fc_N297A-Ra-MM5-DB6, and Fc_N297A-MM5-DB6-Ra (i.e., signaling was reduced by more than 1000-fold compared to MM5-DB6-Fc_N297A). IL-18Ra was able to mask MM5-DB6 and inhibit hIL-18 receptor-mediated signaling, regardless of whether the masking moiety was fused to the N- or C-terminus of MM5-DB6. After MMP9 treatment, the Ra-MM5-DB6-Fc_N297A cleavage, D12-MM5-DB6-Fc_N297A cleavage, Fc_N297A-Ra-MM5-DB6 cleavage, and Fc_N297A-MM5-DB6-Ra cleavage exhibited slightly lower signaling activity than that of MM5-DB6-Fc_N297A or Fc_N297A-MM5-DB6 (see Figures 6A-6D and Table 5), with EC50 values ​​changing within 15-fold (see Table 5). [Table 5]

[0263] Example 5A: Activatable IL-18 polypeptides containing a masking moiety derived from IL-18Rα with variable linker lengths For masked IL-18, the distance between the C-terminus of IL-18 and the N-terminus of IL-18Ra in the Fc-IL18-Ra format was predicted to be 34 nm based on the simulated structure. A linker with 15 AA may result in an extended conformation at the IL-18 and Ra interface. Longer linkers of 25 AA to 30 AA were tested.

[0264] Specifically, Fc-M12-DB6, masked IL-18Ra with variable lengths at the cleavable site (M9 = 15AA, M91 = 30AA, M92 = 25AA), was produced in CHO cells as described above. As shown in Figure 7, C-terminally fused IL-18Ra reduced the activity of Fc-M12-DB6 when linked by all three different linker lengths, but Fc-M12-DB6-Ra-M9 with a 15AA linker exhibited the most reduced activity compared to that of either the 30AA or 25AA linkers.

[0265] Following in vitro cleavage with the protease MMP9, all three Ra-masked Fc-M12-DB6 fragments exhibit significantly restored activity, whereas cleaved Fc-M12-DB6-Ra-M91 and Fc-M12-DB6-Ra-M92 fragments exhibit activity comparable to that of unmasked Fc-M12-DB6, and MMP9-cleaved Fc-M12-DB6-Ra-M9 fragments exhibit partially restored activity. These data confirm that cleavable linkers with lengths of 25 to 30 amino acids result in more desirable restored activity after protease treatment.

[0266] Example 5B: Activatable IL-18 polypeptides containing a masking moiety derived from IL-18Rα with different combinations of cleavable linkers We further designed and investigated masked Fc-M12-DB6 Ra with different combinations of cleavable linkers to improve cleavage probability in the TME. The MMP protease recognition substrate was fused to either the uPA substrate sequence or the legumain cleavable linker, or both, to generate UM (uPA + MMP), ULM (uPA + legumain + MMP), and LM (legumain + MMP) linkers. As shown in Figures 8A and 8B, Fc-M12-DB6-Ra, in which all six sets of linker combinations were tested, exhibited reduced activity comparable to that of Fc-M12-DB6-Ra-M92. Furthermore, after in vitro MMP9 treatment, the six masked Fc-M12-DB6 Ras exhibited significantly restored activity. See Figures 8A and 8B. The data suggest that a cleavable linker between IL-18 and the masking moiety can be designed to be sensitive to multiple proteases without affecting masking efficacy or activity recovery.

[0267] Another multi-protease recognition linker, designated UM-2, was further designed to link IL-18 with a masking moiety. Both Fc-M12-DB6-Ra-UM2 and Fc-MM5-DB6-Ra-UM2 were prepared from CHO cells. The UM-2 linker-fused masked Fc-M12-DB6-Ra and Fc-MM5-DB6-Ra showed significantly reduced activity compared with the corresponding unmasked molecules. In addition to MMP cleavage, MMP-14 and uPA treatments were also performed to remove the masking moiety Ra. The results show that all three tested proteases can efficiently remove the masking moiety, allowing the activity of the IL-18 variants to be restored (Figures 9A and 9B).

[0268] In summary, for masked IL-18 or masked IL-18 variants, cleavable linkers with a length of 15 to 30 AA and multiple protease recognition sequences sufficiently maintained the masking efficacy of the masking moiety. In addition, linkers of appropriate length allowed for good exposure of the protease recognition sequences, resulting in efficient enzymatic cleavage and good recovery activity of IL-18 or IL-18 variants.

[0269] Example 6A. IL-18 variant polypeptides of hIL-18Ra have been constructed. The binding affinities of WT hIL-18 and IL-18 variant polypeptides to hIL-18Rα ECD-Fc are shown in Table 6A. [Table 6A-1] [Table 6A-2]

[0270] The mutations in these IL-18 variant polypeptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM14, corresponding to SEQ ID NOS: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-153) are shown in Table 6B. [Table 6B-1] [Table 6B-2]

[0271] Example 6B: 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.

[0272] 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.

[0273] As shown in Table 7, all IL-18 variant polypeptides tested demonstrated potency 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 50showed 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 7-1] [Table 7-2]

[0274] 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. 5 Cells 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.

[0275] 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 8, the activity of WT hIL-18 was significantly attenuated in the presence of 1 μg / mL of 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 50The results showed that the IL-18 variant polypeptides were significantly less affected by hIL-18BP in terms of hIFNγ induction. [Table 8]

[0276] 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.

[0277] Briefly, freshly isolated mouse splenocytes were cultured at 6.5 × 10 cells per well. 5 Cells 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.

[0278] 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)

[0279] 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.

[0280] M12-SSS was further modified to include a C127S substitution to generate M12-SSSS: YFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDSRDNAPRTIFIISMYKGSGARGMAVTISVKSEKISTLSSENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLASEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 310)

[0281] 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.

[0282] 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 6B. The demonstrated potency of IL-18-SSS-Fc_N297A was comparable to that of wild-type IL-18, whereas M12-SSS-Fc_N297A showed an approximately 7-fold loss in potency compared to M12. 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. 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. Additionally, Fc-fused IL-18 and IL-18 variants are expected to exhibit longer half-lives.

[0283] 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 9]

[0284] The mutation strategies in Table 9 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]

[0285] 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 10 below. [Table 10]

[0286] 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.

[0287] 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.

[0288] 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 Table 11 below, M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A exhibited much higher potency than WT IL-18 in activating IL-18 receptor-mediated signaling. The signaling activity of tested M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, and M12-DB9-Fc_N297A was not inhibited in the presence of 1 μg / ml hIL-18BP-hFc (see Table 11). [Table 11]

[0289] Next, the "DB6" substitutions, i.e., C38I, C68S, and S117C, were introduced into variant MM5 to generate MM5-DB6: MM5-DB6 [ka]

[0290] The "DB6" substitution was also introduced into wild-type IL-18 to generate WT IL18-DB6: WT IL18-DB6 [ka]

[0291] An additional variant, WT-DBo, was designed by introducing a S117C substitution into wild-type IL-18: WT IL-18-DBo [ka]

[0292] 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 12 below, there was no detectable protein for WT IL18-DBo-Fc_N297A after the same expression and purification procedures. 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 12). 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 9), and (iii) a C68 substitution (see Table 9). MM5-DB6-Fc_N297A consistently shows much higher expression yield and purity than MM5-SSS-Fc_N297A (see Table 12). 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.

[0293] 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 12, 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 12]

[0294] 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 6B. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A exhibited potent IL-18 receptor cell activation signals that were stronger than those of WT IL-18 (see Table 13). Fc_N297A-MM5-DB6 exhibited similar potency to WT IL-18, whereas Fc_N297A-M12-DB6 produced a much stronger activation signal on IL-18 reporter cells (see Table 13). 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 Table 13). 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 9) improves the expression profile of IL-18 and IL-18 variants without affecting IL-18BP dependence.

[0295] 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 14). [Table 13]

[0296] For a summary of the above data, see Table 14 below.

[0297] [Table 14]

[0298] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover the modifications and variations of the present disclosure provided they come within the scope of the appended claims and their equivalents.

[0299] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0300] The present invention has been described in terms of specific embodiments discovered or proposed by the inventors to include preferred modes for carrying out the invention. In light of this disclosure, those skilled in the art will recognize that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, changes can be made in the protein structure without affecting the type or amount of biological action. All such modifications are intended to be within the scope of the appended claims. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7] [Table 15-8] [Table 15-9] [Table 15-10] [Table 15-11] Table 15-12 Table 15-13 Table 15-14 Table 15-15 Table 15-16 Table 15-17 Table 15-18 Table 15-19 Table 15-20 Table 15-21 Table 15-22 Table 15-23 Table 15-24 Table 15-25 Table 15-26 Table 15-27 Table 15-28 Table 15-29 Table 15-30 Table 15-31 Table 15-32 Table 15-33 Table 15-34 Table 15-35 Table 15-36 Table 15-37 Table 15-38 Table 15-39 Table 15-40 Table 15-41 Table 15-42 Table 15-43 Table 15-44 Table 15-45 Table 15-46 Table 15-47 Table 15-48 Table 15-49 Table 15-50 Table 15-51 Table 15-52 Table 15-53 Table 15-54 Table 15-55 Table 15-56 Table 15-57 Table 15-58 Table 15-59 Table 15-60 Table 15-61 Table 15-62 Table 15-63 Table 15-64 Table 15-65 Table 15-66 Table 15-67

Claims

1. 1. An activatable interleukin-18 (IL-18) polypeptide, comprising: (a) an IL-18 polypeptide; (b) a masking portion; The activatable interleukin-18 (IL-18) polypeptide, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker.

2. 2. The activatable IL-18 polypeptide of claim 1, wherein said masking moiety, when attached to said IL-18 polypeptide via said cleavable linker, inhibits said IL-18 polypeptide from activating IL-18 receptor-mediated signaling, and optionally said activation of IL-18 receptor is reduced by said masking moiety by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

3. 3. The activatable IL-18 polypeptide of claim 1 or 2, wherein the masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), an extracellular domain of IL-18Rα, an extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any one of the preceding claims, or a variant of any one of the preceding claims.

4. 4. The activatable IL-18 polypeptide of any one of claims 1 to 3, wherein the masking moiety comprises an IL-18 pro-peptide (i.e., a pro-IL-18 pro-peptide), optionally wherein the IL-18 pro-peptide comprises a pro-peptide of human IL-18 (hIL-18), optionally wherein the pro-peptide of hIL-18 comprises the amino acid sequence of SEQ ID NO:

333.

5. 5. The activatable IL-18 polypeptide of claim 4, wherein the masking moiety comprises a truncated pro-peptide of pro-IL-18.

6. 6. The activatable IL-18 polypeptide of claim 5, wherein the truncated pro-peptide of pro-IL-18 comprises the amino acid sequence of SEQ ID NO:

336.

7. 4. The activatable IL-18 polypeptide of claim 1, wherein the masking moiety comprises the extracellular domain of IL-18Rα or a fragment thereof, and optionally the fragment of the extracellular domain of IL-18Ra comprises a) a first and a second domain of the extracellular domain, or b) a third domain of the extracellular domain.

8. 8. The activatable IL-18 polypeptide of claim 3 or claim 7, wherein the masking moiety comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356.

9. 9. The activatable IL-18 polypeptide of any one of claims 1 to 8, wherein the cleavable linker comprises one or more sets of amino acid sequences that are recognized and cleaved by one or more proteases, and optionally the one or more proteases are one or more tumor microenvironment (TME) proteases.

10. 10. The activatable IL-18 polypeptide of claim 9, wherein the one or more proteases are one or more tumor microenvironment (TME) proteases, and the one or more TME proteases are selected from the group consisting of urokinase-type plasminogen activator, matriptase, legumain, prostate-specific antigen, dipeptidyl peptidase, hepsin, matrix metalloproteinase, a disintegrin and metalloproteinase, human leukocyte elastase, proteinase 3, prourokinase, plasminogen, staphylokinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase.

11. 11. The activatable IL-18 polypeptide of claim 10, wherein the one or more TME proteases are matrix metalloproteases selected from the group consisting of matrix metalloprotease 1, matrix metalloprotease 2, matrix metalloprotease 3, matrix metalloprotease 8, matrix metalloprotease 9, matrix metalloprotease 10, matrix metalloprotease 12, and matrix metalloprotease 14.

12. 12. The activatable IL-18 polypeptide of any one of claims 1 to 11, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 14, urokinase-type plasminogen activator, matriptase, and legumain.

13. 13. The activatable IL-18 polypeptide of any one of claims 1 to 12, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases, and optionally the one or more amino acid sequences are recognized and cleaved by a) both legumain and MMP9 / MMP2 / MMP14, b) both uPA and MMP9 / MMP2 / MMP14, or c) all of uPA, legumain, MMP2, MMP14, and MMP9.

14. 14. The activatable IL-18 polypeptide of any one of claims 1 to 13, wherein the cleavable linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339, and SEQ ID NOs:372-377.

15. 15. The activatable IL-18 polypeptide of any one of claims 1 to 14, wherein the cleavable linker further comprises a spacer sequence, and optionally the cleavable linker comprises two spacer sequences flanking both the N-terminus and the C-terminus of the amino acid sequence that is recognized and cleaved by one or more proteases.

16. 16. The activatable IL-18 polypeptide of any one of claims 1 to 15, wherein the cleavable linker comprises two or more sets of amino acid sequences that are recognized and cleaved by one or more proteases.

17. 17. The activatable IL-18 polypeptide of claim 16, wherein at least one of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is flanked by two spacer sequences in the cleavable linker, and each of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is flanked by two spacer sequences in the cleavable linker.

18. 18. The activatable IL-18 polypeptide of any one of claims 1 to 17, wherein the spacer sequence comprises a GS linker, and optionally the GS linker has a length of no more than about 10, 9, 8, 7, 6, or 5 amino acids.

19. 19. The activatable IL-18 polypeptide of any one of claims 1 to 18, wherein the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids.

20. 20. The activatable IL-18 polypeptide of any one of claims 1 to 19, wherein the cleavable linker consists of about 10, 15, 20, or 25 or more amino acids.

21. activatable IL-18 polypeptide, wherein the cleavable linker consists of about 25 to about 30 amino acids, and wherein the masking portion comprises the extracellular domain of IL-18Rα, and further optionally, wherein the masking portion comprises the amino acid sequence of any one of SEQ ID NOs: 353-356, or a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 353-356.

22. 22. The activatable IL-18 polypeptide of any one of claims 1 to 21, wherein the cleavable linker comprises any one of the amino acid sequences of SEQ ID NOs: 399-407.

23. 23. The activatable IL-18 polypeptide of any one of claims 1 to 22, wherein the IL-18 polypeptide comprises wild-type IL-18, optionally wherein the wild-type IL-18 comprises wild-type human IL-18, optionally wherein the wild-type human IL-18 comprises the amino acid sequence of SEQ ID NO:

1.

24. 24. The activatable IL-18 polypeptide of any one of claims 1 to 23, wherein the IL-18 polypeptide comprises an IL-18 variant polypeptide.

25. 25. The activatable IL-18 polypeptide of claim 24, wherein the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18, or (ii) no binding to IL-18BP.

26. 26. The activatable IL-18 polypeptide of claim 24 or 25, wherein the IL-18 variant polypeptide exhibits increased binding to IL-18Rα compared to the wild-type IL-18.

27. 27. The activatable IL-18 polypeptide of any one of claims 24 to 26, wherein the IL-18 variant polypeptide comprises at least three, four, five, or six mutations at G3, E6, D54, Q56, P57, N91, and R104 of the IL-18 polypeptide, the amino acid positions being relative to wild-type (WT) human IL-18 as set forth in SEQ ID NO:

1.

28. 28. The activatable IL-18 polypeptide of any one of claims 24 to 27, wherein the IL-18 variant polypeptide comprises a cysteine ​​at position 117 and a cysteine ​​at position 76, said amino acid positions relative to wild-type human IL-18 set forth in SEQ ID NO: 1, and optionally, the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38 and / or the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68.

29. 29. The activatable IL-18 polypeptide of any one of claims 24-28, wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 and 307-318, and optionally the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150.

30. 30. The activatable IL-18 polypeptide of any one of claims 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking portion, wherein the masking portion comprising a truncated pro-peptide of pro-IL-18 comprises the amino acid sequence of SEQ ID NO: 336, b) a cleavable linker, and c) an IL-18 variant polypeptide comprising any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150, and optionally wherein the cleavable linker has a length of about 50, 40, 30, 25, 20, 15, 12, or 10 or less amino acids.

31. 30. The activatable IL-18 polypeptide of any one of claims 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking moiety comprising the amino acid sequence of any one of SEQ ID NOs: 353-356, b) a cleavable linker, and c) an IL-18 variant polypeptide comprising any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150, and optionally wherein the cleavable linker has a length of about 10-40, 10-30, 20-30, or 25-30 amino acids.

32. 30. The activatable IL-18 polypeptide of any one of claims 1-29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) an IL-18 variant polypeptide comprising any one of SEQ ID NOs: 1-299 and 307-318, optionally wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; b) a cleavable linker; and c) a masking moiety comprising the amino acid sequence of any one of SEQ ID NOs: 353-356, wherein optionally the cleavable linker has a length of about 10-40, 10-30, 20-30, or 25-30 amino acids.

33. 33. The activatable IL-18 polypeptide of any one of claims 1 to 32, wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 340-343 and 345-352, or a) a functional variant thereof comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 340-343 and 345-352.

34. 34. The activatable IL-18 polypeptide of any one of claims 1 to 33, wherein the IL-18 polypeptide comprises a fusion protein comprising (1) a wild-type IL-18 or an IL-18 variant polypeptide, and (2) a half-life prolonging domain, optionally wherein the half-life prolonging domain comprises an albumin binding moiety or an antibody Fc domain or variant thereof, and optionally wherein the half-life prolonging domain is a human IgG Fc domain (such as hIgG1 Fc).

35. 35. The activatable IL-18 polypeptide of claim 34, wherein the IL-18 variant polypeptide of the fusion protein specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18, or (ii) no binding to IL-18BP.

36. 36. The activatable IL-18 polypeptide of claim 34 or 35, wherein the IL-18 variant polypeptide of the fusion protein exhibits increased binding to IL-18Rα compared to the wild-type IL-18.

37. 37. The activatable IL-18 polypeptide of any one of claims 34-36, wherein the human IgG1 Fc domain variant of the fusion protein comprises a N297A mutation (EU numbering), and optionally the human IgG1 Fc domain variant comprises the amino acid sequence of SEQ ID NO: 371 or 390.

38. 38. The activatable IL-18 polypeptide of any one of claims 34 to 37, wherein the C-terminus of the IL-18 variant polypeptide of the fusion protein is fused to the N-terminus of the human IgG Fc domain or variant thereof of the fusion protein.

39. 38. The activatable IL-18 polypeptide of any one of claims 34 to 37, wherein the C-terminus of the human IgG Fc domain or variant thereof of the fusion protein is fused to the N-terminus of the IL-18 variant polypeptide of the fusion protein.

40. 40. The activatable IL-18 polypeptide of any one of claims 34 to 39, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332, 378-389, and 408-418.

41. 41. The activatable IL-18 polypeptide of any one of claims 1 to 40, comprising the amino acid sequence of any one of SEQ ID NOs: 340-343, 345-352, 357-362, 364, 366-370, 391-398, and 408-418.

42. A dimer comprising two activatable IL-18 polypeptides according to any one of claims 1 to 41.

43. 43. The dimer of claim 42, wherein the dimer is a homodimer.

44. 43. The dimer of claim 42, wherein the dimer is a heterodimer.

45. A nucleic acid encoding an activatable IL-18 polypeptide according to any one of claims 1 to 41.

46. A vector comprising the nucleic acid of claim 45.

47. 47. A host cell comprising the nucleic acid of claim 45 or the vector of claim 46.

48. 1. A method for producing an activatable IL-18 polypeptide, comprising: (a) culturing the host cell of claim 47 under conditions in which the activatable IL-18 polypeptide is expressed; (b) recovering the activatable IL-18 polypeptide produced by the host cell.

49. 49. The method of claim 48, further comprising purifying the activatable IL-18 polypeptide.

50. A pharmaceutical composition comprising an activatable IL-18 polypeptide according to any one of claims 1 to 41, a nucleic acid according to claim 45, or a vector according to claim 46.

51. 51. A method of treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 50.

52. 52. The method of claim 51, wherein the disease is cancer.

53. 51. A method of activating IL-18 receptor-mediated signaling in an individual, comprising administering to said individual an effective amount of the pharmaceutical composition of claim 50.

54. 51. 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 50.

55. 55. The method of claim 54, wherein the stimulation comprises increasing the activity and / or number of immune cells that have experienced the antigen.

56. 56. The method of any one of claims 51 to 55, wherein the individual is a human.

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